# Patent

## Title
Pharmaceutical salts of macrocyclic modulators of the ghrelin receptor

## Abstract

The present invention provides novel conformationally-defined macrocyclic compounds that have been demonstrated to be selective modulators of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R1a and subtypes, isoforms and variants thereof). Methods of synthesizing the novel compounds are also described herein. These compounds are useful as agonists of the ghrelin receptor and as medicaments for treatment and prevention of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, genetic disorders, hyperproliferative disorders and inflammatory disorders.

## Background
RELATED APPLICATION INFORMATION
This application is a continuation under 35 U.S.C. §120 of U.S. patent application Ser. No. 11/149,731, filed Jun. 10, 2005, now U.S. Pat. No. 7,476,653, issued Jan. 13, 2009, which claims the benefit under 35 U.S.C. §120 of U.S. patent application Ser. No. 10/872,142, filed Jun. 18, 2004, now U.S. Pat. No. 7,521,420, issued Apr. 21, 2009, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/479,223, filed Jun. 18, 2003. This continuation application also claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/621,642, filed Oct. 26, 2004, U.S. Provisional Patent Application Ser. No. 60/622,005, filed Oct. 27, 2004, and U.S. Provisional Patent Application Ser. No. 60/642,271, filed Jan. 7, 2005. The disclosures of the above-referenced applications are incorporated herein by reference in their entireties.


FIELD OF THE INVENTION
The present invention relates to novel conformationally-defined macrocyclic compounds that bind to and/or are functional modulators of the ghrelin (growth hormone secretagogue) receptor including GHS-R1a and subtypes, isoforms and/or variants thereof. The present invention also relates to intermediates of these compounds, pharmaceutical compositions containing these compounds and methods of using the compounds. These novel macrocyclic compounds are useful as therapeutics for a range of disease indications. In particular, these compounds are useful for treatment and prevention of gastrointestinal disorders including, but not limited to, post-operative ileus, gastroparesis, including diabetic gastroparesis, opioid bowel dysfunction, chronic intestinal pseudo-obstruction, short bowel syndrome and functional gastrointestinal disorders.
BACKGROUND OF THE INVENTION
The improved understanding of various physiological regulatory pathways enabled through the research efforts in genomics and proteomics has begun to impact the discovery of novel pharmaceutical agents. In particular, the identification of key receptors and their endogenous ligands has created new opportunities for exploitation of these receptor/ligand pairs as therapeutic targets. For example, ghrelin is a recently characterized 28-amino acid peptide hormone isolated originally from the stomach of rats with the orthologue subsequently identified in humans. (Kojima, M.; Hosoda, H. et al. Nature 1999, 402, 656-660.) The existence of this peptide in a range of other species suggests a conserved and important role in normal body function. This peptide has been demonstrated to be the endogenous ligand for a previously orphan G protein-coupled receptor (GPCR), type 1 growth hormone secretatogue receptor (hGHS-R1a) (Howard, A. D.; Feighner, S. D.; et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996, 273, 974-977.) found predominantly in the brain (arcuate nucleus and ventromedial nucleus in the hypothalamus, hippocampus and substantia nigra) and pituitary. (U.S. Pat. No. 6,242,199; Intl. Pat. Appl. Nos. WO 97/21730 and WO 97/22004) The receptor has also been detected in other areas of the central nervous system (CNS) and in peripheral tissues, for instance adrenal and thyroid glands, heart, lung, kidney, and skeletal muscles. This receptor was identified and cloned prior to the isolation and characterization of the endogenous peptide ligand and is distinct from other receptors: involved in the regulation of growth hormone (GH) secretion, in particular, the growth hormone-releasing hormone (GHRH) receptor.
A unique characteristic of both the rat and human peptides is the presence of the n-octanoyl (Oct) moiety on Ser3. However, the des-acyl form predominates in circulation, with approximately 90% of the hormone in this form. This group is derived from a post-translational modification and appears relevant for bioactivity and possibly also for transport into the CNS. (Banks, W. A.; Tschöp, M.; Robinson, S. M.; Heiman, M. L. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J. Pharmacol. Exp. Ther. 2002, 302, 822-827.) In a GH-releasing assay, the des-octanoyl form of the hormone was at least 100-fold less potent than the parent peptide, although it has been suggested that the des-acyl species may be responsible for some of the other biological effects associated with ghrelin. This des-acyl form has also been postulated to be primarily responsible for the cardiovascular and cell proliferation effects attributed to ghrelin, while the acylated form participates in maintenance of energy balance and growth hormone release. (Baldanzi, G., Filighenddu, N.; Cutrupi, S.; et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI-3 kinase/AKT. J. Cell Biol. 2002, 159, 1029-1037) Similarly, des-Gln14-ghrelin and its octanoylated derivative have been isolated as endogenous forms of the hormone arising from alternative splicing of the ghrelin gene, but both are found to be inactive in stimulating GH release in vivo. (Hosoda, H.; Kojima, M.; Matsuo, H.; Kangawa, K. Purification and characterization of rat des-Gln14-ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor. J. Biol. Chem. 2000, 275, 21995-2120.). Other minor forms of ghrelin produced by post-translational processing have been observed in plasma, although no specific activity has been attributed to them. (Hosoda, H.; Kojima, M.; et al. Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing. J. Biol. Chem. 2003, 278, 64-70.)
Even prior to the isolation of this receptor and its endogenous peptide ligand, a significant amount of research was devoted to finding agents that can stimulate GH secretion. The proper regulation of human GH has significance not only for proper body growth, but also a range of other critical physiological effects. Since GH and other GH-stimulating peptides, such as GHRH and growth hormone releasing factor (GRF), as well as their derivatives and analogues, are administered via injection, to better take advantage of these positive effects, attention was focused on the development of orally active therapeutic agents that would increase GH secretion, termed GH secretagogues (GHS). Additionally, use of these agents was expected to more closely mimic the pulsatile physiological release of GH.
Beginning with the identification of the growth hormone-releasing peptides (GHRP) in the late 1970's, (Bowers, C. Y. Growth hormone-releasing peptides: physiology and clinical applications. Curr. Opin. Endocrinol. Diabetes 2000, 7, 168-174; Camanni, F.; Ghigo, E.; Arvat, E. Growth hormone-releasing peptides and their analogs. Front. Neurosci. 1998, 19, 47-72; Locatelli, V.; Torsello, A. Growth hormone secretagogues: focus on the growth hormone-releasing peptides. Pharmacol. Res. 1997, 36, 415-423.) a host of agents have been studied for their potential to act as GHS. In addition to their stimulation of GH release and concomitant positive effects in that regard, GHS were projected to have utility in the treatment of a variety of other disorders, including wasting conditions (cachexia) as seen in HIV patients and cancer-induced anorexia, musculoskeletal frailty in the elderly, and growth hormone deficient diseases. Many efforts over the past 25 years have yielded a number of potent, orally available GHS. (Smith, R. G.; Sun, Y. X.; Beatancourt, L.; Asnicar, M. Growth hormone secretagogues: prospects and pitfalls. Best Pract. Res. Clin. Endocrinol. Metab. 2004, 18, 333-347; Fehrentz, J.-A.; Martinez, J.; Boeglin, D.; Guerlavais, V.; Deghenghi, R. Growth hormone secretagogues: Past, present and future. IDrugs 2002, 5, 804-814; Svensson, J. Exp. Opin. Ther. Patents 2000, 10, 1071-1080; Nargund, R. P.; Patchett, A. A.; et al. Peptidomimetic growth hormone secretagogues. Design considerations and therapeutic potential. J. Med. Chem. 1998, 41, 3103-3127; Ghigo, E; Arvat, E.; Camanni, F. Orally active growth hormone secretagogues: state of the art and clinical perspective. Ann. Med. 1998, 30, 159-168; Smith, R. G.; Van der Ploeg, L. H. T.; Howard, A. D.; Feighner, S. D.; et al. Peptidomimetic regulation of growth hormone secretion. Endocr. Rev. 1997, 18, 621-645.) These include small peptides, such as hexarelin (Zentaris) and ipamorelin (Novo Nordisk), and adenosine analogues, as well as small molecules such as carpomorelin (Pfizer), L-252,564 (Merck), MK-0677 (Merck), NN703 (Novo Nordisk), G-7203 (Genentech), S-37435 (Kaken) and SM-130868 (Sumitomo), designed to be orally active for the stimulation of growth hormone. However, clinical testing with such agents have rendered disappointing results due to, among other things, lack of efficacy over prolonged treatment or undesired side effects, including irreversible inhibition of cytochrome P450 enzymes (Zdravkovic M.; Olse, A. K.; Christiansen, T.; et al. Eur. J. Clin. Pharmacol. 2003, 58, 683-688.) Therefore, there remains a need for pharmacological agents that could effectively target this receptor for therapeutic action.
Despite its involvement in GH modulation, ghrelin is primarily synthesized in the oxyntic gland of the stomach, although it is also produced in lesser amounts in other organs, including the kidney, pancreas and hypothalamus. (Kojima, M.; Hsoda, H.; Kangawa, K. Purification and distribution of ghrelin: the natural endogenous ligand for the growth hormone secretagogue receptor. Horm. Res. 2001, 56 (Suppl. 1), 93-97; Ariyasu, H.; Takaya, K.; Tagami, T.; et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J. Clin. Endocrinol. Metab. 2001, 86, 4753-4758) In addition to its role in stimulating GH release, the hormone has a variety of other endocrine and non-endocrine functions (Broglio, F.; Gottero, C.; Arvat, E.; Ghigo, E. Endocrine and non-endocrine actions of ghrelin. Horm. Res. 2003, 59, 109-117) and has been shown to interact with a number of other systems in playing a role in maintaining proper energy balance. (Horvath, T. L.; Diano, S.; Sotonyi, P.; Heiman, M.; Tschop, M. Ghrelin and the regulation of energy balance—a hypothalamic perspective. Endocrinology 2001, 142, 4163-4169; Casanueva, F. F.; Dieguez, C. Ghrelin: the link connecting growth with metabolism and energy homeostasis. Rev. Endocrinol. Metab. Disord. 2002, 3, 325-338). In particular, the peptide ghrelin plays a role as an orexigenic signal in the control of feeding, in which it acts to counteract the effects of leptin. Indeed, it was the first gut peptide proven to have such orexigenic properties. (Kojima, M.; Kangawa, K. Ghrelin, an orexigenic signaling molecule from the gastrointestinal tract. Curr. Opin. Pharmacology 2002, 2, 665-668.) The hormone also is implicated in the hypothalamic regulation of the synthesis and secretion of a number of other neuropeptides involved in appetite and feeding behavior. Levels of ghrelin are elevated in response to fasting or extended food restriction. (Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.; et al. A role for ghrelin in the central regulation of feeding. Nature 2001, 409, 194-198) For example, subjects suffering with anorexia or bulimia exhibit elevated ghrelin levels. Circulating levels of the hormone have been found to rise before meals and fall after meals. In addition, diet-induced weight loss leads to increased ghrelin levels, although obese subjects who have gastric bypass surgery do not likewise experience such an increase. (Cummings, D. E.; Weigle, D. S.; Frayo, R. S.; et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N. Engl. J. Med. 2002, 346, 1623-1630)
This intimate involvement of ghrelin in control of food intake and appetite has made it an attractive target for obesity research. Indeed, few other natural substances have been demonstrated to be involved in the modulation of both GH secretion and food intake.
An additional effect of ghrelin that has not to date been exploited for therapeutic purposes is in modulating gastric motility and gastric acid secretion. The pro-kinetic activity appears to be independent of the GH-secretory action and is likely mediated by the vagal-cholinergic muscarinic pathway. The dose levels required are equivalent to those necessary for the hormone's GH and appetite stimulation actions. It is noteworthy that, in contrast to its inactivity for ghrelin's other actions, the des-Gln14 peptide demonstrated promotion of motility as well. (Trudel, L.; Bouin, M.; Tomasetto, C.; Eberling, P.; St-Pierre, S.; Bannon, P.; L'Heureux, M. C.; Poitras, P. Two new peptides to improve post-operative gastric ileus in dog. Peptides 2003, 24, 531-534; Trudel, L.; Tomasetto, C.; Rio, M. C.; Bouin, M.; Plourde, V.; Eberling, P.; Poitras, P. Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rats. Am. J. Physiol. 2002, 282, G948-G952; Peeters, T. L. Central and peripheral mechanisms by which ghrelin regulates gut motility. J. Physiol. Pharmacol. 2003, 54(Supp. 4), 95-103.)
Ghrelin also has been implicated in various aspects of reproduction and neonatal development. (Arvat, E.; Gianotti, L.; Giordano, R.; et al. Growth hormone-releasing hormone and growth hormone secretagogue-receptor ligands. Focus on reproductive system. Endocrine 2001, 14, 35-43) Also of significance are the cardiovascular effects of ghrelin, since the peptide is a powerful vasodilator. As such, ghrelin agonists have potential for the treatment of chronic heart failure (Nagaya, N.; Kangawa, K. Ghrelin, a novel growth hormone-releasing peptide, in the treatment of chronic heart failure. Regul. Pept. 2003, 114, 71-77; Nagaya, N.; Kangawa, K. Ghrelin improves left ventricular dysfunction and cardiac cachexia in heart failure. Curr. Opin. Pharmacol 2003, 3, 146-151; Bedendi, I.; Alloatti, G.; Marcantoni, A.; Malan, D.; Catapano, F.; Ghe, C.; et al. Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin. Eur. J. Pharmacol. 2003, 476, 87-95) Intl. Pat. Appl. Publ. WO 2004/014412 describes the use of ghrelin agonists for the protection of cell death in myocardial cells and as a cardioprotectant treatment for conditions leading to heart failure. Lastly, evidence has been obtained that ghrelin may have implications in anxiety and other CNS disorders as well as the improvement of memory. (Carlini, V. P., Monzon, M. E., Varas, M. M., Cragnolini, A. B., Schioth, H. B., Scimonelli, T. N., de Barioglio, S. R. Ghrelin increases anxiety-like behavior and memory retention in rats. Biochem. Biophys. Res. Commun. 2002, 299, 739-743)
The myriad effects of ghrelin in humans have suggested the existence of subtypes for its receptor, although none have as yet been identified. (Torsello, A.; Locatelli, Y.; Melis, M. R.; Succu, S.; Spano, M. S.; Deghenghi, R.; Muller, E. E.; Argiolas, A.; Torsello, A.; Locatelli, V.; et al. Differential orexigenic effects of hexarelin and its analogs in the rat hypothalamus: indication for multiple growth hormone secretagogue receptor subtypes. Neuroendocrinology 2000, 72, 327-332.) However, a truncated, inactive form of GHS-R1a, termed GHS-R1b, was isolated and identified at the same time as the original characterization. Evidence is mounting that additional receptor subtypes could be present in different tissues to explain the diverse effects displayed by the endogenous peptides and synthetic GHS. For instance, high affinity binding sites for ghrelin and des-acyl ghrelin have also been found in breast cancer cell lines, cardiomyocytes, and guinea pig heart that are involved in mediating the antiproliferative, cardioprotective and negative cardiac inotropic effects of the peptides. Similarly, specific GHS binding sites besides GHS-R1a and GHS-R1b have been found in prostate cancer cells. Further, ghrelin and des-acyl ghrelin exert different effects on cell proliferation in prostate carcinoma cell lines. (Cassoni, P.; Ghé, C.; Marrocco, T.; et al. Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur. J. Endocrinol. 2004, 150, 173-184) These various receptor subtypes may then be implicated independently in the wide array of biological activities displayed by the endogenous peptides and synthetic GHS. Indeed, recently, the existence of receptor subtypes was offered as an explanation for the promotion of fat accumulation by ghrelin, despite its potent stimulation of the lipolytic hormone, growth hormone. (Thompson, N. M.; Gill, D. A. S.; Davies, R.; Loveridge, N.; Houston, P. A.; Robinson, I. C. A. F.; Wells, T. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 2004, 145, 234-242.) Further, this work suggested that the ratio of ghrelin and des-acyl ghrelin production could help regulate the balance between adipogenesis and lipolysis in response to nutritional status.
The successful creation of peptidic ghrelin analogues that separate the GH-modulating effects of ghrelin from the effects on weight gain and appetite provides strong evidence for the existence and physiological relevance of other receptor subtypes. (Halem, H. A.; Taylor, J. E.; Dong, J. Z.; Shen, Y.; Datta, R.; Abizaid, A.; Diano, S.; Horvath, T.; Zizzari, P.; Bluet-Pajot, M.-T.; Epelbaum, J.; Culler, M. D. Novel analogs of ghrelin: physiological and clinical implications. Eur. J. Endocrinol. 2004, 151, S71-S75.) BIM-28163 functions as an antagonist at the GHS-R1a receptor and inhibits receptor activation by native ghrelin. However, this same molecule is a full agonist with respect to stimulating weight gain and food intake. Additionally, the existence of a still uncharacterized receptor subtype has been proposed based on binding studies in various tissues that showed differences between peptidic and non-peptidic GHS. (Ong, H.; Menicoll, N.; Escher, F.; Collu, R.; Deghenghi, R.; Locatelli, V.; Ghigo, E.; Muccioli, G.; Boghen, M.; Nilsson, M. Endocrinology 1998, 139, 432-435.) Differences between overall GHS-R expression and that of the GHS-R1a subtype in rat testis have been reported. (Barreiro, M. L.; Suominen, J. S.; Gaytan, F.; Pinilla, L.; Chopin, L. K.; Casanueva, F. F.; Dieguez, C.; Aguilar, E.; Toppari, J.; Tena-Sempere, M. Developmental, stage-specific, and hormonally regulated expression of growth hormone secretagogue receptor messenger RNA in rat testis. Biol. Reproduction 2003, 68, 1631-1640) A GHS-R subtype on cholinergic nerves is postulated as an explanation for the differential actions of ghrelin and a peptidic GHS on neural contractile response observed during binding studies at the motilin receptor. (Depoortere, I.; Thijs, T.; Thielemans, L.; Robberecht, P.; Peeters, T. L. Interaction of the growth hormone-releasing peptides ghrelin and growth hormone-releasing peptide-6 with the motilin receptor in the rabbit gastric antrum. J. Pharmacol. Exp. Ther. 2003, 305, 660-667.)
The variety of activities associated with the ghrelin receptor could also be due to different agonists activating different signaling pathways as has been shown for ghrelin and adenosine, both of which interact as agonists at GHS-R1a (Carreira, M. C.; Camina, J. P.; Smith, R. G.; Casanueva, F. F. Agonist-specific coupling of growth hormone secretagogue receptor type 1a to different intracellular signaling systems. Role of adenosine. Neuroendocrinology 2004, 79, 13-25.)
The functional activity of a GPCR has been shown to often require the formation of dimers or other multimeric complexes with itself or other proteins. (Park, P. S.; Filipek, S.; Wells, J. W.; Palczewski, K. Oligomerization of G protein-coupled receptors: past, present, and future. Biochemistry 2004, 43, 15643-15656; Rio, C. D.; Jordan, B. A.; Gomes, I.; Devi, L. A. G-protein-coupled receptor dimerization: modulation of receptor function. Pharmacol. Ther. 2001, 92, 71-87; Devi, L. A. Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking. Trends Pharmacol. Sci. 2001, 22, 532-537.) Likewise, the activity of the ghrelin receptor might also be at least partially governed by such complexes. For example, certain reports indicate that interaction of GHS-R1a with GHRH (Cunha, S. R.; Mayo, K. E. Ghrelin and growth hormone (GH) secreatagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3′,5′-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors. Endocrinology 2002, 143, 4570-4582; Malagón, M. M.; Luque, R. M.; Ruiz-Guerrero, E.; Rodríguez-Pacheco, F.; García-Navarro, S.; Casanueva, F. F.; Gracia-Navarro, F.; Castafño, J. P. Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes Endocrinology 2003, 144, 5372-5380) or between receptor subtypes (Chan, C. B.; Cheng, C. H. K. Identification and functional characterization of two alternatively spliced growth hormone secretagogue receptor transcripts from the pituitary of black seabream Acanthopagrus schlegeli. Mol. Cell. Endocrinol. 2004, 214, 81-95) may be involved in modulating the function of the receptor.
The vast majority of reported approaches to exploiting the ghrelin receptor for therapeutic purposes have focused on modulating metabolic functions. Similarly, the vast majority of literature on GHS focuses on conditions that can be treated via its GH promoting actions. Some embodiments of the invention described herein, in particular, take advantage of selective activation of the ghrelin receptor to provide an avenue for the treatment of diseases characterized by GI dysmotility. The improved GI motility observed with ghrelin demonstrates that ghrelin agonists may be useful in correcting conditions associated with reduced or restricted motility (Murray, C. D. R.; Kamm, M. A.; Bloom, S. R.; Emmanuel, A. V. Ghrelin for the gastroenterologist: history and potential. Gastroenterology 2003, 125, 1492-1502; Fujino, K.; Inui, A.; Asakawa, A.; Kihara, N.; Fujimura, M.; Fujimiya, M. Ghrelin induces fasting motor activity of the gastrointestinal tract in conscious fed rats. J. Physiol. 2003, 550, 227-240; Edholm, T.; Levin, F.; Hellstrom, P. M.; Schmidt, P. T. Ghrelin stimulates motility in the small intestine of rats through intrinsic cholinergic neurons. Regul. Pept. 2004, 121, 25-30.)
Included among these conditions is post-operative ileus (POI, Luckey, A.; Livingston, E.; Taché, Y. Mechanisms and treatment of postoperative ileus. Arch. Surg. 2003, 138, 206-214; Baig, M. K.; Wexner, S. D. Postoperative ileus: a review. Dis. Colon Rectum 2004, 47, 516-526). POI is defined as the impairment of GI motility that routinely occurs following abdominal, intestinal, gynecological and pelvic surgeries. In the U.S. alone, 4.3 million surgeries annually induce POI, accounting for an economic impact of over $1 billion. POI is considered a deleterious response to surgical manipulation with a variable duration that generally persists for 72 hours. It is characterized by pain, abdominal distention or bloating, nausea and vomiting, accumulation of gas and fluids in the bowel, and delayed passage of stool. Patients are neither able to tolerate oral feeding nor to have bowel movements until gut function returns. POI leads to numerous undesirable consequences, including increased patient morbidity, the costly prolongation of hospital stays and, further, is a major cause of hospital readmission. In addition, opiate drugs given as analgesics after surgery exacerbate this condition due to their well-recognized side effect of inhibiting bowel function.
Surgical manipulation of the stomach or intestine causes a disorganization of the gut-brain signaling pathways, impairing GI activity and triggering POI. Ghrelin acts locally in the stomach to stimulate and coordinate the firing of vagal afferent neurons and thereby modulate gut motility. Thus, ghrelin accelerates gastric emptying in humans and is a potent agent proven to treat POI in animal models. Ghrelin agonists duplicate the effects of ghrelin, thus targeting directly the underlying cause of POI to accelerate normalization of gut function and enable more rapid discharge from the hospital. Intravenous administration is often the preferred route of treatment for POI due to the impaired GI motility in these patients that impedes oral therapy. No agent is currently approved by the U.S. FDA specifically for the treatment of POI.
Another major motility disorder is gastroparesis, a particular problem for both type I and type II diabetics. (Camilleri, M. Advances in diabetic gastroparesis. Rev. Gastroenterol. Disord. 2002, 2, 47-56; Tack et al. Gastroenterology 2004; 126: A485; Moreaux, B.; VandenBerg, J.; Thielmans, L.; Meulemans, A.; Coulie, B. Activation of the GHS receptor accelerates gastric emptying in the dog. Digestive Disease Week, 15-20 May 2004, New Orleans, La., USA Abstract M1009; Tack et al. Gastroenterology 2004, 126: A74) Gastroparesis (“stomach paralysis”) is a syndrome characterized by delayed gastric emptying in the absence of any mechanical obstruction. It is variably characterized by abdominal pain, nausea, vomiting, weight loss, anorexia, early satiety, malnutrition, dehydration, gastroesophageal reflux, cramping and bloating. This chronic condition can lead to frequent hospitalization, increased disability and decreased quality of life. Severe, symptomatic gastroparesis is common in individuals suffering from diabetes, affecting from 5-10% of diabetics for a total patient population of 1 million in the U.S. alone. Neuropathy is a frequent, debilitating complication of diabetes. Visceral neuropathy results in GI dysfunction, especially involving the stomach, and leading to impaired gastric motility. Ghrelin promotes gastric emptying both by stimulating the vagus nerve and via direct prokinetic action at the gastric mucosa. Moreover, a recent clinical study indicates that intravenous administration of the natural ghrelin peptide is an effective acute therapy in diabetic gastroparesis patients. A ghrelin agonist would therefore be highly effective in overcoming the fundamental motility barrier faced by gastroparesis patients and correcting this condition. As with POI, no accepted or efficacious therapy for diabetic gastroparesis is available and most current therapies aim to provide only symptomatic relief. Further, many of the therapeutics in development have a mechanism of action similar to earlier products that have failed in this indication. Surgical procedures may ameliorate the disease process, but offer no possibility of cure.
Opioid-induced bowel dysfunction (OBD, Kurz, A.; Sessler, D. J. Opioid-Induced Bowel Dysfunction. Drugs 2003, 63, 649-671.) is the term applied to the confluence of symptoms involving the reduced GI motility that results from treatment with opioid analgesics. Approximately 40-50% of patients taking opioids for pain control experience OBD. It is characterized by hard, dry stools, straining, incomplete evacuation, bloating, abdominal distension and increased gastric reflux. In addition to the obvious short-term distress, this condition leads to physical and psychological deterioration in patients undergoing long term opioid treatment. Further, the dysfunction can be so severe as to become a dose-limiting adverse effect that actually prevents adequate pain control. As with POI, a ghrelin agonist can be expected to counteract the dysmotility resulting from opioid use.
Two less common syndromes may also be helped through the GI motility stimulation effects of ghrelin and ghrelin agonists. Short bowel syndrome is a condition that occurs after resection of a substantial portion of small intestine and is characterized by malnutrition. Patients are observed to have decreased ghrelin levels resulting from loss of the ghrelin-producing neuroendocrine cells of the intestine. It is possible the short bowel feeds back on the release of the hormone. (Krsek, M.; Rosicka, M.; Haluzik, M.; et al. Plasma ghrelin levels in patients with short bowel syndrome. Endocr. Res. 2002, 28, 27-33.) Chronic intestinal pseudo-obstruction is a syndrome defined by the presence of chronic intestinal dilation and dysmotility in the absence of mechanical obstruction or inflammation. Both genetic and acquired causes are known to result in this disorder, which affects high numbers of individuals worldwide annually. (Hirano, I.; Pandolfino, J. Chronic intestinal pseudo-obstruction. Dig. Dis. 2000, 18, 83-92.)
Other conditions and disorders that could be addressed through stimulation of the ghrelin receptor are: emesis such as caused by cancer chemotherapy, constipation such as associated with the hypomotility phase of irritable bowel syndrome (IBS), delayed gastric emptying associated with wasting conditions, gastroesophageal reflux disease (GERD), gastric ulcers (Sibilia, V.; Rindi, G.; Pagani, F.; Rapetti, D.; Locatelli, V.; Torsello, A.; Campanini, N.; Degenghi, R.; Netti, C. Ghrelin protects against ethanol-induced gastric ulcers in rats: studies on the mechanism of action. Endocrinology 2003, 144, 353-359.) and Crohn's disease.
Additionally, GI dysmotility is a significant problem in other mammals as well. For example, the motility dysfunction termed ileus or colic is the number one cause of mortality among horses. Further, ileus is one of the most common complications of equine intestinal surgery, in other words, post-operative ileus. This condition may also have a non-surgical etiology. Some horses may be predisposed to ileus based upon the anatomy and functioning of their digestive tract. Virtually any horse is susceptible to colic with only minor differences based upon age, sex and breed. Additionally, ileus may affect other animals, for example canines. (Roussel, A. J., Jr.; Cohen, N. D.; Hooper, R. N.; Rakestraw, P. C. Risk factors associated with development of postoperative ileus in horses. J. Am. Vet. Med. Assoc. 2001, 219, 72-78; Van Hoogmoed, L. M.; Nieto, J. E.; Snyder, J. R.; Harmon, F. A. Survey of prokinetic use in horses with gastrointestinal injury. Vet. Surg. 2004, 33, 279-285.)
Importantly, for most of the above conditions, no specific, approved therapeutics exist and most therapies simply address symptomatic relief. However, specific modulation of the ghrelin receptor will provide an opportunity to directly target the site of pathophysiological disturbance to better treat the underlying condition and improve clinical outcome. Further, unlike other agents that interact at the GHS-R1a receptor, the compounds of the invention are believed not to stimulate concurrent GH secretion. This separation of the gastrointestinal and GH effects has not previously been reported for any modulators of this receptor. However, as already mentioned, the existence of analogues that separate the appetite control and GH modulatory effects associated with ghrelin has been recently reported (Eur. J. Endocrinol. 2004, 151, S71-S75.)
WO 01/00830 reports on short gastrointestinal peptides (SGIP) that secrete growth hormone and also promote GI motility, but these were not shown to be due to action at the ghrelin receptor. U.S. Pat. No. 6,548,501 discloses specific compounds, but as GHS, useful for stimulation of GI motility. Moreover, other endogenous factors are known to stimulate secretion of GH, but do not promote GI motility. Indeed, many actually inhibit this physiological function. Specific receptor agonists such as the compounds of the present invention have much better potential to be selective and effective therapeutic agents.
Work has continued at the development of potent and selective GHS with a number of small molecule derivatives now being known as has been recently summarized. (Carpino, P. Exp. Opin. Ther. Patents 2002, 12, 1599-1618.) Specific GHS are described in the following U.S. Pat. Nos. and Intl. Pat. Appl. Publs. WO 89/07110; WO 89/07111; WO 92/07578; WO 93/04081; WO 94/11012; WO 94/13696; WO 94/19367; WO 95/11029; WO 95/13069; WO 95/14666; WO 95/17422; WO 95/17423; WO 95/34311; WO 96/02530; WO 96/15148; WO 96/22996; WO 96/22997; WO 96/24580; WO 96/24587; WO 96/32943; WO 96/33189; WO 96/35713; WO 96/38471; WO 97/00894; WO 97/06803; WO 97/07117; WO 97/09060; WO 97/11697; WO 97/15191; WO 97/15573; WO 97/21730; WO 97/22004; WO 97/22367; WO 97/22620; WO 97/23508; WO 97/24369; WO 97/34604; WO 97/36873; WO 97/38709; WO 97/40023; WO 97/40071; WO 97/41878; WO 97/41879; WO 97/43278; WO 97/44042; WO 97/46252; WO 98/03473; WO 98/10653; WO 98/18815; WO 98/22124; WO 98/46569; WO 98/51687; WO 98/58947; WO 98/58948; WO 98/58949; WO 98/58950; WO 99/08697; WO 99/09991; WO 99/36431; WO 99/39730; WO 99/45029; WO 99/58501; WO 99/64456; WO 99/65486, WO 99/65488; WO 00/01726; WO 00/10975; WO 01/47558; WO 01/92292; WO 01/96300; WO 01/97831; U.S. Pat. No. 3,239,345; U.S. Pat. No. 4,036,979; U.S. Pat. No. 4,411,890; U.S. Pat. No. 5,492,916; U.S. Pat. No. 5,494,919; U.S. Pat. No. 5,559,128; U.S. Pat. No. 5,663,171; U.S. Pat. No. 5,721,250; U.S. Pat. No. 5,721,251; U.S. Pat. No. 5,723,616; U.S. Pat. No. 5,726,319; U.S. Pat. No. 5,767,124; U.S. Pat. No. 5,798,337; U.S. Pat. No. 5,830,433; U.S. Pat. No. 5,919,777; U.S. Pat. No. 6,034,216; U.S. Pat. No. 6,548,501; U.S. Pat. No. 6,559,150; U.S. Pat. No. 6,576,686; U.S. Pat. No. 6,686,359; and U.S. Pat. Appl. Nos. 2002/0168343; 2003/100494; 2003/130284; 2003/186844.
Despite this immense body of work, cyclic compounds have rarely been found to act at the receptor. When they have, antagonist activity has been more prevalent. For example, the 14-amino acid compound, vapreotide, an SRIH-14 agonist and somatostatin mimetic, was demonstrated to be a ghrelin antagonist. (Deghenghi R, Papotti M, Ghigo E, et al. Somatostatin octapeptides (lanreotide, octreotide, vapreotide, and their analogs) share the growth hormone-releasing peptide receptor in the human pituitary gland. Endocrine 2001, 14, 29-33.) The binding and antagonist activities of analogues of cortistatin, a cyclic neuropeptide known to bind nonselectively to somatostatin receptors, to the growth hormone secretagogue receptor have been reported (Intl. Pat. Appl. WO 03/004518). (Deghenghi R, Broglio F, Papotti M, et al. Targeting the ghrelin receptor—Orally active GHS and cortistatin analogs. Endocrine 2003, 22, 13-18) In particular, one of these analogues, EP01492 (cortistatin-8) has been advanced into preclinical studies for the treatment of obesity as a ghrelin antagonist. These compounds exhibit an IC50 of 24-33 nM. In addition, these cyclic compounds and their derivatives, plus their use with metal binding agents have been described for their ability to be useful for radiodiagnostic or radiotherapeutic use in the treatment of tumors and acromegaly.
Cyclic and linear analogues of growth hormone 177-191 have been studied as treatments for obesity (WO 99/12969), with one particular compound, AOD9604, having entered the clinic for this indication. A compound already studied that is most similar to the molecules of the present invention is the GHS, G-7203 (EC50=0.43 nM), the cyclic peptide analogue of the growth hormone releasing peptide, GHRP-2 (Elias, K. A.; Ingle, G. S.; Burnier, J. P.; Hammonds, G.; McDowell, R. S.; Rawson, T. E.; Somers, T. C.; Stanley, M. S.; Cronin, M. J. In vitro characterization of four novel classes of growth hormone-releasing peptide. Endocrinol. 1995, 136, 5694-5699) However, simplification of this cyclic derivative led to still potent, linear compounds, whereas, for compounds of the invention, linear analogues have been found to be devoid of ghrelin receptor activity.
The macrocyclic compounds of the invention possess agonist activity. As previously mentioned, however, unlike other agonists of the hGHS-R1a receptor, the compounds of the invention unexpectedly have an insignificant stimulatory effect on the release of growth hormone. Accordingly, the compounds of the present invention can exhibit selective action in the GI tract or for metabolic disorders without side effects due to GH release.
SUMMARY OF THE INVENTION
The present invention provides novel conformationally-defined macrocyclic compounds. These compounds can function as modulators, in particular agonists, of the ghrelin (growth hormone secretagogue) receptor (GHS-R1a).
According to aspects of the present invention, the present invention relates to compounds according to formula I, II and/or III:




or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof,

wherein:

R1 is hydrogen or the side chain of an amino acid, or alternatively R1 and R2 together form a 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8 as defined below, or alternatively R1 and R9 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R2 is hydrogen or the side chain of an amino acid, or alternatively, R1 and R2 together form a 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8 as defined below; or alternatively R2 and R9 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R3 is hydrogen or the side chain of an amino acid, or alternatively R3 and R4 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O or S atom in the ring, wherein the ring is optionally substituted with R8 as defined below, or alternatively, R3 and R7 or R3 and R11 together form a 4-, 5-, 6-, 7- or 8-membered heterocyclic ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R4 is hydrogen or the side chain of an amino acid, or alternatively R4 and R3 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O or S atom in the ring, wherein the ring is optionally substituted with R8 as defined below, or alternatively R4 and R7 or R4 and R11 together form a 4-, 5-, 6-, 7- or 8-membered heterocyclic ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R5 and R6 are each independently hydrogen or the side chain of an amino acid or alternatively R5 and R6 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R7 is hydrogen, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, or a substituted heterocyclic group, or alternatively R3 and R7 or R4 and R7 together form a 3-, 4-, 5-, 6-, 7- or 8-membered heterocyclic ring optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as described below;
R8 is substituted for one or more hydrogen atoms on the 3-, 4-, 5-, 6-, 7- or 8-membered ring structure and is independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido, or, alternatively, R8 is a fused cycloalkyl, a substituted fused cycloalkyl, a fused heterocyclic, a substituted fused heterocyclic, a fused aryl, a substituted fused aryl, a fused heteroaryl or a substituted fused heteroaryl ring when substituted for hydrogen atoms on two adjacent atoms;
X is O, NR9 or N(R10)2+;

    
    
        wherein R9 is hydrogen, lower alkyl, substituted lower alkyl, sulfonyl, sulfonamido or amidino and R10 is hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R9 and R1 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined above;
    
    


Z1 is O or NR11,

    
    
        wherein R11 is hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R3 and R11 together or R4 and R11 together form a 4-, 5-, 6-, 7- or 8-membered heterocyclic ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined above;
    
    


Z2 is O or NR12, wherein R12 is hydrogen, lower alkyl, or substituted lower alkyl;
m, n and p are each independently 0, 1 or 2;
T is a bivalent radical of formula IV:

—U—(CH2)d—W—Y—Z—(CH2)e—  (IV)

    
    
        wherein d and e are each independently 0, 1, 2, 3, 4 or 5; Y and Z are each optionally present; U is —CR21R22— or —C(═O)— and is bonded to X of formula I; W, Y and Z are each independently selected from the group consisting of —O—, —NR23—, —S—, —Se, —SO2—, —C(═O)—O—, —O—C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —SO2—NH—, —NH—SO2—, —CR24R25—, —CH═CH— with the configuration Z or E, —C≡C and the ring structures below:
    
    






    
    
        
        
            wherein G1 and G2 are each independently a covalent bond or a bivalent radical selected from the group consisting of —O—, —NR39—, —S—, —SO—, —SO2—, —C(═O)—, —C(═O)—, —(C(═O), —C(═O)NH—, —NH—C(═O)—, —SO2—NH—, —NH—SO2—, —CR40R41—, —CH═CH— with the configuration Z or E, and —C≡C; with GI being bonded closest to the group U, wherein any carbon atom in the rings not otherwise defined, can be replaced by N, with the proviso that the ring cannot contain more than four N atoms; K1, K2, K3, K4 and K5 are each independently O, NR42 or S, wherein R42 is as defined below;
            R21 and R22 are each independently hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R21 and R22 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N, wherein the ring is optionally substituted with R8 as defined above;
            R23, R39 and R42 are each independently hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, amidino, sulfonyl or sulfonamido;
            R24 and R25 are each independently hydrogen, lower alkyl, substituted lower alkyl, RAA, wherein RAA is a side chain of an amino acid such as a standard or unusual amino acid, or alternatively R24 and R25 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N; or alternatively one of R24 or R25 is hydroxy, alkoxy, aryloxy, amino, mercapto, carbamoyl, amidino, ureido or guanidino while the other is hydrogen, lower alkyl or substituted lower alkyl, except when the carbon to which R24 and R25 are bonded is also bonded to another heteroatom;
            R26, R31, R35 and R38 are each optionally present and, when present, are substituted for one or more hydrogen atoms on the indicated ring and each is independently selected from the group consisting of halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido;
            R27 is optionally present and is substituted for one or more hydrogen atoms on the indicated ring and each is independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido;
            R28, R29, R30, R32, R33, R34, R36 and R37 are each optionally present and, when no double bond is present to the carbon atom to which it is bonded in the ring, two groups are optionally present, and when present, is substituted for one hydrogen present in the ring, or when no double bond is present to the carbon atom to which it is bonded in the ring, is substituted for one or both of the two hydrogen atoms present on the ring and each is independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl, sulfonamido and, only if a double bond is present to the carbon atom to which it is bonded, halogen; and
            R40 and R41 are each independently hydrogen, lower alkyl, substituted lower alkyl, RAA as defined above, or alternatively R40 and R41 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N wherein the ring is optionally substituted with R8 as defined above, or alternatively one of R40 and R41 is hydroxy, alkoxy, aryloxy, amino, mercapto, carbamoyl, amidino, ureido or guanidino, while the other is hydrogen, lower alkyl or substituted lower alkyl, except when the carbon to which R40 and R41 are bonded is also bonded to another heteroatom;
        
        
        with the proviso that T is not an amino acid residue, dipeptide fragment, tripeptide fragment or higher order peptide fragment including standard amino acids;
    
    






or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof,

wherein:

R50 is —(CH2)ssCH3, —CH(CH3)(CH2)ttCH3, —(CH2)uuCH(CH3)2, —C(CH3)3, —(CHR55)vv—R56, or —CH(OR57)CH3, wherein ss is 1, 2 or 3; tt is 1 or 2; uu is 0, 1 or 2; and vv is 0, 1, 2, 3 or 4; R55 is hydrogen or C1-C4 alkyl; R56 is amino, hydroxy, alkoxy, cycloalkyl or substituted cycloalkyl; and R57 is hydrogen, alkyl, acyl, amino acyl, sulfonyl, carboxyalkyl or carboxyaryl;
R51 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted with hydroxy or alkoxy;
R52 is —(CHR58)wwR59, wherein ww is 0, 1, 2 or 3; R58 is hydrogen, C1-C4 alkyl, amino, hydroxy or alkoxy; R59 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl or substituted cycloalkyl;
R53 is hydrogen or C1-C4 alkyl;
X2 is O, NR9 or N(R10)2+;

    
    
        wherein R9 is hydrogen, lower alkyl, substituted lower alkyl, sulfonyl, sulfonamido or amidino and R10 is hydrogen, lower alkyl, or substituted lower alkyl;
    
    


Z5 is O or NR12, wherein R12 is hydrogen, lower alkyl, or substituted lower alkyl; and
T2 is a bivalent radical of formula V:

—Ua—(CH2)d—Wa—Ya—Za—(CH2)e—  (V)

    
    
        wherein d and e are independently 0, 1, 2, 3, 4 or 5; Ya and Za are each optionally present; Ua is —CR60R61— or —C(═O)— and is bonded to X2 of formula II, wherein R60 and R61 are each independently hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R21 and R22 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N, wherein the ring is optionally substituted with R8 as defined above; Wa, Ya and Za are each independently selected from the group consisting of: —O—, —NR62—, —S—, —SO—, —SO2—, —C(═O)—O—, —O—C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —SO2—NH—, —NH—SO2—, —CR63R64—, —CH═CH— with the configuration Z or E, —C≡C—, and the ring structures depicted below:
    
    






    
    
        
        
            wherein G1 and G2 are defined above, and wherein any carbon atom in the ring is optionally replaced by N, with the proviso that the aromatic ring cannot contain more than four N atoms and the cycloalkyl ring cannot contain more than two N atoms;
            
                R62 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, amidino, sulfonyl or sulfonamido;
                R63 and R64 are each independently hydrogen, lower alkyl, substituted lower alkyl or RAA; or alternatively R63 and R64 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N; or alternatively one of R63 and R64 is hydroxy, alkoxy, aryloxy, amino, mercapto, carbamoyl, amidino, ureido or guanidino, while the other is hydrogen, lower alkyl or substituted lower alkyl, except when the carbon to which R63 and R64 are bonded is also bonded to another heteroatom; and RAA indicates the side chain of an amino acid such as a standard or unusual amino acid;
                R65 and R68 are each optionally present, and, when present are substituted for one or more hydrogen atoms on the ring and each is independently halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl or sulfonamido;
                R66 and R67 are each optionally present, and when no double bond is present to the carbon atom to which it is bonded in the ring, two groups are optionally present, and, when present, each is substituted for one hydrogen present in the ring, or when no double bond is present to the carbon atom to which it is bonded in the ring, is substituted for one or both of the two hydrogen atoms present on the ring and each is independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl, sulfonamide and, only if a double bond is present to the carbon atom to which it is bonded, halogen;
                R69 is optionally present, and when present is substituted for one or more hydrogen atoms on the ring and each is independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl or sulfonamido;
                K6 is O or S; and
                ff is 1, 2, 3, 4 or 5;
            
            
            with the proviso that T2 is not an amino acid residue, dipeptide fragment, tripeptide fragment or higher order peptide fragment including standard amino acids;
            or
        
        
    
    






or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof, wherein:

R70 is hydrogen, C1-C4 alkyl or alternatively R70 and R71 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8a as defined below;
R71 is hydrogen, —(CH2)aaCH3, —CH(CH3)(CH2)bbCH3, —(CH2)ccCH(CH3)2, —(CH2)dd—R76 or —CH(OR77)CH3 or, alternatively R71 and R70 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8a as defined below; wherein aa is 0, 1, 2, 3, 4 or 5; bb is 1, 2 or 3; cc is 0, 1, 2 or 3; and dd is 0, 1, 2, 3 or 4; R76 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl or substituted cycloalkyl; R77 is hydrogen, alkyl, acyl, amino acyl, sulfonyl, carboxyalkyl or carboxyaryl;
R72 is C1-C4 alkyl; or alternatively R72 and R73 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O or S atom in the ring, wherein the ring is optionally substituted with R8b as defined below;
R73 is hydrogen, or alternatively R73 and R72 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8b as defined below;
R74 is hydrogen or C1-C4 alkyl or alternatively R74 and R75 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8s as defined below;
R75 is —(CHR78)R79 or alternatively R75 and R74 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8, as defined below; wherein R78 is hydrogen, C1-C4 alkyl, amino, hydroxy or alkoxy, and R79 is selected from the group consisting of the following structures:




    
    
        wherein E1, E2, E3, E4 and E5 are each optionally present and when present are each independently selected from the group consisting of halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, cyano, sulfinyl, sulfonyl and sulfonamido, and represent substitution at one or more available positions on the monocyclic or bicyclic aromatic ring, wherein said substitution is made with the same or different selected group member, and J1 and J2 are each independently O or S;
    
    


R8a, R8b and R8c are each independently substituted for one or more hydrogen atoms on the 3-, 4-, 5-, 6- or 7-membered ring structure and are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido, or, alternatively, R8a, R8b and R8c, are each independently a fused cycloalkyl, a substituted fused cycloalkyl, a fused heterocyclic, a substituted fused heterocyclic, a fused aryl, a substituted fused aryl, a fused heteroaryl or a substituted fused heteroaryl ring when substituted for hydrogen atoms on two adjacent atoms;
X3 is O, NR9 or N(R10)2+;

    
    
        wherein R9 is hydrogen, lower alkyl, substituted lower alkyl, sulfonyl, sulfonamido or amidino and R10 is hydrogen, lower alkyl, or substituted lower alkyl;
    
    


Z10 is O or NR12, wherein R12 is hydrogen, lower alkyl, or substituted lower alkyl; and
T3 is the same as defined for T2 with the exception that Ua is bonded to X3 of formula III.
According to further aspects of the present invention, the compound is a ghrelin receptor agonist or a GHS-R1a receptor agonist.
Further aspects of the present invention provide pharmaceutical compositions comprising: (a) a compound of the present invention; and (b) a pharmaceutically acceptable carrier, excipient or diluent.
Additional aspects of the present invention provide kits comprising one or more containers containing pharmaceutical dosage units comprising an effective amount of one or more compounds of the present invention packaged with optional instructions for the use thereof.
Aspects of the present invention further provide methods of stimulating gastrointestinal motility, modulating GHS-R1a receptor activity in a mammal and/or treating a gastrointestinal disorder comprising administering to a subject in need thereof an effective amount of a modulator that modulates a mammalian GHS-R1a receptor. In particular embodiments, interaction of the modulator and the GHS-R1a receptor does not result in a significant amount of growth hormone release. In still other embodiments, the modulator is a compound of formula I, II and/or III.
Additional aspects of the present invention provide methods of diagnosing tumors and/or acromegaly, comprising administering compounds of the present invention and a radiolabeled metal binding agent and detecting the binding of the composition to a biological target, and treating tumors and/or acromegaly comprising administering a therapeutically effective amount of a composition comprising a compound of the present invention.
Further aspects of the present invention relate to methods of making the compounds of formula I, II and/or III.
Aspects of the present invention further relate to methods of preventing and/or treating disorders described herein, in particular, gastrointestinal disorders, including post-operative ileus, gastroparesis, such as diabetic and post-surgical gastroparesis, opioid-induced bowel dysfunction, chronic intestinal pseudo-obstruction, short bowel syndrome, emesis such as caused by cancer chemotherapy, constipation such as associated with the hypomotility phase of irritable bowel syndrome (IBS), delayed gastric emptying associated with wasting conditions, gastroesophageal reflux disease (GERD), gastric ulcers, Crohn's disease, gastrointestinal disorders characterized by dysmotility and other diseases and disorders of the gastrointestinal tract.
The present invention also relates to compounds of formula I, II and/or III used for the preparation of a medicament for prevention and/or treatment of the disorders described herein.
The foregoing and other aspects of the present invention are explained in greater detail in the specification set forth below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a scheme presenting a general synthetic strategy to provide conformationally-defined macrocycles of the present invention.
FIG. 2 shows a general thioester strategy for making macrocyclic compounds of the present invention.
FIG. 3 shows a general ring-closing metathesis (RCM) strategy for macrocyclic compounds of the present invention.
FIG. 4 (panels A through E) shows competitive binding curves for binding of exemplary compounds of the present invention to the hGHS-R1a receptor.
FIG. 5 (panels A through E) shows concentration-response curves for activation of the hGHS-R1a receptor by exemplary compounds of the present invention.
FIG. 6 shows graphs depicting pharmacokinetic parameters for exemplary compounds of the present invention, specifically after oral administration of 8 mg/kg compound 298 (panel A), after subcutaneous injection of 2 mg/kg compound 298 with cyclodextrin (panel B), after intravenous administration of 2 mg/kg compound 25 with cyclodextrin (panel C) and after intravenous administration of 2 mg/kg compound 298 with cyclodextrin (panel D).
FIG. 7 (panels A and B) shows graphs presenting effects on gastric emptying for exemplary compounds of the present invention.
FIG. 8 shows a graph presenting effects of postoperative ileus for an exemplary compound of the present invention.
FIG. 9 (panels A through D) shows graphs depicting the effect on pulsatile growth hormone release for an exemplary compound of the present invention.
FIG. 10 shows a competitive binding curve for binding of an exemplary compound of the present invention to the hGHS-R1a receptor.
FIG. 11 shows an activation curve demonstrating the agonism of an exemplary compound of the present invention.
FIG. 12 shows a graph depicting agonism and lack of growth hormone release for an exemplary compound of the present invention.
FIG. 13 shows graphs depicting receptor desentization associated with binding of an exemplary compound of the present invention to the hGHS-R1a receptor.
FIG. 14 (panels A and B) shows graphs presenting effects on gastric emptying for an exemplary compound of the present invention.
FIG. 15 shows a graph presenting effects on postoperative ileus for an exemplary compound of the present invention.
FIG. 16 shows graphs depicting reversal of morphine-delayed gastric emptying (panel A) and morphine-delayed gastrointestinal transit (panel B) for an exemplary compound of the present invention.
FIG. 17 (panels A and B) shows graphs depicting effects on gastroparesis for exemplary compounds of the present invention.



DETAILED DESCRIPTION
The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, as used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
All publications, U.S. patent applications, U.S. patents and other references cited herein are incorporated by reference in their entireties.
The term “alkyl” refers to straight or branched chain saturated or partially unsaturated hydrocarbon groups having from 1 to 20 carbon atoms, in some instances 1 to 8 carbon atoms. The term “lower alkyl” refers to alkyl groups containing 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, tert-butyl, 3-hexenyl, and 2-butynyl. By “unsaturated” is meant the presence of 1, 2 or 3 double or triple bonds, or a combination of the two. Such alkyl groups may also be optionally substituted as described below.
When a subscript is used with reference to an alkyl or other hydrocarbon group defined herein, the subscript refers to the number of carbon atoms that the group may contain. For example, C2-C4 alkyl indicates an alkyl group with 2, 3 or 4 carbon atoms.
The term “cycloalkyl” refers to saturated or partially unsaturated cyclic hydrocarbon groups having from 3 to 15 carbon atoms in the ring, in some instances 3 to 7, and to alkyl groups containing said cyclic hydrocarbon groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopropylmethyl, cyclopentyl, 2-(cyclohexyl)ethyl, cycloheptyl, and cyclohexenyl. Cycloalkyl as defined herein also includes groups with multiple carbon rings, each of which may be saturated or partially unsaturated, for example decalinyl, [2.2.1]-bicycloheptanyl or adamantanyl. All such cycloalkyl groups may also be optionally substituted as described below.
The term “aromatic” refers to an unsaturated cyclic hydrocarbon group having a conjugated pi electron system that contains 4n+2 electrons where n is an integer greater than or equal to 1. Aromatic molecules are typically stable and are depicted as a planar ring of atoms with resonance structures that consist of alternating double and single bonds, for example benzene or naphthalene.
The term “aryl” refers to an aromatic group in a single or fused carbocyclic ring system having from 6 to 15 ring atoms, in some instances 6 to 10, and to alkyl groups containing said aromatic groups. Examples of aryl groups include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl and benzyl. Aryl as defined herein also includes groups with multiple aryl rings which may be fused, as in naphthyl and anthracenyl, or unfused, as in biphenyl and terphenyl. Aryl also refers to bicyclic or tricyclic carbon rings, where one of the rings is aromatic and the others of which may be saturated, partially unsaturated or aromatic, for example, indanyl or tetrahydronaphthyl (tetralinyl). All such aryl groups may also be optionally substituted as described below.
The term “heterocycle” or “heterocyclic” refers to saturated or partially unsaturated monocyclic, bicyclic or tricyclic groups having from 3 to 15 atoms, in some instances 3 to 7, with at least one heteroatom in at least one of the rings, said heteroatom being selected from O, S or N. Each ring of the heterocyclic group can contain one or two O atoms, one or two S atoms, one to four N atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. The fused rings completing the bicyclic or tricyclic heterocyclic groups may contain only carbon atoms and may be saturated or partially unsaturated. The N and S atoms may optionally be oxidized and the N atoms may optionally be quaternized. Heterocyclic also refers to alkyl groups containing said monocyclic, bicyclic or tricyclic heterocyclic groups. Examples of heterocyclic rings include, but are not limited to, 2- or 3-piperidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl. All such heterocyclic groups may also be optionally substituted as described below
The term “heteroaryl” refers to an aromatic group in a single or fused ring system having from 5 to 15 ring atoms, in some instances 5 to 10, which have at least one heteroatom in at least one of the rings, said heteroatom being selected from O, S or N. Each ring of the heteroaryl group can contain one or two O atoms, one or two S atoms, one to four N atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. The fused rings completing the bicyclic or tricyclic groups may contain only carbon atoms and may be saturated, partially unsaturated or aromatic. In structures where the lone pair of electrons of a nitrogen atom is not involved in completing the aromatic pi electron system, the N atoms may optionally be quaternized or oxidized to the N-oxide. Heteroaryl also refers to alkyl groups containing said cyclic groups. Examples of monocyclic heteroaryl groups include, but are not limited to pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to carbazolyl, benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, and xanthenyl. All such heteroaryl groups may also be optionally substituted as described below.
The term “hydroxy” refers to the group —OH.
The term “alkoxy” refers to the group —ORa, wherein Ra is alkyl, cycloalkyl or heterocyclic. Examples include, but are not limited to methoxy, ethoxy, tert-butoxy, cyclohexyloxy and tetrahydropyranyloxy.
The term “aryloxy” refers to the group —ORb wherein Rb is aryl or heteroaryl. Examples include, but are not limited to phenoxy, benzyloxy and 2-naphthyloxy.
The term “acyl” refers to the group —C(═O)—Rc, wherein Rc is alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Examples include, but are not limited to, acetyl, benzoyl and furoyl.
The term “amino acyl” indicates an acyl group that is derived from an amino acid.
The term “amino” refers to an —NRdRe, group wherein Rd and Re are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl. Alternatively, Rd and Re together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term “amido” refers to the group —C(═O)—NRfRg wherein Rf and Rg are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl. Alternatively, Rf and Rg together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term “amidino” refers to the group —C(═NRh)NRiRj wherein Rh is selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl; and Ri and Rj are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl. Alternatively, Ri and Rj together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term “carboxy” refers to the group —CO2H.
The term “carboxyalkyl” refers to the group —CO2Rk, wherein Rk is alkyl, cycloalkyl or heterocyclic.
The term “carboxyaryl” refers to the group —CO2Rm, wherein Rm is aryl or heteroaryl.
The term “cyano” refers to the group —CN.
The term “formyl” refers to the group —C(═O)H, also denoted —CHO.
The term “halo,” “halogen” or “halide” refers to fluoro, fluorine or fluoride, chloro, chlorine or chloride, bromo, bromine or bromide, and iodo, iodine or iodide, respectively.
The term “oxo” refers to the bivalent group ═O, which is substituted in place of two hydrogen atoms on the same carbon to form a carbonyl group.
The term “mercapto” refers to the group —SRn wherein Rn is hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl.
The term “nitro” refers to the group —NO2.
The term “trifluoromethyl” refers to the group —CF3.
The term “sulfinyl” refers to the group —S(═O)Rp wherein Rp is alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl.
The term “sulfonyl” refers to the group —S(═O)2—Rq1 wherein Rq1 is alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl.
The term “aminosulfonyl” refers to the group —NRq2—S(═O)2—Rq3 wherein Rq2 is hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; and 3 is alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl.
The term “sulfonamido” refers to the group —S(═O)2—NRrRs wherein Rr and Rs are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Alternatively, Rr and Rs together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term “carbamoyl” refers to a group of the formula —N(Rt)—C(═O)—ORu wherein Rt is selected from hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; and Ru is selected from alkyl, cycloalkyl, heterocylic, aryl or heteroaryl.
The term “guanidino” refers to a group of the formula —N(Rv)—C(═NRw)—NRxRy wherein Rv, Rw, Rx and Ry are independently selected from hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Alternatively, Rx and Ry together form a heterocyclic ring or 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term “ureido” refers to a group of the formula —N(Rz)—C(═O)—NRaaRbb wherein Rz, Raa and Rbb are independently selected from hydrogen, alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl. Alternatively, Raa and Rbb together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N.
The term “optionally substituted” is intended to expressly indicate that the specified group is unsubstituted or substituted by one or more suitable substituents, unless the optional substituents are expressly specified, in which case the term indicates that the group is unsubstituted or substituted with the specified substituents. As defined above, various groups may be unsubstituted or substituted (i.e., they are optionally substituted) unless indicated otherwise herein (e.g., by indicating that the specified group is unsubstituted).
The term “substituted” when used with the terms alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl refers to an alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl group having one or more of the hydrogen atoms of the group replaced by substituents independently selected from unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, halo, oxo, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino, ureido and groups of the formulas —NRccC(═O)Rdd, —NeeC(═NRff)Rgg, —OC(═O)NRhhRii, —OC(═O)Rjj, —OC(═O)ORkk, —NRmmSO2Rnn, or —NRppSO2NqqRrr wherein Rcc, Rdd, Ree, Rff, Rgg, Rhh, Rjj, Rmm, Rpp, Rqq and Rrr, are independently selected from hydrogen, unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl or unsubstituted heteroaryl; and wherein Rkk and Rnn are independently selected from unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl or unsubstituted heteroaryl. Alternatively, Rgg and Rhh, Rjj and Rkk or Rpp and Rqq together form a heterocyclic ring of 3 to 8 members, optionally substituted with unsubstituted alkyl, unsubstituted cycloalkyl, unsubstituted heterocyclic, unsubstituted aryl, unsubstituted heteroaryl, hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, carboxyaryl, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, guanidino or ureido, and optionally containing one to three additional heteroatoms selected from O, S or N. In addition, the term “substituted” for aryl and heteroaryl groups includes as an option having one of the hydrogen atoms of the group replaced by cyano, nitro or trifluoromethyl.
A substitution is made provided that any atom's normal valency is not exceeded and that the substitution results in a stable compound. Generally, when a substituted form of a group is present, such substituted group is preferably not further substituted or, if substituted, the substituent comprises only a limited number of substituted groups, in some instances 1, 2, 3 or 4 such substituents.
When any variable occurs more than one time in any constituent or in any formula herein, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
A “stable compound” or “stable structure” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity and formulation into an efficacious therapeutic agent.
The term “amino acid” refers to the common natural (genetically encoded) or synthetic amino acids and common derivatives thereof, known to those skilled in the art. When applied to amino acids, “standard” or “proteinogenic” refers to the genetically encoded 20 amino acids in their natural configuration. Similarly, when applied to amino acids, “unnatural” or “unusual” refers to the wide selection of non-natural, rare or synthetic amino acids such as those described by Hunt, S. in Chemistry and Biochemistry of the Amino Acids, Barrett, G. C., Ed., Chapman and Hall: New York, 1985.
The term “residue” with reference to an amino acid or amino acid derivative refers to a group of the formula:




wherein RAA is an amino acid side chain, and n=0, 1 or 2 in this instance.

The term “fragment” with respect to a dipeptide, tripeptide or higher order peptide derivative indicates a group that contains two, three or more, respectively, amino acid residues.
The term “amino acid side chain” refers to any side chain from a standard or unnatural amino acid, and is denoted RAA. For example, the side chain of alanine is methyl, the side chain of valine is isopropyl and the side chain of tryptophan is 3-indolylmethyl.
The term “agonist” refers to a compound that duplicates at least some of the effect of the endogenous ligand of a protein, receptor, enzyme or the like.
The term “antagonist” refers to a compound that inhibits at least some of the effect of the endogenous ligand of a protein, receptor, enzyme or the like.
The term “growth hormone secretagogue” (GHS) refers to any exogenously administered compound or agent that directly or indirectly stimulates or increases the endogenous release of growth hormone, growth hormone-releasing hormone, or somatostatin in an animal, in particular, a human. A GHS may be peptidic or non-peptidic in nature, in some instances, with an agent that can be administered orally. In some instances, the agent can induce a pulsatile response.
The term “modulator” refers to a compound that imparts an effect on a biological or chemical process or mechanism. For example, a modulator may increase, facilitate, upregulate, activate, inhibit, decrease, block, prevent, delay, desensitize, deactivate, down regulate, or the like, a biological or chemical process or mechanism. Accordingly, a modulator can be an “agonist” or an “antagonist.” Exemplary biological processes or mechanisms affected by a modulator include, but are not limited to, receptor binding and hormone release or secretion. Exemplary chemical processes or mechanisms affected by a modulator include, but are not limited to, catalysis and hydrolysis.
The term “variant” when applied to a receptor is meant to include dimers, trimers, tetramers, pentamers and other biological complexes containing multiple components. These components can be the same or different.
The term “peptide” refers to a chemical compound comprised of two or more amino acids covalently bonded together.
The term “peptidomimetic” refers to a chemical compound designed to mimic a peptide, but which contains structural differences through the addition or replacement of one of more functional groups of the peptide in order to modulate its activity or other properties, such as solubility, metabolic stability, oral bioavailability, lipophilicity, permeability, etc. This can include replacement of the peptide bond, side chain modifications, truncations, additions of functional groups, etc. When the chemical structure is not derived from the peptide, but mimics its activity, it is often referred to as a “non-peptide peptidomimetic.”
The term “peptide bond” refers to the amide [—C(═O)—NH—] functionality with which individual amino acids are typically covalently bonded to each other in a peptide.
The term “protecting group” refers to any chemical compound that may be used to prevent a potentially reactive functional group, such as an amine, a hydroxyl or a carboxyl, on a molecule from undergoing a chemical reaction while chemical change occurs elsewhere in the molecule. A number of such protecting groups are known to those skilled in the art and examples can be found in “Protective Groups in Organic Synthesis,” Theodora W. Greene and Peter G. Wuts, editors, John Wiley & Sons, New York, 3rd edition, 1999 [ISBN 0471160199]. Examples of amino protecting groups include, but are not limited to, phthalimido, trichloroacetyl, benzyloxycarbonyl, tert-butoxycarbonyl, and adamantyloxycarbonyl. In some embodiments, amino protecting groups are carbamate amino protecting groups, which are defined as an amino protecting group that when bound to an amino group forms a carbamate. In other embodiments, amino carbamate protecting groups are allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc) and α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz). For a recent discussion of newer nitrogen protecting groups: Theodoridis, G. Tetrahedron 2000, 56, 2339-2358. Examples of hydroxyl protecting groups include, but are not limited to, acetyl, tert-butyldimethylsilyl (TBDMS), trityl (Trt), tert-butyl, and tetrahydropyranyl (THP). Examples of carboxyl protecting groups include, but are not limited to methyl ester, tert-butyl ester, benzyl ester, trimethylsilylethyl ester, and 2,2,2-trichloroethyl ester.
The term “solid phase chemistry” refers to the conduct of chemical reactions where one component of the reaction is covalently bonded to a polymeric material (solid support as defined below). Reaction methods for performing chemistry on solid phase have become more widely known and established outside the traditional fields of peptide and oligonucleotide chemistry.
The term “solid support,” “solid phase” or “resin” refers to a mechanically and




chemically stable polymeric matrix utilized to conduct solid phase chemistry. This is denoted by “Resin,” “P-” or the following symbol:

Examples of appropriate polymer materials include, but are not limited to, polystyrene, polyethylene, polyethylene glycol, polyethylene glycol grafted or covalently bonded to polystyrene (also termed PEG-polystyrene, TentaGelTM, Rapp, W.; Zhang, L.; Bayer, E. In Innovations and Persepetives in Solid Phase Synthesis. Peptides, Polypeptides and Oligonucleotides; Epton, R., Ed.; SPCC Ltd.: Birmingham, UK; p 205), polyacrylate (CLEARTM), polyacrylamide, polyurethane, PEGA [polyethyleneglycol poly(N,N-dimethylacrylamide) co-polymer, Meldal, M. Tetrahedron Lett. 1992, 33, 3077-3080], cellulose, etc. These materials can optionally contain additional chemical agents to form cross-linked bonds to mechanically stabilize the structure, for example polystyrene cross-linked with divinylbenezene (DVB, usually 0.1-5%, preferably 0.5-2%). This solid support can include as non-limiting examples aminomethyl polystyrene, hydroxymethyl polystyrene, benzhydrylamine polystyrene (BHA), methylbenzhydrylamine (MBHA) polystyrene, and other polymeric backbones containing free chemical functional groups, most typically, —NH2 or —OH, for further derivatization or reaction. The term is also meant to include “Ultraresins” with a high proportion (“loading”) of these functional groups such as those prepared from polyethyleneimines and cross-linking molecules (Barth, M.; Rademann, J. J Comb. Chem. 2004, 6, 340-349). At the conclusion of the synthesis, resins are typically discarded, although they have been shown to be able to be reused such as in Frechet, J. M. J.; Haque, K. E. Tetrahedron Lett. 1975, 16, 3055.
In general, the materials used as resins are insoluble polymers, but certain polymers have differential solubility depending on solvent and can also be employed for solid phase chemistry. For example, polyethylene glycol can be utilized in this manner since it is soluble in many organic solvents in which chemical reactions can be conducted, but it is insoluble in others, such as diethyl ether. Hence, reactions can be conducted homogeneously in solution, then the product on the polymer precipitated through the addition of diethyl ether and processed as a solid. This has been termed “liquid-phase” chemistry.
The term “linker” when used in reference to solid phase chemistry refers to a chemical group that is bonded covalently to a solid support and is attached between the support and the substrate typically in order to permit the release (cleavage) of the substrate from the solid support. However, it can also be used to impart stability to the bond to the solid support or merely as a spacer element. Many solid supports are available commercially with linkers already attached.
Abbreviations used for amino acids and designation of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247, 977-983. This document has been updated: Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; and in Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pp 39-67. Extensions to the rules were published in the JCBN/NC-IUB Newsletter 1985, 1986, 1989; see Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pp 68-69.
The term “effective amount” or “effective” is intended to designate a dose that causes a relief of symptoms of a disease or disorder as noted through clinical testing and evaluation, patient observation, and/or the like, and/or a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and/or further quantified by one skilled in the art for the relevant mechanism or process. As is generally understood in the art, the dosage will vary depending on the administration routes, symptoms and body weight of the patient but also depending upon the compound being administered.
Administration of two or more compounds “in combination” means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two compounds can be administered simultaneously (concurrently) or sequentially. Simultaneous administration can be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration. The phrases “concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
The term “pharmaceutically active metabolite” is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound.
The term “solvate” is intended to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates, without limitation, include compounds of the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
1. Compounds
Novel macrocyclic compounds of the present invention include macrocyclic compounds comprising a building block structure including a tether component that undergoes cyclization to form the macrocyclic compound. The building block stricture can comprise amino acids (standard and unnatural), hydroxy acids, hydrazino acids, aza-amino acids, specialized moieties such as those that play a role in the introduction of peptide surrogates and isosteres, and a tether component as described herein. The tether component can be selected from the following:







wherein (Z2) is the site of a covalent bond of T to Z2, and Z2 is as defined below for formula I, and wherein (X) is the site of a covalent bond of T to X, and X is as defined below for formula I; L7 is —CH2— or —O—; U1 is —CR101R102— or —C(═O)—; R100 is lower alkyl; R101 and R102 are each independently hydrogen, lower alkyl or substituted lower alkyl; xx is 2 or 3; yy is 1 or 2; zz is 1 or 2; and aaa is 0 or 1.
Macrocyclic compounds of the present invention further include those of formula I, formula II and/or formula III:




or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof,

wherein:
R1 is hydrogen or the side chain of an amino acid, or alternatively R1 and R2 together form a 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8 as defined below, or alternatively R1 and R9 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R2 is hydrogen or the side chain of an amino acid, or alternatively, R1 and R2 together form a 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8 as defined below; or alternatively R2 and R9 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R3 is hydrogen or the side chain of an amino acid, or alternatively R3 and R4 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O or S atom in the ring, wherein the ring is optionally substituted with R8 as defined below, or alternatively, R3 and R7 or R3 and R11 together form a 4-, 5-, 6-, 7- or 8-membered heterocyclic ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R4 is hydrogen or the side chain of an amino acid, or alternatively R4 and R3 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O or S atom in the ring, wherein the ring is optionally substituted with R8 as defined below, or alternatively R4 and R7 or R4 and R11 together form a 4-, 5-, 6-, 7- or 8-membered heterocyclic ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R5 and R6 are each independently hydrogen or the side chain of an amino acid or alternatively R5 and R6 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8 as defined below;
R7 is hydrogen, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, or a substituted heterocyclic group, or alternatively R3 and R7 or R4 and R7 together form a 3-, 4-, 5-, 6-, 7- or 8-membered heterocyclic ring optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as described below;
R8 is substituted for one or more hydrogen atoms on the 3-, 4-, 5-, 6-, 7- or 8-membered ring structure and is independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido, or, alternatively, R8 is a fused cycloalkyl, a substituted fused cycloalkyl, a fused heterocyclic, a substituted fused heterocyclic, a fused aryl, a substituted fused aryl, a fused heteroaryl or a substituted fused heteroaryl ring when substituted for hydrogen atoms on two adjacent atoms;
X is O, NR9 or N(R10)2;

    
    
        wherein R9 is hydrogen, lower alkyl, substituted lower alkyl, sulfonyl, sulfonamido or amidino and R10 is hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R9 and R1 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined above;
    
    


Z1 is O or NR11,

    
    
        wherein R11 is hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R3 and R11 together or R4 and R11 together form a 4-, 5-, 6-, 7- or 8-membered heterocyclic ring, optionally comprising an O, S or additional N atom in the ring, wherein the ring is optionally substituted with R8 as defined above;
    
    


Z2 is O or NR12, wherein R12 is hydrogen, lower alkyl, or substituted lower alkyl;
m, n and p are each independently 0, 1 or 2;
T is a bivalent radical of formula IV:

—U—(CH2)d—W—Y—Z—(CH2)e—  (IV)

    
    
        wherein d and e are each independently 0, 1, 2, 3, 4 or 5; Y and Z are each optionally present; U is —CR21R22— or —C(═O)— and is bonded to X of formula I; W, Y and Z are each independently selected from the group consisting of —O—, —NR23—, —S—, —SO—, —SO2—, —C(═O)—O—, —O—C(═O)—, C(═O)—NH—, —NH—C(═O)—, —SO2—NH—, —NH—SO2—, —CR24R25—, —CH═CH— with the configuration Z or E, —C≡C and the ring structures below:
    
    






    
    
        
        
            wherein G1 and G2 are each independently a covalent bond or a bivalent radical selected from the group consisting of —O—, —NR39—, —S—, —SO—, —SO2—, —C(═O)—, —C(═O)—O—, —O—C(═O)—, —C(═O)NH—, —NH—C(═O)—, —SO2—NH—, —NH—SO2—, —CR40R41—, —CH═CH— with the configuration Z or E, and —C≡C—; with GI being bonded closest to the group U, wherein any carbon atom in the rings not otherwise defined, can be replaced by N, with the proviso that the ring cannot contain more than four N atoms; K1, K2, K3, K4 and K5 are each independently O, NR42 or S, wherein R42 is as defined below;
            R21 and R22 are each independently hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R21 and R22 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N, wherein the ring is optionally substituted with R8 as defined above;
            R23, R39 and R42 are each independently hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, amidino, sulfonyl or sulfonamido;
            R24 and R25 are each independently hydrogen, lower alkyl, substituted lower alkyl, RAA, wherein RAA is a side chain of an amino acid such as a standard or unusual amino acid, or alternatively R24 and R25 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N; or alternatively one of R24 or R25 is hydroxy, alkoxy, aryloxy, amino, mercapto, carbamoyl, amidino, ureido or guanidino while the other is hydrogen, lower alkyl or substituted lower alkyl, except when the carbon to which R24 and R25 are bonded is also bonded to another heteroatom;
            R26, R31, R35 and R38 are each optionally present and, when present, are substituted for one or more hydrogen atoms on the indicated ring and each is independently selected from the group consisting of halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido;
            R27 is optionally present and is substituted for one or more hydrogen atoms on the indicated ring and each is independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido;
            R28, R29, R30, R32, R33, R34, R36 and R37 are each optionally present and, when no double bond is present to the carbon atom to which it is bonded in the ring, two groups are optionally present, and when present, is substituted for one hydrogen present in the ring, or when no double bond is present to the carbon atom to which it is bonded in the ring, is substituted for one or both of the two hydrogen atoms present on the ring and each is independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl, sulfonamido and, only if a double bond is present to the carbon atom to which it is bonded, halogen; and
            R40 and R41 are each independently hydrogen, lower alkyl, substituted lower alkyl, RAA as defined above, or alternatively R40 and R41 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N wherein the ring is optionally substituted with R8 as defined above, or alternatively one of R40 and R41 is hydroxy, alkoxy, aryloxy, amino, mercapto, carbamoyl, amidino, ureido or guanidino, while the other is hydrogen, lower alkyl or substituted lower alkyl, except when the carbon to which R40 and R41 are bonded is also bonded to another heteroatom;
        
        
        with the proviso that T is not an amino acid residue, dipeptide fragment, tripeptide fragment or higher order peptide fragment including standard amino acids;
    
    






or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof,

wherein:

R50 is —(CH2)ssCH3, —CH(CH3)(CH2)ttCH3, —(CH2)uuCH(CH3)2, —C(CH3)3, —(CHR55)vv—R56, or —CH(OR57)CH3, wherein ss is 1, 2 or 3; tt is 1 or 2; uu is 0, 1 or 2; and vv is 0, 1, 2, 3 or 4; R55 is hydrogen or C1-C4 alkyl; R56 is amino, hydroxy, alkoxy, cycloalkyl or substituted cycloalkyl; and R57 is hydrogen, alkyl, acyl, amino acyl, sulfonyl, carboxyalkyl or carboxyaryl;
R51 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted with hydroxy or alkoxy;
R52 is —(CHR58)wwR59, wherein ww is 0, 1, 2 or 3; R58 is hydrogen, C1-C4 alkyl, amino, hydroxy or alkoxy; R59 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl or substituted cycloalkyl;
R53 is hydrogen or C1-C4 alkyl;
X2 is O, NR9 or N(R10)2+;

    
    
        wherein R9 is hydrogen, lower alkyl, substituted lower alkyl, sulfonyl, sulfonamido or amidino and R10 is hydrogen, lower alkyl, or substituted lower alkyl;
    
    


Z5 is O or NR12, wherein R12 is hydrogen, lower alkyl, or substituted lower alkyl; and
T2 is a bivalent radical of formula V:

Ua—(CH2)d—Wa—Ya—Za—(CH2)e—  (V)

    
    
        wherein d and e are independently 0, 1, 2, 3, 4 or 5; Ya and Za are each optionally present; Ua is —CR6OR61— or —C(═O)— and is bonded to X2 of formula II, wherein R60 and R6, are each independently hydrogen, lower alkyl, or substituted lower alkyl, or alternatively R21 and R22 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N, wherein the ring is optionally substituted with R8 as defined above; Wa, Ya and Za are each independently selected from the group consisting of: —O—, —NR62—, —S—, —SO—, —SO2—, —C(═O)O—, —O—C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —SO2—NH—, —NH—SO2—, —CR63R64—, —CH═CH— with the configuration Z or E, —C≡C—, and the ring structures depicted below:
    
    






    
    
        
        
            wherein G1 and G2 are defined above, and wherein any carbon atom in the ring is optionally replaced by N, with the proviso that the aromatic ring cannot contain more than four N atoms and the cycloalkyl ring cannot contain more than two N atoms;
            
                R62 is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, amidino, sulfonyl or sulfonamido;
                R63 and R64 are each independently hydrogen, lower alkyl, substituted lower alkyl or RAA; or alternatively R63 and R64 together form a 3- to 12-membered cyclic ring optionally comprising one or more heteroatoms selected from the group consisting of O, S and N; or alternatively one of R63 and R64 is hydroxy, alkoxy, aryloxy, amino, mercapto, carbamoyl, amidino, ureido or guanidino, while the other is hydrogen, lower alkyl or substituted lower alkyl, except when the carbon to which R63 and R64 are bonded is also bonded to another heteroatom; and RAA indicates the side chain of a standard or unusual amino acid;
                R65 and R68 are each optionally present, and, when present are substituted for one or more hydrogen atoms on the ring and each is independently halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl or sulfonamido;
                R66 and R67 are each optionally present, and when no double bond is present to the carbon atom to which it is bonded in the ring, two groups are optionally present, and, when present, each is substituted for one hydrogen present in the ring, or when no double bond is present to the carbon atom to which it is bonded in the ring, is substituted for one or both of the two hydrogen atoms present on the ring and each is independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amino, mercapto, sulfinyl, sulfonyl, sulfonamide and, only if a double bond is present to the carbon atom to which it is bonded, halogen;
                R69 is optionally present, and when present is substituted for one or more hydrogen atoms on the ring and each is independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl or sulfonamido;
                K6 is O or S; and
                ff is 1, 2, 3, 4 or 5;
            
            
        
        
        with the proviso that T2 is not an amino acid residue, dipeptide fragment, tripeptide fragment or higher order peptide fragment including standard amino acids; or
    
    






or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof, wherein:

R70 is hydrogen, C1-C4 alkyl or alternatively R70 and R71 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8a as defined below;
R71 is hydrogen, —(CH2)aaCH3, —CH(CH3)(CH2)bbCH3, —(CH2)ccCH(CH3)2, —(CH2)dd—R76 or —CH(OR77)CH3 or, alternatively R71 and R70 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8a as defined below; wherein aa is 0, 1, 2, 3, 4 or 5; bb is 1, 2 or 3; cc is 0, 1, 2 or 3; and dd is 0, 1, 2, 3 or 4; R76 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl or substituted cycloalkyl; R77 is hydrogen, alkyl, acyl, amino acyl, sulfonyl, carboxyalkyl or carboxyaryl;
R72 is C1-C4 alkyl, or alternatively R72 and R73 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O or S atom in the ring, wherein the ring is optionally substituted with R8b as defined below;
R73 is hydrogen, or alternatively R73 and R72 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, S or N atom in the ring, wherein the ring is optionally substituted with R8b as defined below;
R74 is hydrogen or C1-C4 alkyl or alternatively R74 and R75 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8c as defined below;
R75 is —(CHR78)R79 or alternatively R75 and R74 together form a 3-, 4-, 5-, 6- or 7-membered ring, optionally comprising an O, N or S atom in the ring, wherein the ring is optionally substituted with R8c as defined below; wherein R78 is hydrogen, C1-C4 alkyl, amino, hydroxy or alkoxy, and R79 is selected from the group consisting of the following structures:




    
    
        wherein E1, E2, E3, E4 and E5 are each optionally present and when present are each independently selected from the group consisting of halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, cyano, sulfinyl, sulfonyl and sulfonamido, and represent substitution at one or more available positions on the monocyclic or bicyclic aromatic ring, wherein said substitution is made with the same or different selected group member, and J1 and J2 are each independently O or S;
    
    


R8a, R8b and R8c are each independently substituted for one or more hydrogen atoms on the 3-, 4-, 5-, 6- or 7-membered ring structure and are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mereapto, sulfinyl, sulfonyl and sulfonamido, or, alternatively, R8a, R8b and R8c are each independently a fused cycloalkyl, a substituted fused cycloalkyl, a fused heterocyclic, a substituted fused heterocyclic, a fused aryl, a substituted fused aryl, a fused heteroaryl or a substituted fused heteroaryl ring when substituted for hydrogen atoms on two adjacent atoms;
X3 is O, NR9 or N(R10)2+;

    
    
        wherein R9 is hydrogen, lower alkyl, substituted lower alkyl, sulfonyl, sulfonamido or amidino and R10 is hydrogen, lower alkyl, or substituted lower alkyl;
    
    


Z10 is O or NR12, wherein R12 is hydrogen, lower alkyl, or substituted lower alkyl; and
T3 is the same as defined for T2 with the exception that Ua is bonded to X3 of formula III.
In some embodiments of the present invention, the compound can have one of the following structures:






















or an optical isomer, enantiomer, diastereomer, racemate or stereochemical mixture thereof.

The present invention includes isolated compounds. An isolated compound refers to a compound that, in some embodiments, comprises at least 10%, at least 25%, at least 50% or at least 70% of the compounds of a mixture. In some embodiments, the compound, pharmaceutically acceptable salt thereof or pharmaceutical composition containing the compound exhibits a statistically significant binding and/or antagonist activity when tested in biological assays at the human ghrelin receptor.
In the case of compounds, salts, or solvates that are solids, it is understood by those skilled in the art that the inventive compounds, salts, and solvates may exist in different crystal or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulas.
The compounds of formula I, II and/or III disclosed herein have asymmetric centers. The inventive compounds may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the scope of the present invention. In particular embodiments, however, the inventive compounds are used in optically pure form. The terms “S” and “R” configuration as used herein are as defined by the IUPAC 1974 Recommendations for Section E, Fundamentals of Stereochemistry (Pure Appl. Chem. 1976, 45, 13-30.)
Unless otherwise depicted to be a specific orientation, the present invention accounts for all stereoisomeric forms. The compounds may be prepared as a single stereoisomer or a mixture of stereoisomers. The non-racemic forms may be obtained by either synthesis or resolution. The compounds may, for example, be resolved into the component enantiomers by standard techniques, for example formation of diastereomeric pairs via salt formation. The compounds also may be resolved by covalently bonding to a chiral moiety. The diastereomers can then be resolved by chromatographic separation and/or crystallographic separation. In the case of a chiral auxiliary moiety, it can then be removed. As an alternative, the compounds can be resolved through the use of chiral chromatography. Enzymatic methods of resolution could also be used in certain cases.
As generally understood by those skilled in the art, an “optically pure” compound is one that contains only a single enantiomer. As used herein, the term “optically active” is intended to mean a compound comprising at least a sufficient excess of one enantiomer over the other such that the mixture rotates plane polarized light. Optically active compounds have the ability to rotate the plane of polarized light. The excess of one enantiomer over another is typically expressed as enantiomeric excess (e.e.). In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes “d” and “1” or (+) and (−) are used to denote the optical rotation of the compound (i.e., the direction in which a plane of polarized light is rotated by the optically active compound). The “1” or (−) prefix indicates that the compound is levorotatory (i.e., rotates the plane of polarized light to the left or counterclockwise) while the “d” or (+) prefix means that the compound is dextrarotatory (i.e., rotates the plane of polarized light to the right or clockwise). The sign of optical rotation, (−) and (+), is not related to the absolute configuration of the molecule, R and S.
A compound of the invention having the desired pharmacological properties will be optically active and, can be comprised of at least 90% (80% e.e.), at least 95% (90% e.e.), at least 97.5% (95% e.e.) or at least 99% (98% e.e.) of a single isomer.
Likewise, many geometric isomers of double bonds and the like can also be present in the compounds disclosed herein, and all such stable isomers are included within the present invention unless otherwise specified. Also included in the invention are tautomers and rotamers of formula I, II and/or III.
The use of the following symbols at the right refers to substitution of one or more hydrogen atoms of the indicated ring with the defined substituent R.




The use of the following symbol indicates a single bond or an optional double bond: 
Embodiments of the present invention further provide intermediate compounds formed through the synthetic methods described herein to provide the compounds of formula I, II and/or III. The intermediate compounds may possess utility as a therapeutic agent for the range of indications described herein and/or a reagent for further synthesis methods and reactions.
2. Synthetic Methods
The compounds of formula I, II and/or II can be synthesized using traditional solution synthesis techniques or solid phase chemistry methods. In either, the constriction involves four phases: first, synthesis of the building blocks comprising recognition elements for the biological target receptor, plus one tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations. The precursors from the assembly are then cyclized in the third stage to provide the macrocyclic structures. Finally, the post-cyclization processing fourth stage involving removal of protecting groups and optional purification provides the desired final compounds. Synthetic methods for this general type of macrocyclic structure are described in Intl. Pat. Appls. WO 01/25257, WO 2004/111077, WO 2005/012331 and WO 2005/012332, including purification procedures described in WO 2004/111077 and WO 2005/012331.
In some embodiments of the present invention, the macrocyclic compounds of formula I, II and/or III may be synthesized using solid phase chemistry on a soluble or insoluble polymer matrix as previously defined. For solid phase chemistry, a preliminary stage involving the attachment of the first building block, also termed “loading,” to the resin must be performed. The resin utilized for the present invention preferentially has attached to it a linker moiety, L. These linkers are attached to an appropriate free chemical functionality, usually an alcohol or amine, although others are also possible, on the base resin through standard reaction methods known in the art, such as any of the large number of reaction conditions developed for the formation of ester or amide bonds. Some linker moieties for the present invention are designed to allow for simultaneous cleavage from the resin with formation of the macrocycle in a process generally termed “cyclization-release.” (van Maarseveen, J. H. Solid phase synthesis of heterocycles by cyclization/cleavage methodologies. Comb. Chem. High Throughput Screen. 1998, 1, 185-214; Ian W. James, Linkers for solid phase organic synthesis. Tetrahedron 1999, 55, 4855-4946; Eggenweiler, H.-M. Linkers for solid-phase synthesis of small molecules: coupling and cleavage techniques. Drug Discovery Today 1998, 3, 552-560; Backes, B. J.; Ellman, J. A. Solid support linker strategies. Curr. Opin. Chem. Biol. 1997, 1, 86-93. Of particular utility in this regard for compounds of the invention is the 3-thiopropionic acid linker. (Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117; Zhang, L.; Tam, J. J. Am. Chem. Soc. 1999, 121, 3311-3320.)
Such a process provides material of higher purity as only cyclic products are released from the solid support and no contamination with the linear precursor occurs as would happen in solution phase. After sequential assembly of all the building blocks and tether into the linear precursor using known or standard reaction chemistry, base-mediated intramolecular attack on the carbonyl attached to this linker by an appropriate nucleophilic functionality that is part of the tether building block results in formation of the amide or ester bond that completes the cyclic structure as shown (Scheme 1). An analogous methodology adapted to solution phase can also be applied as would likely be preferable for larger scale applications.




Although this description accurately represents the pathway for one of the methods of the present invention, the thioester strategy, another method of the present invention, that of ring-closing metathesis (RCM), proceeds through a modified route where the tether component is actually assembled during the cyclization step. However, in the RCM methodology as well, assembly of the building blocks proceeds sequentially, followed by cyclization (and release from the resin if solid phase). An additional post-cyclization processing step is required to remove particular byproducts of the RCM reaction, but the remaining subsequent processing is done in the same manner as for the thioester or analogous base-mediated cyclization strategy.
Moreover, it will be understood that steps including the methods provided herein may be performed independently or at least two steps may be combined. Additionally, steps including the methods provided herein, when performed independently or combined, may be performed at the same temperature or at different temperatures without departing from the teachings of the present invention.
Novel macrocyclic compounds of the present invention include those formed by a novel process including cyclization of a building block structure to form a macrocyclic compound comprising a tether component described herein. Accordingly, the present invention provides methods of manufacturing the compounds of the present invention comprising (a) assembling building block structures, (b) chemically transforming the building block structures, (c) cyclizing the building block structures including a tether component, (d) removing protecting groups from the building block structures, and (e) optionally purifying the product obtained from step (d). In some embodiments, assembly of the building block structures may be sequential. In further embodiments, the synthesis methods are carried out using traditional solution synthesis techniques or solid phase chemistry techniques.
A. Amino Acids
Amino acids, Boc- and Fmoc-protected amino acids and side chain protected derivatives, including those of N-methyl and unnatural amino acids, were obtained from commercial suppliers [for example Advanced ChemTech (Louisville, Ky., USA), Bachem (Bubendorf, Switzerland), ChemImpex (Wood Dale, Ill., USA), Novabiochem (subsidiary of Merck KGaA, Darmstadt, Germany), PepTech (Burlington, Mass., USA), Synthetech (Albany, Oreg., USA)] or synthesized through standard methodologies known to those in the art. Ddz-amino acids were either obtained commercially from Orpegen (Heidelberg, Germany) or Advanced ChemTech (Louisville, Ky., USA) or synthesized using standard methods utilizing Ddz-OPh or Ddz-N3. (Birr, C.; Lochinger, W.; Stahnke, G.; Lang, P. The α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz) residue, an N-protecting group labile toward weak acids and irradiation. Justus Liebigs Ann. Chem. 1972, 763, 162-172.) Bts-amino acids were synthesized by known methods. (Vedejs, E.; Lin, S.; Klapara, A.; Wang, J. “Heteroarene-2-sulfonyl Chlorides (BtsCl, ThsCl): Reagents for Nitrogen Protection and >99% Racemization-Free Phenylglycine Activation with SOCl2.” J. Am. Chem. Soc. 1996, 118, 9796-9797. Also WO 01/25257, WO 2004/111077) N-Alkyl amino acids, in particular N-methyl amino acids, are commercially available from multiple vendors (Bachem, Novabiochem, Advanced ChemTech, ChemImpex). In addition, N-alkyl amino acid derivatives were accessed via literature methods. (Hansen, D. W., Jr.; Pilipauskas, D. J. Org. Chem. 1985, 50, 945-950.)
B. Tethers
Tethers were obtained from the methods previously described in Intl. Pat. Appl. WO 01/25257, WO 2004/111077, WO 2005/012331 and U.S. Provisional Patent Application Ser. No. 60/622,055. Procedures for synthesis of tethers as described herein are presented in the Examples below. Exemplary tethers (T) include, but are not limited to, the following:







and intermediates in the manufacture thereof, wherein (Z) is the site of a covalent bond of T to Z2, Z5 or Z10 and Z2, Z5 and Z10 are defined above for formula I, II and III, respectively, and wherein (X) is the site of a covalent bond of T to X, X2 or X3 and X, X2 and X3 are defined above for formula I; II and III, respectively, L7 is —CH2— or —O—; U1 is —CR101R102— or —C(═O)—; R100 is lower alkyl; R101 and R102 are each independently hydrogen, lower alkyl or substituted lower alkyl; xx is 2 or 3; yy is 1 or 2; zz is 1 or 2; and aaa is 0 or 1.

C. Solid Phase Techniques

Specific solid phase techniques for the synthesis of the macrocyclic compounds of the invention have been described in WO 01/25257, WO 2004/111077, WO 2005/012331 and WO 2005/012332. Solution phase synthesis routes, including methods amenable to larger scale manufacture, were described in U.S. Provisional Patent Application Ser. Nos. 60/622,055 and 60/642,271.
In certain cases, however, the lability of protecting groups precluded the use of the standard basic medium for cyclization in the thioester strategy discussed above. In these cases, either of two acidic methods was employed to provide macrocyclization under acid conditions. One method utilized HOAc, while the other method employed HOAt (Scheme 2). For example, the acetic acid cyclization was used for compound 219.
After executing the deprotection of the Ddz or Boc group on the tether, the resin was washed sequentially with DCM (2×), DCM-MeOH (1:1, 2×), DCM (2×), and DIPEA-DCM (3:7, 1×). The resin was dried under vacuum for 10 min, then added immediately to a solution of HOAc in degassed DMF (5% v/v). The reaction mixture was agitated at 50-70° C. O/N. The resin was filtered, washed with THF, and the combined filtrate and washes evaporated tinder reduced pressure (water aspirator, then oil pump) to afford the macrocycle.




For a representative macrocycle with tether T1, AA3=Leu, AA2=Leu, AA1=Phe, the application of the HOAt method shown in Scheme 2 provided the cyclic peptidomimetic in 10% yield, while the acetic acid method was more effective, and gave 24% overall yield of the same macrocycle. This latter methodology was particularly effective for compounds containing His(Mts) residues. For example, with tether T8, AA3=Phe, AA2=Acp, AA1=His(Mts), the macrocycle was obtained in 20% overall yield, but the majority of the product no longer had the Mts group on histidine (15:1 versus still protected).
Synthesis of representative macrocyclic compounds of the present invention are shown in the Examples below. Table 1A below presents a summary of the synthesis of 228 representative compounds of the present invention. The reaction methodology employed for the construction of the macrocyclic molecule is indicated in Column 2 and relates to the particular scheme of the synthetic strategy, for example, use of the thioester strategy as shown in FIG. 2 or the RCM approach as shown in FIG. 3. Column 3 indicates if any substituents are present on NBB1. Columns 4-6 and 8 indicate the individual building blocks employed for each compound, amino acids, hydroxy acids or tether utilizing either standard nomenclature or referring to the building block designations presented elsewhere in this application. Column 7 indicates the method used for attachment of the tether, either a Mitsunobu reaction (previously described in WO 01/25257) or reductive amination (previously described in WO 2004/111077). The relevant deprotection and coupling protocols as appropriate for the nature of the building block employ standard procedures and those described in WO 2004/111077 for the assembly of the cyclization precursors. The building blocks are listed in the opposite order from which they are added in order to correlate the building block number with standard peptide nomenclature. Hence BB3 is added first, followed by BB2, then BB1, finally the tether (T). In the case of the RCM, the tether is not formed completely until the cyclization step, but the portion of the tether attached to BB1 is still added at this stage of the sequence. The final macrocycles are obtained after application of the appropriate deprotection sequences. If any reaction was required to be carried out post-cyclization, it is listed in Column 9. All of the macrocycles presented in Table 1A were purified and met internal acceptance criteria. Yields (Column 10) are either isolated or as calculated based upon CLND analysis. It should be noted that compounds 58 and 99 were not cyclized to provide the linear analogues of compounds 10 and 133, respectively. The lack of binding potency observed with these linear analogues illustrates the importance of the macrocyclic structural feature for the desired activity.







TABLE 1A





Synthesis of Representative Compounds of the Present Invention





















Macrocyclic






Compound
Assembly Method
NBB1-R
BB1
BB2
BB3





1
Thioester Strategy
H
Bts-Nle
Boc-Sar
Boc-(D)Phe


2
Thioester Strategy
H
Bts-Ile
Boc-(D)Ala
Boc-(D)Phe


3
Thioester Strategy
H
Bts-Val
Boc-Sar
Boc-(D)Phe


4
Thioester Strategy
H
Bts-Nva
Boc-(D)NMeAla
Boc-(D)Phe


5
Thioester Strategy
H
Bts-Nva
Boc-NEtGly
Boc-(D)Phe


6
Thioester Strategy
H
Bts-Nva
Ddz-Sar
Ddz-(D)Trp(Boc)


7
Thioester Strategy
H
Bts-Nva
Ddz-Sar
Ddz-(D)Tyr(But)


8
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


9
Thioester Strategy
H
Bts-Val
Boc-Acp
Boc-Phe


10
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


11
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


12
Thioester Strategy
H
Bts-(D)Val
Boc-Nle
Boc-Nle


13
Thioester Strategy
H
Bts-(D)Val
Boc-Nva
Boc-Phe


14
Thioester Strategy
H
Bts-Ile
Boc-(D)Ala
Boc-Phe


15
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


16
Thioester Strategy
H
Bts-allo-Ile
Boc-(D)NMeAla
Boc-(D)Phe


17
Thioester Strategy
H
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe


18
Thioester Strategy
H
Bts-Acp
Boc-Acp
Boc-Phe


19
Thioester Strategy
H
Bts-Val
Boc-(D)NMeAla
Boc-Phe


20
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe(2-Cl)


21
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe(3-Cl)


22
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-1Nal


23
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(2-Cl)


24
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(3-Cl)


25
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)


26
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


27
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Tyr(OMe)


28
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Bip


29
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Dip


30
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)1Nal


31
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)2Nal


32
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)2Pal


33
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)4-ThzAla


34
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)2-Thi


35
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


36
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


37
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


38
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


39
Thioester Strategy
H
Bts-Nva
Boc-(D)NMeAla
Boc-(D)Phe


40
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


41
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAbu
Boc-(D)Phe


42
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


43
Thioester Strategy
H
Bts-Ile
Boc-(D)NEtAla
Boc-(D)Phe


44
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


45
Thioester Strategy
H
Bts-Leu
Ddz-Acp
Ddz-Glu(OBut)


46
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Val


47
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Leu


48
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Nva


49
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Ala


50
Thioester Strategy
H
Bts-Nva
Ddz-Sar
Ddz-(D)Glu(OBut)


51
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-Gly


52
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Nle


53
Thioester Strategy
H
Bts-Nva
Ddz-Sar
Ddz-(D)Orn(Boc)


54
Thioester Strategy
H
Bts-Nva
Ddz-Sar
Ddz-(D)Ser(But)


55
Thioester Strategy
H
Bts-(D)Nva
Boc-Sar
Boc-(D)Phe


56
Thioester Strategy
H
Bts-(D)Nva
Boc-Sar
Boc-Phe


57
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-Phe


58
Thioester Strategy,
Ac
Bts-Nva
Boc-Sar
Boc-(D)Phe



linear


59
Thioester Strategy
H
Bts-Nva
Boc-Ala
Boc-(D)Phe


60
Thioester Strategy
H
Bts-Nva
Boc-(D)Ala
Boc-(D)Phe


61
Thioester Strategy
H
Bts-Nva
Boc-Gly
Boc-(D)Phe


62
Thioester Strategy
H
Bts-Nva
Boc-Leu
Boc-(D)Phe


63
Thioester Strategy
H
Bts-Nva
Boc-(D)Leu
Boc-(D)Phe


64
Thioester Strategy
H
Bts-Nva
Boc-Phe
Boc-(D)Phe


65
Thioester Strategy
H
Bts-Nva
Boc-(D)Phe
Boc-(D)Phe


66
Thioester Strategy
H
Bts-Nva
Boc-Aib
Boc-(D)Phe


67
Thioester Strategy
H
Bts-Nva
Boc-Acp
Boc-(D)Phe


68
Thioester Strategy
H
Bts-Nva
Ddz-Lys
Boc-(D)Phe


69
Thioester Strategy
H
Bts-Nva
Ddz-(D)Lys(Boc)
Boc-(D)Phe


70
Thioester Strategy
H
Bts-Nva
Ddz-Glu(OBut)
Boc-(D)Phe


71
Thioester Strategy
H
Bts-Nva
Ddz-(D)Glu(OBut)
Boc-(D)Phe


72
Thioester Strategy
H
Bts-Ala
Boc-Sar
Boc-(D)Phe


73
Thioester Strategy
H
Bts-Glu
Boc-Sar
Boc-(D)Phe


74
Thioester Strategy
H
Bts-Lys
Boc-Sar
Boc-(D)Phe


75
Thioester Strategy
H
Bts-Phe
Boc-Sar
Boc-(D)Phe


76
Thioester Strategy
H
Bts-Ser
Boc-Sar
Boc-(D)Phe


77
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


78
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


79
Thioester Strategy
H
Bts-Nva
Boc-NMeAla
Boc-(D)Phe


80
Thioester Strategy
H
Bts-Gly
Boc-Sar
Boc-(D)Phe


81
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


82
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


83
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


84
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


85
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


86
Thioester Strategy
H
Bts-Nva
Boc-Sar
Boc-(D)Phe


87
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Ala


88
Thioester Strategy
H
Bts-Leu
Ddz-Acp
Ddz-Tyr(But)


89
Thioester Strategy
H
Bts-Leu
Ddz-Acp
Ddz-Trp(Boc)


90
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Hfe


91
Thioester Strategy
H
Bts-Leu
Ddz-Acp
Ddz-Lys(Boc)


92
Thioester Strategy
H
Bts-Leu
Ddz-Acp
Ddz-Glu(OBut)


93
Thioester Strategy
H
Bts-Leu
Boc-Ala
Boc-Phe


94
Thioester Strategy
H
Bts-Leu
Boc-(D)Ala
Boc-Phe


95
Thioester Strategy
H
Bts-Leu
Boc-Aib
Boc-Phe


96
Thioester Strategy
H
Bts-(D)Leu
Boc-Acp
Boc-Phe


97
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-(D)Phe


98
Thioester Strategy
H
Bts-(D)Leu
Boc-Acp
Boc-(D)Phe


99
Thioester Strategy,
Ac
Bts-Leu
Boc-Acp
Boc-Phe



linear


100
Thioester Strategy
H
Bts-Ala
Boc-Acp
Boc-Phe


101
Thioester Strategy
H
Bts-Nle
Boc-Acp
Boc-Phe


102
Thioester Strategy
H
Bts-Phe
Boc-Acp
Boc-Phe


103
Thioester Strategy
H
Bts-Lys
Boc-Acp
Boc-Phe


104
Thioester Strategy
H
Bts-Glu
Boc-Acp
Boc-Phe


105
Thioester Strategy
H
Bts-Ser
Boc-Acp
Boc-Phe


106
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


107
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


108
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


109
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


110
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Gly


111
Thioester Strategy
H
Bts-Leu
Boc-Acc
Boc-Phe


112
Thioester Strategy
H
Bts-Gly
Boc-Acp
Boc-Phe


113
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


114
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


115
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe


116
Thioester Strategy
H
Bts-Leu
Ddz-Acp
Ddz-Glu(Et)


117
Thioester Strategy
H
Bts-Abu
Boc-(D)NMeAla
Boc-(D)Phe


118
Thioester Strategy
H
Bts-Leu
Boc-(D)NMeAla
Boc-(D)Phe


119
Thioester Strategy
H
Bts-Thr
Boc-(D)NMeAla
Boc-(D)Phe


120
Thioester Strategy
H
Bts-Thr(OMe)
Boc-(D)NMeAla
Boc-(D)Phe


121
Thioester Strategy
H
Bts-Acc
Boc-(D)NMeAla
Boc-(D)Phe


122
Thioester Strategy
H
Bts-Phe(2-Cl)
Boc-Acp
Boc-Phe


123
Thioester Strategy
H
Bts-Phe(3-Cl)
Boc-Acp
Boc-Phe


124
Thioester Strategy
H
Bts-Phe(4-Cl)
Boc-Acp
Boc-Phe


125
Thioester Strategy
H
Bts-Phe(4-F)
Boc-Acp
Boc-Phe


126
Thioester Strategy
H
Bts-Hfe
Boc-Acp
Boc-Phe


127
Thioester Strategy
H
Bts-Tyr(OMe)
Boc-Acp
Boc-Phe


128
Thioester Strategy
H
Bts-Bip
Boc-Acp
Boc-Phe


129
Thioester Strategy
H
Bts-Dip
Boc-Acp
Boc-Phe


130
Thioester Strategy
H
Bts-1Nal
Boc-Acp
Boc-Phe


131
Thioester Strategy
H
Bts-2Nal
Boc-Acp
Boc-Phe


132
Thioester Strategy
H
Bts-3Pal
Boc-Acp
Boc-Phe


133
Thioester Strategy
H
Bts-4Pal
Boc-Acp
Boc-Phe


134
Thioester Strategy
H
Bts-4-ThzAla
Boc-Acp
Boc-Phe


135
Thioester Strategy
H
Bts-2-Thi
Boc-Acp
Boc-Phe


136
Thioester Strategy
H
Bts-Abu
Boc-Acp
Boc-Phe


137
Thioester Strategy
H
Bts-Nva
Boc-Acp
Boc-Phe


138
Thioester Strategy
H
Bts-Ile
Boc-Acp
Boc-Phe


139
Thioester Strategy
H
Bts-Val
Boc-hcLeu
Boc-Phe


140
Thioester Strategy
H
Bts-Val
Boc-hc(4O)Leu
Boc-Phe


141
Thioester Strategy
H
Bts-Val
Boc-(4O)Acp
Boc-Phe


142
Thioester Strategy
H
Bts-Val
Boc-(3-4)InAcp
Boc-Phe


143
Thioester Strategy
H
Bts-Val
Boc-hc(4S)Leu
Boc-Phe


144
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeVal
Boc-(D)Phe


145
Thioester Strategy
H
Bts-Ile
Boc-NMeVal
Boc-(D)Phe


146
Thioester Strategy
H
Bts-Ile
Boc-NMeNva
Boc-(D)Phe


147
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeLeu
Boc-(D)Phe


148
Thioester Strategy
H
Bts-Ile
Boc-NMeLeu
Boc-(D)Phe


149
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeIle
Boc-(D)Phe


150
Thioester Strategy
H
Bts-Ile
Boc-NMeIle
Boc-(D)Phe


151
Thioester Strategy
H
Bts-Ile
Ddz-(D)Ser(But)
Boc-(D)Phe


152
Thioester Strategy
H
Bts-Ile
Ddz-NMeSer(But)
Boc-(D)Phe


153
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe(4-Cl)


154
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Phe(4-F)


155
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Hfe


156
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Tyr(OMe)


157
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Bip


158
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Dip


159
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-2Nal


160
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-2Pal


161
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-3Pal


162
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-4Pal


163
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-4-ThzAla


164
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-2-Thi


165
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Abu


166
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Ile


167
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-allo-Ile


168
Thioester Strategy
H
Bts-Leu
Boc-Acp
Boc-Acp


169
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Hfe


170
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)3Pal


171
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)4Pal


172
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-Abu


173
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Nva


174
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Val


175
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Ile


176
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Leu


177
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


178
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


179
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


180
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


181
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


182
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


183
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


184
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


185
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


186
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


187
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


188
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


189
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


190
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


191
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


192
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


193
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


194
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


195
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe


196
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


197
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


199
Thioester Strategy
H
Bts-Val
Boc-Acc
Boc-Phe


200
Thioester Strategy
H
Bts-Val
Boc-Acp
Boc-Phe


201
Thioester Strategy
Me
Bts-Nva
Boc-(D)NMeAla
Boc-(D)Phe


202
Thioester Strategy
Ac
Bts-Nva
Boc-(D)NMeAla
Boc-(D)Phe


203
Thioester Strategy
Me
Bts-Leu
Boc-Acp
Boc-Phe


204
Thioester Strategy
Ac
Bts-Leu
Boc-Acp
Boc-Phe


205
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Abu


206
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


207
Thioester Strategy
H
Bts-Val
Boc-hc(4N)Leu
Boc-Phe


208
Thioester Strategy
H
Bts-allo-Ile
Boc-Acp
Boc-Phe


209
Thioester Strategy
H
Bts-Ile
Boc-(D)NMeAla
Boc-(D)allo-Ile


210
Thioester Strategy
H
Bts-2Pal
Boc-Acp
Boc-Phe


211
Thioester Strategy
H
Bts-Val
Boc-hc(4N)Leu
Boc-Phe


212
Thioester Strategy
H
Bts-Ile
Boc-NMeAbu
Boc-(D)Phe


213
Thioester Strategy
H
Bts-Ile
Boc-(D)4-Thz
Boc-(D)Phe


214
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe










215

isolated from synthesis of compound 151













216
Thioester Strategy
H
Bts-Val
Boc-Acc
Boc-Phe


218
Thioester Strategy
H
Bts-hcLeu
Boc-Acp
Boc-Phe


219
Acetic Acid
H
Bts-His(Mts)
Boc-Acp
Boc-Phe



Cyclization


220
Thioester Strategy
H
Bts-Nva
Boc-Pro
Boc-(D)Phe


221
Thioester Strategy
H
Bts-Nva
Boc-(D)Pro
Boc-(D)Phe


222
Thioester Strategy
H
Bts-Leu
Boc-Pro
Boc-Phe


223
Thioester Strategy
H
Bts-Leu
Boc-(D)Pro
Boc-Phe


224
RCM Strategy
H
Fmoc-Ile
Fmoc-(D)Hyp(But)
Fmoc-(D)Phe


225
Thioester Strategy
H
Bts-Pro
Boc-(D)NMeAla
Boc-(D)Phe


226
Thioester Strategy
H
Bts-Pip
Boc-(D)NMeAla
Boc-(D)Phe

















Tether Attachment

Additional




Compound
Method
Tether
Reaction**
Yield (%)*







 1
Mitsunobu Reaction
Boc-T9
None
10.1



 2
Mitsunobu Reaction
Boc-T9
None
13.8



 3
Mitsunobu Reaction
Boc-T9
None
10.3



 4
Mitsunobu Reaction
Boc-T9
None
4.6



 5
Mitsunobu Reaction
Boc-T9
None
8.6



 6
Mitsunobu Reaction
Ddz-T9
None
8.1



 7
Mitsunobu Reaction
Ddz-T9
None
8.8



 8
Mitsunobu Reaction
Boc-T8
None
20.9



 9
Mitsunobu Reaction
Boc-T9
None
9.7



 10
 Mitsunobu Reaction
Boc-T9
None
9.9



 11
Mitsunobu Reaction
Boc-T8
None
9.9



 12
Mitsunobu Reaction
Boc-T8
None
2.9



 13
Mitsunobu Reaction
Boc-T8
None
5.8



 14
Mitsunobu Reaction
Boc-T8
None
27.5



 15
Mitsunobu Reaction
Boc-T9
None
19.5



 16
Mitsunobu Reaction
Boc-T9
None
23.9



 17
Reductive Amination
Boc-T9
None
24.8




Reaction



 18
Mitsunobu Reaction
Boc-T8
None
6.8



 19
Mitsunobu Reaction
Boc-T8
None
12.7



 20
Mitsunobu Reaction
Boc-T8
None
22.0



 21
Mitsunobu Reaction
Boc-T8
None
24.7



 22
Mitsunobu Reaction
Boc-T8
None
10.3



 23
Mitsunobu Reaction
Boc-T9
None
32.6



 24
Mitsunobu Reaction
Boc-T9
None
22.4



 25
Mitsunobu Reaction
Boc-T9
None
21.0



 26
Mitsunobu Reaction
Boc-T9
None
15.5



 27
Mitsunobu Reaction
Boc-T9
None
20.2



 28
Mitsunobu Reaction
Boc-T9
None
31.6



 29
Mitsunobu Reaction
Boc-T9
None
26.1



 30
Mitsunobu Reaction
Boc-T9
None
31.9



 31
Mitsunobu Reaction
Boc-T9
None
21.9



 32
Reductive Amination
Boc-T9
None
6.7




Reaction



 33
Mitsunobu Reaction
Boc-T9
None
7.5



 34
Mitsunobu Reaction
Boc-T9
None
14.2



 35
Mitsunobu Reaction
Boc-T33a
None
9.4



 36
Mitsunobu Reaction
Boc-T33b
None
13.0



 37
Mitsunobu Reaction
TA1 + TB4
None
24.6



 38
Mitsunobu Reaction
TA2 + TB1
Hydrogenation
44.2



 39
Mitsunobu Reaction
Boc-T8
None
21.4



 40
Mitsunobu Reaction
Boc-T8
None
18.6



 41
Mitsunobu Reaction
Boc-T9
None
10.6



 42
Mitsunobu Reaction
Boc-T9
None
1.7



 43
Mitsunobu Reaction
Boc-T9
None
0.4



 44
Mitsunobu Reaction
Boc-T1
None
7.8



 45
Mitsunobu Reaction
Ddz-T8
None
11.6



 46
Mitsunobu Reaction
Boc-T8
None
13.6



 47
Mitsunobu Reaction
Boc-T8
None
9.2



 48
Mitsunobu Reaction
Boc-T8
None
17.5



 49
Reductive Amination
Boc-T9
None
7.5




Reaction



 50
Mitsunobu Reaction
Ddz-T9
None
10.1



 51
Mitsunobu Reaction
Boc-T9
None
6.6



 52
Mitsunobu Reaction
Boc-T9
None
8.7



 53
Mitsunobu Reaction
Ddz-T9
None
8.3



 54
Mitsunobu Reaction
Ddz-T9
None
6.2



 55
Mitsunobu Reaction
Boc-T9
None
8.0



 56
Mitsunobu Reaction
Boc-T9
None
9.3



 57
Mitsunobu Reaction
Boc-T9
None
8.9



 58
Mitsunobu Reaction
Boc-T9
No cyclization
5.9



 59
Mitsunobu Reaction
Boc-T9
None
8.0



 60
Mitsunobu Reaction
Boc-T9
None
13.1



 61
Mitsunobu Reaction
Boc-T9
None
8.4



 62
Mitsunobu Reaction
Boc-T9
None
7.0



 63
Mitsunobu Reaction
Boc-T9
None
11.7



 64
Mitsunobu Reaction
Boc-T9
None
8.5



 65
Mitsunobu Reaction
Boc-T9
None
8.6



 66
Mitsunobu Reaction
Boc-T9
None
15.8



 67
Mitsunobu Reaction
Boc-T9
None
11.7



 68
Mitsunobu Reaction
Ddz-T9
None
7.9



 69
Mitsunobu Reaction
Ddz-T9
None
11.2



 70
Mitsunobu Reaction
Ddz-T9
None
10.0



 71
Mitsunobu Reaction
Ddz-T9
None
9.9



 72
Mitsunobu Reaction
Boc-T9
None
5.2



 73
Mitsunobu Reaction
Boc-T9
None
6.8



 74
Mitsunobu Reaction
Boc-T9
None
6.0



 75
Mitsunobu Reaction
Boc-T9
None
9.5



 76
Mitsunobu Reaction
Boc-T9
None
15.1



 77
Mitsunobu Reaction
Boc-T12
None
12.6



 78
Mitsunobu Reaction
Boc-T27
None
6.8



 79
Mitsunobu Reaction
Boc-T9
None
1.9



 80
Mitsunobu Reaction
Boc-T9
None
1.3



 81
Mitsunobu Reaction
Boc-T1
None
5.3



 82
Mitsunobu Reaction
Boc-T3
None
3.9



 83
Mitsunobu Reaction
Boc-T16
None
1.8



 84
Mitsunobu Reaction
Boc-T4
None
2.6



 85
Mitsunobu Reaction
Boc-T5
None
4.7



 86
Mitsunobu Reaction
Boc-T14
None
0.4



 87
Mitsunobu Reaction
Boc-T9
None
4.8



 88
Mitsunobu Reaction
Ddz-T9
None
18.8



 89
Mitsunobu Reaction
Ddz-T9
None
16.5



 90
Mitsunobu Reaction
Boc-T9
None
8.5



 91
Mitsunobu Reaction
Ddz-T9
None
6.8



 92
Mitsunobu Reaction
Ddz-T9
None
9.1



 93
Mitsunobu Reaction
Boc-T9
None
9.2



 94
Mitsunobu Reaction
Boc-T9
None
21.8



 95
Mitsunobu Reaction
Boc-T9
None
19.3



 96
Mitsunobu Reaction
Boc-T9
None
7.0



 97
Mitsunobu Reaction
Boc-T9
None
9.2



 98
Mitsunobu Reaction
Boc-T9
None
15.3



 99
Mitsunobu Reaction
Boc-T9
No cyclization
10.4



100
Mitsunobu Reaction
Boc-T9
None
10.4



101
Mitsunobu Reaction
Boc-T9
None
19.0



102
Mitsunobu Reaction
Boc-T9
None
15.8



103
Mitsunobu Reaction
Boc-T9
None
12.9



104
Mitsunobu Reaction
Boc-T9
None
9.3



105
Mitsunobu Reaction
Boc-T9
None
11.9



106
Mitsunobu Reaction
Boc-T3
None
6.3



107
Mitsunobu Reaction
Boc-T5
None
4.2



108
Mitsunobu Reaction
Boc-T12
None
18.3



109
Mitsunobu Reaction
Boc-T11
None
10.1



110
Mitsunobu Reaction
Boc-T9
None
2.9



111
Mitsunobu Reaction
Boc-T9
None
3.0



112
Mitsunobu Reaction
Boc-T9
None
3.2



113
Mitsunobu Reaction
Boc-T9
None
16.9



114
Mitsunobu Reaction
Boc-T16
None
2.9



115
Mitsunobu Reaction
Boc-T6
None
0.5



116
Mitsunobu Reaction
Ddz-T8
None
11.8



117
Mitsunobu Reaction
Boc-T9
None
19.7



118
Mitsunobu Reaction
Boc-T9
None
21.0



119
Mitsunobu Reaction
Boc-T9
None
12.2



120
Reductive Amination
Boc-T9
None
17.5




Reaction



121
Mitsunobu Reaction
Boc-T9
None
5.8



122
Mitsunobu Reaction
Boc-T8
None
22.1



123
Mitsunobu Reaction
Boc-T8
None
13.6



124
Mitsunobu Reaction
Boc-T8
None
9.8



125
Mitsunobu Reaction
Boc-T8
None
15.8



126
Mitsunobu Reaction
Boc-T8
None
9.8



127
Mitsunobu Reaction
Boc-T8
None
14.5



128
Mitsunobu Reaction
Boc-T8
None
17.8



129
Mitsunobu Reaction
Boc-T8
None
11.0



130
Mitsunobu Reaction
Boc-T8
None
18.8



131
Mitsunobu Reaction
Boc-T8
None
15.0



132
Reductive Amination
Boc-T8
None
17.0




Reaction



133
Reductive Amination
Boc-T8
None
9.5




Reaction



134
Mitsunobu Reaction
Boc-T8
None
12.0



135
Mitsunobu Reaction
Boc-T8
None
4.0



136
Mitsunobu Reaction
Boc-T8
None
13.3



137
Mitsunobu Reaction
Boc-T8
None
19.0



138
Mitsunobu Reaction
Boc-T8
None
13.8



139
Reductive Amination
Boc-T8
None
18.4




Reaction



140
Reductive Amination
Boc-T8
None
16.7




Reaction



141
Reductive Amination
Boc-T8
None
15.7




Reaction



142
Reductive Amination
Boc-T8
None
17.0




Reaction



143
Reductive Amination
Boc-T8
None
16.1




Reaction



144
Reductive Amination
Boc-T9
None
5.7




Reaction



145
Reductive Amination
Boc-T9
None
4.9




Reaction



146
Reductive Amination
Boc-T9
None
23.3




Reaction



147
Reductive Amination
Boc-T9
None
14.4




Reaction



148
Reductive Amination
Boc-T9
None
25.4




Reaction



149
Reductive Amination
Boc-T9
None
11.4




Reaction



150
Reductive Amination
Boc-T9
None
7.0




Reaction



151
Mitsunobu Reaction
Ddz-T9
None
8.2



152
Reductive Amination
Ddz-T9
None
22.1




Reaction



153
Mitsunobu Reaction
Boc-T8
None
13.5



154
Mitsunobu Reaction
Boc-T8
None
14.4



155
Mitsunobu Reaction
Boc-T8
None
13.5



156
Mitsunobu Reaction
Boc-T8
None
13.2



157
Mitsunobu Reaction
Boc-T8
None
20.2



158
Mitsunobu Reaction
Boc-T8
None
11.3



159
Mitsunobu Reaction
Boc-T8
None
20.5



160
Reductive Amination
Boc-T8
None
2.8




Reaction



161
Reductive Amination
Boc-T8
None
16.5




Reaction



162
Reductive Amination
Boc-T8
None
16.7




Reaction



163
Mitsunobu Reaction
Boc-T8
None
10.0



164
Mitsunobu Reaction
Boc-T8
None
12.5



165
Mitsunobu Reaction
Boc-T8
None
13.0



166
Mitsunobu Reaction
Boc-T8
None
11.1



167
Mitsunobu Reaction
Boc-T8
None
15.3



168
Mitsunobu Reaction
Boc-T8
None
4.2



169
Mitsunobu Reaction
Boc-T9
None
17.0



170
Reductive Amination
Boc-T9
None
14.5




Reaction



171
Reductive Amination
Boc-T9
None
16.4




Reaction



172
Mitsunobu Reaction
Boc-T9
None
12.0



173
Mitsunobu Reaction
Boc-T9
None
16.8



174
Mitsunobu Reaction
Boc-T9
None
13.9



175
Mitsunobu Reaction
Boc-T9
None
15.1



176
Mitsunobu Reaction
Boc-T9
None
9.4



177
Mitsunobu Reaction
Boc-T11
None
9.3



178
Mitsunobu Reaction
Boc-T28
None
11.2



179
Mitsunobu Reaction
Boc-T29
None
8.6



180
Mitsunobu Reaction
Boc-T30
None
10.0



181
Mitsunobu Reaction
TA1 + TB7
None
49.5



182
Mitsunobu Reaction
TA1 + TB7
Hydrogenation
47.7



183
Mitsunobu Reaction
TA2 + TB7
None
59.0



184
Mitsunobu Reaction
TA2 + TB7
Hydrogenation
50.6



185
Mitsunobu Reaction
TA1 + TB6
None
12.4



186
Mitsunobu Reaction
TA2 + TB6
None
3.0



187
Mitsunobu Reaction
TA1 + TB3
None
30.9



188
Mitsunobu Reaction
TA2 + TB3
None
34.9



189
Mitsunobu Reaction
TA2 + TB3
Hydrogenation
24.0



190
Mitsunobu Reaction
TA1 + TB4
Hydrogenation
32.5



191
Mitsunobu Reaction
TA2 + TB4
None
32.2



192
Mitsunobu Reaction
TA2 + TB4
Hydrogenation
22.2



193
Mitsunobu Reaction
TA1 + TB1
None
47.7



194
Mitsunobu Reaction
TA1 + TB1
Hydrogenation
23.7



195
Mitsunobu Reaction
TA2 + TB1
None
66.8



196
Mitsunobu Reaction
Ddz-T32(Boc)
None
13.0



197
Mitsunobu Reaction
Ddz-T31(But)
None
10.6



199
Reductive Amination
Boc-T8
None
16.0




Reaction



200
Mitsunobu Reaction
Boc-T8
None
14.7



201
Reductive Amination
Boc-T9
Reductive
32.4




Reaction

amination






reaction with






formaldehyde



202
Reductive Amination
Boc-T9
Acetylation
14.2




Reaction



203
Reductive Amination
Boc-T8
Reductive
7.7




Reaction

amination






reaction with






formaldehyde



204
Reductive Amination
Boc-T8
Acetylation
11.5




Reaction



205
Mitsunobu Reaction
Boc-T9
None
19.9



206
Mitsunobu Reaction
Boc-T34
None
26.2



207
Mitsunobu Reaction
Boc-T9
None
<1



208
Mitsunobu Reaction
Boc-T8
None
16.7



209
Mitsunobu Reaction
Boc-T9
None
8.6



210
Reductive Amination
Boc-T8
None
1.1




Reaction



211
Reductive Amination
Boc-T8
None
<1




Reaction



212
Mitsunobu Reaction
Boc-T9
None
1.2



213
Reductive Amination
Boc-T9
None
1.0




Reaction



214
Mitsunobu Reaction
TA1 + TB3
Hydrogenation
14.9



215



216
Reductive Amination
Boc-T9
None
11.6




Reaction



218
Mitsunobu Reaction
Boc-T8
None
0.1



219
Reductive Amination
Boc-T8
None
19.0




Reaction



220
Mitsunobu Reaction
Boc-T9
None
15.0



221
Mitsunobu Reaction
Boc-T9
None
14.9



222
Mitsunobu Reaction
Boc-T9
None
11.7



223
Mitsunobu Reaction
Boc-T9
None
20.4



224
Mitsunobu Reaction
TA1 + TB2
Hydrogenation
8.2



225
Reductive Amination
Boc-T9
None
10.0




Reaction



226
Reductive Amination
Boc-T9
None
13.5




Reaction







*Overall Yield: based on theoretical resin loading, starting from ~500 mg resin



**Additional reactions conducted post-cyclization, excpet where otherwise noted, to reach the desired product






Table 1B below presents a summary of the synthesis of 122 representative compounds of the present invention, and Table 1C presents the synthesis of an additional 15 representative compounds. For Table 1B, the reaction methodology employed for the construction of the macrocyclic molecule is indicated in the Column 2 and relates to the particular scheme of the synthetic strategy. Columns 3-6 indicate the individual building blocks employed for each compound, amino acids or tether utilizing either standard nomenclature or referring to the building block designations presented elsewhere in this application. Column 7 indicates the method used for attachment of the tether. The building blocks are listed in the opposite order from which they are added in order to correlate the building block number with standard peptide nomenclature. Column 8 indicates if any additional reaction chemistry was applied, such as to remove auxiliary protection or to reduce a double bond (as was performed with many RCM intermediate products). All of the macrocycles in Tables 1B and 1C were purified and met the acceptance criteria. Yields (Column 9-10) are either isolated or as calculated based upon CLND analysis.







TABLE 1B





Synthesis of Representative Compounds of the Present Invention




















Macrocyclic





Compound
Assembly Method
BB1
BB2
BB3





298
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


299
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)


301
Thioester Strategy
Bts-Tyr(But)
Boc-Acp
Boc-Phe(3-Cl)


303
Thioester Strategy
Bts-Val
Boc-(4O)Acp
Boc-Phe


305
Thioester Strategy
Bts-Ile
Boc-(D)NMeAla
Boc-(D)His(Mts)


306
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


307
RCM Strategy
Fmoc-Cpg
Fmoc-(D)NMeAla
Fmoc-(D)Phe(4-F)


308
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)


309
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


310
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)3-Thi


311
Thioester Strategy
Boc-Cpg
Boc-(D)NMeAla
Boc-(D)Tyr(3-tBu)


312
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(2-F)


313
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(3-F)


314
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(2,4-diCl)


315
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(3,4-diCl)


316
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(3,4-diF)


317
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(3,5-diF)


318
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(pentaF)


319
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-Br)


320
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-I)


321
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-CN)


322
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-CF3)


323
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(3,4-diOMe)


324
Thioester Strategy
Bts-Cpg
Boc-(D)NMcAla
Boc-(D)Trp


325
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-F)


326
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Br)


327
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3,5-diF)


328
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-OMe)


329
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-CN)


330
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3,4-diCl)


331
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3,4-diF)


332
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-CF3)


333
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-3-Thi


334
Thioester Strategy
Bts-Acp
Boc-Aib
Boc-Phe(3-Cl)


335
Thioester Strategy
Boc-Thr(OMe)
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


336
Thioester Strategy
Bts-Ser(OMe)
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


337
Thioester Strategy
Boc-Dap(Cbz)
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


338
Thioester Strategy
Bts-Dab(Boc)
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


339
Thioester Strategy
Bts-Orn(Boc)
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


340
Thioester Strategy
Boc-Met
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


341
Thioester Strategy
Bts-3-Thi
Boc-Acp
Boc-Phe(3-Cl)


342
Thioester Strategy
Bts-Phe(2-CN)
Boc-Acp
Boc-Phe(3-Cl)


343
Thioester Strategy
Bts-Phe(2-OMe)
Boc-Acp
Boc-Phe(3-Cl)


344
Thioester Strategy
Bts-Ser(OMe)
Boc-Acp
Boc-Phe(3-Cl)


345
Thioester Strategy
Bts-Ile
Boc-(4O)Acp
Boc-Phe(3-Cl)


346
Thioester Strategy
Bts-Cpg
Boc-Acp
Boc-Phe(3-Cl)


347
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Ser(OBzl)


348
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Ser(OBzl)


349
Thioester Strategy
Bts-Aib
Boc-Acp
Boc-Phe(3-Cl)


350
Thioester Strategy
Bts-Aib
Boc-Aib
Boc-Phe(3-Cl)


351
Thioester Strategy
Bts-Acp
Boc-(D)Ala
Boc-Phe(3-Cl)


352
Thioester Strategy
Bts-Acp
Boc-Ala
Boc-Phe(3-Cl)


353
RCM Strategy
Fmoc-Ile
Fmoc-(D)NMeAla
Fmoc-(D)Phe(4-F)


354
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


355
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


356
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


357
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


358
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


359
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


360
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


361
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


362
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


363
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


364
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


365
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


366
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


367
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


368
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


369
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


370
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


371
RCM Strategy
Fmoc-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


372
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


373
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


374
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


375
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


376
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


377
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


378
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


379
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


380
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


381
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


382
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


383
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


384
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


385
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


386
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


387
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


388
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


389
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


390
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


391
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(3,4,5-triF)


392
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


393
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


394
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


395
RCM Strategy
Fmoc-Ile
Fmoc-(4N)Acp
Fmoc-Phe(3-Cl)


396
Thioester Strategy
Bts-Acp
Boc-(D)NMeAla
Boc-Phe(3-Cl)


397
Thioester Strategy
Bts-Acp
NMeAla
Boc-Phe(3-Cl)


398
RCM Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


399
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


400
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


401
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


402
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)


403
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe


405
Thioester Strategy
Bts-Nva
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


406
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


407
Thioester Strategy
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


408
Thioester Strategy
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


409
Thioester Strategy
Bts-Val
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


410
RCM Strategy
Bts-Nva
Boc-(D)NMeAla
Boc-(D)Phe


415
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)


417
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)


430
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)


431
Thioester Strategy
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe


432
Thioesier Strategy
Bts-Ile
Boc-(D)NMeAla
Boc-(D)Phe(4-Cl)



















Tether
Additional
Amount
Yield



Compound
Tether
Attachment
Reaction**
(mg)*
(%)*







298
Boc-T33a
Mitsunobu Reaction
None
29.7
12



299
Boc-T9
Mitsunobu Reaction
None
54.1
17



301
Ddz-T8
Mitsunobu Reaction
None
36.5
10



303
Boc-T8
Mitsunobu Reaction
None
60
16



305
Boc-T9
Reductive Animation
None
110
31





Reaction



306
Boc-T11
Mitsunobu Reaction
None
51
8



307
TA2 + TB6
Mitsunobu Reaction
None
13.6
10



308
Boc-T8
Mitsunobu Reaction
None
43.8
14



309
Boc-T9
Mitsunobu Reaction
None
38.2
13



310
Boc-T9
Mitsunobu Reaction
None
33.3
11



311
Boc-T9
Reductive Amination
None
18.6
5.1





Reaction



312
Boc-T9
Mitsunobu Reaction
None
42.9
14



313
Boc-T9
Mitsunobu Reaction
None
38.2
13



314
Boc-T9
Mitsunobu Reaction
None
39.7
12



315
Boc-T9
Mitsunobu Reaction
None
35.3
11



316
Boc-T9
Mitsunobu Reaction
None
40.7
13



317
Boc-T9
Mitsunobu Reaction
None
37.6
12



318
Boc-T9
Mitsunobu Reaction
None
36.1
11



319
Boc-T9
Mitsunobu Reaction
None
37.5
11



320
Boc-T9
Mitsunobu Reaction
None
43.4
12



321
Boc-T9
Mitsunobu Reaction
None
34.5
11



322
Boc-T9
Mitsunobu Reaction
None
40.8
12



323
Boc-T9
Mitsunobu Reaction
None
27.3
8



324
Boc-T9
Mitsunobu Reaction
None
38.6
12



325
Boc-T8
Mitsunobu Reaction
None
33.7
10



326
Boc-T8
Mitsunobu Reaction
None
37.5
10



327
Boc-T8
Mitsunobu Reaction
None
35.2
11



328
Boc-T8
Mitsunobu Reaction
None
31.5
10



329
Boc-T8
Mitsunobu Reaction
None
26.9
8



330
Boc-T8
Mitsunobu Reaction
None
38.4
11



331
Boc-T8
Mitsunobu Reaction
None
37
11



332
Boc-T8
Mitsunobu Reaction
None
30.6
9



333
Boc-T8
Mitsunobu Reaction
None
49.6
18



334
Boc-T8
Mitsunobu Reaction
None
32
11



335
Boc-T9
Reductive Amination
None
62.2
18





Reaction



336
Boc-T9
Mitsunobu Reaction
None
37.7
12



337
Boc-T9
Reductive Animation
Hydrogenolysis
67.5
7





Reaction



338
Boc-T9
Mitsunobu Reaction
None
60
20



339
Boc-T9
Mitsunobu Reaction
None
63
20



340
Boc-T9
Reductive Animation
None
14.4
4





Reaction



341
Boc-T8
Mitsunobu Reaction
None
48
14



342
Boc-T8
Mitsunobu Reaction
None
37.7
10



343
Boc-T8
Mitsunobu Reaction
None
91.3
25



344
Boc-T8
Mitsunobu Reaction
None
22.1
7



345
Boc-T8
Mitsunobu Reaction
None
48
13



346
Boc-T8
Mitsunobu Reaction
None
52.1
16



347
Boc-T8
Mitsunobu Reaction
None
17.1
6



348
Boc-T8
Mitsunobu Reaction
None
104.4
33



349
Boc-T8
Mitsunobu Reaction
None
23.6
7



350
Boc-T8
Mitsunobu Reaction
None
44
15



351
Boc-T8
Mitsunobu Reaction
None
39.1
13



352
Boc-T8
Mitsunobu Reaction
None
15.7
5



353
TA1 + TB4
Mitsunobu Reaction
None
47.8
25



354
Boc-T65
Mitsunobu Reaction
None
26.8
9



355
Boc-T70
Mitsunobu Reaction
None
36.8
12



356
Boc-T72
Mitsunobu Reaction
None
10
3



357
Ddz-T74(Boc)
Mitsunobu Reaction
None
41.8
11



358
TA1 + TB4
Mitsunobu Reaction
None
26.1
26



359
Boc-T58
Mitsunobu Reaction
None
43.6
12



360
TA2 + TB6
Mitsunobu Reaction
None
36.3
18



361
TA2 + TB4
Mitsunobu Reaction
None
36.3
32



362
TA2 + TB1
Mitsunobu Reaction
Hydrogenation
59.4
57



363
TA2 + TB7
Mitsunobu Reaction
Hydrogenation
41.8
44



364
TA2 + TB7
Mitsunobu Reaction
Hydrogenation
49.1
51



365
TA1 + TB10
Mitsunobu Reaction
Hydrogenation
31.2
35



366
TA1 + TB7
Mitsunobu Reaction
Hydrogenation
33.3
37



367
Boc-T33b
Mitsunobu Reaction
None
21.1
6



368
Boc-T33a
Mitsunobu Reaction
None
21.8
10



369
Boc-T9
Mitsunobu Reaction
None
21.1
4



370
TA2 + TB6
Mitsunobu Reaction
Hydrogenation
8.9
NA



371
TA2 + TB4
Mitsunobu Reaction
Hydrogenation
9.9
NA



372
Boc-T69
Mitsunobu Reaction
None
30.9
10



373
Boc-T71
Mitsunobu Reaction
None
34.9
11



374
Ddz-T73(Boc)
Mitsumobu Reaction
None
42.7
12



375
Boc-T39
Mitsunobu Reaction
None
22.3
7



376
Boc-T40
Mitsunobu Reaction
None
7.5
2



377
Boc-T10
Mitsunobu Reaction
None
14.6
5



378
Boc-T58
Mitsunobu Reaction
None
65.3
21



379
Boc-T67
Mitsunobu Reaction
None
36.3
12



380
Boc-T66
Mitsunobu Reaction
None
16.5
5



381
Boc-T65
Mitsunobu Reaction
None
22.5
7



382
Boc-T70
Mitsunobu Reaction
None
24.5
7



383
Boc-T69
Mitsunobu Reaction
None
25.2
7



384
Boc-T71
Mitsunobu Reaction
None
21.9
6



385
Boc-T11
Mitsunobu Reaction
None
23.3
7



386
Boc-T39
Mitsunobu Reaction
None
12
4



387
Boc-T68
Mitsunobu Reaction
None
17.1
5



388
Boc-T67
Mitsunobu Reaction
None
30
9



389
Boc-T68
Mitsunobu Reaction
None
16.1
5



390
Boc-T18
Mitsunobu Reaction
None
28.7
10



391
Boc-T9
Mitsunobu Reaction
None
45.4
14



392
Boc-T40
Mitsunobu Reaction
None
4.3
1



393
Boc-T45
Mitsunobu Reaction
None
2.1
1



394
Boc-T38
Mitsunobu Reaction
None
3.7
1



395
TA1 + TB2
Mitsunobu Reaction
Hydrogenation
0.2
0.2



396
Boc-T8
Mitsunobu Reaction
None
2.3
1



397
Boc-T8
Mitsunobu Reaction
None
1.4
0.4



398
TA2 + TB6
Mitsunobu Reaction
Hydrogenation
3.8
1



399
Boc-T33b
Mitsunobu Reaction
None
5.7
4



400
Boc-T66
Mitsunobu Reaction
None
28.3
9



401
Boc-T8
Mitsunobu Reaction
None
31.5
11



402
Boc-T8
Mitsunobu Reaction
None
29.1
9



403
Boc-T33a
Mitsunobu Reaction
None
103
11



405
Boc-T33a
Mitsunobu Reaction
None
38.8
12



406
Boc-T75a
Mitsunobu Reaction
None
45
13



407
Boc-T33a
Mitsunobu Reaction
None
138.5
16



408
Boc-T75a
Mitsunobu Reaction
None
146.2
21



409
Boc-T33a
Mitsunobu Reaction
None
125.7
19



410
Boc-T75a
Mitsunobu Reaction
None
36
11



415
Boc-T33a
Mitsunobu Reaction
None
127.5
12



417
Boc-T69
Mitsunobu Reaction
None
45.6
13



430
Boc-T75a
Mitsunobu Reaction
None
50.7
14



431
Boc-T33a
Mitsunobu Reaction
None
57.9
17



432
Boc-T33a
Mitsunobu Reaction
None
141
13







*Overall Yield: based on theoretical resin loading, starting from ~500 mg resin



**Additional reactions conducted post-cyclization to reach the desired product













TABLE 1C





Synthesis of Representative Compounds of the Present Invention




















Macrocyclic





Compound
Assembly Method
BB1
BB2
BB3





435
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe


436
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe


437
Thioester Strategy
Bts-Acp
Boc-Acp
Boc-Phe(3-Cl)


438
Thioester Strategy
Bts-Leu
Boc-Acp
Boc-Phe(3-Cl)


439
Thioester Strategy
Bts-Ile
Boc-(3/4O)Acp
Boc-Phe(3-Cl)


440
RCM Strategy
Bts-Ile
Fmoc-(D)NMeSer(OBzl)
Fmoc-(D)Phe(4-F)


441
Thioester Strategy
Bts-Ile
Ddz-Acp
Ddz-Phe(4-CO2tBu)


442
Thioester Strategy
Bts-Ile
Ddz-Acp
Ddz-Ser(But)


443
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Ser(OMe)


444
Thioester Strategy
Boc-Leu
Boc-Acp
Boc-His(Mts)


445
Thioester Strategy
Bts-Ile
Ddz-(D)NMeAla
Ddz-(D)Tyr(But)


446
Thioester Strategy
Bts-Cpg
Boc-(D)NMeAla
Boc-(D)Phe(4-F)


447
RCM Strategy
Bts-Ile
Fmoc-Acp
Fmoc-Phe(3-Cl)


448
RCM Strategy
Bts-Nva
Fmoc-Sar
Fmoc-(DL)αMePhe


449
Thioester Strategy
Bts-Ile
Boc-Acp
Boc-Phe(3-Cl)

















Tether
Additional
Amount
Yield


Compound
Tether
Attachment
Reaction**
(mg)
(%)





435
Boc-T75a
Mitsunobu Reaction
None
29.7
9


436
Boc-T76
Mitsunobu Reaction
None
37.8
11


437
Boc-T8
Mitsunobu Reaction
None
8.3
2


438
Boc-T33a
Mitsunobu Reaction
None
51.2
5


439
Boc-T8
Mitsunobu Reaction
None
5.9
2


440
TA1 + TB2
Mitsunobu Reaction
Hydrogenation
2.7
2


441
Ddz-T8
Mitsunobu Reaction
None
9.8
3


442
Ddz-T8
Mitsunobu Reaction
None
17.1
6


443
Boc-T8
Mitsunobu Reaction
None
19
7


444
Boc-T8
Reductive Animation
None
21
7




Reaction


445
Boc-T9
Mitsunobu Reaction
None
15.5
5


446
Boc-T45
Mitsunobu Reaction
None
3.2
1


447
TA1 + TB9
Mitsunobu Reaction
Hydrogenation
18.2
21


448
TA1 + TB2
Mitsunobu Reaction
Hydrogenation
4.8
2


449
Boc-T77
Mitsunobu Reaction
None
2.6
1





*Overall Yield: based on theoretical resin loading, starting from ~500 mg resin


**Additional reactions conducted post-cyclization to obtain the desired product






The tables directly below present analytical data obtained for compounds 1-197, 199-216, 218-230 (Table 2A), compounds 298, 299, 301, 303, 304-403, 405-410, 415, 417 and 430-432 (Table 2B) and compounds 435-449 (Table 2C), as determined by LC-MS analysis of the purified products. These compounds were further examined for their ability to interact at the human ghrelin receptor utilizing the biological test methods described below.








TABLE 2A







Analytical Characterization for Representative


Compounds of the Present Invention












MW



Compound
Molecular Formula
Calc (g/mol)
MS [(M + H)+] Found













1
C29H40N4O4
508.7
509


2
C29H40N4O4
508.7
509


3
C28H38N4O4
494.6
495


4
C29H40N4O4
508.7
509


5
C29H40N4O4
508.7
509


6
C30H39N5O4
533.7
534


7
C28H38N4O5
510.6
511


8
C32H42N4O4
546.7
547


9
C31H42N4O4
534.7
535


10
C28H38N4O4
494.6
495


11
C28H36N4O4
492.6
493


12
C28H45N4O4
501.7
502


13
C30H40N4O4
520.7
521


14
C29H38N4O4
506.6
507


15
C30H42N4O4
522.7
523


16
C30H42N4O4
522.7
523


17
C29H38N4O4
506.6
507


18
C32H40N4O4
544.7
545


19
C29H38N4O4
506.6
507


20
C32H41N4O4Cl
581.1
581


21
C32H41N4O4Cl
581.1
581


22
C36H44N4O4
596.8
597


23
C30H41N4O4Cl
557.1
557


24
C30H41N4O4Cl
557.1
557


25
C30H41N4O4Cl
557.1
557


26
C30H41N4O4F
540.7
541


27
C31H44N4O5
552.7
553


28
C36H46N4O4
598.8
599


29
C36H46N4O4
598.8
599


30
C34H44N4O4
572.7
573


31
C34H44N4O4
572.7
573


32
C29H41N5O4
523.7
524


33
C27H39N5O4S
529.7
530


34
C28H40N4O4S
528.7
529


35
C31H44N4O4
536.7
537


36
C31H44N4O4
536.7
537


37
C31H42N4O3
518.7
519


38
C31H44N4O3
520.7
521


39
C29H38N4O4
506.6
507


40
C30H40N4O4
520.7
521


41
C31H44N4O4
536.7
537


42
C30H42N4O4
522.7
523


43
C31H44N4O4
536.7
537


44
C25H38N4O4
458.6
459


45
C28H40N4O6
528.6
529


46
C28H42N4O4
498.7
499


47
C29H44N4O4
512.7
513


48
C28H42N4O4
498.7
499


49
C22H34N4O4
418.5
419


50
C24H36N4O6
476.6
477


51
C21H32N4O4
404.5
405


52
C25H40N4O4
460.6
461


53
C24H39N5O4
461.6
462


54
C22H34N4O5
434.5
435


55
C28H38N4O4
494.6
495


56
C28H38N4O4
494.6
495


57
C28H38N4O4
494.6
495


58
C30H43N5O5
553.7
554


59
C28H38N4O4
494.6
495


60
C28H38N4O4
494.6
495


61
C27H36N4O4
480.6
481


62
C31H44N4O4
536.7
537


63
C31H44N4O4
536.7
537


64
C34H42N4O4
570.7
571


65
C34H42N4O4
570.7
571


66
C29H40N4O4
508.7
509


67
C31H42N4O4
534.7
535


68
C31H45N5O4
551.7
552


69
C31H45N5O4
551.7
552


70
C30H40N4O6
552.7
553


71
C30H40N4O6
552.7
553


72
C26H34N4O4
466.6
467


73
C28H36N4O6
524.6
525


74
C29H41N5O4
523.7
524


75
C32H38N4O4
542.7
543


76
C26H34N4O5
482.6
483


77
C31H36N4O3S
544.7
545


78
C23H34N4O4
430.5
431


79
C29H41N4O4
509.7
510


80
C25H33N4O4
453.6
454


81
C21H33N4O4
405.5
406


82
C23H33N4O3
413.5
414


83
C23H35N4O3
415.5
416


84
C25H33N4O3
437.6
438


85
C26H35N4O3
451.6
452


86
C22H30N5O3S
444.6
445


87
C26H40N4O4
472.6
473


88
C32H44N4O5
564.7
565


89
C34H45N5O4
587.8
588


90
C33H46N4O4
562.7
563


91
C29H47N5O4
529.7
530


92
C28H42N4O6
530.7
531


93
C29H40N4O4
508.7
509


94
C29H40N4O4
508.7
509


95
C30H42N4O4
522.7
523


96
C32H44N4O4
548.7
549


97
C32H44N4O4
548.7
549


98
C32H44N4O4
548.7
549


99
C34H49N5O5
607.8
608


100
C29H38N4O4
506.6
507


101
C32H44N4O4
548.7
549


102
C35H42N4O4
582.7
583


103
C32H45N5O4
563.7
564


104
C31H40N4O6
564.7
565


105
C29H38N4O5
522.6
523


106
C27H38N4O3
466.6
467


107
C30H40N4O3
504.7
505


108
C35H42N4O3S
598.8
599


109
C31H43N5O4
549.7
550


110
C25H39N4O4
459.6
460


111
C30H40N4O4
520.7
521


112
C28H37N4O4
493.6
494


113
C32H45N4O4
549.7
550


114
C27H41N4O3
469.6
470


115
C30H41N4O3
505.7
506


116
C30H44N4O6
556.7
557


117
C28H38N4O4
494.6
495


118
C30H42N4O4
522.7
523


119
C28H38N4O5
510.6
511


120
C29H40N4O5
524.7
525


121
C28H36N4O4
492.6
493


122
C35H39N4O4Cl
615.2
615


123
C35H39N4O4Cl
615.2
615


124
C35H39N4O4Cl
615.2
615


125
C35H39N4O4F
598.7
599


126
C36H42N4O4
594.7
595


127
C36H42N4O5
610.7
611


128
C41H44N4O4
656.8
657


129
C41H44N4O4
656.8
657


130
C39H42N4O4
630.8
631


131
C39H42N4O4
630.8
631


132
C34H39N5O4
581.7
582


133
C34H39N5O4
581.7
582


134
C32H37N5O4S
587.7
588


135
C33H38N4O4S
586.7
587


136
C30H38N4O4
518.6
519


137
C31H40N4O4
532.7
533


138
C32H42N4O4
546.7
547


139
C32H42N4O4
546.7
547


140
C31H40N4O5
548.7
549


141
C30H38N4O5
534.6
535


142
C35H40N4O4
580.7
581


143
C31H40N4O4S
564.7
565


144
C32H46N4O4
550.7
551


145
C32H46N4O4
550.7
551


146
C32H46N4O4
550.7
551


147
C33H48N4O4
564.8
565


148
C33H48N4O4
564.8
565


149
C33H48N4O4
564.8
565


150
C33H48N4O4
564.8
565


151
C29H40N4O5
524.7
525


152
C30H42N4O5
538.7
539


153
C32H41N4O4Cl
581.1
581


154
C32H41N4O4F
564.7
565


155
C33H44N4O4
560.7
561


156
C33H44N4O5
576.7
577


157
C38H46N4O4
622.8
623


158
C38H46N4O4
622.8
623


159
C36H44N4O4
596.8
597


160
C31H41N5O4
547.7
548


161
C31H41N5O4
547.7
548


162
C31H41N5O4
547.7
548


163
C29H39N5O4S
553.7
554


164
C30H40N4O4S
552.7
553


165
C27H40N4O4
484.6
485


166
C29H44N4O4
512.7
513


167
C29H44N4O4
1.0
2


168
C29H42N4O4
510.7
511


169
C31H44N4O4
536.7
537


170
C29H41N5O4
523.7
524


171
C29H41N5O4
523.7
524


172
C25H40N4O4
460.6
461


173
C26H42N4O4
474.6
475


174
C26H42N4O4
474.6
475


175
C27H44N4O4
488.7
489


176
C27H44N4O4
488.7
489


177
C29H41N5O4
523.7
524


178
C29H40N4O4
508.7
509


179
C30H42N4O3
506.7
507


180
C31H44N4O3
520.7
521


181
C26H40N4O3
456.6
457


182
C26H42N4O3
458.6
459


183
C27H42N4O3
470.6
471


184
C27H44N4O3
472.7
473


185
C25H38N4O4
458.6
459


186
C26H40N4O4
472.6
473


187
C30H40N4O3
504.7
505


188
C31H42N4O3
518.7
519


189
C31H44N4O3
520.7
521


190
C31H44N4O3
520.7
521


191
C32H44N4O3
532.7
533


192
C32H46N4O3
534.7
535


193
C30H40N4O3
504.7
505


194
C30H42N4O3
506.7
507


195
C31H42N4O3
518.7
519


196
C31H44N6O4
564.7
565


197
C31H42N4O6
566.7
567


199
C29H36N4O4
504.6
505


200
C31H40N4O4
532.7
533


201
C30H42N4O4
522.7
523


202
C31H42N4O5
550.7
551


203
C33H44N4O4
560.7
561


204
C34H44N4O5
588.7
589


205
C25H40N4O4
460.6
461


206
C31H46N6O5
582.7
583


207
C31H43N5O4
549.7
550


208
C32H42N4O4
546.7
547


209
C27H44N4O4
488.7
489


210
C34H39N5O4
581.7
582


211
C31H41N5O4
547.7
548


212
C31H44N4O4
536.7
537


213
C30H40N4O4S
552.7
553


214
C30H42N4O3
506.7
507


215
C33H48N4O5
580.8
581


216
C29H38N4O4
506.6
507


218
C33H42N4O4
558.7
559


219
C32H38N6O4
570.7
571


220
C30H40N4O4
520.7
521


221
C30H40N4O4
520.7
521


222
C31H42N4O4
534.7
535


223
C31H42N4O4
534.7
535


224
C31H42N4O5
550.7
551


225
C29H38N4O4
506.6
507


226
C30H40N4O4
520.7
521


227
C30H40N4O4
520.7
521


228
C30H40N4O4
520.7
521


229
C31H42N4O4
534.7
535


230
C31H42N4O4
534.7
535





Notes


1. Molecular formulas and molecular weights are calculated automatically from the structure via ActivityBase software (IDBS, Guildford, Surrey, UK).


2. M + H obtained from LC-MS analysis using standard methods.


3. All analyses conducted on material after preparative purification by the methods described above.













TABLE 2B







Analytical Characterization for Representative


Compounds of the Present Invention












MW



Compound
Molecular Formula
Calc (g/mol)
MS [(M + H)+] Found













298
C30H39N4O4F
538.7
539


299
C29H37N4O4Cl
541.1
541


301
C35H39N4O5Cl
631.2
631


303
C30H38N4O5
534.6
535


305
C27H40N6O4
512.6
513


306
C28H36N5O4F
525.6
526


307
C25H35N4O4F
474.6
475


308
C29H35N4O4Cl
539.1
539


309
C29H37N4O4F
524.6
525


310
C27H36N4O4S
512.7
513


311
C33H46N4O5
578.7
579


312
C29H37N4O4F
524.6
525


313
C29H37N4O4F
524.6
525


314
C29H36N4O4Cl2
575.5
575


315
C29H36N4O4Cl2
575.5
575


316
C29H36N4O4F2
542.6
543


317
C29H36N4O4F2
542.6
543


318
C29H33N4O4F5
596.6
597


319
C29H37N4O4Br
585.5
585


320
C29H37N4O4I
632.5
633


321
C30H37N5O4
531.6
532


322
C30H37N4O4F3
574.6
575


323
C31H42N4O6
566.7
567


324
C31H39N5O4
545.7
546


325
C32H41N4O4F
564.7
565


326
C32H41N4O4Br
625.6
625


327
C32H40N4O4F2
582.7
583


328
C33H44N4O5
576.7
577


329
C33H41N5O4
571.7
572


330
C32H40N4O4Cl2
615.6
616


331
C32H40N4O4F2
582.7
583


332
C33H41N4O4F3
614.7
615


333
C30H40N4O4S
552.7
553


334
C30H37N4O4Cl
553.1
553


335
C29H39N4O5F
542.6
543


336
C28H37N4O5F
528.6
529


337
C27H36N5O4F
513.6
514


338
C28H38N5O4F
527.6
528


339
C29H40N5O4F
541.7
542


340
C29H39N4O4FS
558.7
559


341
C33H37N4O4SCl
621.2
621


342
C36H38N5O4Cl
640.2
640


343
C36H41N4O5Cl
645.2
645


344
C30H37N4O5Cl
569.1
569


345
C31H39N4O5Cl
583.1
583


346
C31H37N4O4Cl
565.1
565


347
C33H44N4O5
576.7
577


348
C31H42N4O5
550.7
551


349
C30H37N4O4Cl
553.1
553


350
C28H35N4O4Cl
527.1
527


351
C29H35N4O4Cl
539.1
539


352
C29H35N4O4Cl
539.1
539


353
C31H41N4O3F
536.7
537


354
C29H33N4O4F
520.6
521


355
C29H36N4O4F2
542.6
543


356
C30H36N4O4F4
592.6
593


357
C30H40N5O6FS
617.7
618


358
C33H43N4O3Cl
579.2
579


359
C34H47N4O4Cl
611.2
611


360
C28H41N4O4Cl
533.1
533


361
C34H45N4O3Cl
593.2
593


362
C33H45N4O3Cl
581.2
581


363
C29H45N4O3Cl
533.1
533


364
C29H43N4O3Cl
531.1
531


365
C27H41N4O3Cl
505.1
505


366
C28H43N4O3Cl
519.1
519


367
C30H39N4O4F
538.7
539


368
C33H45N4O4Cl
597.2
597


369
C32H43N4O4Cl
583.2
583


370
C28H43N4O4Cl
535.1
535


371
C34H47N4O3Cl
595.2
595


372
C29H36N4O4F2
542.6
543


373
C29H36N4O4FCl
559.1
559


374
C30H40N5O6FS
617.7
618


375
C30H39N4O4F
538.7
539


376
C30H39N4O4F
538.7
539


377
C28H35N4O5F
526.6
527


378
C31H41N4O4F
552.7
553


379
C30H37N4O4F
536.6
537


380
C32H41N4O4Cl
581.1
581


381
C32H39N4O4Cl
579.1
579


382
C32H42N4O4FCl
601.2
601


383
C32H42N4O4FCl
601.2
601


384
C32H42N4O4Cl2
617.6
617


385
C31H42N5O4Cl
584.1
584


386
C33H45N4O4Cl
597.2
597


387
C33H43N4O4Cl
595.2
595


388
C33H43N4O4Cl
595.2
595


389
C30H37N4O4F
536.6
537


390
C26H40N5O3Cl
506.1
506


391
C29H35N4O4F3
560.6
561


392
C33H45N4O4Cl
597.2
597


393
C27H41N4O5Cl
537.1
537


394
C30H39N4O4F
538.7
539


395
C31H42N5O4Cl
584.1
584


396
C30H37N4O4Cl
553.1
553


397
C30H37N4O4Cl
553.1
553


398
C25H37N4O4F
476.6
477


399
C33H45N4O4Cl
597.2
597


400
C29H35N4O4F
522.6
523


401
C29H35N4O4F
522.6
523


402
C32H41N4O4Cl
581.1
581


403
C30H40N4O4
520.7
521


405
C30H41N4O4F
540.7
541


406
C30H38N4O4F2
556.6
557


407
C31H43N4O4F
554.7
555


408
C31H42N4O4F2
572.7
573


409
C30H41N4O4F
540.7
541


410
C30H42N4O4
522.7
523


415
C30H39N4O4Cl
555.1
555


417
C29H36N4O4FCl
559.1
559


430
C30H38N4O4FCl
573.1
573


431
C31H44N4O4
536.7
537


432
C31H43N4O4Cl
571.2
571





Notes


1. Molecular formulas and molecular weights are calculated automatically from the structure via ActivityBase software (IDBS, Guildford, Surrey, UK).


2. M + H obtained from LC-MS analysis using standard methods.


3. All analyses conducted on material after preparative purification by the methods described above.













TABLE 2C







Analytical Characterization for Representative


Compounds of the Present Invention












MW



Compound
Molecular Formula
Calc (g/mol)
MS [(M + H)+] Found













435
C30H39N4O4F
538.7
539


436
C31H40N4O4
532.7
533


437
C32H39N4O4Cl
579.1
579


438
C33H45N4O4Cl
597.2
597


439
C32H39N4O5Cl
595.1
595


440
C37H47N4O5F
646.8
647


441
C33H42N4O6
590.7
591


442
C26H38N4O5
486.6
487


443
C27H40N4O5
500.6
501


444
C29H40N6O4
536.7
537


445
C30H42N4O5
538.7
539


446
C24H35N4O5F
478.6
479


447
C26H39N4O3Cl
491.1
492


448
C29H40N4O4
508.7
509


449
C31H42N5O4Cl
584.1
584





Notes


1. Molecular formulas and molecular weights are calculated automatically from the structure via ActivityBase software (IDBS, Guildford, Surrey, UK).


2. M + H obtained from LC-MS analysis using standard methods.


3. All analyses conducted on material after preparative purification by the methods described above.






D. Chiral Purity Determination

General methods for the HPLC determination of stereoisomeric purity were employed according to techniques known to those skilled in the art and further optimized for the compounds of the present invention.
Method Chiral A: Grad35A-05 (Column: Chiralcel AS-RH, 0.46 cm×15 cm):


    1. Isocratic plateau of 40 min at 35% ACN, 65% of a 50 mM solution of CH3COONH4 in H2O.
    2. 5 min gradient to 70% ACN, 30% of a 50 mM solution of CH3COONH4 in H2O.
    3. Isocratic plateau of 10 min at 70% ACN, 30% of a 50 mM solution of CH3COONH4 in H2O.
    4. 5 min gradient to 35% ACN, 65% of a 50 mM solution of CH3COONH4 in H2O.
    5. Isocratic plateau of 10 min at 35% ACN, 65% of a 50 mM solution of CH3COONH4 in H2O.
    6. Flow: 0.5 mL/min
    7. Column temperature: room temperature
    8. Sample temperature: room temperature

Method Chiral B: Grad40A-05 (Column: Chiralcel OD-RH, 0.46 cm×15 cm):

    1. Isocratic plateau of 40 min at 40% ACN, 60% of a solution 50 mM of CH3COONH4 in H2O.
    2. 5 min gradient to 70% ACN, 30% of a solution 50 mM of CH3COONH4 in H2O.
    3. Isocratic plateau of 10 min at 70% ACN, 30% of a solution 50 mM of CH3COONH4 in H2O.
    4. 5 min gradient to 40% ACN, 60% of a solution 50 mM of CH3COONH4 in H2O.
    5. Isocratic plateau of 10 min at 40% ACN, 60% of a solution 50 mM of CH3COONH4 in H2O.
    6. Flow: 0.5 mL/min
    7. Column temperature: room temperature
    8. Sample temperature: room temperature

Method Chiral C: Grad 55A-05 (Column: Chiralcel OD-RH, 0.46 cm×15 cm):

    1. 40 min isocratic 55%/45% of ACN/50 mM CH3COONH4 in H2O
    2. 5 min gradient to 70%/30% of ACN/50 mM CH3COONH4 in H2O
    3. 10 min isocratic 70%/30% of ACN/50 mM CH3COONH4 in H2O
    4. 5 min gradient to 55%/44% of ACN/50 mM CH3COONH4 in H2O
    5. 10 min isocratic 55%/45% of ACN/50 mM CH3COONH4 in H2O
    6. Flow: 0.5 mL/min
    7. Column temperature: room temperature
    8. Sample temperature: room temperature

Method Chiral D: Grad Iso100B—05 (Column: Chiralcel OD-RH, 0.46 cm×15 cm)

    1. 40 min isocratic 27%/73% of ACN/50 mM CH3COONH4 in H2O
    2. 5 min gradient to 70%/30% of ACN/50 mM CH3COONH4 in H2O
    3. 10 min isocratic 70%/30% of ACN/50 mM CH3COONH4 in H2O
    4. 5 min gradient to 27%/73% of ACN/50 mM CH3COONH4 in H2O
    5. 10 min isocratic 27%/73% of ACN/50 mM CH3COO in H2O
    6. Flow: 0.5 mL/min
    7. Column temperature: room temperature
    8. Sample temperature: room temperature

3. Biological Methods



The compounds of the present invention were evaluated for their ability to interact at the human ghrelin receptor utilizing a competitive radioligand binding assay, fluorescence assay or Aequorin functional assay as described below. Such methods can be conducted in a high throughput manner to permit the simultaneous evaluation of many compounds.
Specific assay methods for the human (GHS-R1a), swine and rat GHS-receptors (U.S. Pat. No. 6,242,199, Intl. Pat. Appl. Nos. WO 97/21730 and 97/22004), as well as the canine GHS-receptor (U.S. Pat. No. 6,645,726), and their use in generally identifying agonists and antagonists thereof are known.
Appropriate methods for determining the functional activity of compounds of the present invention that interact at the human ghrelin receptor are also described below.
A. Competitive Radioligand Binding Assay (Ghrelin Receptor)
The competitive binding assay at the human growth hormone secretagogue receptor (hGHS-R1a) was carried out analogously to assays described in the literature. (Bednarek M A et al. Structure-function studies on the new growth hormone-releasing peptide ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a; J. Med. Chem. 2000, 43, 4370-4376; Palucki, B. L. et al. Spiro(indoline-3,4′-piperidine) growth hormone secretagogues as ghrelin mimetics; Bioorg. Med. Chem. Lett. 2002, 11, 1955-1957.)
Materials
Membranes (GHS-R/HEK 293) were prepared from HEK-293 cells stably transfected with the human ghrelin receptor (hGHS-R1a). These membranes were provided by PerkinElmer BioSignal (#RBHGHSM, lot#1887) and utilized at a quantity of 0.71 μg/assay point.

    1. [125I]-Ghrelin (PerkinElmer, #NEX-388); final concentration: 0.0070-0.0085 mM
    2. Ghrelin (Bachem, #H-4864); final concentration: 1 μM
    3. Multiscreen Harvest plates-GF/C (Millipore, #MAHFC1H60)
    4. Deep-well polypropylene titer plate (Beckman Coulter, #267006)
    5. TopSeal-A (PerkinElmer, #6005185)
    6. Bottom seal (Millipore, #MATAH0P00)
    7. MicroScint-0 (PerkinElmer, #6013611)
    8. Binding Buffer: 25 mM Hepes (pH 7.4), 1 mM CaCl2, 5 mM MgCl2, 2.5 mM EDTA, 0.4% BSA

Assay Volumes



Competition experiments were performed in a 300 μl filtration assay format.

    1. 220 μL of membranes diluted in binding buffer
    2. 40 μL of compound diluted in binding buffer
    3. 40 μL of radioligand ([125I]-Ghrelin) diluted in binding buffer

Final test concentrations (N=1) for compounds of the present invention:

10, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, 0.001 μM.

Compound Handling



Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at −80° C. until the day of testing. On the test day, compounds were allowed to thaw at rt O/N and then diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximal final DMSO concentration in the assay was 0.1%.
Assay Protocol
In deep-well plates, 220 μL of diluted cell membranes (final concentration: 0.71 μg/well) were combined with 40 μL of either binding buffer (total binding, N=5), 1 μM ghrelin (non-specific binding, N=3) or the appropriate concentration of test compound (N=2 for each test concentration). The reaction was initiated by addition of 40 μL of [125I]-ghrelin (final conc. 0.0070-0.0085 nM) to each well. Plates were sealed with TopSeal-A, vortexed gently and incubated at rt for 30 min. The reaction was arrested by filtering samples through Multiscreen Harvest plates (pre-soaked in 0.5% polyethyleneimine) using a Tomtec Harvester, washed 9 times with 500 μL of cold 50 mM Tris-HCl (pH 7.4, 4° C.), and then plates were air-dried in a fumehood for 30 min. A bottom seal was applied to the plates prior to the addition of 25 μL of MicroScint-0 to each well. Plates were than sealed with TopSeal-A and counted for 30 sec per well on a TopCount Microplate Scintillation and Luminescence Counter (PerkinElmer) using a count delay of 60 sec. Results were expressed as counts per minute (cpm).
Data were analyzed by GraphPad Prism (GraphPad Software, San Diego, Calif.) using a variable slope non-linear regression analysis. Ki values were calculated using a Kd value of 0.01 nM for [125I]-ghrelin (previously determined during membrane characterization).
Dmax values were calculated using the following formula:




  
    D
    max
  
  =
  
    1
    -
    
      
        
          
            
              
                test
                ⁢
                
                  
                
                ⁢
                concentration
                ⁢
                
                  
                
                ⁢
                with
              
            
          
          
            
              
                
                  
                    
                      
                        maximal
                        ⁢
                        
                          
                        
                        ⁢
                        displacement
                      
                      -
                    
                  
                
                
                  
                    
                      non
                      ⁢
                      
                        -
                      
                      ⁢
                      specific
                      ⁢
                      
                        
                      
                      ⁢
                      binding
                    
                  
                
              
            
          
        
        
          
            total
            ⁢
            
              
            
            ⁢
            binding
          
          -
          
            non
            ⁢
            
              -
            
            ⁢
            specific
            ⁢
            
              
            
            ⁢
            binding
          
        
      
      ×
      100
    
  




where total and non-specific binding represent the cpm obtained in the absence or presence of 1 μM ghrelin, respectively.

Binding activity at the gherlin receptor for representative compounds of the present invention is shown below in Table 3A through 3D. Compound structures for Tables 3A, 3B and 3D are presented with the various groups as defined for the general structure of formula I. For Tables 3B and 3D, in all entries, m, n and p are 0; X, Z1 and Z2 are each NH. For Table 3B, R1 is H for all entries. The tethers (T) are illustrated with the bonding to X and Z2 as indicated. The compounds themselves are shown for Table 3C. Competitive binding curves for representative compounds 1, 2, 3, 4 and 25 are shown in FIG. 4.







TABLE 3A







Binding Activity at the Human Ghrelin Receptor for Compounds of the Invention















Cmpd
X
R1
R2
m
R7
R3
R4
n





 1
N—H
H




0
CH3
H
H
0


 


 2
N—H
H




0
H
CH3
H
0


 


 3
N—H
H




0
CH3
H
H
0


 


 4
N—H
H




0
CH3
H
CH3
0


 


 5
N—H
H




0
CH2CH3
H
H
0


 


 6
N—H
H




0
CH3
H
H
0


 


 7
N—H
H




0
CH3
H
H
0


 


 8
N—H
H




0
H





0


 


 9
N—H
H




0
H





0


 


 10
N—H
H




0
CH3
H
H
0


 


 11
N—H
H




0
CH3
H
H
0


 


 12
N—H




H
0
H
H




0


 


 13
N—H




H
0
H
H




0


 


 14
N—H
H




0
H
CH3
H
0


 


 15
N—H
H




0
CH3
CH3
H
0


 


 16
N—H
H




0
CH3
CH3
H
0


 


 17
N—H
H




0
CH3
CH3
H
0


 


 18
N—H





0
H





0


 


 19a
N—H
H




0
CH3
CH3
H
0


 


 19b

diastereomer








 


 20
N—H
H




0
H





0


 


 21
N—H
H




0
H





0


 


 22
N—H
H




0
H





0


 


 23
N—H
H




0
CH3
CH3
H
0


 


 24
N—H
H




0
CH3
CH3
H
0


 


 25
N—H
H




0
CH3
CH3
H
0


 


 26
N—H
H




0
CH3
CH3
H
0


 


 27
N—H
H




0
CH3
CH3
H
0


 


 28
N—H
H




0
CH3
CH3
H
0


 


 29
N—H
H




0
CH3
CH3
H
0


 


 30
N—H
H




0
CH3
CH3
H
0


 


 31
N—H
H




0
CH3
CH3
H
0


 


 32
N—H
H




0
CH3
CH3
H
0


 


 33
N—H
H




0
CH3
CH3
H
0


 


 34
N—H
H




0
CH3
CH3
H
0


 


 35
N—H
H




0
CH3
CH3
H
0


 


 36
N—H
H




0
CH3
CH3
H
0


 


 37a
N—H
H




0
CH3
CH3
H
0


 


 37b

diastereomer








 


 38
N—H
H




0
CH3
CH3
H
0


 


 39
N—H
H




0
CH3
H
H
0


 


 40
N—H
H




0
CH3
CH3
H
0


 


 41
N—H
H




0
CH3




H
0


 


 42
N—H
H




0
CH3
CH3
H
0


 


 43
N—H
H




0
CH2CH3
CH3
H
0


 


 44
N—H
H




0
H





0


 


 45
N—H
H




0
H





0


 


 46
N—H
H




0
H





0


 


 47
N—H
H




0
H





0


 


 48
N—H
H




0
H





0


 


 49
N—H
H




0
CH3
H
H
0


 


 50
N—H
H




0
CH3
H
H
0


 


 51
N—H
H




0
CH3
H
H
0


 


 52
N—H
H




0
CH3
H
H
0


 


 53
N—H
H




0
CH3
H
H
0


 


 54
N—H
H




0
CH3
H
H
0


 


 55
N—H




H
0
CH3
H
H
0


 


 56
N—H




H
0
CH3
H
H
0


 


 57
N—H
H




0
CH3
H
H
0


 


 58
N—Ac
H




0
CH3
H
H
0


 


 59
N—H
H




0
H
H
CH3
0


 


 60
N—H
H




0
H
CH3
H
0


 


 61
N—H
H




0
H
H
H
0


 


 62
N—H
H




0
H
H




0


 


 63
N—H
H




0
H




H
0


 


 64
N—H
H




0
H
H




0


 


 65
N—H
H




0
H




H
0


 


 66
N—H
H




0
H
CH3
CH3
0


 


 67
N—H
H




0
H





0


 


 68
N—H
H




0
H
H




0


 


 69
N—H
H




0
H




H
0


 


 70
N—H
H




0
H
H




0


 


 71
N—H
H




0
H




H
0


 


 72
N—H
H
CH3
0
CH3
H
H
0


 


 73
N—H
H




0
CH3
H
H
0


 


 74
N—H
H




0
CH3
H
H
0


 


 75
N—H
H




0
CH3
H
H
0


 


 76
N—H
H




0
CH3
H
H
0


 


 77
N—H
H




0
CH3
H
H
0


 


 78
N—H
H




0
CH3
H
H
0


 


 79
N—H
H




0
CH3
H
CH3
0


 


 80
N—H
H
H
0
CH3
H
H
0


 


 81
N—H
H




0
CH3
H
H
0


 


 82
N—H
H




0
CH3
H
H
0


 


 83
N—H
H




0
CH3
H
H
0


 


 84
N—H
H




0
CH3
H
H
0


 


 85
N—H
H




0
CH3
H
H
0


 


 86
N—H
H




0
CH3
H
H
0


 


 87
N—H
H




0
H





0


 


 88
N—H
H




0
H





0


 


 89
N—H
H




0
H





0


 


 90
N—H
H




0
H





0


 


 91
N—H
H




0
H





0


 


 92
N—H
H




0
H





0


 


 93
N—H
H




0
H
H
CH3
0


 


 94
N—H
H




0
H
CH3
H
0


 


 95
N—H
H




0
H
CH3
CH3
0


 


 96
N—H




H
0
H





0


 


 97
N—H
H




0
H





0


 


 98
N—H




H
0
H





0


 


 99
N—Ac
H




0
H





0


 


100
N—H
H
CH3
0
H





0


 


101
N—H
H




0
H





0


 


102
N—H
H




0
H





0


 


103
N—H
H




0
H





0


 


104
N—H
H




0
H





0


 


105
N—H
H




0
H





0


 


106
N—H
H




0
H





0


 


107
N—H
H




0
H





0


 


108
N—H
H




0
H





0


 


109
N—H
H




0
H





0


 


110
N—H
H




0
H





0


 


111
N—H
H




0
H





0


 


112
N—H
H
H
0
H





0


 


113
N—H
H




0
H





0


 


114
N—H
H




0
H





0


 


115
N—H
H




0
H





0


 


116
N—H
H




0
H





0


 


117
N—H
H




0
CH3
CH3
H
0


 


118
N—H
H




0
CH3
CH3
H
0


 


119
N—H
H




0
CH3
CH3
H
0


 


120
N—H
H




0
CH3
CH3
H
0


 


121
N—H





0
CH3
CH3
H
0


 


122
N—H
H




0
H





0


 


123
N—H
H




0
H





0


 


124
N—H
H




0
H





0


 


125
N—H
H




0
H





0


 


126
N—H
H




0
H





0


 


127
N—H
H




0
H





0


 


128
N—H
H




0
H





0


 


129
N—H
H




0
H





0


 


130
N—H
H




0
H





0


 


131
N—H
H




0
H





0


 


132
N—H
H




0
H





0


 


133
N—H
H




0
H





0


 


134
N—H
H




0
H





0


 


135
N—H
H




0
H





0


 


136a
N—H
H




0
H





0


 


136b

diastereomer








 


137
N—H
H




0
H





0


 


138
N—H
H




0
H





0


 


139
N—H
H




0
H





0


 


140
N—H
H




0
H





0


 


141
N—H
H




0
H





0


 


142
N—H
H




0
H





0


 


143
N—H
H




0
H





0


 


144
N—H
H




0
CH3




H
0


 


145a
N—H
H




0
CH3
H




0


 


145b

diastereomer








 


146a
N—H
H




0
CH3
H




0


 


146b

diastereomer








 


147
N—H
H




0
CH3




H
0


 


148
N—H
H




0
CH3
H




0


 


149
N—H
H




0
CH3




H
0


 


150a
N—H
H




0
CH3
H




0


 


150b

diastereomer








 


151
N—H
H




0
H




H
0


 


152a
N—H
H




0
CH3
H




0


 


152b

diastereomer








 


153
N—H
H




0
H





0


 


154
N—H
H




0
H





0


 


155
N—H
H




0
H





0


 


156
N—H
H




0
H





0


 


157
N—H
H




0
H





0


 


158
N—H
H




0
H





0


 


159
N—H
H




0
H





0


 


160a
N—H
H




0
H





0


 


160b

diastereomer








 


161a
N—H
H




0
H





0


 


161b

diastereomer








 


162a
N—H
H




0
H





0


 


162b

diastereomer








 


163
N—H
H




0
H





0


 


164
N—H
H




0
H





0


 


165
N—H
H




0
H





0


 


166
N—H
H




0
H





0


 


167
N—H
H




0
H





0


 


168
N—H
H




0
H





0


 


169
N—H
H




0
CH3
CH3
H
0


 


170
N—H
H




0
CH3
CH3
H
0


 


171
N—H
H




0
CH3
CH3
H
0


 


172
N—H
H




0
CH3
CH3
H
0


 


173
N—H
H




0
CH3
CH3
H
0


 


174
N—H
H




0
CH3
CH3
H
0


 


175
N—H
H




0
CH3
CH3
H
0


 


176
N—H
H




0
CH3
CH3
H
0


 


177
N—H
H




0
CH3
CH3
H
0


 


178
N—H
H




0
CH3
CH3
H
0


 


179
N—H
H




0
CH3
CH3
H
0


 


180
N—H
H




0
CH3
CH3
H
0


 


181
N—H
H




0
CH3
CH3
H
0


 


182a
N—H
H




0
CH3
CH3
H
0


 


182b

diastereomer








 


183
N—H
H




0
CH3
CH3
H
0


 


184
N—H
H




0
CH3
CH3
H
0


 


184

diastereomer








 


185
N—H
H




0
CH3
CH3
H
0


 


186
N—H
H




0
CH3
CH3
H
0


 


187
N—H
H




0
CH3
CH3
H
0


 


188
N—H
H




0
CH3
CH3
H
0


 


189a
N—H
H




0
CH3
CH3
H
0


 


189b

diastereomer








 


190
N—H
H




0
CH3
CH3
H
0


 


191
N—H
H




0
CH3
CH3
H
0


 


192
N—H
H




0
CH3
CH3
H
0


 


193
N—H
H




0
CH3
CH3
H
0


 


194a
N—H
H




0
CH3
CH3
H
0


 


194b

diastereomer








 


195
N—H
H




0
CH3
CH3
H
0


 


196
N—H
H




0
CH3
CH3
H
0


 


197
N—H
H




0
CH3
CH3
H
0


 


199
N—H
H




0
H





0


 


200
N—H
H




0
H





0


 


201
N-Me
H




0
CH3
CH3
H
0


 


202
N—Ac
H




0
CH3
CH3
H
0


 


203
N-Me
H




0
H





0


 


204
N—Ac
H




0
H





0


 


205
N—H
H




0
CH3
CH3
H
0


 


206
N—H
H




0
CH3
CH3
H
0


 


207
N—H
H




0
H





0


 


208a
N—H
H




0
H





0


 


208b

diastereomer








 


209
N—H
H




0
CH3
CH3
H
0


 


210
N—H
H




0
H





0


 


211
N—H
H




0
H





0


 


212
N—H
H




0
CH3
H




0


 


213
N—H
H




0
H




H
0


 


214
N—H
H




0
CH3
CH3
H
0


 


215
N—H
H




0
H




H
0


 


216
N—H
H




0
H





0


 


218
N—H





0
H





0


 


219
N—H
H




0
H





0
























Ki


Cmpd
Z1
R5
R6
p
Z2
T
(nM)





 1
N—H




H
0
N—H




B


 


 2
N—H




H
0
N—H




C


 


 3
N—H




H
0
N—H




C


 


 4
N—H




H
0
N—H




B


 


 5
N—H




H
0
N—H




C


 


 6
N—H




H
0
N—H




C


 


 7
N—H




H
0
N—H




C


 


 8
N—H
H




0
N—H




B


 


 9
N—H
H




0
N—H




C


 


 10
N—H




H
0
N—H




B


 


 11
N—H




H
0
N—H




B


 


 12
N—H
H




0
N—H




C


 


 13
N—H
H




0
N—H




C


 


 14
N—H
H




0
N—H




C


 


 15
N—H




H
0
N—H




A


 


 16
N—H




H
0
N—H




A


 


 17
N—H




H
0
N—H




A


 


 18
N—H
H




0
N—H




B


 


 19a
N—H
H




0
N—H




A


 


 19b






C


 


 20
N—H
H




0
N—H




A


 


 21
N—H
H




0
N—H




A


 


 22
N—H
H




0
N—H




B


 


 23
N—H




H
0
N—H




A


 


 24
N—H




H
0
N—H




A


 


 25
N—H




H
0
N—H




A


 


 26
N—H




H
0
N—H




A


 


 27
N—H




H
0
N—H




A


 


 28
N—H




H
0
N—H




B


 


 29
N—H




H
0
N—H




B


 


 30
N—H




H
0
N—H




A


 


 31
N—H




H
0
N—H




A


 


 32
N—H




H
0
N—H




B


 


 33
N—H




H
0
N—H




C


 


 34
N—H




H
0
N—H




B


 


 35
N—H




H
0
N—H




B


 


 36
N—H




H
0
N—H




B


 


 37a
N—H




H
0
N—H




B


 


 37b






B


 


 38
N—H




H
0
N—H




B


 


 39
N—H




H
0
N—H




B


 


 40
N—H




H
0
N—H




A


 


 41
N—H




H
0
N—H




B


 


 42
N—H




H
0
N—H




A


 


 43
N—H




H
0
N—H




B


 


 44
N—H
H




0
N—H




G


 


 45
N—H
H




0
N—H




G


 


 46
N—H
H




0
N—H




G


 


 47
N—H
H




0
N—H




C


 


 48
N—H
H




0
N—H




G


 


 49
N—H
CH3
H
0
N—H




G


 


 50
N—H




H
0
N—H




G


 


 51
N—H
H
H
0
N—H




G


 


 52
N—H




H
0
N—H




C


 


 53
N—H




H
0
N—H




G


 


 54
N—H




H
0
N—H




G


 


 55
N—H




H
0
N—H




D


 


 56
N—H
H




0
N—H




G


 


 57
N—H
H




0
N—H




C


 


 58
N—H




H
0
N—H




G


 


 59
N—H




H
0
N—H




D


 


 60
N—H




H
0
N—H




C


 


 61
N—H




H
0
N—H




C


 


 62
N—H




H
0
N—H




D


 


 63
N—H




H
0
N—H




G


 


 64
N—H




H
0
N—H




G


 


 65
N—H




H
0
N—H




D


 


 66
N—H




H
0
N—H




C


 


 67
N—H




H
0
N—H




C


 


 68
N—H




H
0
N—H




D


 


 69
N—H




H
0
N—H




G


 


 70
N—H




H
0
N—H




G


 


 71
N—H




H
0
N—H




G


 


 72
N—H




H
0
N—H




D


 


 73
N—H




H
0
N—H




G


 


 74
N—H




H
0
N—H




D


 


 75
N—H




H
0
N—H




C


 


 76
N—H




H
0
N—H




G


 


 77
N—H




H
0
N—H




C


 


 78
N—H




H
0
N—H




G


 


 79
N—H




H
0
N—H




C


 


 80
N—H




H
0
N—H




G


 


 81
N—H




H
0
N—H




G


 


 82
N—H




H
0
N—H




G


 


 83
N—H




H
0
N—H




G


 


 84
N—H




H
0
N—H




D


 


 85
N—H




H
0
N—H




G


 


 86
N—H




H
0
N—H




G


 


 87
N—H
H
CH3
0
N—H




G


 


 88
N—H
H




0
N—H




D


 


 89
N—H
H




0
N—H




D


 


 90
N—H
H




0
N—H




D


 


 91
N—H
H




0
N—H




G


 


 92
N—H
H




0
N—H




G


 


 93
N—H
H




0
N—H




D


 


 94
N—H
H




0
N—H




D


 


 95
N—H
H




0
N—H




D


 


 96
N—H
H




0
N—H




G


 


 97
N—H




H
0
N—H




C


 


 98
N—H




H
0
N—H




G


 


 99
N—H
H




0
N—H




G


 


100
N—H
H




0
N—H




C


 


101
N—H
H




0
N—H




C


 


102
N—H
H




0
N—H




C


 


103
N—H
H




0
N—H




G


 


104
N—H
H




0
N—H




G


 


105
N—H
H




0
N—H




C


 


106
N—H
H




0
N—H




G


 


107
N—H
H




0
N—H




G


 


108
N—H
H




0
N—H




D


 


109
N—H
H




0
N—H




D


 


110
N—H
H




0
N—H




G


 


111
N—H
H




0
N—H




C


 


112
N—H
H




0
N—H




D


 


113
N—H
H




0
N—H




C


 


114
N—H
H




0
N—H




G


 


115
N—H
H




0
N—H




G


 


116
N—H
H




0
N—H




D


 


117
N—H




H
0
N—H




B


 


118
N—H




H
0
N—H




B


 


119
N—H




H
0
N—H




C


 


120
N—H




H
0
N—H




B


 


121
N—H




H
0
N—H




G


 


122
N—H
H




0
N—H




C


 


123
N—H
H




0
N—H




C


 


124
N—H
H




0
N—H




D


 


125
N—H
H




0
N—H




G


 


126
N—H
H




0
N—H




C


 


127
N—H
H




0
N—H




C


 


128
N—H
H




0
N—H




G


 


129
N—H
H




0
N—H




G


 


130
N—H
H




0
N—H




D


 


131
N—H
H




0
N—H




C


 


132
N—H
H




0
N—H




F


 


133
N—H
H




0
N—H




F


 


134
N—H
H




0
N—H




C


 


135
N—H
H




0
N—H




C


 


136a
N—H
H




0
N—H




B


 


136b






C


 


137
N—H
H




0
N—H




B


 


138
N—H
H




0
N—H




B


 


139
N—H
H




0
N—H




C


 


140
N—H
H




0
N—H




C


 


141
N—H
H




0
N—H




C


 


142
N—H
H




0
N—H




C


 


143
N—H
H




0
N—H




C


 


144
N—H




H
0
N—H




C


 


145a
N—H




H
0
N—H




C


 


145b






F


 


146a
N—H




H
0
N—H




F


 


146b






F


 


147
N—H




H
0
N—H




F


 


148
N—H




H
0
N—H




F


 


149
N—H




H
0
N—H




D


 


150a
N—H




H
0
N—H




C


 


150b






G


 


151
N—H




H
0
N—H




F


 


152a
N—H




H
0
N—H




C


 


152b






C


 


153
N—H
H




0
N—H




B


 


154
N—H
H




0
N—H




B


 


155
N—H
H




0
N—H




E


 


156
N—H
H




0
N—H




B


 


157
N—H
H




0
N—H




C


 


158
N—H
H




0
N—H




F


 


159
N—H
H




0
N—H




B


 


160a
N—H
H




0
N—H




F


 


160b






F


 


161a
N—H
H




0
N—H




F


 


161b






F


 


162a
N—H
H




0
N—H




D


 


162b






G


 


163
N—H
H




0
N—H




G


 


164
N—H
H




0
N—H




C


 


165
N—H
H




0
N—H




G


 


166
N—H
H




0
N—H




G


 


167
N—H
H




0
N—H




G


 


168
N—H





0
N—H




C


 


169
N—H




H
0
N—H




B


 


170
N—H




H
0
N—H




B


 


171
N—H




H
0
N—H




B


 


172
N—H
H




0
N—H




G


 


173
N—H




H
0
N—H




C


 


174
N—H




H
0
N—H




C


 


175
N—H




H
0
N—H




C


 


176
N—H




H
0
N—H




B


 


177
N—H




H
0
N—H




B


 


178
N—H




H
0
N—H




C


 


179
N—H




H
0
N—H




C


 


180
N—H




H
0
N—H




C


 


181
N—H




H
0
N—H




G


 


182a
N—H




H
0
N—H




G


 


182b






G


 


183
N—H




H
0
N—H




G


 


184
N—H




H
0
N—H




C


 


184






C


 


185
N—H




H
0
N—H




C


 


186
N—H




H
0
N—H




C


 


187
N—H




H
0
N—H




C


 


188
N—H




H
0
N—H




F


 


189a
N—H




H
0
N—H




C


 


189b






C


 


190
N—H




H
0
N—H




B


 


191
N—H




H
0
N—H




C


 


192
N—H




H
0
N—H




B


 


193
N—H




H
0
N—H




C


 


194a
N—H




H
0
N—H




C


 


194b






C


 


195
N—H




H
0
N—H




B


 


196
N—H




H
0
N—H




G


 


197
N—H




H
0
N—H




C


 


199
N—H
H




0
N—H




C


 


200
N—H
H




0
N—H




B


 


201
N—H




H
0
N—H




C


 


202
N—H




H
0
N—H




G


 


203
N—H
H




0
N—H




D


 


204
N—H
H




0
N—H




G


 


205
N—H




H
0
N—H




G


 


206
N—H




H
0
N—H




G


 


207
N—H
H




0
N—H




G


 


208a
N—H
H




0
N—H




B


 


208b



0
N—H




B


 


209
N—H




H
0
N—H




C


 


210
N—H
H




0
N—H




F


 


211
N—H
H




0
N—H




F


 


212
N—H




H
0
N—H




C


 


213
N—H




H
0
N—H




F


 


214
N—H




H
0
N—H




C


 


215
N—H




H
0
N—H




D


 


216
N—H
H




0
N—H




D


 


218
N—H
H




0
N—H




B


 


219
N—H
H




0
N—H




C





Binding activity determined using standard method, expressed as follows:


A = 0.1-10 nM;


B = 10-100 nM;


C = 0.1-1.0 μM;


D = 1-10 μM;


E > 500 nM (highest concentration tested);


F > 1 μM (highest conscentration tested);


G > 10 μM (or no activity at highest concentration tested)













TABLE 38







Binding Activity at the Human Ghrelin Receptor for Representative Compounds of the Invention















Compound
R2
R3
R4
R7
R5
R6
Tether
Ki (nm)





298




CH3
H
CH3




H




B


 


299




CH3
H
CH3




H




A


 














301








H
H








B


 


303








H
H








B


 















305




CH3
H
CH3




H




C


 


  306a




CH3
H
CH3




H




B


 









 306b
diastereomer
B


 















307




CH3
H
CH3




H




C


 


308




CH3
H
CH3




H




A


 


309




CH3
H
CH3




H




A


 


310




CH3
H
CH3




H




B


 


311




CH3
H
CH3




H




B


 


312




CH3
H
CH3




H




A


 


313




CH3
H
CH3




H




B


 


314




CH3
H
CH3




H




A


 


315




CH3
H
CH3




H




A


 


316




CH3
H
CH3




H




B


 


317




CH3
H
CH3




H




B


 


318




CH3
H
CH3




H




A


 


319




CH3
H
CH3




H




A


 


320




CH3
H
CH3




H




A


 


321




CH3
H
CH3




H




B


 


322




CH3
H
CH3




H




A


 


323




CH3
H
CH3




H




C


 


324




CH3
H
CH3




H




B


 














325








H
H








B


 


326








H
H








B


 


  327a








H
H








B


 









 327b
diastereomer
C


 














328








H
H








B


 


329








H
H








B


 


330








H
H








A


 


  331a








H
H








B


 









 331b
diastereomer
C


 














  332a








H
H








B


 









 332b
diastereomer
C


 














333








H
H








C


 















335




CH3
H
CH3




H




B


 


336




CH3
H
CH3




H




C


 


337




CH3
H
CH3




H




C


 


338




CH3
H
CH3




H




C


 


339




CH3
H
CH3




H




C


 


340




CH3
H
CH3




H




B


 














341








H
H








B


 


342








H
H








C


 


343








H
H








C


 


344








H
H








C


 


  345a








H
H








C


 









 345b
diastereomer
B


 














346








H
H








B


 


347








H
H








C


 















  348a




CH3
CH3
H
H








C


 









 348b
diastereomer
C


 















  353a




CH3
H
CH3




H




B


 









 353b
diasteromer
B


 















354




CH3
H
CH3




H




B


 


355




CH3
H
CH3




H




B


 


356




CH3
H
CH3




H




C


 


357




CH3
H
CH3




H




C


 














  358a








H
H








B


 









 358b
diastereoamer
C


 














359








H
H








C


 


360








H
H








C


 


361








H
H








C


 


362








H
H








C


 


363








H
H








C


 


364








H
H








C


 


365








H
H








C


 


366








H
H








C


 















367




CH3
H
CH3




H




B


 














  368a








H
H








B


 









 368b
diastereomer
B


 














369








H
H








B


 


370








H
H








C


 


371








H
H








B


 















372




CH3
H
CH3




H




A


 


373




CH3
H
CH3




H




B


 


374




CH3
H
CH3




H




B


 


375




CH3
H
CH3




H




C


 


376




CH3
H
CH3




H




C


 


377




CH3
H
CH3




H




C


 


378




CH3
H
CH3




H




C


 


379




CH3
H
CH3




H




B


 














380








H
H








C


 


381








H
H








B


 


382








H
H








B


 


383








H
H








C


 


384








H
H








C


 


385








H
H








C


 


386








H
H








C


 


387








H
H








C


 


388








H
H








A


 















  389a




CH3
H
CH3




H




B


 









 389b
diastereomer
B


 














390








H
H








C


 















391




CH3
H
CH3




H




A


 














392








H
H








B


 


393








H
H








C


 















394




CH3
H
CH3




H




A


 














395








H
H








B


 















398




CH3
H
CH3




H




C


 














  399a








H
H








C


 









 399b
diastereomer
A


 















400




CH3
H
CH3




H




B


 


401




CH3
H
CH3




H




A


 














  402a








H
H








B


 









 402b
diastereomer
B





Binding activity determined using standard method, expressed as follows: A = 0.1-10 nM; B = 10-100 nM; C = 0.1-1.0 μM













TABLE 3C







Binding Activity at the Human Ghrelin Receptor for


Representative Compounds of the Invention









Com-

Ki


pound
Structure
(nM)












18




B


 


334




B


 


349




B


 


350




C


 


351




B


 


352




C


 


396




B


 


397




C
















TABLE 3D







Binding Activity at the Human Ghrelin Receptor for Representative Compounds of the Invention
















Compound
R1
R2
R3
R4
R7
R5
R6
Tether 
Ki (nM)





435
H




CH3
H
CH3




H




B


 


436
H




CH3
H
CH3




H




B


 














437








H
H








A


 















438
H








H
H








D


 


439
H








H
H








D


 
















440
H








H
CH3









C


 















441
H








H
H








D


 


  442a
H








H
H








E


 









 442b
diastereomer
E


 















  443a
H








H
H








E


 









 443b
diastereomer
E


 















  444a
H








H
H








E


 









 444b
diastereomer
E


 
















445
H




CH3
H
CH3




H




B


 


  446a
H




CH3
H
CH3




H




D


 









 446b
diastereomer
D


 















447
H








H
H








D


 
















448
H




H
H
CH3
H








D


 















449
H








H
H








D





For all compounds, designations are based upon formula I, X = Z1 = Z2 = NH, m = n = p = 0


Binding activity determined using standard method, expressed as follows: A = 0.1-10 nM; B = 10-100 nM; C = 0.1-1.0 μM; D = 1.0-10 μM; E > 10 μM













TABLE 3E







Binding Activity at the Human Ghrelin Receptor for


Representative Compounds of the Invention










Compound
Ki











D



220



 







C



221



 







D



222



 







D



223



 







G



224



 







C



225



 







B



226



 







C



227



 







G



228



 







B



229



 







C



230



 



230 diasteromer
D







Binding activity determined using standard method, expressed as follows: A = 0.1-10 nM; B = 10-100 nM; C = 0.1-1.0 μM; D = 1-10 μM; E > 500 nM (highest concentration tested); F > 1 μM (highest concentration tested); G > 10 μM (or no activity at highest concentration tested)






B. Aequorin Functional Assay (Ghrelin Receptor)

The functional activity of compounds of the invention found to bind to the GHS-R1a receptor can be determined using the method described below which can also be used as a primary screen for ghrelin receptor activity in a high throughput fashion. (LePoul, E.; et al. Adaptation of aequorin functional assay to high throughput screening. J. Biomol. Screen. 2002, 7, 57-65; Bednarek, M. A.; et al. Structure-function studies on the new growth hormone-releasing peptide ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J. Med. Chem. 2000, 43, 4370-4376; Palucki, B. L.; et al. Spiro(indoline-3,4′-piperidine) growth hormone secretagogues as ghrelin mimetics. Bioorg. Med. Chem. Lett. 2001, 11, 1955-1957.)
Materials
Membranes were prepared using AequoScreenTM (EUROSCREEN, Belgium) cell lines expressing the human ghrelin receptor (cell line ES-410-A; receptor accession #60179). This cell line is typically constructed by transfection of the human ghrelin receptor into CHO-K1 cells co-expressing Gα16 and the mitochondrially targeted Aequorin (Ref #ES-WT-A5).

    1. Ghrelin (reference agonist; Bachem, #H-4864)
    2. Assay buffer: DMEM (Dulbecco's Modified Eagles Medium) containing 0.1% BSA (bovine serum albumin; pH 7.0).
    3. Coelenterazine (Molecular Probes, Leiden, The Netherlands).

Final test concentrations (N=8) for compounds of the invention:

10, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001 μM.

Compound Handling



Stock solutions of compounds (10 mM in 100% DMSO) were provided frozen on dry ice and stored at −20° C. prior to use. From the stock solution, mother solutions were made at a concentration of 500 μM by 20-fold dilution in 26% DMSO. Assay plates were then prepared by appropriate dilution in DMEM medium containing 0.1% BSA. Under these conditions, the maximal final DMSO concentration in the assay was <0.6%.
Cell Preparation
AequoScreenTM cells were collected from culture plates with Ca2+ and Mg2+-free phosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelleted for 2 min at 1000×g, re-suspended in DMEM—Ham's F12 containing 0.1% BSA at a density of 5×106 cells/mL, and incubated O/N at rt in the presence of 5 μM coelenterazine. After loading, cells were diluted with assay buffer to a concentration of 5×105 cells/mL.
Assay Protocol
For agonist testing, 50 μL of the cell suspension was mixed with 50 μL of the appropriate concentration of test compound or ghrelin (reference agonist) in 96-well plates (duplicate samples). Ghrelin (reference agonist) was tested at several concentrations concurrently with the test compounds in order to validate the experiment. The emission of light resulting from receptor activation in response to ghrelin or test compounds was recorded using the Hamamatsu FDSS 6000 reader (Hamamatsu Photonics K.K., Japan).
Analysis and Expression of Results
Results were expressed as Relative Light Units (RLU). Concentration response curves were analyzed using GraphPad Prism (GraphPad Software, San Diego, Calif.) by non-linear regression analysis (sigmoidal dose-response) based on the equation E=Emax/(1+EC50/C)n where E was the measured RLU value at a given agonist concentration (C), Emax was the maximal response, EC50 was the concentration producing 50% stimulation and n was the slope index. For agonist testing, results for each concentration of test compound were expressed as percent activation relative to the signal induced by ghrelin at a concentration equal to the EC80 (i.e. 3.7 nM). EC50, Hill slope and % Emax values are reported.
The data show that the representative compounds examined act as agonists at the ghrelin receptor and are devoid of antagonist activity at the concentrations studied. In addition, these compounds were demonstrated to have high selectivity for the ghrelin receptor versus its closest counterpart, the motilin receptor, with which it has 52% sequence homology. (Feighner, S. D.; Tan, C. P.; McKee, K. K.; Palyla, O. C.; Hreniuk, D. L.; Pong, S.-S.; Austin, C. P.; Figueroa, D.; MacNeil, D.; Cascieri, M. A.; Nargund, R.; Bakshi, R.; Abramovitz, M.; Stocco, R.; Kargman, S.; O'Neill, G.; van der Ploeg, L. H. T.; Evans, J.; Patchett, A. A.; Smith, R. G.; Howard, A. D. Receptor for motilin identified in the human gastrointestinal system. Science 1999, 284, 2184-2188.) The endogenous peptides themselves have 36% of residues in common and ghrelin was even identified at one point as motilin-related peptide. (Tomasetto, C.; Karam, S. M.; Ribieras, S.; Masson, R.; Lefebvre, O.; Staub, A.; Alexander, G.; Chenard, M. P.; Rio, M. C. Identification and characterization of a novel gastric peptide hormone: the motilin-related peptide. Gastroenterology 2000, 119, 395-405.) Ghrelin does not interact appreciably at the motilin receptor, although GHRP-6 does. (Depoortere, I.; Thijs, T.; Thielemans, L.; Robberecht, P.; Peeters, T. L. Interaction of the growth hormone-releasing peptides ghrelin and growth hormone-releasing peptide-6 with the motilin receptor in the rabbit gastric antrum. J. Pharmacol. Exp. Ther. 2003, 305, 660-667.) On the other hand, motilin itself as been demonstrated to have some GH-releasing effects. (Samson, W. K.; Lumpkin, M. D.; Nilayer, G.; McCann, S. M. Motilin: a novel growth hormone releasing agent. Brain Res. Bull. 1984, 12, 57-62.)
The level of agonist activity and selectivity for representative compounds of the invention are shown below in Table 4. Concentration-response results for exemplary compounds 1-5 are presented in FIG. 5.







TABLE 4







Functional Assay at the Human Ghrelin Receptor and Selectivity Results












Compounda
Ki (nM)*
EC50 (nM)**
Selectivityb







 1
B
BB
 142/1



 2
C
BB
nd



 3
C
BB
nd



 4g
Bc
AA
3012/1



 5
C
BB
nd



 6
C
AA
 71/1



 7
C
AA
>100/1



 8f
Bd
AA
 200/1



 9g
Ce
BB
 117/1



 10
B
AA
 304/1



 11f
B
BB
nd



 15
A
nd
>1700/1  



 16
A
nd
>2000/1  



 17
A
AA
2500/1



 18
B
AA
 222/1



 19
C
nd
>1700/1  



 20
A
AA
1044/1



 21
A
AA
1078/1



 23
A
AA
30,000/1  



 24
A
nd
3039/1



 25
A
AA
28,000/1  



 26
A
AA
>7700/1  



 27e
A
AA
>7100/1  



 28
B
AA
nd



 30
A
AA
13,000/1  



 31
A
AA
4900/1



 34
B
nd
>1000/1  



 35
B
AA
nd



 36
B
BB
nd



 37a
B
AA
>800/1



 37b
B
BB
nd



 38
B
BB
nd



 39f
A
BB
3400/1



 40
A
AA
>3300/1  



 42
A
nd
4300/1



 43
B
nd
3700/1



 47
C
AA
nd



 97
B
BB
nd



111
B
BB
nd



113g
B
BB
nd



140
C
BB
nd



141
C
AA
nd



153
B
AA
nd



154
B
AA
nd



156
B
AA
nd



168
C
CC
nd



170
B
BB
nd



176
B
AA
 105/1



177
B
AA
>100/1



178
C
BB
nd



184a
C
BB
 28/1



184b
Ce
BB
nd



186
C
BB
nd



191
C
BB
nd



192
B
BB
nd



193
C
BB
nd



194a
C
BB
nd



194b
C
BB
nd



195
B
AA
nd



197
C
CC
 100/1



214
C
BB
nd



226
B
CC
nd



298
B
AA
3100/1



299
A
AA
nd



306a
B
AA
 714/1



311
B
nd
 21/1



314
A
AA
>5500/1  



318
A
AA
nd



322
A
AA
nd



334
B
AA
 346/1



345a
B
AA
>159/1



346
B
AA
nd



351
B
AA
 450/1



354
B
AA
nd



358a
B
AA
nd



363
C
nd
 35/1



367
B
AA
nd



368a
A
CC
nd



372
A
AA
2500/1



374
B
AA
 250/1



382
B
BB
 74/1



388
A
AA
 400/1



389a
B
BB
 450/1



394
A
BB
1700/1



399a
A
CC
 300/1



445
B
AA
nd







aAll compounds were tested as their TFA salts unless otherwise noted.



bVersus the human motilin receptor (nd = not determined)



cAverage of six (6) experiments



dAverage of four (4) experiments



eAverage of two (2) experiments



fHCl salt



gFormate salt



*Binding activity determined using standard method and expressed as A = 0.1-10 nM; B = 10-100 nM; C = 100-1000 nM



**Functional activity determined using standard method and expressed as AA = 1-100 nM; BB = 100-1000 nM; CC >1000 nM;



nd = not determined






C. Cell Culture Assay for Growth Hormone Release

Cell culture assays for determining growth hormone release can be employed as described in Cheng, et al. Endocrinology 1989, 124, 2791-2798. In particular, anterior pituitary glands are obtained from male Sprague-Dawley rats and placed in cold culture medium. These pituitaries are sectioned, for example into one-eighth sections, then digested with trypsin. Cells are collected after digestion, pooled, and transferred into 24 well plates (minimum 200,000 cells per well). After a monolayer of cells has formed, generally after at least 4 d in culture, the cells are washed with medium prior to exposure to the test samples and controls. Varying concentrations of the test compounds and of ghrelin as a positive control were added to the medium. The cells are left for 15 min at 37° C., then the medium removed and the cells stored frozen. The amount of GH release was measured utilizing a standard radioimmunoassay as known to those in the art.
D. Pharmacokinetic Analysis of Representative Compounds of the Invention
The pharmacokinetic behavior of compound of the invention can be ascertained by methods well known to those skilled in the art. (Wilkinson, G. R. “Pharmacokinetics: The Dynamics of Drug Absorption, Distribution, and Elimination” in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Tenth Edition, Hardman, J. G.; Limbird, L. E., Eds., McGraw Hill, Columbus, Ohio, 2001, Chapter 1.) The following method was used to investigate the pharmacokinetic parameters (elimination half-life, total plasma clearance, etc.) for intravenous, subcutaneous and oral administration of compounds of the present invention.
Collection of Plasma
Rats: male, Sprague-Dawley ( ̃250 g)
Rats/Treatment Group: 6 (2 subsets of 3 rats each, alternate bleeds)
Each sample of test compound was sent in solution in a formulation (such as with cyclodextrin) appropriate for dosing. It will be appreciated by one skilled in the art that appropriate modifications to this protocol can be made as required to adequately test the properties of the compound under analysis.

Typical Dose

    1. Intravenous (i.v.): 2 mg/kg
    2. Subcutaneous (s.c). 2 mg/kg
    3. Oral (p.o.): 8 mg/kg









TABLE 5







Representative Intravenous Blood Sampling Schedule.








Subset
Time (min.) relative to Dose Administration

















ID
Pre-dose
1
5
20
60
90
120
180
240
300





Subset
✓

✓

✓

✓

✓



A


Subset

✓

✓

✓

✓

✓


B
















TABLE 6







Representative Subcutaneous & Oral Blood Sampling Schedule.








Subset
Time (min.) relative to Dose Administration

















ID
Pre-dose
5
15
30
60
90
120
180
270
360





Subset
✓

✓

✓

✓

✓



A


Subset

✓

✓

✓

✓

✓


B









Plasma Collection

    1. Same protocol for all dosing groups
    2. For each group, 2 subsets (A and B) of 3 rats/subset


At the time intervals indicated above, 0.7 mL of blood were collected from each animal. It is expected that this volume of blood will yield a sample of at least 0.3 mL of plasma. EDTA was used as an anti-coagulant for whole blood collection. Whole blood samples were chilled and immediately processed by centrifugation to obtain plasma.
Plasma samples were stored frozen (−70° C.) until analysis. Analytical detection of parent compound in plasma samples performed by LC-MS after an appropriate preparation protocol: extraction using solid phase extraction (SPE) cartridges (Oasis MCX, Oasis HLB) or liquid-liquid extraction.
HPLC-MS Method
Column: Atlantis dC18 from Waters 2.1×30 mm
Mobile phases:
A: 95% MeOH, 5% water, 0.1% TFA
B: 95% water, 5% MeOH) 0.1% TFA
Flow: 0.5 mL/min
Gradient (linear):












Time (min)
A
B

















0
30%
70%


0.5
30%
70%


2.8
100%
0%


3.8
100%
0%


4.0
30%
70%


5.0
30%
70%









The analyte was quantitated based upon a standard curve and the method validated with internal standards







TABLE 7







Pharmacokinetic Parameters for Representative Compounds of the


Invention














Clearance




Mode of
Elimination
(mL/
Bioavailability


Compound
Administrationa
(t1/2, min)
min/kg)
(oral)b














25
i.v.
31
67
na


298
i.v.
75
17
na


298
s.c.
66
15
na


298
p.o.
312
14
29%





ai.v. = intravenous (10 time points over 150 min); s.c. = subcutaneous (10 time points over 360 min), p.o. = oral (10 time points over 240 min)


bna = not applicable






Results of the time courses for these studies are provided in FIGS. 6A-6D.
E. Gastric Emptying
To examine the effects of compounds of the invention in a model for gastroparesis, compounds were evaluated for possible effects on gastric emptying in fasted rats. For example, compounds 25 and 298 at 100 μg/kg caused a significant increase (≧30%) in gastric emptying relative to the vehicle control group. The relative efficacy (39% increase) of compounds 25 and 298 at 100 μg/kg i.v. was similar to concurrently run positive reference agents GHRP-6 at 20 μg/kg i.v. (40% increase) and metoclopramide at 10 mg/kg i.v. (41% increase). Accordingly, compounds 25 and 298 at a dose of 100 μg/kg demonstrated gastrokinetic activity in rats, with efficiency similar to GHRP-6 at 20 μg/kg and metoclopramide at 10 mg/kg. Further, compound 25 also demonstrated gastric emptying at 30 μg/kg. This is significantly more potent than other compounds interacting at this receptor previously found to enhance GI motility, which were unable to promote gastric emptying at 100 μg/kg (U.S. Pat. No. 6,548,501).
Test Substances and Dosing Pattern
GHRP-6 and test samples were dissolved in vehicle of 9% HPBCD/0.9% NaCl. Immediately following oral administration of methylcellulose (2%) containing phenol red (0.05%) (2 mL/rat), test substances or vehicle (9% HPBCD/0.9% NaCl) were each administered intravenously (i.v.) at a dosing volume of 5 mL/kg.
Animals
Male Wistar rats were provided by LASCO (A Charles River Licensee Corporation, Taiwan). Space allocation for 6 animals was 45×23×15 cm. Animals were housed in APEC® cages and maintained in a controlled temperature (22° C.-24° C.) and humidity (60%-80%) environment with 12 h light, 12 h dark cycles for at least one week in the laboratory prior to being used. Free access to standard lab chow for rats (Lab Diet, Rodent Diet, PMI Nutrition International, USA) and tap water was granted. All aspects of this work including housing, experimentation and disposal of animals were performed in general accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996).
Chemicals
Glucose (Sigma, USA), Metoclopramide-HCl (Sigma, USA), Methylcellulose (Sigma, USA), NaOH (Sodium Hydroxide, Wako, Japan), Pyrogen free saline (Astar, Taiwan), Phenol Red-Sodium salt (Sigma, USA) and Trichloroacetic acid (Merck, USA). Equipment
8-well strip (Costar, USA), 96-well plate (Costar, USA), Animal case (ShinTeh, R. O. C.), Centrifugal separator (Kokusan, H-107, Japan), Glass syringe (1 mL, 2 mL, Mitsuba, Japan), Hypodermic needle (25G×1′′, TOP Corporation, Japan), Microtube (Treff, Switzerland), pH-meter (Hanna, USA), Pipetamam (P100, Gilson, France), Pipette tips (Costar, USA), Rat oral needle (Natsume, Japan), Spectra Fluor plus (Austria), Stainless scissors (Klappencker, Germany) and Stainless forceps (Klappencker, Germany).
Assay
Test substances were each administered intravenously to a group of 5 O/N-fasted Wistar derived male rats weighing 200±20 g immediately after methylcellulose (2%) containing phenol red (0.05%) was administered orally at 2 mL/animal. The animals were then sacrificed 15 minutes later. The stomach was immediately removed, homogenized in 0.1 N NaOH (5 mL) and centrifuged. Following protein precipitation by 20% trichloroacetic acid (0.5 mL) and re-alkalization of the supernatant with 0.1 N NaOH, total phenol red remaining in the stomach was determined by a colorimetric method at 560 nm. A 30 percent or more (≧30%) increase in gastric emptying, detected as the decrease in phenol red concentration in the stomach relative to the vehicle control group, is considered significant.
Results for two representative compounds of the invention are shown in FIG. 7 and in the Examples below.
F. Gastric Emptying and Intestinal Transit in Rat Model of Postoperative Ileus
This clinically relevant model for POI is adapted from that of Kalff. (Kalff, J. C.; Schraut, W. H.; Simmons, R. L.; Bauer, A. J. Surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus. Ann. Surg 1998, 228, 652-663.) Other known models can also be used to study the effect of compounds of the invention. (Trudel, L.; Bouin, M.; Tomasetto, C.; Eberling, P.; St-Pierre, S.; Bannon, P.; L'Heureux, M. C.; Poitras, P. Two new peptides to improve post-operative gastric ileus in dog. Peptides 2003, 24, 531-534; (b) Trudel, L.; Tomasetto, C.; Rio, M. C.; Bouin, M.; Plourde, V.; Eberling, P.; Poitras, P. Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rats. Am. J. Physiol. 2002, 282, G948-G952.)
Animals


    1. Rat, Sprague-Dawley, male,  ̃300 g.
    2. Fasted O/N prior to study.

Induction of post-operative ileus (POI)

    1. Isofluorane anaesthesia under sterile conditions.
    2. Midline abdominal incision.
    3. Intestines and caecum were eviscerated and kept moist with saline.
    4. The intestines and caecum were manipulated along its entire length with moist cotton applicators analogous to the ‘running of the bowel’ in the clinical setting. This procedure was timed to last for 10 min.
    5. Intestines were gently replaced into the abdomen and the abdominal wound was stitched closed under sterile conditions.

Dosing

    1. Rat was allowed to recover from isofluorane anaesthesia.
    2. Test compounds (or vehicle) were administered intravenously via previously implanted jugular catheter.
    3. Immediate intragastric gavage of methylcellulose (2%) labeled with radioactive 99mTc, t=0.

Experimental

    1. At t=15 min, animal was euthanized with CO2.
    2. Stomach and 10 cm sections along the small intestine were immediately ligated, cut and placed in tubes for measuring of 99mTc in gamma counter.
    3. Stomach emptying and small intestinal transit were measured by calculation of the geometric mean.

Geometric mean=Σ(% total radioactivity×number of segment)/100



Results are depicted in the graph in FIG. 8 and indicate that Compound 298 at 100 μg/kg (i.v. n=5) significantly improves postoperative ileus in comparison to POI+vehicle treated rats. Further results are presented in the Examples below.
G. Growth Hormone Response to Test Compounds
The compounds of the invention likewise can be tested in a number of animal models for their effect on GH release. For example, rats (Bowers, C. Y.; Momany, F.; Reynolds, G. A.; Chang, D.; Hong, A.; Chang, K. Endocrinology 1980, 106, 663-667), dogs (Hickey, G.; Jacks, T.; Judith, F.; Taylor, J.; Schoen, W. R.; Krupa, D.; Cunningham, P.; Clark, J.; Smith, R. G. Endocrinology 1994, 134, 695-701; Jacks, T.; Hickey, G.; Judith, F.; Taylor, J.; Chen, H.; Krupa, D.; Feeney, W.; Schoen, W. R.; Ok, D.; Fisher, M.; Wyvratt, M.; Smith, R. J. Endocrinology 1994, 143, 399-406; Hickey, G. J.; Jacks, T. M.; Schleim, K. D.; Frazier, E.; Chen, H. Y.; Krupa, D.; Feeney, W.; Nargund, R. P.; Patchett, A. A.; Smith, R. G. J. Endocrinol. 1997, 152, 183-192), and pigs (Chang, C. H.; Rickes, E. L.; Marsilio, F.; McGuire, L.; Cosgrove, S.; Taylor, J.; Chen, H. Y.; Feighner, S.; Clark, J. N.; Devita, R.; Schoen, W. R.; Wyvratt, M.; Fisher, M.; Smith, R. G.; Hickey, G. Endocrinology 1995, 136, 1065-1071; (b) Peschke, B.; Hanse, B. S. Bioorg. Med. Chem. Lett. 1999, 9, 1295-1298) have all been successfully utilized for the in vivo study of the effects of GHS and would likewise be applicable for investigation of the effect of ghrelin agonists on GH levels. The measurement of ghrelin of GH levels in plasma after appropriate administration of compounds of the invention can be performed using radioimmunoassay via standard methods known to those in the art. (Deghenghi, R.; et al. Life Sciences 1994, 54, 1321-1328.) Binding to tissue can be studied using whole body autoradiography after dosing of an animal with test substance containing a radioactive label. (Ahnfelt-Rønne, I.; Nowak, J.; Olsen, U. B. Do growth hormone-releasing peptides act as ghrelin secretagogues? Endocrine 2001, 14, 133-135.)
The following method is employed to determine the temporal pattern and magnitude of the growth hormone (GH) response to test compounds, administered either systemically or centrally. Results for compound 298 demonstrating its lack of effect on GH release are presented graphically in FIG. 9. Compound 25 gave similar results. Further results are presented in the Examples below.
Dosing and Sampling Procedures for In Vivo Studies of GH Release
Adult male Sprague Dawley rats (225-300 g) were purchased from Charles River Canada (St. Constant, Canada) and individually housed on a 12-h light, 12-h dark cycle (lights on, time: 0600-1800) in a temperature (22±1° C.)- and humidity-controlled room. Purina rat chow (Ralston Purina Co., St. Louis, Mo.) and tap water were freely available. For these studies, chronic intracerebroventricular (icv) and intracardiac venous cannulas were implanted under sodium pentobarbital (50 mg/kg, ip) anesthesia using known techniques. The placement of the icv cannula was verified by both a positive drinking response to icv carbachol (100 ng/10 μl) injection on the day after surgery and methylene blue dye at the time of sacrifice. After surgery, the rats were placed directly in isolation test chambers with food and water freely available until body weight returned to preoperative levels (usually within 5-7 d). During this time, the rats were handled daily to minimize any stress associated with handling on the day of the experiment. On the test day, food was removed 1.5 h before the start of sampling and was returned at the end. Free moving rats were iv injected with either test sample at various levels (3, 30, 300, 1000 μg/kg) or normal saline at two different time points during a 6-h sampling period. The times 1100 and 1300 were chosen because they reflect typical peak and trough periods of GH secretion, as previously documented. The human ghrelin peptide (5 μg, Phoenix Pharmaceuticals, Inc., Belmont, Calif.) was used as a positive control in the experiments and was diluted in normal saline just before use. To assess the central actions of test compounds on pulsatile GH release, a 10-fold lower dose of the test sample or normal saline was administered icv at the same time points, 1100 and 1300. Blood samples (0.35 mL) were withdrawn every 15 min over the 6-h sampling period (time: 1000-1600) from all animals. To document the rapidity of the GH response to the test compound, an additional blood sample was obtained 5 min after each injection. All blood samples were immediately centrifuged, and plasma was separated and stored at −20° C. for subsequent GH assay. To avoid hemodynamic disturbance, the red blood cells were resuspended in normal saline and returned to the animal after removal of the next blood sample. All animal studies were conducted under procedures approved by an animal care oversight committee.
GH Assay Method
Plasma GH concentrations were measured in duplicate by double antibody RIA using materials supplied by the NIDDK Hormone Distribution Program (Bethesda, Md.). The averaged plasma GH values for 5-6 rats per group are reported in terms of the rat GH reference preparation. The standard curve was linear within the range of interest; the least detectable concentration of plasma GH under the conditions used was approximately 1 ng/mL. All samples with values above the range of interest were reassayed at dilutions ranging from 1:2 to 1:10. The intra- and interassay coefficients of variation were acceptable for duplicate samples of pooled plasma containing a known GH concentration.
4. Pharmaceutical Compositions
The macrocyclic compounds of the present invention or pharmacologically acceptable salts thereof according to the invention may be formulated into pharmaceutical compositions of various dosage forms. To prepare the pharmaceutical compositions of the invention, one or more compounds, including optical isomers, enantiomers, diastereomers, racemates or stereochemical mixtures thereof, or pharmaceutically acceptable salts thereof as the active ingredient is intimately mixed with appropriate carriers and additives according to techniques known to those skilled in the art of pharmaceutical formulations.
A pharmaceutically acceptable salt refers to a salt form of the compounds of the present invention in order to permit their use or formulation as pharmaceuticals and which retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable. Examples of such salts are described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wermuth, C. G. and Stahl, P. H. (eds.), Wiley-Verlag Helvetica Acta, Zürich, 2002 [ISBN 3-906390-26-8]. Examples of such salts include alkali metal salts and addition salts of free acids and bases. Examples of pharmaceutically acceptable salts, without limitation, include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates, ethane sulfonates, propanesulfonates, toluenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
If an inventive compound is a base, a desired salt may be prepared by any suitable method known to those skilled in the art, including treatment of the free base with an inorganic acid, such as, without limitation, hydrochloric acid, hydrobromic acid, hydroiodic, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, including, without limitation, formic acid, acetic acid, propionic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, stearic acid, ascorbic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, cyclohexyl-aminosulfonic acid or the like.
If an inventive compound is an acid, a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine, lysine and arginine; ammonia; primary, secondary, and tertiary amines such as ethylenediamine, N,N′-dibenzylethylenediamine, diethanolamine, choline, and procaine, and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
The carriers and additives used for such pharmaceutical compositions can take a variety of forms depending on the anticipated mode of administration. Thus, compositions for oral administration may be, for example, solid preparations such as tablets, sugar-coated tablets, hard capsules, soft capsules, granules, powders and the like, with suitable carriers and additives being starches, sugars, binders, diluents, granulating agents, lubricants, disintegrating agents and the like. Because of their ease of use and higher patient compliance, tablets and capsules represent the most advantageous oral dosage forms for many medical conditions.
Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, elixirs, and the like with suitable carriers and additives being water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like. Typical preparations for parenteral administration comprise the active ingredient with a carrier such as sterile water or parenterally acceptable oil including polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may also be included. In the case of a solution, it can be lyophilized to a powder and then reconstituted immediately prior to use. For dispersions and suspensions, appropriate carriers and additives include aqueous gums, celluloses, silicates or oils.
The pharmaceutical compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, topical (i.e., both skin and mucosal surfaces, including airway surfaces), transdermal administration and parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intrathecal, intracerebral, intracranially, intraarterial, or intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used.
Compositions for injection will include the active ingredient together with suitable carriers including propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP), emulPhorTM-alcohol-water, cremophor-ELTM or other suitable carriers known to those skilled in the art. These carriers may be used alone or in combination with other conventional solubilizing agents such as ethanol, propylene glycol, or other agents known to those skilled in the art.
Where the macrocyclic compounds of the present invention are to be applied in the form of solutions or injections, the compounds may be used by dissolving or suspending in any conventional diluent. The diluents may include, for example, physiological saline, Ringer's solution, an aqueous glucose solution, an aqueous dextrose solution, an alcohol, a fatty acid ester, glycerol, a glycol, an oil derived from plant or animal sources, a paraffin and the like. These preparations may be prepared according to any conventional method known to those skilled in the art.
Compositions for nasal administration may be formulated as aerosols, drops, powders and gels. Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a physiologically acceptable aqueous or non-aqueous solvent. Such formulations are typically presented in single or multidose quantities in a sterile form in a sealed container. The sealed container can be a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single use nasal inhaler, pump atomizer or an aerosol dispenser fitted with a metering valve set to deliver a therapeutically effective amount, which is intended for disposal once the contents have been completely used. When the dosage form comprises an aerosol dispenser, it will contain a propellant such as a compressed gas, air as an example, or an organic propellant including a fluorochlorohydrocarbon or fluorohydrocarbon.
Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth or gelatin and glycerin.
Compositions for rectal administration include suppositories containing a conventional suppository base such as cocoa butter.
Compositions suitable for transdermal administration include ointments, gels and patches.
Other compositions known to those skilled in the art can also be applied for percutaneous or subcutaneous administration, such as plasters.
Further, in preparing such pharmaceutical compositions comprising the active ingredient or ingredients in admixture with components necessary for the formulation of the compositions, other conventional pharmacologically acceptable additives may be incorporated, for example, excipients, stabilizers, antiseptics, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavoring agents, isotonicity-agents, buffering agents, antioxidants and the like. As the additives, there may be mentioned, for example, starch, sucrose, fructose, dextrose, lactose, glucose, mannitol, sorbitol, precipitated calcium carbonate, crystalline cellulose, carboxymethylcellulose, dextrin, gelatin, acacia, EDTA, magnesium stearate, talc, hydroxypropylmethylcellulose, sodium metabisulfite, and the like.
In some embodiments, the composition is provided in a unit dosage form such as a tablet or capsule.
In further embodiments, the present invention provides kits including one or more containers comprising pharmaceutical dosage units comprising an effective amount of one or more compounds of the present invention.
The present invention further provides prodrugs comprising the compounds described herein. The term “prodrug” is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active. The “prodrug” can be a compound of the present invention that has been chemically derivatized such that, (i) it retains some, all or none of the bioactivity of its parent drug compound, and (ii) it is metabolized in a subject to yield the parent drug compound. The prodrug of the present invention may also be a “partial prodrug” in that the compound has been chemically derivatized such that, (i) it retains some, all or none of the bioactivity of its parent drug compound, and (ii) it is metabolized in a subject to yield a biologically active derivative of the compound. Known techniques for derivatizing compounds to provide prodrugs can be employed. Such methods may utilize formation of a hydrolyzable coupling to the compound.
The present invention further provides that the compounds of the present invention may be administered in combination with a therapeutic agent used to prevent and/or treat metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, genetic disorders, hyperproliferative disorders and inflammatory disorders. Exemplary agents include analgesics (including opioid analgesics), anesthetics, antifungals, antibiotics, antiinflammatories (including nonsteroidal anti-inflammatory agents), anthelmintics, antiemetics, antihistamines, antihypertensives, antipsychotics, antiarthritic, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents (such as DNA-interactive agents, antimetabolites, tubulin-interactive agents, hormonal agents, and agents such as asparaginase or hydroxyurea), corticoids (steroids), antidepressants, depressants, diuretics, hypnotics, minerals, nutritional supplements, parasympathomimetics, hormones (such as corticotrophin releasing hormone, adrenocorticotropin, growth hormone releasing hormone, growth hormone, thyrptropin-releasing hormone and thyroid stimulating hormone), sedatives, sulfonamides, stimulants, sympathoinimetics, tranquilizers, vasoconstrictors, vasodilators, vitamins and xanthine derivatives.
Subjects suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably mammalian. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, and the like, and mammals in utero. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects are preferred. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention.
Illustrative avians according to the present invention include chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo.
The present invention is primarily concerned with the treatment of human subjects, but the invention can also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and for drug screening and drug development purposes.
In therapeutic use for treatment of conditions in mammals (i.e. humans or animals) for which a modulator, such as an agonist, of the ghrelin receptor is effective, the compounds of the present invention or an appropriate pharmaceutical composition thereof may be administered in an effective amount. Since the activity of the compounds and the degree of the therapeutic effect vary, the actual dosage administered will be determined based upon generally recognized factors such as age, condition of the subject, route of delivery and body weight of the subject. The dosage can be from about 0.1 to about 100 mg/kg, administered orally 1-4 times per day. In addition, compounds can be administered by injection at approximately 0.01-20 mg/kg per dose, with administration 1-4 times per day. Treatment could continue for weeks, months or longer. Determination of optimal dosages for a particular situation is within the capabilities of those skilled in the art.
5. Methods of Use
The compounds of formula I, II and/or III of the present invention can be used for the prevention and treatment of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, central nervous system disorders, genetic disorders, hyperproliferative disorders, inflammatory disorders and combinations thereof where the disorder may be the result of multiple underlying maladies. In particular embodiments, the disease or disorder is irritable bowel syndrome (IBS), non-ulcer dyspepsia, Crohn's disease, gastroesophogeal reflux disorders, constipation, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, carcinoid syndrome, malabsorption syndrome, diarrhea, diabetes including diabetes mellitus (type II diabetes), obesity, atrophic colitis, gastritis, gastric stasis, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, celiac disease, an eating disorder or obesity. In other embodiments, the disease or disorder is congestive heart failure, ischemic heart disease or chronic heart disease. In still other embodiments, the disease or disorder is osteoporosis and/or frailty, congestive heart failure, accelerating bone fracture repair, metabolic syndrome, attenuating protein catabolic response, cachexia, protein loss, impaired or risk of impaired wound healing, impaired or risk of impaired recovery from burns, impaired or risk of impaired recovery from surgery, impaired or risk of impaired muscle strength, impaired or risk of impaired mobility, altered or risk of altered skin thickness, impaired or risk of impaired metabolic homeostasis or impaired or risk of impaired renal homeostasis. In other embodiments, the disease or disorder involves facilitating neonatal development, stimulating growth hormone release in humans, maintenance of muscle strength and function in humans, reversal or prevention of frailty in humans, prevention of catabolic side effects of glucocorticoids, treatment of osteoporosis, stimulation and increase in muscle mass and muscle strength, stimulation of the immune system, acceleration of wound healing, acceleration of bone fracture repair, treatment of renal failure or insufficiency resulting in growth retardation, treatment of short stature, treatment of obesity and growth retardation, accelerating the recovery and reducing hospitalization of burn patients, treatment of intrauterine growth retardation, treatment of skeletal dysplasia, treatment of hypercortisolism, treatment of Cushing's syndrome, induction of pulsatile growth hormone release, replacement of growth hormone in stressed patients, treatment of osteochondrodysplasias, treatment of Noonans syndrome, treatment of schizophrenia, treatment of depression, treatment of Alzheimer's disease, treatment of emesis, treatment of memory loss, treatment of reproduction disorders, treatment of delayed wound healing, treatment of psychosocial deprivation, treatment of pulmonary dysfunction, treatment of ventilator dependency; attenuation of protein catabolic response, reducing cachexia and protein loss, treatment of hyperinsulinemia, adjuvant treatment for ovulation induction, stimulation of thymic development, prevention of thymic function decline, treatment of immunosuppressed patients, improvement in muscle mobility, maintenance of skin thickness, metabolic homeostasis, renal homeostasis, stimulation of osteoblasts, stimulation of bone remodeling, stimulation of cartilage growth, stimulation of the immune system in companion animals, treatment of disorders of aging in companion animals, growth promotion in livestock, and/or stimulation of wool growth in sheep.
According to a further aspect of the invention, there is provided a method for the treatment of post-operative ileus, cachexia (wasting syndrome), such as that caused by cancer, AIDS, cardiac disease and renal disease, gastroparesis, such as that resulting from type I or type II diabetes, other gastrointestinal disorders, growth hormone deficiency, bone loss, and other age-related disorders in a human or animal patient suffering therefrom, which method comprises administering to said patient an effective amount of at least one member selected from the compounds disclosed herein having the ability to modulate the ghrelin receptor. Other diseases and disorders treated by the compounds disclosed herein include short bowel syndrome, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, celiac disease, and hyperproliferative disorders such as tumors, cancers, and neoplastic disorders, as well as premalignant and non-neoplastic or non-malignant hyperproliferative disorders. In particular, tumors, cancers, and neoplastic tissue that can be treated by the present invention include, but are not limited to, malignant disorders such as breast cancers, osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas, leukemias, lymphomas, sinus tumors, ovarian, uretal, bladder, prostate and other genitourinary cancers, colon, esophageal and stomach cancers and other gastrointestinal cancers, lung cancers, myelomas, pancreatic cancers, liver cancers, kidney cancers, endocrine cancers, skin cancers and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas.
In particular embodiments, the macrocyclic compounds of the present invention can be used to treat post-operative ileus. In other embodiments, the compounds of the present invention can be used to treat gastroparesis. In still other embodiments, the compounds of the present invention can be used to treat diabetic gastroparesis. In another embodiment, the compounds of the present invention can be used to treat opioid-induced bowel dysfunction. In further embodiments, the compounds of the present invention can be used to treat chronic intestinal pseudo obstruction.
The present invention further provides methods of treating a horse or canine for a gastrointestinal disorder comprising administering a therapeutically effective amount of a modulator having the structure of formula I, II and/or III. In some embodiments, the gastrointestinal disorder is ileus or colic.
As used herein, “treatment” is not necessarily meant to imply cure or complete abolition of the disorder or symptoms associated therewith.
The compounds of the present invention can further be utilized for the preparation of a medicament for the treatment of a range of medical conditions including, but not limited to, metabolic and/or endocrine disorders, gastrointestinal disorders, cardiovascular disorders, obesity and obesity-associated disorders, genetic disorders, hyperproliferative disorders and inflammatory disorders.
Further embodiments of the present invention will now be described with reference to the following examples. It should be appreciated that these examples are for the purposes of illustrating embodiments of the present invention, and do not limit the scope of the invention.
Example 1
Synthesis of Tethers
A. Standard Procedure for the Synthesis of Tether T9




Step T9-1: To a solution of 2-iodophenol (T9-0, 200 g, 0.91 mol, 1.0 eq) in DMF (DriSolv®, 560 mL) is added sodium hydride 60% in mineral oil (3.64 g, 0.091 mol, 0.1 eq) by portions (hydrogen is seen to evolve). The reaction is heated for 1 h at 100° C. under nitrogen, then ethylene carbonate is added and the reaction mixture heated O/N at 100° C. The reaction is monitored by TLC (conditions: 25/75 EtOAc/hex; Rf: 0.15, detection: UV, CMA). The reaction mixture is allowed to cool, then the solvent evaporated under reduced pressure. The residual oil is diluted in Et2O (1.5 L), then washed sequentially with 1 N sodium hydroxide (3×) and brine (2×), dried with MgSO4, filtered and the filtrate evaporated under reduced pressure. The crude product is distilled under vacuum (200 μm Hg) at 110-115° C. to provide T9-1.

Step T9-2: A solution of T9-1 (45.1 g, 0.171 mol, 1.0 eq) and Ddz-propargylamine (synthesized by standard protection procedures, 59.3 g, 0.214 mol, 1.25 eq) in acetonitrile (DriSolv®, 257 mL) was degassed by passing argon through the solution for 10-15 min. To this was added Et3N (85.5 mL, stirred O/N with CaH2, then distilled) and the mixture was again purged by bubbling with argon, this time for 5 min. Recrystallized copper (I) iodide (1.14 g, 0.006 mol, 0.035 eq) and trans-dichloro-bis(triphenylphosphine) palladium (II) (Strem Chemicals, 3.6 g, 0.0051 mol, 0.03 eq) are added and the reaction mixture stirred for 4 h under argon at rt. After 5-10 min, the reaction mixture turned black. The reaction was monitored by TLC (conditions: 55/45 EtOAc/hex). When complete, the solvent was removed under reduced pressure until dryness, then the residual oil diluted with 1 L of a 15% DCM in Et2O solution. The organic phase is washed with citrate buffer pH 4-5 (3×), saturated aqueous sodium bicarbonate (2×), and brine (1×), then dried with MgSO4, filtered and the filtrate evaporated under reduced pressure. The crude product thus obtained is purified by a dry pack column starting with 30% EtOAc/Hex (4-8 L) then increasing by 5% EtOAc increments until 55% EtOAc/Hex to give T9-2 as a brown syrup (yield: 65.8 g, 93.2%).

Step T9-3: To a solution of Ddz-amino-alcohol T9-2 (65.8 g, 0.159 mol, 1.0 eq) in 95% ethanol under nitrogen was added platinum (IV) oxide (3.6 g, 0.016 mol, 0.1 eq) and then hydrogen gas bubbled into the solution for 2 h. The mixture was stirred O/N, maintaining an atmosphere of hydrogen using a balloon. The reaction was monitored by 1H NMR until completion. When the reaction is complete, nitrogen was bubbled for 10 min to remove the excess hydrogen. The solvent is evaporated under reduced pressure, then diluted with EtOAc, filtered through a silica gel pad and the silica washed with EtOAc until no further material was eluted as verified by TLC. (55/45 EtOAc/hex) The combined filtrates were concentrated under reduced pressure. The residue is diluted in DCM (500 mL) and 4 eq of scavenger resin was added and the suspension stirred O/N. For this latter step, any of three different resins were used. MP-TMT resin (Argonaut Technologies, Foster City, Calif., 0.73 mmol/g) is preferred, but others, for example, PS-TRIS (4.1 mmol/g) and Si-Triamine (Silicycle, Quebec City, QC, 1.21 mmol/g) can also be employed effectively. The resin was filtered and washed with DCM, the solvent evaporated under reduced pressure, then dried further under vacuum (oil pump) to provide the product. The yield of Ddz-T9 from T9-0 on a 65 g scale was 60.9 g (91%)

1H NMR (CDCl3): δ 7.19-7.01, (m, 2H), 6.92-9.83 (m, 2H), 6.53 (bs, 2H), 6.34 (t, 1H), 5.17 (bt, 1H), 4.08 (m, 2H), 3.98 (m, 2H), 3.79 (s, 6H), 3.01 (bq, 2H), 2.66 (t, 3H), 1.26 (bs, 8H);
13C NMR (CDCl3): 6160.9, 156.8, 155.6, 149.6, 130.4, 127.5, 121.2, 111.7, 103.2, 98.4, 80.0, 69.7, 61.6, 55.5, 40.3, 30.5, 29.3, 27.4 ppm.
Tether T9 can also be synthesized from another tether molecule by reduction as in step T9-3 or with other appropriate hydrogenation catalysts known to those in the art.
B. Standard Procedure for the Synthesis of Tether T33a and T33b




The construction to the (R)-isomer of this tether (T33a) was accomplished from 2-iodophenol (33-0) and (S)-methyl lactate (33-A). Mitsunobu reaction of 33-0 and 33-A proceeded with inversion of configuration in excellent yield to give 33-1. Reduction of the ester to the corresponding alcohol (33-2) also occurred in high yield and was followed by Sonagashira reaction with Ddz-propargylamine. The alkyne in the resulting coupling product, 33-3, was reduced with catalytic hydrogenation. Workup with scavenger resin provided the desired product, Ddz-T33a.
The synthesis of the (S)-enantiomer (Ddz-T33b) was carried out in an identical manner in comparable yield starting from (R)-methyl lactate (33-B)




C. Standard Procedure for the Synthesis of Tether Precursor RCM-TA1





Step A1-1. To a solution of diol A1-0 (50 g, 567 mmol, 1.0 eq) in CH2Cl2 (1.5 L) were added Et3N (34.5 mL, 341 mmol, 0.6 eq) and DMAP (1.73 g, 14.2 mmol, 0.025 eq). TBDMSCl (42.8 g, 284 mmol, 0.5 eq) in CH2Cl2 (100 mL) was added to this mixture at rt over 4 h with a syringe pump. The reaction was monitored by TLC [EtOAc/hexanes (30:70); detection: KMnO4; Rf=0.39], which revealed starting material, mono-protected compound and di-protected compound. The mixture was stirred O/N, washed with H2O, saturated NH4Cl (aq) and brine, then dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexanes, 30:70) to give the desired mono-protected alcohol A1-1 (yield: 31%).

Step A1-2. To a solution of alcohol A1-1 (26.5 g, 131 mmol, 1.0 eq) in THF (130 mL) at 0° C. was added PPh3 (44.7 g, 170 mmol, 1.3 eq). A freshly prepared and titrated 1.3 M solution of HN3 (149 mL, 157 mmol, 1.5 eq) was added slowly to this mixture, then DIAD (32 mL, 163 mmol, 1.25 eq) also added slowly. This was an exotheric reaction. The resulting mixture was stirred at 0° C. for 1 h with monitoring of the reaction by TLC [EtOAc/hexanes (30:70); detection: KMnO4; Rf=0.77]. Compound A1-2 was obtained, but was not isolated and instead used directly for the next step in solution.

Step A1-3. PPh3 (51 g, 196 mmol, 1.5 eq) was added by portion to the solution of A1-2 and the resulting mixture was stirred at 0° C. for 2 h, allowed to warm to rt and maintained there for 3 h, then H2O (24 mL, 1331 mmol, 10 eq) added. This mixture was heated at 60° C. O/N. The reaction was monitored by TLC [EtOAc/hexanes (1:9); detection: KMnO4; Rf=baseline]. After cooling, a solution of 2 N HCl (327 mL, 655 mmol, 5.0 eq) was added and the resulting mixture stirred at rt for 2 h to obtain compound A1-3 in solution, which was used directly in the next step. TLC [DCM/MeOH/30% NH4OH (7:3:1); detection: KMnO4; Rf=0.32].

Step A1-4. For the next transformation, THF was evaporated under reduced pressure from the above reaction mixture and the remaining aqueous phase extracted with Et2O (5×150 mL) and CHCl3 (3×150 mL). The organic phases were monitored by TLC and if any A1-3 was observed, the organic phase was then extracted with 2 N HCl. The aqueous phase was neutralized cautiously to pH 8 with 10 N NaOH. CH3CN (400 mL) was added to this aqueous solution and Fmoc-OSu (41.9 g, 124 mmol, 0.95 eq) in CH3CN (400 mL) added slowly over 50 min. The solution was stirred at rt O/N. The reaction progress was monitored by TLC [EtOAc/hexanes (1:1); detection: ninhydrin; Rf=0.27]. The aqueous phase was extracted with Et2O, then the combined organic phase dried over MgSO4 and concentrated under reduced pressure. The solid residue obtained was mixed with H2O (120 mL), stirred 30 min, filtered (to remove succinimide byproduct) and dried O/N under vacuum (oil pump). The solid was purified by flash chromatography [gradient: EtOAc/hexanes (50:50) to EtOAc/hexanes (70:30), with the change of eluent once Fmoc-OSu was removed as indicated by TLC] to give compound TA1 as a white solid (yield: 71%).

1H NMR (CDCl3, ppm): 7.8 (d, 2H), 7.6 (d, 2H), 7.4 (t, 2H), 7.3 (t, 2H), 5.9-5.7 (1H, m), 5.6-5.5 (1H, m), 5.0 (1H, broad), 4.4 (2H, d), 4.2 (2H, d), 3.9 (2H, broad), 2.1 (1H, broad).
13C NMR (CDCl3, ppm): 156.8, 144.1, 141.5, 131.9, 128.3, 127.9, 127.3, 125.2, 120.2, 67.0, 58.0, 47.4, 38.0.
D. Standard Procedure for the Synthesis of Tether Precursor RCM-TA2




This material was accessed through application of the cross metathesis reaction shown to construct the carbon backbone. The resulting nitrile was reduced to the amine, which was protected in situ with Fmoc or other appropriate protecting group prior to attachment to the resin, which was performed using standard solid phase chemistry procedures known to those in the art. This standard procedure would also be applicable to homologues of TA2.

E. Standard Procedure for the Synthesis of Tether Precursor RCM-TB1





Step B1-1. To 2-bromobenzyl alcohol (B1-0, 30 g, 160 mmol) in DCM (DriSolv®, 530 mL) as an approximately 0.3 M solution, was added dihydropyran (B1-A, 22 mL, 241 mmol). Pyridinium p-toluenesulfonate (PPTS, 4.0 g, 16 mmol) was added and the reaction mixture stirred vigorously at rt O/N. A saturated solution of Na2CO3 (aq, 200 mL) was then added and the mixture stirred for 30 min. The DCM layer was separated, washed successively with saturated Na2CO3 (aq, 2×100 mL) and brine (2×50 mL), and dried over anhydrous MgSO4. The solvent was evaporated under reduced pressure and the crude residue was purified by dry-pack silica-gel column. [EtOAc/hexanes (1:9); before loading the crude material, the silica was neutralized by flushing with 1% Et3N in DCM] This afforded B1-1 as a colorless oil (42 g, 97%). TLC [EtOAc/hexanes (1:9); Rf=0.56]

Step B1-2. Magnesium turnings (2.21 g, 90 mmol) were added to an approximately 0.8 M solution of B1-1 (from which several portions of toluene were evaporated to remove traces of water, 22.14 g, 81.8 mmol) in anhydrous THF (distilled from sodium benzopheneone ketyl, 100 mL) under an atmosphere of nitrogen. The reaction was initiated by adding iodine chips (50 mg, 0.002 equiv). The reaction mixture was heated to reflux for 2 h, during which time most of the Mg turnings disappeared. The reaction was allowed to cool to rt. In a separate flame-dried round-bottomed flask, freshly distilled allyl bromide (6.92 mL, 81.8 mmol) was diluted with anhydrous THF (50 mL) under a nitrogen atmosphere and cooled to 0° C. using an ice-water bath. To this was gradually transferred the now cooled Grignard solution over a period of 20-30 min using a cannula ensuring that the unreacted magnesium turnings remained in the source flask. The contents of the Grignard preparation flask were washed (2×5 mL dry THF) and the washings transferred via cannula to the allyl bromide solution as well. The resulting mixture was stirred O/N under N2 while allowing it to gradually warm to rt. The reaction was quenched by adding saturated NH4Cl (aq) solution, then diluted with 100 mL Et2O and the layers separated. The aqueous phase was extracted with Et2O (3×100 mL) and the combined organic layers dried over MgSO4, then concentrated under reduced pressure to provide B1-2 (18.54 g, 98%). TLC [EtOAc/hexanes (1:9), Rf=0.53]. This material was utilized in the next step without further purification.

Step B1-3. 2-(2-Propenyl)benzyl alcohol (TB1). The crude THP ether B1-2 (18.54 g, 80 mmol) was dissolved in MeOH (160 mL) and p-toluenesulfonic acid monohydrate (PTSA, 1.52 g, 8 mmol) added. The resulting mixture was stirred at rt O/N, then concentrated under reduced pressure and the residue diluted with Et2O (100 mL). The organic layer was sequentially washed with 5% NaHCO3 (aq) solution (3×50 mL) and brine (1×50 mL), then dried over MgSO4. The solvent was evaporated under reduced pressure and the residue purified by flash chromatography (EtOAc/hexanes, 1:9), to obtain TB1 as a pale-yellow oil (9.2 g, 78%). TLC [EtOAc/hexanes (1:9), detection: UV, PMA; Rf=0.24].

F. Standard Procedure for the Synthesis of Tether Precursor RCM-TB2





Step B2-1. To a suspension of MePPh3Br (85.7 g, 240 mmol, 2.2 eq) in THF (500 mL) was added t-BuOK in portions (26.9 g, 240 mmol, 2.2 eq) and the resulting mixture stirred at rt for 2 h during which time it became yellow. The reaction mixture was cooled to −78° C., 2-hydroxybenzaldehyde (B2-0, 11.6 mL, 109 mmol, 1.0 eq) added over 10 min, then it was stirred O/N at rt. The reaction progress was monitored by TLC [EtOAc/hexanes (20:80); detection: UV, CMA; Rf=0.25]. A saturated NH4Cl (aq) solution was added and the resulting aqueous phase extracted with Et2O (3×). The combined organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexanes, 30:70) to give B2-1 as a yellow oil. The identity and purity were confirmed by 1H NMR (yield: 1100%).

Step B2-2. To a solution of alcohol B2-1 (2.0 g, 16.7=mmol, 1.0 eq) in DMF at 0° C. was added cesium carbonate (1.1 g, 3.34 mmol, 0.2 eq) and the mixture stirred at 0° C. for 15 min. The reaction was warmed to 100° C. and ethylene carbonate added. The resulting mixture was stirred at 100° C. O/N. The reaction was monitored by TLC [EtOAc/hexanes (30:70); detection: LUV, CMA; Rf 0.21]. The solution was cooled to rt and H2O added. The resulting aqueous phase was extracted with Et2O (3×). The organic phase was extracted with brine (3×), dried with MgSO4, filtered and concentrated under reduced pressure. A yellow syrup (TB2) was obtained (yield: 96%), which was of sufficient purity (as assessed by NMR) for further use without additional purification. Note that this product proved to be unstable in the presence of acid.

1H NMR (CDCl3, ppm): 7.50 (1H, dd, Ph), 7.22 (1H, td, Ph), 7.05 (dd, 1H, PhCH═CH2), 6.98 (1H, t, Ph), 7.90 (1H, d, Ph), 5.75 (1H, dd, PhCH═CHH), 5.30 (1H, dd, PhCH—CHH), 4.15-4.10 (2H, m, PhOCH2CH2OH), 4.05-3.95 (2H, m, PhOCH2CH2OH), 2.05 (1H, s, OH).
G. Standard Procedure for the Synthesis of Tether Precursor RCM-TB3




To a solution of 2′-bromophenethylalcohol (B3-0, 2.0 mL, 14.9 mmol, 1.0 eq) in toluene (50 μL) were added tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4, 347 mg, 0.30 mmol, 0.02 eq) and vinyltributyltin (6.5 mL, 22.4=mol, 1.5 eq). The resulting mixture was stirred at reflux for 24 h under N2. Monitoring reaction progress by TLC was difficult since the starting material and product possessed the same Rf [EtOAc/hexanes (30:70)]. The reaction mixture was cooled to rt and saturated KF (aq) solution added at which time a precipitate was formed. The solid was optionally removed by filtration and the aqueous phase extracted with DCM (4×). The combined organic phase was extracted with brine, dried over MgSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexanes, 30:70) to give TB3 as a colorless oil. The identity and purity were confirmed by 1H NMR (yield: 100%).

1H NMR (CDCl3, ppm): 7.57-7.45 (1H, m, Ph), 7.30-7.15 (3H, m, Ph), 7.05 (dd, 1H, PhCH—CH2), 5.65 (1H, dd, PhCH═CHH), 5.32 (1H, dd, PhCH═CHH), 4.85 (2H, t, PhCH2CH2OH), 2.98 (2H, t, PhCH2CH2OH), 1.50 (1H, s, OH).
H. Standard Procedure for the Synthesis of Tether Precursor RCM-TB4




Step B4-1. 1,2-Dihydronaphthalene (B4-0, 5.0 g, 38.4 mmol, 1.0 eq) was dissolved in 200 mL of DCM:MeOH (1:1) and the solution cooled to −78° C. Ozone (O3) was bubbled through the solution until a blue color developed. The reaction was monitored by TLC [EtOAc/hexanes (30:70); detection: UV, CMA; Rf=0.25]. Excess O3 was then removed by bubbling N2 through the solution until the blue color had dissipated. Sodium borohydride (2.9 g, 76.8 mmol, 2.0 eq) was added slowly to the mixture, then it was stirred at rt for 1 h. The reaction was monitored by TLC [EtOAc/hexanes (30:70); detection: UV, CMA; Rf=0.06]. A saturated NH4Cl (aq) solution was added slowly, then the aqueous phase was extracted with DCM (3×). The combined organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. B4-1 was obtained as a yellow oil (yield: 100%). The identity and purity of the compound was confirmed by NMR analysis and typically was of sufficient purity to be used without further manipulation.

Step B4-2. To a solution of the diol B4-1 (6.38 g, 38.4 mmol, 1.0 eq) in benzene (200 HL) was added MnO2 (85%, 16.7 g, 192 mmol, 5.0 eq) and the resulting mixture stirred 1 h at rt. The reaction was monitored by TLC [EtOAc/hexanes (50:50); detection: UV, CMA; Rf=0.24] and more MnO2 (5 eq) added each 1 h period until the reaction was completed, typically this required 2-3 such additions. The MnO2 was filtered through a Celite pad, which was then washed with EtOAc. The combined filtrate and washes were evaporated under reduced pressure to give B4-2. A 1H NMR was taken to check the purity of the resulting compound, which typically contained small amounts of impurities. However, this was sufficiently pure for use in the next step, which was preferably performed on the same day as this step since the aldehyde product (B4-2) had limited stability.

Step B4-3. To a suspension of MePPh3Br (30.2 g, 84.5 mmol, 2.2 eq) in THF (200 mL) was added t-BuOK in portions (9.5 g, 84.5 mmol, 2.2 eq) and the resulting mixture stirred at rt for 2 h during which time the solution became yellow. The reaction mixture was cooled to −78° C., B4-2 [6.3 g, 38.4 mmol, 1.0 eq (based on the theoretical yield)] added over 10 min, then the mixture stirred O/N at rt. The reaction was monitored by TLC [EtOAc/hexanes (50:50); detection: UV, CMA; Rf=0.33]. A saturated NH4Cl (aq) solution was added and the resulting aqueous phase extracted with EtOAc (3×). The combined organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexanes, 40:60) to give TB4 as a yellow oil. NMR was used to confirm the identity and purity of the product (yield: 73%, 2 steps).

1H NMR (CDCl3, ppm): 7.55-7.45 (1H, m, Ph), 7.25-7.10 (3H, m, Ph), 7.05 (dd, 1H, PhCH═CH2), 5.65 (1H, dd, PhCH═CHH), 5.30 (1H, dd, PhCH═CHH), 3.70 (2H, t, PhCH2CH2CH2OH), 2.80 (2H, t, PhCH2CH2CH2OH), 1.90-1.80 (2H, m, PhCH2CH2CH2OH), 1.45 (1H, s, OH).
I. Standard Procedure for the Synthesis of Tether T45




The protected version of this tether was obtained through standard transformations involving monoprotection of triethyleneglycol (45-0) followed by conversion of the remaining alcohol to a mesylate, displacement with azide and catalytic reduction in the presence of di-t-butyl dicarbonate.
J. Standard Procedure for the Synthesis of Tether T65




See the preparation of T9-2 as this tether is actually an intermediate in the synthesis of tether T9.
1H NMR (CDCl3): δ 7.38-7.35 (bd, 1H), 7.30-7.19 (m, 1H), 6.92 (dd, 2H), 4.88 (bs, 1H), 4.16-4.11 (bt, 4H), 3.98-3.95 (t, 2H), 1.46 (s, 9H).
13C NMR (CDCl3): δ 156.7, 155.8, 133.6, 130.0, 121.3, 114.8, 113.1, 112.9, 90.2, 70.8, 61.4, 28.6
K. Standard Procedure for the Synthesis of Tether T66




To a solution of alkyne (Boc-T65, 13.1 g, 45.1 mmol, 1.0 eq) in EtOH/AcOEt (5:1) under N2 is added quinoline (106 μl, 0.9 mmol, 0.02 eq) and the Lindlar catalyst (1.3 g, 10% wt), then hydrogen is bubbled into the mixture. The reaction is monitored (each 30-40 min) by 1H NMR until the reaction is complete. Then, the reaction is filtered through a Celite pad and rinsed with AcOEt until there is no more material eluting. The solvent is removed under reduced pressure. The crude product is purified by flash chromatography with 15% AcOEt/Hex to 40% AcOEt/Hex to give Boc-T66 an oil. (Yield: 7.8 g, 59%) TLC (45/55 AcOEt/Hex): Rf: 0.15; detection: UV, KMnO4.
1H NMR (CDCl3): δ 7.27-7.21 (td, 1H), 7.15-7.10 (dd, 1H), 7.00.6.85, (m, 2H), 6.62-6.58 (bd, 1H), 5.77-5.70 (dt, 1H), 4.13-4.03 (m, 2H), 3.97-3.95 (m, 2H), 3.9-3.88 (bd, 2H), 1.46, (s, 9H)
L. Standard Procedure for the Synthesis of Tether T67




To a solution of Et2Zn (1 M in hexanes, 153 mL, 153.6 mmol, 3.0 eq) in CH2Cl2 (150 mL) at −20° C. was added CH2I2 (12.4 mL, 153.6 mmol, 3.0 eq) (CAUTION: Pressure can develop.) and the mixture stirred at −20° C. for 15 min. Boc-T8 (15.0 g, 51.2 mmol, 1.0 eq) in CH2Cl2 (100 mL) was then added and the mixture stirred at room temperature O/N. The reaction was monitored by TLC [(60% AcOEt: 40% hexane); detection: UV and CMA; Rf=0.39]. The solution was treated with aqueous NH4Cl (saturated) and the aqueous phase was extracted with CH2Cl2. The organic phase was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (60% AcOEt: 40% hexane) to give Boc-T67 as a yellow oil (yield: 57%).
1H NMR (CDCl3, ppm): 7.18 (1H, t), 7.03 (1H, d), 6.88 (2H, t), 4.23-4.04 (4H, m), 3.73-3.70 (2H, m), 1.48 (1H, broad), 1.28 (9H, s), 1.12-1.06 (1H, m), 1.0-0.93 (1H, m), 0.76 (2H, dt).
M. Standard Procedure for the Synthesis of Tether T68




To a solution of Et2Zn (1 M in hexanes, 49.2 mL, 49.2 mmol, 3.0 eq) in CH2Cl2 (30 mL) at −20° C. was added CH2I2 (3.9 mL, 49.2 mmol, 3.0 eq) and the mixture stirred at −20° C. for 15 min. The alkene (Boc-T66, 4.8 g, 16.4 mmol, 1.0 eq) in CH2Cl2 (50 mL) was then slowly added and the mixture stirred at room temperature for 2 h. The solution was treated with aqueous NH4Cl (saturated) and the aqueous phase extracted with CH2Cl2 (1×) then washed with brine (1×). The organic phase was dried over MgSO4, filtered and the solvent removed under reduced pressure. The crude product is purified by flash chromatography (gradient: 40%, then 50% and finally 60% AcOEt in hexanes) to give Boc-T68 as a yellow oil (yield: 90.7%). TLC (60% AcOEt: 40% hexanes): Rf: 0.4; detection: UV, ninhydrin.
1H NMR (CDCl3): δ 7.32-7.20 (td, 2H), 7.10-6.85, (m, 2H), 4.25-4.13 (m, 2H), 4.10-3.99 (m, 2H), 3.41-3.36 (dd, 1H), 2.15-2.02 (m, 1H), 1.38 (s, 9H), 1.04-0.96 (dq, 1H), 0.78-0.73 (q, 1H)
13C NMR (CDCl3): δ 158.0, 130.7, 130.4, 127.9, 127.5, 127.1, 121.2, 121.0, 111.6, 111.2, 79.5 69.8, 61.5, 28.7, 17.8, 16.8, 7.2
N. Standard Procedure for the Synthesis of Tether T69




TLC (25/75 AcOEt/Hex): Rf: 0.03; detection: UV, ninhydrin
1H NMR (CDCl3): δ 7.06-7.00 (bt, 1H), 6.61-6.52 (m, 4H), 6.35 (m, 1H), 5.12 (bt, 1H), 4.03 (m, 2H), 3.95 (m, 2H), 3.77 (s, 6H), 3.11-3.04 (bq, 2H), 2.60 (bt 2H) 1.75 (m, 8H)
13C NMR (CDCl3): δ 163.9, 160.9, 160.6, 157.6, 157.5, 155.6, 149.5, 130.8, 130.6, 125.9, 107.26, 106.9, 103.2, 98.4, 80.8, 77.5, 69.9, 61.3, 60.9, 60.6, 55.4, 40.3, 30.4, 29.3, 26.9,
LC-MS (Grad_A4) tR: 8.37 min
O. Standard Procedure for the Synthesis of Tether T70




TLC (25/75 AcOEt/Hex): Rf. 0.03; detection: UV, ninhydrin
1H NMR (CDCl3): δ 6.84-6.75 (m, 3H), 6.52 (bs, 2H), 6.34 (m, 1H), 5.17 (bt, 1H), 4.01 (m, 2H), 3.93 (m, 2H), 3.77 (s, 6H), 3.10 (bq, 2H), 2.63 (bt, 2H), 1.74 (m, 8H)
13C NMR (CDCl3): δ 160.9, 158.9, 155.8, 155.6, 152.9, 152.9, 149.5, 132.4, 132.3, 1117.1, 116.8, 112.7, 112.6, 103.2, 98.4, 80.8, 70.4, 61.6, 55.5, 40.2, 30.3, 29.3, 27.4.
LC-MS (Grad_A4) tR: 8.29 min
P. Standard Procedure for the Synthesis of Tether T71




TLC (25/75 AcOEt/Hex): Rf: 0.03; detection: UV, ninhydrin
1H NMR (CDCl3): δ 7.12-7.08 (bd, 2H), 6.76-6.73 (d, 1H), 6.52 (m, 2H), 6.33 (bs, 1H), 5.15 (bt, 1H), 4.02 (m, 2H), 3.95 (m, 2H), 3.79 (s, 6H), 3.09 (bq, 2H), 2.61 (bt, 2H), 1.74 (m, 8H)
13C NMR (CDCl3): δ 160.8, 155.6, 155.4, 149.5, 132.4, 130.1, 127.0, 126.0, 112.8, 103.2, 98.4, 80.8, 70.0, 61.4, 55.5, 40.3, 30.2, 29.3, 24.5, 27.4
LC-MS (Grad_A4) tR: 9.60 min
Q. Standard Procedure for the Synthesis of Tether T72




TLC (1/1, Hex/AcOEt): Rf: 0.16
1H NMR (ppm): 1.49 (Boc), 1.8 (CH2), 2.7 (CH2), 3.1 (CH2), 4.0 (CH2), 4.1 (CH2), 4.9 (NH), 6.9 (CH aromatic), 7.35 (CH aromatic), 7.4 (CH aromatic)
13C NMR (ppm): 29, 30, 40, 61, 70, 110, 124, 128, 132, 160
R. Standard Procedure for the Synthesis of Tether T73




TLC (60/40 AcOEt/Hex): Rf: 0.11; detection: UV, ninhydrin,
1H NMR (CDCl3): δ 7.06-6.99, (m, 2H), 6.84-6.81 (m, 1H), 6.5 (m, 2H), 6.32 (m, 1H), 5.11 (bt, 1H), 4.07 (m, 2H), 3.90 (bt, 2H), 3.79 (s, 6H), 3.39 (s, 3H), 3.09 (bt, 2H), 2.64 (bt, 2H), 1.85-1.74 (m, 8H), 1.46 (bs, 9H)
13C NMR (CDCl3): δ 160.8, 157.1, 155.6, 151.9, 149.5, 131.3, 131.0, 128.43, 128.37, 111.6, 103.2, 98.4, 84.8, 80.8, 69.9, 61.4, 60.6, 55.5, 41.8, 40.2, 30.0, 29.3, 28.1, 27.3 ppm.
LC-MS (Grad_A4) tR: 8.26 min.
S. Standard Procedure for the Synthesis of Tether T74




TLC (50/50 AcOEt/Hex): Rf: 0.09; Detection: UV, CMA
1H NMR (DMSO-d6): δ 7.14 (bd, 1H), 6.76-6.71 (m, 2H), 6.53 (m, 2H), 6.33 (bs, 1H), 5.15 (bt, 1H), 4.08 (m, 2H), 3.95 (m, 2H, 3.79 (s, 6H), 3.41 (s, 3H), 3.01 (bq, 2H), 2.64 (bt, 2H), 1.75 (m, 8H), 1.47 (s, 9H)
13C NMR (DMSO-d6): δ 156.1, 152.3, 150.8, 147.0, 144.7, 129.8, 126.9, 125.6, 116.8, 108.4, 98.5, 93.6, 80.3, 76.1, 65.1, 56.7, 50.7, 37.1, 35.6, 25.3, 24.5, 23.4, 22.6
LC-MS (Grad_A4) tR: 8.21 min
T. Standard Procedure for the Synthesis of Tether T75a and T75b




The synthesis of the fluorinated derivative, tether T75, was carried out in an analogous matter to that of the related tether T33 starting from 33-A [(S)-methyl lactate] and appropriately substituted phenol 75-0 to provide 4.1 g of Ddz-T75a as a pale yellow solid. Although the first two steps, Mitsunobu reaction and DIBAL reduction, were high yielding, 91% and 98% respectively, isolation of the final product proved difficult after Sonagashira coupling and hydrogenation, lowering the overall yield to 17%. Again, the corresponding (R)-enantiomer, Ddz-T75b, is accessible by substituting (R)-methyl lactate (33-B) in the above procedure.




U. Standard Procedure for the Synthesis of Tether T76





Step T76-1. 3-Bromo-2-hydroxy-benzaldehyde. In a manner analogous to that of the literature (Hofslokken et al. Acta. Chemica Scand. 1999, 53, 258), a stirred suspension of 2-bromophenol (76-0, 3.5 g, 20 mmol) and paraformaldehyde (8.1 g, 270 mmol) in 100 mL of dry acetonitrile at room temperature was treated with MgCl2 (2.85 g, 30 mmol) and triethylamine (TEA, 10.45 ml, 75 mmol). The mixture was stirred vigorously at reflux O/N. After this period of time, the mixture was cooled to room temperature, then 30 mL of 5% HCl was added and the product extracted with Et2O to give 4.0 g (95%) of 76-1.

TLC (hexanes/dichloromethane, 3:1): Rf=0.3; detection: CMA and UV
Step 76-2. 2-Bromo-6-vinyl-phenol. To a stirred solution of CH3PPh3Br (72 g, 0.033 mol) at room temperature was added, over 5 min, a solution of tBuOK (4.1 g, 0.03 mol) in THF (50 mL). The mixture was cooled to −78° C. and 76-1 (3 g, 0.015 mol) was added dropwise over 15 min. The reaction mixture was allowed to warm to room temperature and stirred for 24 h. After this time, the solvent was removed in vacuo and the residue purified by flash chromatography using hexanes/dichloromethane (3:1) as eluent to afford 76-2 as a colorless oil (2.2 g, 75%).
TLC (hexanes/dichloromethane, 3:1): Rf=0.5; detection: CMA and UV
Step 76-3. The tosylate 76-A was synthesized using the literature method (Buono et al. Eur. J. Org. Chem. 1999, 1671) and then utilized for 76-3 (Manhas, M. S. J. Am. Chem. Soc. 1975, 97, 461-463. Nakano, J. Heterocycles 1983, 20, 1975-1978). To a solution of 76-2 (2.5 g, 12 mmol), Ph3P (4.6 g, 18 mmol) and 76-A (4.3 g, 18 mmol) in 150 mL of THF was slowly added diethylazodicarboxylate (DEAD, 3.5 mL, 18 mmol) at room temperature. The mixture was stirred at room temperature for 6 h until the reaction was complete as indicated by TLC analysis (hexanes/ethyl acetate, 8:2; Rf=0.6; detection: CMA and UV). The solvent was removed under high vacuum and the residue was purified by flash chromatography to obtain 76-3 as a pale brown liquid (4.6 g, 88%).

Step 76-4. 76-3 (3.4 g, 8 mmol) was treated with second generation Grubbs catalyst (0.02 mol %) in 50 mL of DCM (Grubbs, R. J. Org. Chem. 1998, 63, 864-866. Gross, J. Tet. Lett. 2003, 44, 8563-8565. Hoveyda, A. J. Am. Chem. Soc. 1998, 120, 2343-2351). The resulting mixture was stirred at room temperature for 12 h The solvent was then removed under high vacuum and the residue purified by flash column chromatography to obtain 76-4 as a pale brown liquid (2.15 g, 70%). TLC (hexanes/ethyl acetate, 8:2; Rf 0.4; detection: CMA and UV).

Step 76-5. To a solution of 76-4 (1.43 g, 0.023 mol) in dry DMF (50 mL) was added cesium acetate (2.09 g, 0.0109 mol) under an argon atmosphere. The solution was stirred at 50° C., O/N. After this time, the solvent was removed under high vacuum and the residue purified by flash chromatography to obtain 76-5 as a pale brown liquid (0.7 g, 70%). TLC (hexanes/ethyl acetate, 8:2; Rf=0.6; detection: CMA and UV).

Step 76-6 (8-Bromo-2H-chromen-2-yl)-methanol. To a solution of 76-5 (5.5 g, 0.023 mol) in dry MeOH (150 mL) was added sodium metal in a catalytic amount under an argon atmosphere. The solution was then stirred at room temperature for 60 min. After this time, Amberlite IRA-120 (H+) resin was added to neutralize (pH=7) excess sodium methoxide and the mixture was vigorously stirred for 10 min. The resin was removed by filtration and the filtrate evaporated in vacuo. Pure compound 76-6 was recovered as a colorless oil (4.5 g, 98%).

TLC (hexanes/ethyl acetate, 7:3): Rf=0.3; detection: CMA and UV
Step 76-7. 76-6 (4.5 g, 18 mmol) and Ddz-propargyl amine (76-B, 15.16 g, 55.8 mmol) were dissolved in dioxane (150 mL) and diisopropylamine (27 mL). The reaction mixture was degassed by bubbling argon through the solution. PdCl2(PhCN)2 (430 mg, 1.11 mmol, 0.06 eq), CuI (220 mg, 1.11 mmol, 0.06 eq) and tributylphosphine (10% in hexane, 4.4 mL, 2.23 mmol) were added and the mixture was warmed to 70° C. and stirred O/N. The solvent was removed under high vacuum and the residue purified by flash column chromatography to obtain 76-7 as a pale brown liquid (3.2 g, 80%).
TLC (hexanes/ethyl acetate, 1:1): Rf=0.3; detection: CMA and UV
Step 76-8. The acetylene 76-7 (4.5 g, 0.2 mol) was dissolved in EtOH (150 mL), then purged with nitrogen for 10 min. PtO2 (10 mol %, 450 mg) was added, and the mixture purged with a balloon full of hydrogen gas. The mixture was then charged into a Parr bomb, flushed with hydrogen (simply fill with hydrogen at 60 psi, then release and refill, repeat this fill-release-refill cycle 3×), and reacted with hydrogen at 60 psi at room temperature O/N. The reaction mixture was filtered through a pad of Celite (use methanol for washing the pad) and the filtrate concentrated to afford a practically pure (clean by 1H NMR), but colored sample of Ddz-T76 in quantitative yield. Further purification was achieved by subjecting this material to flash chromatography. TLC (hexanes/ethyl acetate, 1:1; Rf=0.3; detection: CMA and UV). Since the product Ddz-T76 has the same Rf as the starting material (76-7), 1H NMR is the best way to distinguish them.
1H NMR (CDCl3): δ 1.73 (s, 6H), 1.75-1.95 (m, 4H), 2.60 (m, 2H), 2.70-2.90 (m, 2H), 3.10 (m, 2H), 3.72 (s, 6H), 3.75 (m, 2H), 4.12 (m, 1H), 5.20 (m, 1H), 6.35 (s, 1H), 6.50 (s, 2H), 6.80 (m, 1H), 6.90 (m, 2H).
13C NMR (CDCl3): δ 23.93 (CH2), 24.97 (CH2), 27.07 (CH2), 29.35 (CH3), 30.45 (CH2), 40.23 (CH2), 55.47 (CH3), 65.76 (CH2), 80.72 (CH), 98.44 (CH), 103.22 (CH), 120.29 (CH), 121.90 (Cq), 127.76 (CH), 128.14 (CH), 129.42 (Cq), 149.56 (Cq), 152.55 (Cq), 155.56 (Cq), 160.84 (Cq).
LC-MS (Grad_A4): tR: 9.46 min; Mass found: 443
V. Standard Procedure for the Synthesis of Tether T77




Step T77-1. 3-Bromo-pyridin-2-ol. A stirred suspension of 2-pyridone (77-0, 19 g, 200 mmol) in 200 mL of 1 M aqueous KBr at room temperature was treated over 15 min with bromine (32 g, 200 mmol; CAUTION: Large quantities of Br2 should be handled carefully!) in 200 mL of 1 M aqueous KBr, then stirred vigorously at room temperature O/N. After 24 h, this solution deposited crystals which were filtered off and then recrystallized from acetonitrile to give 27.2 g (78%) of 3-bromo-pyridin-2-ol. (77-1) [J. Am. Chem. Soc. 1982, 104, 4142-4146; Bioorg. Med. Chem. Lett. 2002, 12, 197-200; J Med Chem. 1979, 22, 1284-1290.]

Molecular weight calcd. for C5H4BrNO: 173; (M+H)+ found: 174
Step T77-2. To a solution of 3-bromo-pyridin-2-ol (77-1, 5 g, 0.028 mol), Ph3P (11 g, 0.04 mol) and 2-(tert-butyldimethylsilanyloxy)-ethanol (77-A, 7 g, 0.04 mol) in 50 mL of THF was slowly added diethylazodicarboxylate (8.1 g, 0.04 mol) at room temperature. The progress of the reaction was easily monitored by TLC [hexanes/ethyl acetate (4:1); Rf=0.5; detection: CMA]. The mixture was stirred at room temperature for 24 h at which point the reaction was complete by TLC analysis. The solvent was removed under high vacuum and the residue purified by flash chromatography to obtain 77-2 as a pale brown liquid (6.3 g, 68%). [Tetrahedron Lett. 1994, 35, 2819-2822; Tetrahedron Lett. 1995, 36, 8917-8920; Synlett, 1995, 845-846. Heetrocycles 1990, 31, 819-824.
Molecular weight calcd. for C13H22BrNO2Si 331; (M+H)+ found: 332
Step T77-3. The protected alcohol 77-2 (3 g, 9.1 mmol) was dissolved in diisopropylamine (50 mL) and the reaction mixture degassed by bubbling argon through the solution. PdCl2(PPh3)2 (410 mg, 0.61 mmol, 0.06 eq), CuI (74 mg, 0.4 mmol, 0.04 eq) and triphenylphosphine (310 mg, 1.12 mmol) were added, then the mixture was warmed to 70° C. and stirred O/N. The solvent was removed under high vacuum and the residue was purified by flash chromatography to obtain 77-3 as a pale brown liquid (3.36 g, 70%) [Org. Lett. 2003, 5, 2441-2444; J. Chem. Soc. Perkin. Trans I 1999, 1505-1510; J. Org. Chem. 1993, 58, 2232-2243; J. Org. Chem. 1999, 58, 95-99; Org. Lett. 2000, 2, 2291-2293; Org. Lett. 2002, 4, 2409-2412]
TLC (hexanes/ethyl acetate, 1:3): Rf: =0.3; detection: CMA
Molecular weight calcd. for C28H40N2O6Si: 528; (M+H)+ found: 529
Step T77-4. The acetylene 77-3 (3 g, 5.67 mmol) was dissolved in EtOH (30 mL) and purged with nitrogen for 10 min. PtO2 (10 mol %, 300 mg) was added and the mixture purged with a balloon full of hydrogen gas. The mixture was then charged into a Parr bomb, flushed with hydrogen (fill with hydrogen at 80 psi then release and refill, repeat this fill-release-refill cycle 3×), and maintained with hydrogen at 80 psi at room temperature O/N. The reaction mixture was filtered through a pad of Celite (use methanol for washing the residue on the Celite) and the filtrate plus washings was concentrated under reduced pressure to afford a practically pure (clean 1H NMR), but colored sample of 77-4 in a quantitative yield. Further purification was achieved by subjecting this material to flash chromatography. The product 77-4 has the same Rf as the starting material (77-3), hence, 1H NMR is the best way to distinguish them.
TLC [(hexanes/ethyl acetate, 1:3); Rf=0.3 detection: CMA]
Molecular weight calcd. for C28H44N2O6Si: 532, (M+H)+ found: 533
Step T77-5. 77-4 (3 g, 5.6 mmol) was dissolved in anhydrous THF (200 mL). To the clear solution was added TBAF (6.7 mmol, 7 mL) and the mixture stirred for 2 h at room temperature. The solution was then poured into ice water. The aqueous solution was extracted with dichloromethane (3×200 mL). The organic layer was washed sequentially with saturated citrate buffer (1×200 mL), water (200 mL) and brine (200 mL). The washed organic extract was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure to give an oily residue. This syrup was purified by flash chromatography (hexanes/AcOEt, 1:2) to give Ddz-T77 as a syrup (2.10 g, yield 90%).
TLC (hexanes/AcOEt, 1:2): Rf=0.3; detection: ninhydrin
1H NMR (CDCl3): δ 1.73 (s, 6H), 1.75 (m, 2H), 2.65 (m, 2H), 3.15 (m, 2H), 3.75 (s, 6H), 3.90 (m, 2H), 4.50 (m, 2H), 5.01 (sb, 1H), 6.30 (s, 1H), 6.50 (s, 2H), 6.80 (m, 1H), 7.40 (m, 1H), 8.01 (m, 1H).
13C NMR (CDCl3): δ 27.23 (CH2), 29.24 (CH3), 29.71 (CH2), 40.17 (CH2), 55.44 (CH3), 62.76 (CH2), 69.11 (CH2), 80.76 (Cq), 98.24 (CH), 103.24 (CH), 117.54 (CH), 124.68 (Cq), 138.82 (CH), 144.17 (CH), 149.45 (Cq), 155.50 (Cq), 160.84 (Cq), 162.03 (Cq).
Molecular weight calcd. for C22H30N2O6: 418; (M+H)+ found: 419
Example 2
Synthesis of Representative Macrocyclic Compounds
The following are provided as representative examples for the macrocyclic compounds of the invention. For solid phase methods, all yields are reported starting from 300-325 mg of PS-aminomethyl resin (loading 2.0 mmol/g) unless otherwise noted. Attachment of the first building block, BB3, varies from 100% to 55% for the more difficult residues, typically sterically crowded structures such as Ile or Val. The remaining couplings for BB2 and BB1 proceed in an average yield of 80-90%. Attachment of the tether using the Mitsunobu reaction yields from 50-90% of the desired linear precursor. The macrocyclization itself proceeds in an average yield of 20-50%. Minimal loss of yield occurs in post-cyclization processing.
All the retention time values presented herein are based on the UV portion of the HPLC data. In the HPLC procedure, ELSD and CLND data (not listed) were also procured to further assess purity of the final products, and for quantification (CLND). All compounds were analyzed using the same HPLC conditions. The details for the HPLC procedure used was as follows: Column: XTerra MS C18 4.6×50 mm, 3.5 μm, from Waters, HPLC: Alliance 2695 from Waters; MS: Platform LC from Micromass/Waters; CLND: 8060 from Antek; PDA: 996 from Waters; Gradient_B4. (i) 0 to 50% MeOH 0.1% aqueous TFA in 6 min, (ii) 3 min at 50% MeOH: 0.1% aqueous TFA; (iii) 50 to 90% MeOH: 0.1% aqueous TFA in 5 min; (iv) 3 min at 90% MeOH: 0.1% aqueous TFA. Retention time (tR) for the compound is listed.
Modifications were made to the standard methods for compounds 58, 99, 201, 203 and 215.
Compound 1
Yield: 33.4 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6): δ 8.53, 8.41, 8.34 (doublets J=8.7 Hz for all, 1H); 8.13-8.06, 7.82-7.75 (multiplets, 1H); 7.30-7.05 (m, 8H); 6.90-6.77 (m, 2H); 4.58-4.46, 4.40-4.29, 4.27-4.16 (multiplets, 1H); 4.09-3.99, 3.97-3.82 (multiplets, 2H); 3.77-3.44 (m, 2H); 3.37-3.19 (m, 4H); 3.15, 3.08 (2s, 2H); 2.98-2.86 (m, 5H); 2.52 (s, 3H); 1.94-1.75, 1.60-1.30 (multiplets, 2H); 1.22 (br s, 4H); 0.86-0.75 (m, 3H).
HRMS calc. for C29H40N4O4; 508.3049. found 508.3040±0.0015.
HPLC tR=8.94 min.
Compound 3
Yield: 33.0 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6): δ 8.54 (d, J=9.4 Hz), 8.43-8.36 (m), and 8.12 (br t, J=5.65 Hz) (1H); 7.90 (d, J=6.6 Hz), 7.79-7.72 (m) (1H); 7.30-7.05 (m, 6H); 6.90-6.76 (m, 3H); 4.60-4.50 (m), 4.43 (d, J=18.3 Hz), 4.26-4.16 (m) (1H); 4.13-4.02 (m, 1H); 4.01-3.84 (m, 2H); 3.74-3.41 (m, 2H); 3.17, 3.09 (2s, 3H); 2.99-2.86 (m, 5H); 2.43-2.18 (m, 1H); 1.97-1.75 (m, 3H); 1.72-1.39 (m, 1H); 0.96 (d, 5.76 Hz, 3H); 0.93-0.77 (m, 2H); 0.68 (d, 5.76 Hz, 3H).
HRMS calc. for C28H38N4O4; 494.2893. found 494.2888±0.0015.
HPLC tR=8.11 min.
Compound 4
Yield: 15.3 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, CD3CN); δ 7.48-7.19 (m, 6H); 7.13-6.98 (m, 3H); 4.71-4.51 (m, 3H); 4.48-4.32 (m, 1H); 4.26-4.01 (m, 1H); 3.79-3.57 (m, 2H); 3.48-3.20 (m, 3H); 3.19-3.06 (m, 5H); 3.01-2.89 (m, 2H); 2.80-2.62 (m, 2H); 2.09-1.96 (m, 3H); 1.94-1.70 (m, 1H); 1.57-1.36 (m, 4H); 1.32-1.26 (m, 1H); 1.08-0.97 (m, 3H).
HRMS calcd for C29H40N4O4; 508.3049. found 508.3045±0.0015
HPLC tR=8.37 min
Compound 6
Yield: 28.2 mg macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6): δ 10.80 (s, 1H); 8.46 (d, J=9.65 Hz), 8.36-8.28 (m), 8.14-8.07 (m), and 8.02 (d, J=9.65 Hz) (1H); 7.73-7.65 (m), 7.59 (d, 8.2 Hz), and 7.51 (d, J=8.2 Hz) (1H); 7.3 (d, J=8.2 Hz, 1H); 7.16-6.91 (m, 5H); 6.89-6.76 (m, 2H); 4.62-4.49 (m) and 4.42-4.24 (m) (1H); 4.15-3.81 (m, 2H); 3.77-3.43 (m, 2H); 3.41-3.19 (m, 6H); 3.22-2.85 (m, 6H); 2.52 (s, 3H); 1.89-1.69 (m, 1H); 1.59-1.02 (m, 4H); 0.88-0.74 (m, 3H).
HRMS calc. for C30H39N5O4; 533.3002. found 533.2990±0.0016.
HPLC tR=8.22 min.
Compound 8
Yield: 74.9 mg pure macrocycle was obtained (CLND quantification) from 600-650 mg starting resin
1H NMR (300 MHz, DMSO-d6): δ 9.47 (br s), 9.07 (s) (1H) and 8.32 (br s) (2H); 7.94 (d, 6.6 Hz, 1H); 7.60-7.42 (m, 2H); 7.38 (d, 9.0 Hz, 1H); 7.28-7.04 (m, 7H); 6.93 (t, 8.1 Hz, 1H); 6.60 (d, J=14.4 Hz) and 6.39-6.27 (m) (1H); 4.51-4.38 (m, 1H); 4.29-4.08 (m, 2H); 3.87-3.63 (m, 2H); 3.40-3.13 (m, 2H); 2.94 (t, J=14.1 Hz, 1H); 2.53-2.50 (m, 1H); 2.32-2.17 (m, 1H); 1.86-1.06 (m, 10H); 0.95-0.79 (m, 6H).
HRMS calc. for C32H42N4O4; 546.3206. found 546.3198±0.0016.
HPLC tR=9.02 min.
Compound 9
Yield: 33.7 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6): δ 8.48 (s, 1H); 7.92 (d, J=5.3 Hz, 1H); 7.81 (d, J=8.5 Hz, 1H); 7.26-7.08 (m, 7H); 6.88-6.75 (m, 2H); 4.30 (br t, J=10.1 Hz, 1H); 4.0 (t, J=8.6 Hz, 1H); 3.87 (br d, J=8.6 Hz, 1H); 3.70-3.58 (m, 1H); 3.4-3.25 (m, 1H); 3.04-2.85 (m, 3H); 2.73 (d, 7.67 Hz, 1H); 2.53 (s, 3H); 2.35-2.09 (m, 2H); 1.92-1.44 (m, 8H); 1.42-1.18 (m, 2H); 0.85, 0.81 (2 doublets, J=6.76 Hz, 6H).
13C NMR (75 MHz, DMSO-d6): δ 176.15; 173.20; 171.27; 157.18; 140.08; 130.72; 130.52; 129.71; 128.64; 127.87; 126.62; 120.88; 111.44; 68.29; 67.10; 66.99; 55.24; 48.42; 41.11; 41.03; 39.36; 36.93; 35.77; 34.65; 32.38; 30.55; 29.96; 23.83; 22.65; 19.87.
HRMS calc. for C31H42N4O4; 534.3206. found 534.2139±0.0016.
HPLC tR=9.29 min.
Compound 10
Yield: 19.2 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6): δ 8.53, 8.41, 8.38 (doublets, J=8.8, 8.5, 8.5 Hz, 1H); 8.16-8.05, 7.87-7.71 (multiplets, 1H); 7.31-7.04 (m, 7H); 6.91-6.75 (m, 2H); 4.60-4.45, 4.39-4.30, 4.28-4.16 (m, 1H), 4.10-4.00, 3.97-3.83 (m, 2H); 3.73-3.46 (m, 2H); 3.22-3.20 (m 1H), 3.16, 3.09 (2s, 3H), 2.45-2.39 (m, 1H); 2.99-2.86 (m, 1H); 2.85-2.58 (m, 5H); 2.48-2.22 (m, 1H); 2.07 (s, 1H), 1.95-1.78 (m, 1H), 1.75-1.42 (m, 1H), 1.42-1.17 (m, 4H), 0.88-0.77 (m, 3H).
HRMS calc. for C28H38N4O4; 494.2893. found 494.2888±0.0015
HPLC tR=8.27 min.
Compound 221
Yield: 50.3 mg macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6): δ 7.86 (d, J=6.7 Hz) and 7.65-7.58 (m) (1H); 7.28-7.06 (m, 7H); 6.88 (d, 8.06 Hz, 1H); 6.81 (t, J=6.7 Hz, 1H); 4.07-3.91 (m, 3H); 3.77-3.65 (m, 1H); 3.56-3.38 (m, 2H); 3.35-3.25 (m, 3H); 3.25-3.07 (m, 2H); 3.04-2.63 (m, 3H); 2.52 (s, 3H); 2.01-1.71 (m, 4H); 1.66-1.49 (m, 2H); 1.47-1.17 (m, 4H); 0.90-0.78 (m, 3H).
13C NMR (75 MHz, DMSO-d6): δ 172.15; 170.81; 170.74; 157.29; 139.62; 130.76; 130.56; 129.56; 128.82; 61.73; 59.29; 56.37; 47.90; 41.11; 41.03; 39.36; 35.81; 35.43; 30.23; 30.03; 29.63; 25.12; 19.15; 14.66.
HRMS calc. for C30H40N4O4; 520.3049. found 520.3041±0.0016.
HPLC tR=8.30 min.
Example 3
Alternative Synthetic Strategies
Alternative synthetic strategies amenable to larger scale synthesis of compounds of the present invention are discussed below.
A. Method LS1 for Representative Large Scale Synthesis of Compounds of the Invention







Step LS1-A: Synthesis of LS1-8





To alcohol Cbz-T33a (2.4 g, 7.0 mmol, 1.0 eq) in CH2Cl2 (50 mL) were added NBS (1.5 g, 8.4 mmol, 1.2 eq) and PPh3 (2.2 g, 8.4 mmol, 1.2 eq). The mixture was stirred at room temperature O/N and a saturated aqueous NCl solution was added. The aqueous phase was extracted with CH2Cl2 (2×) and the combined organic phases were extracted with a saturated aqueous NH4Cl solution to remove succinimide byproduct. The organic phase was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (20% AcOEt, 80% hexanes) to give bromide LS1-8a as a yellow oil (2.6 g, 91%).
TLC (30% AcOEt, 70% hexanes); Rf=0.56; detection: UV and CMA
1H NMR (CDCl3): δ 7.37-7.26 (5H, m, Ph), 7.19-7.13 (2H, m, Ph), 6.90 (1H, t, Ph), 6.83 (1H, d, Ph), 5.10 (2H, s, NHC(O)OCH2Ph), 4.96 (1H, broad, NHCbz), 4.59 (1H, sextuplet, PhOCH(CH3)CH2Br), 3.58-3.47 (2H, m, CH2Br), 3.19 (2H, q, CH2NHCbz), 2.67 (2H, t, PhCH2CH2), 1.78 (2H, quint, PhCH2CH2), 1.44 (3H, d, CHCH3).
LC/MS (Grad_A4): tR=11.15 min
Step LS1-B1: Synthesis of LS1-10




The hydrochloride salt of H-Nva-OMe was dissolved in an aqueous solution of Na2CO3 (1 M) and saturated with NaCl to ensure extraction of all of the free amine. The aqueous solution was extracted with AcOEt (3×). The combined organic phases were extracted with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The free amine, H-Nva-OMe, was recovered in 90% yield. It is important to perform the alkylation with the free amine (H-Nva-OMe) to eliminate chloride formation (OTs to Cl) as a side reaction. In a dried round-bottomed flask, bromide LS1-8a (740 mg, 1.83 mmol, 1.0 eq) and H-Nva-OMe (479 mg, 3.60 mmol, 2.0 eq) were added. Degassed (by stirring under vacuum for 30 min) DMF (3.7 mL), anhydrous Na2CO3 (232 mg, 2.19 mmol, 1.2 eq) and KI (61 mg, 0.37 mmol, 0.2 eq) were added and the mixture stirred at 110° C. O/N. Water was added and the aqueous phase was extracted with Et2O (3×). The combined organic phases were extracted with water (2×), then brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (30% AcOEt: 70% hexanes) to give secondary amine LS1-10 as a yellow oil (709 mg, 85%).
TLC (30% AcOEt, 70% hexanes); Rf=0.32; detection: UV and CMA,
1H NMR (CDCl3): δ 7.35-7.29 (5H, m, Ph), 7.17-7.12 (2H, m, Ph), 6.91-6.84 (2H, m, Ph), 5.51 (1H, broad, CH2NHCHRR′), 5.09 (2H, s, OCH2Ph), 4.67-4.51 (1H, m, PhOCH(CH3)R), 3.65 (3H, s, C(O)OCH3), 3.24-3.10 (3H, m, NHCH(Pr)CO2Me and CH2NHCbz), 2.87-2.41 (4H, m, PhCH2CH2 and NHCH2CH(Me)OPh), 1.86-1.76 (2H, m, PhCH2CH2), 1.70-1.63 (2H, m, CH3CH2CH2), 1.36-1.28 (2H, m, CH3CH2CH2), 1.23 (3H, d, CHCH3), 0.90 (3H, t, CH3CH2CH2).
13C NMR (CDCl3): δ 176.44, 156.88, 155.58, 137.14, 131.16, 130.57, 128.68, 128.34, 128.21, 127.33, 120.79, 112.62, 73.16, 66.62, 61.30, 54.21, 51.95, 40.86, 36.02, 30.60, 27.88, 19.20, 17.80, 14.07.
LC/MS (Grad_A4): tR=6.76 min
Step LS1-B2: Alternative Synthesis of LS1-10
To a solution of alcohol Cbz-T33a (8.5 g, 24.7 mmol, 1.0 eq) in CH2Cl2 (125 mL) were added Et3N (10.4 mL, 74.1 mmol, 3.0 eq), TsCl (5.2 g, 27.2 mmol, 1.1 eq) and DMAP (302 mg, 2.47 mmol, 0.1 eq). The mixture was stirred O/N at room temperature and then an aqueous solution of saturated NH4Cl was added. The aqueous phase was extracted with CH2Cl2 (2×) and the combined organic phases were dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (30% AcOEt, 70% hexanes) to give tosylate LS1-8b as an oil (9.4 g, 90%).
TLC (50% AcOEt, 50% hexanes); Rf=0.47; detection: UV and CMA
1H NMR (CDCl3): δ 7.74 (2H, d, Ph), 7.36-7.26 (7H, m, Ph), 7.14-7.08 (2H, m, Ph), 6.88 (1H, t, Ph), 6.74 (1H, d, Ph), 5.10 (2H, s, NHC(O)OCH2Ph), 4.97 (1H, broad, NHCbz), 4.61-4.55 (1H, m, PhOCH(CH3)CH2OTs), 4.19-4.05 (2H, m, CH2OTs), 3.15 (2H, q, CH2NHCbz), 2.56 (2H, td, PhCH2CH2), 2.42 (3H, s, PhCH3) 1.74 (2H, quint, PhCH2CH2), 1.27 (3H, d, CHCH3)
13C NMR (CDCl3): δ 156.67, 155.05, 145.20, 137.04, 133.02, 131.16, 130.65, 130.11, 128.72, 128.28, 128.23, 128.10, 127.39, 121.50, 112.87, 71.99, 71.42, 66.68, 40.79, 30.32, 27.57, 21.87, 16.74.
LC-MS (Grad_A4): tR=11.02 min
Application of the procedure in Step LS1-BI, substituting the tosylate LS1-8b as alkylating agent gave 73% yield of LS1-10 with 2 eq of H-Nva-OMe.
Step LS1-C1: Synthesis of LS1-7




To a solution of amine LS1-10 (697 mg, 1.53 mmol, 1.0 eq) in THF/H2O (1:1, 15 mL) at 0° C. were added Na2CO3 (244 mg, 1.68 mmol, 1.5 eq) and (Boc)2O (366 mg, 1.68 mmol, 1.1 eq), then the mixture stirred at room temperature for 36-48 h. THF was evaporated under reduced pressure and the aqueous phase was extracted with Et2O (3×). The combined organic phases were extracted with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The Boc compound was obtained as a yellow oil and used without further purification for the next reaction.
TLC (30% AcOEt, 70% hexane): Rf=0.49; detection: UV and CMA
To a solution of the crude Boc compound in THF/H2O (1:1, 15 mL) was added LiOH (309 mg, 7.35 mmol, 5.0 eq) and the mixture stirred O/N at rt. THF was evaporated under reduced pressure and the remaining aqueous basic phase was then acidified with 1 M HCl to pH 3 (pH paper). The aqueous phase was extracted with AcOEt and the combined organic phases were extracted with water and brine. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. Carboxylic acid LS1-7 was obtained as a yellow oil (687 mg, 83%, 2 steps).
TLC (50% AcOEt, 50% hexane); Rf=0.32; detection: UV and CMA
13C NMR (CDCl3): δ 176.11, 156.81, 155.51, 155.18, 136.93, 131.13, 130.37, 128.72, 128.31, 127.44, 121.20, 113.70, 81.36, 73.40, 66.79, 61.99, 40.80, 32.83, 31.56, 30.33, 28.48, 27.48, 20.10, 17.53, 14.11.
LC/MS (Grad_A4): tR=12.50 min
Step LS1-C2: Divergent Synthetic Route (No Amine Protection)




The H-Nva-OtBu.HCl was dissolved in an aqueous solution of Na2CO3 (1 M) and saturated with NaCl to ensure extraction of all of the free amine. This aqueous solution was extracted with AcOEt (3×). The combined organic phases were extracted with brine, dried over MgSO4, filtered and concentrated under reduced pressure. About 90% of the free amine, H-Nva-OtBu, was recovered. It is important to perform the alkylation with the free amine (H-Nva-OtBu) to eliminate chloride side product formation (OTs-> Cl).
In a dried round-bottomed flask, tosylate LS1-8b (1.0 g, 2.01 mmol, 1.0 eq) and H-Nva-OtBu (752 mg, 4.02 mmol, 2.0 eq) were added. Degassed (by stirring under vacuum for 30 min) DMF (4 mL) and anhydrous Na2CO3 (256 mg, 2.41 mmol, 1.2 eq, note that other bases were less effective) were added and the mixture stirred at 110° C. O/N. Water was added and the aqueous phase extracted with Et2O (3×). The combined organic phases were extracted with water (2×) and brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (30% AcOEt: 70% hexanes) to give the amine, LS1-12, as a yellow oil (683 mg, 75%). This crude secondary amine (1.0 eq) was dissolved in 4 M HCl/dioxane (10 eq) and the mixture stirred O/N at room temperature. The solvent was evaporated under reduced pressure and Et2O added to the residue. A white precipitate was formed upon addition of heaxnes to this mixture. The precipitate was filtered and rinsed with cold hexanes to give the desired amino acid, LS1-13, as a white solid.
TLC (50% AcOEt, 50% hexane); Rf=0.71; detection: UV and CMA
LS1-13, despite the presence of the free amine, has been used in the remaining part of the synthetic scheme to successfully access the desired macrocycle.
Step LS1-D: Synthesis of Dipeptide LS1-6




The tosylate salt of H-(D)Phe-OBn was dissolved in an aqueous solution of 1 M Na2CO3 and the aqueous solution extracted with AcOEt (3×). The combined organic phases were extracted with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The free amine H-(D)Phe-OBn was recovered in 90% yield. To a solution of H-(D)Phe-OBn (3.0 g, 11.76 mmol, 1.0 eq) in THF/CH2Cl2 1/1 (60 mL) were added Boc-(D)NMeAla-OH (2.5 g, 12.35 mmol, 1.05 eq), 6-Cl HOBt (2.0 g, 11.76 mmol, 1.0 eq) and DIPEA (10.2 mL, 58.8 mmol, 5.0 eq). The mixture was cooled to 0° C. and EDCI (2.48 g, 12.94=mol, 1.1 eq) was added. The mixture was stirred 1 h at 0° C. and at room temperature O/N. Solvent was evaporated under reduced pressure and the residue dissolved in AcOEt. The organic phase was washed sequentially with an aqueous 1 M solution of citrate buffer (pH 3.5, 2×), an aqueous solution of saturated NaHCO3 (2×) and brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The dipeptide was obtained as a yellow oil and used as obtained for the next step (5.3 g, 100%). The dipeptide was dissolved in a solution of HCl/dioxane (4 M, 30 mL, 10 eq), 50 mL of dioxane were then added to facilitate the agitation and the mixture stirred for 1 h at room temperature; a heterogeneous solution was obtained. The mixture was concentrated under reduced pressure and dried further on mechanical vacuum pump. The dipeptide hydrochloride salt LS1-6 was obtained as pale yellow solid (4.4 g, 100%).
1H NMR (DMSO-d6): δ 9.40-8.70 (3H, d and 2 broads, C(O)NH and CH3NH2+Cl−), 7.39-7.17 (10H, m, Ph), 5.11 (2H, s, C(O)OCH2Ph), 4.69-4.61 (1H, m, CHCH3), 3.69 (1H, dd, CHCH2Ph), 3.31 (3H, s, CH3NH2+Cl−), 3.17-3.11 and 2.97-2.90 (CHCH2Ph), 1.28 (3H, d, CHCH3)
13C NMR (DMSO-d6): δ 171.33, 169.18, 137.63, 136.31, 129.92, 129.11, 128.95, 128.83, 128.63, 127.30, 67.00, 56.57, 54.38, 36.98, 31.11, 16.47.
LC/MS (Grad_A4): tR=6.17 min
Step LS1-E: Synthesis of Amino Acid LS1-5




To a solution of acid LS1-7 (1.45 g, 2.67 mmol, 1.05 eq) in THF/CH2Cl2 1/1 (13 mL) at 0° C. were added hydrochloride salt LS1-6 (958 mg, 2.55 mmol, 1.0 eq), DIPEA (2.2 mL, 12.8 mmol, 5.0 eq) and HATU (1.07 g, 2.81 mmol, 1.1 eq). The mixture was stirred at room temperature O/N. Solvent was evaporated and the residue was dissolved in AcOEt. The organic phase was washed sequentially with an aqueous solution of 1 M citrate buffer (pH=3.5, 2×), aqueous solution of saturated NaHCO3 (2×), then with brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (gradient: 20% AcOEt, 80% hexanes to 30% AcOEt, 70% hexanes) to give the desired fully protected tripeptide as a pale yellow gummy foam (1.6 g, 73%).
TLC (50% AcOEt, 50% hexanes): Rf=0.78; detection: UV and CMA
LC/MS (Grad_A4): tR=15.15 min
To a solution of the protected, alkylated tripeptide (1.5 g, 1.75 mmol, 1.0 eq) in AcOEt (23 μL) was added 10% Pd/C (20% by weight, 315 mg) and then hydrogen was bubbled through the solution. The mixture was stirred O/N under a hydrogen atmosphere. Nitrogen was bubbled through the reaction, then the mixture filtered on a Celite pad and rinsed with AcOEt. The combined filtrate was evaporated under reduced pressure to give LS1-5 as a white solid (1.1 g, quantitative).
TLC (50% AcOEt, 50% hexanes): Rf=0.52; detection: UV and CMA
LCMS (Grad_A4): tR=8.23 min
Step LS1-F: Macrocyclization and Final Deprotection




To a solution of cyclization precursor LS1-5 (50 mg, 0.08 mmol, 1.0 eq) in THF (3.2 mL, for a concentration of 25 mM) was added DIPEA (68 μL, 0.39 mmol, 5.0 eq) and DEPBT (28 mg, 0.094 mmol, 1.2 eq) and the mixture stirred at room temperature O/N. Solvent was evaporated under reduced pressure and the residue purified by flash chromatography (1% MeOH, 99% CH2Cl2) to give Boc-protected macrocycle LS1-11 as a white solid (40 mg, 0.064 mmol, 80%). On a 1 g scale of precursor LS1-5 at a reaction concentration of 25 mM, the yield was 73%.
TLC (5:95 MeOH:DCM): Rf=0.43; detection: UV and CMA
1H NMR (DMSO-d6 60° C.): δ 7.62 (1H, d, NH), 7.47 (1H, broad, NH), 7.27-7.08 (7H, m, Ph), 6.85-6.79 (2H, m, Ph), 4.78 (1H, broad), 4.51-4.38 (1H, m), 4.11-4.02 (2H, m), 3.62-3.56 (1H, m), 3.32-3.04 (5H, m), 2.92 (3H, s, N—CH3), 2.72-2.46 (2H, m), 1.90-1.59 (4H, m), 1.46 (9H, s, C(CH3)3), 1.28-1.06 (8H, m), 0.65 (3H, t, CH2CH3).
13C NMR (DMSO-d6): δ 172.03, 171.07, 155.83, 155.60, 139.69, 131.82, 130.82, 129.69, 128.73, 127.73, 126.75, 121.06, 113.40, 80.66, 74.75, 57.22, 56.66, 50.49, 35.88, 33.72, 32.71, 30.41, 28.68, 19.35, 18.44, 14.95, 14.19.
LC-MS (Grad_A4): tR=12.82 min
Macrocycle LS1-11 (565 mg, 0.91 mmol, 1.0 eq) was dissolved in a solution of 4 M HCl/dioxane (4.6 mL, 20 eq) and the mixture stirred 2 h at room temperature. The mixture was concentrated under reduced pressure and placed under vacuum (oil pump) to give final macrocycle Compound 410 as a white solid (508 mg, 100%).

Chiral HPLC indicated no racemization when compared to its (L)-antipode at position AA3.

1H NMR (DMSO-d6, 60° C.): δ 9.38 (1H, broad), 8.28 (1H, d), 8.13 (1H, broad), 7.81 (1H, t), 7.28-7.13 (7H, m, Ph), 6.93-6.87 (2H, m, Ph), 4.84-4.77 (1H, m), 4.54-4.40 (3H, m), 3.35-3.07 (6H, m), 2.94 (3H, s, N—CH3), 2.90-2.81 and 2.64-2.47 (2H, m), 1.85-1.64 (4H, m), 1.38-1.21 (5H, m), 1.10 (3H, d, CH3), 0.88 (3H, t, CH2CH3).
13C NMR (CDCl3): δ 171.92, 171.46, 170.44, 155.11, 139.07, 131.68, 130.47, 129.87, 128.67, 127.54, 126.90, 121.50, 112.94, 69.83, 67.03, 58.14, 56.33, 55.61, 55.29, 53.88, 50.48, 37.29, 32.29, 31.08, 29.70, 28.58, 18.15, 17.89, 15.20, 14.55.
LC-MS (Grad_A4): tR=6.23 min
LC chiral (Grad35A-05): tR=26.49 min
LC chiral (Grad40A-05): tR=26.54 min
B. Method LS2 for Representative Large Scale Synthesis of Compounds of the Invention




Step LS2-A: Synthesis of Dipeptide LS2-21





A stirred suspension of H-(D)Phe-OtBu.HCl (5 g, 0.02 mol, 1 eq) and Z-(D)NMeAla-OH (4.98 g, 0.021 mol, 1.05 eq) in 130 mL of anhydrous THF-DCM (1:1) at room temperature was treated with DIPEA (17.50 mL, 0.1 mol, 5 eq) and 6-Cl-HOBt (3.40 g, 0.02 mol, 1 eq). The mixture was stirred vigorously at room temperature for several minutes, cooled with an ice bath, then EDCI (4.20 g, 0.022 mol, 1.1 eq) was added and the mixture stirred for 1 h. After this period of time, the ice bath was removed and the reaction was stirred at room temperature O/N. The solvent was removed under reduced pressure and the residue dissolved in 100 mL of AcOEt and washed with citrate buffer solution (1 N, 2×100 mL), saturated NaHCO3 solution (2×100 mL) and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure to give 9.25 g (100%) of a colorless oil, LS2-24.
TLC (hexanes/ethyl acetate, 1:1): Rf 0.3; detection: CMA and UV
1H NMR (CDCl3): δ 1.25 (m, 2H), 1.40 (s, 9H), 2.66 (s. 3H), 2.85 (dd, 1H), 3.15 (dd, 1H), 4.70 (q, 2H), 5.15 (s, 2H), 6.50 (sb, 1H), 7.15 (m, 2H), 7.20 (m, 3H), 7.35 (m, 5H).
13C NMR (CDCl3): δ 28.18, 38.23, 53.61, 53.61, 67.87, 127.12, 128.40, 128.19, 128.40, 128.61, 128.8, 129.53, 170.01.
LC/MS (Grad_A4); tR=9.73 min; Mass found: 440
Dipeptide LS2-24 (6.9 g, 0.015 mol) was dissolved in AcOEt (100 mL), then purged with nitrogen for 10 min. 10% Pd—C (690 mg) was added and the mixture purged with a balloon full of hydrogen gas. The mixture was then hydrogenated under atmospheric pressure using a H2 balloon. After 12 h, the reaction mixture was filtered through a short pad of Celite, and the filter cake washed with AcOEt. The combined filtrate and washings were concentrated under reduced pressure to afford practically pure (clean NMR), colorless, solid compound LS2-21 (4.30 g, 90%) which was used directly in the next step without further purification.
TLC (100% AcOEt): Rf=0.1; detection: CMA and UV.
1H NMR (CDCl3): δ 1.20 (d J=7.03 Hz, 3H) (s, 9H), 2.40 (s, 1H), 3.01-3.20 (m, 3H), 4.80 (q, 1H), 7.20 (m, 5H), 7.60 (m, 1H).
13C NMR (CDCl3): δ 19.64, 28.18, 35.12, 38.46, 53.06, 60.42, 82.29, 127.05, 128.50, 129.71, 136.61, 170.85, 174.28.
LC-MS (Grad_A4): tR=5.86 min; Mass found: 306
Step LS2-B: Synthesis of Tripeptide LS2-22




A stirred suspension of dipeptide LS2-21 (2 g, 6.50 mmol, 1 eq) and Bts-Nva-OH (LS2-28, 2.15 g, 6.85 mmol, 1.05 eq) in 32 mL of anhydrous DCM at 0° C. was treated with DIPEA (4.50 mL, 0.026 mol, 4 eq) and HATU (2.72 g, 7.18 mmol, 1.1 eq). The mixture was stirred vigorously at 0° C. for 1 h. After this period of time, the ice bath was removed and the reaction stirred at room temperature O/N. The solvent was removed in vacuo and the residue dissolved in 30 mL of AcOEt. The organic phase was sequentially washed with 1 N citrate buffer solution (2×30 mL), saturated NaHCO3 solution (2×30 mL) and brine (1×30 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography [ethyl acetate/hexanes (1/1)] to afford LS2-22 as a colorless solid (3.13 g, 80%).
TLC (hexanes/ethyl acetate, 3:2): Rf=0.3; detection: CMA and UV
1H NMR (CDCl3): δ0.95 (m, 3H), 1.20 (d, 2H), 1.40 (s, 9H), 1.42-1.70 (m, 4H), 2.60 (m, 2H), 2.90 (s, 3H), 4.40 (m, 1H), 4.80 (m, 1H), 4.92 (m, 1H), 6.10 (m, 1H), 6.30 (M, 1H), 6.40 (m, 1H), 6.90 (m, 2H), 7.20 (m, 3H), 7.40-7.60 (m, 2H), 7.90 (m, 1H), 8.10 (m, 1H).
13C NMR (CDCl3): δ 23.42, 26.32, 33.12, 48.63, 49.10, 49.85, 77.56, 117.63, 120.67, 122.35, 122.93, 123.11, 123.80, 124.13, 124.68, 124.75, 131.45, 147.67, 165.16, 165.68, 167.66.
LC-MS (Grad_A4): tR=11.48 min; Mass found: 602
Step LS2-C: Synthesis of LS2-23




A stirred suspension of tripeptide LS2-22 (0.4 g, 0.66 mmol) and tether bromide LS2-9 (0.5 g, 1.32 mmol, synthesized as in Step LS1-A for the corresponding Cbz derivative) in 1.33 mL of anhydrous DMF at room temperature was treated with KI (0.12 g, 0.66 mmol) and K2CO3 (0.185 g, 1.32 mmol). The mixture was stirred vigorously at 80° C. for 24 hours. After this period of time, this mixture was cooled to room temperature, then 20 ml of water was added and the product extracted with Et2O (3×30 mL). The combined organic layer was washed with brine (2×30 mL), dried over magnesium sulfate and concentrated under vacuum. The residue was purified by flash chromatography [hexanes/ethyl acetate (1:2)] to afford LS2-25 as a white solid (70%).
TLC (hexanes/ethyl acetate, 2:1): Rf=0.4; detection: CMA and UV
1H NMR (DMSO-d6): δ0.5 (m, 1H), 0.70 (m, 1H), 1.01-1.40 (m,) 1.60 (m, 3H), 1.80 (m, 1H), 2.55 (m,), 2.95 (m, 4H), 3.1 (m, 2), 3.30 (m, 2H), 3.60 (m, 1H), 3.90 (m, 1H), 4.30 (m, 1H), 4.80 (m,), 6.80 (m, 3H), 7.05 (m, 6H), 7.60 (2H), 7.95 (m, 1H), 8.20 (m, 1H), 8.25 (m, 1H), 8.90 (s, 2H).
13C NMR (CDCl3): δ 13.84, 15.36, 17.40, 17.70, 19.40, 22.17, 27.52, 28.14, 28.67, 30.29, 31.27, 33.27, 38.01, 40.35, 51.02, 53.08, 54.35, 56.72, 70.25, 73.13, 81.10, 113.49, 120.94, 122.28, 125.44, 127.01, 127.19, 127.19, 127.68, 127.68, 127.79, 128.64, 129.57, 130.06, 136.2, 137.10, 165.10, 170.10, 171.10.
LC-MS (Grad_A4): tR=15.10 min; Mass found: 892
100 mg of alkylated tripeptide LS2-25 (100 mg, 0.11 μmol) was treated with 2 mL of 50% TFA, 3% triethylsilane (TES) in DCM, then the mixture stirred for 1 h at room temperature. After this period of time, all solvents were removed under reduced pressure. The crude compound LS2-23 was dried using vacuum pump for 1 h and used directly in the next step without further purification.
LC/MS (Grad_A4): tR=8.55 min; Mass found: 737
Step LS2-D: Synthesis of LS2-26 (Macrolactamization)




To a stirred suspension of alkylated-tripeptide 23 (0.12 mmol) and DIPEA (0.100 mL, 0.56 mmol) in 11.22 mL of anhydrous THF at room temperature was added DEPBT (41 mg, 0.14 mmol). The mixture was stirred vigorously at room temperature O/N. The reaction was then concentrated to dryness under reduced pressure and the residue dissolved in 10 mL of AcOEt. The organic solution was sequentially washed with citrate buffer solution (1 N, 2×30 mL), saturated NaHCO3 (2×30 mL) and brine (1×30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography using [ethyl acetate/hexanes (3:1)] to afford LS2-26 (Bts-410) as a white solid (80 mg, 98%).
TLC (ethyl acetate/hexanes, 3:1): Rf=0.3; detection: CMA and UV
1H NMR (CDCl3): δ 0.64 (m, 3H), 0.87 (m, 1H), 1.02 (m, 2H), 1.20 (m, 6H), 1.40 (m, 3H), 1.60 (m, 4H), 1.80 (m, 1H), 2.01 (m, 1H), 2.40 (m, 1H), 2.80 (m, 1H), 3.15 (s, 3H), 3.20 (m, 2H), 3.45 (m, 1H), 3.60-3.80 (m, 2H), 4.40-4.60 (dd, 2H), 4.70 (m, 2H), 5.01 (m, 1H), 5.90 (m, 1H), 6.80 (m, 2H), 6.90 (m, 1H), 7.15-7.25 (m, 7H), 7.60 (m, 2H), 8.01 (m, 1H), 8.10 (m, 1H).
13C NMR (CDCl3): δ 13.28, 13.55, 18.75, 18.98, 28.89, 29.92, 29.92, 33.19, 36.81, 36.98, 39.55, 51.94, 53.83, 55.25, 59.51, 74.64, 111.66, 120.64, 122.51, 125.15, 127.10, 127.37, 127.84, 128.07, 128.86, 129.47, 130.51, 136.55, 137.30, 152.58, 155.86, 165.33, 169.75, 170.09, 171.66.
LC/MS (Grad_A4): tR=13.17 min; Mass found: 719
LC Chiral (column ODRH, Grad 55A-05): tR=42.059.
Step LS2-E: Synthesis of Compound 410




To a stirred suspension of macrocycle LS2-26 (40 mg, 0.003 mmol) in 0.110 mL of DMF was added 23 mg of K2CO3 and 10 μl of mercaptopropanoic acid at room temperature, then the reaction left O/N. The reaction was concentrated to dryness under reduced pressure and the crude residue dissolved in 10 mL of AcOEt. The organic solution was washed with a saturated solution of NaHCO3 (2×30 mL), ten brine (1×30 mL). The organic layer was dried over anhydrous sodium, sulfate, filtered and evaporated to dryness under reduced pressure. Compound 410 was thus isolated in 90% yield.
TLC (100% AcOEt): Rf=0.2; detection: CMA and UV
1H NMR (DMSO-d6): δ 0.79 (m, 3H), 1.20 (m, 9H), 1.30 (M, 1H), 1.60 (m, 1H), 1.90 (m, 1H), 2.10 (Sb, 1H), 2.35 (ddd, J=4.98, 4.95, 4.69 Hz, 1H), 2.56 (Sb, 1H), 2.63 (m, 1H), 2.80 (ddd, J=4.99, 4.69, 4.40 Hz, 1H), 3.01-3.15 (m, 5H), 3.25 (dd, J=4.69, 4.11 Hz, 1H), 3.30 (s, 2H), 3.55 (sb, 1H), 3.95 (q, J=7.33, 7.04 Hz, 1H), 4.50 (sb, 1H), 6.80 (m, 1H), 6.90 (m, 1H), 7.10-7.30 (m, 7H), 7.70 (m, 2H).
13C NMR (DMSO-d6): δ 14.60, 14.84, 18.46, 18.85, 29.80, 29.96, 34.03, 35.84, 36.31, 40.68, 54.79, 55.67, 57.77, 58.11, 73.42, 112.26, 120.58, 126.84, 127.81, 128.80, 129.73, 131.10, 140.10, 158.10, 172.10, 172.40, 176.10.
LC/MS (Grad_A4): tR=6.19 min; Mass found: 522
Example 4
Synthesis and Biological Results for Representative Compound 298
A. Solution Synthesis of Compound 298










Step LS3-1. Synthesis of cyclopropylglycine methyl ester hydrochloride salt. To a suspension of H-Cpg-OH (LS3-A, 20.0 g, 174 mmol, 1.0 eq) in anhydrous MeOH (350 mL) at 0° C. was slowly added freshly distilled (from PCl5) acetyl chloride (185 mL, 2.6 mol, 15 eq) over 45 min. The mixture was allowed to warm to room temperature and stirred 16-18 h. The reaction was monitored by TLC [MeOH/NH4OH/AcOEt (10:2:88); detection: ninhydrin; Rf=0.50]. The mixture was then concentrated under vacuum, azeotroped with toluene (3×) and dried under high vacuum 16-18 h to give LS3-1 as a pale yellow solid (30.0 g, >100% crude yield).

1H NMR (CD3OD): δ 4.88 (3H, s, NH3+), 3.85 (3H, s, CH3O), 3.36-3.33 (1H, d, NH3+CHCH3O), 1.19-1.10 (1H, m, CH(CH2)2), 0.83-0.53 (4H, m, CH(CH2)2).
Step LS3-2. Synthesis of tether bromide. To crude alcohol Cbz-T33a (21.5 g, 62.6 mmol, 1.0 eq) in anhydrous CH2Cl2 (250 mL) were added NBS (12.8 g, 72.0 mmol, 1.15 eq, larger amounts of NBS lead to dibrominated side product) and PPh3 (18.9 g, 72.0 mmol, 1.15 eq). The round bottom flask was protected from light with foil and the mixture stirred at room temperature 16-18 h with monitoring by TLC [AcOEt/Hexanes (3:7); detection: UV and CMA; Rf=0.42]. A saturated aqueous NCl solution (200 mL) was added and the aqueous phase extracted with CH2Cl2 (2×150 mL). The combined organic phases were washed with a saturated aqueous NH4Cl solution (2×200 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (AcOEt:hexanes, gradient, 5:95 to 15:85) to give bromide LS3-2 as a slightly yellow oil (22.2 g, 88.4%).
1H NMR (CDCl3): δ 7.37-7.26 (5H, m, Ph), 7.19-7.13 (2H, m, Ph), 6.92-6.88 (1H, t, Ph), 6.84-6.81 (1H, d, Ph), 5.10 (2H, s, NHC(O)OCH2Ph), 4.96 (1H, broad, NHCbz), 4.62-4.56 (1H, sextuplet, PhOCH(CH3)CH2Br), 3.58-3.45 (2H, m, CH2Br), 3.22-3.16 (2H, q, CHNHCbz), 2.69-2.64 (2H, t, PhCH2CH2), 1.83-1.78 (2H, quint, PhCH2CH2), 1.45 (3H, d, CHCH3).
13C NMR (CDCl3): δ 156.66, 155.08, 136.99, 131.28, 130.77, 128.75, 128.32, 128.28, 127.49, 121.56, 113.03, 73.12, 66.76, 40.69, 36.12, 30.45, 27.48, 19.00.
LC/MS (Grad_A4): tR=11.04 min
Step LS3-3. The hydrochloride salt LS3-1 was dissolved in an aqueous solution of Na2CO3 (1 M, 275 mL, 0.272 mol, 1.5 eq). The basic aqueous phase was saturated with NaCl and extracted with AcOEt/CH2Cl2 (2:1) (5×100 mL). TLC [MeOH/NH4OH/AcOEt (10:2:88); detection: ninhydrin; Rf=0.50]. The combined organic phases were dried over MgSO4, filtered and concentrated under low vacuum at room temperature to give free amino-ester LS3-3 as a yellow oil (19.1 g, 85%, 2 steps). LS3-3 is volatile and should not be left on a mechanical vacuum pump for extended periods of time. To minimize diketopiperazine formation, Step LS3-4 should occur immediately after isolation of LS3-3.
1H NMR (CDCl3): δ 3.70 (3H, s, CH3O), 2.88-2.85 (1H, d, NH2CHCH3O), 1.54 (1H, s, NH2), 1.04-0.97 (1H, m, CH(CH2)2), 0.56-0.27 (4H, m, CH(CH)2).
Step LS3-4. In a dried round-bottom flask, bromide LS3-2 (47.2 g, 117 mmol, 1.0 eq) and freshly prepared LS3-3 (19.1 g, 148 mmol, 1.2 eq) were added. Degassed anhydrous DMF (117 mL), anhydrous Na2CO3 (14.8 g, 140 mmol, 1.2 eq) and KI (19.4 g, 117 mmol, 1.0 eq) were added and the mixture was stirred at 100° C. under a nitrogen atmosphere for 16-18 h. Reaction progress was monitored by LC-MS and/or TLC. The mixture was cooled down to room temperature and water (200 mL) added and the aqueous phase extracted with MTBE (3×100 mL). The combined organic phases were washed sequentially with water (2×100 mL) and brine (1×100 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography [hexanes/AcOEt/DCM, gradient (85:10:5) to (50:45:5)] to give LS3-4 as an orange oil (43.1 g, 81%).
TLC [hexanes/AcOEt (1:1)]: Rf=0.35; detection: UV and CMA
1H NMR (CDCl3): δ 7.31-7.22 (5H, m, Ph), 7.07-7.03 (2H, m, Ph), 6.80-6.74 (2H, m, Ph), 5.48 (1H, broad, CH2NHCHRR′), 5.00 (2H, s, OCH2Ph), 4.49-4.43 (1H, m, PhOCH(CH3)R), 3.56 (3H, s, C(O)OCH3), 3.18-3.11 (3H, m, NHCH(Pr)CO2Me and CH2NHCbz), 2.75-2.50 (4H, m, PhCH2CH2 and NHCH2CH(Me)OPh), 1.76-1.68 (2H, m, PhCH2CH2), 1.19-1.14 (3H, d, PhOCH(CH3)R), 0.88-0.80 (1H, m, CH(CH2)2), 0.46-0.13 (4H, m, CH(CH2)2).
LC/MS (Grad_A4): tR=6.63 min
Step LS3-5. To a solution of secondary amine LS3-4 (43.0 g, 94.7 mmol, 1.0 eq) in THF/H2O (1:1, 475 mL) at 0° C. were added Na2CO3 (15.1 g, 113.7 mmol, 1.5 eq) and (Boc)2O (24.8 g, 142.1 mmol, 1.2 eq). The mixture was allowed to warm to room temperature and stirred 24 h. Reaction was monitored by LC/MS and/or TLC. THF was evaporated under vacuum and the residual aqueous phase was extracted with MTBE (3×100 mL). The combined organic phases were washed with brine (1×100 mL), dried over MgSO4, filtered and evaporated under vacuum to give the crude LS3-5 as an orange oil (59.1 g, >100% crude yield).
TLC [hexanes/AcOEt (1:1)]: Rf 0.57; detection: UV and CMA
LC/MS (Grad_A4): 12.98 min.
Step LS3-6. To a solution of LS3-5 (52.5 g, 94.7 mmol, 1.0 eq.) in THF/H2O (1:1, 475 mL) at room temperature was added LiOH monohydrate (19.9 g, 474 mmol, 5.0 eq.). The mixture was stirred 16-18 h at room temperature. The reaction was monitored by LC/MS (Grad_A4): tR=12.21 min. TLC [Hexanes/AcOEt (1:1); detection: UV and CMA; Rf baseline]. The reaction mixture was acidified with citrate buffer (1M, pH 3.5) and THF was then evaporated under vacuum. The residual aqueous phase was extracted with AcOEt (3×150 mL), then the combined organic phases washed with brine (1×100 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give carboxylic acid LS3-6 as a white gummy solid (47.3 g, 93% for 2 steps).
LC/MS (Grad_A4): tR=12.16 min
Step LS3-7. To a suspension of H-(D)Phe(4F)—OH (LS3-B, 55.6 g, 0.30 mol, 1.0 eq) in benzene (1.2 L) was added p-TSA (69.4 g, 0.37 mol, 1.2 eq) and benzyl alcohol (157 mL, 1.52 mol, 5.0 eq). The mixture was stirred at reflux 16-18 h in a Dean-Stark apparatus during which a homogeneous solution was obtained. The mixture was cooled down to room temperature and a white precipitate formed. The precipitate was diluted with Et2O (500 mL), filtered and triturated with Et2O (3×500 mL). The solid was dried under vacuum to give LS3-7 as a white solid (126 g, 93.1%). Substitution of toluene for benzene resulted in reduced reaction time, 2-3 h.
1H NMR (DMSO-d6): δ 8.40 (3H, bs, NH3Cl), 7.47-7.36 (2H, d, Ph), 7.37-7.06 (11H, m, Ph), 5.15 (2H, s, OCH2Ph), 4.37 (1H, bt, CHCH2Ph), 3.09-3.05 (2H, m, CHCH2Ph), 2.27 (3H, s, CH3Ph).
13C NMR (DMSO-d6): δ 169.52 163.83, 160.62, 140.01, 138.56, 135.48, 132.16, 132.04, 131.33, 131.28, 129.09, 129.05, 128.84, 128.72, 127.09, 126.20, 116.18, 115.89, 67.83, 53.88, 35.83, 21.47.
LC/MS (Grad_A4): tR=6.12 min
Melting point (uncorrected): 165-167° C.
Step LS3-8._The tosylate salt LS3-7 (122 g) was taken up in an aqueous solution of Na2CO3 (1 M, 500 mL). The resulting basic aqueous solution was extracted with AcOEt (4×500 mL) and the combined organic phases were washed with brine (1×250 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give the amino-ester LS3-8 as a white solid (74.4 g, 99%).
1H NMR (CDCl3): δ 7.38-7.28 (5H, m, OCH2Ph), 7.10-7.06 (2H, m, Ph(4F)), 6.96-6.90 (2H, m, Ph(4F)), 5.13 (2H, d, OCH2Ph), 3.76-3.71 (1H, t, CHCH2Ph), (2H, dq, CHCH2Ph), 1.53 (2H, s, NH2)
Step LS3-9. To a solution of LS3-8 (74.4 g, 0.27 mol, 1.0 eq) in anhydrous THF/CH2Cl2 (1:1, 1120 mL) were added Boc-(D)NMeAla-OH (LS3-C, 57.1 g, 0.28 mol, 1.03 eq), 6-Cl-HOBt (46.2 g, 0.27 mol, 1.0 eq) and DIPEA (238 mL, 1.37 mol, 5.0 eq). The mixture was cooled to 0° C. and EDCI (57.6 g, 0.3 mol, 1.1 eq) was added. The mixture was stirred 1 h at 4° C., allowed to warm to room temperature and stirred 18 h. The solvent was evaporated in vacuo and the residue dissolved in AcOEt (1000 mL). The organic phase was washed sequentially with an aqueous solution of citrate buffer (1 M, pH 3.5, 2×500 mL), H2O (1×500 mL), an aqueous solution of saturated NaHCO3 (CAUTION: CO2 is evolved, 2×500 mL) and brine (1×500 mL). The organic phase was dried over MgSO4 (180 g), filtered and concentrated under reduced pressure to give crude dipeptide LS3-9 as a yellow oil. (127 g, >100% crude yield).

Step LS3-10. The oil LS3-9 was dissolved in 150 mL of dioxane, then a solution of 4 M HCl in dioxane (1360 mL, 20 eq) added and the mixture stirred for 1 h at room temperature. Reaction was monitored by TLC [AcOEt/Hexanes (3:2)]; Rf=baseline; detection: UV and ninhydrin]. The mixture was concentrated under reduced pressure and the resulting residue co-evaporated with Et2O (2×500 mL), then dried under vacuum. The crude LS3-10 was obtained as a slightly yellow solid (96 g, 89.7%). This was dissolved in hot 95% EtOH (200 mL), then MTBE (900 mL) added. The mixture was cooled down to room temperature, then put in a freezer (−20° C.) for 18 h. The resulting crystals were collected by filtration and washed with MTBE (2×200 mL), then dried under vacuum to give crystalline dipeptide hydrochloride LS3-10 (62 g, 64.5% recovery).

1H NMR (DMSO-d6): δ 9.31-9.28 (1H, d, C(O)NH), 7.38-7.26 (7H, m, Ph), 7.09-7.04 (2H, m, Ph), 5.10 (2H, s, C(O)OCH2Ph), 4.65-4.57 (1H, m, CHCH3), 3.76-3.69 (1H, d, CHCH2Ph), 3.15-3.08 and 2.99-2.91 (CHCH2Ph), 2.221 (3H, s, CH3NH2+Cl−), 1.31-1.28 (3H, d, CHCH3).
13C NMR (DMSO-d6): δ 171.33, 169.18, 137.63, 136.31, 129.92, 129.11, 128.95, 128.83, 128.63, 127.30, 67.00, 56.57, 54.38, 36.98, 31.11, 16.47.
LC/MS (Grad_A4): tR=6.26 min
LC Chiral (Iso100B—05): tR=29.6 min. 97% UV
Melting point (uncorrected): 140-142° C.
Step LS3-11. To a solution of carboxylic acid LS3-6 (47.3 g, 87.6 mmol, 1.0 eq) and dipeptide hydrochloride salt LS3-10 (36.2 g, 91.9 mmol, 1.05 eq) in anhydrous THF/CH2Cl2 (1:1) (438 mL) at 0° C. were added DIPEA (92 mL, 526 mmol, 6.0 eq) and HATU (34.9 g, 91.9 mmol, 1.05 eq). The mixture was allowed to warm to room temperature and stirred 16-18 h. Reaction was monitored by TLC [AcOEt/Hex (1:1); Rf=0.48; detection: UV and CMA] The mixture was concentrated under reduced pressure and the residue dissolved in AcOEt (250 mL). The organic phase was washed sequentially with an aqueous solution of citrate buffer (1 M, pH 3.5, 3×150 mL), H2O (1×150 mL), an aqueous solution of saturated NaHCO3 (2×150 mL) and brine (1×150 mL). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography [AcOEt:hexanes, gradient (10:90) to (50:50)] to give LS3-11 as a white gummy solid (70.0 g, 90%).
LC/MS (Grad_A4): tR=15.06 min
Step LS3-12. To a suspension of 10% Pd/C (13.8 g, 20% by weight) in AcOEt (150 mL) was added a solution of alkylated tripeptide LS3-11 (69.0 g, 78.4 mmol, 1.0 eq) in AcOEt (375 mL), then hydrogen was bubbled through the solution for 16-18 h. The reaction was monitored by TLC [AcOEt/hexanes (1:1); Rf=0.22; detection: UV and CMA]. The mixture was purged by nitrogen bubbling, filtered through a Celite pad and rinsed with AcOEt (3×). The combined filtrate and washings were evaporated under reduced pressure to give LS3-12 as a white solid (51.4 g, 100%).
LC/MS (Grad_A4): tR=8.05 min
Step LS3-13. To LS3-12 (51.4 g, 78.4 mmol, 1.0 eq) was added a solution of 3.0 M HCl in dioxane/H2O (75:25, 525 mL, 1.57 mol, 20 eq) and the mixture stirred at room temperature 1.5 h. The solvent was evaporated under vacuum, then the residue was azeotroped with toluene (3×) and dried under vacuum to give crude LS3-13 as an off-white solid (58.0 g, >100% yield).
LC/MS (Grad_A4): tR=5.38 min.
Step LS3-14. To a solution of macrocyclic precursor LS3-13 (78.4 mmol based on LS3-12, 1.0 eq) in anhydrous THF (1.57 L, 50 mM) were added DIPEA (68.0 mL, 392 mmol, 7.0 eq) and DEPBT (25.8 g, 86.2 mmol, 1.1 eq). The mixture was stirred at room temperature 16-18 h. The reaction was monitored by TLC [MeOH/AcOEt (1:9); Rf=0.38; detection: UV and CMA]. At the end of the reaction, significant quantities of DIPEA salts were in suspension in the solution. Prior to evaporation, these salts were filtered and washed with THF to avoid excessive bumping of the solution during evaporation. The solvent was evaporated under vacuum and the residue taken up in an aqueous solution of Na2CO3 (1 M, 500 mL) and AcOEt (250 mL). The separated basic aqueous phase was extracted with AcOEt (2×250 mL). The combined organic phases were washed with brine (2×250 mL), dried over MgSO4, filtered and evaporated under reduced pressure. The crude material so obtained was purified by flash chromatography [AcOEt:MeOH, gradient (100:0) to (90:10)] to give macrocycle compound 298 as a pale yellow solid (35.0 g, 83%, 2 steps).
LC/MS (Grad_A4): tR=6.19 min
Step LS3-15. To crude compound 298 (18.5 g, 34.4 mmol, 1.0 eq) in anhydrous EtOH (100 mL) was slowly added 1.25 M HCl in EtOH (41.2 mL, 51.5 mmol, 1.5 eq). The mixture was stirred 5 min, cooled down to 0° C. and filtered while still cold. The white precipitate was washed with cold anhydrous EtOH (3×75 mL) and dried under vacuum to give compound 298 hydrochloride as an amorphous white solid (15.3 g, 88% recovery, corrected).

Purification of Compound 298. Amorphous compound 298 hydrochloride (14.2 g, 24.7 mmol) was dissolved in a hot mixture of EtOH/H2O (9:1, 215 mL). The solution was cooled down to room temperature and then placed in a freezer (−20° C.) for 16-18 h. The crystals were collected by filtration and washed with cold anhydrous EtOH (3×75 mL) to give compound 298 hydrochloride as a crystalline white solid (12.4 g, 86% recovery). Crystalline compound 298 hydrochloride (11.4 g, 19.9 mmol) was taken up in 1 M

Na2CO3/AcOEt (1:1, 200 mL) and stirred until complete dissolution of the solid. The separated basic aqueous phase was extracted with AcOEt (2×50 mL). The combined organic phases were washed with brine (1×50 mL), dried over MgSO4, filtered and evaporated under vacuum. The oily residue was dissolved in a minimum amount of AcOEt, then hexanes was added until a white precipitate formed. The mixture was evaporated and dried under vacuum to give compound 298 as a white amorphous solid (11.1 g, 100% recovery).
LC/MS (Grad_A4): 6.18 min; Purity (UV/ELSD/CLND); 100/100/100.
This reaction sequence has been repeated in comparable yields starting from 1 kg Cbz-T33a, 518 g LS3-A and 1 kg LS3-B to yield over 400 g of the desired macrocyclic product compound 298 and/or the corresponding HCl salt form. Similar procedures can be applied for other compounds of the invention.
As an alternative, the t-butyl ester of Cpg (LS3-14), produced under standard conditions, can be utilized as was described in Step LS3-4 to provide alkylated Cpg LS3-15 by reaction with Cbz-T33a. This, without protection of the secondary amine on LS3-16 produced by standard acid deprotection of the t-butyl ester of LS3-15, then undergoes chemoselective coupling with dipeptide LS3-10 to prepare LS3-17. Straightforward simultaneous hydrogenolysis of both Cbz and benzyl protecting groups then leads to intermediate LS3-13 in a more efficient approach that avoids two steps.




Step LS3-17. To the hydrochloride salt of carboxylic acid LS3-16 (2.1 g, 4.41 mmol, 1.0 eq) and LS3-10 (1.7 g, 4.59 mmol, 1.05 eq) in anhydrous THF/CH2Cl2 (1:1, 22 mL) at 0° C. were added DIPEA (5.3 mL, 30.6 mmol, 7.0 eq) and HATU (1.7 g, 4.59 mmol, 1.05 eq). The mixture was allowed to warm to room temperature and stirred 16-18 h. The reaction was monitored by LC-MS. The mixture was concentrated under reduced pressure and the residue dissolved in AcOEt (150 mL). The organic phase was washed sequentially with an aqueous solution of citrate buffer (1 M, pH 3.5, 3×25 mL), H2O (1×25 mL), an aqueous solution of saturated NaHCO3 (2×25 mL) and brine (1×25 mL). The organic phase was dried over MgSO4, filtered and concentrated under vacuum to give LS3-17 as a white solid (3.5 g, >100% crude yield).

LC/MS (Grad_A4): tR=12.09 min.
Step LS3-18. To a suspension of 10% Pd/C (596 mg, 20% by weight) in 95% EtOH (10 mL) was added a solution of alkylated tripeptide LS3-17 (3.0 g, 3.82 mmol, 1.0 eq) in AcOEt (15 mL) and hydrogen bubbled through the solution for 2 h. The mixture was then stirred under a hydrogen atmosphere for 16-18 h. The reaction was monitored by TLC [100% AcOEt; Rf=Baseline; detection: UV and CMA]. The mixture was purged by nitrogen bubbling, filtered through a Celite pad and rinsed with 95% EtOH (3×20 mL). The combined filtrate and rinses were evaporated under reduced pressure to give LS3-13 as a white solid (2.0 g, 94%).
LC/MS (Grad_A4): tR=5.40 min.
B. Biological Results
1. Radioligand Binding Assay on Ghrelin Receptor (human clone, hGHS-R1a)
Objective


    
    
        1. To demonstrate that compound 298 has a direct, high affinity interaction with hGHS-R1a

Key Aspects of Method

        1. Binding performed on membranes prepared from HEK293 expressing the transfected, cloned human ghrelin receptor (hGHS-R1a).
        2. [125I]Ghrelin was used as the radioligand for displacement (Kd=0.01 nM, test concentration=0.007 nM).
        3. Ghrelin (unlabeled, 1 μM) was used to determine non-specific binding.
        4. Compound 298 tested in duplicate samples over an 11-point concentration curve.

Results

    
    


Compound 298 binding to hGHS-R1a has been run multiple times. A representative binding inhibition curve as shown in FIG. 10 demonstrates that compound 298 binds competitively, reversibly, and with high affinity to hGHS-R1a.
2. Cell-based, Functional Assays on Ghrelin Receptor (Human Clone, hGHS-R1a)
Objectives


    
    
        1. To demonstrate that compound 298 is a full agonist at hGHS-R1a.
        2. To measure the potency of compound 298 agonist activity at hGHS-R1a.

Key Aspects of Method

        1. Assay performed on CHO-K1 cells expressing the transfected, cloned human ghrelin receptor (hGHS-R1a) and Gα16 
        2. Suspended cells incubated O/N with coelenterazine.
        3. Stimulation of hGHS-R1a activates Gα16, causing intercellular Ca2+ release which ultimately leads to the oxidation of coelenterazine and the emission of a quantitative luminescent signal.
        4. Ghrelin was used as the positive control.
        5. Compound 298 tested in duplicate samples over an 8-point concentration curve.

Results

    
    


Compound 298 activates hGHS-R1a with an EC50=25 nM as shown in FIG. 11. Compound 298 is a full agonist based on its similar, maximal efficacy to the ghrelin peptide (positive control).
3. Compound 298 (i.v.) Effect on Growth Hormone (GH) Release in Conscious, Freely-Moving Rats.
Ghrelin (and analogues thereof) is known to potently stimulate GH release from the pituitary in various species including rat following intravenous dosing.
Objectives


    
    
        1. To determine whether compound 298 stimulates GH release in rat.
        2. To determine whether compound 298 modulates ghrelin-induced GH release in rat.

Method

        1. Model adapted from Tannenbaum et al. (2003), Endocrinology 144:967-974.
        2. Rats implanted with chronic, intravenous (i.v.) cannulae.
        3. Rats allowed to move freely even while dosing drug or sampling blood to minimize stress-induced changes in GH release.
        4. Compound 298 administered at GH peak and trough levels to measure:
        
            a. Stimulatory effect, if any, on GH release; and
            b. Whether any stimulatory effect is sustained with repeated dosing.
        
        
        5. Blood samples are drawn at defined, 15-minute intervals throughout the test day and growth hormone (GH) measured directly by radioimmunoassay.
        6. Compound 298 tested at 3, 30, 300, 1000 μg/kg (i.v., N=5-6/rats per group).
        7. Ghrelin (positive control) tested at 5 μg (i.v.).

Results

    
    


Compound 298 at doses up to 1000 μg/kg causes no significant difference in pulsatile GH release in comparison to vehicle controls (FIG. 12A for 300 μg/kg). Ghrelin at a dose of 5 μg causes a significant increase in GH release when dosed at both peak and trough levels (positive control). Compound 298 dosed 10 min. prior to ghrelin neither inhibits nor augments ghrelin-induced GH release (FIG. 12B). As a secondary indicator of GH release, the effects of compound 298 on the levels of IGF-1 were also examined at the 1000 μg/kg dose. No changes in IGF-1 levels upon treatment with compound 298 were observed.
4. Compound 298 Effect on hGHS-R1a Receptor Desensitization
G-protein coupled receptors can undergo receptor desensitization upon agonist stimulation, where the degree of receptor desensitization is partly characteristic of the agonist. Lesser receptor desensitization is desirable because this correlates with lesser development of tolerance with chronic use of drug. This factor, among others, has been implicated in the poor clinical performance of GHS.
Objective


    
    
        1. To determine the extent to which Compound 298 causes desensitization of the ghrelin receptor (human clone, hGHS-R1a).

Method

        1. Studies by FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices).
        2. Assay performed on HEK293 cells expressing hGHS-R1a.
        3. Compound 298 agonist potency was measured using duplicate samples over a 12-point concentration curve; EC50 for compound 298 established.
        4. In a separate experiment, cells expressing hGHS-R1a are exposed to a range of concentrations of compound 298 (1, 10, 100, 1000 nM) for 3 minutes. Compound 298 washed out, then cells treated with a concentration of ghrelin (EC100) that elicits maximal stimulation at non-desensitized receptors.
        5. A DC50 value is calculated. The DC50 value is defined as the pre-treatment concentration of compound 298 that desensitizes the ghrelin (EC100) response by 50%.

Results

    
    


Compound 298 is a full agonist (EC50=5 nM; FIG. 13A). Increasing pre-treatment concentrations of compound 298 desensitize the maximal response to EC100 ghrelin (DC50=32 nM; FIG. 13B). The DC50 value is >6-fold less potent than the EC50 value, thus compound 298 stimulates the receptor more potently than it desensitizes the receptor. Compound 298 desensitizes the receptor  ̃10-fold less potently than other ghrelin agonists (i.e. ghrelin peptide and the GHS capromorelin [Pfizer]; FIG. 13C).
Compound 298 has a favorable desensitization profile since it (1) stimulates the receptor 6-fold more potently that it desensitizes the receptor and (2) elicits desensitization at a 10-fold lower potency than the endogenous ligand (i.e. ghrelin) and alternate, small-molecule ghrelin agonists. Accordingly, compound 298 may elicit less tolerance than alternate ghrelin agonists with chronic dosing.
5. Compound 298 Effect on Gastric Emptying of a Solid Meal in Naïve Rat
Objectives


    
    
        1. To ascertain data for compound 298 as a prokinetic agent with potent effects on gastric emptying, a model for gastroparesis.

Methods

        1. Overnight-fasted rats (male, Wistar,  ̃200 g, N=5/group) were given a meal of methylcellulose (2%) by intragastric gavage. The meal was labeled with phenol red (0.05%).
        2. Test articles (i.e. vehicle, compound 298, metoclopramide, etc.) were administered by intravenous injection immediately after meal.
        3. Animals were sacrificed 15 minutes later; the stomach was immediately removed and homogenized in 0.1 N NaOH and centrifuged.
        4. Total phenol red remaining in the stomach was quantified by a calorimetric method at 560 nm.
        5. A >30% increase in gastric emptying, detected based on the phenol red concentration in comparison to the control group, is considered significant.

Results

    
    


Metoclopramide (marketed gastroparesis product), ghrelin and GHRP-6 (reference peptide agonists at hGHS-R1a) all demonstrated significant gastric emptying (FIG. 14A). Compound 298 caused significant gastric emptying in a dose-dependent manner with  ̃100-fold superior potency to metoclopramide (FIG. 14B). Compound 298 potently stimulated gastric emptying of a solid meal in naïve rats with a 100-fold superior potency to metoclopramide, a currently used drug with prokinetic activity.
6. Effect of Compound 298 in the Treatment of Post-operative Ileus in Rat
Objective
To measure the therapeutic utility of compound 298 in a rat model of post-operative ileus (POI).
Methods


    
    
        1. Model adapted from Kälff et al. (1998), Ann Surg 228:652-63.
        2. Rats (male, Sprague-Dawley, 250-300 g) were implanted with jugular vein catheters to accommodate dosing of test articles.
        3. Rats were fasted O/N, anesthetized with isofluorane and subjected to abdominal surgery.
        4. Following an abdominal incision, the small intestine caecum and large intestine were eviscerated for a period of 15 min and kept moist with saline.
        5. A “running of the bowel” was performed, a clinically-relevant manipulation of the intestines characterized by first pinching the upper small intestine and continuing this manipulation down through the large intestine.
        6. Rats are allowed a 15 min recovery beginning after the disappearance of any effects of the isofluorane anesthesia.
        7. Rats are dosed with vehicle or compound 298 (30, 100, or 300 μg/kg, i.v., N=6/gp) followed by intragastric gavage of 99mTc methylcellulose (2%) meal.
        8. After 15 min, the rats were euthanized and the stomach and consecutive 10 cm segments of the intestine were isolated. Radioactivity (99mTc) in each tissue isolate was measured as a means of measuring the transit of the meal.

Results

    
    


In FIG. 15, the distribution of the bars indicates the distribution of the meal in the stomach (‘ST’) and consecutive 10 cm segments of the small intestine at 15 min post-oral gavage. Abdominal surgery coupled with a running of the bowel caused a significant ileus in rats as determined by comparison of the naïve (i.e. unoperated) and POI treatment groups. Compound 298 significantly increased gastric emptying and intestinal transit at test concentrations of 100 and 300 μg/kg (i.v.). The data corresponding to the 100 μg/kg dose is presented in FIG. 15. At 100 μg/kg (i.v.), compound 298 significantly promoted GI transit by 2.7× as measured by the geometric center of the meal in comparison to the POI+vehicle treatment group. Compound 298 significantly improved gastric emptying and intestinal transit in rats with post-operative ileus. Compound 298 can effectively treat an existing, post-surgical ileus; thus, prophylactic use prior to surgery is not required as is the case for opioid antagonists in clinical development.
7. The Effect of Compounds of the Invention on Gastric Emptying and Gastrointestinal Transit in a Model of Opioid-Delayed Gastric Emptying
Opioid analgesics, such as morphine, are well known to delay gastrointestinal transit which is an important side-effect for this class of drugs. The clinical term for this syndrome is opioid bowel dysfunction (OBD). Importantly, patients recovering from abdominal surgery experience post-operative ileus that is further exacerbated by concomitant opioid therapy for post-surgical pain.
Objective


    1. To determine whether compounds of the invention may have therapeutic utility in the treatment of opioOBD.

Methods

    1. Rats (male, Sprague-Dawley, 250-300 g) are implanted with jugular vein catheters to accommodate dosing of test articles.
    2. Overnight-fasted rats are administered morphine (3 mg/kg s.c.).
    3. After 30 min, rats are to be dosed with vehicle or compound 298 (300 or 1000 μg/kg, i.v., n=4-to-6/gp) followed by intragastric gavage of 99mTc methylcellulose (2%) meal.
    4. After 15 min, the rats are euthanized and the stomach and consecutive 10 cm segments of the intestine are isolated. Radioactivity (99mTc) in each tissue isolate is measured as a means of measuring the transit of the meal.

Results



Morphine (3 mg/kg, s.c.) significantly delayed gastric emptying and intestinal transit in rats (FIG. 16A). Opioid-delayed gastrointestinal transit was effectively reversed in a dose-dependent manner by treatment with compound 298 (iv.) (FIG. 16B).
8. Metabolic Stability in Human Plasma
Drugs are susceptible to enzymatic degradation in plasma through the action of various proteinases and esterases. Thus, plasma stability is often performed as a metabolic screen in the early phases of drug discovery. The aim of this study is to measure the metabolic stability of compounds of the invention in human plasma.
Experimental Method
The stability of compound 298 in human plasma at 37° C. has been measured at 2 and 24 h. Two forms of compound 298 have been studied: free amine and corresponding HCl salt. Also, the stability of compound 298 has been established in plasma alone and in plasma buffered with phosphate-buffered saline (PBS) where the ratio of plasma to phosphate buffer (pH 7.0) is 20:1. Assays were both performed and analyzed in triplicate samples. Compound 298 was extracted from plasma matrix using an SPE technique (Oasis MCX cartridge). Sample analysis is done using LC-MS in APCI+ mode. The level of compound 298 in plasma samples is compared to the level of compound 298 in a spiked sample stored at −60° C. from the same pool of plasma. Results are presented as a percent recovery of compound 298.







TABLE 8







Percent Recovery of Compound 298 Following Incubation in Human


Plasma (37° C.).













Free Amine +





Free amine
PBS
HCl Salt
HCl Salt + PBS
















2
24
2
24
2
24
2
24



Hours
Hours
Hours
Hours
Hours
Hours
Hours
Hours


Triplicates
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)


















Assay #1
101.0
105.5
98.3
97.9
100.2
96.6
102.9
97.8


Assay #2
100.3
95.6
100.4
100.8
99.1
104.3
97.4
101.9


Assay #3
101.3
100.9
98.3
101.9
101.6
102.3
99.4
98.5


Mean
100.9
100.7
99.0
100.2
100.3
101.1
99.9
99.4


Standard
0.5
4.9
1.2
2.1
1.3
4.0
2.7
2.2


Deviation


RSD
0.5
4.9
1.3
2.1
1.3
4.0
2.7
2.2









As shown in Table 8, compound 298 is stable in human plasma at 37° C. for at least 24 hours independent of compound form (i.e. free amine or salt) or whether or not the plasma samples are pH buffered with PBS.
9. Compound 298 Interaction Profile at Nine Human Cytochrome P450 Enzyme Subtypes
Compound 298 (0.0457 to 100 μM) has minimal inhibitory activity at all cyp450 enzymes tested, except cyp3A4, and has moderate inhibitory activity at cyp3A4. The inhibitory activity observed for compound 298 at cyp3A4 was not anticipated to be physiologically relevant based on the low doses of compound 298 required for therapeutic activity. Also, there was no indication that compound 298 would undergo a drug-drug interaction with opioid analgesics that may be co-administered to POI patients.
10. Compound 298 Profile in hERG Channel Inhibition
Compound 298 (1, 10 μM) had no significant effect on hERG channel function in comparison to vehicle (0.1% DMSO) controls. E-4031 (positive control) completely inhibited hERG channel currents at 500 nM.
Example 5
Gastroparesis Animal Model
High caloric meals are well known to impede gastric emptying. This observation has recently been exploited by Megens, A. A.; et al. (unpublished) to develop a rat model for delayed gastric emptying as experienced in gastroparesis.
Materials


    1. Wistar rats, male, 200-250 g
    2. Chocolate test meal: 2 mL Clinutren ISO® (1.0 kcal/mL, Nestle S A, Vevey Switzerland)

Method



The test meal is given to the subjects by oral gavage at time=0. After 60 min, the subjects are sacrificed, the stomachs excised and the contents weighed. Untreated animals experienced a significant delay in gastric emptying as denoted by the higher residual stomach content.
Test compounds were administered intravenously as aqueous solutions, or solutions in normal saline, at time=0 at three dose levels (0.08 mg/kg; 0.30-0.31 mg/kg, 1.25 mg/kg). When necessary, for example compounds 21, 299 and 415, 10% cyclodextrin (CD) was added to solubilize the material. Test compounds examined utilizing subcutaneous injection are administered at time=−30 min. Four to five (4-5) rats were tested per group, except in the case of the cyclodextrin control in which ten (10) rats comprised the group.
Results are reported as percentage relative to the stomach weight for injection only of solvent as a control as shown in FIGS. 17A and 17B and illustrate the gastric emptying capability of the compounds of the present invention. These results are applicable for the utility of these compounds for the prevention and/or treatment of gastroparesis and/or postoperative ileus.
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

## Claims

1. A pharmaceutically acceptable salt of a macrocyclic compound having the following structure:

2. The pharmaceutically acceptable salt of claim 1, wherein the salt is an amorphous form.

3. The pharmaceutically acceptable salt of claim 1, wherein the salt is a crystalline form.

4. The pharmaceutically acceptable salt of claim 1, wherein the salt is a hydrochloride, hydrobromide or hydroiodide salt.

5. The pharmaceutically acceptable salt of claim 1, wherein the salt is a hydrochloride salt.

6. A process for preparing a pharmaceutically acceptable salt of a compound having the following structure:




The process comprising:
(a) dissolving a macrocyclic compound having the following structure:





in a solution of an alcohol to form solution A;
(b) adding an acid to solution A to form acidified solution A;
(c) optionally cooling acidified solution A; and
(d) separating a precipitated salt from acidified solution A;
(e) dissolving the precipitated salt from (d) in a hot mixture of an alcohol and water to form solution C;
(f) cooling solution C; and
(g) separating a precipitated salt from solution C.

7. The process of claim 6, wherein the alcohol is ethanol.

8. The process of claim 6, wherein the acid is hydrochloric acid.

9. The process of claim 6, wherein the precipitated salt is an amorphous form.

10. The process of claim 6, wherein the precipitated salt is a hydrochloride salt.

11. The process of claim 6, wherein the precipitated salt from solution C is a crystalline form.

12. A pharmaceutical composition comprising:
(a) a pharmaceutically acceptable salt of claim 1; and
(b) a pharmaceutically acceptable carrier, excipient or diluent.

13. The pharmaceutical composition of claim 12 further comprising a growth hormone secretagogue.

14. The pharmaceutical composition of claim 13, wherein the growth hormone secretagogue is hexarelin, GHRP-1, GHRP-2, GHRP-6, ipamorelin, MK-0677, NN703, capromorelin, G7039, G7134, G7203, G7502, SM-130686, RC-1291, L-692429, L-692587, L-739943, L-163255, L-163540, L-163833, L-166446, CP-424391, EP-51389, LY-444711, NNC-26-0235, NNC-26-0323, NNC-26-0610, NNC-26-0722, NNC-26-1089, NNC-26-1136, NNC-26-1137, NNC-26-1187, NNC-26-1291, growth hormone releasing factor, IGF-I or IGF-II.