CA2086428A1 - Immunosuppressive compounds - Google Patents

Immunosuppressive compounds

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Publication number
CA2086428A1
CA2086428A1 CA002086428A CA2086428A CA2086428A1 CA 2086428 A1 CA2086428 A1 CA 2086428A1 CA 002086428 A CA002086428 A CA 002086428A CA 2086428 A CA2086428 A CA 2086428A CA 2086428 A1 CA2086428 A1 CA 2086428A1
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CA
Canada
Prior art keywords
compound
composition
straight
benzyl
hydrogen
Prior art date
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Abandoned
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CA002086428A
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French (fr)
Inventor
David M. Armistead
Joshua S. Boger
Harold V. Meyers
Jeffrey O. Saunders
Roger D. Tung
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Vertex Pharmaceuticals Inc
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Individual
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Application filed by Individual filed Critical Individual
Publication of CA2086428A1 publication Critical patent/CA2086428A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/56—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having carbon atoms of carboxamide groups bound to carbon atoms of carboxyl groups, e.g. oxamides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/60—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Abstract

This invention relates to a novel class of immunosuppressive 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 inhibit T cell activation. As such, 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 for use in the treatment of a wide variety of autoimmune diseases in humans and other mammals.

Description

WO 92/00278 PCI`/US91/04694 J ~ 2 8 NC)VE~ IMMI~NOSUPPRESSI~IE COMPOUNDS

Back~round o~ the Invention Poet operative graft rejectione are a major com-pl~cation affecting the success of bone marrow and organ transplantatione. However, through the use of immuno-suppressive drug therapy, gra~t re~ection in organ transplantation can be significantly reduced.
A wide variety of diseases can be characterized as ~autoimmune diseases". Such diseaees are similar to graft rejection, except that the rejection is of self tissue. Immunosuppressive therapy can al50 be of use in preventing this inappropriate self rejection.
One widely acc-pted im~unosupprcseant for the prevention o2 graft re~ection ~- ¢y¢losporin A (C~A). It ~g a nAtural product of ~ungal motabolism and has bcen demonstrated to have potent lmmunosuppre~eive activity in clinical organ transplantations. Calne, R.Y. et al., Br.
Med. J. 2 :934-936 (1981); White, D.J.C. Druqs 24:322-334 (1982). Although CsA is widely used in immuno-~uppressant therapy, its usage (particularly in highdosage) is often accompanied ~y side e~fects which include nephrotoxicity, hepatotoxicity and other central nervous ~ystem disorders.
~he following diseases have been treated with cyclosporin A with positive resulte, confirming the importance o~ the autoimmune component ~n these diseases and their e~fective treatment with compounde working ~y selective T-cell i~mune suppression ~imilar to c~clo-sporin A.

W092/00278 PCT/US9l/~s4 ~8~8 -2-1) 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).
8ehcet's disease.
Sanders, N. et al., Lancet 454-455 (1983).
Behcet's disease.
Note: Cyclosporin A is currently approved in Japan for the treatment o~ Behcet's disease, the first autoimmune disease indication ~or 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. Dermatol. 123:166-167 (1987). Atopic s~in disease.
Appleboom, T. et al., Amer. 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.
2 :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 red cell aplasia tPRCA).

W092t00278 PCT/US9t/~694 _3- ,Jv8~2~

Stryckmans, P.A. et al., New Enql. J. Med.
3 :6~5-6~6 (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 autoim~une diseases.
Wiesner, R.H. et al., He~atoloav 7:1025, Abst. #9, (1987). Primary biliary cirrhosis.
Hyams, J.S. et al., GastroenteroloqY 93:890-893 (1987). Autoimmune hepatitis.
~llison, M.C. et al., Lancet, 902-903 ~1984).
Crohn's disease.
Brynskov, J. et al., Ga~troenterolooY 92:1330 ~lg87). Crohn's disease.
Porro, G.B. et al., Ital. J. Gastroenterol.
I9:40-41 (1987). Ulcerative colitis.
5) Neurology: Amyotrophic lateral sclerosis (ALS, nLou Gehr~g's disease"), myasthenia gravis and multiple sclerosis.
Appel, S.H. et al., Arch. Neurol. 45:381-386 (1988). ALS.
Tindall, R.S.A. et 1., New Enql. J. Med.
316:719-724 (1987). Myasthenia gravis.
Ann. Neurol. 24, No. 1, p. 169,m Abstract P174 (1988). Multiple sclerosis.
Dommasch, D. et al., Neuroloav 38 Suppl. 2, 28-29 (1988). ~ultiple sclerosis.

W092/002~8 PCT/US9l/04694 '~0~ 4-
6~ Nephrotic Syndrome: Nephrotic 6yndrome, membrano-prol~erative glomerulonephritis (MPGN) and related diseases.
Watzon, A.R. et al., Clin. Ne~hrol. 25:273-274 (1986). Nephrotic syndrome.
Te~ani, A. et al., Kidnev 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 Ri~thoven, A.W. et al., Ann. Rheu~. Dis.
45:726-731 (1986). RA.
Dougados, M. et al., Ann. Rheum. Dis. 47:127-133 (1988). RA.
8) Tnsulin-Dependent Diabetes Méllitus (IDDM) Stiller, C.R. et al., Science 223:1362-1367 (1984). IDDM.
Assan, R. et al., Lancet, 67-71 (1985). IDDM.
Bougneres, P.F. et al., New Enql. J. Med.
318:663-670 (1988). IDDM.
Diabetes 37:1574-1582 (1988).
IDDM.
Many veterinary diseases are also characterized as autoimmune diseases. Autoimmune di~eases such as those listed above have been observed in mammals. Papa, FØ
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 W092t00278 PCT/US91/~694 ~5~ 2 ~ ~ jJ ~ ~48 498-505 (1986) immune mediated diseases of czts and dogs;
George, L.W. and S.L. White, Vet. Clin. Nortr. Amer.
6:203-213 (1984) autoimmune skin diseases in large mammals; Bennett, D., In. Pract. 6:74-86 (1984) auto-immune diseaQes 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 r~.ease of lymphokines, such as interleukin 2 (IL-2) tBunjes, D.
et al., Eur. J. Immunol. 11:657-661 (1981)~ and pr~vents clonal expansion of helper and cytotoxic T cells ~Lar~son, E. J. Immunol. 124:2828-2833 (1980)~. CsA has been ehown to bind the cytosol~c prot-in, cyclophilin, and inhibit the prolyl-pept~dyl cis-trans isomer~se (PPIase) activity of that protein. Fischer, G. et al., Nature 337:476-478 ~1989); Takahashi, N. et al., Nature 337:473-475 ~I989). The PPIasQs may mediate T cell acti-vation by catalyzing the rotomerization of peptide bonds of prolyl residues.
Recently, a second natural product isolated from Stre~tomvces, referred to as FR-506, has bQen 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 ~nd inhibits cytotoxic T-cell generation in ~itro at 100 times lower concentration than cyciosporin A. Rino, T. et al., J. Antibiot. 15:~256_ 1265 ~1987). FK-506 al60 inhibits PPIase acti~ity, but is structurally different ~rom CsA and binds to a binding w092/00278 PCT/US91/~69~

2 ~ 6-protein (F~P) distinct from cyclophilin. ~arding, M.W.
et al., Nature 34l:758-760 ~l989); Siekierka, J.J., Nature 341:755-757 (1989).

Summarv of the lnvention Ihis invention relates to a novel class of immuno-suppresgive compounds having an affinity for the FK-506 binding protein (FK~P). Once bound to this protein, the immunosuppressive compounds inhibit the prolyl peptidyl cis-trans isomerase (rotamaQe) actlvity of the FXBP 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 auto~mmune disease in humans and other mammal~.

Brief DescriPtion of the Flqure Figure l 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-~uppressive compounds represented ~y the formula I:
~I-w092/00278 PCT/US9l/~694 _7~ 2 8 ~nd pharmaceutically acceptable salts thereof, whereln A i5 O, N~, or N-(C~-C4 alkyl);
wherein B is hydrogen, CHL-Ar, tCl-C6)-straight or branched alkyl, (Cl-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl, (CS-C7)-cycloalkenyl or Ar substi-tuted (Cl-C6)-alkyl or alkenyl, or ~l ~
:0,~
~ , wherein ~ and Q ~re independently hydrogen, (Cl-C6)-~traight or branched alkyl or (Cl-C6)-straight or br~nch-d ~lk-nyl;
where~n T is Ar or substituted cyclohexyl with ~ubstituent~ ~t positions 3 ~nd 4 which ~re independently ~elected ~rom the group consi~ting of hydrogen, hydroxyl, O-(C1-C4)-alkyl or O-(C1-C4)-alkenyl and c~rbonyl;
wherein Ar is selected from the group consisting of l-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 2-pyridyl, 3-pyri~yl, 4-pyridyl and phenyl having one to three substituents which are independently selected Srom the group consisting of hydroqen, halo, hydroxyl, nitro, CF3, (Cl-C6)-straight or branched alkyl or (C1-C6)-20 ` straight or branched alkenyl, O-(Cl-C4)-straight or branched alkyl or O-(C1-C4)-str~ight or branched ~lkenyl, O-benzyl, O-phenyl, amino and phenyl;
wherein D i5 either hydrogen or U; E is either oxygen ox CK-U, provided that if D is hydrogen, then E is CH-U or if E is oxygen then D is U;

W092/00278 PCT/US91/~694 ~ ?~ -8-wherein U is hydrogen, O-(C1-C4)-straight or branched alkyl or O-(Cl-C4)-straight or branched alkenyl, ~Cl-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl or (C5-C7) -cyclo-S alkenyl substituted with (C1-C4)-straight or branched alkyl or (Cl-C4)-straight or ~ranched alkenyl, 2-indolyl, 3-indolyl, t(Cl-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 X may be taken together to form a 5-7 membered heterocyclic ring which may contain an oxygen (O), sulfur (S), SO or SO2 substituent therein.
The ~tereochemistry at position 1 (For~ula I) ~s (R) or ~S), with (S) preferred.
The conpounds o~ the present invention can be used in the form of 6alts derived from inorganic or organic acids and bases. Included among such acid ~alts 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 W092t00278 PCT/US91/04694 2 ~ 3 ~ 2 ~

such as calcium and magnesium salts, salt with organic bases such as dicyclohexylamine ~alt~, N-methyl-D-glucamine, and salts with _mino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quaternized with ~uch agents aslower alkyl ha}ides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl ~ulfates 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.
Pre~erably, the compounds w~ll have a molecular weight below about 750 atomic mass units (a.m.u.) and most pre~erably below about 500 a.m.u. Examples o~
compounds in which the J and K substituents are taken together to ~orm a heterocyclic ring are shown in Table~
1 and 2. Examples o~ other preferred compounds o~ this invention are listed in ~ables 3 and 4.

WO 92/00278 pcr/us91/o4694 2~3~d~2~ ' ~L
~ H
~r , ~o. B D nXi Xd 2 Benzyl Phenyl 1 25~1M >5.01~M
3 Benzyl Phenyl 2 1.5~1M ~2.011M
4 Allyl Phenyl 2 8~uM ND
S l-Naphthyl Ph-nyl 2 0.9~.M ND
6 2-Naphthyl Phenyl 2 7.01 M 1.0 ~M
7 Benzyl 2-Metl~ylpropyl 2 O.9~M ND
8 Benzyl 2-Methoxyphenyl 2 171 M >75/uM
9 Benzyl 3-Methoxyphenyl 2 0.3~ M >1.31 M
Benzyl 4-Methoxyphenyl 2 5.0f~M 5.0f~M
11 Benzyl 3,5-Dimethoxyphenyl 2 2.0~uM 0.61 M
12 Benzyl 2,6-Dimethox~phenyl 2 50 I~M 25~-M
13 Benzyl 3,4,5-~rimethoxy-phenyl 2 0.l~ M 2~M
14 Benzyl - 4-Fluorophenyl 2 4.0/uM 10~ N
Benzyl 3-Nitrophenyl 2 16D~M ~751~M
16 Benzyl 4-Nitrophenyl 2 160~M lOOl M
17 Benzyl 2-Pyridyl 2 130fuM ~1OOOIuM
18 Benzyl 2-Pyridyl-N-oxide 2 >500~M lO~lM
19 tert-Butyl 2-Furyl 1 200~uM ~500/~M

W092/00278 PCT/US91/~694 s v ~ 2 8 TAB~E 1 (continued) No. B D n Xi Kd 20 Benzyl 2-Furyl 2 3~M >12~M
21 Benzyl 3-Indolyl 2 25pN 20~M
22 Benzyl 2-Thiophenyl 2 0.8~M 4~M
23 E-3-Phenyl-2-methyl-prop-2-enyl Phenyl 2 1.S~M ND
24 E-3-(4-Hydroxy- Phenyl 2 6~M ND
phenyl)-2-methyl-prop-2-enyl E-3-tc~s-(4- Phenyl 2 0.6~M ND
hydroxycyclo-hexyl)~-2-methyl-prop-2-enyl 26 E-3-ttrans-(4- Phenyl 2 0.5~M ND
Hydroxycyclo-hexyl)]-2-methyl-prop-2-enyl 27 Benzyl 2-Nitrobenzyl 1 26~M >25~M
28 Hydrogen Methoxy 2 ND ND
29 tert-Butyl Methoxy 1 600~M >500~N
Allyl Methoxy 2 l90~M >25~M
31 Benzyl Methoxy 2 80~M ~50~M
32 2-Cyclohexylethyl Methoxy 2 45~M >40~M
33 3-Cyclohexylpropyl Methoxy 2 20~M 12~M
34 4-Cyclohexylbutyl Methoxy 2 6~M 2-3~M

W092~00278 PCT/US91/04694 2 '~ 3 ~

TAB~E 1 (continued) No. B D n Ki Kd 35 -Cyclopentyl- Methoxy 2 35~M ND
propyl 36 E-3-(4-Methoxy- Methoxy 2 40~M >30~M
phenyl)-2-methyl-prop-2-enyl 37 ~-3-(3,4-Dime- Methoxy 2 lO~M ND
thoxyphenyl)-2-methyl-prop-2-enyl 38 E-3-(4-Hydroxy- Methoxy 2 60~M ND
phenyl~-2-methyl-prop-2-enyl 39 E-3-lci~-~4- Methoxy 2 70~M >20~M
Hydroxycyclo-hexyl)]-2-methyl-prop-2-enyl 40 Benzyl Cyclohexyl 2 1.3~M 3~M
41 Benzyl Ethyl 1 400~M >300~M
42 8enzyl 3~Methoxyphenyl 1 5 I~M 80 llM
43 8ereyl 2~Pyridyl 1 300 ILM ND
44 Benzyl 3,4-Dllluorophenyl 2 3 ~enzyl (E)-2-(4-Methoxyphenyl~ 2 1 ethenyl 46 Bereyl l Hydroxy-1-cycbhexyl 2 1~ 2~M
47 Benzyl 2-Naphthjl 2 ~ ~M 0.3 ~M
48 8enzyl 1 Naphthyl 2 1 ~M 2 ~M
~9 (S~ Methylbenzyl Phenyl 2 0.5 IIM 0.6~LM
~0 ~enzyl 2-Hydroxy 2- 2 ~2 llM 0.35 ~M
1etrahydropyr~nyl WO 92/00278 Pcr/us91/o4694 -13- ~ 2 ~

TA8LE 1 (continued) No. B D n Xi Rd 51 tA~-Mothylhnzyl Phenyl 2 1 5 IlM1 llM
52 Benzyl 3 Tr~luoro nethylphonyl 2 15 ~IU 1 3 ~ M
53 Bonzyl 3 Benzyloxyphenyl 2 0 ~ ~M0 2 ~M
54 Benzyl tE~ 2 tert Butylethenyl 2 9 IIM 3 ~uM
Benzyl 2-Trltluoromothylphenyl 2 6 ~M ND
56 4 Cyclohexyl~utyl Phenyl 2 0 4 ~M ND
57 44 ydohexylbutyl 3,4,5 Trimethoxyphonyl 2 0 04 IlM 01 llM
58 4 Phonylb~nzyl Phenyl 2 5 ~IU ND
5~ 4~Ph nylb~n2yl 3,4,5Trim~thoxyphonyl 2 2IlM ND
Benzyl 3 Rhoxyphonyl 2 0~6~M ND
61 3 Ph noxyb nzyl 3,4,5Trlrnothoxyphonyl 2 0018 IlM 0036 tlM
62 3~Phenoxybon2yl Phonyl 2 O O9~M015 I~M
63 4 Phenylbutyl 3,~,5-Trlmothoxyphenyl 2 0 019 ~M 01 ~lM
64 4 Phenylbutyl Phenyl 2 0 35 IlMND
Ben2yl 3 (3-Prop~nyloxy)phenyl 2 1 ~M ND
66 Bonzyl 3-(2-P~opoxy)phenyl 2 0 5 ~ ND
67 Benzyl 1-Me~hylpn~pyl 2 1~1M ND
68 2 Phenylethyl Phonyl 2 11 ~M ND
69 6 PhenylhexYl Phenyl 2 0 5 llM ND
5 Phenylpentyl 3,4,5-Trimethoxyphonyl 2 0 07 ~M ND
7~ 6 Phenylhexyl 3,4,5-Tr~rnethoxyphonyl 2 01 ~M 0 05 ~M
72 wyclohoxylhexyl 3,4,5-Trirnethoxyphenyl 2 0 0~ ~M 0,5 73 4-Phenoxybenzyl 3,~,5 Trlrnethoxyphenyl 2 0 8 ~M ND

WO 92/00278 PCI/US9t/04694 ~a~ 2~

TABLE 1 (cont~nued) No. B D n Xi ~d -74 5-Cl~cbhexylpo~dyl 3,4,5-Trlmethoxyph~nyl 2 0 09 ~IM 0 08 ~IM
Bon~ylS (1-Butoxy)phonyl 2 036pM ND
76 ~-Phenylbutyl 3-(2-Propoxy~phenyl 2 01 ~M ND
77 4-(4-lodophenyl)butyl 3,4,5-Trlmethoxyphenyl 2 D 016 ~lM 0 06 ~M
78 ~ lodober¢yl S,4,5Trim-thoxyphenyl 2 1 4IlM ND
79 2 (2 NapMhyl)ethyl 3,4,5~Trimethoxyphonyl 2 0 22 ~M ND
2 (1 NapMhyl)01hyl 3,4,5 Trhnothoxyphonyl 2 0 5 llM ND
B1 ~ ~nylbu~ 4 bdophonyl 2 O B ~IM 025 ~M
82 4 ~honyuNly~ 3 bdophonyl 2 0 ~3 ~M 02 pM
83 3~hnylp~opyl 3,4,5Trimothoxyphenyl 2 011~M ND
84 3 ~1ndolynp opyl 3,4,5 Trimothoxyphonyl 2 0 017 IIM 0 054 ~lM
4 (4 Mothoxyphenyl)butyl 3,4,5-Trimethoxyphonyl 2 O Ot3 llM 0 049 pM
~6 ~ Phenylbut 2-onyl 3,4,5 Trlmethoxyphonyl 2 O B ~
87 ~-Phonylbut-3~nyl 3,~"5-Trim~hoxyphonyl 2 0 3 ~M ND
8B 4~4 Al oeaminophor~l)prowl 3,4,5-Trimothoxyphonyl 2 0 011 IIM 0 07 ~M
89 4 ~nyipropyl 1-Cyclohoxonyl 2 0 78 ~
4(~11ethoxyphenyl)but3ony1 3,4,5-Trhnothoxyphonyl 2 0011IIM 06011M
~1 4 Phenylpropyt 1-Fluoro 1-eyclohexyl 2 0 84 ~
92 ~Phor~ropyl 3 8utoxyphenyl 2 t 4 ~M ND
93 3 13-(N Fo~ylindoly~lpropyl 3,4,5-Trirne1hoxyphonyl 2 0 01~ IlM 0 06 ~M
94 ~indolyl)butyl 3,4,5 Trirnethoxyphenyl 2 0 01~ ~M 0 05 ~M
4-Phenylbutyl Ben2yl 2 0~5 ILM ND

W0 92/00278 Pcr/us9l/04694 -15- ~, V ~ ~J - s~, TA~Læ 1 (continued) No. 8 D n Xi l~d 96 4-~nylbutyt 3-Biphenyl 2 Q0411M0.033 ~M
9; 4-~henylbutyl 4-tert Butylphenyl 2 0.6 ~LMND
9B 4-Pheny~utyl Cycbhexyl 2 0.08 ~lM 0.1B llM
99 4-Pheny~utyl Cychhexylmethyl 2 0.12 ~tND
100 4 Phenyî~utyl 3,4-Melhylenedioxyphenyl 2 0.25 ~M ND
101 4-Phenyt~utyl 4-T tr hydropyranyl 2 0.~4IIMND
102 4 Phenylbutyl 3-Cycbhexy~-methoxy- 2 14 ~LM ND
phenyl 103 4phenylbutyl 4 ~4 M thoxybenzyloxy 2 0.71~M ND

104 4 phenylbuty~ Butyl 2 0.1B llM ND
105 4phenytbutyl Rhyl 2 ~.6 ~M ND
106 3 (N~Benzhridazolyl)propyl 3.4S-Trimethoxyphenyl 2 o.~ M ND
107 S (N~Pur~propyl 3,4,~-Trimethoxyphenyl 2 0.13 ~M ND
108 ~S.s) 2 Methyl 3~hydroxy 4 J,4,~T~nethoxyphenyl 2 0.~ M ND
phenylPrWI

ND Indicates not d~1em~ined.

W092/00278 PCT/US91/~694 2~ 28 -16-~ABLE 2 ,~
~' No B U n X~ Xd 109 Benzyl 3,4-Methylenedioxyphenyl 1 3pM>lS~M
110 Benzyl 3,4-Methylenedioxyphenyl 2 3pN>4pM
111 B-nzyl 4-Methoxyphenyl 1 6pM>30~M
1~2 B-nzyl 4-M-thoxyphenyl 2 4~M>8~M
113 Benzyl 2,5-D~methoxyph-nyl 1 lO~M ND
114 B-nzyl 2,4,5-Trimethoxyphenyl 1 25~M ND
115 B-nzyl 3,4,5-Trimethoxyphenyl 1 4SOpM >25pM
116 Bénzyl 4-Dimethyl~minophenyl 2 20~M ~5pM
117 Benzyl 4-Nitrophenyl 2 14~M >5pM
118 Benzyl 2-Furyl 2 2 SpM ND
119 Benzyl 3-Furyl 2 2 5pM ND
120 Benzyl 3-Indolyl 2 >60pM >8pM
121 8enzyl 3-Pyridyl 2 25pM ND
122 Benzyl Hydrogen 2 300pM ND
123 Benzyl Phenyl 2 ll~M ND

WOs2/00278 PCT/US91/~6g4 2~g'j~8 J ~

No B D J X Ki Kd 124 Benzyl Methoxy Methyl Hydrogen - lOOO~N ~200~M
125 Benzyl Methoxy Methyl S-Methyl 400~M >200~M
126 Benzyl Methoxy Methyl S-I~opropyl 170yM ~200~M
127 E*hyl Methoxy Benzyl Hydrogen ~1200~M ~300~M
128 tert- Methoxy Ethyl S-Methyl >400~M ~500~M
Butyl K _ ~N~

~U
No B U J X Xi ~d 129 Benzyl 4-Methoxy- Methyl . S-Methyl 80~M >150~M
phenyl 130 Benzyl 4-Methoxy- Heth,yl S-Isopropyl 30~M ~20~M
phenyl 131 Benzyl 3,4-Methylene- Methyl S-Meth~l 50~M ND
dioxyphenyl 132 Benzyl 3,4-Methylene- Hydrogen S-Methyl 60~M ND
dioxyphenyl W092/00278 PCT/US91/~694 2G8 b~ 18-The immunosuppressive compounds of this invention have an affinity for the FK-506 binding protein which is }ocated in the cytosol of lymphocytes, particularly T
lymphocytes. When the immunosuppressive compounds are bound to the FXBP, 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 FXBP.
Compounds of this invention, however, may have an affinity for other FX-506 binding proteins. Inhibition of the prolyl p-ptidyl ci~-tran~ $~omera~e may further be indicative of binding to an FX-506 binding protein.
Human FX-506 binding protein can be obtained as described by Harding, M.W. et al., Nature 341:758-760 ~1989). Values for the apparent Kd can be determined from a competitive LH-20 binding assay performed as described by Harding et al., using 32-tl-14C~-benzoyl FX-506 as a reporting ligand; or using t3H~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 FXBP are reported in Tables 1-4. The data was obtained using ~e latter method, where the ability of an unlabeled compound to compete with the binding of ~3H~dihydro-FR-506 to FX-506 binding protein was measured.
The inhibition of the PPIase trotamase) enzyme activity of the FXBP (apparent "Ki" values) can also be measured according to the methods described by either W092/00278 PCT/US91/~694 h ~ 3 'J `~ ~ ~

~arding, M.W. et al., Nature 341:758-760 (1989) or SiekierXa, J.J. et al., Nature 341:7SS-757 (1989). The ci~-trans isomerization o~ the proline-alanine peptide bond in a model su~strate, N-~ucc~nyl-Ala-Ala-Pro-Phe-p-nitroanilide, is monitored spectrophotometrically in acoupled ~ssay 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 e~fect of the addition of different concentrations of 1~ lnhibitor 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 o~ the ~pparent Xi value. The extent o~ enzyme inhibition (Ki) o~ some preSerred compounds i~ shown in lS Tables 1-4.
The compounds of the present invention can be ~urther characterized in cellular biological experiments in vitro where their re iblance in function and use to cyclosporin A and to FK- j6 is apparent. (See Tables 5 and 6) 2~; 4~ -20-Assays and IC50Cyclosporln Value ~or Druas A RaPamvcin FK-506 1) Human PB~ I <l~g/ml <'~g/ml <l~g/ml 2) T-Cell Hybridoma <l~g/ml <l~g/ml <l~g/ml + TCR/CD2 3) Apoptosis Blocks Inactive Blocks at at at l~g/ml l~g/ml l~g/ml 4) CTL~ Proll~era~ g/ml ~O.O~g/ml ~ g/ml tion ~ IL-2 WO 92/00278 PCr/US91/04694 - 21~ ?

TABLE 6: C~llular A~s-y Re~ults No. PMA (~M) OKT3 (~M) LB (~M) JVM (~M) CTLL(~M) 3 7.6 4.6 ~10 ~10 ~8.5 ~10 ~10 ~10 ~10 ~10 7 ~10 a10 ~10 ~10 ~10 ~0 6.5 ~10 ~10 ~10 ND ND ND ND ND

13 ~10 5.9 ~10 ~10 ~10 ND ND ND ND ND
16 ~10 ~10 ~10 ~10 ~10 ND ND ND ND ND

WO 92/00278 PCr/US91/04694 2~8~

~ABLE 6 (Corlt. ) 22 ~10 ~10 ~10 ~10~10 23 ~10 ~10 ~10 ~10~10 ~10 ~10 ~10 ~10~10 ~10 ~10 ~10 >10~10 26 ~10 6.5 ~10 ~0~10 ND ND ND ND ND

32 ND .ND ND ND ND

3~ ND ND ND ND ND

7.0 1.0 ~10 ~10~10 WO g2/00278 PCrtUS91/04694 s~

TABLE 6 (Cont. ) ~10 ~10 ~10 ~10 ~10 46 ~10 ~10 ~10 ~10 ~10 47 ~10 ~10 ~10 ~10 ~10 48 ~10 ~10 ~10 >10 ~10 49 ~10 6.2 ~10 ~10 ~10 52 ~10 ~10 ~10 ~0 ~10 53 ~10 8;0 ~10 ~10 8.0 ND ND ND ND ND
56 s10 ~10 ~10 6.5 5.0 57 4.0 4.5 ~10 8.0 6.0 ~8 ND ND ND ND ND
~9 ND ND ND ND ND
~10 ~10 ~10 ~10 ~10 W O 92/00278 PC~r/US91/04694 2 a ~ ~J ~'~ 8 -24-TABLE 6 (Cont.) 61 4.0 8.0 >10 >10 3.2 62 6.~ >10 >10 >10 >10 63 6.0 3.1 1 0 8.5 3.8 64 1 0 6.0 >10 >10 >10 66 >10 ~10 ~10 ~10 ~1 0 66 ~10 ~10 ~10 ~10 ~10 67 >10 ~10 ~10 >10 >10 68 >10 ~10 >1O >10 ~1 0 69 6.1 >10 ~10 ~10 >10 7.0 4.5 >10 9.0 4.2 71 6.0 6,6 7.~ 8.0 3.8 ~2 9.0 >10 ~10 >~0 ~.~
7 3 8.0 >10 4.~ 6.0 7.0 74 8.0 9,0 1 0 10 ~,o 7~ 8.0 ~10 9.0 '10 4.5 ?6 ~.0 1 0 9.0 >1 0 6.0 77 4.6 8.0 6.0 7.0 2.1 7 8 ~10 >10 ~1 0 ~10 >10 79 1 0 2.6 >10 >10 8.0 3.0 4.0 1 0 1 0 6.0 Pcr/usgl /04694 -25- 2 ~ g ~ ~ ~ 8 ~ABLE 6 (Cont.) 81 7.0 ~10 >10 ~10 ~10 82 1 0 ~10 ~10 ~10 ~10 83 5.~ 5.5 8.5 ~ .0 84 4.5 6.0 6.0 ~10 2.0 8~ 4.5 4.5 7.0 1 0 1.~
86 9.0 ~10 ~1 0 ~10 ~10 87 7.0 ~10 ~10 ~10 ~10 88 2.2 2.2 2.~ 4.~ 4.0 89 8.0 ~10 ~10 ~10 ~10 8.0 ~10 ~10 ~0 7.0 91 :~10 ~10 ~`10 ~10 ~10 92 9.0 ~10 ~10 ~10 ~10 93 6.0 7.0 1 0 8.7 3.7 94 5.0 5.5 ~10 7.0 4.0 9~ ~10 ~10 ~10 ~10 ~10 96 ~10 ~10 ~10 ~1 0 ~10 97 ~10 ~1 0 ~10 ~10 6.0 98 ~10 ~1 0 ~10 ~10 ~tO
99 ~10 ~10 ~10 ~10 ~10 100 ~10 ~10 ~10 ~10 ~10 101 ~10 ~10 ~10 ~10 ~10 1 02 ~10 ~10 1 0 10 ~1 0 WO 92/00278 PCI~/US91/04694 2 ,~ 2 ~

IABLE 6 ( Cont . ) 103 7.0 10 10 ~10 10 tO4 4.0 ~10 ~10 ~10 ~10 tO5 ~tO ~10 ~tO ~tO ~10 106 7.0 ~10 ~tO ~10 3.0 107 6.5 ~10 ~10 ~10 ~10 108 ~10 ~10 8.0 ~10 ~10 1t2 ND ND ND ND ND

11~ ND ND ND ND ND

t21 ND ND ND ND ND

WO g2/00278 PCl`/US91/04694 -27- 2~f3d2~3 TABLE 6 (Con. ) All o~ the compounds In Table 6 show~d toxicity at hi~her concentrations than ~ 1eir immunosuppresive aotivi'qr and were ~pically concentrations ~ 10 llM.
PMA and OKT3 - mitogens used to stimulate proliferation of human peripheral blood b,~mphocytes (PBC). Compounds are evaluated on their ability to inhibit proliferation.
LB and JVM - human viral transfo~ed B tymphoblastoid cell lines sffmulated to prolifsra1e in a mixed Iymphocyte reaction (MLR). The compounds are evaluated on their ability to inhibit this proliferation.
CTLJ_ - inhibition of proliferation of cytotoxic T c~lls stimulated by IL-2.

W092/002~8 PCT/US91/~694 '~S ~ 28-1) Assay ~imilar to Yoshimura, N. et al., Trans-plantation 47:356-359 (1989). Assay uses fresh human peripheral blood lymphocyt~s isolated by Ficoll-Hypaque density centrifugation, stimulated by the OKT3 antibody tanti-CD3) which stimulates via interaction with CD3.
Stimulation is measured by incorporation of radioactive thymidine t(3H)TdR] into proliferating cells, with an uninhibited control signal of 48,000-75,000 cpm. IC50 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 t(3H)TdR~ into pro-li~erating cells, with an uninhiblted control signal of23,000 cpm. IC50 values are estimated ~rom inhibitions of proliferation observed at various drug concentrations.
3) Assay according to 8hi , 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 FX-506-like mechanism of action. Note that the chemically related, but mechanistically distinct, im~uno-suppressant rapamycin is inactive in this assay.

W092/00278 PCT/US9l/04694 -29- hv3 ù~i~8 4) Assay according to DuMont, F. et al., J.
Immunol. 144:251_258 (1990). The assay ~easures the ~timulation of CTLL cells in response to IL-2. Prolifer-ation is measured by incorpoation of ~3H)TdR. Immuno-S suppressants which work by a ~imilar mechanism to cyclo-sporin A and FX-506 will not in~ibit 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-suppressant, rapamycin, is active in this assay.
These assays can be used to profile the cellular activity o~ the compounds of the present invention.
ThUB, it i~ clear from these results that the compounds resemble both cyclosporin A and FK-506 in its cellular activity, including immunosuppression, in contrast to the mechanist~cally dissimilar immunosuppressant agent rapamycin. Furthermore, the observed cellular activity is consistent guantitatively 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 ~an 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 WO 92/00278 PCr/US91/04694 2~

various autoimmune diseases. The compounds of this invention can also be administered to ma als other than humans for treatment of ~arious 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 re;ection, 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 disea~e~ 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, amyotrophic lateral sclerosis, myasthenia gravis, multiple sclerosis, nephrotic syndrome, membranoproliferative glomerulonephrit~s, 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 ~nd transition to complete dependence on external insulin.
For these purposes the compounds of the present invention may be administered orally, parenterally, by -31- ~ ~ 3 ~

inhalation #pray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir in dosage formu-lations containing conventional non-toxic pharma-ceutically-acceptable carriers, adjuvant~ 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 ~terile in~ectable sulution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
Among the accQptable vehicles and solvents that may be employed are water, Ringer's solut~on and isotonic ~odium chloride solution. In addition, ~terile, fixed oil~ are conventionally employed as a solvent or ~uspending 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 injecta~les, 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 ~s Ph. Helv or similar alcohol.
The compounds may be administered orally, in the form of capsules or tablets, for example, or as an ~ 32-aqueous suspension or solution. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, ~uch as magnesium stearate, are also typically added. For oral S administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous ~uspensions 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 co~pounds 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 ~uitable non-irritat-~n~ excipient which is ~olid at room temperature but~iguid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, bee~wax 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 i~otonic, pH adjusted ~terile saline, either with or without a W092/00278 PCT/US9l/04694 -33- ~ 2 3 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 ~kin, 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 pe~rolatum, 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, ~orbitan monostearate, polysorbate 60, cstyl e~ters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and wàter.
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 ~g/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 ~ingle dosage form will v~ry depending upon the host treated and the particular ~ode of administration.
It ~s 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 w092/00278 PCT/US91/~694 2 ~ 34 of excretion, drug combination and the severity of the particular dicease being treated.
The compound can also be administered in combination with a ~teroid, such as methyl prednisalone ~cetate, for additional immunosuppressive effect. The steroid is administered orally, intravenously, rectally, topically or by inhalation. Dosages (based upon ~ethyl pre-dnisalone acetate) of 0.1-5 mg/Xg/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-~uppres3ant drugs, such ag rapamycin, azathiopr~ne, 15-deoxyspergualin, cyclosporin, FX-506 or combinations o2 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 ~y way of the following examples, which are not intended to be limiting in any way.

~ 3 ~ 8 EXANPLES

General Proton nuclear magnetic resonance (lH NMR) spectra were recorded at 300 MHz on a Bruker AC 300 or at 500 NHz S on a Bruker AMX 500. Chemical shifts for proton resonances are reported in parts per million (~) relative to Me4Si (~ 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 Assoc~ates 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~ '3P4/~20; B ' 0-1% H3PO4/CH3CN. A linear gradient ~ 95~ A/5t B to 100% B ov r 15 minut-s followed by 1.5 minutes at 100% B was used, with detection at 214 nM.
The compounds described below are illustrated in Figure 1.
EXAMP~E 1 Svnthesis of (E)-3-rcis-4-(hvdroxvcYclohexvl)l-2-methvlProp-2-enyl N- (Phenylqlyoxyl) -Pipecolate (25).

1. (S)-BenzYl PiPecolate (133) To a ~lurry of 7.3 g (26.14 mmol) of the tartrate salt of (S)-pipecolic acid (Eg~ertson M. and S.J.
Danishefsky, J. Orq. Chem. 54:11 (1989) in 75 m~ of dry benzene was added 13.5 mL (0.13 mol) of benzyl _lcohol and 5.48 g (28.8 mmol) of p-toluenesulfonic acid monohy-drate. The reaction mixture was heated at re1ux W092/00278 PCT/US91/~694 ~V~ f~ -36-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 ~tirred overnight at 4-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). lH NMR
t300 MHz, D20) ~ 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 MgS04 and evaporated to yield (S)-B~nzyl pipecolate (133) as a pale yellow oil.

2. (S)-N-tPhenYlqlvoxYl)~iPecolic 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 0C 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 0C 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 ~L of methylene chloride containing a catalytic amount of dimethylformamide. The reaction mixture was stirred at 25C overnight at which time the mixture was poured into -37- `~i~ 3 -J '~ 2 8 1.0 N HCl. The agueous layer was discarded and the organic l~yer was washed twice with saturated sodium bicar~onate. The combined agueous layers were wa~hed with methylene chloride, acidified to pH 2.0 with con-centratQd HCl, and then extracted repeatedly with ether.
Fla~h chromatography (Still, W.C. et al., J. Orq. Chem.
43.2923 (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. lH NMR (500 MHz, CDC13) ~ 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), ~.68-1.38 (m).

3. cis-and trans-4-~tert-~ut~ldimethYlsilYloxy) cYclohexane-1-ol (136) and (137) To a solution of 3.43 g (21.7 mmol) of ~i~- 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 0C was added 3.0 mL
(25.0 mmol) of 2,6-lutidine followed by 5.5 m~ (23.8) mmol of tert-butyldimethylsilyl trifluoromethane-sulfonate. The ice bath was removed and the reaction mixture was allowed to stir at 25C for 2 h at which time the solution was poured into saturated ~odium bicarbonate. The layers were partitioned and the organic layer was washed with saturated copper sul~ate and water and then dried over MgS04 to give 5.9 g of the crude méthyl 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 W092/00278 PCT/US9t/04694 2 a ~

mixture was stirred at 25C for 0.5 h and was then guenched 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 S twice with ethyl acetate. T~e combined organic extracts were dried over MgS04 and concentrated to give 4.9 g of the diastereomeric alcohols. Flash chromatography (elution with 1:5 ethyl acetate-hexane) gave 6~0 mg of (136), 1.10 g of tl37) and 2.40 g of a mixture of the two. Data for (136): lH NMR (300 MHz, CDC13) ~ 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): 1H NMR (300 MHz, CDCl3) ~ 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).
5 4. (E)-Eth~l 3-~cis-(4-tert-Butvlaimethvlsilvl-oxvcYclohexYl)1-2-methYl~ro~-2-enoate ~138) To a -78C solution of oxalyl chloride (465 yL, 5.33 mmol) in 5.0 mL of methylene chloride was added dimethyl-sulfoxide (755 ~L, 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 -78C for 45 min at which time 2.2 mL (16.0 ~mol) 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 MgS04 and evaporated to dryness to give 620 mg of the intermediate aldehyde which was treated WO 92/00278 PCI/llS91tO4694 ~33~i~2g directly with 1.22 g (3.36 mmol) of (carbethoxyethyl-idine)triphenylpho~phorane in 5.0 m~ 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 MgS04 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-ButYldimethvlsilvloxYcY
hexYl)1-2-methYl~ro~-2-en-1-ol (139) To a solution o~ 300 mg ~0.95 mmol) o~ enoate (138) in ~.o m~ of anhydrous tetrahydrofuran at 25'C was added 18 mg (0.43 mmol) o~ 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 wa~ extracted with two portions of ethyl acetate. The combined organic extracts were washed with water and brine and then dried over MgS04. Evaporation and flash chromatography (elution with 1:10 ethyl acetate-hexane) gave 220 mg of the allyic alcohol (139)~ lH NMR (300 MHz, CDC13) ~ 5.34 (d), 3.96 (d), 3.85 (m~, 2.26-2.18 (m), 1.64 (d), 1.61-1.34 (m), 1.82 (s), O.o (s).

W092/00278 PCT/US91/~694 2~ q~ 40-6. (E)-3-rcis-(4-tert-ButyldimethvlsilYloxYcYclo-hexvl)l-2-methvlProP-2-enyl N-(Phenvl~lyoxYl)-PiPecolate ~140) To a solution of 68 mg (0.24 mmol) of allylic alcohol (139), 42.3 mg ~0.~6 mmol) of acid ~135) and 39.8 mg ~0.20 mmol) of 1-~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 o~er MgS04 and concentrated to yield a yellow oil.
lS Flash chromatography ~elùtion with 15% ethyl acetate in hexane) gave 9.2 mg of the ester (140) as a rotameric mixture. lH NMR (500 MHz, CDC13) ~ 8.01 (d), 7.94 (d), 7.59-7.S2 (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-(hYdroxvcvclohexYl)1-2-methvl~ro~-2-en~l N-(PhenYlqlYoxYl)-PiPecolate (25) To a solution of 9.2 mg (0.02 mmol) of ester (140) in 1.0 mL of acetonitrile at 25C was added dropwise a ~olution consisting of a 95:5 mixture of acetonitrile:
48% hydrofluoric acid and the reaction was stirred until *hin layer chromatography (TLC) indicated the dis-appearance of starting material. The reaction was W092/00278 PCT/US91/~6~4 -41- 2~8~i~2~

quenched by the addition of saturated potassium car-bonate. The reaction mixture was extracted with three portions of ethyl acetate, dried over MgS04 and concen-trated. Flash chromatography ~elution with 35% ethyl acetate in hexane) yielded 6.1 mq (82%) of the ester (2S) ~s a rotameric mixture. lH NMR t500 MHz, CDC13) ~ 8.02 (dd), 7.97 (dd), 7.67-7.59 (m), 7.56-7.48 (m), 5.49 (d), 5.44 (d), 4.61 (ABg), 4.44 (s), 4.41 (d), 3.98-3.91 (m), 3.S (br d), 3.26 (ddd), 3.11 (ddd), 2.43-2.18 (m), 1.86-1.37 (m), 1.72 (d), Rf 0.57 (3:1 ethyl acetate-hexane).

Svnthesis of (S)-BenzYl N-(~henYlqlYoxvl)~i~ecolate (3) To a solut~on of 43 mg (0.19 mmol) of freshly generated (S)-Benzyl pipecolate (133) (described in Example 1) in 2.0 m~ of anhydrous methylene chloride was added 44 mg (0.29 mmol) of benzoylformic acid and 56 mg (0.29 mmol) of 1-(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 ~queous layer was extracted twice with methylene chloride. The combined organic extracts were dried over MgS04 and concentrated to yield a yell~w oil.
Flas~ chromatography (elution with 1:3 ether-hexane) gave 49 mg (72%) of the keto-amide (3) as a rotameric ~ixture.
lH NMR (500 NHz, C~C13) ~ 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), Rf O.S5 (1:1 ether-hexane).

W092/00278 PCT/US91/~694 Svnthesis of (S)-Benzvl N-t(3-methoxv~henyl)alvoxvl) pi~ecolate (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 (~981)) as described in Example 2. Flash chromatography (elution with ~:4 ether-hexane) gave 73 mg (93%) of (9) as a rotameric mixture. lH NMR (300 MHz, CDC13) ~
7.59-7.10 (m), 5.42 (d), 5.23 (ABg), 5.09 (ABq), 4.59 ~dd), 4.38 (d), 3.82 (s), 3.81 (5), 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), Rf 0.45 (1:1 ether-hexane).

SYnthesis of (S)-Benzvl N-(2-furvlqlvoxvl1Pi~ecolate (20) ~o a 0C 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 ~L (1.14 mmol) of diisopropylethylamine, 174 mg (1.24 mmol) of ~-oxo-2-furanacetic acid, 579 mg (1.24 mmol) of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and then 216 ~L (1.24 ~mol) of diisopropylethylamine.
The resulting reaction mixture was stirred at ~mbient temperature for 14 h and then evaporated to dryness. The residue was dissolved into 150 mL of ethyl acetate, washed seguentially with 50 mL of 0.5 N HCl, 50 mL of saturated NaHCO3, 50 mL of brine and was then dried over W092/00278 PCT/US~l/~694 ~43~ 2~g~3-12~

MgS04 and concentrated. Flash chromatography (elution with 2~ ether in methylene chloride) provided 163 mg (46~) of the keto-amide (20) as an oil. The ~ NMR
~pectrum of this compound (300 MHz, CDC13) was consistent with the product as a mixture of rotamers. Rf 0.2 (2%
ether ln methylene chloride). ~PLC, Rt=12.83 m~n.

SYnthesis of (S)-Benzvl N-(4-Methoxvcinnamovl)~ipecolate (112) ~o a soiution of 145 mg (0.37 mmol) of (S)-Benzyl pipecolate salt (134) (described in Example 1) in 8.0 mL
o~ methylene chloride was added 102 mg tO.57 m~ol) o~
4-methoxycinnamic acid, 107 mg (0.55 mmol) of 1-t3-di-methylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 130 ~L (0.74 mmol) of diisopropylethylamine.
The resulting ~olution was ~tirred 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 colorless oil. lH NMR (300 MHz, CDC13) ~ 7.66 (d), 7.50 (d), 7.3S tm), 6.90 td), 6.82 td), 6.63 (d), 5.55 (d), 5.20 tbr ~), 4.86 (br s), 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). Rf 0.40 (1:1 ether-hexane~.

w092/00278 PCT/US91/~694 _44_ Svnthesis of (S)-3-(3~4-MethvlenedioxYphenvl)-proP-2-enoYlproline benzvl ester (109) and ~5)-3-(3,4-Methvlene-dioxYDhenYl)-~roD-2-enoYlalanine benzYl ester (132) A ~olution of 192 mg (1.0 mmol) of 3,4-methylene-dioxycinnamic acid, 121 ~g (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 ~acuo. The residue was dissolved in 10 mL of dichloromethane and poured into 3 ~olumes o~ diethyl ether. The mixture was washed sequentially with water, lOS potassium bisulfate colution, water, ~aturated 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 ~ilica gel colu~n chromatography, using a stepwise gradient of 30%, 35%, 40%, and 4S% ethyl acetate in hexane as eluant. Ester (109) (142 mg) was obtained as a colorless oil, Rf ~ 0.4 (40% ethyl acetate/hexane);
HPLC, Rt = 12.26; lH NMR (300 MHz) consistent with structure. Ester (132) (162 mg) was obtained as a colorle~s oil; TLC, Rf ~ 0.22 (40% ethyl acetate/
hexane); HPLC, Rt ~ 11.84 min; lH NMR (300 MHz) consistent with structure.

W092/00278 PCT/US91/~694 ~45~ 2u~; ~2~

SYnthesis of (S)-N-3-(4-methoxvPhenYl)-ProD-2-enovl-N-methvlalanine benzYl ester (129) ~. ~S)-N-MethYlalanine benzYl ester ~-toluene sulfonic acid R~lt (141) A suspension of 1.55 g (15.0 mmol) of (S)-N-methyl-alanine and 9.31 (9o.0 mmol) o~ benzyl alcohol in 30 mL
of toluene was treated with 3.00 g (15.8 mmol) of p-toluenesulfonic 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 lnto 200 mL of ether, precipitating a yellow o$1. The solv-nts were decantQd and the residue wa6 taken up into ethyl acetate and concentrated to yield a viscous, light yellow oil (141); T~C: R~ - 0.34, 95:5:0.5 CH2C12/MeO~/concentrat-d ammonium hydroxide; 1H NMR (300 MHz) consistent with structure.

2. (S)-N-3-(4-methoxvDhenYl)-~ro~-2-enovl-N-methyl-alanine benzYl ester (129) A suspension of 96 mg (0.5 mmol) of 4-methoxy-cinnamic acid ~nd 121 mg (0.5 mmol) of (141) in 6 mL of dichloromethane was cooled in an ice/water bath under nitrogen. ~he 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 seguentially with water, 10 W092/00278 PCT/US91/~694 2 ~
potassium bisulfate solution, water, ~aturated 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, Rf - 0.24 (35% ethyl acetate/hexane); HPLC, Rt ~ 13.34 min; lH NMR (300 MHz) consistent with structure.

Svnthesis of ~S)-N-MethYloxalvl-N-ethylalanine benzYl ester (127) . ~s)-N-9-FluorenvlmethoxYcarbonYl-N-ethYlalanine benzYlester ~142) 15A suspension of 848 mg (2.5 mmol) of (S)-N-Fmoc-N-ethylalanine in 10 mL of dichloromethane was treated with 436 ~L (5.0 mmol) of oxalyl chloride followed by a catalytic ~mount (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 80C oil bath with vigorous stirring for 20 minutes, then cooled and filtered through a pad of diatomaceous earth. The filtrate was concen-trated, and the residue was purified by ~ilica gel column chromatography, using 10% acetone in hexane as eluant.
Ester (142) (810 mg) was obtained as a colorless oil;
TLC: Rf = 0.28, 15% acetone/hexane; lH NMR (300 MHz) con-sistent with structure.

W092/00278 PCT/US91/~694 _47_ ~3s~

2. (S)-N-Methvloxalvl-N-ethYlalanine benzvl ester (127) A solution of 0.25 g (0.S8 mmol) of (142) in 3 mL of acetonitrile was treated with 3 mL of diethylamine and the ~ixture was allowed to ~tand for 20 min. The mixture S 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 lll ~L (0.64 mmol) of diiso-propylethylamine followed, during approximately 1 min.with 54 ~L (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 seguentially with water, 10%
pota~sium biaul~ate solution, wat~r, saturated sodium b~carbonate solution, water, and saturated sodium chloride solution. The organic layer was dried o~er magnesium sul~ate, ~iltered, and concentrated ~n vacuo.
The residue was purified by silica gel column chromato-graphy using 1:7 acetone: hexane as eluant. Ester (127) (147 mg) was obtained as a colorless oil, Rf - 0.36, 35%
acetone/hexane; HPLC, Rt ' 12.19 min; lH NMR (300 MHz) consistent with struct~re.

~XA~LE 9 Svnthesis of tS)-3-cyclopentvlpropvl N-(2-MethvloxalYl)-piPecolate (135) 1. (S)-N-(MethYloxalYl)Pipecolic Acid (143) The acid (143) was prepared from methyl oxalyl chloride as described in Example 1 for the production of W092/00278 PCT/US9l/~694 ~Id~&4~ -48-(S)-N-(Phenylglyoxyl)pipecolic acid (135). Thus, 3.16 g (11.32 ~mol) o~ the tartrate salt of (S)-pipecolic acid and 1.19 mL (12.45 mmol) of methyl oxalyl chloride gave 1.25 g (Sl%) of the acid (143) as a tan solid. lH NMR
(300 MHz, CDC13) ~ 5.31 (d), 4.62 (d), 4.49 (br d), 3.61 (br d), 3.90 (s), 3.88 (s), 3.46 (dt), 2.97 (dt), 2.40-1.40 (m).

2. ~5)-3-CvcloPentvlProPvl N-(2-Methvloxalvl)PiPecolate (3s) The ester (35) was prepared from 3-cyclopentyl~
propan-1-ol and the acid (143) as described in Example 2.
Flash chromatography (elution with 2% ether in methylene chloride) gave 72 mg (48%) of ~35) as a colorless oil.
The lH NMR spectrum of this compound (300 M~z, CDC13) was consi~tent with the product as rotamers. Rf 0.56 (10%
ether in methylene chloride). HPLC, Rt ' 14.30 min.

DISCUSSION OF ASSAYS
:
Cell Source and Culture 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 TI8214, JUgXAT CLONE
E6-1 ATCC TIB152). The human allogeneic B cell lines used for aotivation of the fresh PBLs are EBV-transformed lymphocytes from normal healthy adult donors with two -49- ~,'j~ ,;~

completely different HIA haplotypes. All cell lines were routinely tested for the presence of Mvco~lasma contami-nation using the Gibco Nycotect test ~it ~nd are MYco~lasma-free. Culture medium consists of RPMI 1640 (Gibco, Grand Island, NY) containing penicillin (50 U/ml) and ~treptomycin (50 ~g/ml), L-glutamine 2 mM, 2 mer-captoethanol (5 x 10 5), 10% heat-inactivated FCS and 10 mM HEPES.

Compound Solutions and Titrations All che~i~al stocks were dissolved in DMS0.
Titrations 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, uslng multiple three-fold dilutions from 1 ~M or 10 ~M stock solutions.

MTT Assav The MTT assay is a colorimetric technique to deter-mine 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 dif~erent concentrations of test compounds in serum-free medium (B 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 assay culture period, 20 ~l of MTT dye (5 mg/ml in p~ 7.2 PBS) were added to earh microtiter well. At the end of the incubation time, most of the cult~re media was carefully aspirated out of each W092/00278 PCT/US9l/04694 23.),~ 12') ~o well. Then 100 ~1 of acidified isopropyl alcohol (0.04 N
~Cl) was added to solubil~ze the dye and optical density is read at 570 D minus OD at 630 nm (Molecu}ar Devices Thermomax plate reader ~nd Softmax software program, Menlo Park, CA). Results were compared w~th mean OD in controls (medium with no drugs) and doses causing 50%
toxicity (TC50) were calculated.

Mitoqenesis Assavs ("PMA" and "OKT3") The inhibitory effect of test compounds on the proliferation of human PBLs in response to mitogens (Waithe, W.K. and X. Hirschhorn, Handbook of ExDerimenta}
ImmunoloqY, 3d Ed. Blackwell Scientific Publications, Oxford (1978); Mishell, B.B. and S.M. Shiigi, Selected Methods in Cellular Immunoloqv W.~. Freeman and Co., San Francisco, CA ~1980)) was assessed by stimulation of 5 x 104 cells with OXT3 (10 4 dilution final) or PMA
(lOng/ml) plus ionomycin (250 nglml) in the presence or absence of dif~erent concentrations of test compounds and control drugs (CsA, FK506, Pagamycin) in final volume of 200 ~l per well in 96 well round bottomed plates. After 48 h incubation ~37C, 5% CO2), cells were pulsed with 1 ~Ci of 3H-thymidine, harvested 24 h later with a Tom Tek cell harvester, and counted in LXB ~-scintillation counter. Results (cpm) were compared with controls with medium alone, and concentrations causing 50% reduction in counts ~IC50) were calculated~

MIR Bioassa~s ("LB" and "JVM") Antigen activated proliferation of PBLs in a primary mixed lymphocyte reaction was assessed in the presence or W092/00278 PCT/US91/~694 -51- ~J;~J~

ab~ence oS different concentrations of tested compounds and control drugs. 5 x 104 fresh PBLs were stimulated with S x 10 of Mitomycin C treated-allogeneic EBV-transformed p-lymphoglastoid cells, LB and JVM, in a final volume of 200 ~1 per well in 96-well round-bottomed plates (Mishell, B.B. and S.M. Shiigi, Selected Methods in Cellular Immunoloqv W.H. Freeman and Co., SAn Francisco, CA (1980); Nelson, P.A. et al., Trans-plantation 50:286 (1990)). Cultures were pulsed on day 6, harvested 24 h later and counted as in previous section.

_L-2 MicroassaY ("CTLL") To determine i~ test compounds inhibit the later T
¢ell activation proces~ of cytoklne utilizat~on, the proliferative response of the lL-2 dependent CTLL-20 murine T cell line (ATCC) was ~ssessed (Gillis, S. et al., J. Immunoloov 20:2027 (1978)). CsA and FK506 inhibit the production of IL-2 by activated T cells, 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. Immunoloqv 44:251 (1990)). 3 x 103 CTLLs were exposed to different concentrations of test compounds and control drugs in the presence of 1 U/m of human recombinant IL-2 (Genzyme, rIL-2) for 24 h. Fo~r h after adding drugs, cells were pulsed with 1 ~CI of 3H-thymidine, incubated for an additional 20 h ~37C, 5%
C02), ~nd then harvested and counted as pre~iously described.

Claims (31)

1. An immunosuppressant compound having an affinity for FR-506 binding protein and a molecular weight below about 750 amu.
2. An immunosuppressant compound of Claim 1, capable of inhibiting the prolyl peptidyl cis-trans isomerase activity of the FK-506 binding protein.
3. An immunosuppressant compound of Claim 1, having a molecular weight below about 500 amu.
4. A compound having immunosuppressive activity, represented by the formula:

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 substituted (C1-C6)-alkyl or alkenyl, or , wherein L and Q are independently hydrogen, (C1-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl;
wherein T is Ar or substituted cyclohexyl with substituents at positions 3 and 4 which are inde-pendently selected from the group consisting of hydrogen, hydroxyl, O-(C1-C4)-alkyl or O-(C1-C4)-alkenyl and carbonyl;
wherein Ar is selected from the group consis-ting of 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl and phenyl having one to three substituents which are inde-pendently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, CF3, (C1-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl, O-(C1-C4)-straight or branched alkyl or O-(C1-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, O-(C1-C4)-straight or branched alkyl or O-(C1-C4)-straight or branched alkenyl, (C1-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl or (C5-C7)-cycloalkenyl substituted with (C1-C4)-straight or branched alkyl (C1-C4)-straight or branched or alkenyl, 2-indolyl, 3-indolyl, [C1-C4)-alkyl or (C1-C4)-alkenyl]-Ar or Ar;
wherein J is hydrogen or C1 or C2 alkyl; K is (C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl; or wherein J and K may be taken together to form a 5-7 membered heterocyclic ring which may contain an O, S, SO or SO2 substituent therein, and wherein the stereochemistry at carbon position 1 is R or S.
5. An immunosuppressant compound of Claim 4, having an affinity for FK-506 binding protein.
6. An immunosuppressant compound of Claim 4, capable of inhibiting the prolyl peptidyl cis-trans isomerase activity of the FK-506 binding protein.
7. An immunosuppressant compound of Claim 4, having a molecular weight below about 750 amu.
8. An immunosuppressant compound of Claim 7, having a molecular weight below about 500 amu.
9. An immunosuppressant compound of Claim 4, wherein the stereochemistry at carbon position 1 is S.
10. An immunosuppressant compound of Claim 4, wherein J
and K are taken together and is represented by the formula:

wherein n is 1 or 2.
11. An immunosuppressant compound of Claim 10, wherein B
is selected from the group consisting of benzyl, alkyl, naphthyl, tert-butyl, hydrogen, E-3-phenyl-2-methyl-prop-2-enyl, E-3-(4-hydroxyphenyl)-2-methyl-prop-2-enyl, E-3-(4-hydroxycyclohexyl)-2-methyl-prop-2-enyl, cyclohoxylethyl, cyclohexyl-propyl, S-sec-phenethyl, cyclohexylbutyl, cyclo-pentylpropyl, E-3-(4-methoxyphenyl)-2-methyl-prop-2-enyl, E-3-(3,4-dimethoxyphenyl)-2-methyl-prop-2-enyl and E-3-[cis]-(4-hydroxycyclohexyl)]-2-methyl-prop-2- enyl; and D is selected from the group consisting of phenyl, methoxyphenyl, cyclohexyl, ethyl, methoxy, nitr?oenzyl, thiophenyl, indolyl, furyl, pyridyl, pyridyl-N-oxide, nitrophenyl, fluorophenyl, trimsthoxyphenyl and dimethoxyphenyl.
12. An immunosuppressant compound of Claim 4, wherein J
and X are taken together and is represented by the formula:

wherein n is 1 or 2.
13. An immunosuppressant compound of Claim 12, wherein U
is selected from the group consisting of methoxy-phenyl, hydrogen, dimethoxyphenyl, trimethoxyphenyl, dimethylaminophenyl, nitrophenyl, furyl, indolyl, pyridyl and methylenedioxyphenyl.
14. A compound having immunosuppressive activity repre-sented by any of the structures shown in Figure 1 and having an affinity for FK-506 binding protein.
15. A composition for suppressing an immune response in an individual, comprising an immunosuppressive amount of an immunosuppressant compound having an affinity for FK-506 binding protein and having a molecular weight below about 750 amu, in a phyaio-logically acceptable vehicle.
16. A composition of Claim 15, wherein the immune reponse to be suppressed is an autoimmune response.
17. A composition of Claim 15, wherein the immune response to be suppressed is an immune response associated with graft rejection.
18. A composition of Claim 15, further comprising an immunosuppressant selected from the group consisting of cyclosporin, rapamycin, FK-506, 15-deoxyspergualin, OKT3 and azathioprine.
19. A composition of Claim 15, further comprising a steroid.
20. A composition for suppressing an immune response in an individual, comprising an immunosuppressive amount of a compound of Claim 4 having an affinity for FK-506 binding protein and having a molecular weight below about 750 amu, in a physiologically acceptable vehicle.
21. A composition of Claim 20, wherein the immuno-suppressant compound is represented by any of the compounds recited in Tables 1, 2, 3 or 4.
22. A composition of Claim 20, wherein the immuno-suppressant compound is represented by the struc-tures shown in Figure 1.
23. A composition of Claim 20, wherein the immune reponse to be suppressed is an autoimmune response.
24. A composition of Claim 20, wherein the immune response to be suppressed is an immune response associated with graft rejection.
25. A composition for preventing or significantly reducing graft rejection in a bone marrow or organ transplant in an individual, comprising an immuno-suppressive amount of a compound of Claim 4 having an affinity for FK-506 binding protein and having a molecular weight below about 750 amu, in a physio-logically acceptable vehicle.
26. A composition of Claim 25, wherein the immunosup-pressant compound is represented by the structures shown in Figure 1.
27. A composition of Claim 25, further comprising an immunosuppressant selected from the group consisting of cyclosporin, rapamycin, FK506, 15-deoxy-spergualin, OKT3 and azathioprine.
28. A composition of Claim 15, further comprising a steroid.
29. A composition of preventing or significantly reducing an autoimmune response in a mammal, com-prising an immunosuppressive amount of a compound of Claim 4 having an affinity for FK-506 binding protein and having a molecular weight below about 750 amu, in a physiologically acceptable vehicle.
30. A composition of Claim 29, wherein the immunosup-pressant compound is represented by the structures shown in Figure 1.
31. A composition of Claim 29, wherein the mammal is a human.
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US5665774A (en) 1997-09-09
WO1992000278A1 (en) 1992-01-09
AU8285591A (en) 1992-01-23
KR100197306B1 (en) 1999-06-15
BR1100732A (en) 2000-06-06
BR1100764A (en) 2000-02-08

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