Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/92—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with a hetero atom directly attached to the ring nitrogen atom
  • C07D211/96—Sulfur atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
  • C07D207/48—Sulfur atoms

Definitions

• autoimmune diseases A wide variety of diseases can be characterized as "autoimmune diseases". Such diseases are similar to graft rejection, except that the rejection is of self tissue. Immunosuppressive therapy can also be of use in preventing this inappropriate self rejection.
• CsA cyclosporin A
• Gastroenterology/Hepatology Primary cirrhosis, autoimmune hepatitis, ulcerative colitis, Crohn's disease and other gastrointestinal autoimmune diseases.
• ALS Amyotrophic lateral sclerosis
• myasthenia gravis myasthenia gravis and multiple sclerosis.
• Nephrotic Syndrome Nephrotic syndrome, membrano- proliferative glomerulonephritis (MPGN) and related diseases.
• IPDM Insulin-Pependent Piabetes Mellitus
• IPPM Assan, R. et al.. Lancet, 67-71 (1985). IPPM. Bougneres, P.F. et al., New Engl. J. Med.
• IPPM Piabetes 37:1574-1582 (1988). IPPM.
• autoimmune diseases are also characterized as autoimmune diseases. Autoimmune diseases such as those listed above have been observed in mammals. Papa, F.O. et al. , Equine Vet. J. 22:145-146 (1990) infertility of autoimmune origin in the stallion; Gorman, N.T. and L.L.
• CsA causes immunosuppression.
• CsA inhibits the release of ly phokines, such as interleukin 2 (IL-2) [Bunjes, D. et al. , Eur. J. Immuno1. 13.:657-661 (1981)] and prevents clonal expansion of helper and cytotoxic T cells [Larsson, E. J. Immunol. 124:2828-2833 (1980)].
• IL-2 interleukin 2
• CsA has been shown to bind the cytosolic protein, cyclophilin, and inhibit the prolyl-peptidyl cis-trans isomerase (PPIase) activity of that protein.
• PPIase prolyl-peptidyl cis-trans isomerase
• the PPIases may mediate T cell acti ⁇ vation by catalyzing the rotomerization of peptide bonds of prolyl residues.
• FK-506 a second natural product isolated from Streptomyces, referred to as FK-506, has been demon ⁇ strated to be a potent immunosuppresive agent. Tanaka, H. et al., J. Am. Chem. Soc. 109:5031-5033 (1987). FK-506 inhibits IL-2 production, inhibits mixed lympho ⁇ cyte culture response and inhibits cytotoxic T-cell generation jLn vitro at 100 times lower concentration than cyclosporin A. Kino, T. et al. , J. Antibiot. 15:1256- 1265 (1987) .
• FK-506 also inhibits PPIase activity, but is structurally different from CsA and binds to a binding protein (FKBP) distinct from cyclophilin.
• FKBP binding protein
• This invention relates to a novel class of immuno ⁇ suppressive compounds having an affinity for the FK-506 binding protein (FKBP) .
• FKBP FK-506 binding protein
• the immunosuppressive compounds inhibit the prolyl peptidyl cis-trans isomerase (rotamase) activity of the FKBP and lead to inhibition of T cell activation.
• the compounds of this invention can be used as immunosuppressive drugs to prevent or significantly reduce graft rejection in bone marrow and organ transplantations and in the treat ⁇ ment of autoimmune disease in humans and other mammals.
• This invention relates to a novel class of immuno ⁇ suppressive compounds having a sulfonamide substituent and which is represented by the formula I:
• A is CH,, oxygen, NH or N-(C1-C4 alkyl); wherein B and P are independently Ar, hydrogen, (C1-C6)-straight or branched alkyl, (C1-C6)-straight or branched alkenyl, (C1-C6)-straight or branched alkyl or alkenyl that is substituted with a (C5-C7)-cycloalkyl, (C1-C6)-straight or branched alkyl or alkenyl that is substituted with a (C5-C7)-cycloalkenyl, or Ar substituted (C1-C6)-straight or branched alkyl or alkenyl, wherein, in each case, one or two of the CH_ groups of the alkyl or alkenyl chains may contain 1-2 heteroatoms selected from the group consisting of oxygen, sulfur, SO and S0_ in chemically reasonable substitution patterns, or
• both B and P are not hydrogen; wherein Q is hydrogen, (C1-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl; wherein T is Ar or substituted 5-7 membered cyclo- alkyl with substituents at positions 3 and 4 which are independently selected from the group consisting of hydrogen, hydroxyl, 0-(Cl-C4)-alkyl, 0-(Cl-C4)-alkenyl and carbonyl; wherein Ar is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, monocyclic and bicyclic heterocyclic ring systems with individual ring sizes being 5 or 6 which may contain in either or both rings a total of 1-4 heteroatoms independently selected from 0, N and S; wherein Ar may
• the stereochemistry at position 1 (Formula I) is (R) or (S) , with (S) preferred.
• the stereochemistry at position 2 is (R) or (S) .
• the compounds of the present invention can be used in the form of salts derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulf te butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydro ⁇ hloride, hydrobro ide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate,
• Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salt with organic bases such as dicyclohexylamine salts, N-methyl-P- glucamine, and salts with amino acids such as arginine, lysine, and so forth.
• the basic nitrogen- containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
• lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides
• dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates
• long chain halides such as decy
• the compounds will have a molecular weight below about 750 atomic mass units (a.m.u.) and most preferably below about 500 a.m.u.
• the immunosuppressive compounds of this invention have an affinity for the FK-506 binding protein which is located in the cytosol of lymphocytes, particularly T lymphocytes. When the immunosuppressive compounds are bound to the FKBP, they act to inhibit the prolyl- peptidyl cis-trans isomerase activity of the binding protein and inhibit lymphocyte activation mediated by FKBP.
• FK-506 binding protein has been identified by Harding, M.W. et al. , Nature 341:758-760 (1989) and can be used as the standard by which to evaluate binding affinity of the compounds for FKBP.
• Compounds of this invention may have an affinity for other FK-506 binding proteins. Inhibition of the prolyl peptidyl cis-trans isomerase may further be indicative of binding to an FK-506 binding protein.
• Human FK-506 binding protein can be obtained as described by Harding, M.W. et al. , Nature 341:758-760 (1989) . Values for the apparent K, can be determined from a competitive LH-20 binding assay performed as described by Harding et al. , using 32-[1- 14C]-benzoyl FK-
• FK-506 binding protein was measured.
• the inhibition of the PPIase (rotamase) enzyme activity of the FKBP can also be measured according to the methods described by either Harding, M.W. et al. , Nature 341:758-760 (1989) or Siekierka, J.J. et al. , Nature 341:755-757 (1989).
• the cis-trans isomerization of the proline-alanine peptide bond in a model substrate, N-succinyl-Ala-Ala-Pro-Phe-p- nitroanilide is monitored spectrophotometrically in a coupled assay with chymotrypsin, which releases 4-nitro- anilide from the trans form of the substrate.
• the compounds of the present invention can be further characterized in cellular biological experiments in vitro where their resemblance in function and use to cyclosporin A and to FK-506 is apparent. (See Table 2) .
• CTLL Prolifera ⁇ »l ⁇ g/ml * 0.01 ⁇ g/ml »l ⁇ g/ral tion + IL-2 1) Assay similar to Yoshimura, N. et al. , Transplantation 47:356-359 (1989). Assay uses fresh human peripheral blood lymphocytes isolated by Ficoll-Hypaque density centrifugation, stimulated by the 0KT3 antibody (anti-CP3) which stimulates via interaction with CP3. Stimulation is measured by incorporation of radioactive thymidine [( H)TdR] into proliferating cells, with an uninhibited control signal of 48,000-75,000 cpm. IC 50 values are estimated from inhibitions of prolifer ⁇ ation observed at various drug concentrations.
• T-cell clone stimulated with antibody to the T-cell receptor (TCR) and antibody to CP2.
• Stimulation is measured by incorpor- ation of radioactive thymidine [( H)TdR] into prolifer ⁇ ating cells, with an uninhibited control signal of 23,000 cpm.
• IC 5f values are estimated from inhibitions of proliferation observed at various drug concentrations.
• the assay uses a T-cell hybridoma similar to that described.
• the assay measures activation-induced (anti-CP3) cell death (evaluated by counting viable cells after staining as described) in a T-cell hybridoma that mimics the effect known to occur in immature thymocytes.
• the ability of cyclosporin A and FK-506 to inhibit this cell death is herein used as a sensitive indication of compounds with cyclosporin-like and/or FK-506-like mechanism of action. Note that the chemically related, but mechanistically distinct, immunosuppressant rapamycin is inactive in this assay.
• IL-2 is provided to overcome this block. Note that the chemically related, but mechanistically distinct immuno- suppressant, rapamycin, is active in this assay.
• the compounds can be used as immunosuppres ⁇ sants for prophylaxis of organ rejection or treatment of chronic graft rejection and for the treatment of auto ⁇ immune diseases.
• the immunosuppressive compounds of this invention can be periodically administered to a patient undergoing bone marrow or organ transplantation or for another reason in which it is desirable to substantially reduce or suppress a patient's immune response, such as in various autoimmune diseases.
• the compounds of this invention can also be administered to mammals other than humans for treatment of various mammalian autoimmune diseases.
• the novel compounds of the present invention possess an excellent degree of activity in suppression of antigen-stimulated growth and clonal expansion of T-cells, especially those T-cells characterized as "helper" T-cells. This activity is useful in the primary prevention of organ transplant rejection, in the rescue of transplanted organs during a rejection episode, and in the treatment of any of several autoimmune diseases known to be associated with inappropriate autoimmune responses.
• autoimmune diseases include: uveitis, Behcet's disease. Graves ophthalmopathy, psoriasis, acute dermato- myositis, atopic skin disease, scleroderma, eczema, pure red cell aplasia, aplastic anemia, primary cirrhosis, autoimmune hepatitis, ulcerative colitis, Crohn's disease, amyotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, nephrotic syndrome, membrano- proliferative glomerulonephritis, rheumatoid arthritis and insulin-dependent diabetes mellitus.
• Graves ophthalmopathy Graves ophthalmopathy, psoriasis, acute dermato- myositis, atopic skin disease, scleroderma, eczema, pure red cell aplasia, aplastic anemia, primary cirrhosis
• treatment is effective to reduce the symptoms and slow progression of the disease.
• treatment as described below is most effective when instituted before the complete cessation of natural insulin production and transition to complete dependence on external insulin.
• the compounds of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir in dosage formu ⁇ lations containing conventional non-toxic pharma- ceutically-acceptable carriers, adjuvants and vehicles.
• parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrasternal and intracranial injection or infusion techniques.
• the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example as a sterile injectible aqueous or oleagenous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
• a non-toxic parenterally-acceptable diluent or solvent for example as a solution in 1,3-butanediol.
• acceptable vehicles and solvents that may be employed are water. Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or di-glycerides.
• Fatty acids such as oleic acid and its glyceride deri ⁇ vatives find use in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
• oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
• These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or similar alcohol.
• the compounds may be administered orally, in the form of capsules or tablets, for example, or as an aqueous suspension or solution.
• carriers which are commonly used include lactose and corn starch.
• Lubricating agents, such as magnesium stearate, are also typically added.
• useful diluents include lactose and dried corn starch.
• aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
• the compounds of this invention may also be ad ⁇ ministered in the form of suppositories for rectal administration of the drug.
• compositions can be prepared by mixing the drug with a suitable non-irritat ⁇ ing excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug.
• suitable non-irritat ⁇ ing excipient include cocoa butter, beeswax and polyethylene glycols.
• the compounds of this invention may also be admin ⁇ istered topically, especially when the conditions addressed for treatment involve areas or organs readily accessible by topical application, including autoimmune diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily pre ⁇ pared for each of these areas.
• the compounds can be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
• the compounds may be formulated in an ointment such as petrolatum.
• the compounds can be formulated in a suitable ointment containing the compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
• the compounds can be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in, for example, a ixture of one or more of the following: mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
• Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.
• Posage levels on the order of 0.01 to 100 mg/kg per day of the active ingredient compound are useful in the treatment of the above conditions.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• a specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination and the severity of the particular disease being treated.
• the compound can also be administered in combination with a steroid, such as methyl prednisalone acetate, for additional immunosuppressive effect.
• a steroid such as methyl prednisalone acetate
• the steroid is administered orally, intravenously, rectally, topically or by inhalation.
• Posages (based upon methyl pre ⁇ dnisalone acetate) of 0.1-5 mg/kg/day may be employed.
• An initial loading dose of 100-500 mg may be employed.
• Steroid doses may be decreased with time from the higher toward the lower doses as the clinical situation indicates.
• the compounds can be administered with other immuno ⁇ suppressant drugs, such as rapamycin, azathioprine, 15-deoxyspergualin, cyclosporin, FK-506 or combinations of these, to increase the immunosuppressive effect.
• OKT3 which is a murine monoclonal antibody to CP3 surface antigen of human T lymphocytes, can also be coadministered intravenously with compounds of the present inventions for rescue and reversal of acute allograft rejections, particularly in renal transplan ⁇ tations.
• H NMR proton nuclear magnetic resonance
• the reaction mixture was concentrated and flash chromatographed (elution with a gradient of 5% ethyl acetate in hexane to 100% ethyl acetate) to give 29 mg of the sulfonamide (5) as an oil.
• the compound is illustrated below.
• Fresh peripheral blood lymphocytes (PBLs) from LeukoPak cells or whole blood from random normal blood donors (tested HIV-negative and hepatitis negative) are isolated and separated by density centrifugation over Histopaque 1077 (Sigma Chemical Co., St. Louis, MO).
• the murine CTLL cytotoxic T cell line and the human Jurkat T cell line are from ATCC (CTLL-2 ATCC TIB214, JURKAT CLONE E6-1 ATCC TIB152) .
• the human allogeneic B cell lines used for activation of the fresh PBLs are EBV-transformed lymphocytes from normal healthy adult donors with two completely different HLA haplotypes.
• Culture medium consists of RPMI 1640 (Gibco, Grand Island, NY) containing penicillin (50 U/ml) and streptomycin (50 /*g/ml) , L-glutamine 2 mM, 2 mmeerrccaappttooeetthhaa:nol (5 x 10 —5) , 10% heat-inactivated FCS and 10 mM HEPES.
• the MTT assay is a colorimetric technique to determine the toxicity of the compounds on growing lymphoid and non-lymphoid cell lines based on reduction of the tetrazolium salt by intact mitochondria (Mossman, T., J. Immunol. Methods 65:55 (1983)). Cell viability in the presence or absence of different concentrations of test compounds in serum-free medium (HB 104, HANA Biologic, Inc.) was assessed using MTT
• Antigen activated proliferation of PBLs in a primary mixed lymphocyte reaction was assessed in the presence or absence of different concentrations of tested compounds
• EBV-transformed j8-lymphoblastoid cells LB and JVM, in a final volume of 200 ⁇ l per well in 96-well round-bottomed plates (Mishell, B.B. and S.M. Shiigi, Selected Methods in Cellular Immunology W.H. Freeman and Co., San
• CTLL IL-2 Microassay
• 3 x 10 CTLLs were exposed to different concentrations of test compounds and control drugs in the presence of 1 U/ml of human recombinant IL-2 (Genzyme, rIL-2) for 24 h.
• IL-2 human recombinant IL-2
• PMA and OKT3 - mitogens used to stimulate proliferation of human peripheral blood lymphocytes (PBC) Compounds are evaluated on their ability to inhibit proliferation.
• LB and JVM - human viral-transformed B lymphoblastoid cell lines stimulated to proliferate in a mixed lymphocyte reaction

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