BISARYLSULFONE AND DIALKYLARYLSULFONE COMPOUNDS AS CALCIUM CHANNEL BLOCKERS

The invention relates to bisarylsulfone and dialkylarylsulfone compounds (e.g., compounds according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) useful in treating conditions associated with calcium channel function, and particularly conditions associated with N-type calcium channel activity. The invention also relates to pharmaceutical compositions that include these bisarylsulfone compounds, as well methods for the treatment of conditions such as cardiovascular disease, epilepsy, cancer and pain.

DETAILED DESCRIPTION

Compounds

The invention features compounds that can inhibit voltage-gated calcium channels (e.g., N- and/or T-type). For example, diarylsulfone compounds can inhibit N-type voltage gated Ca2+channels, and dialkylarylsulfone compounds can inhibit voltage gated N- and T-type calcium channels.

Exemplary compounds are described by any of Formulas (I)-(IX), which include compounds (1)-(227) of Tables 4 and 5. Other embodiments, exemplary methods of synthesis, and uses of these compounds are also described herein.

Utility and Administration

The compounds described herein are useful in the methods of the invention and, while not bound by theory, are believed to exert their desirable effects through their ability to modulate the activity of calcium channels, particularly the activity of N-type calcium channels. This makes them useful for treatment of certain conditions where modulation of N-type calcium channels is desired, including pain, epilepsy, migraine, Parkinson's disease, depression, schizophrenia, psychosis, and tinnitus.

Modulation of Calcium Channels

The entry of calcium into cells through voltage-gated calcium channels mediates a wide variety of cellular and physiological responses, including excitation-contraction coupling, hormone secretion and gene expression (e.g., Miller et al., Science 235:46-52 (1987); Augustine et al.,Annu Rev Neurosci10: 633-693 (1987)). In neurons, calcium channels directly affect membrane potential and contribute to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Calcium entry further affects neuronal functions by directly regulating calcium-dependent ion channels and modulating the activity of calcium-dependent enzymes such as protein kinase C and calmodulin-dependent protein kinase II. An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitter, which also affects neurite outgrowth and growth cone migration in developing neurons.

Calcium channels mediate a variety of normal physiological functions, and are also implicated in a number of human disorders as described herein. For example, calcium channels also have been shown to mediate the development and maintenance of the neuronal sensitization and hyperexcitability processes associated with neuropathic pain, and provide attractive targets for the development of analgesic drugs (reviewed in Vanegas et al.,Pain85: 9-18 (2000)). Native calcium channels have been classified by their electrophysiological and pharmacological properties into T-, L-, N-, P/Q- and R-types (reviewed in Catterall,Annu Rev Cell Dev Biol16: 521-555, 2000; Huguenard,Annu Rev Physiol58: 329-348, 1996). The L-, N- and P/Q-type channels activate at more positive potentials (high voltage-activated) and display diverse kinetics and voltage-dependent properties (Id.).

The modulation of ion channels by the compounds described herein (e.g., a compound according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) can be measured according to methods known in the art (e.g., in the references provided herein). Modulators of ion channels, e.g., voltage gated calcium ion channels, and the medicinal chemistry or methods by which such compounds can be identified, are also described in, for example: Birch et al.,Drug Discovery Today,9(9):410-418 (2004); Audesirk, “Chapter 6-Electrophysiological Analysis of Ion Channel Function,”Neurotoxicology: Approaches and Methods,137-156 (1995); Camerino et al., “Chapter 4: Therapeutic Approaches to Ion Channel Diseases,”Advances in Genetics,64:81-145 (2008); Petkov, “Chapter 16-Ion Channels,” Pharmacology: Principles and Practice, 387-427 (2009); Standen et al., “Chapter 15-Patch Clamping Methods and Analysis of Ion Channels,”Principles of Medical Biology, Vol.7, Part 2, 355-375 (1997); Xu et al.,Drug Discovery Today,6(24):1278-1287 (2001); and Sullivan et al.,Methods Mol. Biol.114:125-133 (1999). Exemplary experimental methods are also provided in the Examples.

Mutations in calcium channel α1 subunit genes in animals can provide important clues to potential therapeutic targets for pain intervention. Genetically altered mice null for the α1B N-type calcium channel gene have been reported by several independent groups (Ino et al.,Proc. Natl. Acad. Sci. USA98:5323-5328 (2001); Kim et al.,Mol Cell Neurosci18:235-245 (2001); Kim et al., Neuron 31:35-45 (2001); Saegusa et al.,Proc. Natl. Acad. Sci. USA97:6132-6137 (2000); and Hatakeyama et al.,NeuroReport12:2423-2427 (2001)). These studies indicate that the N-type channel may be a potential target for mood disorders as well as pain.

Gabapentin (1-(aminomethyl)cyclohexaneacetic acid (Neurontin®)), is an anticonvulsant that also acts on N-type channels. Though not specific for N-type calcium channels, subsequent work has demonstrated that gabapentin is also successful at preventing hyperalgesia in a number of different animal pain models, including chronic constriction injury (CCI), heat hyperalgesia, inflammation, diabetic neuropathy, static and dynamic mechanical allodynia associated with postoperative pain (e.g., Cesena et al.,Neurosci Lett262: 101-104 (1999); Field et al.,Pain80: 391-398 (1999); Cheng et al.,Anesthesiology92: 1126-1131 (2000); and Nicholson,Acta Neurol Scand101: 359-371 (2000)).

T-type channels can be distinguished by having a more negative range of activation and inactivation, rapid inactivation, slow deactivation, and smaller single-channel conductances. There are three subtypes of T-type calcium channels that have been molecularly, pharmacologically, and elecrophysiologically identified: these subtypes have been termed a 1 G, α1H, and α1I (alternately called CaV3.1, CaV3.2 and CaV3.3 respectively).

T-type calcium channels are involved in various medical conditions. In mice lacking the gene expressing the 3.1 subunit, resistance to absence seizures was observed (Kim et al.,Mol. Cell Neurosci.18(2): 235-245 (2001)). Other studies have also implicated the 3.2 subunit in the development of epilepsy (Su et al.,J. Neurosci.22: 3645-3655 (2002)). There is also evidence that some existing anticonvulsant drugs, such as ethosuximide, function through the blockade of T-type channels (Gomora et al.,Mol. Pharmacol.60: 1121-1132 (2001)).

Low voltage-activated calcium channels are highly expressed in tissues of the cardiovascular system. There is also a growing body of evidence that suggests that T-type calcium channels are abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type calcium channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel is minimal. Therefore, selectively blocking calcium channel entry into cancerous cells may be a valuable approach for preventing tumor growth (e.g., PCT Patent Publication Nos. WO 05/086971 and WO 05/77082; Taylor et al.,World J. Gastroenterol.14(32): 4984-4991 (2008); Heo et al.,Biorganic&Medicinal Chemistry Letters18:3899-3901 (2008)).

T-type calcium channels may also be involved in still other conditions. A recent study also has shown that T-type calcium channel antagonists inhibit high-fat diet-induced weight gain in mice. In addition, administration of a selective T-type channel antagonist reduced body weight and fat mass while concurrently increasing lean muscle mass (e.g., Uebele et al.,The Journal of Clinical Investigation,119(6):1659-1667 (2009)). T-type calcium channels may also be involved in pain (see for example: US Patent Publication No. 2003/0086980; PCT Publication Nos. WO 03/007953 and WO 04/000311). In addition to cardiovascular disease, epilepsy (see also US Patent Publication No. 2006/0025397), cancer, and chronic or acute pain, T-type calcium channels have been implicated in diabetes (US Patent Publication No. 2003/0125269), sleep disorders (US Patent Publication No. 2006/0003985), Parkinson's disease and psychosis such as schizophrenia (US Patent Publication No. 2003/0087799); overactive bladder (Sui et al.,British Journal of Urology International99(2): 436-441 (2007); US Patent Publication No. 2004/0197825), renal disease (Hayashi et al.,Journal of Pharmacological Sciences99: 221-227 (2005)), anxiety and alcoholism (US Patent Publication No. 2009/0126031), neuroprotection, and male birth control.

Diseases and Conditions

Epilepsy as used herein includes but is not limited to partial seizures such as temporal lobe epilepsy, absence seizures, generalized seizures, and tonic/clonic seizures.

Acute pain as used herein includes but is not limited to nociceptive pain and post-operative pain. Chronic pain includes but is not limited by: peripheral neuropathic pain such as post-herpetic neuralgia, diabetic neuropathic pain, neuropathic cancer pain, failed back-surgery syndrome, trigeminal neuralgia, and phantom limb pain; central neuropathic pain such as multiple sclerosis related pain, Parkinson disease related pain, post-stroke pain, post-traumatic spinal cord injury pain, and pain in dementia; musculoskeletal pain such as osteoarthritic pain and fibromyalgia syndrome; inflammatory pain such as rheumatoid arthritis and endometriosis; headache such as migraine, cluster headache, tension headache syndrome, facial pain, headache caused by other diseases; visceral pain such as interstitial cystitis, irritable bowel syndrome and chronic pelvic pain syndrome; and mixed pain such as lower back pain, neck and shoulder pain, burning mouth syndrome and complex regional pain syndrome.

In treating osteoarthritic pain, joint mobility can also improve as the underlying chronic pain is reduced. Thus, use of compounds of the present invention to treat osteoarthritic pain inherently includes use of such compounds to improve joint mobility in patients suffering from osteoarthritis.

The compounds described herein can be tested for efficacy in any standard animal model of pain. Various models test the sensitivity of normal animals to intense or noxious stimuli (physiological or nociceptive pain). These tests include responses to thermal, mechanical, or chemical stimuli. Thermal stimuli usually involve the application of hot stimuli (typically varying between 42-55° C.) including, for example: radiant heat to the tail (the tail flick test), radiant heat to the plantar surface of the hindpaw (the Hargreaves test), the hotplate test, and immersion of the hindpaw or tail into hot water. Immersion in cold water, acetone evaporation, or cold plate tests may also be used to test cold pain responsiveness. Tests involving mechanical stimuli typically measure the threshold for eliciting a withdrawal reflex of the hindpaw to graded strength monofilament von Frey hairs or to a sustained pressure stimulus to a paw (e.g., the Ugo Basile analgesiometer). The duration of a response to a standard pinprick may also be measured. When using a chemical stimulus, the response to the application or injection of a chemical irritant (e.g., capsaicin, mustard oil, bradykinin, ATP, formalin, acetic acid) to the skin, muscle joints or internal organs (e.g., bladder or peritoneum) is measured.

In addition, various tests assess pain sensitization by measuring changes in the excitability of the peripheral or central components of the pain neural pathway. In this regard, peripheral sensitization (i.e., changes in the threshold and responsiveness of high threshold nociceptors) can be induced by repeated heat stimuli as well as the application or injection of sensitizing chemicals (e.g., prostaglandins, bradykinin, histamine, serotonin, capsaicin, or mustard oil). Central sensitization (i.e., changes in the excitability of neurons in the central nervous system induced by activity in peripheral pain fibers) can be induced by noxious stimuli (e.g., heat), chemical stimuli (e.g., injection or application of chemical irritants), or electrical activation of sensory fibers.

Various pain tests developed to measure the effect of peripheral inflammation on pain sensitivity can also be used to study the efficacy of the compounds (Stein et al.,Pharmacol. Biochem. Behav. (1988) 31: 445-451; Woolf et al.,Neurosci. (1994) 62: 327-331). Additionally, various tests assess peripheral neuropathic pain using lesions of the peripheral nervous system. One such example is the “axotomy pain model” (Watson,J. Physiol. (1973) 231:41). Other similar tests include the SNL test which involves the ligation of a spinal segmental nerve (Kim and Chung,Pain(1992) 50: 355), the Seltzer model involving partial nerve injury (Seltzer,Pain(1990) 43: 205-18), the spared nerve injury (SNI) model (Decosterd and Woolf,Pain(2000) 87:149), chronic constriction injury (CCI) model (Bennett (1993)Muscle Nerve16: 1040), tests involving toxic neuropathies such as diabetes (streptozocin model), pyridoxine neuropathy, taxol, vincristine, and other antineoplastic agent-induced neuropathies, tests involving ischaemia to a nerve, peripheral neuritis models (e.g., CFA applied peri-neurally), models of post-herpetic neuralgia using HSV infection, and compression models.

In all of the above tests, outcome measures may be assessed, for example, according to behavior, electrophysiology, neurochemistry, or imaging techniques to detect changes in neural activity.

Exemplary models for the treatment of pain and epilepsy include, but are not limited to, the following.

Models of Pain

L5/L6 Spinal Nerve Ligation (SNL)-Chung Pain Model

The Spinal Nerve Ligation is an animal model representing peripheral nerve injury generating a neuropathic pain syndrome. In this model, experimental animals develop the clinical symptoms of tactile allodynia and hyperalgesia. L5/L6 Spinal nerve ligation (SNL) injury can be induced using the procedure of Kim and Chung (Kim et al.,Pain50:355-363 (1992)) in male Sprague-Dawley rats.

Assessment of Tactile Allodynia—Von Frey

The assessment of tactile allodynia can consist of measuring the withdrawal threshold of the paw ipsilateral to the site of nerve injury in response to probing with a series of calibrated von Frey filaments (innocuous stimuli). Animals can be acclimated to the suspended wire-mesh cages for 30 minutes before testing. Each von Frey filament can be applied perpendicularly to the plantar surface of the ligated paw of rats for 5 seconds. A positive response may be indicated by a sharp withdrawal of the paw. Measurements can be taken before and after administration of test articles. The paw withdrawal threshold can be determined by the non-parametric method of Dixon (Dixon,Ann. Rev. Pharmacol. Toxicol.20:441-462 (1980)), in which the stimulus was incrementally increased until a positive response was obtained, and then decreased until a negative result was observed. The protocol can be repeated until three changes in behaviour are determined (“up and down” method) (Chaplan et al.,J. Neurosci. Methods53:55-63 (1994)). For example, the 50% paw withdrawal threshold can be determined as (10[Zf+kδ])/10,000, where Xf=the value of the last von Frey filament employed, k=Dixon value for the positive/negative pattern, and δ=the logarithmic difference between stimuli. The cut-off values for rats can be no less than 0.2 g and no higher than 15 g (5.18 filament); for mice no less than 0.03 g and no higher than 2.34 g (4.56 filament). A significant drop of the paw withdrawal threshold compared to the pre-treatment baseline is considered tactile allodynia.

Assessment of Thermal Hypersensitivity—Hargreaves

The method of Hargreaves and colleagues (Hargreaves et al.,Pain32:77-8 (1988)) can be employed to assess paw-withdrawal latency to a noxious thermal stimulus. Rats may be allowed to acclimate within a Plexiglas enclosure on a clear glass plate for 30 minutes. A radiant heat source (e.g., halogen bulb coupled to an infrared filter) can then be activated with a timer and focused onto the plantar surface of the affected paw of treated rats. Paw-withdrawal latency can be determined by a photocell that halts both lamp and timer when the paw is withdrawn. The latency to withdrawal of the paw from the radiant heat source can be determined prior to L5/L6 SNL, 7-14 days after L5/L6 SNL but before drug, as well as after drug administration. A maximal cut-off of 33 seconds is typically employed to prevent tissue damage. Paw withdrawal latency can be thus determined to the nearest 0.1 second. A significant drop of the paw withdrawal latency from the baseline indicates the status of thermal hyperalgesia. Antinociception is indicated by a reversal of thermal hyperalgesia to the pre-treatment baseline or a significant (p<0.05) increase in paw withdrawal latency above this baseline. Data can be converted to % anti hyperalgesia or % anti nociception by the formula: (100×(test latency−baseline latency)/(cut-off−baseline latency) where cut-off is 21 seconds for determining anti hyperalgesia and 40 seconds for determining anti nociception.

Models of Epilepsy

6 Hz Psychomotor Seizure Model of Partial Epilepsy

Compounds can also be evaluated for the protection against seizures induced by a 6 Hz, 0.2 ms rectangular pulse width of 3 s duration, at a stimulus intensity of 32 mA (CC97) applied to the cornea of male CF1 mice (20-30 g) according to procedures described by Barton et al, “Pharmacological Characterization of the 6 Hz Psychomotor Seizure Model of Partial Epilepsy,”Epilepsy Res.47(3):217-27 (2001). Seizures can be characterized by the expression of one or more of the following behaviours: stun, forelimb clonus, twitching of the vibrissae and Straub-tail immediately following electrical stimulation. Animals can be considered “protected” if following pre-treatment with a compound the 6 Hz stimulus failed to evoke a behavioural response as describe above.

To assess a compound's undesirable side effects (toxicity), animals can be monitored for overt signs of impaired neurological or muscular function. In mice, the rotarod procedure (Dunham and Miya,J. Am. Pharmacol. Assoc.46:208-209 (1957)) is used to disclose minimal muscular or neurological impairment (MMI). When a mouse is placed on a rod that rotates at a speed of 6 rpm, the animal can maintain its equilibrium for long periods of time. The animal is considered toxic if it falls off this rotating rod three times during a 1-min period. In addition to MMI, animals may exhibit a circular or zigzag gait, abnormal body posture and spread of the legs, tremors, hyperactivity, lack of exploratory behavior, somnolence, stupor, catalepsy, loss of placing response and changes in muscle tone.

Male Wistar rats (P6 to P9 for voltage-clamp and P15 to P18 for current-clamp recordings) can be anaesthetized through intraperitoneal injection of Inactin (Sigma). The spinal cord can then be rapidly dissected out and placed in an ice-cold solution protective sucrose solution containing (in mM): 50 sucrose, 92 NaCl, 15 D-Glucose, 26 NaHCO3, 5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgSO4, 1 kynurenic acid, and bubbled with 5% CO2/95% O2. The meninges, dura, and dorsal and ventral roots can then removed from the lumbar region of the spinal cord under a dissecting microscope. The “cleaned” lumbar region of the spinal cord may be glued to the vibratome stage and immediately immersed in ice cold, bubbled, sucrose solution. For current-clamp recordings, 300 to 350 μm parasagittal slices can be cut to preserve the dendritic arbour of lamina I neurons, while 350 to 400 μm transverse slices can be prepared for voltage-clamped NaVchannel recordings. Slices may be allowed to recover for 1 hour at 35° C. in Ringer solution containing (in mM): 125 NaCl, 20 D-Glucose, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 1 kynurenic acid, 0.1 picrotoxin, bubbled with 5% CO2/95% O2. The slice recovery chamber can then returned to room temperature (20 to 22° C.) for recordings.

Neurons may be visualized using IR-DIC optics (Zeiss Axioskop 2 FS plus, Gottingen, Germany), and neurons from lamina I and the outer layer of lamina II can be selected based on their location relative to the substantia gelatinosa layer. Neurons can be patch-clamped using borosilicate glass patch pipettes with resistances of 3 to 6 MΩ. Current-clamp recordings of lamina I/II neurons in the intact slice, the external recording solution was the above Ringer solution, while the internal patch pipette solution contained (in mM): 140 KGluconate, 4 NaCl, 10 HEPES, 1 EGTA, 0.5 MgCl2, 4 MgATP, 0.5 Na2GTP, adjusted to pH 7.2 with 5 M KOH and to 290 mOsm with D-Mannitol (if necessary). Tonic firing neurons can be selected for current-clamp experiments, while phasic, delayed onset and single spike neurons may be discarded (22). Recordings can be digitized at 50 kHz and low-pass filtered at 2.4 kHz.

In addition to being able to modulate a particular calcium channel (e.g., CaV2.2, CaV3.1, CaV3.2, or CaV3.3), it may be desirable that the compound has very low activity with respect to the hERG K+channel, which is expressed in the heart: compounds that block this channel with high potency may cause reactions which are fatal. See, e.g., Bowiby et al., “hERG (KCNH2 or KV11.1 K+Channels: Screening for Cardiac Arrhythmia Risk,”Curr. Drug Metab.9(9):965-70 (2008)). Thus, for a compound that modulates calcium channel activity, it may also be shown that the hERG K+channel is not inhibited or only minimally inhibited as compared to the inhibition of the primary channel targeted. Similarly, it may be desirable that the compound does not inhibit cytochrome p450, an enzyme that is required for drug detoxification. Such compounds may be particularly useful in the methods described herein.

The compounds of the invention modulate the activity of calcium channels; in general, said modulation is the inhibition of the ability of the channel to transport calcium. As described below, the effect of a particular compound on calcium channel activity can readily be ascertained in a routine assay whereby the conditions are arranged so that the channel is activated, and the effect of the compound on this activation (either positive or negative) is assessed. Exemplary assays are also described in the Examples.

Pharmaceutical Compositions

For use as treatment of human and animal subjects, the compounds of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired—e.g., prevention, prophylaxis, or therapy—the compounds are formulated in ways consonant with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

The compounds described herein (e.g., a compound according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) may be present in amounts totaling 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, gastrointestinal, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

In general, for use in treatment, the compounds described herein (e.g., a compound according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) may be used alone, as mixtures of two or more compounds or in combination with other pharmaceuticals. An example of other pharmaceuticals to combine with the compounds described herein (e.g., a compound according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) would include pharmaceuticals for the treatment of the same indication. For example, in the treatment of pain, a compound may be combined with another pain relief treatment such as an NSAID, or a compound which selectively inhibits COX-2, or an opioid, or an adjuvant analgesic such as an antidepressant. Another example of a potential pharmaceutical to combine with the compounds described herein (e.g., a compound according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) would include pharmaceuticals for the treatment of different yet associated or related symptoms or indications. Depending on the mode of administration, the compounds will be formulated into suitable compositions to permit facile delivery. Each compound of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.

The compounds of the invention may be prepared and used as pharmaceutical compositions comprising an effective amount of a compound described herein (e.g., a compound according to any of Formulas (I)-(IX) or compounds (1)-(227) of Tables 4 and 5) and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, the composition includes at least two different pharmaceutically acceptable excipients or carriers.

Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. The compounds can be administered also in liposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See, for example, U.S. Pat. No. 5,624,677, which is herein incorporated by reference.

Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the invention. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

For administration to animal or human subjects, the dosage of the compounds of the invention may be, for example, 0.01-50 mg/kg (e.g., 0.01-15 mg/kg or 0.1-10 mg/kg). For example, the dosage can be 10-30 mg/kg.

Each compound of a combination therapy, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately.

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Generally, when administered to a human, the oral dosage of any of the compounds of the combination of the invention will depend on the nature of the compound, and can readily be determined by one skilled in the art. Typically, such dosage is normally about 0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and more desirably about 5 mg to 500 mg per day. Dosages up to 200 mg per day may be necessary. Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the patient. Chronic, long-term administration may be indicated.

Examples

Synthesis of the Invention Compounds

The following reaction schemes and examples are intended to illustrate the synthesis of a representative number of compounds. Accordingly, the following examples are intended to illustrate but not to limit the invention. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein. Exemplary compounds prepared according to methods known in the art and described herein are provided in Tables 4 and 5.

Purification of crude organic mixtures was conducted by a High Throughput Organic Purification (HiTOP) Laboratory using reversed phase preparative HPLC. Two approaches were utilized depending on the nature of the target; a low pH approach (Table 1) or a high pH approach (Table 2). Analytical scale chromatography, as known in the art, was used to determine the type of preparative method required for each sample as well as to conduct final purity checks and product confirmation on collected final material.

Preparative HPLC was performed using the following method specific parameters and the assigned “Narrow” method (Table 3).

TABLE 3NARROW METHOD PARAMETERSINJECTIONAim to load a maximum of 100 mg of crudeVOLUME:materialCOLUMNAmbientTEMPERATURE:GRADIENTGradient of Solvents A and B (as below)PROFILE:Narrow MethodTimeFlowSolvent B (%)Step(min)(mL/min)ABCDEF10.042.510152739516321.542.510152739516332.042.5—25374961—49.542.5404759718373510.542.5409595959595611.542.5959595959595TOTAL RUN11.5 minutes (Run can be terminated early onceTIME:target is collected)Scan mode:PDA @ 220 nm and MS Scan from from 220 m/zto 700 m/z

Procedure for the synthesis of 2-methyl-2-((3-(trifluoromethyl)phenyl) sulfonyl)propanoic acid (5)

Preparation of ethyl 2-methyl-2-(3-(trifluoromethyl)phenylthio)propanoate (3)

Preparation of ethyl 2-methyl-2-(3-(trifluoromethyl)phenylsulfonyl)propanoate (4)

Preparation of 2-methyl-2-(3-(trifluoromethyl)phenylsulfonyl)propanoic acid (5)

Procedure for the synthesis of 3-(methylsulfonyl)-5-(trifluoromethyl)picolinic acid (7)

Procedure for the synthesis of 2-(methylsulfonyl)-6-(trifluoromethyl) nicotinic acid (9)

Procedure for the synthesis of 2-(isopropylsulfonyl)-6-(trifluoromethyl) nicotinic acid (10)

Procedure for the synthesis of 2-(methylsulfonyl)-6-(trifluoromethyl) isonicotinic acid (13)

Preparation of 2-bromo-4-iodo-6-(trifluoromethyl)pyridine (12)

Preparation of 2-(methylsulfonyl)-6-(trifluoromethyl)isonicotinic acid (13)

Procedure for the synthesis of (4-((3-(trifluoromethyl)phenyl) sulfonyl)phenyl)methanamine (17)

Preparation of 4-((3-(trifluoromethyl)phenyl)thio)benzonitrile (15)

Preparation of 4-((3-(trifluoromethyl)phenyl)sulfonyl)benzonitrile (16)

Preparation of (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (17)

A slurry of Raney nickel was washed twice with MeOH to remove water and provide a enough catalytic material for the reaction. 4-((3-(Trifluoromethyl)phenyl) sulfonyl)benzonitrile (16) (8.01 g, 25.73 mmol) in MeOH (200 mL) was added to the catalyst, and the solution saturated with NH3(gas). The reaction was hydrogenated using a Parr apparatus at 55 PSI for 2 hours. The reaction was then filtered, and the filtrate was concentrated in vacuo to give (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl) methanamine (17) (7.93 g, 98%). The product was confirmed by positive ion mode LCMS and FIA MS and used without further purification.

(2-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (18) and (3-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (19) were prepared in an analogous fashion to (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (17) using the appropriately substituted fluorobenzonitrile.

(5-((3-(trifluoromethyl)phenyl)sulfonyl)pyridin-2-yl)methanamine (20) was prepared in an analogous fashion to (4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl) methanamine (17) using 5-chloropicolinonitrile.

Procedure for the synthesis of (4-((3-(trifluoromethyl)phenyl)sulfonyl) pyridin-2-yl)methanamine (24)

Preparation of 4-((3-(trifluoromethyl)phenyl)thio)picolinonitrile (22)

Preparation of 4-O-(trifluoromethyl)phenyl)sulfonyl)picolinonitrile (23)

Preparation of (4-((3-(trifluoromethyl)phenyl)sulfonyl)pyridin-2-yl)methanamine (24)

(4-((3-(trifluoromethyl)phenyl)sulfonyl)pyridin-2-yl)methanamine (24) was prepared in an analogous fashion to (4-((3-(trifluoromethyl)phenyl)sulfonyl) phenyl)methanamine (17) using (4-((3-(trifluoromethyl)phenyl)sulfonyl)picolinonitrile (23).

Procedure for the synthesis of 2-(trifluoromethyl)-6-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (28)

Preparation of ethyl 2-methyl-6-(trifluoromethyl)nicotinate (26)

Preparation of ethyl 2-(bromomethyl)-6-(trifluoromethyl)nicotinate (27)

Preparation of 2-(trifluoromethyl)-6-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (28)

Procedure for the synthesis of 2-(trifluoromethyl)-5-(3-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (34)

Preparation of methyl 3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (32)

Preparation of methyl 1-(2-bromoethyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (33)

Preparation of 2-(trifluoromethyl)-5-(3-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (34)

Methyl 1-(2-bromoethyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (33) (100 mg, 0.33 mmol), DIPEA (0.29 mL, 1.67 mmol) and (3-((3-(trifluoromethyl) phenyl)sulfonyl)phenyl)methanamine (18) (97 mg, 0.33 mmol) were stirred in DMF (3 mL) in a sealed vessel at 200° C. for 45 minutes in a microwave reactor. The reaction was concentrated in vacuo, and the residue purified by mass directed reverse phase HPLC to give 2-(trifluoromethyl)-5-(3-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (34)

2-(trifluoromethyl)-5-(2-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (35) was prepared in an analogous manner using (2-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (19)

General Coupling Protocols for Diarylsulfone Compounds

Stoichiometries given are to be considered exemplary and can be varied. Suitable organic bases may be used as alternates to TEA (e.g., DIPEA). Suitable coupling agents may be used as an alternative to HATU (e.g. EDC/HOBt). For HCl salts, at least one additional equivalent of base to that described must be employed. DCM may be substituted for DMF as solvent.

a. General Coupling Protocol for the Synthesis of Compounds with General Structure (36)

Exemplified by the synthesis 2-(methylsulfonyl)-4-(trifluoromethyl)-N-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)benzamide (38)

B. General protocol for BOC amino acids amide coupling exemplified by the synthesis (R)—N-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)pyrrolidine-2-carboxamide (40)

(4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (17) (100 mg, 0.34 mmol), HATU (178 mg, 0.48 mmol), TEA (197 μL, 1.41 mmol), and (R)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (40a) (87 mg, 0.34 mmol) were stirred in DMF (1 mL) at room temperature for 16 hours. The reaction was concentrated in vacuo, and the residue was treated with 2M HCl in Et2O at room temperature for 5 hours. The reaction was then quenched with NaHCO3saturated solution, and the organics were separated, dried, and concentrated in vacuo. The residue was purified by mass directed reverse phase HPLC to give (R)—N-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl) pyrrolidine-2-carboxamide (40).

C. B. General protocol for BOC amino acids amide coupling exemplified by the synthesis 2-(1-aminocyclohexyl)-N-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl) acetamide (41)

(4-((3-(trifluoromethyl)phenyl)sulfonyl)phenyl)methanamine (17) (720 mg, 2.28 mmol), EDC (570 mg, 2.99 mmol), HOBT (410 mg, 2.99 mmol), DIPEA (640 μL, 3.89 mmol), and 2-(1-((tert-butoxycarbonyl)amino)cyclohexyl)acetic acid (588 mg, 2.28 mmol) were stirred in DMF (10 mL) at room temperature for 16 hours. The reaction was concentrated in vacuo. The residue was diluted with ethyl acetate (100 ml) and then washed sequentially with saturated NH4Cl and saturated NaHCO3. The organics were dried (Na2SO4) and then concentrated in vacuo, and the residue was purified by column chromatography using EtOAc:Hexane (1:1) to give the pure intermediate (41a). The material was further dissolved in ethyl acetate, and HCl gas was bubbled for two minutes to give the final product 2-(1-aminocyclohexyl)-N-(4-((3-(trifluoromethyl)phenyl)sulfonyl)benzyl)acetamide (41) with >98% purity.

Procedure for the synthesis of 3-(methylsulfonyl)-5-(trifluoromethyl) picolinic acid (43)

Procedure for the synthesis of 2-(methylsulfonyl)-6-(trifluoromethyl) nicotinic acid (44)

Procedure for the synthesis of 2-(isopropylsulfonyl)-6-(trifluoromethyl) nicotinic acid (46)

Procedure for the synthesis of 2-(methylsulfonyl)-6-(trifluoromethyl) isonicotinic acid (49)

Preparation of 2-bromo-4-iodo-6-(trifluoromethyl)pyridine (48)

Preparation of 2-(methylsulfonyl)-6-(trifluoromethyl)isonicotinic acid (49)

2-Bromo-4-iodo-6-(trifluoromethyl)pyridine (48) (2.70 g, 7.67 mmol) was stirred under argon in dry THF (30 mL) at −10° C.iPrMgCl (2.0 M, THF, 4.5 mL, 9.0 mmol) was added, and the mixture was stirred at 0° C. for 30 minutes. CO2was bubbled through the reaction, and stirring continued for 1.5 hours while allowing to warm to room temperature. The reaction was concentrated in vacuo, taken up in DMF (20 mL), and stirred with NaSMe (0.90 g, 19 mmol) at 100° C. for 2 hours. The reaction was concentrated in vacuo, the residue was taken up in MeOH (50 mL) and H2O (50 mL) with oxone monopersulfate (30 g, 49 mmol), and the reaction stirred at room temperature for 3 hours. The reaction was filtered, the filtrate basified with 10% NaOH for 30 minutes, and the MeOH removed in vacuo. The aqueous residue was acidified with 6 N HCl and extracted with EtOAc (3×50 mL). The organics were dried (Na2SO4), concentrated in vacuo, and the residue recrystallized from EtOAc/hexanes with the presence of 1 eq. DMF to give 2-(methylsulfonyl)-6-(trifluoromethyl)isonicotinic acid (49) (1.70 g, 51%);1H NMR (300 MHz, CD3OD) δ 2.88 (s, 3H, DMF), 3.01 (s, 3H, DMF), 3.34 (s, 3H), 8.00 (s, 1H, DMF), 8.52 (s, 1H), 8.73 (s, 1H).

General procedure for the preparation of 6-phenoxypyridin-3-amines (12)

Exemplified by the procedure for 6-(3-chloro-4-fluorophenoxy)pyridin-3-amine (15)

Preparation of 2-(3-chloro-4-fluorophenoxy)-5-nitropyridine (52A)

Preparation of 6-(3-chloro-4-fluorophenoxy)pyridin-3-amine (53A)

General procedure for the synthesis of 2-methyl-2-(3-(substituted) phenylsulfonyl)propanoic acid exemplified by the synthesis of 2-methyl-2-((3-(trifluoromethyl)phenyl)sulfonyl)propanoic acid (58a)

Preparation of ethyl 2-methyl-2-(3-(trifluoromethyl)phenylthio)propanoate (56)

Preparation of ethyl 2-methyl-2-(3-(trifluoromethyl)phenylsulfonyl)propanoate (57)

Preparation of 2-methyl-2-(3-(trifluoromethyl)phenylsulfonyl)propanoic acid (58a)

Procedure for the synthesis of 2-methyl-2-((3-(trifluoromethyl)phenyl) sulfonyl)propan-1-amine (60)

Preparation of 2-methyl-2-O-(trifluoromethyl)phenyl)sulfonyl)propanamide (59)

2-Methyl-2-(3-(trifluoromethyl)phenylsulfonyl)propanoic acid (58a) (4.86 g, 16.4 mmol) and oxalyl chloride (4.3 mL, 48.5 mmol) were stirred in dry CH2Cl2(100 mL) at room temperature under Ar. DMF (cat) was added, and the reaction was stirred at room temperature for 1 hour. The solvent was removed in vacuo, dried under high vacuum for 2 hours, and the residue was then taken up in dry CH2Cl2(50 mL). NH3(gas) was bubbled through the reaction for 10 minutes, and the reaction was then stirred at room temperature for 16 hours. The reaction was diluted with DCM (50 mL) and washed sequentially with 1 N HCl, NaHCO3(saturated solution), and brine. The organics were dried (Na2SO4) and concentrated in vacuo to give 2-methyl-2-((3-(trifluoromethyl)phenyl) sulfonyl)propanamide (59) (4.83 g, 100%);1H NMR (300 MHz-CD3Cl) δ 1.54 (s, 6H), 5.75 (bs, 1H), 6.83 (bs, 1H), 7.67 (t, 1H, J=7.83 Hz), 7.89 (d, 1H, J=7.77 Hz). 8.02 (d, 1H, J=7.86 Hz), 8.08 (s, 1H). The product was used without further purification.

Preparation of 2-methyl-2-((3-(trifluoromethyl)phenyl)sulfonyl)propan-1-amine (60)

2-Methyl-2-((3-(trifluoromethyl)phenyl)sulfonyl)propanamide (59) (4.83 g, 16.4 mmol) and BH3.THF (1M solution; 52 ml, 52 mmol) were stirred in dry THF (75 mL) under Ar at reflux for 3 hours. The reaction was cooled, 6 N HCl (26 mL) added, and then the reaction was heated at reflux for 1 hour. The reaction was concentrated in vacuo, and the residue was taken up in H2O (30 mL) and washed with Et2O. The aqueous layer was filtered, and the filtrate basified with NaOH (7 g). The reaction was extracted with DCM, and the organics were dried (Na2SO4) and concentrated in vacuo to give 2-methyl-2-((3-(trifluoromethyl)phenyl)sulfonyl)propan-1-amine (60) (2.9 g, 63%);1H NMR (300 MHz-CD3Cl) δ 1.31 (s, 6H), 2.98 (s, 2H), 7.74 (m, 1H), 7.95 (m, 1H), 8.12 (m, 2H). The product was used without further purification.

Procedure for the synthesis of N,3-dimethyl-3-((3-(trifluoromethyl) phenyl)sulfonyl)butan-1-amine hydrochloride (71)

Preparation of 3-methyl-3-((3-(trifluoromethyl)phenyl)thio)butanoic acid (62)

Preparation of 3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butanoic acid (63)

Preparation of 3-methyl-3-O-(trifluoromethyl)phenyl)sulfonyl)butan-1-ol (65)

Preparation of 1-((4-azido-2-methylbutan-2-yl)sulfonyl)-3-(trifluoromethyl)benzene (67)

Preparation of 3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butan-1-amine (68)

1-((4-Azido-2-methylbutan-2-yl)sulfonyl)-3-(trifluoromethyl)benzene (67) (1 g, 3.1 mmol) and Pd(OH)2(10% w/w) were taken up in EtOH and hydrogenated in a Parr apparatus under an H2atmosphere (50 PSI) for 1 hour. The catalyst was removed by multiple filtrations, and the filtrate was concentrated in vacuo to give 3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butan-1-amine (68) (800 mg, 90%). The product was confirmed with positive ion mode LCMS and FIA MS and used without further purification.

tert-Butyl methyl(3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butyl)carbamate (70) (690 mg, 1.68 mmol) was taken up in EtOAc (40 mL). HCl gas was passed through the solution at room temperature for 5 minutes, and stirring then continued for 15 minutes. The reaction was concentrated in vacuo to give N,3-dimethyl-3-((3-(trifluoromethyl)phenyl) sulfonyl)butan-1-amine hydrochloride (71) (472 mg, 82%). The product was confirmed with positive ion mode LCMS and FIA MS and used without further purification.

Procedure for the synthesis of 3-((3-fluoro-5-(trifluoromethyl)phenyl) sulfonyl)-3-methylbutan-1-amine (72)

Preparation of N 3-(3-fluoro-5-(trifluoromethyl)phenylthio)-3-methylbutanoic acid (75)

Preparation of 3-fluoro-5-(trifluoromethyl)benzenethiol magnesium bromide (74)

Mg ribbon (1.09 g, 44.9 mmol) (cleaned with hexane/Et2O) and I2(initiator) was stirred in dry THF (75 mL) at room temperature. 1-Bromo-3-fluoro-5-(trifluoromethyl)benzene (73) (10.0 g, 41.2 mmol) was added dropwise, and the reaction stirred for 2 hours at room temperature (reaction initiated with heat gun). Sulfur (1.32 g, 41.2 mmol) was added, and the reaction stirred at room temperature for 2 hours. The reaction was filtered, and the filtrate concentrated in vacuo to give crude 3-fluoro-5-(trifluoromethyl)benzenethiol magnesium bromide (74) which was used without purification.

Preparation of 3-(3-fluoro-5-(trifluoromethyl)phenylthio)-3-methylbutanoic acid (75)

Preparation of 3-(3-fluoro-5-(trifluoromethyl)phenylsulfonyl)-3-methylbutan-1-amine (72)

3-(3-Fluoro-5-(trifluoromethyl)phenylsulfonyl)-3-methylbutan-1-amine (72) was prepared in analogous fashion to afford 3-methyl-3-(3-(trifluoromethyl)phenylsulfonyl) butan-1-amine (68) using 3-(3-fluoro-5-(trifluoromethyl)phenylthio)-3-methylbutanoic acid (75).

Procedure for the synthesis of 6-(2-methyl-2-((3-(trifluoromethyl) phenyl)sulfonyl)propyl)-2-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (79)

Preparation of ethyl 2-methyl-6-(trifluoromethyl)nicotinate (77)

Preparation of ethyl 2-(bromomethyl)-6-(trifluoromethyl)nicotinate (78)

Preparation of 6-(2-methyl-2-((3-(trifluoromethyl)phenyl)sulfonyl)propyl)-2-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (79)

Procedure for the synthesis of 6-(3-methyl-3-((3-(trifluoromethyl)phenyl) sulfonyl)butyl)-2-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (80)

6-(3-Methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butyl)-2-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (81) was synthesized in an analogous manner to 6-(2-methyl-2-((3-(trifluoromethyl)phenyl)sulfonyl)propyl)-2-(trifluoromethyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (79) using 3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butan-1-amine (68)

Procedure for the synthesis of 5-(3-methyl-3-((3-(trifluoromethyl) phenyl)sulfonyl)butyl)-2-(trifluoromethyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (85)

Preparation of methyl 3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (83)

3-(Trifluoromethyl)-1H-pyrazole-5-carboxylic acid (82) (1.0 g, 8.33 mmol) was stirred in MeOH (50 mL) at room temperature. AcCl (1.18 mL, 16.67 mmol) was added dropwise, and the reaction stirred at reflux for 2 hours. The reaction was concentrated in vacuo and partitioned between EtOAc and saturated NaHCO3solution. The organics were dried (Na2SO4) and concentrated in vacuo to give methyl 3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (83) (1.0 g, 93%); NMR (300 MHz, CDCl3) δ 3.98 (s, 3H), 7.10 (s, 1H). The product was used without purification.

Preparation of methyl 1-(2-bromoethyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (84)

Preparation of 5-(3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butyl)-2-(trifluoromethyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-oneamine (85)

Methyl 1-(2-bromoethyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxylate (84) (100 mg, 0.33 mmol), DIPEA (0.29 mL, 1.67 mmol) and 3-methyl-3-((3-(trifluoromethyl) phenyl)sulfonyl)butan-1-amine (68) (97 mg, 0.33 mmol) were stirred in DMF (3 mL) in a sealed vessel at 200° C. for 45 minutes in a microwave reactor. The reaction was concentrated in vacuo, and the residue purified by mass directed reverse phase HPLC to give 5-(3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butyl)-2-(trifluoromethyl)-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (85).

General Coupling Protocols

Stoichiometries given are to be considered exemplary and can be varied. Suitable organic bases may be used as alternates to TEA (e.g., DIPEA). Suitable coupling agents may be used as an alternative to HATU (e.g. EDC/HOBt). For HCl salts, at least one additional equivalent of base to that described must be employed. DMF may be substituted for CH2Cl2as solvent.

(A) General Coupling Protocol for the Synthesis of Compounds with General Structure (86)

Exemplified by the synthesis N-(6-(4-fluoro-3-(trifluoromethyl)phenoxy)pyridin-3-yl)-2-methyl-2-((3-(trifluoromethoxy)phenyl)sulfonyl)propanamide (88)

Preparation of N-(6-(3-chloro-4-fluorophenoxy)pyridin-3-yl)-2-methyl-2-((3-(trifluoromethoxy)phenyl)sulfonyl)propanamide (88)

(B) General protocol for BOC amino acids amide coupling exemplified by the synthesis of 2-(1-aminocyclohexyl)-N-(3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl) butyl)acetamide hydrochloride (91)

3-Methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butan-1-amine (68) (100 mg, 0.34 mmol), HATU (178 mg, 0.48 mmol), TEA (197 μL, 1.41 mmol), and 2-(1-((tert-butoxycarbonyl)amino)cyclohexyl)acetic acid (89) (87 mg, 0.34 mmol) were stirred in DMF (1 mL) at room temperature for 16 hours to afford (90). The reaction was concentrated in vacuo, the residue treated with 2M HCl in Et2O at room temperature for 5 hours, and quenched with NaHCO3saturated solution. The organics were separated, dried, and concentrated in vacuo. The residue was purified by mass directed reverse phase HPLC to give 2-(1-aminocyclohexyl)-N-(3-methyl-3-((3-(trifluoromethyl)phenyl)sulfonyl)butyl)acetamide hydrochloride (91).

N- and T-Type Channel Blocking Activities

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (1.4×106cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2incubator at 29° C.

Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in either the 2 mM KCl buffer or 12.5 mM KCl buffer and incubated for 20 minutes at 29° C. in 5% CO2. Calcium entry was evoked with an addition of 130 mM KCl stimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 130 KCl, with the pH adjusted to 7.4 with NaOH) for both the closed-state or inactivated-state assay. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA™ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing the fluorescence signal in the presence of compound and fitted with a logistic function (1) to obtain the concentration that inhibited 50% (IC50) of the RLU signal using OriginPro v.7.5 software (OriginLab, Northampton, Mass.).

To assess the quality of the FLIPR assays the Z-factor (2) was used to quantify the suitability of the assay conditions using the following equation:

Data are expressed as mean and standard deviation (SD).

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (2.0×106cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2incubator at 29° C.

(Sigma-Aldrich), was loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with the following low Ca2+buffer (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 0.001 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in the buffer containing low Ca2+and incubated for 20 minutes at 29° C. in 5% CO2. Calcium entry was evoked with an addition of (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 6 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA™ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing the fluorescence signal in the presence of compound and fitted with a logistic function (1) to obtain the concentration that inhibited 50% (IC50) of the RLU signal using OriginPro v.7.5 software (OriginLab, Northampton, Mass.).

To assess the quality of the FLIPR assays the Z-factor (2) was used to quantify the suitability of the assay conditions using the following equation:

Data are expressed as mean and standard deviation (SD).

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 1004 of cells (1.2×106cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2incubator at 29° C.

NaOH. 4.4 μM of the fluorescent indicator dye Fluo-4 (Invitrogen) prepared in pluronic acid (Sigma-Aldrich) was loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with either a 2 mM KCl closed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 2 KCl, with the pH adjusted to 7.4 with NaOH) when performing the closed-state assay or 7.6 mM KCl inactivated-state buffer (in mM: 130.9 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 7.6 mM KCl, with the pH adjusted to 7.4 with NaOH) when performing the inactivated-state assay. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich), diluted in either the 2 mM KCl buffer or 7.6 mM KCl buffer, and incubated for 20 minutes at 29° C. in 5% CO2. Calcium entry was evoked with an addition of either 12 mM KCl stimulation buffer (in mM: 128.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 12 KCl, with the pH adjusted to 7.4 with NaOH) or 14.5 mM KCl stimulation buffer (in mM: 126 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 14.5 KCl, with the pH adjusted to 7.4 with NaOH) for the closed-state or inactivated-state assay respectively. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA™ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing the fluorescence signal in the presence of compound and fitted with a logistic function (1) to obtain the concentration that inhibited 50% (IC50) of the RLU signal using OriginPro v.7.5 software (OriginLab, Northampton, Mass.).

To assess the quality of the FLIPR assays the Z-factor (2) was used to quantify the suitability of the assay conditions using the following equation:

Data are expressed as mean and standard deviation (SD).

Exemplary data obtained according to these procedures are shown in Tables 4 and 5.

Other Embodiments