# Patent

## Title
N-arylsulfonyl aza-bicyclic derivatives as potent cell adhesion inhibitors

## Abstract

Compounds of Formula I are antagonists of VLA-4 and/or alpha4/beta7, and as such are useful in the inhibition or prevention of cell adhesion and cell-adhesion mediated pathologies. These compounds may be formulated into pharmaceutical compositions and are suitable for use in the treatment of AIDS-related dementia, allergic conjunctivitis, allergic rhinitis, Alzheimer's disease, asthma, atherosclerosis, autologous bone marrow transplantation, certain types of toxic and immune-based nephritis, contact dermal hypersensitivity, inflammatory bowel disease including ulcerative colitis and Crohn's disease, inflammatory lung diseases, inflammatory sequelae of viral infections, meningitis, multiple sclerosis, multiple myeloma, myocarditis, organ transplantation, psoriasis, pulmonary fibrosis, restenosis, retinitis, rheumatoid arthritis, septic arthritis, stroke, tumor metastasis, uveititis, and type I diabetes.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is based on, and claims priority from, U.S provisional application No. 60/277,233, filed Mar. 20, 2001, which is hereby incorporated by reference in its entirety.


SUMMARY OF THE INVENTION
The compounds of the present invention are antagonists of the VLA-4 integrin (“very late antigen-4”; CD49d/CD29; or α4β1), the α4β7 integrin (LPAM-1 and α4βp), and/or the α9β1 integrin, and are useful in the treatment, prevention and suppression of diseases mediated by VLA-4-, α4β7-, and/or α9β1-binding and cell adhesion and activation.
BACKGROUND OF THE INVENTION
The present invention relates to potent substituted N-arylsulfonylated-proline derivatives which are useful for the inhibition and prevention of leukocyte adhesion and leukocyte adhesion-mediated pathologies. This invention also relates to compositions containing such compounds and methods of treatment using such compounds.
Many physiological processes require that cells come into close contact with other cells and/or extracellular matrix. Such adhesion events may be required for cell activation, migration, proliferation and differentiation. Cell-cell and cell-matrix interactions are mediated through several families of cell adhesion molecules (CAMs) including the selecting, integrins, cadherins and immunoglobulins. CAMs play an essential role in both normal and pathophysiological processes. Therefore, the targeting of specific and relevant CAMs in certain disease conditions without interfering with normal cellular functions is essential for an effective and safe therapeutic agent that inhibits cell-cell and cell-matrix interactions.
The integrin superfamily is made up of structurally and functionally related glycoproteins consisting of a and b heterodimeric, transmembrane receptor molecules found in various combinations on nearly every mammalian cell type. VLA-4 (“very late antigen-4”; CD49d/CD29; or α4β1) is an integrin expressed on all leukocytes, except platelets and mature neutrophils, including dendritic cells and macrophage-like cells and is a key mediator of the cell-cell and cell-matrix interactions of these cell types. The ligands for VLA-4 include vascular cell adhesion molecule-1 (VCAM-1) and the CS-1 domain of fibronectin (FN). VCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells at sites of inflammation. VCAM-1 is produced by vascular endothelial cells in response to pro-inflammatory cytokines The CS-1 domain is a 25 amino acid sequence that arises by alternative splicing within a region of fibronectin. A role for VLA-4/CS-1 interactions in inflammatory conditions has been proposed (see M. J. Elices, “The integrin α4β1 (VLA-4) as a therapeutic target” in Cell Adhesion and Human Disease, Ciba Found. Symp., John Wiley & Sons, NY, 1995, p. 79).
α4β7 (also referred to as LPAM-1 and α4βp) is an integrin expressed on leukocytes and is a key mediator of leukocyte trafficking and homing in the gastrointestinal tract. The ligands for α4β7 include mucosal addressing cell adhesion molecule-1 (MadCAM-1) and, upon activation of α4β7, VCAM-1 and fibronectin (Fn). MadCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells of gut-associated mucosal tissues of the small and large intestine (“Peyer's Patches”) and lactating mammary glands. MadCAM-1 can be induced in vitro by proinflammatory stimuli. MadCAM-1 is selectively expressed at sites of lymphocyte extravasation and specifically binds to the integrin, α4β7.
The α9β1 integrin is found on airway smooth muscle cells, non-intestinal epithelial cells, and neutrophils, and, less so, on hepatocytes and basal keratinocytes. Neutrophils, in particular, are intimately involved in acute inflammatory responses. Attenuation of neutrophil involvement and/or activation would have the effect of lessening the inflammation. Thus, inhibition of α9β1 binding to its respective ligands would be expected to have a positive effect in the treatment of acute inflammatory conditions.
Neutralizing anti-α4 antibodies or blocking peptides that inhibit the interaction between VLA-4 and/or α4β7 and their ligands have been shown efficacious both prophylactically and therapeutically in several animal models of disease, including i) experimental allergic encephalomyelitis, a model of neuronal demyelination resembling multiple sclerosis; ii) bronchial hyperresponsiveness in sheep and guinea pigs as models for the various phases of asthma; iii) adjuvant-induced arthritis in rats as a model of inflammatory arthritis; iv) adoptive autoimmune diabetes in the NOD mouse; v) cardiac allograft survival in mice as a model of organ transplantation; vi) spontaneous chronic colitis in cotton-top tamarins which resembles human ulcerative colitis, a form of inflammatory bowel disease; vii) contact hypersensitivity models as a model for skin allergic reactions; viii) acute nephrotoxic nephritis; ix) tumor metastasis; x) experimental autoimmune thyroiditis; xi) ischemic tissue damage following arterial occlusion in rats; and xii) inhibition of TH2 T-cell cytokine production including IL-4 and IL-5 by VLA-4 antibodies which would attenuate allergic responses (J.Clinical Investigation 100, 3083 (1997). The primary mechanism of action of such antibodies appears to be the inhibition of lymphocyte and monocyte interactions with CAMs associated with components of the extracellular matrix, thereby limiting leukocyte migration to extravascular sites of injury or inflammation and/or limiting the priming and/or activation of leukocytes. Animal models of these diseases may also be used to demonstrate efficacy of small molecule VLA-4 antagonists
There is additional evidence supporting a possible role for VLA-4 interactions in other diseases, including rheumatoid arthritis; various melanomas, carcinomas, and sarcomas, including multiple myeloma; inflammatory lung disorders; acute respiratory distress syndrome (ARDS); pulmonary fibrosis; atherosclerotic plaque formation; restenosis; uveitis; and circulatory shock (for examples, see A. A. Postigo et al., “The α4β1/VCAM-1 adhesion pathway in physiology and disease.”, Res. Immunol., 144, 723 (1994) and J.-X. Gao and A. C. Issekutz, “Expression of VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines.” Immunol. 89, 375 (1996)).
At present, there is a humanized monoclonal antibody (Antegren®, Athena Neurosciences/Elan ) against VLA-4 in clinical development for the treatment of multiple sclerosis and Crohn's disease and a humanized monoclonal antibody (ACT-1®/LDP-02 LeukoSite) against α4β7 in clinical development for the treatment of inflammatory bowel disease. There are also several VLA-4 antagonists in early clinical trials for treatment of asthma and arthritis. There still remains a need for potent low molecular weight inhibitors of VLA-4-, α4β7- and/or α9β1 dependent cell adhesion that have pharmacokinetic and pharmacodynamic properties suitable for use as human pharmaceuticals.
PCT Application No. WO98/53818 discloses compounds having activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4, and having the formula: 




PCT Application No. WO98/53814 discloses compounds having activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4, and having the formula: 




PCT Application No. WO98/53814 discloses compounds having activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4, and having the formula: 




PCT Application Nos. WO99/06390, WO99/06431, WO99/06432, WO99/06433, WO99/06434, WO99/06435, WO99/06436, and WO99/06437 disclose compounds having activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4, and having the formula: 






DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds of Formula I: 




or a pharmaceutically acceptable salt thereof wherein:

    
    
        Ring A is cycloalkyl or heterocyclyl,
        wherein ring A is optionally substituted with one to four substituents independently selected from Rc;
        X and Y are independently selected from
        
            1) halogen,
            2) C1-3alkyl,
            3) C1-3alkoxy;
        
        
        Z is
        
            1) N,
            2) N+—O—;
        
        
        R1 is
        
            1) hydrogen,
            2) C1-10alkyl,
            3) aryl-C1-10alkyl;
        
        
        R2 is
        
            1) hydrogen or
            2) C1-10alkyl;
        
        
        R3 and R4 are independently selected from
        
            1) hydrogen,
            2) C1-10alkyl,
            3) —ORd,
            4) —CO2Rd,
            5) —C(O)NRdRe,
            6) —NRdRe,
            7) —NRdS(O)mRe,
            8) —NRdC(O)Re,
            9) —NRdC(O)ORe,
            10) —NRdC(O)NRdRe,
        
        
        wherein alkyl is optionally substituted with one to four substituents independently selected from Ra;
        R5 is
        
            1) hydrogen,
            2) OH,
            3) OCH3; or
            4) NH2;
        
        
        Ra is
        
            1) —ORd,
            2) —NRdS(O)mRe,
            3) —NO2,
            4) halogen
            5) —S(O)pRd,
            6) —SRd,
            7) —S(O)2ORd,
            8) —S(O)mNRdRe,
            9) —NRdRe,
            10) —O(CRfRg)nNRdRe,
            11) —C(O)Rd,
            12) —CO2Rd,
            13) —CO2(CRfRg)nCONRdRe,
            14) —OC(O)Rd,
            15) —CN,
            16) —C(O)NRdRe,
            17) —NRdC(O)Re,
            18) —OC(O)NRdRe,
            19) —NRdC(O)ORe,
            20) —NRdC(O)NRdRe,
            21) —CRd(N—ORe),
            22) CF3,
            23) —OCF3,
            24) C3-8cycloalkyl, or
            25) heterocyclyl;
        
        
        wherein cycloalkyl and heterocyclyl are optionally substituted with one to four groups independently selected from Rc;
        Rc is
        
            1) halogen,
            2) amino,
            3) carboxy,
            4) C1-4alkyl,
            5) C1-4alkoxy,
            6) aryl,
            7) aryl C1-4alkyl,
            8) hydroxy,
            9) CF3,
            10) OC(O)C1-4alkyl,
            11) OC(O)NRfRg, or
            12) aryloxy;
        
        
        Rd and Re are independently selected from hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, Cy and Cy C1-10alkyl, wherein alkyl, alkenyl, alkynyl and Cy are optionally substituted with one to four substituents independently selected from Rc; or
        Rd and Re together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and N—Rh;
        Rf and Rg are independently selected from hydrogen, C1-10alkyl, Cy and Cy-C1-10alkyl; or
        Rf and Rg together with the carbon to which they are attached form a ring of 5 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and nitrogen;
        Rh is selected from Rf and —C(O)Rf;
        Cy is selected from cycloalkyl, heterocyclyl, aryl, and heteroaryl;
        Ar1 is selected from phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl each optionally substituted with one or two groups independently selected from Rc;
        Ar2 is 1,4-phenylene or 2,5-pyridylene;
        p is 1 or 2;
        m is 0, 1 or 2;
        n is 0, 1 or 2.
    
    


In one embodiment of formula I Ar1 is pyridyl optionally substituted with C1-3alkyl, or phenyl optionally substituted with one to two groups independently selected from halogen, C1-3alkyl, carboxy, cyano, trifluoromethyl, and trifluoromethoxy. In one subset of this embodiment Ar1 is 3-substituted phenyl optionally having a second substituent on the 4- or 5-position wherein the substituents are independently selected from chloro, fluoro, bromo, methyl, trifluoromethyl and trifluoromethoxy. In another subset Ar1 is 3-pyridyl, 3-chlorophenyl, 3-carboxy-phenyl, or 3,5-dichlorophenyl. Examples of Ar1 include phenyl, 3-carboxylphenyl, 3-bromophenyl, 3-chlorophenyl, 3-fluorophenyl, 3-trifluoromethylphenyl, 3-trifluoromethoxphenyl, 4-chlorophenyl, 4-methylphenyl, 3,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-bis(trifluoromethyl)phenyl, 3,5-dimethylphenyl, 3-pyridyl, 5-methyl-3-pyridyl.
In another embodiment of formula I, Ar2 is 1,4-phenylene. Examples of Ar2 include 1,4-phenylene and 2,5-pyridylene as shown below. 




In another embodiment of formula I one of X and Y is halogen and the other is selected from halogen, C1-3alkyl and C1-3alkoxy. In one subset of this embodiment one of X and Y is chloro and the other is chloro or methoxy. In another subset X and Y are each chloro.
In another embodiment of formula I, R3 is hydrogen, carboxy, or substituted amino.
In another embodiment of formula I, R4 is hydrogen, carboxy, or substituted amino.
One embodiment of formula I provides compounds of formula Ia: 




wherein Z is N or N+O—;

    
    
        Ring A is C3-6cycloalkyl, pyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl or dioxyl,
        R2 is H or methyl;
        R3 is selected from H, C1-6alkyl, carboxyl, or —NRdRe;
        Rc is selected from hydrogen, C1-4alkyl; halogen, cyano, carboxyl, trifluoromethyl, or trifluoromethoxy; or
        a pharmaceutically acceptable salt thereof.
    
    


In one subset of formula Ia, Ring A is cyclopentyl.
In another subset of formula Ia, Ring A is cyclohexyl.
In yet another subset of formula Ia, Rc is chloro.
In another aspect the present invention provides a method for the prevention or treatment of diseases, disorders, conditions or symptoms mediated by cell adhesion in a mammal which comprises administering to said mammal an effective amount of a compound of formula I.
In one embodiment said disease or disorder is selected from asthma, allergic rhinitis, multiple sclerosis, atherosclerosis, inflammatory bowel disease, rheumatoid arthritis, and organ transplantation.
In another aspect the present invention provides a method for preventing the action of VLA-4 in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula I.
Another aspect of the present invention provides a pharmaceutical composition which comprises a compound of formula I and a pharmaceutically acceptable carrier.
“Alkyl”, as well as other groups having the prefix “alk”, such as alkoxy, alkanoyl, means carbon chains which may be linear or branched or combinations thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like.
“Alkenyl” means carbon chains which contain at least one carbon-carbon double bond, and which may be linear or branched or combinations thereof. Examples of alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.
“Alkynyl” means carbon chains which contain at least one carbon-carbon triple bond, and which may be linear or branched or combinations thereof. Examples of alkynyl include ethynyl, propargyl, 3-methyl-1-pentynyl, 2-heptynyl and the like.
“Cycloalkyl” means mono- or bicyclic saturated carbocyclic rings, each of which having from 3 to 10 carbon atoms. The term also includes monocyclic rings fused to an aryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.
“Aryl” means mono- or bicyclic aromatic rings containing only carbon atoms. The term also includes aryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion. Examples of aryl include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, and the like.
“Heteroaryl” means a mono- or bicyclic aromatic ring containing at least one heteroatom selected from N, O and S, with each ring containing 5 to 6 atoms. Examples of heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.
“Heterocyclyl” means mono- or bicyclic saturated rings containing at least one heteroatom selected from N, S and O, each of said ring having from 3 to 10 atoms in which the point of attachment may be carbon or nitrogen. The term also includes monocyclic heterocycle fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion. Examples of “heterocyclyl” include pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2,3-dihydrofuro(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, dihydroindolyl, and the like. The term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4-pyridones attached through the nitrogen or N-substituted-(1H,3H)-pyrimidine-2,4-diones (N-substituted uracils).
“Halogen” includes fluorine, chlorine, bromine and iodine.
Optical Isomers—Diastereomers—Geometric Isomers—Tautomers
Compounds of Formula I contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of Formula I.
Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed with compounds of Formula I.
Compounds of the Formula I may be separated into diastereoisomeric pairs of enantiomers by, for example, fractional crystallization from a suitable solvent, for example MeOH or ethyl acetate or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active amine as a resolving agent or on a chiral HPLC column.
Alternatively, any enantiomer of a compound of the general Formula I or Ia may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
Salts
The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.
It will be understood that, as used herein, references to the compounds of Formula I are meant to also include the pharmaceutically acceptable salts.
Utilities
The ability of the compounds of Formula I to antagonize the actions of VLA-4 and/or α4β7 integrin makes them useful for preventing or reversing the symptoms, disorders or diseases induced by the binding of VLA-4 and or α4β7 to their various respective ligands. Thus, these antagonists will inhibit cell adhesion processes including cell activation, migration, proliferation and differentiation. Accordingly, another aspect of the present invention provides a method for the treatment (including prevention, alleviation, amelioration or suppression) of diseases or disorders or symptoms mediated by VLA-4 and/or α4β7 binding and cell adhesion and activation, which comprises administering to a mammal an effective amount of a compound of Formula I. Such diseases, disorders, conditions or symptoms are for example (1) multiple sclerosis, (2) asthma, (3) allergic rhinitis, (4) allergic conjunctivitis, (5) inflammatory lung diseases, (6) rheumatoid arthritis, (7) septic arthritis, (8) type I diabetes, (9) organ transplantation rejection, (10) restenosis, (11) autologous bone marrow transplantation, (12) inflammatory sequelae of viral infections, (13) myocarditis, (14) inflammatory bowel disease including ulcerative colitis and Crohn's disease, (15) certain types of toxic and immune-based nephritis, (16) contact dermal hypersensitivity, (17) psoriasis, (18) tumor metastasis, (19) atherosclerosis, and (20) hepatitis.
The utilities of the present compounds in these diseases or disorders may be demonstrated in animal disease models that have been reported in the literature. The following are examples of such animal disease models:
i) experimental allergic encephalomyelitis, a model of neuronal demyelination resembling multiple sclerosis (for example, see T. Yednock et al., “Prevention of experimental autoimmune encephalomyelitis by antibodies against α4β1 integrin.” Nature, 356, 63 (1993) and E. Keszthelyi et al., “Evidence for a prolonged role of α4 integrin throughout active experimental allergic encephalomyelitis.” Neurology, 47, 1053 (1996));
ii) bronchial hyperresponsiveness in sheep and guinea pigs as models for the various phases of asthma (for example, see W. M. Abraham et al., “α4-Intergrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.” J. Clin. Invest. 93, 776 (1993) and A. A. Y. Milne and P. P. Piper, “Role of VLA-4 integrin in leucocyte recruitment and bronchial hyperresponsiveness in the gunea-pig.” Eur. J. Pharmacol., 282, 243 (1995));
iii) adjuvant-induced arthritis in rats as a model of inflammatory arthritis (see C. Barbadillo et al., “Anti-VLA-4 mAb prevents adjuvant arthritis in Lewis rats.” Arthr. Rheuma. (Suppl.), 36 95 (1993) and D. Seiffge, “Protective effects of monoclonal antibody to VLA-4 on leukocyte adhesion and course of disease in adjuvant arthritis in rats.” J. Rheumatol., 23, 12 (1996));
iv) adoptive autoimmune diabetes in the NOD mouse (see J. L. Baron et al., “The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between α4-integrins and vascular cell adhesion molecule-1.”, J. Clin. Invest., 93, 1700 (1994), A. Jakubowski et al., “Vascular cell adhesion molecule-Ig fusion protein selectively targets activated α4-integrin receptors in vivo: Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.” J. Immunol., 155, 938 (1995), and X. D. Yang et al., “Involvement of beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MadCAM-1) in the development of diabetes in nonobese diabetic mice”, Diabetes, 46, 1542 (1997));
v) cardiac allograft survival in mice as a model of organ transplantation (see M. Isobe et al., “Effect of anti-VCAM-1 and anti-VLA-4 monoclonal antibodies on cardiac allograft survival and response to soluble antigens in mice.”, Tranplant. Proc., 26, 867 (1994) and S. Molossi et al., “Blockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteripathy in rabbit cardiac allografts.” J. Clin Invest., 95, 2601 (1995));
vi) spontaneous chronic colitis in cotton-top tamarins which resembles human ulcerative colitis, a form of inflammatory bowel disease (see D. K. Podolsky et al., “Attenuation of colitis in the Cotton-top tamarin by anti-α4 integrin monoclonal antibody.”, J. Clin. Invest., 92, 372 (1993));
vii) contact hypersensitivity models as a model for skin allergic reactions (see T. A. Ferguson and T. S. Kupper, “Antigen-independent processes in antigen-specific immunity.”, J. Immunol., 150, 1172 (1993) and P. L. Chisholm et al., “Monoclonal antibodies to the integrin a-4 subunit inhibit the murine contact hypersensitivity response.” Eur. J. Immunol., 23, 682 (1993));
viii) acute nephrotoxic nephritis (see M. S. Mulligan et al., “Requirements for leukocyte adhesion molecules in nephrotoxic nephritis.”, J. Clin. Invest., 91, 577 (1993));
ix) tumor metastasis (for examples, see M. Edward, “Integrins and other adhesion molecules involved in melanocytic tumor progression.”, Curr. Opin. Oncol., 7, 185 (1995));
x) experimental autoimmune thyroiditis (see R. W. McMurray et al., “The role of α4 integrin and intercellular adhesion molecule-1 (ICAM-1) in murine experimental autoimmune thyroiditis.” Autoimmunity, 23, 9 (1996);
xi) ischemic tissue damage following arterial occlusion in rats (see F. Squadrito et al., “Leukocyte integrin very late antigen-4/vascular cell adhesion molecule-1 adhesion pathway in splanchnic artery occlusion shock.” Eur. J. Pharmacol., 318, 153 (1996; and
xii) inhibition of TH2 T-cell cytokine production including IL-4 and IL-5 by VLA-4 antibodies which would attenuate allergic responses (J. Clinical Investigation 100, 3083 (1997).
Dose Ranges
The magnitude of prophylactic or therapeutic dose of a compound of Formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of Formula I and its route of administration. It will also vary according to the age, weight and response of the individual patient. In general, the daily dose range lie within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.005 mg to about 10 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases.
For use where a composition for intravenous administration is employed, a suitable dosage range is from about 0.001 mg to about 25 mg (preferably from 0.01 mg to about 1 mg) of a compound of Formula I per kg of body weight per day and for cytoprotective use from about 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 1 mg to about 10 mg) of a compound of Formula I per kg of body weight per day.
In the case where an oral composition is employed, a suitable dosage range is, e.g. from about 0.01 mg to about 100 mg of a compound of Formula I per kg of body weight per day, preferably from about 0.1 mg to about 10 mg per kg and for cytoprotective use from 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 10 mg to about 100 mg) of a compound of Formula I per kg of body weight per day.
In the case where an aerosol composition is employed, a suitable dosage range is, e.g. from about 0.001 mg to about 1 mg of a compound of Formula I per kg of body weight per day, preferably from about 0.005 mg to about 0.5 mg per kg of a compound of Formula I per kg of body weight per day.
For the treatment of diseases of the eye, ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of Formula I in an acceptable ophthalmic formulation may be used.
Pharmaceutical Compositions
Another aspect of the present invention provides pharmaceutical compositions which comprises a compound of Formula I and a pharmaceutically acceptable carrier. The term “composition”, as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of Formula I, additional active ingredient(s), and pharmaceutically acceptable excipients.
Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The pharmaceutical compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.
The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (aerosol inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers. The compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device. The preferred delivery systems for inhalation are metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a compound of Formula I in suitable propellants, such as fluorocarbons or hydrocarbons and dry powder inhalation (DPI) aerosol, which may be formulated as a dry powder of a compound of Formula I with or without additional excipients.
Suitable topical formulations of a compound of formula I include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like.
In practical use, the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
In addition to the common dosage forms set out above, the compounds of Formula I may also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200 and 4,008,719.
Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
The following are examples of representative pharmaceutical dosage forms for the compounds of Formula I:
















Injectable Suspension (I.M.)
mg/mL







Compound of Formula I
10



Methylcellulose
5.0



Tween 80
0.5



Benzyl alcohol
9.0



Benzalkonium chloride
1.0



Water for injection to a total volume of 1 mL







Tablet
mg/tablet







Compound of Formula I
25



Microcrystalline Cellulose
415



Povidone
14.0



Pregelatinized Starch
43.5



Magnesium Stearate
2.5




500







Capsule
mg/capsule







Compound of Formula I
25



Lactose Powder
573.5



Magnesium Stearate
1.5




600







Aerosol
Per canister















Compound of Formula I
24
mg



Lecithin, NF Liq. Conc.
1.2
mg



Trichlorofluoromethane, NF
4.025
g



Dichlorodifluoromethane, NF
12.15
g










Combination Therapy
Compounds of Formula I may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of Formula I are useful. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of Formula I. When a compound of Formula I is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of Formula I is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of Formula I. Examples of other active ingredients that may be combined with a compound of Formula I, either administered separately or in the same pharmaceutical compositions, include, but are not limited to: (a) other VLA-4 antagonists such as those described in U.S. Pat. No. 5,510,332, WO97/03094, WO97/02289, WO96/40781, WO96/22966, WO96/20216, WO96/01644, WO96/06108, WO95/15973 and WO96/31206; (b) steroids such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants; (d) antihistamines (H1-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine pyrilamine, astemizole, terfenadine, loratadine, cetirizine, fexofenadine, descarboethoxyloratadine, and the like; (e) non-steroidal anti-asthmatics such as b2-agonists (terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, bitolterol, salmeterol and pirbuterol), theophylline, cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists (zafirlukast, montelukast, pranlukast, iralukast, pobilukast, SKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-1005); (f) non-steroidal antiinflammatory agents (NSAIDs) such as propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone); (g) cyclooxygenase-2 (COX-2) inhibitors such as celecoxib, rofecoxib, and parecoxib; (h) inhibitors of phosphodiesterase type IV (PDE-IV); (i) antagonists of the chemokine receptors, especially CCR-1, CCR-2, and CCR-3; (j) cholesterol lowering agents such as HMG-CoA reductase inhibitors (lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and other statins), sequestrants (cholestyramine and colestipol), nicotinic acid, fenofibric acid derivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), and probucol; (k) anti-diabetic agents such as insulin, sulfonylureas, biguanides (metformin), a-glucosidase inhibitors (acarbose) and glitazones (troglitazone, pioglitazone, englitazone, MCC-555, BRL49653 and the like); (1) preparations of interferon beta (interferon beta-1a, interferon beta-1b); (m) anticholinergic agents such as muscarinic antagonists (ipratropium nad tiatropium); (n) other compounds such as 5-aminosalicylic acid and prodrugs thereof, antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeutic agents.
The weight ratio of the compound of the Formula I to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the Formula I is combined with an NSAID the weight ratio of the compound of the Formula I to the NSAID will generally range from about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. Combinations of a compound of the Formula I and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
Abbreviations used in the following Schemes and Examples:













4-DMAP:
4-dimethylaminopyridine


Ac2O:
acetic anhydride


AcCN:
acetonitrile


Ag2O:
silver(I) oxide


AIBN:
2,2′-azobisisobutyronitrile


BF3-Et2O:
borontrifluoride etherate


BH3-DMS:
borane dimethylsulfide complex


Bn:
benzyl


BOC:
tert-butoxycarbonyl


BOC-ON
2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile


BOP:
benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium



hexafluorophosphate


brine:
saturated sodium chloride solution


CBZ:
benzyloxycarbonyl


Cy3P:
tricyclohexylphosphine


DBU:
1,8-diazobicyclo[5.4.0]undec-7-ene


DCC:
dicyclohexylcarbodiimide


DIBAL-H:
diisobutylaluminum hydride


DIPEA:
N,N-diisopropylethylamine


DME:
1,2-dimethoxyethane


DMF:
dimethylformamide


DMPU:
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone


DMSO:
dimethylsulfoxide


EDC:
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide



hydrochloride


Et:
ethyl


Et2O:
diethyl ether


EtOAc:
ethyl acetate


EtOH:
ethanol


FMOC:
9-fluorenylmethoxylcarbonyl


g or gm:
gram


h or hr:
hours


HATU:
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium



hexafluorophosphate


HBTU:
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium



hexafluorophosphate


HOAc:
acetic acid


HOAt:
1-hydroxy-7-azabenzotriazole


HOBt:
1-hydroxybenzotriazole


HPLC:
high pressure liquid chromatography


in vacuo:
rotoevaporation


KOAc:
potassium acetate


LDA:
lithium diisopropylamide


LiHMDS:
lithium hexamethyldisilylamide


mCPBA:
meta-chloroperbenzoic acid


Me:
methyl


MeI:
methyl iodide


MeOH:
methanol


mg:
milligram


MHz:
megahertz


min:
minutes


mL:
milliliter


mmol:
millimole


MPLC:
medium pressure liquid chromatography


MS or ms:
mass spectrum


MsCl:
methanesulfonyl chloride


NBS:
N-bromosuccinimide


NMO:
4-methyl-morpholine-N-oxide


Pd2dba3:
tris(dibenzylideneacetone)dipalladium(0)


Ph:
phenyl


Ph3P:
triphenylphosphine


pTSA:
para-toluenesulfonic acid


PyBOP:
(benzotriazol-1-yloxy)tripyrrolidinophosphonium



hexafluorophosphate


rt:
room temperature


TBAF:
tetrabutylammonium fluoride


TBSCl:
tert-butyldimethylsilyl chloride


t-Bu3P:
tri-tert-butylphosphine


TEA:
triethylamine


TFA:
trifluoroacetic acid


THF:
THF


TLC:
thin layer chromatography


TMSCHN2:
trimethylsiliyldiazomethane


TMSCl:
trimethylsilyl chloride


TMSI:
trimethylsilyl iodide


TPAP:
tetrapropylammonium perruthenate


TsCl:
para-toluene sulfonyl chloride









Compounds of the present invention may be prepared by procedures illustrated in the accompanying schemes. In the first method (Scheme 1), a substituted pyridyl-4-carboxylic acid derivative A is treated with thionyl chloride to make the carboxylic acid chloride derivative which is subsequently reacted with a 4-amino-(L)-phenylalanine derivative to yield the amide B. The N-BOC-protecting group in B is removed with strong acid (TFA or HCl) to afford the free amine C. An appropriately substitued fused cyclic pyrrolidine or piperidine-carboxylate D is sulfonylated with a substitued arylsulfonyl chloride in the presence of base (DIPEA or Na2CO3) to yield sulfonamide E which, if containing an ester protecting group, is treated with hydroxide to afford the free acid. Amine C and acid E are reacted together in the presence of an appropriate coupling agent (eg., PyBOP, HBTU/HOAt, premake the acid chloride of E, etc.) to afford amide F. The ester in F can be hydrolyzed with hydroxide (if R1 is n-alkyl) or TFA or HCl (if R1 is tert-butyl) to acid G. 




Compounds of the present invention may be prepared by procedures detailed in the following examples. The examples are provided to illustrative the present invention and are not to be construed as limiting its scope in any manner.
REFERENCE EXAMPLE 1
3,5-Dichloroisonicotinic acid
To a solution of 3,5-dichloropyridine (10.00 g, 67.57 mmol) in 70 mL of THF was added 35.4 mL of a 2.0 M solution of LDA in THF at −78° C. The reaction was stirred for 1 h, then CO2 gas was bubbled through the solution for 20 mins. The reaction was allowed to warm to rt over 1 h then quenched with 1N NaOH (100 mL) and washed with Et2O (50 mL). The aqueous layer was acidified with conc HCl which caused a precipitate to form. The precipitate was collected by filtration and recrystallized from EtOH to give the title compound as a pale yellow solid (7.1 g, 36.97 mmol, 55%).
1H NMR (500 MHz, DMSO-d6): δ 8.73 (s, 2H).
REFERENCE EXAMPLE 2
4-((3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester hydrochloride
Step A N-(BOC)-4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester
A slurry of 3,5-dichloroisonicotinic acid (3.1 g, 16.11 mmol) in 10 mL of CH2Cl2 was treated with DMF (50 μL) and thionyl chloride (1.23 mL, 16.91 mmol) and heated to reflux for 5 h. The reaction was concentrated to give a yellow oil. This oil was dissolved in 5 mL of CH2Cl2 and added to N-(BOC)-4-amino-(L)-phenylalanine, methyl ester (4.00 g, 14.39 mmol) and 4-methylmorpholine (2.7 mL, 24.21 mmol) in 25 mL of CH2Cl2 at 0° C. After stirring for 2 h at this temperature, the reaction was quenched with water (50 mL) and extracted into CH2Cl2 (3×100 mL). The combined organics were combined, dried over anhydrous MgSO4 and concentrated in vacuo to give a yellow solid. Trituration with CH2Cl2 gave 5.5 g of a while solid.
1H NMR (500 MHz, CDCl3): δ 8.63 (s, 2H); 7.58 (d, J=8.2 Hz, 2H); 7.23 (d, J=8.2 Hz, 2H); 6.91 (d, J=8.4 Hz, 1H); 4.39 (m, 1H); 3.70 (s, 3H); 3.11 (m, 1H); 2.91 (m, 1H); 2.00 (s, 9H);
MS m/e 468.20 (M+).
Step B 4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester hydrochloride
N-(BOC)-4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (2.50 g, 5.34 mmol) was dissolved in EtOAc (40 mL) and treated with HCl (gas). Concentration in vacuo gave the title compound as a yellow solid (2.05 g, 4.59 mmol, 86%).
1H NMR (500 MHz, CD3OD): δ 8.69 (s, 2H); 7.68 (d, J=8.5 Hz, 2H); 7.31 (m, J=8.5 Hz, 2H); 4.35 (t, J=6.9 Hz, 1H); 3.83 (s, 3H); 3.29 (m, 1H); 3.21 (m, 1H); MS m/e 368.13 (M+).
EXAMPLE 1
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisionicotinoyl)amino]-(L)-phenylalanine





Step A Octahydroisoindole-1-carboxylic acid
N-BOC-octahydroisoindole-1-carboxylic acid (18.6 mmol) was dissolved in 4 M HCl in dioxane (46.5 mL) at rt. After stirring for 1 hr, the reaction was determined to be complete by TLC. The reaction mixture was concentrated in vacuo, triturated with Et2O (2×70 mL) and then dried in vacuo to afford octahydroisoindole-1-carboxylic acid (4.13 g) as a white solid.
500 MHz 1HNMR (CD3OD) δ 4.40 (d, J=5.5 Hz, 1H); 3.39 (m, 1H); 3.31 (m, 1H); 2.70 (m, 1H); 2.58; (m, 1H); 1.78-1.14 (m, 8H).
Step B N-(3,5-dichlorobenzenesulfonyl)-octahydroisoindole-1-carboxylic acid
Octahydroisoindole-1-carboxylic acid (4.12 gm) was dissolved in a solution of water (125 mL) containing 5.32 g of Na2CO3. Upon complete dissolution, 4.93 g of 3,5-dichlorobenzenesulfonyl chloride was added. The reaction was stirred at rt over night. The reaction mixture was then washed with EtOAc (2×100 mL). The aqueous layer was isolated and acidified to pH=3 by the dropwise addition of a concentrated HCl solution. A solid precipitate formed in the aqueous layer and was filtered and dried in vacuo to afford N-(3,5-dichlorobenzenesulfonyl)-octahydro-isoindole-1-carboxylic acid (14.64 g) as a white solid.
500 MHz 1HNMR (CD3OD) δ 7.88 (s, 2H); 7.77 (s, 1H); 4.39 (d, J=7 Hz, 1H); 3.47 (m, 1H); 3.37 (m, 1H); 2.48 (m, 1H); 2.12 (m, 1H); 1.69-1.21 (m, 8H).
Step C N-(3,5-Dichlorobenzenesulfonyl)-octahydroisoindole-1-carboxylic acid chloride
N-(3,5-Dichlorobenzenesulfonyl)-octahydroisoindole-1-carboxylic acid (1 gm, 2.64 mmol) was dissolved in CH2Cl2 (5 mL) containing DMF (1 drop) and SOCl2 (770 μL, 10.56 mmol). The reaction mixture was refluxed under nitrogen for  ̃1 hr. The reaction mixture was monitored by TLC for the formation of the acid chloride. The reaction mixture was then concentrated in vacuo and azeotroped with toluene (1×5 mL) to afford N-(3,5-dichlorobenzenesulfonyl)-octahydroisoindole-1-carboxylic acid chloride which was used without further purification in the subsequent step.
Step D N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
4-((3,5-Dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester hydrochloride from Reference Example 2 (2.19 mmol) was dissolved in CH2Cl2 (10 mL) and then the solution was cooled to 0° C. N-methylmorpholine (3.6 mmol) was added, followed by a solution of N-(3,5-dichlorobenzenesulfonyl)-octahydro-isoindole-1-carboxylic acid chloride in CH2Cl2 (10 mL). The reaction proceeded at 0° C. for 30 min and then at rt for another 30 min. The reaction mixture was then diluted with 75 mL of EtOAc. This solution was washed with 1N HCl (2×50 mL), NaHCO3 (1×50 mL), brine (1×50 mL), and dried with anhydrous Na2SO4. The solvent was removed in vacuo to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-octahydroisindole-1-carbonyl)-4[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (1.42 gm) as a brownish solid.
MS, m/e 727 (M+H+)
Step E Separation of Diastereomers
Step D was repeated several times to afford a total of 22 gm of N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroinsonicotinoyl)amino]-(L)-phenylalanine, methyl ester which was heated in refluxing EtOH. It was found that the filtrate solution had a 3:1 ratio of the two diastereomers. The solids that remained from the EtOH heating had a 1:2 ratio of the respective diastereomers. These solids were further heated with refluxing EtOAc. This filtrate solution gave a 3:1 of isomers. The filtrates were combined and concentrated to afford approximately 11 g of solid that was subsequently purified by preparative HPLC (77 mm chiralpak OD column, eluent 4:1 EtOH:heptane; flow rate 100 mL/min; 15-20 mL injections every 10 min). The fractions containing the major isomer which eluted first were combined and concentrated in vacuo to yield 8.82 g of N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester as a white solid.
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.81 (d, J=1.5 Hz, 2H); 7.80 (t, J=1.5 Hz, 1H); 7.63 (d, J=9.0 Hz, 2H); 7.31 (d, J=9.0 Hz, 2H); 4.86 (dd, J=8.5, 5.5 Hz, 1H); 4.14 (d, J=7.0 Hz, 1H); 3.75 (s, 3H); 3.47 (dd, J=11.0, 8.0 Hz, 1 H); 3.41 (dd, J=11.0, 11.0 Hz, 1H); 3.25 (dd, J=14.0, 5.0 Hz, 1H); 3.06 (dd, J=14.0, 9.0 Hz, 1H); 2.28 (m, 1H); 1.79 (m, 1H); 1.65-0.97 (m, 8H).
MS, m/e 727 (M+H)+.
The minor isomer could also be isolated.
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.84 (d, J=1.5 Hz, 2H); 7.79 (t, J=1.5 Hz, 1H); 7.61 (d, J=8.5 Hz, 2H); 7.30 (d, J=8.5 Hz, 2H); 4.76 (dd, J=10.5, 4.5 Hz, 1H); 4.17 (d, J=6.5 Hz, 1H); 3.78 (s, 3H); 3.43 (dd, J=11.5, 8.0 Hz, 1H); 3.37 (dd, J=11.5, 11.5 Hz, 1H); 3.25 (dd, J=14.0, 4.5 Hz, 1H); 2.91 (dd, J=14.0, 10.5 Hz, 1H); 2.10 (m, 1H); 1.82 (m, 1H); 1.64-0.86 (m, 7H); 0.47 (m, 1H).
MS, m/e 727 (M+H)+.
Step F N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (8.8 gm, 12.1 mmol) was dissolved in 110 mL of CH3CN and 22 mL of H2O and cooled in an ice-water bath. LiOH-H2O (4.41 g, 105 mmol) was added. Reaction mixture was stirred at 0° C. for 30 min and then at rt for 45 min. The reaction mixture was cooled in an ice-water bath again and 1N HCl solution was added dropwise until pH=2. The reaction mixture was then extracted with water (100 mL) and EtOAc (3×150 mL). The EtOAc washes were combined and washed with brine(1×100 ml) and dried over anhydrous Na2SO4. The solvents were removed in vacuo to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (7.7 gm) as a white solid.
500 MHz 1HNMR (CD3OD) δ 8.63 (s, 1H); 7.87 (m, 4H); 7.64 (d, J=8.5 Hz, 2H); 7.35 (d, J=8.5 Hz, 2H); 4.18 (d, J=6.5 Hz, 1H); 4.82 (m, 1H); 3.49 (m, 3H); 3.10 (m, 1H); 2.30 (m, 2H); 1.80-1.05 (m, 8H).
MS, m/e 713 (M+H+).
Likewise the minor isomer from Step E could also be hydrolyzed to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1(R)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
1H NMR (400 MHz, CD3OD): δ 8.63 (s, 2H); 7.82 (d, 2H); 7.69 (t, 1H); 7.62 (d, 2H); 7.33 (d, 2H); 4.78 (dd, 1H); 4.18 (d, 1H); 3.46-3.22 (m, 3H); 2.94 (dd, 1H); 2.08 (m, 1H); 1.80 (m, 1H); 1.66-0.80 (m, 7H); 0.44 (m, 1H).
MS, m/e 713 (M+H)+.
EXAMPLE 2
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine
A solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydro-isoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (170 mg, 0.238 mmol) and mCPBA (205 mg, 0.35 mmol) in EtOAc (2.5 mL) was heated to 50° C. for 3 hr. The reaction was cooled and the solvent removed in vacuo. The residue was purified by preparative C18-HPLC eluted with a gradient of 30% to 90% AcCN in water to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydro-isoindole-1(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine (64.5 mg) as a white solid.
500 MHz 1HNMR (CD3OD) δ 8.54 (s, 1H); 7.81 (m, 4H); 7.64 (d, J=8.5 Hz, 2H); 7.35 (d, J=8.5 Hz, 2H); 4.17 (d, J=7 Hz, 1H); 4.81 (m, 1H); 3.48 (m, 3H); 3.10 (m, 1H); 2.30 (m, 2H); 1.80-1.5 (m, 8H).
MS, m/e 731 (M+H+).
EXAMPLE 3
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinolyl-3(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine





Step A N-BOC-[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid, methyl ester
A solution of N-BOC-1,2,3,4-tetrahydroisoquinoline-3(S)-carboxylic acid, methyl ester (2.83 g, 9.2 mmol), 5% Rh/Al2O3 (1.415 g) in 33 mL of MeOH was shaken at 60° C. for 36 hrs under an atmosphere of 40 psi hydrogen. The reaction was filtered through celite and concentrated in vacuo to afford N-BOC-[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid, methyl ester which was used without further purification in the subsequent step.
Step B [4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid
N-BOC-[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid, methyl ester (2.8 g, 9.2 mmol) was dissolved in 6N HCl (50 mL). The reaction was refluxed overnight. The reaction mixture was concentrated in vacuo and azeotroped with toluene to afford [4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid which was used without further purification in the subsequent step.
Step C N-(N-[(3,5-Dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid
[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid was reacted with 3,5-dichlorobenzenesulfonyl chloride in the presence of Na2CO3 according to the procedure described in Example 1, Step B to afford N-(N-[(3,5-dichlorobenzene)-sulfonyl]-[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid which was used in the subsequent step.
Step D N-(N-[(3,5-Dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinolyl-3(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinoline-3(S)-carboxylic acid, 3,5-(dichloroisonicotinoyl) amino-(L)-phenylalanine, methyl ester (1 mmol), and HOAt (1.5 mmol) in CH2Cl2 (2 mL) at 0° C. was added DIPEA (2 mmol)) and HATU (1.1 mmol). The reaction was allowed to warm to rt overnight. The reaction was diluted with EtOAc, then washed with 1M HCl, NaHCO3, and brine, dried over anhydrous MgSO4 and concentrated in vacuo. The crude mixture was purified by flash column chromatography on silica gel with gradual elutions of 75% Hexanes/Et2O to 100% Et2O to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinolyl-3(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester which was used in the subsequent step.
Step E N-(N-[(3,5-Dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinolyl-3(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinolyl-3(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester was treated with LiOH-H2O according to the procedure described in Example 1, Step F to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-[4a(S),8a(S)]-decahydroisoquinolyl-3(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
MS, m/e 729.2 (M+).
EXAMPLE 4
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine





Step A N-[(3,5-Dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carboxylic acid
(3a(S),7a(S))-octahydro-indole-2(S)-carboxylic acid (Bachem, 2.0 g, 12 mmol) was reacted with 3,5-dichlorobenzenesulfonyl chloride according to the procedure described in Example 1, Step B to afford N-[(3,5-dichlorobenzene)-sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carboxylic acid (4.2 g, 93%).
1H NMR (400 MHz, CD3OD): δ 7.86 (d, 2H); 7.78 (t, 1H); 4.22 (dd, 1H), 3.72 (m, 1H); 2.30-1.15 (m, 11H).
Step B N-(N-[(3,5-Dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
To a suspension of N-[(3,5-dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carboxylic acid (0.21 mmol), 4-(3,5-(dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester hydrochloride from Reference Example 2 (0.21 mmol)), and PyBOP (0.21 mmol) in CH2Cl2 (2 mL) was added DIPEA (0.42 mmol)). The reaction was stirred at rt for 20 h then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with hexane/ethyl acetate (1:1) to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)-amino]-(L)-phenylalanine, methyl ester (0.12 g, 76%).
1H NMR (400 MHz, CD3OD): δ 8.63 (s, 2H), 7.84 (d, 2H); 7.79 (t, 1H); 7.62 (d, 2H); 7.24 (dd, 2H), 4.83 (m, 1H); 4.12 (dd, 1H); 3.77 (s, 3H); 3.70 (m, 1H); 3.26 (dd, 1H); 3.13 (dd, 1H); 2.10-1.10 (m, 11H).
MS, m/e 727 (M+H)+.
Step C N-(N-[(3,5-Dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (Step B, 0.11 g, 0.15 mmol) was treated with LiOH-H2O according to the procedure described in Example 1, Step F with following modifications: the reaction mixture was partitioned between brine, 0.5 M sodium hydrogen sulfate and ethyl acetate. The product was extracted with EtOAc (3×), and the combined extracts were dried over anhydrous MgSO4 and concentrated in vacuo to afford N-(N-[(3,5-dichlorobenzene)-sulfonyl]-(3a(S),7a(S))-octahydro-indole-2(S)-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (0.10 mg, 94%).
1H NMR (400 MHz, CD3OD): δ 8.62 (s, 2H); 7.86 (d, 2H); 7.80 (t, 1H); 7.62 (d, 2H); 7.30 (d, 2H); 4.78 (dd, 1H); 4.12 (dd, 1H); 3.72 (m, 1H); 3.36-3.26 (m, 1H); 3.15 (dd, 1H); 2.2-1.1 (m, 11H).
MS, m/e 713 (M+H)+.
EXAMPLE 5
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3,4-cis-isopropylidenedioxy-2-methyl-(L)-prolyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine





Step A N-BOC-4(R)-hydroxy-2-methyl-(L)-proline, methyl ester
To a solution of N-BOC-4(R)-hydroxyproline, methyl ester (Bachem, 17 g, 69 mmol) and MeI (276 mmol) in 250 mL of anhydrous THF at −30° C. was added LDA (Aldrich, 1.5 M in cyclohexane, 250 mmol). The reaction was allowed to warm up to rt over 4 h. The reaction was cooled to −30° C., and was quenched with saturated aqueous ammonium chloride (50 mL). The resulting mixture was partitioned between EtOAc and brine, and the product was extracted with EtOAc (3×100 mL). The combined extracts were dried over anhydrous MgSO4 and concentrated in vacuo. The solvent was removed in vacuo and the residue was purified by repeated flash column chromatography on silica gel eluted with 20:1 to 10:1 hexane/acetone to afford N-BOC-4(R)-hydroxy-2-methyl-(L)-proline, methyl ester (slower eluting and major isomer, 10 g, 58%). The stereochemistry was assigned by NMR comparisons with literature reports (Noe, C R et al Pharmazie 1996, 51, 800).
1H NMR (400 MHz, CD3OD): δ (mixture of two rotamers) 4.40-4.32 (m, 1H); 3.69/3.65 (s, 3H); 3.68-3.60 (m, 1H); 3.30-3.28 (m, 1H); 2.40-2.28 (m, 1H); 2.00-1.88 (m, 1H); 1.64 (s, 3H); 1.44/1.38 (s, 9H).
Step B N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-proline, methyl ester
N-BOC-4(R)-hydroxy-2-methyl-(L)-proline, methyl ester was reacted with 3,5-dichlorobenzenesulfonyl chloride and Na2CO3 according to the procedure described in Example 1, Step B to afford N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-proline, methyl ester (4.5 g, 49%).
1H NMR (400 MHz, CD3OD): δ 7.80 (m, 2H); 7.74 (m, 1H); 4.41 (m, 1H); 3.71 (s, 3H); 3.67 (m, 1H); 3.43 (m, 1H); 2.43 (m, 1H); 2.00 (dd, 1H); 1.72 (s, 3H).
Step C N-[(3,5-Dichlorobenzene)sulfonyl]-3,4-dehydro-2-methyl-(L)-proline, methyl ester
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-proline, methyl ester (3.5 g, 9.5 mmol) in anhydrous CH2Cl2 (20 mL) was added pyridine (2.3 mL, 29 mmol) and mesyl chloride (1.5 mL, 19 mmol) at 0° C. The reaction was allowed to warm up to rt over 7 h, and the reaction mixture was diluted with EtOAc (100 mL) and was washed with dilute cupric sulfate (2×), brine, and dried over MgSO4. The solution was filtered and concentrated in vacuo to dryness to give the crude mesylate which was used without further purification.
Thus, the residue was dissolved in DMF (20 mL) and was added to a solution of sodium phenylselenide (prepared from 1.7 g of diphenyldiselenide (5.5 mmol) and 0.46 g of sodium borohydride (12 mmol) in t-butanol (20 mL) and EtOH (10 mL) following the procedure of Robinson, J K et al. (Tetrahedron 1998, 54, 981)) at rt. After stirring at 60° C. for 4 h, the resulting mixture was partitioned between EtOAc and water. The product was extracted with EtOAc (3×100 mL). The combined extracts were washed with brine, dried over anhydrous MgSO4. The solution was filtered, concentrated in vacuo and the residue was purified by flash column chromatography on silica gel eluted with 10:1 hexane/EtOAc to yield N-[(3,5-dichlorobenzene)sulfonyl]-4(S)-phenylseleno-2-methyl-(L)-proline, methyl ester (3.4 g, 71%), which was used immediately.
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-4(S)-phenylseleno-2-methyl-(L)-proline, methyl ester (3.4 g, 6.7 mmol) and pyridine (0.81 mL, 10 mmol) in CH2Cl2 (40 mL) at 0° C. was added hydrogen peroxide (30%, 1.4 mL, 17 mmol). The reaction was warmed to rt over 2 h and then diluted with EtOAc (200 mL). The solution was washed with 0.5 M KHSO4 (2×), saturated NaHCO3 and brine. It was dried over anhydrous Na2SO4, filtered and concentrated in vacuo to dryness. The residue was purified by flash column chromatography on silica gel eluted with 10:1 hexane/EtOAc to yield N-[(3,5-dichlorobenzene)sulfonyl]-3,4-dehydro-2-methyl-(L)-proline, methyl ester (1.4 g, 44%).
1H NMR (500 MHz, CD3OD): δ 7.80 (d, J=1.5 Hz, 2H); 7.76 (br d, J=1.5 Hz, 1H); 5.96 (dt, J=6.5, 2.0 Hz, 1H); 5.65 (dt, J=6.5, 2.0 Hz, 1H); 4.23 (ABqdd, 2H); 3.68 (s, 3H); 1.70 (s, 3H).
Step D N-[(3,5-Dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-proline, methyl ester (two isomers)
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-3,4-dehydro-2- methyl-(L)-proline, methyl ester (0.19 g, 0.54 mmol) in acetone/t-butanol/water (0.5 mL each) was added 4-methylmorpholine N-oxide (0.16 g, 1.4 mmol) and a crystal of osmium tetroxide. After stirring at rt for 3.5 h, the reaction was quenched with the addition of potassium bisulfite (0.2 g), and the resulting mixture was partitioned between EtOAc and brine. The product was extracted with EtOAc (2×50 mL). The combined extracts were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with 1:1 hexane/EtOAc to afford N-[(3,5-dichlorobenzene)sulfonyl]-cis-3,4-dihydroxy-2-methyl-(L)-proline, methyl ester (0.20 g, 97%) as a 3:2 mixture of diastereomers (based on NMR analysis). This diol was then dissolved in acetone (0.8 mL), and was treated with 2,2-dimethoxypropane (0.20 mL) and a drop of perchloric acid (75%). After stirring at rt for 1 h, the reaction was quenched with the addition of TEA (0.5 mL), and the resulting mixture was loaded onto a chromatography column of silica gel and eluted with 10:1 hexane/EtOAc to yield the two diastereomers of N-[(3,5-dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-proline, methyl ester (48 mg of faster eluting isomer, 120 mg of slower eluting isomer, 24 mg of mixture of two isomers; total yield: 84%).
Faster eluting isomer:
1H NMR (500 MHz, CD3OD): δ 7.75 (br s, 3H); 4.79 (m, 1H); 4.64 (d, J=6.0 Hz, 1H); 3.80 (ABq, 2H); 3.63 (s, 3H); 1.55 (s, 3H); 1.38 (s, 3H); 1.30 (s, 3H).
Slower eluting isomer:
1H NMR (500 MHz, CD3OD): δ 8.01 (d, J=1.8 Hz, 2H); 7.74 (t, J=1.8 Hz, 1H); 4.87 (dd, J=6.0, 5.5 Hz, 1H); 4.56 (d, J=6.0 Hz, 1H); 3.68 (s, 3H); 3.64 (dd, J=12.0, 4.5 Hz, 1H); 3.48 (d, J=12.0 Hz, 1H); 1.68 (s, 3H); 1.37 (s, 3H); 1.26 (s, 3H).
Step E N-[(3,5-Dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-proline (two isomers)
The two isomers of N-[(3,5-dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-proline were separately treated with LiOH-H2O according to the procedure described in Example 1, Step F to afford their respective carboxylic acids.
The faster eluting isomer from Step D (Isomer 1):
1H NMR (500 MHz, CD3OD): δ 7.76 (t, J=1.5 Hz, 2H); 7.72 (d, J=1.5 Hz, 1H); 4.78 (dd, J=6.0, 5.0 Hz, 1H); 4.66 (d, J=6.0 Hz, 1H); 3.83 (dd, J=11.5, 4.5 Hz, 1H); 3.74 (d, J=11.5 Hz, 1H); 1.55 (s, 3H); 1.37 (s, 3H); 1.30 (s, 3H).
The conversion of the slower eluting isomer from Step D to the corresponding carboxylic acid required heating at 50° C. for 2 days. (Isomer 2).
1H NMR (500 MHz, CD3OD): δ 8.04 (d, J=2.0 Hz, 2H); 7.70 (d, J=2.0 Hz, 1H); 4.87 (dd, J=10.5, 5.5 Hz, 1H); 4.55 (d, J=5.5 Hz, 1H); 3.64 (dd, J=11.5, 4.5 Hz, 1H); 3.46 (d, J=11.5 Hz, 1H); 1.67 (s, 3H); 1.40 (s, 3H); 1.27 (s, 3H).
Step F N-(N-[(3,5-Dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-prolyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
The individual isomers of N-[(3,5-dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-proline from Step E were separately coupled to 4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (Reference Example 2). Thus, to a suspension of N-[(3,5-dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-proline, PyBOP (1 mol equivalent) and 4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (1 mol equivalent) in CH2Cl2 (5 mL) was added DIPEA (2 mol equivalents). The reaction was stirred at rt for 20 h then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with hexane/ethyl acetate (1:1) to afford the separate isomers of N-(N-[(3,5-dichlorobenzene)sulfonyl]-3,4-cis-isopropylidenedioxy-2-methyl-(L)-prolyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester.
Isomer 1 from isomer 1 of Step E (55 mg, 55%).
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.77 (d, J=1.8 Hz, 2H); 7.73 (t, J=1.8 Hz, 1H); 7.61 (d, J=6.5 Hz, 2H); 7.27 (d, J=6.5 Hz, 2H); 4.69 (d, J=6.0 Hz, 1H); 4.68 (dd, J=9.0, 5.5 Hz, 1H); 4.56 (dd, J=9.5, 9.0 Hz, 1H); 3.78 (d, J=12.5 Hz, 1H); 3.75 (s, 3H); 3.39 (dd, J=12.5, 4.5 Hz, 1H); 3.28 (dd, J=14.0, 5.5 Hz, 1H); 3.05 (dd, J=14.0, 9.0 Hz, 1H); 1.51 (s, 3H); 1.24 (s, 3H); 1.16 (s, 3H).
MS: m/e 759 (M+H)30 .
Isomer 2 from isomer 2 of Step E (100 mg, 55%)
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.93 (d, J=1.8 Hz, 2H); 7.77 (t, J=1.8 Hz, 1H); 7.57 (d, J=6.5 Hz, 2H); 7.19 (d, J=6.5 Hz, 2H); 4.90 (dd, J=5.0, 5.0 Hz, 1H); 4.69 (dd, J=6.0, 6.0 Hz, 1H); 4.51 (d, J=6.0 Hz, 1H); 3.74 (s, 3H); 3.64 (dd, J=11.5, 5.0 Hz, 1H); 3.38 (d, J=11.5 Hz, 1H); 3.24 (dd, J=14.0, 7.0 Hz, 1H); 3.14 (dd, J=14.0, 5.5 Hz, 1H); 1.56 (s, 3H); 1.27 (s, 3H); 1.26 (s, 3H).
MS: calculated for C31H30Cl4N4O8S 758, observed m/e 759 (M+H)30 .
Step G N-(N-[(3,5-Dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-prolyl-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
The individual isomers of N-(N-[(3,5-dichlorobenzene)sulfonyl]-cis 3,4-isopropylidenedioxy-2-methyl-(L)-prolyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (Step F) were separately reacted with LiOH-H2O (6 eq) in 1:1:1 THF/MeOH/water (equal volume) at 0° C. for 2 h. The reaction mixture was partitioned between EtOAc/brine/0.5 M potassium hydrogen sulfate, and the product was extracted with EtOAc (3×). The combined extracts were dried over anhydrous Na2SO4, filtered and concentrated in vacuo to yield the separate isomers of N-(N-[(3,5-dichlorobenzene)sulfonyl]-cis-3,4-isopropylidenedioxy-2-methyl-(L)-prolyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
Isomer 1 from isomer 1 of Step F (60 mg, 100%)
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.77 (d, J=1.8 Hz, 2H); 7.73 (t, J=1.8 Hz, 1H); 7.61 (d, J=8.5 Hz, 2H); 7.29 (d, J=8.5 Hz, 2H); 5.46 (dd, J=6.0, 2.0 Hz, 1H); 4.74 (d, J=6.0 Hz, 1H); 4.67 (m, 1H); 4.55 (dd, J=5.5, 5.0 Hz, 1H); 3.77 (d, J=12.5 Hz, 1H); 3.35 (dd, J=12.5, 4.5 Hz, 1H); 3.05 (dd, J=14.0, 9.0 Hz, 1H); 1.51 (s, 3H); 1.23 (s, 3H); 1.14 (s, 3H).
MS: m/e 745 (M+H)30 .
Isomer 2 from isomer 2 of Step F (100 mg, 55%)
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.95 (d, J=1.8 Hz, 2H); 7.74 (t, J=1.8 Hz, 1H); 7.55 (d, J=8.0 Hz, 2H); 7.24 (d, J=6.5 Hz, 2H); 4.89 (dd, J=6.0, 6.0 Hz, 1H); 4.61 (m, 1H); 4.50 (d, J=6.0 Hz, 1H); 3.62 (dd, J=11.5, 5.5 Hz, 1H); 3.32-3.20 (m, 2H); 3.18 (dd, J=13.5, 4.5 Hz, 1H); 1.56 (s, 3H); 1.27 (s, 3H); 1.26 (s, 3H).
MS: m/e 745 (M+H)30 .
EXAMPLE 6
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (Isomer 3)
Step A N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carboxylic acid, methyl ester
To a solution of N-(3,5-dichlorobenzenesulfonyl)-octahydroisoindole-1-carboxylic acid from Example 1, Step B (0.20 g, 0.53 mmol) in MeOH (2 mL) and CH2Cl2 (2 mL) at 0° C. was added over a 10 min period a solution of 1M trimethyl-silyldiazomethane (1.1 mL, 1.1 mmol) in hexanes. The reaction was allowed to come to rt over a period of 3 h. The reaction was quenched with HOAc. Toluene was added and the reaction was concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with 5:1 hexane/ethyl acetate to yield N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1-carboxylic acid, methyl ester as a racemic mixture.
1H NMR (500 MHz, CD3OD): δ 7.87 (d, J=2.0 Hz, 2H); 7.78 (t, J=2.0 Hz, 1H); 4.45 (d, J=7.0 Hz, 1H), 3.75 (s, 3H); 3.45 (dd, J=10.0, 7.5 Hz, 1H); 3.34 (dd, J=10.5, 10.0 Hz, 1H), 2.48 (m, 1H); 2.13 (m, 1H); 1.70-1.15 (m, 8H).
The two enantiomers were separated on a Chiralcel OD column eluted with 5% EtOH/hexane.
Faster eluting isomer:
1H NMR (500 MHz, CD3OD): δ 7.86 (d, J=1.5 Hz, 2H); 7.78 (t, J=1.5 Hz, 1H); 4.44 (d, J=7.0 Hz, 1H), 3.76 (s, 3H); 3.45 (dd, J=10.5, 8.0 Hz, 1H); 3.34 (dd, J=10.5, 10.5 Hz, 1H), 2.48 (m, 1H); 2.13 (m, 1H); 1.70-1.15 (m, 8H).
Slower eluting isomer:
1H NMR (500 MHz, CD3OD): δ 7.86 (d, J=1.5 Hz, 2H); 7.78 (t, J=1.5 Hz, 1H); 4.44 (d, J=7.0 Hz, 1H), 3.76 (s, 3H); 3.45 (dd, J=10.5, 8.0 Hz, 1H); 3.34 (dd, J=10.5, 10.5 Hz, 1H), 2.48 (m, 1H); 2.13 (m, 1H); 1.70-1.15 (m, 8H).
Step B N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carboxylic acid
To a sample of N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1-carboxylic acid, methyl ester (the slower eluting isomer from Step A, 0.16 g, 0.41 mmol) in EtOH (2 mL) and water (1 mL) was added LiOH-H2O (0.16 g, 3.8 mmol). After stirring at 80° C. for 3 h, the reaction mixture was cooled to rt, and was partitioned between brine, 2 M HCl and EtOAc. The product was extracted with EtOAc (3×30 mL), and the combined extracts were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford N-[(3,5-dichlorobenzene)sulfonyl]-octahydroisoindole-1-carboxylic acid (0.16 g, 100%) which was used without further purification.
1H NMR (500 MHz, CD3OD): δ (3:2 mixture of isomers) (selected peaks) 7.85/7.87 (d, 2H); 7.78/7.77 (t, 1H); 4.02/4.38 (d, 1H).
Step C N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carboxylic acid (Step B, 0.16 g, 0.42 mmol) was coupled to 4-[(3′,5′-dichloroisonicotinoyl)-amino]-(L)-phenylalanine, methyl ester hydrochloride according to the procedure described in Example 5, Step F to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.14 g, 45%) as a 3:1 mixture of isomers. The mixture was separated into two pure diastereomers on a Chiralcel OD column eluted with 25% EtOH/hexane.
Faster eluting isomer:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.79 (apparent s, 3H); 7.60 (d, J=8.5 Hz, 2H); 7.28 (d, J=8.5 Hz, 2H); 4.81 (dd, J=9.5, 5.5 Hz, 1H); 3.85 (d, J=4.5 Hz, 1H); 3.75 (s, 3H); 3.44 (dd, J=9.5, 7.0 Hz, 1H); 3.26 (dd, J=14.0, 5.0 Hz, 1H); 3.16 (dd, J=9.0, 8.0 Hz, 1H); 3.05 (dd, J=14.0, 9.5 Hz, 1H); 2.08 (m, 1H); 1.96 (m, 1H); 1.46-1.04 (m, 7H); 0.76 (m, 1H).
MS: m/e 727 (M+H)30 .
Slower eluting isomer:
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.84 (d, J=2.0 Hz, 2H); 7.79 (t, J=2.0 Hz, 1H); 7.61 (d, J=8.5 Hz, 2H); 7.30 (d, J=8.5 Hz, 2H); 4.76 (dd, J=10.5, 4.5 Hz, 1H); 4.17 (d, J=7.0 Hz, 1H); 3.78 (s, 3H); 3.46-3.34 (m, 2H); 3.25 (dd, J=13.5, 5.0 Hz, 1H); 2.91 (dd, J=14.0, 10.5 Hz, 1H); 2.10 (m, 1H); 1.83 (m, 1H); 1.64-0.86 (m, 7H); 0.47 (m, 1H).
MS: calculated for C31H30Cl4N4O6S 726, observed m/e 727 (M+H)30 .
Step D N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (Step C, faster eluting isomer, 78 mg, 0.11 mmol) was reacted with LiOH-H2O according to the procedure described in Example 1, Step F with the following modifications: the reaction mixture was partitioned between brine, 0.5 M potassium hydrogen sulfate and EtOAc. The product was extracted with EtOAc (3×), and the combined extracts were dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford N-(N-[(3,5-Dichlorobenzene)sulfonyl]-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.79 (apparent s, 3H); 7.60 (d, J=8.5 Hz, 2H); 7.30 (d, J=8.5 Hz, 2H); 4.77 (m, 1H); 3.86 (d, J=4.5 Hz, 1H); 3.42 (dd, J=9.5, 6.5 Hz, 1H); 3.30 (dd, 1H); 3.16 (dd, J=9.5, 8.0 Hz, 1H); 3.06 (dd, J=14.0, 9.0 Hz, 1H); 2.08 (m, 1H); 1.98 (m, 1H); 1.46-1.08 (m, 7H); 0.76 (m, 1H).
MS: m/e 713 (M+H)30 .
EXAMPLE 7
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (Isomer 4)
The slower eluting isomer of N-(N-[(3,5-dichlorobenzene)sulfonyl]-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester from Example 6, Step C was reacted with LiOH-H2O according to the procedures described in Example 1, Step F. The reaction mixture was partitioned between brine, 0.5 M potassium hydrogen sulfate and EtOAc. The product was extracted with EtOAc (3×), and the combined extracts were dried over anhydrous MgSO4 and concentrated in vacuo to afford N-(N-[(3,5-dichlorobenzene)-sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (Isomer 4) (67 mg).
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.82 (d, J=1.5 Hz, 2H); 7.78 (t, J=1.5 Hz, 1H); 7.58 (d, J=8.5 Hz, 2H); 7.28 (d, J=8.5 Hz, 2H); 4.74 (dd, J=10.0, 5.0 Hz, 1H); 3.86 (d, J=3.5 Hz, 1H); 3.42 (dd, J=9.0, 7.5 Hz, 1H); 3.29 (dd, 1H); 3.16 (dd, J=9.0, 8.5 Hz, 1H); 2.99 (dd, J=14.0, 5.0 Hz, 1H); 2.21 (m, 1H); 1.78 (m, 1H); 1.60-0.86 (m, 7H); 0.54 (m, 1H).
MS: m/e 713 (M+H)30 .
EXAMPLE 8
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (Isomers 1 and 2)





Step A N-BOC-1,3-dihydro-2H-isoindol-1-carboxylic acid, methyl ester
To a solution of N-BOC-1,3-dihydro-2H-isoindol-1-carboxylic acid (SNPE, 10 g, 38 mmol) in CH2Cl2 (100 mL) and MeOH (100 mL) at 0° C. was added trimethylsilyldiazomethane dropwise until the yellow color persisted. After stirring at rt for 15 min, the reaction mixture was concentrated in vacuo. The residue was azeotroped with toluene to afford N-BOC-1,3-dihydro-2H-isoindol-1-carboxylic acid, methyl ester which was used without further purification.
Step B N-BOC-1-methyl-1,3-dihydro-2H-isoindol-1-carboxylic acid, methyl ester
To a solution N-BOC-1,3-dihydro-2H-isoindol-1-carboxylic acid, methyl ester (38 mmol) and MeI (10 mL, 160 mmol) in 150 mL of anhydrous THF at −30° C. was added LDA (Aldrich, 1.5 M in cyclohexane, 51 mL, 76.5 mmol). The reaction was allowed to warm up to rt over 4 h. The reaction was cooled to −30° C., and was quenched with saturated aqueous solution of NH4Cl (50 mL). The resulting mixture was partitioned between EtOAc and brine, and the product was extracted with EtOAc (3×100 mL). The combined extracts were dried with anhydrous MgSO4 and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with 10:1 to 5:1 hexane/ether to afford N-BOC-1-methyl-1,3-dihydro-2H-isoindol-1-carboxylic acid, methyl ester.
1H NMR (500 MHz, CD3OD): δ (mixture of rotamers) 7.40-7.10 (m, 4H); 4.80-4.64 (m, 2H); 3.65/3.62 (s, 3H); 1.84/1.82 (s, 3H); 1.51/1.47 (s, 9H).
Step C N-BOC-1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester
A sample of N-BOC-1-methyl-1,3-dihydro-2H-isoindol-1-carboxylic acid, methyl ester (2.0 g, 6.9 mmol) and 5% Rh on alumina (1 g) in EtOH (20 mL) was hydrogenated (40 psi) at 60° C. overnight. The catalyst was filtered through a pad of celite and the filtrate was concentrated in vacuo to yield N-BOC-1-methyl-octahydroisonindole-1-carboxylic acid, methyl ester which was used without further purification.
Step D 1-Methyl-octahydroisoindole-1-carboxylic acid, methyl ester hydrochloride
N-BOC-1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester was treated with HCl according to the procedure described in Example 1, Step A to afford 1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester hydrochloride which was used without further purification.
Step E N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester
1-Methyl-octahydroisoindole-1-carboxylic acid, methyl ester hydrochloride (0.77 g, 3.7 mmol) was reacted with 3,5-dichlorobenzenesulfonyl chloride according to the procedure described in Example 1, Step B. The product was purified by flash column chromatography on silica gel eluted with 10:1 hexane/Et2O to afford N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester as one major diastereomer along with at least one other minor diastereomer (slower eluting).
Step F N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid
The slower eluting isomer of N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester from Step E (0.55 g, 1.4 mmol) was reacted with LiOH-H2O according to the procedure described in Example 1, Step F. After stirring at rt overnight, the reaction mixture was diluted with water (40 mL), and was extracted with Et2O (3×50 mL). The combined extracts were dried over anhydrous MgSO4, filtered, and concentrated in vacuo afford starting material (0.32 g, 58%) as a single isomer by NMR.
1H NMR (500 MHz, CD3OD): δ 7.90 (d, J=1.5 Hz, 2H); 7.74 (t, J=1.5 Hz, 1H); 3.74 (s, 3H); 3.50 (dd, J=8.5, 8.5 Hz, 1H); 3.19 (dd, J=9.0, 8.5 Hz, 1H); 2.64 (m, 1H); 2.14 (m, 1H); 1.77 (s, 3H); 1.74-1.10 (m, 8H).
The aqueous fraction was then acidified to pH 3 with concentrated HCl and extracted with EtOAc (3×50 mL). The combined extracts were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to dryness to give a residue (0.19 g) which contained a single minor isomer of N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid along with some minor contaminants.
1H NMR (500 MHz, CD3OD): δ (selected peaks) 1.58 (s, 3H).
Step G N-(N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
The single minor isomer of N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid isolated in Step F (0.19 g, 0.47 mmol) was coupled to 4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester hydrochloride according to the procedure described in Example 5, Step F to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.26 g, 74%). Further purification on reverse phase HPLC eluted with AcCN and water (containing 0.1% formic acid) and then on a Chiralcel AD column gave N-(N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester as two pure single diastereomers.
Faster eluting isomer on the AD column:
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.79 (d, J=2.0 Hz, 2H); 7.52 (t, J=2.0 Hz, 1H); 7.61 (d, J=8.5 Hz, 2H); 7.28 (d, J=8.5 Hz, 2H); 4.78 (dd, J=9.5, 4.5 Hz, 1H); 3.77 (s, 3H); 3.47 (dd, J=9.0, 9.0 Hz, 1H); 3.30 (dd, 1H); 3.26 (dd, J=14.0, 4.5 Hz, 1H); 3.05 (dd, J=14.0, 9.0 Hz, 1H); 2.53 (m, 1H); 1.91 (m, 1H); 1.65-0.84 (m, 7H); 1.49 (s, 3H); 0.55 (m, 1H).
Slower eluting isomer on the AD column:
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.83 (d, J=1.5 Hz, 2H); 7.78 (t, J=1.5 Hz, 1H); 7.62 (d, J=8.5 Hz, 2H); 7.26 (d, J=8.5 Hz, 2H); 4.78 (dd, J=10.5, 4.5 Hz, 1H); 3.79 (s, 3H); 3.40-3.28 (m, 3H); 3.01 (dd, J=14.0, 10.5 Hz, 1H); 2.05 (m, 1H); 1.72-0.84 (m, 8H); 1.42 (s, 3H); 0.46 (m, 1H).
Step H N-(N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
The two single isomers of N-(N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester from Step G were separately reacted with LiOH-H2O according to the procedure described in Example 1, Step F. The reaction mixture was partitioned between brine, 0.5 M sodium hydrogen sulfate and EtOAc. The product was extracted with EtOAc (3×), and the combined extracts were dried over anhydrous Na2SO4 and concentrated in vacuo to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
Isomer from the faster eluting isomer of Step G:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.79 (d, J=2.0 Hz, 2H); 7.79 (t, J=1.5 Hz, 1H); 7.59 (d, J=8.5 Hz, 2H); 7.31 (d, J=8.5 Hz, 2H); 4.67 (m, 1H); 3.51 (m, 1H); 3.36-3.22 (m, 2H); 3.05 (m, 1H); 2.18 (m, 1H); 1.97 (m, 1H); 1.66-0.88 (m, 7H); 1.48 (s, 3H); 0.53 (m, 1H).
MS: m/e 727 (M+H)30 .
Isomer from the slower eluting isomer of Step G:
1H NMR (400 MHz, CD3OD): δ 8.64 (s, 2H); 7.84 (d, J=1.5 Hz, 2H); 7.77 (t, J=1.5 Hz, 1H); 7.60 (d, J=8.5 Hz, 2H); 7.26 (d, J=8.5 Hz, 2H); 4.69 (m, 1H); 3.39 (d, J=14.0, 5.0 Hz, 1H), 3.36-3.26 (m, 2H); 3.03 (dd, J=14.0, 9.5 Hz, 1H); 2.08 (m, 1H); 1.72-0.84 (m, 8H); 1.43 (s, 3H); 0.46 (m, 1H).
MS: m/e 727 (M+H)30 .
EXAMPLE 9
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (Isomers 3 and 4)
Step A N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid
To a sample of the faster eluting isomer of N-[(3,5-dichlorobenzene)-sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid, methyl ester from Example 8, Step E (1.8 g, 4.4 mmol) in DMSO (60 mL) at rt was added KOH solid (3.9 g, 69 mmol) in water (10 mL). After stirring at 90° C. for 1 h, the reaction mixture was cooled and partitioned between Et2O/water. The layers were separated and the aqueous layer was treated concentrated HCl to pH 3. This aqueous layer was extracted with Et2O (2×200 mL) and the combined extracts were dried over anhydrous MgSO4, filtered, and concentrated in vacuo to dryness to afford the other isomers of N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid (1.8 g) which were used without further purification.
1H NMR (500 MHz, CD3OD): δ 7.92 (d, J=2.0 Hz, 2H); 7.71 (t, J=2.0 Hz, 1H); 3.50 (dd, J=8.5, 8.5 Hz, 1H); 3.16 (dd, J=10.0, 9.5 Hz, 1H); 2.08 (m, 1H); 1.778 (s, 3H); 1.75-1.05 (m, 9H).
Step B N-(N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
To a suspension of the isomers of N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carboxylic acid from Step A (1.8 g, 4.4 mmol) in CH2Cl2 (10 mL) was added SOCl2 (1.3 mL, 18 mmol). After heating at 40° C. for 2 h, the reaction mixture was cooled and concentrated in vacuo to dryness, and the residue was azeotroped with toluene (1×). This residue was dissolved in CH2Cl2 (10 mL) and added to a solution of 4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester hydrochloride (1.8 g, 4.1 mmol), N-methylmorpholine (1.5 mL, 14 mmol) in 10 mL of CH2Cl2 (10 mL) at 0° C. The reaction mixture was allowed to warm to rt and stirred for 2 days. The resulting mixture was loaded onto a silica gel column and eluted with 1:1 hexane/EtOAc to afford a 1:1 mixture of diastereomers. Pure isomers were obtained by eluting on Chiralpak AD and Chiralcel OD columns as in Example 8, Step G.
Faster eluting isomer on the AD column:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.93 (d, J=2.0 Hz, 2H); 7.78 (t, J=2.0 Hz, 1H); 7.62 (d, J=8.5 Hz, 2H); 7.19 (d, J=8.5 Hz, 2H); 4.70 (dd, J=10.0, 5.0 Hz, 1H); 3.77 (s, 3H); 3.55 (dd, J=9.0, 8.0 Hz, 1H); 3.29 (dd, 1H); 3.18 (dd, J=11.5, 9.5 Hz, 1H); 2.95 (dd, J=14.0, 10.0 Hz, 1H); 2.52 (m, 1H); 1.95 (m, 1H); 1.70 (s, 3H); 1.66-0.88 (m, 7H); 0.71 (m, 1H).
Slower eluting isomer on the AD column:
1H NMR (500 MHz, CD3D): δ 8.64 (s, 2H); 7.84 (d, J=2.0 Hz, 2H); 7.71 (t, J=2.0 Hz, 1H); 7.60 (d, J=8.5 Hz, 2H); 7.32 (d, J=8.5 Hz, 2H); 4.76 (dd, J=9.0, 4.5 Hz, 1H); 3.77 (s, 3H); 3.49 (dd, J=9.0, 9.0 Hz, 1H); 3.26 (dd, J=14.0, 5.0 Hz, 1H); 3.18 (dd, J=10.5, 10.5 Hz, 1H); 3.06 (dd, J=13.5, 9.0 Hz, 1H); 2.57 (m, 1H); 1.83 (m, 1H); 1.80 (s, 3H); 1.68-1.12 (m, 6H); 0.95 (m, 1H); 0.64 (m, 1H).
MS: m/e 741 (M+H)30 .
Step C N-(N-[(3,5-Dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
The two single isomers of N-(N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester from Step B were separately reacted with LiOH-H2O according to the procedure described in Example 1, Step F. The reaction mixture was partitioned between brine, 0.5 M sodium hydrogen sulfate and EtOAc. The product was extracted with EtOAc (3×), and the combined extracts were dried over anhydrous Na2SO4 and concentrated in vacuo to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-1-methyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
Isomer from the faster eluting isomer of Step B:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.77 (d, 2H); 7.78 (t, 1H); 7.60 (d, 2H); 7.20 (d, 2H); 4.66 (m, 1H); 3.57 (dd, 1H); 3.32 (m. 1H); 3.16 (dd, 1H); 2.96 (dd, 1H); 2.55 (m, 1H); 1.95 (m, 1H); 1.72 (s, 3H); 1.70-0.60 (m, 8H).
MS: m/e 727 (M+H)30 .
Isomer from the slower eluting isomer of Step B:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.78 (d, J=2.0 Hz, 2H); 7.70 (t, J=2.0 Hz, 1H); 7.57 (d, J=8.5 Hz, 2H); 7.36 (d, J=8.5 Hz, 2H); 4.70 (m, 1H); 3.50 (dd, J=9.0, 8.5 Hz, 1H); 3.26 (dd, J=14.0, 5.0 Hz, 1H); 3.18 (dd, J=10.5, 10.0 Hz, 1H); 3.10 (dd, J=13.5, 8.0 Hz, 1H); 2.57 (m, 1H); 1.87 (m, 1H); 1.81 (s, 3H); 1.70-0.84 (m, 7H); 0.95 (m, 1H).
MS: m/e 727 (M+H)30 .
EXAMPLE 10
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (Isomers 1-4)





Step A N-Acetyl-3-azabicyclo[3.3.0]octane-2,2-dicarboxylic acid, diethyl ester
The title compound (1.9 g, 50%) was prepared from cyclopenten-1-aldehyde (1.2 g, 13 mmol; prepared following the procedure of Brown et al J. Chem. Soc. 1950, 3634) and diethyl acetamidomalonate (Aldrich, 2.8 g, 13 mmol) following the procedure of Chung et al (J. Org. Chem. 1990, 55, 270).
1H NMR (400 MHz, CD3OD): δ 4.19 (q, 2H), 4.18 (q, 2H); 3.92 (dd, 1H), 3.35 (dd, 1H); 2.94 (m, 2H); 2.04 (s, 3H); 1.95-1.50 (m, 6H); 1.24 (t, 3H); 1.23 (t, 3H).
MS: calculated for C15H23NO5 297, observed m/e 298 (M+H)+.
Step B 3-Azabicyclo[3.3.0]octane-2-carboxylic acid, hydrobromide
A solution of N-acetyl-3-azabicyclo[3.3.0]octane-2,2-dicarboxylic acid, diethyl ester (1.9 g, 6.3 mmol) in aqueous HBr (48%, 10 mL) and HOAc (2 mL) was heated at 120° C. under nitrogen for 18 h. The reaction mixture was cooled, concentrated in vacuo, and the residue was azeotroped with acetonitrile (2×) and Et2O/toluene (1×) to yield 3-azabicyclo[3.3.0]octane-2-carboxylic acid, hydrobromide (1.4 g, 93%).
MS: m/e 156 (M+H)30 .
Step C N-[(3,5-Dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carboxylic acid
3-Azabicyclo[3.3.0]octane-2-carboxylic acid, hydrobromide (0.86 g, 3.6 mmol) was reacted with 3,5-dichlorobenzenesulfonyl chloride according to the procedure described in Example 1, Step B (0.5 additional equivalent of sodium carbonate was used) to yield N-[(3,5-dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]-octane-2-carboxylic acid. (1.3 g, 100%).
1H NMR (400 MHz, CD3OD): δ (3:2 mixture of isomers) (selected peaks) 4.50/4.08 (d, 1H); 3.72/3.62 (dd, 1H).
Step D N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
N-[(3,5-Dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carboxylic acid (0.20 g, 0.55 mmol) was coupled to 4-[(3′,5′-dichloroisonicotinoyl)-amino]-(L)-phenylalanine, methyl ester according to the procedure described in Example 4, Step B to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-azabicyclo-[3.3.0]octane-2-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester as a mixture of four isomers (0.14 g, 73%). This mixture was separated on Chiralcel OD and Chiralpak AD columns into the four pure diastereomers.
Isomer 1 (order of elution):
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.74 (apparent s, 3H); 7.56 (d, J=8.5 Hz, 2H); 7.23 (d, J=8.5 Hz, 2H); 4.73 (dd, J=10.5, 5.0 Hz, 1H); 3.96 (d, J=3.0 Hz, 1H); 3.80 (s, 3H); 3.60 (dd, J=9.5, 8.0 Hz, 1H); 3.26 (dd, J=14.0, 5.0 Hz, 1H); 3.12 (dd, J=10.0, 4.0 Hz, 1H); 2.91 (dd, J=14.0, 10.5 Hz, 1H); 2.54 (m, 1H); 2.01 (m, 1H); 1.82-1.68 (m, 2H); 1.58-1.42 (m, 2H); 1.30-1.16 (m, 2H).
MS: m/e 712 (M+H)30 .
Isomer 2:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.76 (t, J=1.5 Hz, 1H); 7.73 (d, J=1.5 Hz, 2H); 7.63 (d, J=8.5 Hz, 2H); 7.30 (d, J=8.5 Hz, 2H); 4.75 (dd, J=8.5, 6.0 Hz, 1H); 4.38 (d, J=8.5 Hz, 1H); 3.73 (s, 3H); 3.72 (dd, 1H); 3.24 (dd, J=14.5, 5.5 Hz, 1H); 3.08-3.00 (m, 2H); 2.84 (m, 1H); 2.40 (m, 1H); 1.70-0.80 (m, 6H).
MS: m/e 712 (M+H)30 .
Isomer 3:
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.76 (t, J=2.0 Hz, 1H); 7.73 (d, J=2.0 Hz, 2H); 7.60 (d, J=8.5 Hz, 2H); 7.29 (d, J=8.5 Hz, 2H); 4.69 (dd, J=9.0, 6.0 Hz, 1H); 3.99 (d, J=3.5 Hz, 1H); 3.72 (s, 3H); 3.66 (dd, J=10.0, 8.0 Hz, 1H); 3.21 (dd, J=14.0, 5.5 Hz, 1H); 3.12-3.03 (m, 2H); 2.64 (m, 1H); 2.41 (m, 1H); 1.90-0.80 (m, 6H).
MS: m/e 712 (M+H)30 .
Isomer 4:
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.77 (d, J=2.0 Hz, 2H); 7.76 (t, J=2.0 Hz, 1H); 7.59 (d, J=8.5 Hz, 2H); 7.28 (d, J=8.5 Hz, 2H); 4.73 (dd, J=10.0, 4.5 Hz, 1H); 4.36 (d, J=9.0 Hz, 1H); 3.78 (s, 3H); 3.70 (dd, J=11.0, 8.5 Hz, 1H); 3.25 (dd, J=14.0, 4.5 Hz, 1H); 3.03 (dd, J=10.5, 9.0 Hz, 1H); 2.94 (dd, J=14.0, 10.0 Hz, 1H); 2.73 (m, 1H); 2.42 (m, 1H); 1.63-1.52 (m, 2H); 1.42-1.32 (m, 2H); 1.06-0.95 (m, 2H).
MS: m/e 712 (M+H)30 .
Step E N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
The individual isomers of N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester from Step D were separately converted to their respective free acids according to the procedure described in Example 1, Step F. The reaction mixture was partitioned between brine, 1 M HCl and EtOAc. The product was extracted with EtOAc (3×), and the combined extracts were dried over anhydrous MgSO4 and concentrated in vacuo to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-azabicyclo[3.3.0]octane-2-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
Isomer 1 (according to Step D):
1H NMR (400 MHz, CD3OD): δ 8.63 (s, 2H); 7.76-7.70 (m, 3H); 7.56 (d, 2H); 7.24 (d, 2H); 4.71 (m, 1H); 3.98 (d, 1H); 3.59 (dd, 1H); 3.32-3.24 (m, 1H); 3.13 (dd, 1H); 2.92 (dd, 1H); 2.53 (m, 1H); 2.00 (m, 1H); 1.82-0.82 (m, 6H).
MS: m/e 699 (M+H)30 .
Isomer 2:
1H NMR (400 MHz, CD3OD): δ 8.63 (s, 2H); 7.77 (t, 1H); 7.76 (d, 2H); 7.62 (d, 2H); 7.34 (d, 2H); 4.64 (m, 1H); 4.40 (d, 1H); 3.72 (dd, 1H); 3.23 (dd, 1H); 3.12-2.98 (m, 2H); 2.84 (m, 1H); 2.39 (m, 1H); 1.70-0.80 (m, 6H).
MS: m/e 699 (M+H)30 .
Isomer 3:
1H NMR (400 MHz, CD3OD): δ 8.63 (s, 2H); 7.76 (t, 1H); 7.73 (d, 2H); 7.60 (d, 2H); 7.32 (d, 2H); 4.66 (dd, 1H); 4.12 (d, 1H); 3.64 (dd, 1H); 3.24 (dd, 1H); 3.12-3.00 (m, 2H); 2.64 (m, 1H); 2.42 (m, 1H); 1.90-0.80 (m, 6H).
MS: m/e 699 (M+H)30 .
Isomer 4:
1H NMR (500 MHz, CD3OD): δ 8.64 (s, 2H); 7.79 (d, 2H); 7.76 (t, 1H); 7.59 (d, 2H); 7.30 (d, 2H); 4.74 (dd, 1H); 4.38 (d, 1H); 3.69 (dd, 1H); 3.25 (dd, 1H); 3.02 (dd, 1H); 2.98(dd, 1H); 2.74 (m, 1H); 2.40 (m, 1H); 1.63-0.95 (m, 6H).
MS: m/e 699 (M+H)30 .
EXAMPLE 11
N-{N-[(4-methylbenzene)sulfonyl]octahydroisoindole-1-carbonyl}-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
Step A N-(4-methylbenzenesulfonyl)-octahydroisoindole-1-carboxylic acid
Octahydroisoindole-1-carboxylic acid (0.125 g; 0.74 mmol) from Example 1, Step A, p-toluenesulfonyl chloride (0.155 g; 0.814 mmol), and sodium carbonate (0.157 g; 1.48 mmol) were dissolved in water (3 mL). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with water and washed with EtOAc. The water layer was acidified with concentrated. HCl and extracted with CH2Cl2 (2×50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford N-(4-methylbenzenesulfonyl)-octahydroisoindole-1-carboxylic acid (0.15 g).
MS: m/e 324.2
Step B N-(N-[(4-methylbenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
N-(4-methylbenzenesulfonyl)-octahydroisoindole-1-carboxylic acid (0.03 g; 0.093 mmol), N-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, t-butyl ester (0.042 g; 0.102 mmol), and PyBOP (0.058 g; 0.112 mmol) were dissolved in CH2Cl2 (1 ml). DIPEA (0.043 mL; 0.23 mmol) was added and the reaction mixture was stirred at rt for 18 hr. The residue was purified by HPLC. The resulting oil was dissolved in CH2Cl2 (0.5 mL) and TFA (0.5 mL). The reaction mixture was stirred at rt for 4 hr. The reaction was purified by HPLC to yield N-(N-[(4-methyl-benzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)-amino]-(L)-phenylalanine (0.019 g).
MS m/e 659.3
EXAMPLE 12
N-(3-Methylbenzenesulfonyl)-octahydroisoindole-1-carbonyl-(4-(N-(3,5-dichloroisonicotinoyl))amino)-phenylalanine
Step A N-(N-(BOC)-octahydroisoindole-1-carbonyl)-(4-(N-(3,5-dichloroisonicotinoyl))amino)-phenylalanine, methyl ester
N-(t-BOC)-octahydroisoindole-2-carboxylic acid (200 mg, 0.744 mmol) and 4-(N-(3,5-dichloroisonicotinoyl))amino)-phenylalanine, methyl ester hydrochloride (0.0648 mmol, 26 mg) were dissolved in 7.4 mL of CH2Cl2. DIPEA (1.86 mmol, 240 mg) was added followed by O-(benzotriazol-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate (benzotriazol-1-yl)oxy-tris-pyrrodino-phosphonium hexafluorophosphate (0.818 mmol, 426 mg). After stirring at rt for 2.5 days, the solvents were removed in vacuo and the residue was purified by flash column chromatography on silica gel eluted with a gradient of hexane and EtOAc to yield N-(N-(BOC)-octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)-amino)-(L)-phenylalanine, methyl ester (40 mg, 83%) as a white foam (homogeneous by TLC (Rf=0.15, 1:1 hexane:ethyl acetate).
500 MHz 1H NMR (CDCl3): δ 1.45 (s, 9H), 1.0-1.9 (m, 8H), 2.2-2.4 (m, 2H), 3.0-3.2 (m, 2H), 3.2-3.7 (m, 2H), 3.71, 3.74 (2s, 3H), 4.05-4.13 (m, 1H), 4.85-4.95 (m, 1H), 6.45 (d, J=8H, 1H), 7.22 (dd, J=8.3, 1.7 Hz, 2H), 7.6 (m, 2H), 7.8 (m, 1H), 8.60 (s, 2H).
MS: (LC/MS ESI) m/e 519 (M−CO2-t-Bu)+.
Step B N-(Octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)-amino)-(L)-phenylalanine, methyl ester
N-(N-(BOC)-octahydroisoindole-1-carbonyl)-(4-(N-(3,5-dichloroisonicotinoyl))amino)phenylalanine, methyl ester (120 mg) was treated with 4 mL of a 2M solution of HCl in EtOAc. After being stirred at rt for 5 hr, TLC (1:1 hexane: ethyl acetate) indicated disappearance of all starting material. The excess HCl was removed by a stream of nitrogen and the volatiles were removed in vacuo to afford N-(octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester which was used without further purification in the subsequent reaction.
500 MHz 1H NMR (CDCl3): δ 0.8-1.7 (m, 8H), 2.15-2.65 (m, 2H), 2.8-2.95 (m, 1H), 3.05-3.4 (m, 3H), 3.9 (s, 3H), 4.35-4.35 (m, 1H), 4.65-4.80 (m, 1H), 7.13 (d, J=8.2H, 1H), 7.20 (d, J=8.3, 1H), 7.55 (m, 2H), 8.51, 8.53 (2s, 2H).
Mass spectrum (LC/MS ESI) m/e 519 (M+1)+.
Step C N-(N-(3-Methylbenzenesulfonyl)-octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester
N-(Octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (30 mg) was dissolved in 0.15 each of CH2Cl2 and THF. To this was added m-toluenesulfonyl chloride (0.051 mmol, 9.7 mg), followed by DIPEA (0.153 mmol, 20 mg) and 4-DMAP (0.01 mmol, 1.24 mg). This mixture was stirred at rt overnight when TLC (1:2 hexane:ethyl acetate) indicated the disappearance of all starting material. The volatiles were removed in vacuo and the residue was purified by flash column chromatography on silica gel eluted with a gradient of hexane and EtOAc to yield N-(N-(3-methylbenzenesulfonyl)-octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (35 mg) as a sticky foam (homogeneous by TLC (1:2 hexane:ethyl acetate)).
500 MHz 1H NMR (CDCl3): δ 0.75-1.65 (m, 8H), 2.0 (m, 1H), 2.2-2.3 (m, 1H), 2.43 (s, 3H), 3.0-3.2 (m, 1H), 3.2-3.5 (m, 3H), 3.75, 3.80 (2s, 3H), 4.0-4.2 (m, 1H), 4.95-5.05 (m, 1H), 7.10-7.20 (m, 1H), 7.26 (d, J=8 Hz, 1H), 7.38 (d, J=8.3 Hz, 1H), 7.56-7.68 (m, 3H), 7,80, 7.86 (2 br s, 1H), 8.58 (s, 2H).
Mass spectrum (LC/MS ESI) m/e 673 (M+1)+.
Step D N-(N-(3-Methylbenzenesulfonyl)octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine
N-(N-(3-Methylbenzenesulfonyl)-octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (0.036 mmol, 24 mg) was dissolved 0.089 mL of MeOH and 0.089 mL of a 0.4 N solution of NaOH in MeOH. The resulting mixture was stirred overnight at rt. Volatiles were removed in vacuo and the residue was partitioned between 5% citric acid and CH2Cl2. The aqueous layer was extracted with CH2Cl2 (2×). The organic extracts were combined and washed with brine, and dried over anhydrous Na2SO4. The residue obtained after filtration and evaporation of volatiles was purified by flash column chromatography on silica gel eluted with a mixture of CH2Cl2 and MeOH to provide N-(N-(3-methylbenzenesulfonyl)-octahydroisoindole-1-carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)-amino)-(L)-phenylalanine (18 mg, 67%) as a white foam (homogeneous by TLC (9:1 dichloromethane: methanol)).
500 MHz 1H NMR (CD3OD): δ 0.80-1.65 (m, 9H), 2.1-2.3 (m, 1H), 2.43, 2.44 (2s, 3H), 3.0-3.15 (m, 1H), 3.2-3.55 (m, 6H), 4.04, 4.12 (2d, J=7.1, 6.8 Hz, 1H), 4.64-4.70 (m, 1H), 7.33 (d, J=8.5 Hz, 1H), 7.37 (d, J=8.5 Hz, 1H), 7.46-7.55 (m, 2H), 7.58-7.62 (m, 2H), 7.65-7.72 (m, 2H), 8.62 (s, 2H).
Mass spectrum (LC/MS ESI) m/e 659 (M+1)+.
The following compounds were prepared by the method described above.












Example

Mass


Number
Name
Spectrum*







13)
N-(N-(3-Bromobenzenesulfonyl)-octahydro-
725



isoindole-1-carbonyl)-4-(N-(3,5-dichloroisonico-



tinoyl)amino)-(L)-phenylalanine


14)
N-(N-(benzenesulfonyl)-octahydroisoindole-1-
645



carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-



(L)-phenylalanine


15)
N-(N-(3-Trifluoromethylbenzenesulfonyl)-
713



octahydro-isoindole-1-carbonyl)-4-(N-(3,5-di-



chloroisonicotinoyl)-amino)-(L)-phenylalanine


16)
N-(N-(3-Trifluoromomethoxybenzenesulfonyl)-
729



octahydro-isoindole-1-carbonyl)-4-(N-(3,5-di-



chloroisonicotinoyl)-amino)-(L)-phenylalanine


17)
N-(N-(2-Chlorobenzenesulfonyl)-octahydroiso-
681



indole-1-carbonyl)-4-(N-(3,5-dichloroisonicotin-



oyl)amino)-(L)-phenylalanine


18)
N-(N-(3-Cyanobenzenesulfonyl)-octahydroisoin-
670



dole-1-carbonyl)-4-(N-(3,5-dichloroisonico-



tinoyl)amino)-(L)-phenylalanine


19)
N-(N-(3,5-Dimethyl-benzenesulfonyl)-octahydro-
673



isoindole-1-carbonyl)-4-(N-(3,5-dichloroisonico-



tinoyl)amino)-(L)-phenylalanine


20)
N-(N-(5-Methyl-3-pyridinesulfonyl)-octahydro-
660



isoindole-1-carbonyl)-4-(N-(3,5-dichloroisonico-



tinoyl)amino)-(L)-phenylalanine


21)
N-(N-(3-Fluorobenzenesulfonyl)-octahydroisoin-
663



dole-1-carbonyl)-4-(N-(3,5-dichloroisonicotin-



oyl)amino)-(L)-phenylalanine


22)
N-(N-(3,5-Bis(trifluoromethyl)benzenesulfonyl)-
781



octahydroisoindole-1-carbonyl)-4-(N-(3,5-



dichloroisonicotinoyl)-amino)-(L)-phenylalanine


23)
N-(N-(4-Chlorobenzenesulfonyl)-octahydroiso-
681



indole-1-carbonyl)-4-(N-(3,5-dichloroisonicotin-



oyl)amino)-(L)-phenylalanine


24)
N-(N-(3-Pyridinesulfonyl)-octahydroisoindole-1-
646



carbonyl)-4-(N-(3,5-dichloroisonicotinoyl)amino)-



(L)-phenylalanine


25)
N-(N-(2-Bromobenzenesulfonyl)-octahydroisoin-
725



dole-1-carbonyl)-4-(N-(3,5-dichloroisonicotin-



oyl)amino)-(L)-phenylalanine


26)
N-(N-(3-Chlorobenzenesulfonyl)-octahydroiso-
681



indole-1-carbonyl)-4-(N-(3,5-dichloroisonicotin-



oyl)amino)-(L)-phenylalanine


27)
N-(N-(3,4-Dichlorobenzenesulfonyl)-octahydro-
715



isoindole-1-carbonyl)-4-(N-(3,5-dichloroisonico-



tinoyl)amino)-(L)-phenylalanine


28)
N-(N-(3-Carboxybenzenesulfonyl)-octahydroiso-
688



indole-1-carbonyl)-4-(N-(3,5-dichloroisonicotin-



oyl)amino)-(L)-phenylalanine









EXAMPLE 29
N-{N-[(3-Chlorobenzene)sulfonyl]octahydroisoindol-1-carboxyl}-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine
Step A N-(BOC)-4-[(3′,5′-Dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester
N-(BOC)-4-[(3′,5′-Dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.3 g, 0.64 mmol) and mCPBA (0.22 g; 1.28 mmol) were dissolved in CH2Cl2. The reaction mixture was stirred at rt overnight. TLC showed starting material remaining so an additional amount of mCPBA (0.22 g; 1.28 mmol) was added. The reaction was heated at 45° C. for 2 hours. The solution was concentrated in vacuo and the residue purified by flash column chromatography on silica gel eluted with 90% EtOAc/hexane to afford N-(BOC)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester (0.34 g).
MS m/e 428.0 (M−BOC)+.
Step B 4-[(3′,5′-Dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester hydrochloride
N-(BOC)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester (0.7 mmol) was dissolved in EtOAc. HCl gas was bubbled through the reaction mixture for several minutes. The resulting solution was stirred at rt for 15 min. The reaction mixture was concentrated in vacuo to afford 4-[(3′,5′-dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester, hydrochloride (0.25 g) which was used without further purification in the next reaction.
Step C N-(N-[(3-Chlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine
[N-(3-Chlorobenzene)sulfonyl]-octahydroisoindole-1-carboxylic acid (0.035 g; 0.1 mmol), N-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine, methyl ester hydrochloride (0.043 g; 0.11 mmol) and PyBOP (0.062 g; 0.12 mmol) were dissolved in CH2Cl2 (1 ml). DIPEA (0.055 mL; 0.3 mmol) was added and the reaction mixture was stirred at rt for 3 hr. The residue was purified by HPLC. The resulting oil was dissolved in MeOH (0.5 mL). An aqueous solution of 1N NaOH ( ̃1 mL) was added. The reaction mixture was stirred at rt for 2 hr. The reaction was acidified with TFA and purified by HPLC to yield N-(N-[(3-chlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine (0.025 g).
MS m/e 697.0
EXAMPLE 30
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloro-2′-hydroxy-isonicotinoyl)amino]-(L)-phenylalanine
Step A N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine, methyl ester
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (196 mg, 0.270 mmol) from Example 1, Step D was reacted with mCPBA according to the procedure described in Example 29, Step A to yield N-(N-[(3,5-dichlorobenzene)-sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester (122 mg, 61%).
400 MHz 1H NMR (CD3OD) δ 8.54 (br, 2H); 8.26 (d, J=6.4 Hz, 1H); 7.97 (d, J=6.4 Hz, 1H); 7.83 (s, 2H); 7.79 (s, 1H); 7.62 (d, J=6.8 Hz, 2H); 7.31 (d, J=6.8 Hz, 2H); 4.87 (m, 1H); 4.15 (d, J=5.2 Hz, 1H); 3.74 (s, 3H); 3.44 (m, 2H); 3.28 (m, 1H); 3.05 (m, 1H); 2.29 (m, 1H); 1.78 (m, 1H); 1.60 (m, 1H); 1.52-1.36 (m, 4H); 1.05 (m, 4H).
Step B N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(2′-acetoxy-3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
A solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine, methyl ester (60 mg, 0.081 mmol) in 1.2 ml of acetic anhydride was heated to 100° C. for 6.5 hr. LC-MS showed approximately 1:1 starting material and desired product. After cooling to rt, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with CH2Cl2/MeOH/HOAc (95:5:0.5) to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-octahydroisoindole-1-carbonyl)-4-[(2′-acetoxy-3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (25 mg (39%).
400 MHz 1H NMR (CD3OD) δ 8.49 (br, 2H); 7.82 (s, 1H); 7.88 (s, 1H); 7.47 (d, J=6.4 Hz, 2H); 7.35 (d, J=6.8 Hz, 2H); 4.21 (d, J=5.2 Hz, 1H); 3.72 (s, 3H); 3.44 (d, J=7.6 Hz, 2H); 3.34 (s, 3H); 3.30 (m, 2H); 3.18 (m, 1H); 2.37 (m, 1H); 1.60-1.28 (m, 8H).
Step C N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloro-2′-hydroxy-isonicotinoyl)amino]-(L)-phenylalanine
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(2′-acetoxy-3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (25 mg, 0.0314 mmol) in 0.2 ml of MeOH was added NaOH (0.20 ml, 0.5 M). The reaction mixture was stirred at rt for 4 hr. The reaction was quenched by addition of TFA (1 drop). The solvent was removed in vacuo and the residue purified by HPLC eluted with 30-90% acetonitrile in water (0.1% TFA) at 10 ml/min over 15 min to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloro-2′-hydroxy-isonicotinoy)amino]-(L)-phenylalanine (22 mg, 94% yd).
400 MHz 1H NMR (CD3OD) δ 8.53 (s, 2H); 7.80 (s, 1H); 7.81 (s, 1H); 7.64 (d, J=6.8 Hz, 2H); 7.34 (d, J=6.8 Hz, 2H); 4.14 (d, J=5.2 Hz, 1H); 3.43 (m, 2H); 3.31 (m, 2H); 3.08 (m, 1H); 2.24 (m, 1H); 1.79 (m, 1H); 1.60-1.03 (m, 8H).
EXAMPLE 31
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-7a-carboxyl-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine





Step A N-benzyl-aziridine-2-carboxylic acid, benzyl ester
To a solution of 2,3-dibromopropionyl chloride (5.00 g, 19.97 mmol) in THF (25 ml) at 0° C. was added dropwise benzyl alcohol (2.10 ml, 19.97 mmol). After stirring for 1 h, the reaction was concentrated in vacuo and the resulting oil dissolved in CH2Cl2 (25 ml) and treated with a solution of benzyl amine (2.20 ml, 19.97 mmol) and TEA (5.80 ml, 41.94 mmol) in CH2Cl2 (10 ml) at 0° C. After stirring for 3 h, the reaction was filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluted with hexane/ethyl acetate (4:1) to afford N-benzyl-aziridine-2-carboxylic acid, benzyl ester as a colorless oil (2.35 g).
500 MHz 1HNMR (CDCl3) δ 7.20-7.50 (m, 10H); 7.77 (s, 1H); 5.23 (d, J=12.8 Hz, 1H); 5.15 (d, J=12.4 Hz, 1H); 3.61 (d, J=13.5 Hz, 1H); 3.54 (d, J=13.5, 1H); 2.31 (m, 1H); 2.26 (dd, J=2.9, 6.4 Hz, 1H); 1.79 (d, J=6.4 Hz, 1H).
Step B N-benzyl-octahydroisoindole-1,7a-dicarboxylic acid, 1-benzyl ester, 7a-methyl ester
A solution of N-benzyl-aziridine-2-carboxylic acid benzyl ester (0.50 g) was dissolved in 1-cyclohexene-1-carboxylate, methyl ester in a sealed tube under nitrogen and was heated to 175° C. After 18 h, the reaction was cooled and the solution placed onto a silica gel flash chromatography column and eluted with hexane/ethyl acetate (10:1). N-benzyl-octahydroisoindole-1,7a-dicarboxylic acid, 1-benzyl ester, 7a-methyl ester was isolated (0.35 g) as a 3:1 mixture of diastereomers as a white solid.
MS m/e 408.2 (M+).
Step C Octahydroisoindole-1,7a-dicarboxylic acid, 7a-methyl ester
A solution of N-benzyl-octahydroisoindole-1,7a-dicarboxylic acid, 1-benzyl ester, 7a-methyl ester (0.35 g) and 10% palladium on carbon (0.3 g) in MeOH (5 ml) was stirred under an atmosphere of hydrogen. After being stirred for 4 h, the reaction was filtered through celite and concentrated in vacuo to yield octahydroisoindole-1,7a-dicarboxylic acid, 7a-methyl ester as a white solid (0.28 g).
MS m/e 228.0 (M+).
Step D N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1,7a-dicarboxylic acid, 7a-methyl ester
To a suspension of octahydroisoindole-1,7a-dicarboxylic acid, 7a-methyl ester (0.2 g, 0.88 mmol) in water (2 ml) was added 3,5-dichlorobenzene-sulfonyl chloride (0.26 g, 1.05 mmol) and Na2CO3 (0.140 g, 1.32 mmol). After being stirred overnight, the reaction was diluted with 2N HCl (10 ml) and extracted with EtOAc (25 ml). The organic layer was washed with saturated aqueous NaCl (15 ml), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by RP-HPLC to afford N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1,7a-dicarboxylic acid, 7a-methyl ester as two separable diastereomers.
First eluting diastereomer
1H NMR (500 MHz, CDCl3): δ 7.82 (d, J=1.8 Hz, 2H); 7.58 (t, J=2.0 Hz, 1H); 4.77 (s, 1H); 3.77 (s, 3H); 3.56 (dd, J=8.1, 10.3 Hz, 1H); 3.41 (t, J=10.3 Hz, 1H); 2.68 (m, 1H); 1.93 (m, 1H); 1.2-1.7 (m, 7H).
MS, m/e 389.8 (M+).
Second eluting diastereomer
1H NMR (500 MHz, CDCl3): δ 7.75 (d, J=1.9 Hz, 2H); 7.58 (t, J=1.9 Hz, 1H); 4.26 (s, 1H); 3.70 (s, 3H); 3.53 (t, J=8.8 Hz, 1H); 3.35 (t, J=9.6 Hz, 1H); 3.1 (m, 1H); 2.12 (m, 1H); 1.1-1.7 (m, 7H).
MS, m/e 389.8 (M+).
Step D N-(N-[(3,5-dichlorobenzene)sulfonyl]-7a-methoxycarbonyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
The diastereomers isolated from Step C were separately reacted according to the following procedure: To a suspension of 4-((3,5-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester hydrochloride from Reference Example 2 (0.023 g, 0.050 mmol), PyBOP (0.029 g, 0.055 mmol) and N-[(3,5-dichlorobenzene)sulfonyl]octahydroisoindole-1,7a-dicarboxylic acid, 7a-methyl ester (0.020 g, 0.046 mmol) in CH2Cl2 (0.30 ml) was added DIPEA (0.025 ml, 0.138 mmol). The reaction was stirred at rt for 20 h then concentrated in vacuo. The residue was purified by preparative HPLC on silica gel and used in the subsequent reaction.
Step E N-(N-[(3,5-dichlorobenzene)sulfonyl]-7a-carboxyl-octahydroisoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine
The resulting white solid from Step D was dissolved in MeOH (0.5 ml) and treated with 1N NaOH (0.2 ml). After stirring at rt for 22 h, the reaction was acidified with TFA, concentrated in vacuo, and purified by preparative HPLC on silica gel to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-7a-carboxyl-octahydro-isoindole-1-carbonyl)-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine as a white solid (0.464 g).
MS m/e 756.6 (M+).
Further purification by preparative HPLC gave samples of the individual diastereriomers.
First eluting diastereomer
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.99 (d, J=8.0 Hz, 1H); 7.87 (d, J=1.9 Hz, 2H); 7.80 (t, J=1.8 Hz, 1H); 7.63 (d, J=8.4 Hz, 2H); 7.34 (d, J=8.5 Hz, 2H); 4.80 (m, 1H); 4.48 (s, 1H); 3.58 (dd, J=7.6, 11.7 Hz, 1H); 3.45 (t, J=11.3 Hz, 1H); 3.28-3.32 (m, 1H); 3.10 (dd, J=9.0, 14.2 Hz); 2.06-2.14 (m, 1H)1; 1.1-1.6 (m, 8H).
MS, m/e 756.9 (M+).
Second eluting diastereomer
1H NMR (500 MHz, CD3OD): δ 8.63 (s, 2H); 7.97 (d, J=8.5 Hz, 1H); 7.86 (d, J=1.8 Hz, 2H); 7.80 (t, J=1.2 Hz, 1H); 7.59 (d, J=8.4 Hz, 2H); 7.34 (d, J=8.5 Hz, 2H); 4.8-4.9 (m, 1H); 4.55 (s, 1H); 3.54 (d, J=9.2 Hz, 2H); 3.21 (dd, J=5.0, 14.2 Hz, 1H); 3.09 (dd, J=5.0, 14.2 Hz, 1H); 2.10-2.14 (m, 1H)1; 1.1-1.6 (m, 8H).
MS, m/e 756.9 (M+).
EXAMPLE 32
N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-3-(6-((3′,5′-dichloroisonicotinoyl)amino)-3-pyridyl)alanine
Step A 2-Bromo-5-bromomethyl-pyridine
2-Bromo-5-methylpyridine (4.01 g, 23.31 mmol), NBS (5.19 g, 29.14 mmol), and AIBN (0.19 g, 1.17 mmol) were dissolved in CCl4 (46.6 mL, 23.31 mmol). The reaction mixture was heated to 75° C. for 4 h. The residue was quenched with water and extracted from EtOAc. The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo in vacuo. The crude mixture was purified by flash column chromatography on silica gel eluted with 7% EtOAc/hexane to give 2.65 g of the title compound. MS m/e 251.9 (M+).
Step B 2-[(N-Diphenylmethylene)amino]-3-(4-bromo-3-pyridinyl)propanoic acid, ethyl ester
To a solution of N-(diphenylmethylene)glycine ethyl ester (4.30 g, 16.11 mmol) in THF (25 mL) at −78° C. was added dropwise a 2.0M solution of LDA in THF (8.0 mL, 16.0 mmol). After being stirred for 30 min at −78° C., a solution of 2-bromo-5-bromomethyl-pyridine in 10 mL of THF was added and the reaction allowed to warm to rt. After 1 h, the reaction was diluted with EtOAc (50 mL) and washed with water (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo to give a yellow oil. The oil was purified by flash column chromatography on silica gel eluted with hexane/EtOAc (10:1) to yield 2-[(N-diphenylmethylene)amino]-3-(4-bromo-3-pyridinyl)propanoic acid, ethyl ester as a pale yellow oil.
Step C 3,5-Dichloropyridine-4-carboxylic acid chloride
3,5-Dichloropyridine-4-carboxylic acid (0.5 g, 2.6 mmol; Reference Example 1) was dissolved in CH2Cl2 (1.75 mL) and DMF (50 μL). Thionyl chloride (0.21 mL, 2.86 mL) was added and the reaction mixture was heated at 50° C. for 20 h. The residue was concentrated in vacuo. The formation of the acid chloride was observed by TLC (50% EtOAc/hexane) and the crude 3,5-dichloropyridine-4-carboxylic acid chloride was used in Step E without further purification.
Step D 3,5-Dichloropyridine-4-carboxamide
3,5-Dichloropyridine-4-carboxylic acid chloride (0.54 g, 2.6 mmol) was dissolved in CH2Cl2 (2.6 mL, 2.6 mmol) and NH3 (2.0 M in dioxane) (6.5 mL) was added. The reaction mixture was cooled to 0° C. for 30 min. The residue was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by preparative HPLC on silica gel to yield 3,5-dichloropyridine-4-carboxamide (0.135 g).
Step E 3-(6-[3′,5′-dichloroisonicotinoyl)amino]-3-pyridinyl)-2-[(N-diphenylmethylene)amino]propanoic acid, ethyl ester
A solution of 2-[(N-diphenylmethylene)amino]-3-(4-bromo-3-pyridinyl)propanoic acid, ethyl ester (2.00 g, 4.57 mmol), 3,5-dichloropyridine-4-carboxamide (0.96 g, 5.03 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.083 g, 0.091 mmol), Cs2CO3 (2.00 g, 6.40 mmol), and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (0.16 g, 0.27 mmol) in THF (10 mL) was heated to 75° C. under nitrogen. After 16 h, the reaction was cooled, diluted with EtOAc (50 mL), washed with saturated aqueous NaCl (50 mL) and dried over anhydrous MgSO4. The solvent was removed in vacuo followed by purification by flash column chromatography on silica gel eluted with hexane/EtOAc (10:1) to yield 3-(6-[3′,5′-dichloroisonicotinoyl)-amino]-3-pyridinyl)-2-[(N-diphenylmethylene)amino]propanoic acid, ethyl ester as a colorless oil (2 g).
MS m/e 574.2 (M+).
Step F 3-(6-(3′,5′-Dichloroisonicotinoyl)amino-3-pyridyl)alanine, ethyl ester
To a solution of 3-(6-[3′,5′-dichloroisonicotinoyl)amino]-3-pyridinyl)-2-[(N-diphenylmethylene)amino]propanoic acid, ethyl ester in THF (10 mL) and water (5 mL) was added glacial HOAc (5 mL). After 3 h, the reaction was diluted with 2N HCl (25 mL) and extracted with EtOAc (2×25 mL) which were discarded. The aqueous layer was made basic (pH=12) with 1N NaOH and the product extracted into EtOAc (3×25 mL). The combined organics were washed with brine (50 mL), dried over anhydrous MgSO4 and concentrated in vacuo to yield 3-(6-(3′,5′-dichloroisonicotinoyl)amino-3-pyridyl)-alanine, ethyl ester as a white foam: MS m/e 383.1 (M+).
1H NMR (500 MHz, CDCl3): δ 8.48 (s, 1H), 8.27 (d, J=8.7 Hz, 1H), 7.62 (dd, J=2.3, 8.5 Hz, 1H), 7.42 (d, J=2 Hz, 1H), 4.16 (q, J=7.1 Hz, 2H), 3.57 (dd, J=4.6, 7.8 Hz, 1H), 2.88 (dd, J=5.3, 14 Hz, 1H), 2.69 (dd, J=7.8, 13.9 Hz, 1H), 1.26 (t, J=7.1 Hz, 3H).
Step G N-(N-[(3,5-Dichlorobenzene)sulfonyl]octahydroisoindole-1-carbonyl)-3-(6-((3′,5′-dichloroisonicotinoyl)amino)-3-pyridyl)alanine
To a suspension of 3-(6-((3′,5′-dichloroisonicotinoyl)amino)-3-pyridyl)alanine, ethyl ester (0.030 g, 0.079 mmol), PyBOP (0.050 g, 0.095 mmol) and N-(3,5-dichlorobenzenesulfonyl)-octahydroisoindole-1-carboxylic acid (0.030 g, 0.079 mmol) in CH2Cl2 (0.25 ml) was added DIPEA (0.021 ml, 0.118 mmol). The reaction was stirred at rt for 19 h then purified via preparative HPLC. The resulting white solid was dissolved in MeOH (0.5 ml) and treated with 1N NaOH (0.2 ml). After stirring at rt for 2 h, the reaction was acidified with TFA, concentrated in vacuo and the residue purified by preparative HPLC to yield N-(N-[(3,5-dichlorobenzene)-sulfonyl]octahydroisoindole-1-carbonyl)-3-(6-((3′,5′-dichloroisonicotinoyl)amino)-3-pyridyl)alanine as a white solid (0.234 g).
MS m/e 714.1 (M+).
EXAMPLE 33
N-(5-(3,5-Dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-yl-carbonyl)-(4-(3,5-dichloro-pyridin-4-yl-carbonyl)amino)-(S)-phenylalanine





Step A 5-Triphenylmethyl-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)-pyrrol-4(S)-carboxylate, methyl ester
A solution of 225 mg (0.53 mmol) of N-triphenylmethyl-3(R)-allyl-4-oxo-(S)-proline, methyl ester in 1.5 ml of CH2Cl2 and 2.3 mL of MeOH was cooled in a −78° C. bath and ozone gas was passed through it until the solution was pale blue. The reaction was purged with nitrogen after a few minutes and 0.15 mL of dimethylsulfide was added. The solution was allowed to warm to rt during the next 1 hr and concentrated. The residue was partitioned between CH2Cl2 and water. The organic layer was dried, concentrated in vacuo and the residue was dissolved in 2 mL of CH2Cl2. To this solution 36 mg (0.55 mmol) of methylamine hydrochloride and 0.1 mL of DIPEA were added. The mixture was sonicated for 2 min to break up methylamine hydrochloride and 0.235 g (1.11 mmol) of sodium triacetoxyborohydride was added. After stirring the reaction overnight, it was diluted with CH2Cl2, washed with water, brine and dried over anhydrous Na2SO4. The filtrate was concentrated in vacuo and the residue was purified on a preparative TLC plate eluted with 5% MeOH−CH2Cl2 to afford 5-triphenylmethyl-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)-pyrrol-4(S)-carboxylate, methyl ester (67 mg).
1H NMR (500 MHz, CDCl3): δ 0.2 (m, 1H), 1.61 (d, 1H), 1.8-2.2 (m, 3H), 2.05 (s, 3H), 2.69 (m, 1H), 2.86 (m, 1H), 3.74 (s, 3H), 3.91 (m, 1H), 3.97 (m, 1H), 7.15-7.7 (m, 15H). LC-MS (Method A): Retention Time: 2.7 min. Mass Spectrum: 427 (M+1).
Step B 5-(3,5-Dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-carboxylic acid
To a solution of 5-triphenylmethyl-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-carboxylate, methyl ester in 3 mL of CH2Cl2, 0.03 mL of TFA was added. After 30 min the reaction was concentrated in vacuo. The residue was diluted with dichloroethane and concentrated in vacuo to remove the remaining TFA. The residual oil was dissolved in 2 mL of MeOH and 0.5 mL of 1N NaOH was added. The mixture was sonicated for 2 hr. The solution was neutralized to pH 7 with 1.2 N HCl and concentrated in vacuo. The residue was dissolved in 1.5 mL of dioxane and 1.5 mL of water containing 20 mg of Na2CO3. To this solution 45 mg (0.18 mmol) of 3,5-dichlorobenzenesulfonyl chloride was added and stirred for 6 hr. The reaction was neutralized to pH 7 with 1.2 N HCl and extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to afford 5-(3,5-dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-carboxylic acid (0.109 g) which was used in the next step without further purification.
LC-MS (Method A): Retention Time: 1.8 min. Mass Spectrum: 379 (M+1).
Step C N-(5-(3,5-Dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-yl-carbonyl)-4-((3′,5′-dichloroisonicotinoyl)-amino)-(L)-phenylalanine, methyl ester
To 109 mg of 5-(3,5-dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-carboxylic acid in 2 mL of CH2Cl2, 45 mg (0.111 mmol) of 4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester, hydrochloride and 0.05 mL (0.36 mmol) of TEA were added. After all the salt had dissolved, 60 mg (0.115 mmol) of PyBOP was added and the mixture was stirred overnight. The reaction was partitioned between CH2Cl2 and water. The CH2Cl2 layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC eluted with 5% MeOH−EtOAc to yield N-(5-(3,5-dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-yl-carbonyl)-4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester (39 mg).
LC-MS (Method A): Retention Time: 2.7 min. Mass Spectrum: 728 (M+1).
Step D N-(5-(3,5-dichlorobenzensulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-yl-carbonyl)-4-((3′,5′-dichloroisonicotinoyl)-amino)-(L)-phenylalanine
A solution of 39 mg (0.053 mmol) of N-(5-(3,5-dichlorobenzene-sulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-yl-carbonyl)-4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester in 1.5 mL of MeOH and 0.5 mL of water was treated with 7 mg (0.167 mmol) of LiOH-H2O. After stirring for 2 hr, the reaction was incomplete so the mixture was sonicated for 15 min. The solution was neutralized to pH 6.5 with 1.2 N HCl and concentrated in vacuo. The residue was purified by reverse phase HPLC eluted with a gradient of 10-90% MeCN-water with 0.1% TFA to afford 32 mg of N-(5-(3,5-dichlorobenzene-sulfonyl)-1-methyl-octahydro-3a(S),6a(R)-pyrrolo(3,4-b)pyrrol-4(S)-yl-carbonyl)-4-((3′,5′-dichloroisonicotinoyl)amino)-(L)-phenylalanine, trifluoroacetate salt as a white solid.
1H NMR (500 MHz, CD3OD): δ 2.0-2.6 (m, 4H), 2.87 (s, 3H), 3.0-3.35 (m, 3H), 3.82 (m, 1H), 3.95 (m, 1H), 4.53 (m, 1H), 4.7 (m, 1H), 4.81 (m, 1H), 7.39 (d, 2H), 7.6 (d, 2H), 7.85 (s, 1H), 7.96 (s, 2H), 8.64 (s, 2H).
LC-MS (Method A): Retention Time: 2.5 min. Mass Spectrum: 716 (M+1).
EXAMPLE 34
Antagonism of VLA-4 Dependent Binding to VCAM-IG Fusion Protein
Step A Preparation of VCAM-Ig
The signal peptide as well as domains 1 and 2 of human VCAM (GenBank Accession no. M30257) were amplified by PCR using the human VCAM cDNA (R & D Systems) as a template. The resulting PCR product of 650 bp was digested with EcoRI and BclI and ligated to expression vector pIg-Tail (R & D Systems, Minneapolis, Minn.) digested with EcoRI and BamHI. The pIg-Tail vector contains the genomic fragment which encodes the hinge region, CH2 and CH3 of human IgG1 (GenBank Accession no. Z17370). The DNA sequence of the resulting VCAM fragment was verified using Sequenase (US Biochemical, Cleveland, Ohio). The fragment encoding the entire VCAM-Ig fusion was subsequently excised from pIg-Tail with EcoRI and NotI and ligated to pCI-neo (Promega, Madison, Wis.) digested with EcoRI and NotI. The resulting vector, designated pCI-neo/VCAM-Ig was transfected into CHO-K1 (ATCC CCL 61) cells using calcium-phosphate DNA precipitation (Specialty Media, Lavalette, N.J.). Stable VCAM-Ig producing clones were selected according to standard protocols using 0.2-0.8 mg/ml active G418 (Gibco, Grand Island, N.Y.), expanded, and cell supernatants were screened for their ability to mediate Jurkat adhesion to wells previously coated with 1.5 mg/ml (total protein) goat anti-human IgG (Sigma, St. Louis, Mo.). A positive CHO-K1/VCAM-Ig clone was subsequently adapted to CHO-SFM serum-free media (Gibco) and maintained under selection for stable expression of VCAM-Ig. VCAM-Ig was purified from crude culture supernatants by affinity chromatography on Protein A/G Sepharose (Pierce, Rockford, Ill.) according to the manufacturer's instructions and desalted into 50 mM sodium phosphate buffer, pH 7.6, by ultrafiltration on a YM-30 membrane (Amicon, Beverly, Mass.).
Step B Preparation of 125I-VCAM-Ig
VCAM-Ig was labeled to a specific radioactivity greater that 1000 Ci/mmole with 125I-Bolton Hunter reagent (New England Nuclear, Boston, Mass.; cat# NEX120-0142) according to the manufacturer's instructions. The labeled protein was separated from unincorporated isotope by means of a calibrated HPLC gel filtration column (G2000SW; 7.5×600 mm; Tosoh, Japan) using uv and radiometric detection.
Step C VCAM-Ig Binding Assay
Compounds of this invention were prepared in DMSO at 100× the desired final assay concentration. Final concentrations were selected from a range between 0.001 nM-100 μM. Jurkat cells were centrifuged at 400×g for five mins and resuspended in binding buffer (25 mM HEPES, 150 mM NaCl, 3 mM KCl, 2 mM glucose, 0.1% bovine serum albumin, pH 7.4). The cells were centrifuged again and resuspended in binding buffer supplemented with MnCl2 at a final concentration of 1 mM. Compounds were assayed in Millipore MHVB multiscreen plates (cat# MHVBN4550, Millipore Corp., Mass.) by making the following additions to duplicate wells: (i) 200 μL of binding buffer containing 1 mM MnCl2; (ii) 20 μL of 125I-VCAM-Ig in binding buffer containing 1 mM MnCl2 (final assay concentration  ̃100 pM); (iii) 2.5 μL of compound solution or DMSO; (iv) and 0.5×106 cells in a volume of 30 mL. The plates were incubated at rt for 30 mins, filtered on a vacuum box, and washed on the same apparatus by the addition of 100 μL of binding buffer containing 1 mM MnCl2. After insertion of the multiscreen plates into adapter plates (Packard, Meriden, Conn., cat# 6005178), 100 μL of Microscint-20 (Packard cat# 6013621) was added to each well. The plates were then sealed, placed on a shaker for 30 seconds, and counted on a Topcount microplate scintillation counter (Packard). Control wells containing DMSO alone were used to determine the level of VCAM-Ig binding corresponding to 0% inhibition. Contol wells in which cells were omitted were used to determine the level of binding corresponding to 100% inhibition. Binding of 125I-VCAM-Ig in the absence of cells was usually less than 5% of that observed using cells in the presence of vehicle. Percent inhibition was then calculated for each test well and the IC50 was determined from a ten point titration using a validated four parameter fit algorithm.
EXAMPLE 35
Antagonism of α4β7 Dependent Binding to VCAM-Ig Fusion Protein
Step A α4β7 Cell Line
RPMI-8866 cells (a human B cell line α4+β1−β7+; a gift from Prof. John Wilkins, University of Manitoba, Canada) were grown in RPMI/10% fetal calf serum/100 U penicillin/100 μg streptomycin/2 mM L-glutamine at 37° C., 5% carbon dioxide. The cells were pelleted at 1000 rpm for 5 mins and then washed twice and resuspended in binding buffer (25 mM Hepes, 150 mM NaCl, 0.1% BSA, 3 mM KCl, 2 mM Glucose, pH 7.4).
Step B α4β7 VCAM-Ig Binding Assay
Compounds of this invention were prepared in DMSO at 100× the desired final assay concentration. Final concentrations were selected from a range between 0.001 nM-100 μM. Compounds were assayed in Millipore MHVB multiscreen plates (Cat# MHVBN4550) by making the following sequential additions to duplicate wells: (i) 100 mL/well of binding buffer containing 1.5 mM MnCl2; (ii) 10 mL/well 125I-VCAM-Ig in binding buffer (final assay concentration <500 pM); (iii) 1.5 mL/well test compound or DMSO alone; (iv) 38 mL/well RPMI-8866 cell suspension (1.25×106 cells/well). The plates were incubated at rt for 45 mins on a plate shaker at 200 rpm, filtered on a vacuum box, and washed on the same apparatus by the addition of 100 mL of binding buffer containing 1 mM MnCl2. After insertion of the multiscreen plates into adapter plates (Packard, Meriden, Conn., cat# 6005178), 100 mL of Microscint-20 (Packard cat# 6013621) was added to each well. The plates were then sealed, placed on a shaker for 30 seconds, and counted on a Topcount microplate scintillation counter (Packard). Control wells containing DMSO alone were used to determine the level of VCAM-Ig binding corresponding to 0% inhibition. Wells in which cells were omitted were used to determine the level of binding corresponding to 100% inhibition. Percent inhibition was then calculated for each test well and the IC50 was determined from a ten point titration using a validated four parameter fit algorithm.

## Claims

1. A compound of Formula I: 




or a pharmaceutically acceptable salt thereof wherein:
Ring A is cycloalkyl or heterocyclyl, 
wherein ring A is optionally substituted with one to four substituents independently selected from Rc; 
X and Y are independently selected from 
1) halogen, 
2) C1-3alkyl, 
3) C1-3alkoxy; 

Z is 
1) N, 
2) N+—O—; 

R1 is 
1) hydrogen, 
2) C1-10alkyl, 
3) aryl-C1-10alkyl; 

R2 is 
1) hydrogen or 
2) C1-10alkyl; 

R3 and R4 are independently selected from 
1) hydrogen, 
2) C1-10alkyl, 
3) —ORd, 
4) —CO2Rd, 
5) —C(O)NRdRe, 
6) —NRdRe, 
7) —NRdS(O)mRe, 
8) —NRdC(O)Re, 
9) —NRdC(O)ORe, 
10) —NRdC(O)NRdRe, 


wherein alkyl is optionally substituted with one to four substituents independently selected from Ra;
R5 is 
1) hydrogen, 
2) OH, 
3) OCH3; or 
4) NH2; 

Ra is 
1) —ORd, 
2) —NRdS(O)mRe, 
3) —NO2, 
4) halogen 
5) —S(O)pRd, 
6) —SRd, 
7) —S(O)2ORd, 
8) —S(O)mNRdRe, 
9) —NRdRe, 
10) —O(CRfRg)nNRdRe, 
11) —C(O)Rd, 
12) —CO2Rd, 
13) —CO2(CRfRg)nCONRdRe, 
14) —OC(O)Rd, 
15) —CN, 
16) —C(O)NRdRe, 
17) —NRdC(O)Re, 
18) —OC(O)NRdRe, 
19) —NRdC(O)ORe, 
20) —NRdC(O)NRdRe, 
21) —CRd(N—ORe), 
22) CF3, 
23) —OCF3, 
24) C3-8cycloalkyl, or 
25) heterocyclyl; 


wherein cycloalkyl and heterocyclyl are optionally substituted with one to four groups independently selected from Rc;
Rc is 
1) halogen, 
2) amino, 
3) carboxy, 
4) C1-4alkyl, 
5) C1-4alkoxy, 
6) aryl, 
7) aryl C1-4alkyl, 
8) hydroxy, 
9) CF3, 
10) OC(O)C1-4alkyl, 
11) OC(O)NRfRg, or 
12) aryloxy; 

Rd and Re are independently selected from hydrogen, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, Cy and Cy C1-10alkyl, wherein alkyl, alkenyl, alkynyl and Cy are optionally substituted with one to four substituents independently selected from Rc; or 
Rd and Re together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and N—Rh; 
Rf and Rg are independently selected from hydrogen, C1-10alkyl, Cy and Cy—C1-10alkyl; or 
Rf and Rg together with the carbon to which they are attached form a ring of 5 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and nitrogen; 
Rh is selected from Rf and —C(O)Rf; 
Cy is selected from cycloalkyl, heterocyclyl, aryl, and heteroaryl; 
Ar1 is selected from phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl each optionally substituted with one or two groups independently selected from Rc; 
Ar2 is 1,4-phenylene or 2,5-pyridylene; 
p is 1 or 2; 
m is 0, 1 or 2; 
n is 0, 1 or 2.

2. A compound of claim 1 wherein Ar1 is pyridyl optionally substituted with C1-3alkyl, or phenyl optionally substituted with one to two groups independently selected from halogen, C1-3alkyl, carboxy, cyano, trifluoromethyl, and trifluoromethoxy.

3. A compound of claim 1 wherein Ar1 is 3-substituted phenyl optionally having a second substituent on the 4- or 5-position wherein the substituents are independently selected from chloro, fluoro, bromo, methyl, trifluoromethyl and trifluoromethoxy.

4. A compound of claim 1 wherein Ar1 is 3,5-dichlorophenyl.

5. A compound of claim 1 wherein Ar2 is 1,4-phenylene.

6. A compound of claim 1 wherein X and Y are each chloro.

7. A compound of claim 1 wherein R3 and R4 are independently selected from hydrogen, carboxy, or substituted amino.

8. A compound of claim 1 having the formula Ia: 




wherein Z is N or N+O—;
Ring A is C3-6cycloalkyl, pyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl or dioxyl, 
R2 is H or methyl; 
R3 is selected from H, C1-6alkyl, carboxyl, or —NRdRe; 
Rc is selected from hydrogen, C1-4alkyl; halogen, cyano, carboxyl, trifluoromethyl, or trifluoromethoxy; or 
a pharmaceutically acceptable salt thereof.

9. A compound of claim 8 wherein Ring A is cyclopentyl.

10. A compound of claim 8 wherein Ring A is cyclohexyl.

11. A compound of claim 8 wherein Rc is chloro.

12. A pharmaceutical composition which comprises a compound of claim 1 and a pharmaceutically acceptable carrier thereof.