Amidophenyl-sulfonylamino-quinoxaline compounds of formula (I)are CCK2 modulators useful in the treatment of CCK2 mediated diseases.

FIELD OF THE INVENTION

There is provided by the present invention compounds that are CCK2 receptor modulators. More particularly, there is provided by the present invention quinoxalines that are CCK2 receptor modulators useful for the treatment of disease states mediated by CCK2 receptor activity.

BACKGROUND OF THE INVENTION

This invention relates to gastrin and cholecystokinin (CCK) receptor ligands. The invention also relates to methods for preparing such ligands and to compounds that are useful intermediates in such methods. The invention further relates to pharmaceutical compositions comprising such ligands and methods for preparing such pharmaceutical compositions.

The gastrins and cholecystokinins are structurally related neuropeptides that exist in gastrointestinal tissue, gastrinomas and, in the case of the cholecystokinins, the central nervous system (J. H. Walsh, Gastrointestinal Hormones, L. R. Johnson, ed., Raven Press, New York, 1994, p. 1).

Several forms of gastrin are found including 34-, 17- and 14-amino acid species with the minimum active fragment being the C-terminal tetrapeptide (TrpMetAspPhe-NH2), which is reported in the literature to have full pharmacological activity (H. J. Tracy and R. A. Gregory, Nature (London), 1964, 204:935-938). Much effort has been devoted to the synthesis of analogs of this tetrapeptide (and the N-protected derivative Boc-TrpMetAspPhe-NH2) in an attempt to elucidate the relationship between structure and activity.

Natural cholecystokinin is a 33 amino acid peptide (CCK-33), the C-terminal 5 amino acids of which are identical to those of gastrin. Also found naturally is the C-terminal octapeptide (CCK-8) of CCK-33. A review of CCK receptors, ligands and the activities thereof may be found in P. de Tullio et al. (Exp. Opin. Invest. Drugs, 2000, 9(1):129-146).

Gastrin and cholecystokinin are key regulators of gastrointestinal function. In addition, cholecystokinin is a neurotransmitter in the brain. Gastrin is one of the three primary stimulants of gastric acid secretion. In addition to the acute stimulation of gastric acid, gastrin has a trophic effect on the gastrointestinal mucosa and is implicated as a trophic hormone of several adenocarcinomas, including pancreatic, colorectal, esophageal and small cell lung.

Cholecystokinin stimulates intestinal motility, gallbladder contraction, and pancreatic enzyme secretion, and is known to have trophic actions on the pancreas thus increasing, inter alia, pancreatic enzyme production. Cholecystokinin also inhibits gastric emptying and has various effects in the central nervous system, including regulation of appetite and pain.

Gastrin acts on CCK2 (otherwise known as gastrin/CCK-B receptors) whereas cholecystokinin acts on both CCK2 and CCK1 receptors (otherwise known as cholecystokinin/CCK-A receptors). Compounds that bind to cholecystokinin and/or gastrin receptors are important because of their potential pharmaceutical use as antagonists of the natural peptides or mimetics of the natural peptides acting as partial or full agonists at the cholecystokinin and/or gastrin receptors. A selective gastrin receptor antagonist has not yet been marketed. However, several are currently undergoing clinical evaluation. JB95008 (gastrazole) is being developed by The James Black Foundation and Johnson & Johnson Pharmaceutical Research & Development LLC for the potential treatment of advanced pancreatic cancer (pancreatic adenocarcinoma), and is currently in Phase II clinical trials. ML Laboratories and Panos are developing L-365,260 (Colycade), which is in Phase II clinical trials for pain. Other potential indications included eating disorders and cancer. YF-476 (formerly YM-220), under joint development by Yamanouchi and Ferring Research Institute, is in Phase I clinical trials for gastro-esophageal reflux disease (GERD). In Phase I trials, Zeria Pharmaceutical is investigating Z-360, an orally available 1,5-benzodiazepine derivative (WO-09825911), as a potential treatment for gastroduodenal ulcers and reflux esophagitis. CR 2945 (itriglumide), an orally active anthranilic acid derivative, has been investigated by Rotta in Phase I trials for anxiety disorders, cancer (particularly colon cancer) and peptic ulcer.

Gastrimmune, Aphton Corporation's anti-gastrin vaccine, which works by chemical neutralization of the hormone, is undergoing late stage clinical trials for cancer indications, in particular, pancreatic and gastric tumors.

In addition to those indications described above, gastrin (CCK2) antagonists have been proposed for the following gastrin-related disorders: gastrointestinal ulcers, Barrett's esophagus, antral G cell hyperplasia, pernicious anaemia, Zollinger-Ellison syndrome, and other conditions in which lower gastrin activity or lower acid secretion is desirable.

Cholecystokinin (CCK1) receptors have been shown to mediate cholecystokinin-stimulated gallbladder contraction, pancreatic enzyme secretion, satiety, gastric emptying inhibition and regulation of peristalsis, indicating a key role in the integrated physiological gastrointestinal response to a meal. In addition, there is evidence that cholecystokinin receptors mediate a mitogenic action of cholecystokinin on some adenocarcinomas. Consequently, selective cholecystokinin receptor antagonists, for example, devazepide (Merck), lorglumide (Rotta), 2-NAP (JBF), dexloxiglumide (Rotta), and lintitript (Sanofi) have been examined in the clinic for potential applications in, inter alia, irritable bowel syndrome, chronic constipation, non-ulcer dyspepsia, acute and chronic pancreatitis, biliary disease and pancreatic cancer. Additional roles of cholecystokinin receptors include the regulation of appetite and metabolism, indicating potential therapeutic applications in the treatment of disorders such as obesity and anorexia nervosa. Other possible uses are in the potentiation of opiate (for example morphine) analgesia and in the treatment of cancers, especially of the pancreas. Moreover, ligands for cholecystokinin/gastrin receptors in the brain have been claimed to possess anxiolytic activity, and gastrin receptor antagonists would be expected to act as neurological agents towards the relief of anxiety and related neuroses and psychoses.

SUMMARY OF THE INVENTION

The invention features a quinoxaline sulfonamide compound of formula (I):

wherein
R1and R2are each independently selected from the group consisting ofa) H, C1-7alkyl, C2-7alkenyl, C2-7alkynyl, C3-7cycloalkyl, C3-7cycloalkenyl, benzo-fusedC4-7cycloalkyl where the point of attachment is a carbon atom adjacent to the ring junction, C3-7cycloalkylC1-7alkyl,b) naphthyl-(CRs2)—, benzoylC0-3alkyl-(CRs2)—, phenyl, said phenyl optionally fused at two adjacent carbon atoms to Rf, phenyl-(CRs2)—, said phenyl optionally fused at two adjacent carbon atoms to Rf,Rfis a linear 3- to 5-membered hydrocarbon moiety having 0 or 1 unsaturated bonds and having 0, 1 or 2 carbon members which is a carbonyl,c) Ar6—(CRs2)—, where Ar6is a 6-membered heteroaryl having carbon as a point of attachment, having 1 or 2 heteroatom members which are —N═ and optionally benzo fused,d) Ar5—(CRs2)—, where Ar5is a 5-membered heteroaryl having carbon as a point of attachment, having 1 heteroatom member selected from the group consisting of O, S, >NH or >NC1-4alkyl, having 0 or 1 additional heteroatom member which is —N═ and optionally benzofused,e) Ar6-6—(CRs2)—, where Ar6-6is phenyl having the point of attachment and fused to a 6-membered heteroaryl having 1 or 2 heteroatom members which are —N═,f) Ar6-5—(CRs2)—, where Ar6-5is phenyl having the point of attachment and fused to a 5-membered heteroaryl having 1 heteroatom member selected from the group consisting of O, S, >NH or >NC1-4alkyl and having 0 or 1 additional heteroatom member which is —N═,g) C1-4alkylO- and HSC1-4alkyl,where R1and R2are not simultaneously H and, except in positions where Rsis indicated, each of a) to g) is substituted with 0, 1, 2, or 3 of Rq,Rqis independently selected from the group consisting of C1-4alkyl, hydroxy, fluoro, chloro, bromo, iodo, trifluoromethyl, aminoC1-4alkyl, C1-4alkylaminoC1-4alkyl, diC1-4alkylaminoC1-4alkyl, HO—C1-4alkyl, C1-4alkylO—C1-4alkyl, HS—C1-4alkyl, C1-4alkylS-C1-4alkyl, C1-4alkoxy and C1-4alkylS-,Rsis independently selected from the group consisting of H, C1-4alkyl, perhaloC1-4alkyl, mono- or di-haloC1-4alkyl, aminoC1-4alkyl, C1-4alkylaminoC1-4alkyl, diC1-4alkylaminoC1-4alkyl, HO—C1-4alkyl, HS—C1-4alkyl, C1-4alkylO—C1-4alkyl, C1-4alkylS-C1-4alkyl and phenyl;or, alternatively,
R1and R2may be taken together with the nitrogen to which they are attached and are selected from the group consisting ofi) 10-oxa-4-aza-tricyclo[5.2.1.02,6]dec-4-yl, optionally mono- or di-substituted with Rp,Rpis independently selected from the group consisting of hydroxy, C1-4alkyl, hydroxyC1-4alkyl, phenyl, mono-, di- or tri-halo substituted phenyl and hydroxyphenyl,ii) a 4-7 membered heterocyclic ring said heterocyclic ring having 0 or 1 additional heteroatom members separated from the nitrogen of attachment by at least one carbon member and selected from O, S, —N═, >NH or >NRp, having 0, 1 or 2 unsaturated bonds, having 0, 1 or 2 carbon members which is a carbonyl, optionally having one carbon member which forms a bridge and having 0, 1 or 2 substituents Rp,iii) a benzo fused 4-7 membered heterocyclic ring said heterocyclic ring having 0 or 1 additional heteroatom members separated from the nitrogen of attachment by at least one carbon member and selected from O, S, —N═, >NH or >NRp, having 0 or 1 additional unsaturated bonds, having 0, 1 or 2 carbon members which is a carbonyl, having 0, 1, 2, or 3 halo substituents on the benzene ring only and having 0, 1 or 2 substituents Rp,iv) a 4-7 membered heterocyclic ring said heterocyclic ring having 0 or 1 additional heteroatom members separated from the nitrogen of attachment by at least one carbon member and selected from O, S, —N═, >NH or >NRp, having 0, 1 or 2 unsaturated bonds, having 0, 1 or 2 carbon members which is a carbonyl and optionally having one carbon member which forms a bridge, the heterocyclic ring fused at two adjacent carbon atoms forming a saturated bond or an adjacent carbon and nitrogen atom forming a saturated bond to a 4-7 membered hydrocarbon ring, having 0 or 1 possibly additional heteroatom member, not at the ring junction, selected from O, S, —N═, >NH or >NRp, having 0, 1 or 2 unsaturated bonds, having 0, 1 or 2 carbon members which is a carbonyl and having 0, 1 or 2 substituents Rp;v) 8-oxo-1,5,6,8-tetrahydro-2H,4H-1,5-methano-pyrido[1,2-a][1,5]diazocin-3-yl, optionally having 0, 1 or 2 substituents Rp;
Rais independently selected from the group consisting of C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl, phenyl, furanyl, thienyl, benzyl, pyrrol-1-yl, —OH, —OC1-6alkyl, —OC3-6cycloalkyl, -Ophenyl, -Obenzyl, —SH, —SC1-6alkyl, —SC3-6cycloalkyl, -Sphenyl, -Sbenzyl, —CN, —NO2, —N(Ry)Rz(wherein Ryand Rzare independently selected from H, C1-4alkyl or C1-6cycloalkylC1-4alkyl), —(C═O)C1-4alkyl, —SCF3, halo, —CF3, —OCF3, and —COOC1-4alkyl, or, alternatively, two adjacent Ra, may be taken together with the carbons of attachment to form a fused ring and selected from the group consisting of phenyl, pyridyl and pyrimidinyl;
or alternatively, R2and one of Racan be taken together to be —CH2— or >C═O and to form a fused ring to the phenyl;
Rbis, independently, selected from the group consisting of C1-4alkyl and halogen;
and enantiomers, diastereomers, hydrates, solvates and pharmaceutically acceptable salts, esters and amides thereof.

The invention also features pharmaceutical compositions containing such compounds and methods of using such compositions in the treatment or prevention of disease states mediated by CCK2 receptor activity.

DETAILED DESCRIPTION

Preferably, R1and R2are independently selected from the group consisting of H,

where each of a) to g) is substituted with 0, 1, 2, or 3 of Rqand for those groups in which Rsis hydrogen, up to one Rsmay be other than hydrogen.

It is preferred that one of R1and R2is H or C1-4alkyl where the other is not H or C1-4alkyl. It is also preferred that one of R1and R2is H, methyl or ethyl.

In another preferred embodiment, at least one of R1and R2are selected from the groups consisting of

with the proviso that said Rsis not hydrogen, said phenyl is optionally fused at two adjacent carbon atoms to Rfand, except in positions where “Rs” or “H” is specifically indicated, each member is substituted with 0, 1, 2, or 3 of Rq.

Preferably, Rfis selected from the group consisting of —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and —(C═O)CH2CH2CH2—.

Most preferably, Rsis selected from the group consisting of H, methyl, ethyl, hydroxymethyl, fluoromethyl and dimethylaminomethyl.

Most preferably, Rqis selected from the group consisting of methyl, hydroxy, fluoro, chloro, bromo, iodo and trifluoromethyl.

Preferably, R1and R2taken together with the nitrogen to which they are attached are selected from the group consisting of

iii) 3,4-dihydro-2H-quinolin-1-yl, 3,4-dihydro-2H-quinolin-2-yl, 2,3-dihydro-indol-1-yl, 1,3-dihydro-isoindol-2-yl, 1-oxo-1,3-dihydro-isoindol-2-yl, tetrahydro-benzo[b, c or d]azepin-1-yl, 2,3-dihydro-benzo[e or f][1,4]oxazepin-4-yl, where each member of iii) in each ring has 0 or 1 unsaturated bond and has 0, 1 or 2 carbon members which are a carbonyl,

iv) decahydro-quinolin-1-yl, octahydro-isoquinolin-2-yl, octahydro-[1 or 2]pyrindin-1 or 2-yl, octahydro-indol-1-yl, octahydro-isoindol2-yl, hexahydro-cyclopenta[b]pyrrol-1-yl, hexahydro-cyclopenta[c]pyrrol-2-yl, (5,6,7 or 8-H or Rp)-decahydro-[1,5 or 1,6 or 1,7 or 1,8]naphthyridin-1-yl, (5,6,7 or 8-H or Rp)-decahydro-[2,5 or 2,6 or 2,7 or 2,8]naphthyridin-2-yl, 1-H or Rp-octahydro-pyrrolo[2,3-c]pyridin-6-yl, 2-H or Rp-octahydro-pyrrolo[3,4-c]pyridin-5-yl, 1-H or Rp-octahydro-pyrrolo[3,2-c]pyridin-5-yl, 1-H or Rp-octahydro-pyrrolo[2,3-b]pyridin-7-yl, 6-H or Rp-octahydro-pyrrolo[3,4-b]pyridin-1-yl, 1-H or Rp-octahydro-pyrrolo[3,2-b]pyridin-4-yl, 5-H or Rp-octahydro-pyrrolo[3,4-c]pyridin-2-yl, 6-H or Rp-octahydro-pyrrolo[2,3-c]pyridin-1-yl, 1-H or Rp-octahydro-pyrrolo[3,4-b]pyridin-6-yl, 7-H or Rp-octahydro-pyrrolo[2,3-b]pyridin-1-yl, octahydro-1,5-methano-pyrido[1,2-a][1,5]diazocin-3-yl, where each member of iv) in each ring has 0, 1 or 2 carbon members which is a carbonyl, each ring of attachment has 0 or 1 unsaturated bonds and each secondary ring has 0, 1 or 2 unsaturated bonds,

where each member of i), ii), iii), iv) or v) is further substituted with 0, 1 or 2 of Rp.

Preferably, there is one Ra. More preferably, there is one Rapositioned on the ring para to the amide substituent.

Preferably, where two adjacent Raare taken together with the carbons of attachment to form a fused ring, the fused ring is phenyl.

Preferably, Rbis absent or selected from the group consisting of methyl, ethyl, I, F, Cl and Br.

Preferred compounds of the present invention are selected from the group consisting of:

Additional preferred compounds of the present invention are selected from the group consisting of:

The features and advantages of the invention are apparent to one of ordinary skill in the art. Based on this disclosure, including the summary, detailed description, background, examples, and claims, one of ordinary skill in the art will be able to make modifications and adaptations to various conditions and usages. Publications described herein are incorporated by reference in their entirety.

The amidophenyl-sulfonylamino-quinoxalines of formula (I) may be produced by a number of reaction schemes. In Scheme A, sulfonylation is the final step of the process and in Scheme B, sulfonylation is the initial step of the process. Persons skilled in the art will recognize that certain compounds are more advantageously produced by one scheme as compared to the other.

Referring to Scheme A, commercially available aminonaphthoic acid A1 is reacted with triphosgene and Hünig's base to produce the benzofused isatoic anhydride species of the genus A2. Various isatoic anhydrides A2 are available commercially. An amine is acylated with the isatoic anhydride A2 to produce a benzamide A5. Benzamide A5 may also be obtained from commercially available anthranilic acid A3 through peptide coupling. Benzamide A5 may additionally be obtained from commercially available nitrobenzoic acid A4 through peptide coupling followed by reduction of the nitro group. In one synthetic pathway, benzamide A5 is sulfonylated with quinoxaline sulfonyl chloride D1 to produce quinoxaline sulfonamide compounds (I). In a second synthetic pathway, benzamide A5 is first sulfonylated with the sulfonyl chloride to produce benzothiadiazole compounds A6. This first step is followed by reduction of the benzothiadiazole to extrude sulfur resulting in phenylene diamine A7, which is condensed with glyoxal to produce quinoxaline sulfonamide compounds (I). Where Raor Rbis a primary or secondary amine or hydroxy, they can be protected with common protecting groups. In the case of the primary or secondary amine, there can be employed Boc or Cbz. In the case of hydroxy, there can be employed TBS, TES or benzyl. Of course, a precursor substituent may be employed in the reaction steps and later transformed into the desired substituent. For example, where A6 is produced with Raas nitro, the nitro may be reduced to the amine, and the amine may be, for example, alkylated, acylated, diazotized, etc.

Referring to Scheme B, aniline B1 is sulfonylated to sulfonamide B2. In the case that R″ is ester or cyano, the ester or cyano is hydrolyzed to the carboxylic acid B3. In a first route, acid B3 undergoes peptide coupling under standard conditions with an amine to produce benzothiadiazole compounds A6. This coupling is followed by reduction of the benzothiadiazole to extrude sulfur resulting in phenylene diamine A7, which is condensed with a two carbon synthon to produce quinoxaline sulfonamide compounds (I). In a second route, acid B3 is reduced to extrude sulfur resulting in phenylene diamine B4, which is condensed with a two-carbon synthon to produce quinoxaline sulfonamide C3. Sulfonamide C3 undergoes peptide couple coupling under standard conditions to produce quinoxaline sulfonamide compounds (I). Where Raor Rbis a primary or secondary amine or hydroxy, it can be protected with common protecting groups. In the case of the primary or secondary amine, there can be employed Boc or Cbz. In the case of hydroxy, there can be employed TBS, TES or benzyl. Of course, a precursor substituent may be employed in the reaction steps and later transformed into the desired substituent. For example, where B4 is produced with Raas nitro, the nitro may be reduced to the amine and the amine may be, for example, alkylated, acylated, diazotized, etc. R′ may be selected from suitable protecting groups, including alkyl protecting groups, benzyl protecting group and silyl protecting groups.

Referring to Scheme C, aniline C1 is sulfonylated to quinoxaline C2. In the case that R″ is an ester or cyano, the ester or cyano is hydrolyzed to the acid C3. Acid C3 undergoes peptide coupling under standard conditions with an amine to produce quinoxaline sulfonamide compounds (I). Where Raor Rbis a primary or secondary amine or hydroxy, it can be protected with common protecting groups. In the case of the primary or secondary amine, there can be employed Boc or Cbz. In the case of hydroxy, there can be employed TBS, TES or benzyl. Of course, a precursor substituent may be employed in the reaction steps and later transformed into the desired substituent. For example, where B4 is produced with Raas nitro, the nitro may be reduced to the amine and the amine may be, for example, alkylated, acylated, diazotized, etc. R′ may be selected from suitable protecting groups, including alkyl protecting groups, benzyl protecting group and silyl protecting groups.

Referring to Scheme D, phenylene diamine is condensed with glyoxal to produce hydroxy quinoxaline. This is followed by acylation with thionocarbamoyl chloride producing a thionocarbamate. The thionocarbamate is isomerized by heating to a thiocarbamate, where good yields are obtained with heating to 240 C for about 45 minutes. Finally, the thiocarbamate is saponified to the corresponding thiol and immediately thereafter oxidized to the sulfonylchloride.

The compounds of the present invention are CCK2 modulators and, as disclosed herein, many are demonstrated CCK2 antagonists. As such, the compounds are useful in the treatment of CCK2 mediated disease states. Particularly, the compounds may be used in the treatment or prevention of pancreatic adenocarcinoma, pain, eating disorders, gastro-esophageal reflux disease, gastroduodenal ulcers, reflux esophagitis, anxiety, colon cancer, peptic ulcers, pancreatic tumors, gastric tumors, Barrett's esophagus, antral G cell hyperplasia, pernicious anaemia and Zollinger-Ellison syndrome. Particularly, CCK2 antagonists are now in development for the treatment or prevention of pancreatic adenocarcinoma, pain, gastro-esophageal reflux disease, gastroduodenal ulcers, reflux esophagitis, anxiety, colon cancer, peptic ulcers, pancreatic tumors and gastric tumors.

It is anticipated that the compounds of the invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical administration, and inhalation. For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension. Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose. Cornstarch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil. For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

Effective doses of the compounds of the present invention may be ascertained by conventional methods. The specific dosage level required for any particular patient will depend on a number of factors, including severity of the condition being treated, the route of administration and the weight of the patient. In general, however, it is anticipated that the daily dose (whether administered as a single dose or as divided doses) will be in the range 0.01 to 1000 mg per day, more usually from 1 to 500 mg per day, and most usually from 10 to 200 mg per day. Expressed as dosage per unit body weight, a typical dose will be expected to be between 0.0001 mg/kg and 15 mg/kg, especially between 0.01 mg/kg and 7 mg/kg, and most especially between 0.15 mg/kg and 2.5 mg/kg.

EXAMPLES

In order to illustrate the invention, the following examples are included. These examples do not limit the invention. They are only meant to suggest a method of practicing the invention. Those skilled in the art may find other methods of practicing the invention, which are obvious to them. However, those methods are deemed to be within the scope of this invention.

Protocol for Preparative Reversed-Phase HPLC

Protocol for HPLC (Reversed-Phase)

Hewlett Packard Series 1100

Mass spectra were obtained on an Agilent series 1100 MSD using electrospray ionization (ESI) in either positive or negative modes as indicated.

NMR spectra were obtained on either a Bruker model DPX400 (400 MHz) or DPX500 (500 MHz) spectrometer. The format of the1H NMR data below is: chemical shift in ppm down field of the tetramethylsilane reference (multiplicity, coupling constant J in Hz, integration).

Alternatively, quinoxaline-5-sulfonic acid [5-iodo-2-(morpholine-4-carbonyl)-phenyl]amide could be prepared by the following procedure:

To a stirred solution of S—(S)-2-methyl-propane-2-sulfinic acid 4-fluoro-benzylideneamide (0.81 g, 3.1 mmol) in DCM (20 mL) at −50° C. was added a solution of methyl magnesium bromide (3.0 M in diethyl ether, 2.4 mL, 7.2 mmol). The reaction mixture was stirred at −50° C. for 1 h then allowed to warm slowly to room temperature overnight. The reaction was quenched by the addition of a satd. aq. NH4Cl solution, and the mixture was poured into H2O and extracted with DCM (3×). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. Purification by flash chromatography (EtOAc/hexanes) provided the title compound as a colorless viscous oil (0.86 g, 98%, 94% de). Major diastereomer:1H NMR (400 MHz, CDCl3): 7.33-7.27 (m, 2H), 7.06-6.98 (m, 2H), 4.56 (dq, J=6.6, 3.2 Hz, 1H), 3.30 (br d, J=2.3 Hz, 1H), 1.52 (d, J=6.7 Hz, 3H), 1.20 (s, 9H).

A. (S)-1-(2,4-Dichloroorophenyl)ethylamine hydrochloride. The amine was prepared according to the procedures described in EXAMPLE 1, Steps H through J, starting with (R)-tert-butanesulfinamide.

A. (S)-3-Methylmorpholine. (S)-3-Methylmorpholine was prepared as described for the (R) enantiomer (EXAMPLE 12, Steps A and B) but starting with (S)-2-aminopropan-1-ol.

A. (S,S)-3,5-Dimethylmorpholine-4-carboxylic acid tert-butyl ester and (S,R)-meso-3,5-Dimethylmorpholine-4-carboxylic acid tert-butyl ester. A mixture of (S)-2-aminopropan-1-ol (8.5 g, 110 mmol), hydroxyacetone (10.9 g, 147 mmol), and PtO2(0.10 g, 0.44 mmol) was combined with methanol (200 mL) in a 1 L Parr bottle. The reaction vessel was placed on a Parr shaker for 14 h under an atmosphere of 30 psi of hydrogen. The catalyst was removed by filtration through a pad of diatomaceous earth, rinsing with excess methanol. The filtrate was concentrated in vacuo to provide a mixture of diastereomeric aminodiols as a viscous yellow liquid [7:5 (S,S):(S,R) based on crude1H NMR]. The crude diol mixture (5.0 g, 37.5 mmol) was stirred in a 150 mL thick-walled sealable reaction vessel as 40 mL concentrated H2SO4was added slowly (significant exotherm observed). The vessel was sealed and heated at 140° C. for 7 h. The dark brown mixture was poured into 100 mL crushed ice, and the flask was rinsed into the reaction mixture with 50 mL of H2O. The resulting mixture was cooled in an ice bath and made basic by the slow addition of 10 N NaOH. The aqueous mixture was extracted with diethyl ether (3×300 mL). Salts began to precipitate from the aqueous layer. The aqueous layer was filtered through a sintered glass funnel, and the precipitated salts were washed with H2O (100 mL). The aqueous filtrate was further extracted with diethyl ether (6×200 mL). The combined organic layers were dried (MgSO4) and concentrated in vacuo to give a mixture of cis- and trans-dimethylmorpholines as an orange liquid (1.8 g, 41%). To a mixture of the unpurified dimethylmorpholine isomers (1.8 g, 16 mmol), NaOH (1.2 g, 30 mmol), and H2O (7 mL) was added di-tert-butyl-dicarbonate (3.2 g, 15 mmol) in one portion at room temperature. The mixture was stirred overnight, then was poured into H2O (30 mL) and extracted with diethyl ether (3×30 mL). The combined organic layers were dried (MgSO4) and concentrated in vacuo to give the mixture of Boc-protected morpholines as an orange liquid. The diastereomers were separated by flash chromatography (EtOAc/petroleum ether) to provide (S,S)-3,5-dimethylmorpholine-4-carboxylic acid tert-butyl ester (2.0 g, 59%). TLC (10% EtOAc/petroleum ether): Rf=0.41.1H NMR (500 MHz, CDCl3): 3.85-3.78 (m, 4H), 3.49-3.43 (m, 2H), 1.47 (s, 9H), 1.29 (d, J=6.4 Hz, 6H). In addition, (S,R)-meso-3,5-dimethylmorpholine-4-carboxylic acid tert-butyl ester (0.90 g, 27%) was obtained. TLC (10% EtOAc/petroleum ether): Rf=0.33.1H NMR (500 MHz, CDCl3): 3.93 (dq, J=7.0, 3.9 Hz, 2H), 3.70 (d, J=11.5 Hz, 2H), 3.55 (dd, J=11.5, 3.9 Hz, 2H), 1.47 (s, 9H), 1.30 (d, J=7.0 Hz, 6H).
B. (S,S)-3,5-Dimethylmorpholine. Hydrogen chloride gas was bubbled into a stirred solution of. (S,S)-3,5-dimethylmorpholine-4-carboxylic acid tert-butyl ester (2.0 g, 9.2 mmol) in methanol (20 mL) at 0° C. over a 10 min period. The reaction was allowed to stir for 20 min at 0° C. then for 5 h at room temperature. The methanol was removed in vacuo, and the residue was partitioned between diethyl ether and 2 N NaOH. The layers were separated, and the aqueous layer was extracted with diethyl ether (4×). The combined organic layers were dried (MgSO4) and concentrated in vacuo to give the title morpholine as a yellow oil (0.64 g, 61%).1H NMR (500 MHz, CDCl3): 3.70 (dd, J=11.0, 3.1 Hz, 2H), 3.31 (dd, J=11.0, 5.7 Hz, 2H), 3.20-3.12 (m, 2H), 1.47 (br s, 1H), 1.12 (d, J=6.7 Hz, 6H).
C. (S,R)-meso-3,5-Dimethylmorpholine. Hydrogen chloride gas was bubbled into a stirred solution of (S,R)-meso-3,5-dimethylmorpholine-4-carboxylic acid tert-butyl ester (0.90 g, 4.2 mmol) in methanol (20 mL) at 0° C. over a 10 min period. The reaction was allowed to stir for 20 min at 0° C. then for 5 h at room temperature. The methanol was removed in vacuo, and the residue was partitioned between diethyl ether and 2 N NaOH. The layers were separated, and the aqueous layer was extracted with diethyl ether (4×). The combined organic layers were dried (MgSO4) and concentrated in vacuo to give the title morpholine as a yellow oil.1H NMR (500 MHz, CDCl3): 3.78-3.68 (m, 2H), 3.02-2.92 (m, 4H), 1.50 (br s, 1H), 0.97 (d, J=7.5 Hz, 6H).
D. (R,S)-Quinoxaline-5-sulfonic acid [2-(3,5-dimethylmorpholine-4-carbonyl)-5-iodophenyl]-amide. A suspension of 4-iodo-2-(quinoxaline-5-sulfonylamino)benzoic acid (EXAMPLE 4, Step D; 0.050 g, 0.14 mmol) was heated at reflux in thionyl chloride (5 mL) for 30 min. The reaction became homogeneous. The thionyl chloride was removed in vacuo, and the residue was re-concentrated from toluene (3×) to remove residual thionyl chloride. The acid chloride was obtained as an off-white solid. The acid chloride was stirred in toluene (5 mL) at 90° C. with (S,R)-meso-3,5-dimethylmorpholine (50 mg, 0.43 mmol) for 1 h. The reaction mixture was poured into 1 N HCl and extracted with DCM (3×). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by flash chromatography (EtOAc/hexanes) to provide 32 mg (50%) of the desired amide as a solid. MS (ESI): mass calculated for C21H21IN4O4S, 552.0; m/z found, 551 [M−H]−. HPLC (reverse phase): RT=8.77 min.1H NMR (400 MHz, CDCl3, rotameric broadening): 9.04 (d, J=1.8 Hz, 1H), 9.00 (d, J=1.8 Hz, 1H), 8.58 (br s, 1H), 8.56 (dd, J=7.0, 1.5 Hz, 1H), 8.38 (dd, J=8.4, 1.4 Hz, 1H), 7.93 (dd, J=8.4, 7.4 Hz, 1H), 7.81 (d, J=1.4 Hz, 1H), 7.38 (dd, J=8.0, 1.6 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 4.15-4.02 (br m, 2H), 3.74 (d, J=11.6 Hz, 2H), 3.56 (dd, J=11.5, 3.6 Hz, 2H), 1.34 (d, J=7.0 Hz, 6H).

Examples 41 through 96 were prepared using the methods described above.

Assay Methods

Binding Assay

Assay Development

Zinc Finger Proteins (ZFP) specific for the CCK2R gene were identified by Sangamo Biosciences. The ZFP domain was fused with the herpes simplex virus VP16 activation domain, and the fusion protein was subsequently cloned into the pcDNA3 mammalian expression vector (Invitrogen, San Diego, Calif.). Tet-inducible cell lines expressing the coding region from the ZFP vector were created using the T-REx-293™ cell line (Invitrogen). After 2 weeks of selection in culture medium containing 400 mg/mL Zeocin (Invitrogen), sixty drug-resistant stable clones were isolated and analyzed for ZFP expression as well as CCK2R induction upon addition of doxycycline to the culture medium. The cell line with the most appropriate CCK2R ZFP construct was used in all further assays and was termed the HEKZFP cell line.
Cell Culture
HEKZFP cells were grown in DMEM supplemented with L-glutamine (2 mM), penicillin (50 units/mL) and streptomycin (50 μg/mL) and 10% FBS (v/v). HEKZFP cells were treated with 2 mM doxycycline (Sigma-Aldrich, MO; USA) for 2 days to de-repress the tet-regulated expression of the CCK2 receptor selective zinc finger proteins and were harvested using a rubber cell scraper.
Membrane Preparation
Membranes were prepared from the HEKZFP cells after induction. Frozen cell pellets (−40° C.) were thawed in 14 mL of buffer A (10 mM HEPES, 130 mM NaCl, 4.7 mM KCl, 5 mM MgCl, 1 mM EGTA and 15.4 mg/100 mL bacitracin at pH 7.2), adapted from E. A. Harper et al. (Br. J. Pharmacol. (1996) 118(7):1717-1726). The thawed pellets were homogenized using a Polytron PT-10 (7×1 s). The homogenates were centrifuged for 5 min at 1500 rpm (600×g), and the resulting pellets were discarded. The supernatants were re-centrifuged in order to collect the receptor-membrane pellets (25 min 15,000 rpm; 39,800×g), which were re-suspended in buffer A.
Incubation Conditions
All assays were conducted in 96-well plates (GF/B millipore filter plates) using buffer A. For the optimal cell number determination experiments, cells in concentrations ranging from 2.5×105to 12.5×105cells/well were incubated with 20 pM [125I]-BH-CCK-8S (50 μL 60 pM solution) in a total volume of 150 μL. Total binding of [125I]-BH-CCK-8S was determined in the presence of 15 μL of buffer A. Non-specific binding of [125I]-BH-CCK-8S was determined in the presence of 15 μL of 10 μM YF476, a CCK-2 receptor selective antagonist that is structurally unrelated to the radioligand [125]-BH-CCK-8S. The assay preparation was incubated for 1 h at 21±3° C., and then the assay was terminated by rapid filtration of the preparation under reduced pressure. The loaded filters were washed three times using undiluted PBS (100 μL), and then 100 μL of scintillation fluid was added to the filter plate. Bound radioactivity was determined using a Topcount (Packard BioScience, Meriden, Conn.) with a count time of 1 min. From these experiments a cell concentration of 1 pellet in 15 mL of buffer was chosen for use in other assays. To validate the radioligand concentration and incubation time for the assay, saturation and kinetic binding studies were also conducted (see M. F. Morton, The Pharmacological Characterization of Cholecystokinin Receptors in the Human Gastrointestinal Tract. PhD Thesis, University of London, 2000). The affinity of novel compounds was estimated by incubating membrane preparations with 15 μL of competing ligand (0.1 pM-1 mM) for 60 min at 21±3° C. The assay was then terminated according to the procedure outlined above.
Data Analysis
The pKi values were determined using the equation of Y.-C. Cheng and W. H. Prusoff (Biochem. Pharmacol., 1973, 22(23):3099-3108):

Ki=IC501+[L]KD
To circumvent problems associated with computer-assisted data analysis of compounds with low affinity, the data obtained in the current study were weighted according to a method described by Morton. In brief, 100% and 0% specific binding were defined independently using total binding and binding obtained in the presence of a high concentration of the reference antagonist, 2-NAP.

TABLE 1EXpKi18.127.737.947.657.867.577.587.497.4107.3117.2127.1136.8146.5157.9167.4177.2187.2197.1207.1216.9226.8236.6246.5256.5266.5276.5286.4296.3306.2327.2337.9348.1357.8367.6376.7386.5397.1406.6427.6438.0457.5487.7507.2537.6547.4567.4577.4587.2617.4627.8637.5647.6667.3676.2717.1727.0737.1747.5757.2767.6777.3787.4796.6927.1946.4966.2
Guinea-Pig Gastric Corpeal Muscle Assay
CCK2 receptor-mediated muscle contraction was measured in an isolated muscle-strip assay of guinea-pig gastric corpeal muscle according to the methods described by Roberts et al. (S. P. Roberts, E. A. Harper, G. F. Watt, V. P. Gerskowitch, R. A. Hull, N. P. Shankley, and J. W. Black, Br. J. Pharmacol., 1996, 118(7):1779-1789). In brief, strips of muscle were dissected and suspended in isolated tissue organ baths for isotonic muscle contraction recording. The baths, containing Krebs-Henseleit solution, were maintained at 24° C. and gassed continuously with 95% O2and 5% CO2. CCK1 receptors known to be present in this assay were blocked using a selective concentration of a suitable CCK1 receptor antagonist (e.g. 2-NAP). The effectiveness of the test compounds was assessed by measuring their effect on contractile concentration-response curves obtained using a well-characterized surrogate for the hormone gastrin (pentagastrin). The title compound of Example 2 behaved as a competitive antagonist in this assay with a pKBvalue of 8.8.