Patent Application
20110118283
The present invention relates to pyrrolidine-2-carboxamide derivatives I which act as antagonists of mdm2 interactions and hence are useful as potent and selective anticancer agents. The present compounds are of the general formula
wherein R1, R2, R3, R3, R4, R5 are as described herein
and enantiomers and pharmaceutically acceptable salts and esters thereof.
p53 is a tumor suppresser protein that plays a central role in protection against development of cancer. It guards cellular integrity and prevents the propagation of permanently damaged clones of cells by the induction of growth arrest or apoptosis. At the molecular level, p53 is a transcription factor that can activate a panel of genes implicated in the regulation of cell cycle and apoptosis. p53 is a potent cell cycle inhibitor which is tightly regulated by MDM2 at the cellular level. MDM2 and p53 form a feedback control loop. MDM2 can bind p53 and inhibit its ability to transactivate p53-regulated genes. In addition, MDM2 mediates the ubiquitin-dependent degradation of p53. p53 can activate the expression of the MDM2 gene, thus raising the cellular level of MDM2 protein. This feedback control loop insures that both MDM2 and p53 are kept at a low level in normal proliferating cells. MDM2 is also a cofactor for E2F, which plays a central role in cell cycle regulation.
The ratio of MDM2 to p53 (E2F) is dysregulated in many cancers. Frequently occurring molecular defects in the p16INK4/p19ARF locus, for instance, have been shown to affect MDM2 protein degradation. Inhibition of MDM2-p53 interaction in tumor cells with wild-type p53 should lead to accumulation of p53, cell cycle arrest and/or apoptosis. MDM2 antagonists, therefore, can offer a novel approach to cancer therapy as single agents or in combination with a broad spectrum of other antitumor therapies. The feasibility of this strategy has been shown by the use of different macromolecular tools for inhibition of MDM2-p53 interaction (e.g. antibodies, antisense oligonucleotides, peptides). MDM2 also binds E2F through a conserved binding region as p53 and activates E2F-dependent transcription of cyclin A, suggesting that MDM2 antagonists might have effects in p53 mutant cells.
The present invention relates to pyrrolidine-2-carboxamide derivatives I which act as antagonists of mdm2 interactions and hence are useful as potent and selective anticancer agents. The present compounds are of the general formula
wherein
Preferred above are compounds of formula I wherein R2 is a substituted aryl, i.e., a substituted phenyl selected from
wherein
where R6, R7 are both methyl, or linked to form a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group,
Preferred are compounds of formula I having a stereochemical structure as shown as formula II
wherein
Preferred above are compounds of formula H wherein R2 is a substituted phenyl selected from
where R6, R7 are both methyl, or linked to form a cyclopropyl, cyclobutyl, cyclopentyl or acyclohexyl group,
Further preferred are compounds in formula II wherein
More preferred of the above formula II compounds are those wherein R2 is a substituted aryl, i.e., a substituted phenyl selected from
where R6, R7 are both methyl, or linked to form a cyclopropyl, cyclobutyl, cyclopentyl or acyclohexyl group,
Especially preferred are compounds selected from the group consisting of
In the specification where indicated the various groups may be substituted by 1-5 or, preferably, 1-3 substituents independently selected from the group consisting of lower alkyl, lower-alkenyl, lower-alkynyl, dioxo-lower-alkylene (forming e.g. a benzodioxyl group), halogen, hydroxy, CN, CF3, NH2, N(H, lower-alkyl), N(lower-alkyl)2, aminocarbonyl, carboxy, NO2, lower-alkoxy, thio-lower-alkoxy, lower-alkylsufonyl, aminosulfonyl, lower-alkylcarbonyl, lower-alkylcarbonyloxy, lower-alkoxycarbonyl, lower-alkyl-carbonyl-NH, fluoro-lower-alkyl, fluoro-lower-alkoxy, lower-alkoxy-carbonyl-lower-alkoxy, carboxy-lower-alkoxy, carbamoyl-lower-alkoxy, hydroxy-lower-alkoxy, NH2-lower-alkoxy, N(H, lower-alkyl)-lower-alkoxy, N(lower-alkyl)2-lower-alkoxy, lower-alkyl-1-oxiranyl-lower-alkoxy-lower-alkyl, 2-oxo-pyrrolidin-1-yl, (1,1-dioxo)-2-isothiazolidine, 3-lower-alkyl sulfinyl, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted aryl ring, a substituted or unsubstituted heteroaryl ring, trifluoro-lower-alkylsulfonylamino-aryl, lower-alkyl sulfonylaminocarbonyl, lower-alkyl sulfonylaminocarbonyl-aryl, hydroxycarbamoyl-phenyl, benzyloxy-lower-alkoxy, mono- or di-lower alkyl substituted amino-sulfonyl and lower-alkyl which can optionally be substituted with halogen, hydroxy, NH2, N(H, lower-alkyl) or N(lower-alkyl)2. Preferred substituents for the cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocycle rings are halogen, lower alkoxy, lower alkyl, hydroxycarbonyl, carboxy, carboxy lower alkoxy, oxo and CN. Preferred substituents for alkyl are alkoxy and N(lower alkyl)2.
The term “alkyl” refers to straight- or branched-chain saturated hydrocarbon groups having from 1 to about 20 carbon atoms, including groups having from 1 to about 7 carbon atoms. In certain embodiments, alkyl substituents may be lower alkyl substituents. The term “lower alkyl” refers to alkyl groups having from 1 to 6 carbon atoms, and in certain embodiments from 1 to 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, and s-pentyl.
As used herein, “cycloalkyl” is intended to refer to any stable monocyclic or polycyclic system which consists of carbon atoms only, any ring of which being saturated, and the term “cycloalkenyl” is intended to refer to any stable monocyclic or polycyclic system which consists of carbon atoms only, with at least one ring thereof being partially unsaturated. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, bicycloalkyls, including bicyclooctanes such as [2.2.2]bicyclooctane or [3.3.0]bicyclooctane, bicyclononanes such as [4.3.0]bicyclononane, and bicyclodecanes such as [4.4.0]bicyclodecane (decalin), or Spiro compounds. Examples of cycloalkenyls include, but are not limited to, cyclopentenyl or cyclohexenyl.
The term “alkenyl” as used herein means an unsaturated straight-chain or branched aliphatic hydrocarbon group containing one double bond and having 2 to 6, preferably 2 to 4 carbon atoms. Examples of such “alkenyl group” are vinyl, ethenyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl.
The term “alkynyl” as used herein means an unsaturated straight-chain or branched aliphatic hydrocarbon group containing one triple bond and having 2 to 6, preferably 2 to 4 carbon atoms. Examples of such “alkynyl group” are ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.
The term “halogen” as used in the definitions means fluorine, chlorine, bromine, or iodine, preferably, fluorine and chlorine.
“Aryl” means a monovalent, monocyclic or bicyclic, aromatic carbocyclic hydrocarbon radical, preferably a 6-10 member aromatic ring system. Preferred aryl groups include, but are not limited to, phenyl, naphthyl, tolyl, and xylyl.
“Heteroaryl” means an aromatic heterocyclic ring system containing up to two rings. Preferred heteroaryl groups include, but are not limited to, thienyl, furyl, indolyl, pyrrolyl, pyridinyl, pyrazinyl, oxazolyl, thiaxolyl, quinolinyl, pyrimidinyl, imidazole and tetrazolyl.
In the case of aryl or heteroaryl which are bicyclic it should be understood that one ring may be aryl while the other is heteroaryl and both may be substituted or unsubstituted.
“Heterocycle” means a substituted or unsubstituted 5 to 8 membered, mono- or bicyclic, non-aromatic hydrocarbon, wherein 1 to 3 carbon atoms are replaced by a hetero atom selected from nitrogen, oxygen or sulfur atom. Examples include pyrrolidin-2-yl; pyrrolidin-3-yl; piperidinyl; morpholin-4-yl and the like.
“Hetero atom” means an atom selected from N, O and S.
“Alkoxy, alkoxyl or lower alkoxy” refers to any of the above lower alkyl groups attached to an oxygen atom. Typical lower alkoxy groups include methoxy, ethoxy, isopropoxy or propoxy, butyloxy and the like. Further included within the meaning of alkoxy are multiple alkoxy side chains, e.g. ethoxy ethoxy, methoxy ethoxy, methoxy ethoxy ethoxy and the like and substituted alkoxy side chains, e.g., dimethylamino ethoxy, diethylamino ethoxy, dimethoxy-phosphoryl methoxy and the like.
“Pharmaceutically acceptable,” such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
“Pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, trifluoro acetic acid and the like.
Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide. Chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.
The compounds of formula I and II as well as their salts that have at least one asymmetric carbon atom may be present as racemic mixtures or different stereoisomers. The various isomers can be isolated by known separation methods, e.g., chromatography. Compounds disclosed herein and covered by formula I and II above may exhibit tautomerism or structural isomerism. It is intended that the invention encompasses any tautomeric or structural isomeric form of these compounds, or mixtures of such forms, and is not limited to any one tautomeric or structural isomeric form depicted in the formulas above.
The compounds of the present invention are useful in the treatment or control of cell proliferative disorders, in particular oncological disorders. These compounds and formulations containing said compounds may be particularly useful in the treatment or control of solid tumors, such as, for example, breast, colon, lung and prostate tumors.
A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration; it may be given as continuous infusion.
Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, as well as the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of a formula I compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, sachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.
“Effective amount” means an amount that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
“IC50” refers to the concentration of a particular compound required to inhibit 50% of a specific measured activity. IC50 can be measured, inter alia, as is described subsequently.
The present invention provides methods for the synthesis of substituted pyrrolidine-2-carboxamide. The compounds of the invention can be prepared by processes known in the art. Suitable processes for synthesizing these compounds are provided in the examples.
Compounds of this invention can be synthesized according to the following general schemes. The key transformation is a convergent [2+3] cylcoaddition of emine II and activated olefin III to generate pyrrolidine-3-carbonitrile compounds IV in a stereoselective and efficient manner.
The starting materials are either commercially available or can be synthesized by methods known to those of ordinary skill in the art. Preparations of intermediates II and III are illustrated in Scheme 1 and 2. In general an appropriately selected aldehyde can be reacted with glycine tert-butyl ester or glycine methyl ester to generate imine II and were used as a crude product (Scheme 1).
An intermediate of formula III can be made from a base-catalyzed condensation reaction of appropriately selected substituted-phenyl acetonitrile and aldehyde The reaction proceeds in a highly stereoselective manner with Z-isomer as the major or exclusive product.
As illustrated in Scheme 3, pyrrolidine of formula IV can be made from intermediates II and III by a convergent 1,3-dipolar cylcoaddition reaction mediated by lewis acid AgF and triethylamine. The [2+3] cycloaddition reactions of azomethine ylides 1,3-dipoles with olefinic dipolarphiles to form pyrrolidine ring formation have been described in published procedures including Jorgensen, K. A. et al (Org. Lett. 2005, Vol 7, No. 21, 4569-4572), Grigg, R. et al (Tetrahedron, 1992, Vol 48, No. 47, 10431-10442; Tetrahedron, 2002, Vol 58, 1719-1737), Schreiber, S. L. et al (J. Am. Chem. Soc., 2003, 125, 10174-10175), and Carretero, J. C. et al (Tetrahedron, 2007, 63, 6587-6602). Compounds IV is subsequently converted to acid V followed by amide formation with various amines using HATU as the coupling reagent to give the compounds of formula I. The amide formation from V to I can also be achieved under other conditions using EDCI and HOBt, or oxalyl chloride, or diphenylphosphinic chloride as the coupling reagent to activate the acid V.
The pyrrolidine compounds I, IV, V are prepared initially as a racemic mixture and can be chirally separated using chiral Super Fluid Chromatography (SFC) or chiral HPLC or chiral column chromatography. For example, racemic mixture of compound Ia and Ia′ can be readily resolved into two optically pure or enriched chiral enantiomers by separation using chiral Super Fluid Chromatography (SFC). (Scheme 4).
The compounds of the present invention may be synthesized according to known techniques. The following examples and references are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.
A mixture of glycine tert-butyl ester (Alfa) (2.71 g, 20.0 mmol) and 3,3-dimethyl-butyraldehyde (Alfa) (2.21 g, 21.0 mmol) in CH2Cl2 (50 mL) was stirred at rt overnight. The reaction mixture was concentrated and the residue was dried in vacuo to give [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester (4.29 g, 100%) as colorless oil which was used in the next step without further purification.
To a solution of 4-bromothiopene-2-acetonitrile (Matrix) (5 g, 24.7 mmol) and 3-chloro-benzaldehyde (Aldrich) (3.36 mL, 29.7 mmol) in methanol (200 mL) was slowly added a methanolic solution (Aldrich, 25 wt. %) of sodium methoxide (6.2 mL, 27.2 mmol). The reaction mixture was heated and stirred at room temperature for 1.5 h. The mixture became cloudy, and was filtered. The light yellow precipitate was washed with cold methanol, and then dried in vacu to give (Z)-2-(4-bromo-thiophen-3-yl)-3-(3-chloro-phenyl)-acrylonitrile as a light yellow solid (6.3 g, 79%).
To a solution of [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester (2.13 g, 10 mmol) and give (Z)-2-(4-bromo-thiophen-3-yl)-3-(3-chloro-phenyl)-acrylonitrile (2.59 g, 8 mmol) in dichloromethane (100 mL) were added triethyl amine (2.73 mL, 19.6 mmol) and AgF (1.51 g, 10 mmol). The mixture was stirred at room temperature for 48 h. The mixture was filtered through a short pad of silica gel. The silica gel was washed with ethyl acate. The filtrate was concentrated. The residue was purified by flash column chromatography (SiO2, 1-20% of EtOAc in hexanes) to give rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester as a light yellow solid (2.6 g, 60%)
A solution of rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester (2.6 g, 4.8 mmol) in dichloromethane (7 mL) was added trifluoroacetic acid (3 mL). The reaction mixture was stirred at room temperature for 1 h, then concentrated. The residue was then triturated with ethyl ether hexanes, concentrated, dried under reduced pressure to give rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid as light yellow solid (2.5 g, 87%).
To a solution of (4S)-(+)-4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane (Aldrich) (21.1 g, 0.14 mol) and triethylamine (40 mL, 0.28 mol) in dichloromethane (250 mL) at 0° C. was added methanesulfonyl chloride (13.4 mL, 0.17 mol) dropwise. The reaction mixture was stirred at 0° C. for 1.5 h, then water was added. The organic layer was separated, washed with water, brine, dried over MgSO4, concentrated to give methanesulfonic acid 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl ester as a yellow oil (31.7 g, 98%).
To a solution of methanesulfonic acid 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl ester (31.7 g, 0.14 mol) in N,N-dimethylformamide (200 mL) was added NaN3 (46 g, 0.71 mol). The reaction mixture was stirred at room temperature for 70 h. Then the mixture was partitioned between ethyl acetate and water. The organic layer was separated, washed with water, brine several times, dried over MgSO4, concentrated to give (S)-4-(2-azido-ethyl)-2,2-dimethyl-[1,3]dioxolane as a yellow oil (21.3 g, 88%).
A suspension of (S)-4-(2-azido-ethyl)-2,2-dimethyl-[1,3]dioxolane as a yellow oil (18.7 g, 0.11 mol) and PtO2 (2.5 g) in ethyl acetate (100 mL) was vigorously shaken in a Parr under atmosphere of H2 (50 psi) for 18 h. The mixture was filtered through a short pad of celite. The filtrate was concentrated to give 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine as a colorless oil (14 g, 88%).
A mixture of rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid (0.3 g, 0.5 mmol) in dichloromethane (10 mL) was added 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (0.22 g, 1.5 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) (0.34 g, 0.9 mmol) and iPr2NEt (0.44 mL, 2.5 mmol) respectively. The reaction mixture was stirred at room temperature for 18 h. The mixture was then diluted with CH2Cl2 and washed with water, brine. The organic phase was separated, filtered and dried over Na2SO4. The mixture was then concentrated and purified by SiO2 flash column chromatography (20-100% of EtOAc in hexanes) to give rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide as a light yellow gum (0.26 g, 84%).
To a solution of rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide (0.26 g, 0.43 mol) in tetrahydrofuran (10 mL) was added aqueous HCl solution (1N, 1 mL). The reaction mixture was stirred at room temperature for 2 h, then concentrated. Then the residue was partitioned between ethyl acetate and water. The organic layer was separated, washed with water, aqueous saturated NaHCO3, brine, dried over MgSO4, concentrated, dried under reduced pressure to give rac-(2R,3R,4R,5S)-4-(4-bromo-thiophen-2-yl)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (0.185 g, 75%).
HRMS (ES+) m/z Calcd for C25H31BrClN3O3S+H[(M+H)+]: 568.1031, found: 568.1032.
In a manner similar to the method described in Example 2, (5-bromopyridin-2-yl)acetonitrile(BetaPharma) (1 g, 5.1 mmol) was reacted with 3-chloro-2-fluorobenzaldehyde (0.96 g, 6.1 mmol), methanolic solution (25 wt %) of sodium methoxide (1.3 mL, 5.6 mmol) in methanol (30 mL) at 50° C. for 3 h to give (Z)-2-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-acrylonitrile as a grey solid (1.2 g, 71%).
In a manner similar to the method described in Example 3, [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester prepared in Example 1 (1.1 g, 5 mmol) was reacted with (Z)-2-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-acrylonitrile (1.2 g, 8.9 mmol) prepared in Example 8, AgF (0.54 g, 4.3 mmol), and triethylamine (1.24 mL, 29 mmol) in dichloromethane (80 mL) at room temperature for 18 h to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester as a light yellow solid (0.85 g, 44%).
In a manner similar to the method described in Example 4, rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester prepared in Example 9 (0.85 g, 1.5 mmol) was reacted with trifluoroacetic acid in dichloromethane at room temperature to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid as a yellow solid (0.8 g, 85%).
In a manner similar to the method described in Example 6, rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid prepared in Example 10 (0.35 g, 0.54 mmol) was reacted with 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (0.24 g, 1.6 mmol), HATU (0.37 g, 0.98 mmol) and iPr2NEt (0.47 mL, 2.7 mmol) in CH2Cl2 at room temperature to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide as a white foam (0.27 g, 75%).
In a manner similar to the method described in Example 7, rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide prepared in Example 11 (0.25 g, 0.4 mmol) was reacted with aqueous HCl solution in tetrahydrofuran at room temperature to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (0.21 g, 91%).
HRMS (ES+) m/z Calcd for C26H31BrClFN4O3+H [(M+H)+]: 581.1325, found: 581.1327.
To the solution of 5-chloro-2-(chloromethyl)pyridine (CGenetech) (0.99 g, 6.1 mmol) in ethanol (8 mL) and H2O (6 mL) was added KCN (1.03 g, 15.9 mmol). The reaction mixture was heated at 100° C. for 1 h. The mixture was cooled, and extracted with ethyl acetate. The organic layer was separated, washed with saturated aqueous NaHCO3 solution, brine, dried over MgSO4, and concentrated. The residue was purified by chromatography (EtOAc:hexanes=1:3) to give (5-chloro-pyridin-2-yl)acetonitrile as a yellow oil (0.64 g, 69%).
In a manner similar to the method described in Example 2, (5-chloropyridin-2-yl)acetonitrile (0.64 g, 4.2 mmol) was reacted with 3-chloro-2-fluorobenzaldehyde (0.8 g, 5.0 mmol), methanolic solution (25 wt %) of sodium methoxide (1.1 mL, 4.6 mmol) in methanol (30 mL) at 50° C. for 3 h to give (Z)-3-(3-chloro-2-fluoro-phenyl)-2-(5-chloro-pyridin-2-yl)-acrylonitrile as a light yellow solid (1.0 g, 81%).
To a solution of [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester prepared in Example 1 (0.95 g, 4.3 mmol) and (Z)-3-(3-chloro-2-fluoro-phenyl)-2-(5-chloro-pyridin-2-yl)-acrylonitrile (1.0 g, 3.4 mmol) prepared in Example 14 in dichloromethane (100 mL) were added triethylamine (1.19 mL, 8.5 mmol) and AgF (0.66 g, 5.2 mmol) sequentially.
The mixture was stirred at room temperature for 18 h. The mixture was then quenched with sat. NH4Cl and extracted with CH2Cl2. The organic phase was separated, filtered through celite and dried over Na2SO4, and concentrated. The residue was dissolved into tert-butanol (20 mL), and DBU (3.3 mL, 24 mmol) was added. The mixture was heated at 100° C. for 18 h, then cooled to room temperature, and concentrated. The residue was partitioned between ethyl acetate and water. The organic layer were separated, dried over MgSO4, and concentrated. The residue was purified by chromatography (EtOAc:hexanes=1;10, 1:5) to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester as a light yellow gum (1.3 g, 87%).
In a manner similar to the method described in Example 4, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester prepared in Example 15 (1.3 g, 2.6 mmol) was reacted with trifluoroacetic acid in dichloromethane at room temperature to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid as a light yellow solid (1.15 g, 79%).
In a manner similar to the method described in Example 6, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid prepared in Example 16 (0.45 g, 0.80 mmol) was reacted with 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (0.35 g, 2.4 mmol), HATU (0.54 g, 1.4 mmol) and iPr2NEt (0.69 mL, 4.0 mmol) in CH2Cl2 at room temperature to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide as a light yellow gum (0.3 g, 65%).
In a manner similar to the method described in Example 7, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide prepared in Example 17 (0.3 g, 0.52 mmol) was reacted with aqueous HCl solution in tetrahydrofuran at room temperature to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (0.25 g, 89%).
HRMS (ES+) m/z Calcd for C26H31Cl2FN4O3+H[(M+H)+]: 537.1830, found: 537.1828.
Rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide (0.22 g) was separated by chiral SFC chromatography to provide chiral-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (63 mg, 29%) and chiral-(2S,3R,4R,5R)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (62 mg, 28%).
HRMS (ES+) m/z Calcd for C26H31Cl2FN4O3+H[(M+H)+]: 537.1830, found: 537.1830.
To a solution of ethyl cyanoacetate (Aldrich) (4 g, 35.4 mmol) in anhydrous DMSO (30 mL) at 0° C. was added slowly NaH (60%, 1.42 g, 35.6 mmol). The mixture was stirred at 0° C. for 0.5 h, then 5-chloro-2,3-difluoropyridine (Combi-Blocks) (5.3 g, 35.6 mmol) was added. The reaction mixture was stirred at room temperature for 18 h. Water was added. The organic layer was separated, the aqueous layer was then extracted with ethyl acetate twice. The combined organic layers were washed with brine, dried over MgSO4, concentrated. The residue was purified by chromatography (EtOAc:hexanes=1:4, 1:1) to give (5-chloro-3-fluoro-pyridin-2-yl)-acetonitrile as a yellow gum (3.2 g, 37%).
To the solution of (5-chloro-3-fluoro-pyridin-2-yl)-acetonitrile (2.8 g, 11.5 mmol) in DMSO (30 mL) was added NaCl (2 g, 34 mmol). The reaction mixture was heated at 170° C. for 2 h. The mixture was partitioned between ethyl acetate and water. Organic layer was separated, the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over MgSO4, and concentrated. The residue was purified by chromatography (EtOAc:hexanes=1:4, 1:3) to give (5-chloro-3-fluoro-pyridin-2-yl)-acetonitrile as a brown oil (0.85 g, 43%).
In a manner similar to the method described in Example 2, (5-chloro-3-fluoro-pyridin-2-yl)-acetonitrile (0.85 g, 5.0 mmol) was reacted with 3-chloro-2-fluorobenzaldehyde (0.8 g, 5.0 mmol), methanolic solution (25 wt %) of sodium methoxide (1.1 g, 5 mmol) in methanol (50 mL) at room temperature for 3 h to give (Z)-3-(3-chloro-2-fluoro-phenyl)-2-(5-chloro-3-fluoro-pyridin-2-yl)-acrylonitrile as a white solid (1.24 g, 80%).
To a solution of [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester prepared in Example 1 (1.1 g, 5 mmol) and (Z)-3-(3-chloro-2-fluoro-phenyl)-2-(5-chloro-3-fluoro-pyridin-2-yl)-acrylonitrile (1.24 g, 4 mmol) prepared in Example 21 in dichloromethane (100 mL) were added triethylamine (1.39 mL, 10 mmol) and AgF (0.77 g, 6.1 mmol) sequentially. The mixture was stirred at room temperature for 18 h. The mixture was then quenched with sat. NH4Cl and extracted with CH2Cl2. The organic phase was separated, filtered through celite and dried over Na2SO4, and concentrated. The residue was dissolved into tert-butanol (10 mL), and DBU (4.8 mL, 32 mmol) was added. The mixture was heated at 100° C. for 18 h, then cooled to room temperature, and concentrated. The residue was partitioned between ethyl acetate and water. The organic layer were separated, dried over MgSO4, and concentrated. The residue was purified by chromatography (EtOAc:hexanes=1;10, 1:5) to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester as a light yellow gum (1.0 g, 48%).
In a manner similar to the method described in Example 4, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester prepared in Example 22 (1.0 g, 1.9 mmol) was reacted with trifluoroacetic acid in dichloromethane at room temperature to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid as a yellow solid (1.0 g, 90%).
In a manner similar to the method described in Example 6, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid prepared in Example 23 (0.45 g, 0.77 mmol) was reacted with 24(S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (0.34 g, 2.3 mmol), HATU (0.53 g, 1.4 mmol) and iPr2NEt (0.67 mL, 3.9 mmol) in CH2Cl2 at room temperature to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide as a light yellow gum (0.2 g, 44%).
In a manner similar to the method described in Example 7, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide prepared in Example 24 (0.2 g, 0.33 mmol) was reacted with aqueous HCl solution in tetrahydrofuran at room temperature to give rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-3-fluoro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (0.1 g, 53%).
HRMS (ES+) m/z Calcd for C26H30Cl2F2N4O3+H[(M+H)+]: 555.1736, found: 555.1736.
To a solution of tert-butyl cyanoacetate (Alfa) (1.5 g, 11 mmol) in anhydrous tetrahydrofuran (100 mL) at 0° C. was added slowly NaH (60%, 1.0 g, 25 mmol). The mixture was stirred at 0° C. for 0.5 h, then 5-bromo-2-chloropyrimidine (TCI-US) (2.5 g, 13 mmol) was added. The reaction mixture was stirred at room temperature for 18 h. Water was added. The mixture was partitioned between ethyl acetate and water. The organic layer was separated, the aqueous layer was then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over MgSO4, concentrated. The residue was trituated with ethyl acetate and hexanes, and the mixture was filtered to give (5-bromo-pyrimidin-2-yl)-cyano-acetic acid tert-butyl ester as a yellow precipitate (3.2 g, 98%).
To the solution of (5-bromo-pyrimidin-2-yl)-cyano-acetic acid tert-butyl ester (3.2 g, 10 mmol) in dichloromethane (30 mL) was added trifluoroacetic acid (10 mL). The reaction mixture was stirred at room temperature for 4 h. The mixture was concentrated. To the residue was added water, and the “pH” of the mixture was adjusted to neutral by saturated aqueous NaHCO3 solution. The mixture was then extracted with ethyl acetate. Organic layer was separated, the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over MgSO4, and concentrated. The residue was purified by chromatography (EtOAc:hexanes=1:3, 1:2, 1;1) to give (5-bromo-pyrimidin-2-yl)-acetonitrile as a white solid (1.2 g, 71%).
In a manner similar to the method described in Example 2, (5-bromo-pyrimidin-2-yl)-acetonitrile (2.2 g, 13 mmol) was reacted with 3-chloro-2-fluorobenzaldehyde (2.2 g, 14 mmol), methanolic solution (25 wt %) of sodium methoxide (3 mL, 13 mmol) in methanol (30 mL) at room temperature for 3 h to give (Z)-2-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-acrylonitrile as a white solid (2.9 g, 66%).
To a solution of [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester prepared in Example 1 (1.1 g, 5 mmol) and (Z)-2-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-acrylonitrile (1.33 g, 4 mmol) prepared in Example 27 in dichloromethane (100 mL) were added triethylamine (1.39 mL, 10 mmol) and AgF (0.77 g, 6.1 mmol) sequentially. The mixture was stirred at room temperature for 18 h. The mixture was then quenched with sat. NH4Cl and extracted with CH2Cl2. The organic phase was separated, filtered through celite and dried over Na2SO4, and concentrated. The residue was dissolved into tert-butanol (10 mL), and DBU (4.8 mL, 32 mmol) was added. The mixture was heated at 100° C. for 18 h, then cooled to room temperature, and concentrated. The residue was partitioned between ethyl acetate and water. The organic layer were separated, dried over MgSO4, and concentrated. The residue was purified by chromatography (EtOAc:hexanes=1:3) to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester as a yellow gum (0.45 g, 20%).
In a manner similar to the method described in Example 4, rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester prepared in Example 28 (0.45 g, 0.82 mmol) was reacted with trifluoroacetic acid in dichloromethane at room temperature to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid as a light yellow solid (0.36 g, 88%).
In a manner similar to the method described in Example 6, rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid prepared in Example 29 (0.36 g, 0.71 mmol) was reacted with 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (0.31 g, 2.1 mmol), HATU (0.48 g, 1.28 mmol) and iPr2NEt (0.62 mL, 3.55 mmol) in CH2Cl2 at room temperature to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide as a white gum (0.28 g, 64%).
In a manner similar to the method described in Example 7, rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide prepared in Example 30 (0.28 g, 0.45 mmol) was reacted with aqueous HCl solution in tetrahydrofuran at room temperature to give rac-(2R,3S,4S,5S)-4-(5-bromo-pyrimidin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a light yellow solid (0.21 g, 81%).
HRMS (ES+) m/z Calcd for C25H30BrClFN5O3+H[(M+H)+]: 582.1278, found: 582.1282.
In a manner similar to the method described in Examples 6, rac-(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid prepared in Example 16 (0.3 g, 0.52 mmol) was reacted with methyl 4-aminobenzoate (Acros)(0.12 g, 0.79 mmol), HATU (0.36 g, 0.96 mmol) and iPr2NEt (0.23 mL, 1.3 mmol) in CH2Cl2 at room temperature to give rac-4-{[(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-benzoic acid methyl ester as a white solid (50 mg, 16%).
HRMS (ES+) m/z Calcd for C30H29Cl2FN4O3+H[(M+H)+]: 583.1674, found: 583.1674.
To a solution of rac-4-{[(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-benzoic acid methyl ester prepared in Example 32 (30 mg, 0.05 mmol) in tetrahydrofuran (3 mL) was added an aqueous solution (1 N) of NaOH (3 mL, 3 mmol) and methanol (1 mL). The reaction mixture was stirred at room temperature for 18 h, and the “pH” of the solution was adjusted to 5-6 by aqueous HCl solution. The mixture was extracted ethyl acetate twice. The combined organic extracts were washed with water, brine, dried over MgSO4, and concentrated. The residue was triturate with dichlormethane and hexanes to give rac-4-{[(2R,3S,4S,5S)-3-(3-chloro-2-fluoro-phenyl)-4-(5-chloro-pyridin-2-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-benzoic acid as a off white solid (28 mg, 82%).
HRMS (ES+) m/z Calcd for C29H27Cl2FN4O3+H[(M+H)+]: 569.1517, found: 569.1518.
In a manner similar to the method described in Examples 6, rac-(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid trifluoroacetic acid prepared in Example 10 (0.24 g, 0.39 mmol) was reacted with methyl 4-aminobenzoate (Acros)(0.59 g, 3.9 mmol), HATU (0.27 g, 0.7 mmol) and iPr2NEt (0.17 mL, 0.98 mmol) in CH2Cl2 at room temperature to give rac-4-{[(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-benzoic acid methyl ester as a white solid (50 mg, 20%).
HRMS (ES+) m/z Calcd for C30H29BrClFN4O3+H[(M+H)+]: 627.1169, found: 627.1167.
To a solution rac-4-{[(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-benzoic acid methyl ester prepared in Example 34 (35 mg, 0.056 mmol) in tetrahydrofuran (3 mL) was added an aqueous solution (1 N) of NaOH (3 mL, 3 mmol) and methanol (1 mL). The reaction mixture was stirred at room temperature for 18 h, and the “pH” of the solution was adjusted to 5-6 by aqueous HCl solution. The mixture was extracted ethyl acetate twice. The combined organic extracts were washed with water, brine, dried over MgSO4, and concentrated to rac-4-{[(2R,3S,4S,5S)-4-(5-bromo-pyridin-2-yl)-3-(3-chloro-2-fluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-benzoic acid as a white solid (28 mg, 82%).
HRMS (ES+) m/z Calcd for C29H27BrClFN4O3+H[(M+H)+]: 627.1169, found: 627.1167.
To a solution of 3-chlorobenzaldehyde (0.7 g, 5 mmol) and 3-pyridylacetonitrile (TCI-Japan) (0.59 g, 5 mmol) in iPrOH (20 mL) was added aqueous solution (2 N) of NaOH (0.3 mL, 0.6 mmol). The reaction mixture was stirred at room temperature for 48 h. The mixture was partitioned between ethyl acetate and water. The organic layer was separated, washed with water, brine, dried over Na2SO4, and concentrated. The residue was purified by flash column chromatography (10-70% of AcOEt in hexanes) to give (Z)-3-(3-chloro-phenyl)-2-pyridin-3-yl-acrylonitrile as a yellow foam (0.33 g, 28%).
In a manner similar to the method described in Example 3, [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester prepared in Example 1 (0.42 g, 2 mmol) was reacted with (Z)-3-(3-chloro-phenyl)-2-pyridin-3-yl-acrylonitrile (0.31 g, 1.3 mmol) prepared in Example 36, AgF (0.25 g, 2 mmol), and triethylamine (0.4 g, 4 mmol) in 1,2-dichloroethane (10 mL) at room temperature for 18 h to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-4-pyridin-3-yl-pyrrolidine-2-carboxylic acid tert-butyl ester as a light yellow solid (0.29 g, 49%).
HRMS(ES+) m/z Calcd for C26H32ClN3O2+H[(M+H): 454.2256; Found: 454.2258.
A solution of rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-4-pyridin-3-yl-pyrrolidine-2-carboxylic acid tert-butyl ester (0.25 g, 0.55 mmol) in conc. H2SO4 (2 mL) was stirred at room temperature for 2 h. The mixture was then poured into ice and extracted with ethyl acetate. The organic layer was separated, dried over Na2SO4, and concentrated. The residue was purified by chromatography (15-25% EtOAc in hexanes) to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-4-pyridin-3-yl-pyrrolidine-2-carboxylic acid as a white solid (0.19 g, 87%).
HRMS(ES+) m/z Calcd for C22H24ClN3O2+H[(M−H):398.1628; Found: 398.1630.
A mixture of rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-4-pyridin-3-yl-pyrrolidine-2-carboxylic acid (80 mg, 0.2 mmol), 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (44 mg, 0.3 mmol), HATU (114 mg, 0.3 mmol) and iPr2NEt (0.1 mL, 1 mmol) in CH2Cl2 (2 mL) was stirred at room temperature for 1 h. The mixture was then diluted with CH2Cl2 and washed with water, brine. The organic phase was separated, filtered and dried over Na2SO4. The mixture was then concentrated and to the residue was added PPTS (cat) and methanol (2 mL). The reaction mixture was heated under microwave irradiation in CEM microwave reactor at 120° C. for 5 min. The mixture was concentrated and the residue was diluted with EtOAc and washed with water, brine. The organic phase was separated, dried over Na2SO4, and concentrated. The residue was purified by SiO2 flash column chromatography (5% of MeOH in EtOAc) to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-4-pyridin-3-yl-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a white amorphous (63 mg, 61%).
HRMS (ES+) m/z Calcd for C26H33ClN4O3+H[(M+H)+]: 485.2314; Found: 485.2311.
To a solution of 3-chlorobenzaldehyde (1.4 g, 10 mmol) and 2-chloro-5-(cyanomethyl)pyridine (Matrix)(1.52 g, 10 mmol) in iPrOH (20 mL) was added aqueous solution (2 N) of NaOH (0.6 mL, 1.2 mmol). The reaction mixture was stirred at room temperature for 20 h. The mixture was partitioned between ethyl acetate and water. The organic layer was separated, washed with water, brine, dried over Na2SO4, and concentrated. The residue was purified by flash column chromatography (10-70% of AcOEt in hexanes) to give (Z)-3-(3-chloro-phenyl)-2-(6-chloro-pyridin-3-yl)-acrylonitrile as a white solid (1.85 g, 67%).
HRMS (ES+) m/z Calcd for C14H8Cl2N2+H[(M+H): 275.0138; Found: 275.0137.
In a manner similar to the method described in Example 3, [3,3-dimethyl-but-(E)-ylideneamino]-acetic acid tert-butyl ester prepared in Example 1 (0.53 g, 2.5 mmol) was reacted with (Z)-3-(3-chloro-phenyl)-2-(6-chloro-pyridin-3-yl)-acrylonitrile (0.55 g, 2 mmol) prepared in Example 40, AgF (0.32 g, 2.5 mmol), and triethylamine (0.5 g, 5 mmol) in 1,2-dichloroethane (20 mL) at room temperature for 18 h to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester as a light yellow solid (0.13 g, 14%).
HRMS(ES+) m/z Calcd for C26H31Cl2N3O2+H[(M+H): 488.1866; Found: 488.1864.
A solution of rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid tert-butyl ester (0.36 g, 0.73 mmol) in conc. H2SO4 (1 mL) and acetonitrile (3 mL) was heated under microwave irradiaition at 120° C. for 10 min. The mixture was then poured into ice-water, and the “pH” was adjusted to neutral by aqueous NaOH solution. The mixture was extracted with ethyl acetate. The organic layer was separated, dried over Na2SO4, and concentrated. The residue was purified by chromatography (15-25% EtOAc in hexanes) to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid as a white amorphous (0.21 g, 24%).
HRMS(ES+) m/z Calcd for C22H23Cl2N3O2+H[(M+H): 432.1240; Found: 432.1241.
In a manner similar to the method described in Example 6, rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid (0.2 g, 0.46 mmol) was reacted with 2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethylamine (0.1 g, 0.69 mmol), HATU (0.26 g, 0.69 mmol) and iPr2NEt (0.12 mL, 0.92 mmol) in CH2Cl2 at room temperature to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide as a white amorphous (0.21 g, 81%).
HRMS(ES+) m/z Calcd for C29H36Cl2N4O3+H[(M+H): 559.2237; Found: 559.2234.
To a solution of rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid [2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-amide (70 mg, 0.27 mmol) in methanol (2 mL) was added PPTS (cat.). The reaction mixture was heated under microwave irradiation in CEM microwave reactor at 120° C. for 5 min. The mixture was concentrated and the residue was diluted with EtOAc and washed with water, brine. The organic phase was separated, dried over Na2SO4, and concentrated. The residue was purified by SiO2 flash column chromatography (50-100% EtOAc in hexanes) to give rac-(2R,3R,4R,5S)-3-(3-chloro-phenyl)-4-(6-chloro-pyridin-3-yl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carboxylic acid ((S)-3,4-dihydroxy-butyl)-amide as a white amorphous (47 mg, 73%).
HRMS(ES+) m/z Calcd C26H32Cl2N4O3+H[(M+H): 519.1924; Found: 519.1925.
To a stirred solution of (2R,3S,4S,5S)-3-(3-chloro-2-fluorophenyl)-4-(5-chloro-3-fluoropyridin-2-yl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid (214 mg, 0.457 mmol) in 4 ml methylene chloride, diphenylphosphinic chloride (Aldrich, 216, 0.174 ml) was added followed by DIPEA (Aldrich, 177 mg, 0.239 ml). The solution was stirred for 10 min. at rt and then methyl 4-amino-3-methoxybenzoate (Aldrich, 91 mg, 0.50 mmol) was added and the mixture was stirred at rt overnight. The solvent was reduced to about 3 ml and loaded on a 40 g silica gel column and eluted on a esco machine (3-5% EtOAc/CH2Cl2) to givea solid, which was again chromatographied on a reverse phase column (50-95% CH3CN/H2O) to give 55 mg white solid.
MS (ES+) m/z Calcd for C31H30Cl2F2N4O4+H[(M+H)+]: 631, found: 631.
In a 25 mL round-bottomed flask, (2R,3S,4S,5S)-3-(3-chloro-2-fluorophenyl)-4-(5-chloro-3-fluoropyridin-2-yl)-4-cyano-5-neopentylpyrrolidine-2-carboxylic acid 2,2,2-trifluoroacetic acid (1:1) salt(150 mg, 258 μmol, Eq: 1.00), was combined with CH2Cl2 (3 ml) to give a suspension. N-ethyl-N-isopropylpropan-2-amine (117 mg, 157 μL, 902 mol, Eq: 3.5) and diphenylphosphinic chloride (Aldrich, 152 mg, 123 μL, 644 μmol, Eq: 2.5) were added and the reaction was stirred at RT for 10 minutes. Methyl 4-amino-3-fluorobenzoate (Aldrich, 43.6 mg, 258 μmol, Eq: 1.00) was added and the reaction mixture was stirred at RT for 4 days and then concentrated on Rotor Vac.
The crude material was dissolved in DMSO and was purified by preparative HPLC (65-100% ACN/H2O, 0.1% TFA). The fractions was combined, concentrated and freeze dried to give a yellow solid (7.5 mg, 4.7% yield) as desired product. MS (ES+) m/z Calcd for C30H27Cl2F3N4O3+H[(M+H)+]: 619, found: 619.
The ability of the compounds to inhibit the interaction between p53 and MDM2 proteins was measured by an HTRF (homogeneous time-resolved fluorescence) assay in which recombinant GST-tagged MDM2 binds to a peptide that resembles the MDM2-interacting region of p53 (Lane et al.). Binding of GST-MDM2 protein and p53-peptide (biotinylated on its N-terminal end) is registered by the FRET (fluorescence resonance energy transfer) between Europium (Eu)-labeled anti-GST antibody and streptavidin-conjugated Allophycocyanin (APC).
Test is performed in black flat-bottom 384-well plates (Costar) in a total volume of 40 uL containing:90 nM biotinylate peptide, 160 ng/ml GST-MDM2, 20 nM streptavidin-APC (PerkinElmerWallac), 2 nM Eu-labeled anti-GST-antibody (PerkinElmerWallac), 0.2% bovine serum albumin (BSA), 1 mM dithiothreitol (DTT) and 20 mM Tris-borate saline (TBS) buffer as follows: Add 10 uL of GST-MDM2 (640 ng/ml working solution) in reaction buffer to each well. Add 10 uL diluted compounds (1:5 dilution in reaction buffer) to each well, mix by shaking. Add 20 uL biotinylated p53 peptide (180 nM working solution) in reaction buffer to each well and mix on shaker. Incubate at 37° C. for 1 h. Add 20 uL streptavidin-APC and Eu-anti-GST antibody mixture (6 nM Eu-anti-GST and 60 nM streptavidin-APC working solution) in TBS buffer with 0.2% BSA, shake at room temperature for 30 minutes and read using a TRF-capable plate reader at 665 and 615 nm (Victor 5, Perkin ElmerWallac). If not specified, the reagents were purchased from Sigma Chemical Co.
Activity data for some of the Example compounds expressed as IC50 :bsa:0.02% are as follows:
This application claims the benefit of U.S. Provisional Application No. 61/261,913, filed Nov. 17, 2009, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61261913 | Nov 2009 | US |
