Background of the Invention

Post operative graft rejections are a major com¬ plication affecting the success of bone marrow and organ transplantations. However, through the use of immuno¬ suppressive drug therapy, graft rejection in organ transplantation can be significantly reduced.

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.

One widely accepted im unosuppressant for the prevention of graft rejection is cyclosporin A (CsA) . It is a natural product of fungal metabolism and has been demonstrated to have potent immunosuppressive activity in clinical organ transplantations. Calne, R.Y. et al., Br. Med. J. 82:934-936 (1981); White, D.J.C. Drugs 24:322- 334 (1982). Although CsA is widely used in immuno- suppressant therapy, its usage (particularly in high dosage) is often accompanied by side effects which include nephrotoxicity, hepatotoxicity and other central nervous system disorders.

The following diseases have been treated with cyclosporin A with positive results, confirming the importance of the autoimmune component in these diseases and their effective treatment with compounds working by selective T-cell immune suppression similar to cyclo¬ sporin A.

1) Ophthalmology: Uveitis, Behcet's disease and

Grave's ophthalmopathy. Weetman, A.P. et al. , Lancet 486-489 (1982).

Grave's ophthalmopathy. Nussenblatt, R.B. et al., Lancet 235-238 (1983).

Uveitis. French-Constant, C. et al. , Lancet 454 (1983).

Behcet'ε disease. Sanders, M. et al. , Lancet 454-455 (1983). Behcet's disease.

Note: Cyclosporin A is currently approved in Japan for the treatment of Behcet's disease, the first autoimmune disease indication for this compound.

2) Dermatology: Various autoimmune skin diseases including psoriasis.

Zabel, P. et al. , Lancet 343 (1984). Acute dermatomyositis. van Joost, T. et al. , Arch. Dermato1. 123:166-167

(1987) . Atopic skin disease. Appleboom, T. et al. , Aroer. J. Med. 82:866-867

(1987) . Scleroderma. Logan, R.A. and R.D.R. Camo, J. Roy. Soc. Med.

81:417-418 (1988). Eczema. Griffiths, C.E.M. et al. , Brit. Med. J. 293_:731-732 (1986). Psoriasis.

Ellis, C.N. et al., J. Amer. Med. Assoc.

256:3110-3116 (1986). Psoriasis.

3) Hematology: Various diseases including anemia. Toetterman, T.H. et al., Lancet, 693 (1984). Pure _r ed cell aplasia (PRCA) .

Stryckmans, P.A. et al., New Engl. J. Med.

310:655-656 (1984). Aplastic anemia. Gluckman, E. et al., Bone Marrow Transplant 3_ Suppl. 1, 241 (1988). Aplastic anemia. 4) Gastroenterology/Hepatology: Primary cirrhosis, autoimmune hepatitis, ulcerative colitis, Crohn's disease and other gastrointestinal autoimmune diseases.

Wiesner, R.H. et al. , Hepatology 7:1025, Abst. #9, (1987) . Primary biliary cirrhosis.

Hyams, J.S. et a^. , Gastroenterology 93:890-893

(1987) . Autoimmune hepatitis. Allison, M.C. et al. , Lancet, 902-903 (1984). Crohn's disease. Brynskov, J. et al. , Gastroenterology 92:1330 (1987) . Crohn's disease. Porro, G.B. et al. , Ital. J. Gastroenterol. 19:40-41 (1987). Ulcerative colitis. 5) Neurology: A yotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), myasthenia gravis and multiple sclerosis. Appel, S.H. et al. , Arch. Neurol. 45:381-386

(1988). ALS. Tindall, R.S.A. et al. , New Engl. J. Med. 3_16:719-724 (1987). Myasthenia gravis. Ann. Neurol. 24, No. 1, p. I69,m Abstract P174 (1988) . Multiple sclerosis. Dommasch, D. et al. , Neurology 38 Suppl. 2, 28-29 (1988). Multipl sclerosis.

6) Nephrotic Syndrome: Nephrotic syndrome, membrano- proliferative glomerulonephritis (MPGN) and related diseases.

Watzon, A.R. et al. , Clin. Nephrol. 25:273-274 (1986) . Nephrotic syndrome.

Tejani, A. et al. , Kidney Int. 33:729-734 (1988).

Nephrotic syndrome. Meyrier, A. et al. , Transplat Proc. 20, Suppl. 4 (Book III) , 259-261 (1988) . Nephrotic syndrome. LaGrue, G. et al. , Nephron. 44:382-382 (1986). MPGN.

7) Rheumatoid Arthritis (RA)

Harper, J.I. et al., Lancet 981-982 (1984). RA Van Rijthoven, A.W. et a^. , Ann. Rheum. Pis. 4_5:726-731 (1986). RA.

Dougados, M. et al. , Ann. Rheum. Pis. 47:127-133 (1988). RA.

8) Insulin-Dependent Piabetes Mellitus (IPPM) Stiller, CR. et al. , Science 223:1362-1367 (1984). IPPM.

Assan, R. et al. , Lancet, 67-71 (1985) . IPPM. Bougneres, P.F. et al. , New Engl. J. Med. 3_18:663-670 (1988). IPPM. Piabetes 37:1574-1582 (1988). IPPM.

Many veterinary 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. Werner, Brit. Vet. J. 142:403-410, 491-497 and

498-505 (1986) immune mediated diseases of cats and dogs; George, L.W. and S.L. White, Vet. Clin. North A er. £« :203-213 (1984) autoimmune skin diseases in large mammals; Bennett, D. , In. Pract. 6:74-86 (1984) auto- immune diseases in dogs; Halliwell, R.E., J. Amer. Vet. Assoc. 181:1088-1096 (1982) autoimmune diseases in domesticated animals.

The mechanism by which CsA causes immunosuppression has been established. In vitro, CsA inhibits the release of lympho j.ner, such as interleukin 2 (IL-2) [Bunjes, D. et al., JBωr. J. Immunol. ljL:657-661 (1981)] and prevents clonal expansion of helper and cytotoxic T cells [Larsson, E. J. Immunol. 124:2828-2833 (1980)]. CsA has been shown to bind the cytosolic protein, cyclophilin, and inhibit the prolyl-peptidyl cis-trans isomerase

(PPIase) activity of that protein. Fischer, G. et al. , Nature 337:476-478 (1989); Takahashi, N. et al. , Nature 23_7 ^:473 - ⁴⁷⁵ (1989). The PPIases may mediate T cell acti¬ vation by catalyzing the rotomerization of peptide bonds of prolyl residues.

Recently, a second natural product isolated from Streptomyces, referred to as FK-506, has been demon¬ strated to be a potent im unosuppresive 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. Harding, M.W. et al., Nature 341:758-760 (1989); Siekierka, J.J., Nature 341:755-757 (1989).

Summary of the Invention This invention relates to a novel class of immuno¬ suppressive compounds having an affinity for the FK-506 binding protein (FKBP) . Once bound to this 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.

Brief Description of the Figure

Figure 1 illustrates some preferred compounds of this invention. The synthesis of each of the preferred compounds is described in detail in the Example section.

Detailed Description of the Invention This invention relates to a novel class of immuno¬ suppressive compounds represented by the formula I:

and pharmaceutically acceptable salts thereof, wherein A is 0, NH, or N-(C1-C4 alkyl); wherein B is hydrogen, CHL-Ar, (C1-C6)-straight or branched alkyl, (C1-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl, (C5-C7)-cycloalkenyl or Ar substi¬ tuted (C1-C6)-alkyl or alkenyl, or

wherein L and Q are independently hydrogen, (C1-C6)- straight or branched alkyl or (C1-C6)-straight o branched alkenyl; wherein T is Ar or substituted cyclohexyl with substituents at positions 3 and 4 which are independently selected from the group consisting of hydrogen, hydroxyl, 0-(Cl-C4)-alkyl or 0-(Cl-C4)-alkenyl and carbonyl; wherein Ar is selected from the group consisting of l-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl,

2-pyridyl, 3-pyridyl, 4-pyridyl and phenyl having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, CF ₃ , (C1-C6)-straight or branched alkyl or (C1-C6)- straight or branched alkenyl, 0-(Cl-C4)-straight or branched alkyl or 0-(Cl-C4)-straight or branched alkenyl, O-benzyl, O-phenyl, amino and phenyl; wherein D is either hydrogen or U; E is either oxygen or CH-U, provided that if D is hydrogen, then E is CH-U or if E is oxygen then D is U;

wherein U is hydrogen, 0-(Cl-C4)-straight or branched alkyl or 0-(Cl-C4)-straight or branched alkenyl, (C1-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl or (C5-C7) -cyclo- alkenyl substituted with (C1-C4)-straight or branched alkyl or (C1-C4)-straight or branched alkenyl, 2-indolyl, 3-indolyl, [ (C1-C4)-alkyl or (C1-C4)-alkenyl) ]-Ar or Ar (Ar as described above) ; wherein J is hydrogen or Cl or C2 alkyl or benzyl; K is (C1-C4)-straight or branched alkyl, benzyl or cyclo- hexylmethyl; or wherein J and K may be taken together to form a 5-7 membered heterocyclic ring which may contain an oxygen (0) , sulfur (S) , SO or SO, substituent therein. The stereochemistry at position 1 (Formula I) is (R) or (S) , with (S) preferred.

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, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, hepta- noate, hexanoate, hydrochloride, hydrobromide, hydro- iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl- propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. 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-D- glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen- containing groups can be guaternized 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.

Preferably, 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. Examples of compounds in which the J and K substituents are taken together to form a heterocyclic ring are shown in Tables 1 and 2. Examples of other preferred compounds of this invention are listed in Tables 3 and 4.

TABLE 1

B n Ki

TABLE 1 (continued)

B Ki K _^

Benzyl 2-Fury1 2 3μM >12μM Benzyl 3-Indolyl 2 25μM 2OμM Benzyl 2-Thiophenyl 2 O.δμM 4μM E-3-Phenyl-2- methyl-prop-

2-enyl Phenyl 2 1.5μM ND E-3-(4-Hydroxy- Phenyl 2 6μM ND phenyl)-2- ethyl-prop-

2-enyl E-3-[cis-(4- Phenyl 0.6μM ND hydroxycyclo- hexyl)]-2- methyl-prop-2- enyl E-3-[trans-(4- Phenyl 0.5μM ND

Hydroxycyclo- hexyl) ]-2-methyl- prop-2-enyl Benzyl 2-Nitrobenzyl 1 26μM >25μM Hydrogen Methoxy 2 ND ND tert-Butyl Methoxy 1 600μM >500μM Allyl Methoxy 2 19OμM >25μM Benzyl Methoxy 2 8OμM >50μM

2-Cyclohexylethy1 Methoxy 2 45μM >40μM 3-Cyclohexylpropyl Methoxy 2 2OμM 12μM 4-Cyclohexylbutyl Methoxy 2 6μM 2-3μM

TABLE 1 (continued)

B Ki

TABLE 1 (continued)

B Ki

TABLE 1 (continued)

No. B Ki

3,4,5-Trimethoxyphenyl 2 0.09 μM 0.08 μM

3-(1-Butoxy)phenyI 2 0.36 μM ND

3-(2-Propoxy)phenyl 2 0.1 μM ND

3,4,5-Trimethoxyphenyl 2 0.016 μM 0.06 μM

3,4,5-Trimethoxyphenyl 2 1.4 μM ND

3,4,5-Trimethoxyphenyl 2 0.22 μM ND

3 ,4 ,5-Trimethoxypheny I 2 0.5 μM ND

4-lodophenyl 2 0.6 μM 0.25 μM

3-lodophenyl 2 0.13 μM 0.2 μM

3,4,5-Trimethoxyphenyl 2 0.11 μM ND 3,4,5-Trimethoxyphenyl 2 0.017 μM 0.054 μM 65 4-(4-Methoxyphenyt)butyl 3,4,5-Trimethoxyphenyl 2 0.013 μM 0.049 μM 86 4-Phenylbut-2-enyl 3,4,5-Trimethoxyphenyl 2 0.8 μM ND 87 4-Phenytout-3-enyI 3,4,5-Trimethoxyphenyl 2 0.5 μM ND

88 4-(4-AIlocaminophenyl)propyl 3,4,5-Trimethoxyphenyl 2 0.011 μM 0.07 μM

89 4-PhenylpropyI 1-CyelohexenyI 2 0.78 μM ND

90 4-(4-Methoxyphenyl)but-3-enyI 3,4,5-Trimethoxyphenyl 2 0.011 μM 0.60 μM

91 4-PhenylpropyI 1-Fluoro-1 -cyclohexyl 2 0.54 μM ND

92 4-Phenylpropyl 3-Butoxyphenyl 2 1.4 μM ND

93 3-[3-(N-Formylindolyl)]propyl 3,4,5-Trimethoxyphenyl 2 0.015 μM 0.06 μM

94 4-(3-lndolyl)butyl 3,4,5-Trimethoxyphenyl 2 0.016 μM 0.05 μM

95 4-Phenylbutyl Benzyl 2 0.35 μM ND

TABLE 1 (continued)

No. B n Ki

ND indicates not determined.

No. B U n Ki K^

TABLE 3

No B Ki

124 Benzyl Methoxy Methyl Hydrogen lOOOμM >200μM

125 Benzyl Methoxy Methyl S-Methyl 40OμM >200μM

126 Benzyl Methoxy Methyl S-Isopropyl 17OμM >200μM

127 Ethyl Methoxy Benzyl Hydrogen >1200μM >300μM

128 tert- Methoxy Ethyl S-Methyl >400μM >500μM Butyl

TABLE 4

No B U Ki

129 Benzyl 4-Methoxy- Methyl . S-Methyl 80μM >150μM phenyl

130 Benzyl 4-Methoxy- Methyl S-Isopropyl 3OμM >20μM phenyl

131 Benzyl 3,4-Methylene- Methyl S-Methyl 50μM ND dioxyphenyl

132 Benzyl 3,4-Methylene- Hydrogen S-Methyl 6OμM ND dioxyphenyl

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. One particular 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, however, 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-[l- C]-benzoyl FK- 506 as a reporting ligand; or using [ H]dihydro-FK-506, as described by Siekierka, J.J. et al. , Nature 341:755- 757 (1989) . The binding affinities for several compounds of this invention for the FKBP are reported in Tables 1-4. The data was obtained using the latter method, where the ability of an unlabeled compound to compete with the binding of [ ³ H]dihydro-FK-506 to FK-506 binding protein was measured.

The inhibition of the PPIase (rotamase) enzyme activity of the FKBP (apparent "Ki" values) 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. Fischer, G. et al., Nature 337:476-478 (1989). The inhibitory effect of the addition of different concentrations of inhibitor on the extent of the reaction is determined, and analysis of the change in first order rate constant as a function of inhibitor concentration yields an estimate of the apparent Ki value. The extent of enzyme inhibition (K.) of some preferred compounds is shown in Tables 1-4.

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 Tables 5 and 6)

TABLE 5

Assays and IC ₅₀ Cyclosporin Value for Drugs A Rapamycin FK-506

1) Human PBL + <lμg/ml <lμg/ml <lμg/ml OKT3

2) T-Cell Hybridoma <lμg/ml <lμg/ml <lμg/ml + TCR/CD2

TABLE 6: Cellular Assay Results

TABLE 6 (Cont.)

TABLE 6 (Cone. )

DA¬

TABLE 6 (Cont.)

TABLE 6 (Cont.)

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Z Z Z Z Z Z Z Z Z Z Z Z

O o T- o -i- o T- o T- _, Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω T- Λ Λ Λ Λ 00 Z Z Z Z Z Z Z Z Z Z Z Z Z Z o υ SO C-4 so

•

W

Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω - Λ Z Z Z Z Z Z Z Z Z Z Z Z Z

I

All of the compounds in Table 6 showed toxicity at higher concentrations than their tmmunosuppresive activity and were typically concentrations > 10 μM.

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 a mixed lymphocyte reaction (MLR). The compounds are evaluated on their ability to inhibi this proliferation.

CTLL - inhibition of proliferation of cytotoxic T cells stimulated by IL-2.

1) Assay similar to Yoshimura, N. et al. , Trans¬ plantation 47:356-359 (1989) . Assay uses fresh human peripheral blood lymphocytes isolated by Ficoll-Hypague density centrifugation, stimulated by the OKT3 antibody (anti-CD3) which stimulates via interaction with CD3.

Stimulation is measured by incorporation of radioactive thymidine [( H)TdR] into proliferating cells, with an uninhibited control signal of 48,000-75,000 cpm. ^c ₅₀ values are estimated from inhibitions of proliferation observed at various drug concentrations.

2) Assay similar to above, but using T-cell clone stimulated with antibody to the T-cell receptor (TCR) and antibody to CD2. Stimulation is measured by incorpor- ation of radioactive thymidine [( H)TdR] into pro- liferating cells, with an uninhibited control signal of 23,000 cpm. I ^C 5o ^{v lues are} estimated from inhibitions of proliferation observed at various drug concentrations.

3) Assay according to Shi, Y. et al. , Nature 339:625-626 (1989). The assay uses a T-cell hybridoma similar to that described. The assay measures acti¬ vation-induced (anti-CD3) 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, immuno- suppressant rapamycin is inactive in this assay.

4) Assay according to DuMont, F. et al. , J.

Immunol. 144:251-258 (1990). The assay measures the stimulation of CTLL cells in response to IL-2. Prolifer-

₃ ation is measured by incorpoation of ( H)TdR. Immuno- suppressants which work by a similar mechanism to cyclo¬ sporin A and FK-506 will not inhibit in this IL-2 driven process, since they function by the inhibition of produc¬ tion of endogenous IL-2. In this assay, exogenous IL-2 is provided to overcome this block. Note that the chemically related, but mechanistically distinct immuno- εuppressant, rapamycin, is active in this assay.

These assays can be used to profile the cellular activity of the compounds of the present invention. Thus, it is clear from these results that the compounds resemble both cyclosporin A and FK-506 in its cellular activity, including immunosuppression, in contrast to the mechanistically dissimilar im unosuppressant agent rapamycin. Furthermore, the observed cellular activity is consistent quantitatively with the activity observed for FKBP binding and inhibition of PPIase (rotamase) activity shown in Table 1.

Thus, the compounds can be used as immunosup¬ pressants for prophylaxis of organ rejection or treatment of chronic graft rejection and for the treatment of autoimmune 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 anti¬ gen-stimulated growth and clonal expansion of T-cells, especially those T-cells characterized as ••helper" T-cells. This activity is useful in the primary pre- vention 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. These autoimmune diseases include: uveitis, Behcet's disease. Graves ophthalmopathy, psoriasis, acute der- matomyositis, atopic skin disease, scleroderma, eczema, pure red cell aplasia, aplastic anemia, primary cirrhosis, autoimmune hepatitis, ulcerative colitis, Crohn's disease, a yotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, nephrotic syndrome, membranoproliferative glomerulonephritis, rheumatoid arthritis and insulin-dependent diabetes mellitus. In all of the above-listed autoimmune diseases, treatment is effective to reduce the symptoms and slow progression of the disease. In the case of insulin-dependent diabetes mellitus, 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.

For these purposes 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. The term 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. Among the acceptable vehicles and solvents that may be employed are water. Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid and its glyceride deriv¬ atives find use in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxy- ethylated 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. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When 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 administered in the form of suppositories for rectal administration of the drug. These 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. Such materials include cocoa butter, beeswax and polyethylene glycols. The compounds of this invention may also be administered 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 prepared for each of these areas.

For ophthalmic use, 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. Alter¬ natively for the ophthalmic uses, the compounds may be formulated in an ointment such as petrolatum.

For application topically to the skin, 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. Alternatively, the compounds can be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in, for example, a mixture 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. Dosage 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.

It is understood, however, that 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. The steroid is administered orally, intravenously, rectally, topically or by inhalation. Dosages (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. Administration of cyclosporin and FK-506 together should be avoided due to contraindications reported resulting from coadministration of these immunosuppressants. The dosage level of other immunosuppressant drugs will depend upon the factors previously stated and the immuno¬ suppressive effectiveness of the drug combination.

OKT3, which is a murine monoclonal antibody to CD3 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.

The invention will be further illustrated by way of the following examples, which are not intended to be limiting in any way.

EXA PLES

General

Proton nuclear magnetic resonance ( H NMR) spectra were recorded at 300 MHz on a Bruker AC 300 or at 500 MHz on a Bruker AMX 500. Chemical shifts for proton resonances are reported in parts per million (δ ) relative to Me.Si (δ 0.0). Analytical high performance liquid chromatography (HPLC) was performed on either a Waters 600 E or a Hewlett Packard 1050 liquid chromatograph. HPLC assessments of compounds were run on a Waters

Associates Delta Pak 5 micron, 15 cm. column at a flow rate of 1.5 mL per minute. The solvent system used was: A = 0.1% H ₃ P0 ₄ /H ₂ 0; B = 0.1% H ₃ P0 ₄ /CH ₃ CN. A linear gradient of 95% A/5% B to 100% B over 15 minutes followed by 1.5 minutes at 100% B was used, with detection at 214 nM.

The compounds described below are illustrated in Figure 1.

EXAMPLE 1

Synthesis of (E)-3-rcis-4-(hydroxycyclohexyl) ]-2- methylprop-2-enyl N-(phenylglyoxyl)-pipecolate (25).

1. (S)-Benzyl Pipecolate (133)

To a slurry of 7.3 g (26.14 mmol) of the tartrate salt of (S)-pipecolic acid (Egbertson M. and S.J. Danishefsky, J. Orq. Chem. 5_4:11 (1989) in 75 mL of dry benzene was added 13.5 mL (0.13 ol) of benzyl alcohol and 5.48 g (28.8 mmol) of p-toluenesulfonic acid onohy- drate. The reaction mixture was heated at reflux

under a Dean-Stark trap for 2 h and then cooled to room temperature. The solution was then diluted with 400 mL of ether and stirred overnight at 4 ^β C. The resulting white solid was collected on a filter, washed with hexane and dried in vacuo to give 9.2 g (90%) of the p-toluene- sulfonic acid salt of benzyl pipecolate (134) . H NMR (300 MHZ, D ₂ 0) δ 7.63 (d) , 7.41 (s) , 7.28 (d) , 5.26 (ABq) , 4.8 (s) , 4.03 (dd) , 3.92 (dd) , 3.51-3.39 (m) , 3.15-2.93 (S) , 2.40 (s) , 2.36-2.24 (m) , 1.98-1.53 (m) . Benzyl pipecolate was routinely generated by treating an ethyl acetate suspension of this salt with saturated sodium bicarbonate until dissolution of the organic material. The aqueous layer was extracted twice with ethyl acetate and the combined organic extracts were dried with MgSO. and evaporated to yield (S)-Benzyl pipecolate (133) as a pale yellow oil.

2. (S)-N-(Phenylglyoxyl)pipecolic Acid (135)

To a solution of 4.95 g (17.72 mmol) of the tartrate salt of L-(S)-pipecolic acid in 18.0 mL of methylene chloride at 0°C was added 20.4 mL (117.10 mmol) of diisopropylethylamine followed by 12.4 mL (97.7 mmol) of chlorotrimethylsilane and the resulting solution was stirred at 0 ^β C for 15 minutes. To this mixture was added 17.72 mmol of benzoylformyl chloride, which was freshly prepared in a separate reaction flask at ambient temperature from 2.66 g (17.72 mmol) of benzoylformic acid and 2.3 mL (26.37 mmol) of oxalyl chloride in 18.0 mL of methylene chloride containing a catalytic amount of dimethylformamide. The reaction mixture was stirred at 25°C overnight at which time the mixture was poured into

1.0 N HC1. The aqueous layer was discarded and the organic layer was washed twice with saturated sodium bicarbonate. The combined aqueous layers were washed with methylene chloride, acidified to pH 2.0 with con¬ centrated HC1, and then extracted repeatedly with ether. Flash chromatography (Still, W.C. et al., J. Org. Chem. 4_ :2923 (1978)) (elution with 1:1 ethyl acetate-hexane containing 1% acetic acid provided 2.3 g of (S)-N- (Phenylglyoxyl)-pipecolic acid (135) as a rotameric mixture. ^X H NMR (500 MHz, CDC1 ₃ ) δ 11.4-11.1 (br s) , 8.02 (d) , 7.98 (d) , 7.65 (t) , 7.58-7.43 (m) , 5.45 (d) , 4.64 (dd) , 4.43 (d), 3.52 (dd) , 3.25 (ddd) , 3.01 (ddd) , 2.42 (d) , 2.24 (d) , 1.86-1.78 (m) , 1.68-1.38 (m) .

3. cis-and trans-4-(tert-Butyldimethylsilyloxy)- cvclohexane-1-ol (136) and (137)

To a solution of 3.43 g (21.7 mmol) of cis- and trans-methyl 4-hydroxycyclohexane carboxylate (Noyce, D.S. and D.B. Denney, J. Am. Chem. Soc. 74:5912 (1952)) in 45 mL of methylene chloride at 0°C was added 3.0 mL (26.0 mmol) of 2,6-lutidine followed by 5.5 mL (23.8) mmol of tert-butyldimethylsilyl trifluoromethane- sulfonate. The ice bath was removed and the reaction mixture was allowed to stir at 25°C for 2 h at which time the solution was poured into saturated sodium bicarbonate. The layers were partitioned and the organic layer was washed with saturated copper sulfate and water and then dried over MgS0 ₄ to give 5.9 g of the crude methyl esters. A solution of 5.72 g (21.0 mmol) of this mixture in 45 mL of anhydrous THF was treated with 400 mg (10.5 mmol) of lithium aluminum hydride. The reaction

mixture was stirred at 25 ^β C for 0.5 h and was then quenched by the slow addition of a saturated solution of Rochelle's salt. The mixture was diluted with ether, the layers were partitioned and the aqueous layer was washed twice with ethyl acetate. The combined organic extracts were dried over MgSO. and concentrated to give 4.9 g of the diastereomeric alcohols. Flash chromatography (elution with 1:5 ethyl acetate-hexane) gave 650 mg of

(136), 1.10 g of (137) and 2.40 g of a mixture of the two. Data for (136): ^X H NMR (300 MHz, CDC1 ₃ ) δ 3.99-3.92 (m) , 3.46 (d) , 1.72-1.58 (m) , 1.57-1.36 (m) , 0.86 (s) , 0.08 (S) . Data for (137): ^Η NMR (300 MHz, CDC1 ) <5 3.47 (dddd), 3.38 (d) , 1.86-1.67 (m) , 1.47-1.16 (m) , 1.05-0.77 (m), 0.72 (s) , -0.02 (s) .

4. (E)-Ethyl 3-rcis-(4-tert-Butyldimethylsilyl- oxycyclohexyl) 1-2-methylprop-2-enoate (138) To a -78°C solution of oxalyl chloride (465 μL, 5.33 mmol) in 5.0 mL of methylene chloride was added dimethyl- sulfoxide (755 μL, 10.65 mmol). The resulting solution was stirred for 5 min and then 650 mg (2.66 mmol) of the alcohol (136) was added in 5.0 mL of methylene chloride. The reaction mixture was stirred at -78°C for 45 min at which time 2.2 mL (16.0 mmol) of triethylamine was added and the solution was allowed to warm to ambient temperature. The reaction was quenched with 1.0 N HCl and the aqueous layer was extracted with three portions of methylene chloride. The.combined organic extracts were dried over MgSO. and evaporated to dryness to give 620 mg of the intermediate aldehyde which was treated

directly with 1.22 g (3.36 mmol) of (carbethoxyethyl- idine)triphenylphosphorane in 5.0 mL of methylene chloride. The resulting reaction mixture was stirred at ambient temperature overnight and was then poured into water. The layers were partitioned and the aqueous layer was extracted twice with methylene chloride. The com¬ bined organic layers were dried over MgSO. and con¬ centrated to yield 1.55 g of crude product. Flash chromatography (elution with 1:20 ether-hexane) gave 300 mg of the enoate (138) as an oil.

5. (E)-3-rcis-(4-tert-Butyldiroethylsilyloxycyclo- hexyl) ]-2-methylprop-2-en-l-ol (139) To a solution of 300 mg (0.95 mmol) of enoate (138) in 2.0 mL of anhydrous tetrahydrofuran at 25°C was added is mg (0.43 mmol) of lithium aluminum hydride and the resulting mixture was allowed to stir for 30 min. The reaction was quenched by the slow addition of saturated Rochelle's salt and diluted with ethyl acetate. The layers were separated and the aqueous layer was extracted with two portions of ethyl acetate. The combined organic extracts were washed with water and brine and then dried over MgSO.. Evaporation and flash chromatography (elution with 1:10 ethyl acetate-hexane) gave 220 mg of the allyic alcohol (139). ^X H NMR (300 MHz, CDC1 ₃ ) δ 5.34 (d), 3.96 (d), 3.85 (m) , 2.26-2.18 (m) , 1.64 (d) , 1.61-;.34 (m) , 1.82 (s) , 0.0 (s) .

6. (E)-3-rcis-(4-tert-Butyldimethylsilyloxycyclo- hexyl)1-2-methylprop-2-enyl N-(phenylglyoxyl)- pipecolate (140)

To a solution of 68 mg (0.24 mmol) of allylic alcohol (139), 42.3 mg (0.16 mmol) of acid (135) and 39.8 mg (0.20 mmol) of l-(3-dimethylamino-propyl)-3-ethyl- carbodiimide hydrochloride (EDC) in 2.0 mL of anhydrous methylene chloride was added a catalytic amount of 4-dimethylaminopyridine and the resulting mixture was stirred overnight at room temperature. The reaction mixture was then poured into water, the layers parti¬ tioned and the aqueous layer was extracted twice with methylene chloride. The combined organic extracts were dried over MgSO. and concentrated to yield a yellow oil. Flash chromatography (elution with 15% ethyl acetate in hexane) gave 9.2 mg of the ester (140) as a rotameric mixture. H NMR (500 MHz, CDC1 ₃ ) δ 8.01 (d) , 7.94 (d) , 7.59-7.52 (m) , 7.46-7.39 (m) , 7.19 (d) , 7.12 (d) , 6.82 (d), 6.51 (s), 6.38 (s) , 5.43 (d) , 4.78 (ABq) , 4.62 (dd) , 4.58 (S), 4.41 (d) , 3.51 (dd) , 3.23 (ddd), 3.01 (ddd), 2.41 (d), 2.24 (d), 1.91 (s) , 1.84-1.76 (m) , 1.65-1.48 (m) , 0.96 (S) , 0.18 (s) .

7. (E)-3-rcis-4-(hydroxycyclohexyl) ]-2-methylprop-2- enyl N-(phenylglyoxyl)-pipecolate (25) O a solution of 9.2 mg (0.02 mmol) of ester (140) in 1.0 mL of acetonitrile at 25 ^β C was added dropwise a solution consisting of a 95:5 mixture of acetonitrile: 48% hydrofluoric acid and the reaction was stirred until thin layer chromatography (TLC) indicated the dis- appearance of starting material. The reaction was

quenched by the addition of saturated potassium car¬ bonate. The reaction mixture was extracted with three portions of ethyl acetate, dried over MgSO. and concen¬ trated. Flash chromatography (elution with 35% ethyl acetate in hexane) yielded 6.1 mg (82%) of the ester (25) as a rotameric mixture. H NMR (500 MHz, CDC1 ₃ ) δ 8.02 (dd), 7.97 (dd), 7.67-7.59 (m) , 7.56-7.48 ( ) , 5.49 (d) , 5.44 (d) , 4.61 (ABq) , 4.44 (s) , 4.41 (d) , 3.98-3.91 (m) , 3.5 (br d) , 3.26 (ddd), 3.11 (ddd), 2.43-2.18 (m) , 1.86-1.37 (m) , 1.72 (d) , R _f 0.57 (3:1 ethyl acetate-hexane) .

EXAMPLE 2

Synthesis of (S)-Benzyl N-(phenylglyoxyl)pipecolate (3) To a solution of 43 mg (0.19 mmol) of freshly generated (S)-Benzyl pipecolate (133) (described in

Example 1) in 2.0 mL of anhydrous methylene chloride was added 44 mg (0.29 mmol) of benzoylformic acid and 56 mg (0.29 mmol) of l-(3-dimethylamino-propyl)-3-ethylcarbo- diimide hydrochloride (EDC) and the resulting mixture was stirred overnight at room temperature. The reaction mixture was then poured into water, the layers par¬ titioned and the aqueous layer was extracted twice with methylene chloride. The combined organic extracts were dried over MgSO. and concentrated to yield a yellow oil. Flash chromatography (elution with 1:3 ether-hexane) gave 49 mg (72%) of the keto-amide (3) as a rotameric mixture. H NMR (500 MHz, CDC1 ₃ ) δ 7.98 (d) , 7.91 (d) , 7.58 (t) , 7.41-7.30 (m), 5.45 (d) , 5.21 (ABq), 5.06 (ABq), 4.61 (dd), 4.42 (d), 3.48 (dd), 3.19 (ddd), 2.96 (ddd), 2.40 (d), 2.21 (d), 1.83-1.72 (m) , 1.61-1.49 (m) , 1.46-1.33 (m) , R _f 0.55 (1:1 ether-hexane).

EXAMPLE 3

Synthesis of (S)-Benzyl N-r(3-methoxyphenyDglyoxyl) ]- pipecolate (9)

The keto-amide (9) was prepared from 45 mg (0.205 mmol) of (S)-Benzyl pipecolate (133) (described in

Example 1) and 55 mg (0.306 mmol) of 3-methoxybenzoyl- formic acid (Barnish, T. et al. , J. Med. Chem. 24:339 (1981)) as described in Example 2. Flash chromatography (elution with 1:4 ether-hexane) gave 73 mg (93%) of (9) as a rotameric mixture. H NMR (300 MHz, CDC1 ₃ ) δ

7.59-7.10 (m)i, 5.42 (d) , 5.23 (ABq), 5.09 (ABq), 4.59 (dd) , 4.38 (d) , 3.82 (S) , 3.81 (s) , 3.48-3.40 (m) , 3.20 (ddd), 2.98 (ddd), 2.39 (d) , 2.21 (d) , 1.82-1.70 (m) , 1.61-1.22 (m) , R _f 0.45 (1:1 ether-hexane).

EXAMPLE 4

Synthesis of (S)-Benzyl N-(2-furylglyoxyl)pipecolate (20)

To a 0°C solution of 412 mg (1.03 mmol) of (S)- Benzyl pipecolate salt (134) (described in Example 1) in 40 mL of acetonitrile was added 198 μL (1.14 mmol) of diisopropylethylamine, 174 mg (1.24 mmol) of α-oxo-2- furanacetic acid, 579 mg (1.24 mmol) of benzotriazol- 1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and then 216 μL (1.24 mmol) of diisopropylethylamine. The resulting reaction mixture was stirred at ambient temperature for 14 h and then evaporated to dryness. The residue was dissolved into 150 mL of ethyl acetate, washed sequentially with 50 mL of 0.5 N HCl, 50 mL of saturated NaHCO,, 50 mL of brine and was then dried over

MgSO. and concentrated. Flash chromatography (elution with 2% ether in methylene chloride) provided 163 mg (46%) of the keto-amide (20) as an oil. The ^X H NMR spectrum of this compound (300 MHz, CDC1 ₃ ) was consistent with the product as a mixture of rotamers. R _f 0.2 (2% ether in methylene chloride). HPLC, R _t =12.83 min.

EXAMPLE 5

Synthesis of (S)-Benzyl N-(4-Methoxycinnamoyl) .:pecolate (112)

To a solution of 145 g (0.37 mmol) of (S)-Benzyl pipecolate salt (134) (described in Example 1) in 8.0 mL of methylene chloride was added 102 mg (0.57 mmol) of 4-methoxycinnamic acid, 107 mg (0.55 mmol) of l-(3-di- methylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 130 μL (0.74 mmol) of diisopropylethylamine.

The resulting solution was stirred at ambient temperature for 12 h and was then concentrated under reduced pres¬ sure. Flash chromatography (elution with 1:1 ethyl acetate-hexane) gave 91 mg (65%) of the amide (112) as a r c-nollnorrll βeeses i o-ii-ϊ 1l . __ * 1H NMR (300 MHz , CDC1 ₃ ) δ 7. 66 (d) , 7.50 (d) , 7.35 (m) , 6.90 (d) , 6.82 (d) , 6. 63 (d) , 5.55 (d) , 5.20 (br ε) , 4.86 (br ε) , 4.67 (br d) 4. 03 (br d) , 3.83 (S) , 3.37 (dt) , 2.78 (dt) 2.33 (br d) , 1.74 (m) , 1.43 (m) . R _f 0.40 (1 : 1 ether-hexane) .

EXAMPLE 6

Synthesis of (S)-3-(3,4-Methylenedioxyphenyl)-prop-2- enoylproline benzyl ester (109) and (S)-3-(3,4-Methylene¬ dioxyphenyl)-prop-2-enoylalanine benzyl ester (132)

A solution of 192 mg (1.0 mmol) of 3,4-methylene- dioxycinnamic acid, 121 mg (0.5 mmol) of proline benzyl ester hydrochloride and 108 mg (0.5 mmol) of alanine benzyl ester hydrochloride in 6 mL of acetonitrile was treated sequentially with 0.35 mL (2.0 mmol) of diiso¬ propylethylamine and 443 mg (1.0 mmol) of benzotriazol- 1-yloxytris(dimethylamino)phosphonium hexafluorophos¬ phate. The mixture was stirred for 16 hrs then concen¬ trated in vacuo. The residue was dissolved in 10 mL of dichloromethane and poured into 3 volumes of diethyl ether. The mixture was waεhed εequentially with water, 10% potassium bisulfate solution, water, saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The organic layer waε dried over magneεium εulfate, filtered and concentrated in vacuo. The reεidue was purified by silica gel column chromatography, using a stepwiεe gradient of 30%, 35%, 40%, and 45% ethyl acetate in hexane as eluant. Ester (109) (142 mg) was obtained as a colorlesε oil, Rf = 0.4 (40% ethyl acetate/hexane) ; HPLC, R _t = 12.26; H NMR (300 MHz) consistent with structure. Ester (132) (162 mg) was obtained as a colorless oil; TLC, R _f - 0.22 (40% ethyl acetate/ hexane); HPLC, R _t - 11.84 min; ^X H NMR (300 MHz) consistent with structure.

EXAMPLE 7

Synthesis of (S)-N-3-(4-methoxyphenyl)-prop-2-enoyl-N- methylalanine benzyl ester (129)

1. (S)-N-Methylalanine benzyl ester p-toluene suli. ^* f c acid salt (141)

A suspension of 1.55 g (15.0 mmol) of (S)-N-methyl- alanine and 9.31 (90.0 mmol) of benzyl alcohol in 30 mL of toluene was treated with 3.00 g (15.8 mmol) of p- tolueneεulfonic acid monohydrate. The mixture was heated for 19 hours under reflux with removal of water via a Dean-Stark trap. After cooling, the reaction solution was poured into <α00 mL of ether, precipitating a yellow oil. The solvents were decanted and the residue was ta^en up into ethyl acetate and concentrated to yield a viscouε, light yellow oil (141); TLC: R- = 0.34, 95:5:0.5 CH-Cl./MeOH/concentrated ammonium hydroxide; H NMR (300 MHz) consistent with structure.

2. (S)-N-3-(4-methoxypheny1)-prop-2-enoy1-N-methyl- alanine benzyl ester (129) A suspension of 96 mg (0.5 mmol) of 4-methoxy- cinnamic acid and 121 mg (0.5 mmol) of (141) in 6 mL of dichloromethane was cooled in an ice/water bath under nitrogen. The mixture was treated with 0.26 mL (1.5 mmol) of diisopropylethylamine and 135 mg (0.53 mmol) of N,N-bis-(2-oxo-3-oxazolidinyl) phosphinic chloride and then stirred for 16 hours, warming slowly to ambient temperature. The mixture was poured into three volumes of diethyl ether and washed sequentially with water, 10%

potassium bisulfate solution, water, saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by preparative thick layer silica gel chromatography using 40% ethyl acetate/hexane as eluant. Ester (129) (40 mg) was obtained as a yellow oil: TLC, R _f ■ 0.24 (35% ethyl acetate/hexane) ; HPLC, R _t - 13.34 min; H NMR (300 MHz) consiεtent with structure.

EXAMPLE 8

Synthesis of (S)-N-Methyloxalyl-N-ethylalanine benzyl ester (127)

1. (S)-N-9-Fluorenylmethoxycarbonyl-N-ethylalanine benzylester (142) A εuεpension of 848 mg (2.5 mmol) of (S)-N-Fmoc- N-ethylalanine in 10 mL of dichloromethane was treated with 436 μL (5.0 mmol) of oxalyl chloride followed by a catalytic amount (1 drop) of dimethylformamide. The mixture was stirred for one hour, then concentrated in vacuo. The yellow, oily residue was treated with 10 mL of toluene followed by 517 mg (0.5 mmol) of benzyl alcohol and 669 mg (5.0 mmol) of silver cyanide. The mixture was heated in an 80°C oil bath with vigorous stirring for 20 minutes, then cooled and filtered through a pad of diatomaceous earth. The filtrate waε concen¬ trated, and the reεidue was purified by silica gel column chromatography, using 10% acetone in hexane as eluant. Ester (142) (810 mg) was obtained as a colorless oil; TLC: R _f = 0.28, 15% acetone/hexane; H NMR (300 MHz) con- sistent with structure.

2. (S)-N-Methyloxalyl-N-ethylalanine benzyl ester (127) A solution of 0.25 g (0.58 mmol) of (142) in 3 mL of acetonitrile was treated with 3 mL of diethyla ine and the mixture was allowed to stand for 20 min. The mixture was concentrated in vacuo and the residue was taken up in 10 mL of acetonitrile and again evaporated. After repeating this process, the residue was dissolved in 4 mL of dichloromethane, cooled in an ice/water bath under nitrogen, and treated with 111 μL (0.64 mmol) of diiso- propylethylamine followed, during approximately 1 min. with 54 μL (0.64 mmol) of methyl oxalyl chloride. The mixture was stirred overnight, warming slowly to ambient temperature, then poured into three volumes of ether. The mixture was washed sequentially with water, 10% potasεium biεulfate solution, water, saturated εodium bicarbonate solution, water, and saturated εodium chloride εolution. The organic layer waε dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue waε purified by silica gel column chromato- graphy using 1:7 acetone: hexane as eluant. Ester (127) (147 mg) was obtained as a colorless oil, R. = 0.36, 35% acetone/hexane; HPLC, R. = 12.19 min; H NMR (300 MHz) consistent with εtructure.

EXAMPLE 9

Syntheεiε of (S)-3-Cyclopentylpropyl N-(2-Methyloxalyl)- pipecolate (135)

1. (S)-N-(MethyloxalyDpipecolic Acid (143)

The acid (143) was prepared from methyl oxalyl chloride as described in Example 1 for the production of

(S)-N-(Phenylglyoxyl)pipecolic acid (135). Thus, 3.16 g (11.32 mmol) of the tartrate salt of (S)-pipecolic acid and 1.19 mL (12.45 mmol) of methyl oxalyl chloride gave 1.25 g (51%) of the acid (143) aε a tan εolid. H NMR (300 MHZ, CDC1 ₃ ) δ 5.31 (d) , 4.62 (d) , 4.49 (br d) , 3.61 (br d), 3.90 (ε), 3.88 (s) , 3.46 (dt) , 2.97 (dt) , 2.40-1.40 (m) .

2. (S)-3-Cyclopentylpropyl N-(2-Methyloxalyl)pipecolate 1351 The ester (35) waε prepared from 3-cyclopentyl- propan-1-ol and the acid (143) as described in Example 2.

Flaεh chromatography (elution with 2% ether in methylene chloride) gave 72 mg (48%) of (35) as a colorless oil.

The H NMR εpectru of thiε compound (300 MHz, CDC1 ₃ ) was consistent with the product as rotamers. R _f 0.56 (10% ether in methylene chloride). HPLC, R _t = 14.30 min.

DISCUSSION OF ASSAYS

Cell Source and Culture

Freεh peripheral blood lymphocyteε (PBLs) from LeukoPak cells or whole blood from random normal blood donors (tested HIV-negative and hepatitiε 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 lineε uεed for activation of the fresh PBLs are EBV-transformed lymphocytes from normal healthy adult donorε with two

completely different HLA haplotypes. All cell lines were routinely tested for the presence of Mycoplasma contami¬ nation using the Gibco Mycotect test kit and are Mycoplasma-free. Culture medium consists of RPMI 1640 (Gibco, Grand Island, NY) containing penicillin (50 U/ml) and streptomycin (50 μg/ml) , L-glutamine 2 mM, 2 mer- ccaappttooeetthhaanol (5 x 10 —5), 10% heat-inactivated FCS and 10 mM HEPES.

Compound Solutions and Titrationε All chemical stocks were dissolved in DMSO.

Titrationε of compounds were made into the medium the individual assay was carried out in, i.e., complete RPMI or HB 104 for final diluted concentrations, uεing multiple three-fold dilutionε from 1 μM or 10 μM εtock εolutionε.

MTT Aεεay

The MTT assay is a colori etric technique to deter¬ mine the toxicity of the compounds on growing lymphoid and non-lymphoid cell lines baεed on reduction of the tetrazolium salt by intact mitochondria (Mosεman, 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 (3-[4,5-dimethyl-thiazoyl- 2-yl]2,5-diphenyl-tetrazolium bromide). At 4 h before the end of the 3-day toxicity asεay culture period, 20 μl of MTT dye (5 mg/ml in pH 7.2 PBS) were added to each microtiter well. At the end of the incubation time, moεt of the culture media was carefully aspirated out of each

well. Then 100 μl of acidified isopropyl alcohol (0.04 N HCl) was added to solubilize the dye and optical density is read at 570 nm minus OD at 630 nm (Molecular Devices Thermomax plate reader and Softmax software program, Menlo Park, CA) . Resultε were compared with mean OD in controls (medium with no drugs) and doses causing 50% toxicity (TC ₅₀ ) were calculated.

Mitogenesis Aεεayε ("PMA" and "0KT3")

The inhibitory effect of teεt compounds on the proliferation of human PBLs in response to mitogens

(Waithe, W.K. and K. Hirschhorn, Handbook of Experimental Immunology, 3d Ed. Blackwell Scientific Publications, Oxford (1978) ; Miεhell, B.B. and S.M. Shiigi, Selected Methodε in Cellular Immunology W.H. Freeman and Co., San Francisco, CA (1980) ) was assesεed by stimulation of 5 x 10 ⁴ cells with 0KT3 (10~ ⁴ dilution final) or PMA (lOng/ml) plus ionomycin (250 ng/ml) in the presence or absence of different concentrationε of test compounds and control drugs (CsA, FK506, Pagamycin) in final volume of 200 μl per well in 96 well round bottomed plates. After

48 h incubation (37°C, 5% CO.), cells were pulεed with 1 μCi of 3H-thymidine, harvested 24 h later with a Tom Tek cell harvester, and counted in LKB 0-scintillation counter. Results (cpm) were compared with controls with medium alone, and concentrations causing 50% reduction in counts (IC__) were calculated.

MLR Bioassayε ("LB" and "JVM")

Antigen activated proliferation of PBLε in a primary mixed lymphocyte reaction waε aεsessed in the preεence or

absence of different concentrations of tested compounds

Λ and control drugs. 5 x 10 fresh PBLs were stimulated

₃ with 5 x 10 of Mitomycin C treated-allogeneic EBV- transformed 9-lymphoglastoid cells, LB and JVM, in a final volume of 200 μl 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

Francisco, CA (1980) ; Nelson, P.A. et al. , Trans- plantation 5£:286 (1990)). Cultureε were pulεed on day 6, harvested 24 h later and counted as in previous section.

IL-2 Microassay ("CTLL")

To determine if test compounds inhibit the later T cell activation process of cytokine utilization, the proliferative reεponse of the IL-2 dependent CTLL-20 murine T cell line (ATCC) was asεessed (Gillis, S. et al., J. Immunology 120:2027 (1978)). CsA and FK506 inhibit the production of IL-2 by activated T cellε, whereas Rapamycin interferes with the utilization of IL-2. Rapamycin thus inhibits IL-2 dependent prolifer¬ ation of the CTLLs, and CsA and FK506 do not (Dumont, F.J. et al. , J. Immunolocry 144:251 (1990)). 3 x 10 ³ CTLLs were exposed to different concentrationε of test compounds and control drugs in the presence of l U/ml of human recombinant IL-2 (Genzyme, rIL-2) for 24 h. Four h after adding drugs, cells were pulsed with 1 μCI of 3H-thymidine, incubated for an additional 20 h (37 ^β C, 5% CO_) , and then harvested and counted as previously described.