WO1999020272A1 - Acides azapeptidiques utilises comme inhibiteurs de l'adhesion cellulaire - Google Patents

Acides azapeptidiques utilises comme inhibiteurs de l'adhesion cellulaire Download PDF

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Publication number
WO1999020272A1
WO1999020272A1 PCT/US1998/022008 US9822008W WO9920272A1 WO 1999020272 A1 WO1999020272 A1 WO 1999020272A1 US 9822008 W US9822008 W US 9822008W WO 9920272 A1 WO9920272 A1 WO 9920272A1
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independently selected
aryl
optionally substituted
ioalkyl
substituents independently
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PCT/US1998/022008
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English (en)
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Stephen E. Delaszlo
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Merck & Co., Inc.
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Priority claimed from GBGB9724874.4A external-priority patent/GB9724874D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU13614/99A priority Critical patent/AU1361499A/en
Publication of WO1999020272A1 publication Critical patent/WO1999020272A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/04—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
    • C07D237/04—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having less than three double bonds between ring members or between ring members and non-ring members

Definitions

  • AZAPEPTIDE ACIDS AS CELL ADHESION INHIBITORS
  • the present invention relates to novel substituted azapeptide acid 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 selectins, integrins, cadherins and immunoglobulins.
  • CAMs play an essential role in both normal and pathophysiological processes.
  • the integrin superfamily is made up of structurally and functionally related glycoproteins consisting of ⁇ and ⁇ heterodimeric, transme brane receptor molecules found in various combinations on nearly every mammalian cell type, (for reviews see: E. C. Butcher, Cell. G7, 1033 (1991); T. A. Springer, Cell. 76, 301 (1994); D. Cox et al., "The Pharmacology of the Integrins.” Medicinal Research Rev. 14. 195 (1994) and V. W.
  • VLA-4 very late antigen-4"; CD49d/CD29; or «4 ⁇ i
  • VLA-4 very late antigen-4"; CD49d/CD29; or «4 ⁇ i
  • VCAM-1 vascular cell adhesion molecule-1
  • FN fibronectin
  • VCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells at sites of inflammation and on dendritic and macrophage-like cells.
  • VCAM-1 is produced by vascular endothelial cells in response to pro-inflammatory cytokines (See A. J. H. Gearing and W. Newman, "Circulating adhesion molecules in disease.”,
  • 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 0C4 ⁇ (VLA-
  • ⁇ 4 ⁇ also referred to as LPAM-1 and (X4 ⁇ p) is an integrin expressed on leukocytes and is a key mediator of leukocyte trafficking and homing in the gastrointestinal tract (see C. M. Parker et al., Proc. Natl. Acad. Sci. USA. f£, 1924 (1992)).
  • the ligands for ⁇ 4 ⁇ 7 include mucosal addressing cell adhesion molecule-1 (MadCAM-1) and, upon activation of ⁇ 4 ⁇ 7, 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. (See M. J. Briskin et al., Nature. 363. 461 (1993); A. Hamann et al., J. Immunol.. 152. 3282 (1994)). MadCAM-1 can be induced in vitro by proinflammatory stimuli (See E. E. Sikorski et al. J. Immunol.. 151. 5239 (1993)). MadCAM-1 is selectively expressed at sites of lymphocyte extravasation and specifically binds to the integrin, ⁇ 4 ⁇ 7.
  • Neutralizing anti- ⁇ 4 antibodies or blocking peptides that inhibit the interaction between VLA-4 and or ⁇ 4 ⁇ 7 and their ligands have proven efficacious both prophylactically and therapeutically in several animal models of disease, including 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 ⁇ 4 ⁇ integrin.” Nature. 356, 6j5 (1993) and E. Keszthelyi et al., "Evidence for a prolonged role of 0C4 integrin throughout active experimental allergic encephalomyelitis.” Neurology.
  • VLA-4 interactions in other diseases including rheumatoid arthritis; various melanomas, carcinomas, and sarcomas; inflammatory lung disorders; atherosclerotic plaque formation; restenosis; and circulatory shock (for examples, see A. A. Postigo et al., "The ⁇ 4 ⁇ ]/VCAM-l 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)).
  • a humanized monoclonal antibody for example, see A. A. Postigo et al., "The ⁇ 4 ⁇ ]/VCAM-l adhesion pathway in physiology and disease.”, Res. Immunol.. 144. 723 (1994) and J.-
  • VLA-4 (Antegren® Athena Neurosciences/Elan ) against VLA-4 in clinical development for the treatment of "flares” associated with multiple sclerosis and a humanized monoclonal antibody (ACT-1® LeukoSite) against ⁇ 4 ⁇ 7 in clinical development for the treatment of inflammatory bowel disease.
  • ACT-1® LeukoSite a humanized monoclonal antibody against ⁇ 4 ⁇ 7 in clinical development for the treatment of inflammatory bowel disease.
  • peptidyl antagonists of VLA-4 have been described (D. Y. Jackson et al., "Potent 4 ⁇ l peptide antagonists as potential anti-inflammatory agents", J. Med. Chem.. 40, 3359 (1997); H. N. Shroff et al., "Small peptide inhibitors of 4 ⁇ 7 mediated MadCAM-1 adhesion to lymphocytes", Bioorg. Med. Chem. Lett.. 6, 2495 (1996); US 5,510,332, WO97/03094, WO
  • the compounds of the present invention are antagonists of the VLA-4 integrin ("very late antigen-4"; CD49d/CD29; or ⁇ 4 ⁇ l) and/or the ⁇ 4 ⁇ 7 integrin (LPAM-1 and ⁇ 4 ⁇ p), thereby blocking the binding of VLA-4 integrin ("very late antigen-4"; CD49d/CD29; or ⁇ 4 ⁇ l) and/or the ⁇ 4 ⁇ 7 integrin (LPAM-1 and ⁇ 4 ⁇ p), thereby blocking the binding of
  • VLA-4 to its various ligands, such as VCAM-1 and regions of fibronectin and/or ⁇ 4 ⁇ 7 to its various ligands, such as MadCAM-1, VCAM-1 and fibronectin.
  • these antagonists are useful in inhibiting cell adhesion processes including cell activation, migration, proliferation and differentiation.
  • VLA-4 and/or ⁇ 4 ⁇ 7 binding and cell adhesion and activation are useful in the treatment, prevention and suppression of diseases mediated by VLA-4 and/or ⁇ 4 ⁇ 7 binding and cell adhesion and activation, such as multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, septic arthritis, type I diabetes, organ transplantation, restenosis, autologous bone marrow transplantation, inflammatory sequelae of viral infections, myocarditis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, certain types of toxic and immune-based nephritis, contact dermal hypersensitivity, psoriasis, tumor metastasis, and atherosclerosis.
  • diseases mediated by VLA-4 and/or ⁇ 4 ⁇ 7 binding and cell adhesion and activation such as multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheum
  • the present invention provides novel compounds of Formula I
  • Rl is 1) Ci-ioalkyl
  • R4 is 1) hydrogen, or 2) a group selected from Rl; or R2 and R ⁇ together with the atoms to which they are attached form a ring of 4 to 7 members containing 0-1 additional heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein said ring may be isolated or benzo-fused, and optionally substituted with one to four substituents independently selected from ⁇ ; R4 is 1) hydrogen,
  • heteroaryl Ci-ioalkyl wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents independently selected from R a , and aryl and heteroaryl are optionally substituted with one to four substituents independently selected from R D ;
  • R6 is 1) hydrogen,
  • Arl-Ar2 wherein Ar ⁇ and Ar 2 are independently aryl or heteroaryl, and each is optionally substituted with one to four substituents independently selected from R ⁇ ; alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents independently selected from R a ; and Cy is optionally substituted with one to four substituents independently selected from R" except aryl and heteroaryl; R a is 1) -CF 3 ;
  • R D is 1) a group selected from R a ,
  • heteroaryl wherein alkyl, alkenyl, alkynyl, aryl, heteroaryl are optionally substituted with a group independently selected from R c ;
  • R c is 1) halogen
  • R d and R e are independently selected from the group consisting of
  • R d and R e together with the atoms to which they are attached form a heterocyclic ring of 5 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and nitrogen;
  • R* and R ⁇ are independently selected from hydrogen, Ci-ioalkyl, Cy and Cy Ci-ioalkyl; or
  • alkyl, alkenyl, alkynyl and aryl are each optionally substituted with one to four substituents independently selected from R c ;
  • Cy is cycloalkyl, heterocyclyl, aryl, or heteroaryl; m is an integer from 0 to 2; n is an integer from 0 to 2;
  • X is 1) -C(O)OR d ,
  • Y is 1) -C(O)-
  • Rl is Cl-l ⁇ alkyl, Cy or Cy-Ci-10 alkyl wherein alkyl is optionally substituted with one to four substituents independently selected from Ra, and Cy is optionally substituted with one to four substituents independently selected from Rb.
  • Rl the preferred Cy is aryl or heteroaryl.
  • Rl examples include phenyl, 3,5-dichlorophenyl, 3-fluorophenyl, 4- fluorophenyl, benzyl, 4-[(2-methylphenyl)urea]benzyl, t-butyl, benzyl, 3- cyanophenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 3-chlorophenyl, 4- (benzamido)phenyl, 4-(phenylacetamido)phenyl and the like.
  • R2 and R are independently hydrogen or Ci-io alkyl optionally substituted with one to four substituents independently selected from Ra, or R2 and R3 together with the atoms to which they are attached form a ring of 5 to 6 members containing 0-1 additional heteroatom selected from the group consisting of oxygen, sulfur and nitrogen, and wherein said ring is optionally substituted with one to four substituents independently selected from Rb.
  • suitable R2/R3 are hydrogen, butyl, an amino acid side chain which is within the definition of R2/R3 ? cyclohexyl, 4-(acetamido)butyl, pyrazolidine, hexahydropyridazine, methylpyrazolidine, and the like.
  • R4 is hydrogen, Ci-l ⁇ alkyl or Cy- Cl-l ⁇ alkyl.
  • suitable R4 are methyl, benzyl, butyl, hydrogen.
  • R5 is hydrogen and R6 is Cl-l ⁇ alkyl, Cy, Cy-l-io alkyl, Arl-Ar 2 -, or Arl-Ar 2 - Cl-10alkyl wherein alkyl, Cy, Ar and Ar 2 are optionally substituted as provided under Formula I.
  • R6 the preferred Cy is aryl or heteroaryl.
  • R6 is an amino acid side chains which is within the definition of R6, methyl, isobutyl, sec-butyl, benzyl, phenyl, n-butyl, 4-fluorophenyl, naphthyl, biphenylmethyl, 2'- (methoxy)-biphenylmethyl, 2'-(cyano)-biphenylmethyl, 2'-(tetrazol-5-yl)- biphenylmethyl, 2'-(l-methyl-tetrazol-5-yl)-biphenylmethyl, 2'-(2-methyl- tetrazol-5-yl)-biphenylmethyl, 4-(t-butoxy)-benzyl, biphenyl, 2'- methoxybiphenyl, 2'-cyanobiphenyl and the like.
  • X is - C(O)OR d .
  • Y is -C(O)- or
  • n is 0 or 1 and m is 0 or 1; preferably n+m is 0 or 1.
  • the present invention provides a compound of Formula la
  • Rl is 1) Ci-ioalkyl
  • heteroaryl-Ci-ioalkyl wherein alkyl is optionally substituted with one to four substituents independently selected from R a ; and aryl or heteroaryl is optionally substituted with one to four substituents independently selected from Rb; R4 is 1) hydrogen, or
  • Ci-ioalkyl optionally substituted with one to four substituents independently selected from R a ;
  • Arl-Ar2- wherein alkyl is optionally substituted with one to four substituents independently selected from R a ; aryl or heteroaryl is optionally substituted with one to four substituents independently selected from Rb except aryl and heteroaryl; Arl and Ar2 are independently aryl or heteroaryl, and each is optionally substituted with from one to four groups independently selected from Rb; R d is 1) hydrogen, 2) Ci-ioalkyl,
  • Ra, Rb ; Rc and Cy are as defined under Formula I.
  • Rl is aryl optionally substituted with one to four halogen
  • R4 is hydrogen
  • R5 is hydrogen
  • R6 is 1) Ci- ⁇ alkyl, 2) aryl,
  • Arl-Ar 2 -Ci-5alkyl wherein alkyl is optionally substituted with one to four substituents independently selected from R a ; aryl is optionally substituted with one to four substituents independently selected from Rb except aryl and heteroaryl; Arl an( j j ⁇ 2 are independently phenyl, and each is optionally substituted with from one to four groups independently selected from Rb.
  • Representative compounds of Formula I include:
  • 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.
  • 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-l-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 ring fused to an aryl group in which the point of attachment is on the non-aromatic portion.
  • cycloalkyl examples include cyclopropyl, 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.
  • aryl include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3- dihydrobenzofuranyl, benzopyranyl, 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.
  • 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.
  • 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.
  • heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2,3- dihydrofuro(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, dihydroindolyl, and the like.
  • Halogen includes fluorine, chlorine, bromine and iodine.
  • 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.
  • tautomers 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 diastereomeric pairs of enantiomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof.
  • a suitable solvent for example methanol 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 acid as a resolving agent.
  • any enantiomer of a compound of the general Formula I or la may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
  • 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.
  • basic ion exchange resins such as
  • salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • 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.
  • 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 ⁇ 4 ⁇ 7 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, and (19) atherosclerosis.
  • 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 0.01 mg to about 50 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.
  • 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.
  • 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.
  • 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.
  • compositions which comprises a compound of Formula I and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (nasal or buccal 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.
  • the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulisers.
  • 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 system for inhalation is a 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.
  • MDI metered dose inhalation
  • Suitable topical formulations of a compound of formula I include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pharmaceutical composition containing such other drugs in addition to the compound of Formula I is preferred.
  • 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:
  • VLA-4 antagonists such as those described in US 5,510,332, WO97/03094, WO97/02289, WO96/40781, WO96/22966, WO96/20216, WO96/01644, WO96/06108, WO95/15973 and WO96/31206;
  • steroids such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone;
  • immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants;
  • antihistamines antihistamines (Hl-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine
  • 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.
  • the synthesis of azapeptides has been reviewed in: J. Gante, Synthesis , 405-413, 1989.
  • alkyl hydrazines are well known to those skilled in the art. A review of the syntheses of alkyl hydrazines may be found by U. Jensen-Korte, Methoden Org. Chem. (Houben -Weyl) 4th ed. 1952-1990, Vol 16a, pp 421-503.
  • tert-butyl carbazate A may be condensed with an appropriate aldehyde or ketone to give, upon reduction by catalytic hydrogenation or treatment with a reducing agent such as borane/THF or sodium cyanoborohydride of the intermediate hydrazone, the protected alkyl hydrazine B.
  • a reducing agent such as borane/THF or sodium cyanoborohydride of the intermediate hydrazone
  • This reagent may be used in the preparation of azapeptides.
  • FMOC analog of B It may be necessary to prepare the FMOC analog of B.
  • This material may be prepared by utilizing FMOC -hydrazine in place of A in the sequence A to B.
  • B may be acylated with benzyloxycarbonyl chloride to give E and the Boc group removed by treatment with trifluoroacetic acid (TFA) to give F.
  • TFA trifluoroacetic acid
  • F Acylation of F with FMOCC1 followed by hydrogenation over palladium and carbon gives G. 1,2-Dialkyl hydrazine syntheses are described in U. Jensen- Korte, Methoden Org. Chem. (Houben -Weyl) 4th ed. 1952-1990, Vol 16a, pp 503-529.
  • the intermediate E may be alkylated in the presence of a base such as sodium hydride and an alkylating agent to give H. Hydrogenolysis of the Cbz group would produce the regiochemically defined protected unsymmetrical hydrazine I. Protecting group manipulation under standard conditions may then provide other suitably protected unsymmetrical hydrazines J. Alternatively, intermediate F may be reductively alkylated with an aldehyde or ketone to give K. K may also be derived from H by treatment with an acid such as TFA. Protecting group manipulation may then give regiochemically defined protected unsymmetrical hydrazine L.
  • a base such as sodium hydride and an alkylating agent
  • the Cbz group of C is removed by hydrogenolysis and the resulting intermediate D may be used in azapeptide formation.
  • D Treatment of D with hydrogen chloride provides E, another useful intermediate for solution or solid phase azapeptide synthesis.
  • the FMOC analog of E, for use in solid phase synthesis may be prepared from D by acylation followed by Boc deprotection under standard conditions to give F.
  • An alternative method of preparing E in a more direct fashion is via the di- Boc cyclic hydrazine G that is in turn prepared from the di-Boc- hydrazine H as shown below.
  • the appropriate alkylating reagents are used. Alkylating agents incorporating other heteroatoms (O, N(protected), S), would provide cyclic hydrazines with additional heteroatoms in the ring.
  • Unsymmetrical substituted pyrazolidines may be prepared as described in Scheme 3. Dialkylation of A with a substituted dibromoalkane provides a mixture of regioisomeric diacyl pyrazolidines. This mixture may be separable. In the event that separation may not be achieved, treatment of the mixture with TFA may give rise to a separable mixture of monoacyl pyrazolidines B and C. The monoacyl pyrazolidines may subsequently be utilized in the preparation of azapeptides in solution phase, or alternatively, following functional group manipulation solid phase technology may be utilized.
  • azapeptides of Formula I are characterized by the presence of a key urea bond as seen in A Scheme 4.
  • the synthesis of the intermediates toward Formula I is outlined in Scheme 4.
  • the protected hydrazine B is reacted with an equivalent of an activating agent such as phosgene (or triphosgene), p-nitrophenylchloroformate or carbonyl diimidazole in the presence of a base such a triethylamine or diisopropylethylamine to give an intermediate of structure C.
  • an activating agent such as phosgene (or triphosgene), p-nitrophenylchloroformate or carbonyl diimidazole
  • a suitably protected amino acid D (protecting groups such as a tert- butyl ester for a carboxylic acid are preferred; however the X group may also be unprotected in the case of initial activation of the hydrazine) may provide compounds of structure A.
  • the amino acid D may be activated to give E which in turn may be combined with B to give A.
  • Removal of the protecting group is, in general, the next step to provide a mono-acylhydrazine F ready for attachment of the R ⁇ Y group and may be accomplished under conditions appropriate to the protecting group being utilized.
  • Activating agent p-nitrophenyl chloroformate, phosgene or carbonyl diimidazole
  • Compounds of Formula I may also be prepared on polystyrene supports, as shown in Scheme 6, thereby permitting the rapid synthesis of analogs through the use of excess reagents to drive reactions to completion and filtration to remove reagents from the resin and the attached products.
  • FMOC protected amino acids A are coupled to an appropriate acid sensitive linker such as 4-hydroxymethyl-3- methoxyphenol which is, in turn, attached to the polystyrene resin B.
  • the resulting resins are treated with 20% piperidine/dimethylformamide (DMF) several times and are then washed with DMF several times followed by the solvent for the next reaction to give C.
  • DMF piperidine/dimethylformamide
  • R ⁇ has a biaryl moiety
  • Scheme 7 Substituted aryl or heteroaryl boronic acids are coupled to A in the presence of a palladium(O) reagent, such as tetrakis(triphenylphosphine)palladium under Suzuki conditions (N. Miyaura et al., Synth. Commun., 1981, 11, 513-519) to yieldB Tyrosine triflate starting materials are prepared by treatment of the tyrosine analog of A with triflic anhydride in pyridine. The protecting group is then removed to give the corresponding compounds of Formula I.
  • a palladium(O) reagent such as tetrakis(triphenylphosphine)palladium under Suzuki conditions (N. Miyaura et al., Synth. Commun., 1981, 11, 513-519)
  • Tyrosine triflate starting materials are prepared by treatment of the tyrosine analog of A with triflic anhydride in
  • the bromide or iodide can be converted into the desired boronic acid by treatment with an alkyllithium reagent in tetrahydrofuran at low temperature followed by addition of trimethyl or triisopropyl borate.
  • Hydrolysis to the boronic acid can be effected by treatment of the intermediate with aqueous base and then acid, aryl boronates which may also be utilized in coupling reactions in place of aryl boronic acids may be prepared by palladium catalyzed boronation of aryl iodides and bromides as described in J. Org Chem , 1995, 60, 7508-7510.
  • the aryl coupling reaction may be performed by application of Stille-type carbon-carbon bond forming conditions
  • Step B The product of Step B was dissolved in 8.0 mL of 50% trifluoroacetic acid/CH2Cl2 and stirred at room temperature for 1 hour.
  • Step D Synthesis of the title compound
  • a solution of 30 mg (0.054 mmol) of the product of Step C in 1 mL of MeOH was added 0.016 mL of a 5N NaOH solution in water (0.081 mmol).
  • the reaction mixture was stirred at room temperature over night.
  • the solution was acidified to pH 2.0, diluted with water and filtered.
  • the solid was washed with ether to provide the desired product.
  • Step A Preparation of l-benzyloxycarbonyl-2-t- butoxycarbonylhydrazine .
  • Step B Preparation of l-benzyloxycarbonyl-2-t- butoxycarbonylpyrazolidine 3.0 g (75 mmol) of 60% sodium hydride in oil was suspended in 50 mL of DMF. 10 g (37.5 mmol) of l-benzyloxycarbonyl-2-t- butoxycarbonylhydrazine was added in 50 mL of DMF dropwise to the mixture. The reaction mixture was stirred at room temperature for 1 hour and then 3.8 mL (37.5 mmol) of 1,3-dibromopropane was added. The reaction mixture was stirred over 48 hours and the solvent was then removed in vacuo. The residue was dissolved in ethyl acetate and washed successively with 5% aqueous citric acid solution, saturated sodium carbonate solution, water and brine and was dried over MgSO4.
  • Step C Preparation of 1-benzyloxycarbonyl-pyrazolidine. trifluoroacetic acid salt. 2.4 g (7.8 mmol) of l-benzyloxycarbonyl-2-t- butoxycarbonylpyrazolidine was dissolved in 20 mL of 25% trifluoroacetic acid/CH2Cl2 at room temperature. The solution was stirred for 4 hours and concentrated in vacuo to give a gum. iH-NMR (400 MHz, CDC13): 2.4 (q, 2H); 3.7 (t, 2H); 3.8 (t,2H); 5.2 (s, 2H); 7.28-7.35 (m, 5H).
  • Step D Preparation of benzyloxycarbonyl- -aza-prolyl-(L)- leucine. tert-butyl ester.
  • reaction mixture was stirred at room temperature over night and then was then diluted with 25 mL of ethyl acetate. The solution was washed with 5% citric acid, water and brine and was dried over MgSO4. The mixture was filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 40% EtOAc/hexanes to give the desired product as a gum.
  • Step E Preparation of benzyloxycarbonyl- -aza-prolyl-(L)-leucine.
  • Step A Preparation of ⁇ -aza-prolyl-(L)-leucine. tert-butyl ester.
  • Step B Preparation of N-phenylsulfonyl- -aza-prolyl-(L)-leucine. tert-butyl ester
  • Example 5 and 6 separated Example 7 - mixture of isomers
  • Step A Preparation of a mixture of l-Boc-2-Cbz-5- methylpyrazolidine and l-Boc-2-Cbz-3-methylpyrazolidine
  • a suspension of 0.88 g (0.022 mol) of 60% sodium hydride in 20 mL of DMF was treated with a solution of 2.66 g (0.01 mol) of l-Cbz-2- Boc-hydrazine in 10 mL of DMF at 0°C.
  • the reaction mixture was stirred for 30 minutes at 0°C and 1 hour at room temperature.
  • 1.3 mL (0.011 mol) of 1,3-dibromobutane was added neat and the mixture was stirred over night.
  • Step B Preparation of l-Cbz-3-methylpyrazolidine and l-Cbz-5-methylpyrazolidine.
  • Example 2 Step C The method of Example 2 Step C was utilized using the more polar product of Step B above and tert-butyl (L)-phenylalanine as starting material. Purification by flash chromatography over silica gel eluting with 30% EtOAc/hexanes gave the title product. H-NMR (CDCI3
  • step E-b Preparation of:
  • Step B The product of Step B was dissolved in ethyl acetate and treated with a solution of hydrogen chloride dissolved in ethyl acetate. A white precipitate formed immediately and was recovered by filtration after 20 minutes and was washed with ethyl acetate to give the desired product.
  • This material may be the mono or dihydrochloride or a mixture of both.
  • Step B Synthesis of l-Boc-2-FMOC-pyrazolidine.
  • 6.0 g (35 mmol) N-Boc-pyrazolidine was dissolved in 20 mL of methylene chloride to which was added 7.2 mL (42 mmol) of diisopropylethyl amine at 0°C.
  • 7.2 mL (42 mmol) of diisopropylethyl amine at 0°C.
  • To this solution was added portionwise 9.0 g of 2-fluorenylmethyl chloroformate. The solution was stirred at room temperature for 3 hours.
  • the reaction mixture was washed with 5% citric acid, water and brine.
  • the organic phase was dried over MgSO4, filtered and concentrated in vacuo to give the product as a white solid.
  • Step C Synthesis of N-FMOC-pyrazolidine. 7.4 grams of N-Boc-N-FMOC-pyrazolidine was dissolved in
  • Step D General procedure for the solid phase synthesis of azapeptides.
  • the solution was stirred for 30 minutes and then added to the previously prepared resin (note: for more than one reaction at a time the cocktail of activated N-FMOC- pyrazolidine was multiplied by the appropriate factor).
  • the resin was mixed for 1 hour with the activated pyrazolidine and then filtered.
  • the resin was washed with 50% CH2CI2/THF 3 times. A small amount of resin was removed (1 mg) and submitted to the Kaiser test to insure that the free amino groups had been successfully acylated.
  • the resin was treated with 20% piperidine/DMF three times followed by washing with DMF 3 times and CH2CI2 twice.
  • the resin was filtered and washed: CH2CI2 3 times, DMF 3 times, CH2CI2 2 times, MeOH, CH2CI2, MeOH, CH2CI2 2 times.
  • the resin was treated with 1.5 mL of a 10% solution of trifluoroacetic acid in methylene chloride 3 times with the filtrate collected after each treatment.
  • the combined filtrates were concentrated in vacuo and azeotroped from toluene to provide the desired products. Lyophilization from 50% acetonitrile/water was also used to provide the products as amorphous solids.
  • the products were analyzed by HPLC and mass spectroscopy.
  • Step A Synthesis of pyrazolidine hydrochloride.
  • Step B 0.1 g (0.02 mmol) of resin was prepared for acylation by treatment with 20% piperidine/DMF as described above. To the resin was added a solution of 0.1 g (0.5 mmol) of p-nitrophenyl chloroformate, 0.064 g (0.65 mmol) diisopropylethyl amine in 1.0 mL of 50% CH2CI2/THF.
  • Example 9 The procedure described in Example 9 was followed, utilizing pyrazolidine dihydrochloride as the hydrazine.
  • the product was purified by prep-TLC over silica gel eluting with 10% chloroform/CH2Cl2 0.1% acetic acid to give the product as a glass.
  • Step E The product of Step E was stirred in 1 mL of 50% TFA/CH 2 C1 2 for 2 hours. The reaction mixture was concentrated in vacuo and the residue was purified by preparatory thin layer chromatography eluting with 7% MeOH/ CH 2 C1 2 1% HOAc to provide the product as a glass.
  • Step A N-(Boc)-(S)-4-iodo-phenylalanine tert-butyl ester
  • a suspension of 7.5 g (0.019 m) of 4-iodophenylalanine tert-butyl ester in 100 mL of dichloromethane was added 2.52 g 0.019 m of diisopropyl ethyl amine followed by 4.14 g of di-tert-butyl-dicarbonate.
  • Step B N-(Boc)-(S)-2'-methoxy-biphenylalanine. tert-butyl ester. 7.97 g (0.018 m) of the product of Step A was dissolved in 160 mL of 2:1 toluene: ethanol. To this solution was added 2.99 g (0.0198 m) 2- methoxyphenylboronic acid, 0.69 g of tetrakistriphenylphosphine palladium (0) and 22.7 mL (0.45 m) of 2.0 M sodium carbonate in water. The reaction mixture was degassed three times and then heated at 90° O for 90 minutes at which time the reaction mixture was black.
  • Step C Preparation of (S)-2'-methoxy-biphenylalanine, tert-butyl ester, hydrochloride.
  • Step E The product of Step E was stirred with 1.5 mL of 50% TFA/CH 2 C1 2 for 1 hour. The reaction mixture was concentrated in vacuo and the residue was purified by preparatory thin layer chromatography over silica gel eluting with 5% MeOH/CH 2 Cl 2 1% HOAc. to give the desired product.
  • FABMS Calc. C 26 H 35 N 3 Cl 2 SO 6 ; 577; Obs.: 578
  • Step A Preparation of CS-1 Coated Plates.
  • Untreated 96 well polystyrene flat bottom plates were coated with bovine serum albumin (BSA; 20 ⁇ g/mL) for 2 hours at room temperature and washed twice with phosphate buffered saline (PBS).
  • BSA bovine serum albumin
  • PBS phosphate buffered saline
  • the albumin coating was next derivatized with 10 ⁇ g/mL 3-(2- pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), a heterobifunctional crosslinker, for 30 minutes at room temperature and washed twice with PBS.
  • SPDP 3-(2- pyridyldithio) propionic acid N-hydroxysuccinimide ester
  • the CS-1 peptide (Cys-Leu-His-Gly-Pro-Glu-Ile- Leu-Asp-Val-Pro-Ser-Thr), which was synthesized by conventional solid phase chemistry and purified by reverse phase HPLC, was next added to the derivatized BSA at a concentration of 2.5 ⁇ g/mL and allowed to react for 2 hours at room temperature. The plates were washed twice with PBS and stored at 4°C.
  • Step B Preparation of Fluorescentlv Labeled Jurkat Cells.
  • Jurkat cells obtained from the American Type Culture Collection (Rockville, MD; cat # ATCC TIB-152) were grown and maintained in RPMI-1640 culture medium containing 10% fetal calf serum (FCS), 50 units/mL penicillin, 50 ⁇ g/mL streptomycin and 2 mM glutamine. Fluorescence activated cell sorter analysis with specific monoclonal antibodies confirmed that the cells expressed both the oc4 and ⁇ l chains of VLA-4. The cells were centrifuged at 400xG for five minutes and washed twice with PBS.
  • FCS fetal calf serum
  • the cells were incubated at a concentration of 2 x 10 cells/mL in PBS containing a 1 ⁇ M concentration of a fluorogenic esterase substrate (2', 7'-bis-(2-carboxyethyl)-5-(and -6)- carboxyfluorescein, acetoxymethyl ester; BCECF-AM; Molecular Probes Inc., Eugene, Oregon; catalog #B-1150) for 30-60 minutes at 37°C in a 5% CO2/air incubator.
  • the fluorescently labeled Jurkat cells were washed two times in PBS and resuspended in RPMI containing 0.25% BSA at a final concentration of 2.0 x 10 cells/mL.
  • Step C Assay Procedure.
  • Compounds of this invention were prepared in DMSO at lOOx the desired final assay concentration. Final concentrations were selected from a range between 0.001 nM-100 ⁇ M.
  • Three ⁇ L of diluted compound, or vehicle alone, were premixed with 300 ⁇ L of cell suspension in 96-well polystyrene plates with round bottom wells. 100 ⁇ L aliquots of the cell /compound mixture were then transferred in duplicate to CS-1 coated wells. The cells were next incubated for 30 minutes at room temperature. The non-adherent cells were removed by two gentle washings with PBS.
  • the signal peptide as well as domains 1 and 2 of human VCAM were amplified by PCR using the human VCAM cDNA (R & D Systems) as template and the following primer sequences: 3'-PCR primer:5'-AATTATAATTTGATCAACTTAC CTGTCAATTCTTTTACAGCCTGCC-3'; 5'-PCR primer: 5'-ATAGGAATTCCAGCTGCCACCATGCCTGGGAAGATGGTCG-3'.
  • the 5'-PCR primer contained EcoRI and PvuII restriction sites followed by a Kozak consensus sequence (CCACC) proximal to the initiator methionine ATG.
  • the 3'-PCR primer contained a Bell site and a splice donor sequence. PCR was performed for 30 cycles using the following parameters: 1 min. at 94 C, 2 min. at 55 C, and 2 min. at 72 C.
  • the resulting PCR product of 650 bp was digested with EcoRI and Bell and ligated to expression vector pig-Tail (R & D Systems, Minneapolis, MN) digested with EcoRI and BamHI.
  • the pig-Tail vector contains the genomic fragment which encodes the hinge region, CH2 and CH3 of human IgGl (GenBank Accession no. Z17370).
  • the DNA sequence of the resulting VCAM fragment was verified using Sequenase (US Biochemical, Cleveland, OH).
  • 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, WI) 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, NJ).
  • Stable VCAM-Ig producing clones were selected according to standard protocols using 0.2-0.8 mg/mL active G418 (Gibco, Grand Island, NY), expanded, and cell supernatants were screened for their ability to mediate Jurkat adhesion to wells previously coated with 1.5 ⁇ g/mL (total protein) goat anti-human IgG (Sigma, St. Louis, MO).
  • VCAM-Ig was purified from crude culture supernatants by affinity chromatography on Protein A/G Sepharose (Pierce, Rockford, IL) 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, MA).
  • Step B Preparation of 125 I-VCAM-Ig.
  • the labeled protein was separated from unincorporated isotope by means of a calibrated HPLC gel filtration column (G2000SW;
  • Step C VCAM-Ig Binding Assay.
  • Compounds of this invention were prepared in DMSO at lOOx the desired final assay concentration. Final concentrations were selected from a range between 0.001 nM-100 ⁇ M.
  • Jurkat cells were centrifuged at 400xG for five minutes and resuspended in binding buffer (25 mM HEPES, 150 mM NaCl, 3 mM KC1, 2 mM glucose, 0.1% bovine serum albumin, pH 7.4). The cells were centrifuged again and resuspended in binding buffer supplemented with MnCl 2 at a final concentration of 1 mM.
  • I- 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 IC 50 was determined from a ten point titration using a validated four parameter fit algorithm.
  • Step A ⁇ Cell line.
  • RPMI-8866 cells (a human B cell line ⁇ ⁇ ; a gift from Prof. John Wilkins, University of Manitoba, Canada) were grown in RPMI/10% fetal calf serum/ 100 U penicillin/100 ⁇ g streptomycin/2 mM L-glutamine at 37° C, 5 % carbon dioxide. The cells were pelleted at 1000 rpm for 5 minutes and then washed twice and resuspended in binding buffer (25 mM HEPES, 150 mM NaCl , 0.1 % BSA, 3 mM KC1, 2 mM Glucose, pH 7.4).
  • binding buffer 25 mM HEPES, 150 mM NaCl , 0.1 % BSA, 3 mM KC1, 2 mM Glucose, pH 7.4
  • Step B VCAM-Ig Binding Assay.
  • Compounds of this invention were prepared in DMSO at lOOx the desired final assay concentration. Final concentrations were selected from a range between 0.001 nM-100 ⁇ M.
  • Compounds were assayed in Millipore MHVB multiscreen plates (Cat# MHVBN4550) by making the following sequential additions to duplicate wells: (i) 100 ⁇ L/well of binding buffer containing 1.5 mM MnCl 2 ; (ii) 10 ⁇ l/well 125 I- VCAM-Ig in binding buffer (final assay concentration ⁇ 500 pM); (iii) 1.5 ⁇ l/well test compound or DMSO alone; (iv) 38 ⁇ l/well RPMI-8866 cell suspension (1.25 x 10 6 cells/well).
  • the plates were incubated at room temperature for 45 minutes on a plate shaker at 200 rpm, filtered on a vacuum box, and washed on the same apparatus by the addition of 100 ⁇ L of binding buffer containing 1 mM MnCl 2 .
  • 100 ⁇ L 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 IC 50 was determined from a ten point titration using a validated four parameter fit algorithm.

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Abstract

Des acides azapeptidiques de formule (I) constituent des antagonistes de VLA-4 et/ou α4β7, et en tant que tels ils sont utiles dans l'inhibition ou la prévention de l'adhésion cellulaire et de pathologie induite par l'adhésion cellulaire. Ces composés peuvent être formulés dans des compositions pharmaceutiques et sont appropriés à une utilisation dans le traitement de l'asthme, d'allergies, de l'inflammation, de la sclérose en plaque ainsi que d'autres troubles inflammatoires et autoimmuns.
PCT/US1998/022008 1997-10-21 1998-10-19 Acides azapeptidiques utilises comme inhibiteurs de l'adhesion cellulaire WO1999020272A1 (fr)

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US6576397P 1997-11-17 1997-11-17
US60/065,763 1997-11-17
GBGB9724874.4A GB9724874D0 (en) 1997-11-26 1997-11-26 Azapeptide acids as cell adhesion inhibitors
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