CA2102178A1 - Novel immunosuppressive compounds - Google Patents

Novel immunosuppressive compounds

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Publication number
CA2102178A1
CA2102178A1 CA 2102178 CA2102178A CA2102178A1 CA 2102178 A1 CA2102178 A1 CA 2102178A1 CA 2102178 CA2102178 CA 2102178 CA 2102178 A CA2102178 A CA 2102178A CA 2102178 A1 CA2102178 A1 CA 2102178A1
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straight
c1
compound
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CA 2102178
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French (fr)
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John P. Duffy
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Vertex Pharmaceuticals Inc
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John P. Duffy
Vertex Pharmaceuticals Incorporated
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D211/00Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/92Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with a hetero atom directly attached to the ring nitrogen atom
    • C07D211/96Sulfur atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D207/00Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C07D207/48Sulfur atoms

Abstract

This invention relates to anovel 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 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

W~9~21313 PCT/US92/04391 21 ~ 8 NOVE~ IMMuNosuppREssrvE CO~POUN~S
~ _ ~ . . , BacXqround o~ th~ Inven~ion .

Post operative graft re~ections are a ma~or compli-cation affecting thQ succe~s of bone marrow and organ transplantations. HowevQr, throu~h thQ uge o~ immuno-~uppres~ive drug therapy, graft re~ection in organ transplantation can bs s~gni~icantly reduced.
A wide var~ety of disQasQs can bs characterized as ~auto~m~une di~ea~es~. Such di~ases ~rQ ~imilar to gra~t re~ection, exc~pt that thQ r~ection i8 0~ ~elf ti~ue. Immunosuppressive therapy can al80 be o~ use in preventing this inappropriate self rejection.
one widely accepted immunosuppressapt for the prQ~Qntion o~ graft rQ~ection is cyclosporin A (CsA). It ~8 a natural product of fungal metaboli~m and has been de~onstrated to havQ pot~nt iounosuppressive activity in clinical organ transplantations~ Calne, R.Y. et al., 8r.
~ed.-J. 282:~34-936 (1981~; White, D.~.C. Drugs 24:322-334 (1982). Although C~A i8 widely used in immuno-suppreQsant therapy, its usage (partiaularly in high - dosage) is o~ten accompanied by side e~fects which include nephrotox~city, hepatotoxicity and other central nervous system disorders.
The following disease~ have been treated with cyclo~porin A with positive result~, confirming th~
~mportanc~ o~ the autoimmun~ ~omponent in these diseases and their e$fective trQatm~nt with compounds wor~in~ ~y ~ ctiue T-cell immun~ suppre~sion ~imilar to cyclo-sporin A.
1) Ophthalmology: Uveitis, Behcet's disease and Grave's ophthalmopathy.

~ i313 210217 8 PCT/US92/04391 -2- ~;

Weetman, A.P. et al., Lancet 486-489 (1982).
Grave'~ opthal~opathy.
Nussenblatt, R.B. Qt al., Lancet 23~-238 (1983).
U~eitis.
Fr~nch-Constant, C. et al., Lancet 454 (1983).
Behcet's di~ease.
Sanders, N. Qt al., Lancet 454-455 tl983).
B-hcet~s dis~as~.
Not : Cy¢lo~por~n A ~ ¢urrently approve~ in J~pan - ~or t~e treatnent of Behc~t'~ disease, thQ first autoimmunQ d~seas~ indication for this compound.
2) Dermatology: Various autoimmune skin diseases inc~uding psoriasi~.
Zabel, P~ et al., Ianc~t 343 (1984). Acute der~ato~yo#itl8 -~an ~005t~ T. ~t al., Arch. Dermatol. 123:166-167 tl987). Atopic dkin dlsea~e.
Appleboom, T. et ~1., Amer. J. Med. 82:866-867 (1987). Scleroder~a.
Logan, R.A. and R.D.R. Camo, J. ~oY. Soc! Med.
81:417-418 t1988). Eczema.
Gri~fiths, C.E.M. et al., Brit. Med. J.
2 :731-732 (1986). Psoriasis.
Elli~, C.N. et al., J. Amer. Med. Assoc.
256:3110-3116 (1986). P~oriasi~.

3) Hematology: Various diseases including anemia.
Toetterman, T.H. et al., Lancet, 693 (1984). Pure red cell ~plasia ~PRCA).
Stryckman~, P.~. et al., N~w Engl. J. Med.
310:6S5-656 ~1984). Aplastic anemia.
Gluckman, E. et al., Bone ~arrow Transplant 3 vo g2/21313 _3_ PCrJU592/04391 Suppl. l, 241 (~988). Aplasti~: an~mia.

4~ ~:astroe~tQrology/~epatology: Primary cirrhosis, autoi~mun~ hepatiti~, ulc~rative coliti~, Crohn's - diseasQ and other gastrointestinal autoioune di~ase~.
Wiesner, R.H. et al., Hepatoloqy 7:1025, Abst. #9, (1987). Pri~ary biliary cirrho~
~Iy~, J.S. et al., ~a~troenterol~ 93:890-~93 ~1987). ~u~oi~un~ hep~t~i~.
Al~ison, ~.C. ~ al., I.ano~, 902-903 ~19~4)., Crohn'~ di~ase.
Brynskov, J. ~t al., Gastroent2roloqy 92 :1330 ~1987). Crohn's disea~e.
Porro, G.B. et al., It~ str~-nterol 19:40-41 ~1987). UlcQrative colitis.

5) Neurology: Amyotroph~c lateral s~lerosis (ALS, ~ou Gdhr~q'~ di~eas~U)~ ~ya~thoni~ gra~i~ ~nd mult~ple sclerosi~.
Appel, S.H. et al., rch. Neurol. 45:381-386 (1988). ALS.
Tindall, R.S.A. at al., New En~l. J. Med.
_ :719-7~4 (1987). Myasthenia gravis.
Ann. Neurol. 24, No. 1, p. 169,m Abstract Pl74~(ls88~. Multiple ~clero~is.
Do~masch, D. et al., Neuroloqy 38 Suppl. 2, 28-29 (198B). ~ultipl~ sclerosis.

6~ Nephrotic 9yndro~e: Nephrotic ~yndrome, m~mbrano-proliferati~e glo~erulonep~rit~s ~NPGN~ and relatad di~ëases.
Watzon, A.R. et al., Clin. Nephrol. 25:273-274 (1986). Nephroti~ syndrome.

W ~21313 2 1 0 2 1 ~ 8 - Pcr/US92/o4391 Tejani, A. et al., Xidney Int. 33:729-734 (1988).
Nephroti¢ syndrom~.
~eyrier, A. et al., Transplant Proc. 20, Suppl. 4 (Book III), 259-261 ~l988). Nephrotic syndrome.
IaGrus, G. et al~, Nephron. 44:382-382 (1986).
NæGN.

7) Rheumatoid Arthriti~ (RA) Harpsr, J.I. et al., ~an¢et 981-982 (1984). RA
Van Ri~thoven, A.W. ot al., Ann. Rh~um. Di~.
- 45:~26-731 (1986). RA.
Dougados, H. et al., Ann. Rheum. Dis. 47:127-133 (198~). RA.

8) Insulin-~epsndent Diabetes Mellitus (IDDM) Stiller, C.R. ~t al., Science 223:136~-1367 (l984). DDM.
As~an, R. et al., Lancet, 67-71 ~l~85). IDDN.
Bougneres, P.F. et al., New Ensl. J. ~ed.
3l8:663-670 (l988). IDDN.
Di~betes 37:lS74-~582 (1g88). IDDM.

Many veterinary diseases are alæo characterized as autoimmune diseases. Autoimmune diseases su~h as those listed above have been observ~d in mammals. Papa, ~Ø
et al., Eouine Vet. J. 22:145-146 (1990) infertility of autoimmune or~qin in the stallion; Gorman, N.T. and ~.L.
Werner, Brit. Vet. J. 142:403-410, 491-497 ~nd 498-505 (1986) immunQ mediatea di8eases o~ cats and dogs; G~orge, L.W. ~nd S~. White, Vet. Cl~n. North Amer. ~:203-213 (1984) sUtoiD une skin disQases ~n large mam~als;
Bennett, D., n. Pract. 6:74-86 (1984) auto~mmune ~.
di~ease~ in dogs; Halliwell, R.E~, ~. Amer. Vet. Assoc.

WOg2/21313 2~0217~8 Pcr/US92/043gl _81:1088-1096 (1982) ~utoi~une di~eases in domesticated animals.
The mecha~is~ by which C~A causes ~mmunosuppression has been e~tablished. In vitro, CsA inhibits the release of lymphokines, ~uch a~ int~rleukin 2 (IL-2~ lBunjes, D.
Qt al., Eur. J. Immunol. 1l:657_661 (1981)1 and prevents clonal eYpansion o~ helper and cytotoxic T cQlls t~arsson, E. J. I D unol. ~24:2828-2833 ~1980)~. CaA has b~Qn sho~m to bind thQ cytosolic protsin, t:yclophilin, and inhibit th~ prolyl-peptidyl cia-tran~ isom~ras~
~PPI~) activity of that protein. Fischer, G. et aI., Nature 33~:476-478 (1989); Takahashi, ~. et al., Nature 337:473-475 (1989). The PPIases may mediate T cell acti-vation by c~talyzing the rotomerization of peptide bonds o~ prolyl re~idue~.
Rec~ntly, a ~econd natural product isolated from strePtomYces, referred to as FX-506, has b~en demon-strat~d to be a potent immunosuppresive agent. Tanaka, H. et aI., J. Am. Chem. Soc. 09:5031-5~33 (1987).
FK-506 $nhibit~ IL-2 production, inhibits mixed lympho-cyte culture response and inhibits cytotoxic T-cell generation in vitro at 100 times lower concentration than cyclosporin A. Xino, T. et al., J. Antibiot. 15:~256_ 1265 (1987). FX-506 also inhibits PPIase acti~ity, but i~ ~tructurally different from CsA and binds to a binding protein (FKBP) distinct from cyclophilin. Harding, M.W.
et al., Nature 34~:758-760 (1989); Siekierka, J.J., Nature 341:755-757 ~1989).
. . .

Summary Or the Invention Th~s inv~ntion relates to a novel class of immuno-~uppressive compo D ds having an affinity for the FK-506 binding protein (~P). once bound to this protein, the W09~21313 2 10 2 1 ~ 8 PCT/US92/04391 i~munosuppressive co.mpound~ lnhibit the prolyl peptidyl cis-trans isomerase (rota~ase) activity of thQ FKBP and lead to inhibition o~ T cQll activation. The compounds o~ this invention can be used as immunosuppregsive drugs to pre~ent or significantly reduce graft re~ection in bone ~arrow ~nd organ transplantation~ and in the treat-~ent of autoi~une disease in humans and other mammals.

Dsta~led DescriPtion o~ the Invention Thi8 invent~on relat~ to a novol class o~ immuno-~uppressive oompounds having a sul~ona~ide substituent and which i8 represented by the for~ula I:

~ N ~ ~ D
O ~:
E .

~nd pharmaoeutically acceptable salts thereof, wherein A is CH2, oxygen, NH or N-(Cl-C4 alkyl);
wherein a and D are independently ~r, hydrogen, (Cl-C6)-straight or branched alkyl, (Cl-C6)-straight or ;~
branched aIkenyl, (Cl-C6)-straight or branched alkyl or alkonyl that i~ ~ubstitut~d with a (C5-C7)-cycloa~kyl, (~l-C6)-straight or branched alkyl or alkenyl that i8 ~ubst$tuted with a ~C5-C7)-¢ycloalkenyl, or Ar . ~. .
substitutod (Cl-C6)-straight or branched alkyl or alk~nyl, wherein, in each ¢ase, on~ or two of th~ CH2 grOUpB O~ the alkyl or alkenyl ¢hain~ ~ay contain 1-2 ~0~2t3~3 Pcr/us~uo43gl 210~1~8 heteroatom~ selected from the group consisting o~ oxygen, ~ulfur, so and S02 in chemically reasonable substitution pattern~, or ~ -.O ~

pxovid~d that both B and D are not hydrogen;
wh~rein Q i8 hydrogen, (Cl-C6)-~traight or bxanch~d alkyl or (Cl-C6)-~traight or branched alkenyl;
wherein ~ i~ Ar or substituted 5-7 membered cyclo-alkyl with ~ubstituents at position~ 3 and 4 which are independently s~lected ~rom th~ group consisting of hydrogen, hydroxyl, O-(Cl-C4)-alkyl, 0-(Cl-C4)-alkenyl and carbonyl;
where~n Ar i~ ~elQcted fro~ th~ group Gon~sting of p enyl, l-naph~hyl, ~-naphthyl, 2-~uryl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, monocyclic and bicyclic heterocyclic ring systems with indiv~dual ring ~izes being S or 6 which may contain in either or both rings a total of 1-4 heteroat~ms `
~ndependently selQcted fro~ 0, N and S; wherein AX ~ay contain one to three substituents which are independently selected from the group ~onæisting of hydrogen, halo, hydroxyl, nitro, trifluor~methyl, trifluoromethoxy, (Cl-C6)-straight or branched alkyl, (C2-C6)-straight or br~nched alkQnyl, O-tCl-C4)-ætraight or branched a~kyl, .. .
O-~C2-C4)-straight or branched alkenyl, O-benzyl, O-phenyl, 1,2-methylenedioxy, amino, carboxyl and phenyl;
wherein E i5 tCl-C6)-straight or branched alkyl, (Cl-C6) straight or branched alkenyl, (C5-C7)-cycloalkyl, (C5-C7)-cycloal~enyl substitutQd with (Cl-~4)-straight or U ~21313 8 PCT/USg~0439l branched alkyl or (Cl-C4)-straight or branohed alkenyl, t(C2-C4)-alkyl or (C2-C4)-alkenyl)]-Ar or Ar (Ar a~
described above);
~ herein J i~ hydrogen or Cl or C2 alkyl or benzyl; X
is (Cl-C4)-straight or branched alkyl, bsnzyl or cyclo-hexyl~ethyl; or wherein J and R may be taken together to ~or~ a 5-7 ~Q~bsr~a heteroc~lic ring which ~ay contain an oxygen, ~ulfur, SO or S02 ~ubstituent therQin; an~
whsrein n i~ 0-3.
The ~tereochemlstry at position 1 (Formula I) is (R) or (S), with (S) preferred. The stereochemist n at position 2 is (R) or (S).
ThQ compounds of the present invention can bs u~sd in the form of ~alt~ derived from inorganic or organic acids and ba8e8. Included among such acid alts are the following: acetate, adipate, alg~nate, aspartate, henzoate, benzenesulfonat~, bisulfate butyrate, citrate, c~mphorate, camphor~ulfonate, cqclopentanepropionate, ~-digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinata, oxalate, pa~oate, pectinate, persulfate, 3-phenylpro-pionate, picrate, pivalate, propionate, succinate, tar~rate, thio~yanate, tosylate and undecanoate. Base salts ~nclude ~mmonium salts, alkali metal salt~ such aæ
~oaium ~nd potas~ium salts, alkalin~ earth m~tal salts BUCh a~ calcium and magne~ium salts, ~alt with organic bases such as dicyclohexylamine sa}ts, N-m~thyl-D-glucamine, and ~alts with amino asids such as arginine, lysine, and so forth. ~150, the basic nitrogen-~ 21313 2 1 0 2 1 7 8 PCT/USg2~91 _9_ containing groups can be quaternized with such agents a~
lower alkyl halides, such as ~Qthyl, ~thyl, propyl, and butyl chloride, bro~ides and iodide~; dialkyl sulfates lik~ di~ethyl, diethyl, dibutyl and diamyl sulfates, long chain halides ~uch as decyl, lauryl, myristyl and ~tearyl chlorides, bromides and iodides, aral~yl halides like b~nzyl and phenethyl bro~ides and others. Water or oil-solubl- or di~per~ible products ar~ thereby obtained.
PrQ~rably, the co~pounds will h~v~ a molecular w ~gh~ b-low about 7S0 ato~ic ~ass unit~ (a.~.u.) and ~ost pref~rably b~low about S00 a.~.u. Examples of compound~ in which the J and X substituents are taken together to form a heterocyclic ring are shown in Table 1. A~ shown in TablQ 1, co~pound contains a fiv -~e~ber0d h~terocyclic ring sy~tem when n~l; and thQ
co~pound contain~ a six-~e~kered heterocyclic ring system wh~n n-2. ;

~'9~21313 210 21 18 PCT~US9~04391 TABLæ 1: coMpsuNDs D

~Of .
``.

No. n m _ B _ D _ _ E _ ~
-2 1 0 Hydrogen Phenyl Phenyl 3 1 0 ~ydrogen Ph~nyl . 4-~ethylphenyl 4 2 0 ~ydrogen Pben~l 4~Me~hylphenyl 5 2 0 Hydrogen 3-Ph~nylpropyl 4-~ethylphenyl 6 2 0 Hydrogen 3-Ph~nylpropyl 4-M~thoxyphenyl ~ 2 0 Hydrogen 3-Phenylpropyl 2-Thienyl 8 2 0 Hydrogen 3-Phenylpropyl 2,4,6-Triisopro-py~phenyl 9 - 2- 0 Hydrogen 3-Phenylpropyl 4-Fluorophenyl 10 2 0 ~ydrogen 3-Phenylpropyl Phenyl 11 2 0 Hydrogen 3-Phenylpropyl 3-~ethoxyphenyl 12 2 0 Hydrogen 3-Phenylpr~pyl 2-Methoxyphenyl .13 2 0 Hydrogen 3 Phenylpropyl 3,5-Dimethoxy-phe~yl 14 2 0 Hydrogen 3-Phenylpropyl 3,4,5-Tximeth-oxyph~nyl 15 2 0 Hydrogen 3-Phenylpropyl M~thyl 16 2 1 ~ydrogen 3-Phenylpropyl l-Naphthyl 17 2 0 Hydrogen 3-Phenylpropyl 8-Quinolyl SUBSrlTUTE SHEEI-~ 21313 210 217 8 PCT/US~2/04391 Tabl~ 1 (continued) No. n m B D _ E

18 2 0 Hydrogen 3-Ph~nylpropyl 1-(5-N,N-Di-~ethylamino)-naphthyl 19 2 0 Hydroqen 4-Phenoxyphenyl 4-~ethox~phenyl 20 2 0 ~ydrogen 4-Phenoxyph~nyl 4-NethylphQnyl 21 Z O Hydroyan 3-Phenoxyphenyl ~-Th~enyl 22 2 0 Hydrogen 3-P~enoxyphenyl 8-Quinolyl :~
23 2 0 Hydrogen 3-Phenoxyphenyl 4-iodophenyl 24 2 0 Hydrogen 3-Phenoxyphenyl 2,496-Tri-methylphenyl 25 2 o ~ydrogen 3-Phenoxyphenyl k~nzyl 26 ~ O Hydrogen 3-Phenoxyphenyl 2-Naphthyl 27 2 0 B~nsyl 3-Phenylpropyl 4-Fluorophenyl 28 2 0 2-~henyl~thyl 3-Phenylpropyl 4-Fluorophenyl 29 2 0 3-Phenylpropyl 3-Phenylpropyl 4-MQthylp~enyl 2 0 3-Phenylpropyl 3-Phenylprop~l 4-Nitrophenyl 31 2 0 3-~henylpropyl 3-Phenylpropyl 3-Nitrophenyl 32 2 0 3-Phenylpropyl 3-Phenylpropy~ 4-Chlorop~enyl 33 2 0 3-Phenylpropyl 3-Phenylpropyl 4-Methoxyphenyl 34 2 0 3-Phenylpropyl 3-Phenylpropyl 4-Fluorophenyl 2 0 3-Phenylpropyl 3-Phenylpropyl 2-Th~enyl 36 2 0 Hydrogen 3-Phenylpropyl E-Styrenyl . SUB8TITUTE SHEET

~ 1313 2 1 0 2 1 7 8 PCT/US92/043gl Th~ immuno~uppressive co~pounds o~ thi~ invention hav~ an affinity for thQ FK-506 binding pro~in which is located in th~ cyto~ol of lymphocytes, particularly T
ly~phocyte~. ~hen tha immunosuppre~iv~ compounds are bound to th~ FXBP, they act to inhibit th~ prolyl-p~ptiayl ci~-tran~ isomera#e activity of the binding prot~in and inhibit lymphocyte activ~tion ~ediated by FXBP. On~ p~rticular FK-506 binding prot~in has been ~dbnt~ied by ~arding, ~.W. et al., Natur- 34l:758-760 ~989) and c~n b~ u~ed a~ th~ standard by ~hich to ~valuat~ binding affinity o~ th~ compoundR ror FXBP.
Compound~ of this invention, however, may h~ve ~n af~inity for other FK-506 binding proteins. Inhibition o~ th~ prolyl peptidyl ci~-trans iso~erase may further be indi¢at~ve of binding to an F~-506 binding protsin.
Hu~an FR-506 binding protein can bQ obtained as described by Harding, ~.W. et al., Nature 341:758-~60 ~l989). Valu for the ~pparent Kd can bs determined fro~ ~ competitiv~ LH-20 binding as~ay p~rformed as db~cribed by Harding et al., using 32-tl-l4C~-benzoyl FK-506 a~ a reporting ligand; or using t3H]dihydro-FX-506, as described by Siekierka, J.J. et al., Nature 34l:755-757 (1989). The binding affinities for two of the compounds of this invention for the FKBP are reported in the ExamplQs Section. The data was obtained using the latter method, where the ability of an unlabeled compound to co~pQte with the binding of t3H~dihydro-FK-506 to FK-506 binding protein wa~ ~Qasured.
The inhibition of the PPIasQ ~rotamasQ) enzy~e activity o~ the FKBP (apparent "Ki" values) can also be ~easured acaording to the methods described by either ~0~21313 Harding, H.W. et al., ature 34l:758-760 (1989) or -~
Siekierk~, J.J. et ~1., Nature 341:755-757 (1989). Th~
¢is-tran~ isom~rization of thQ prolin~-alanine peptide bond in a model subætrate, N-succinyl-ala-Ala-Pro-Phe-p-nitroanilide, iB mon~tored ~pactrophoto~etrically in a coupled a~say with chy~otrypsin, which releases 4-nitro-anilide from the trans form of the substrate. Fischer, G. et al., Nature 337:476-478 (1989). Tha inhibitory ~ect of the addition o~ dif~erQnt conc~ntra~ion~ o~
~nhibitor on ths ~xtent o~ ths reaction i~ determin~d, and analysis of the change in ~irst order rate con~tant a~ a function of inhibitor concentration yields an e~tim~te of the apparent Ki value.
Th~ compounds of the present invention can be frther characterized in cellular biological experiment~
in itro wh r~ their re~emblance in function and USQ to cyclosporin A and to PK-506 iæ apparent. (Se2 Table 2).

Aææayæ and IC Cyclospo~in Value for Dru~ A Rapamycin FK-506 1) Human PBL ~ g/ml <l~g/ml <l~g/ml 2) T-Cell Hybridoma ~lpg/ml <l~g/ml ~l~gtml ~ TCR/CD2 3) Apoptosi~ Blocks Inactive Blockæ
at at at l~g/ml lpg/ml l~g/ml 4) CTLL Proli~era- l~g/ml ~O.Ol~q/ml >~l~g/ml tion ~ IL-2 ~ 1313 2 1 0 2 1 1 ~

1) Assay ~i~ilar to Yoshimura, N. et al., - Transplantat~on 47:356-35g (1g89). Assay uses fresh human p~ripheral blood lymphocytes isolated by Ficoll-HypaguQ density centrifugation, stimulated by the OXT3 ~ntibody ~nti-CD3) which sti~ulates via interaction w~th CD3. Sti~ul~tion i8 ~asured by incorporation o~
r~dio~ct~v~ thymid~n~ t(3H)TdR] into proliferating cells, ~ith ~n uninhibited control sigDal o~ 48,000-75,000 cpm.
IC50 values ~r~ ~sti-ated ~ro~ lnhibitions o~ pro~i~er-ation obsQrved at variou~ drug concentrations.
2) Assay sim~lar to abov~, but using T-cell clone stimulated with antibody to the T-cell receptor (TCR) and antibody to CD2. Stimulation is measured by incorpor-~tion o~ radioactive thymidin~ t(3H)TdRl into prolifer-ating cells, with an uninhib~ted control æignal o~ 23,000 cpm. IC50 values ~re estimated fro~ i~hibitions o~
proliferation observed at various drug concentrations.
3) Ass~y according to Shi, Y. et al., Natur~
339:625-626 (1989). The assay uses a T-cell hybridoma - - similar to that described. The assay measures activation-induced (anti-CD3) cell daath (evaluated by counting viable cells after staining as described) in a T-cell hybridoma that mimics the eXfect ~nown to occur in i~mature thymocytes. The ability o* Gyclospor~n A and FK-506 to inhibit this cell death is herein used as a sensitive indication o~ compounds with cyclosporin-like and/or F~-506-like mechanism of action. Not~ that thQ
che~ically related, but mechanistically distinct, immunosuppressant rapamycin i8 inactive in this assay.
4) Assay according ~o DuMont, F. st al., J.
Immunol. 144:25l-258 ~l990). ~hQ assay measures the st~mulation of CTLL cells in response to IL-2.

~vog2/21313 2 1 0 2 1 7 8 PC~/USg2/04391 Prolifer~tion is ~e~sured by incorpoation o~ (3H)TdR.
I~uno~uppr~ssants which work by a ~imllar mechani~m to cyclosporin A and FX-506 will not inhibit in this IL-2 driven proces~, sin¢~ they function by the inhibition of production of ~ndogenous IL-2. In this ass~y, ~xogenou~
IL-2 is pro~ided to overcom~ this block. NotQ that thQ
c~e~ic~lly r~lat~d, but ~chanist$cally di~tinct i D uno-suppress~nt, rap~ycin, is activ~ in this ~ssay.
Th~ as~ay~ and the ones s~t forth in th~ Exampl~
#oction can b~ u~ed to profil~ th~ cellular ~ctivity of tho co~pounds of the pre~ent invention. It has been ~hown th~t the co~pound~ o~ this invention resemble both cyclosporin A ~nd FR-506 in its cellular activit~, including i~uno~uppre~sion, in contrast to the ~dhanistically di~s~ilar i~muno~uppressant agent rapamyoin. Furtber~or~, th~ observQd cellular activity i~ consi~tent quant~tatively with the activity observed for FXBP binding and inhibition o* PPIase (rotamase) activity.
Thus, the compounds can be used as immunosuppre~-sants for prophylaxis of organ rejection or treatment of chronic graft rQ~ection and for the treatment of auto-i~mun~ dis~as~s.
The i~uno~upprQssive compounds of this invention can be periodically administered to a patient undergoing bonQ ~rrow or organ transplantat~on or for another r~a~on in which it is desirable to ~ubstantially reduce `~
or suppress a patiènt'~ une response, such as in variou~ auto~Y~une di~eases. The co~pounds o~ this in~ntion can also be adm~n~tered to ~ammals other than human~ for troat~ent of variou~ mammalian autoimmune a$se~

~ U21313 7 8 PcT/usg2/0439l Th~ novel compounds o~ the pre~ent invention possess an excellent degree o~ activity in suppression of antigen-~timulated growth snd clonal expansion o~
T-cell8, especially tho~e T-CQ11S characterized as ~helper~ T-cells. This activity i~ useful in the primary prevention of organ transplant re~ection, in the rescue of tsan~planted osgans during a re~ection episode, and in ths troRt~ent of any o~ ~e~esal autoi~mun~ diseases known to bs associated w~th inappropriatQ autoi~une respon~e~.
The&e autoi_un~ di~eases include: uveiti~, Behc~t'Q
disease, Graves ophthal~opathy, p~oriaQi~, acute dermato-myositis, atopic skin disease, scleroderma, eczema, pure red cell aplasia, aplastic ane~ia, prima n cirrhoæis, autoiDune hepatitis, ulcerativ~ colitis, Crohn's disQase, a~yotrophic lateral ~clerosis, myasthenia gravis, multiplQ sclQrosis, nephrotic syndrome, membrano-proliferativQ glomQrulonephritis, rheumatoid arthritis and insulin-dependent diabetes mellitus. In all o~ the ~bove-list0d autoi~munQ diseases, treatment is effectiv~
to reduce the ~ymptoms and slow progression of the dise~se. In the case of insulin-dependent diabetes mellitu~, treatment as described below i5 most ef~ective when instituted before the complete cessation of natural insulin production and transition to complete dependence on external insulin.
For thesQ purposes the compounds of the present invention may be administered orally, parenterally, by inhal~tion spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir in dosage fo D u-lations containing conventional non-toxic pharma- -¢auti¢ally-a¢c~pt~bl~ ¢arriers, ad~uvants and vehic~es.
ThQ ter~ parenteral as usQa her~in includ~ ~ubcutanQous, intra~enous, intra~uscular, intrasternal and intracranial injection or infusion techniques.

r.~

~9~21313 2 1 0 2 1 7 ~ PCT/Usg2/0439l ThQ pharmaceutical c~mpo~itions may bs in the form of a stQrile ~n~ctabl~ pr~paration, ~or examp~8 as a sterile injectibl~ aqueous or oleagenous suspension.
Thi8 ~uspens~on may be formulated a~cording to tachniques known in thQ art u~ing suita~le diFpersing or wetting agent~ and ~u~pending agQnts. The gterile injectable preparation may also bs a ~terile in~ectable solution or ~uspension in a non-toxic parenterally-ac¢eptable diluent or solvent, for exa~pl~ as a ~olution in 1,3-butanediol.
A~on~ thQ a¢oeptablQ vohiclo~ and solvant~ that ~ay b4 employed arQ water, Ringer'~ solution and isotonic sodium chloride golution. In addition, ~terile, fixed oilæ are conventionally employed a~ a solvent or suspending medium. For thi8 purpose any bland fixed oil may b2 employed including ~ynthetic mono- or di-glycerides.
Fatty acids ~uch as olei¢ acid and its glyceride deri-vat~e~ find u~e in the preparation of in~ectable~, as do natur~ ph.~rmaceut~cally-~cceptable oil~, ~uch as olive oil or castor oil, espec$ally in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol aiIuent or dispersant such as P Helv or similar alcohol.
The compounds may be admin~stered orally, in the ~orm of capsule~ or tablets, for example, or as an aqueou~ suspension or solution. In the case of tablets for oral u~e, carrier~ which are commonly u6ed include lactose and corn starch. Lubricating agents, such a3 magne~.ium ~t~arate, are al80 typically added. For oral adm~nistration in a ~psule form, useful diluents ~nclud~
lactose and dried corn star~h. When aqueous suspensions are required for oral use, tha active ~ngredient is combined with emulæifying ~nd #us~ending agent~. If dQsired, certain sweetening and/or flavoring and/or coloring agents may ~e added.

9~21313 PCT/US92/04391 The co~pounds of thi~ in~ntion may alfio b~ ad-min~stered in ~he form of suppo~itories for rectal admini~tration of the drug. These composition~ can be prepared by mixing the drug with a suitable non-irrîtat-ing excipient which i~ solid at room t~mpsratur~ but liquid at the rsctal tsmperature and therefore will melt in the rectum to rel~ase the drug. Such mater~
includ~ cocoa butter, beeswax ~nd polysthylen~ glycols.
The compound~ of this invsntion may also bs admin-istered topic~lly, espe~ially when the ¢onditions addressed for treatment involve areas or organs readily accessible by topical application, including autoimmune :`:
diseases of the eye, the skin, or the lower intestinal -~
tr~ct. Suitable topical formulations are readily pre-pared for each of these areas.
For op~thalmic use, the compounds can be form~lated as micronized suspension~ in isotonic, pH ad~usted sterile saline, or, prefer~bly, ~s solutions in ~sotonic, p~ ad~usted gterile saline, either with or without a preservative 8uch as benzylalkonium chloride. Alter-natively for the ophthalmic uses, ~he compounds may be formulated in an ointment such as petrolatum.
For application topically to the sXin, the compounds can be fo D ulated ~n a suitable ointment containing the compound suspended or dissolved in, for example, a mlxture with one or more of the following: mineral oil, liguid petrolatum, white petrolatum, propylsne glycol, polyoxyethylene polyo~ypropylene compound, emulsify~ng wax and water. Alternatively, the compound~ can be ~ormulated in a suitable lotion or cream containing the active compound ~uspended or dlssolved in, for example, a ~0~2t313 PCT/~S92/043gl 21~217 '~
_~9_ mixture of one or ~or~ of tha following: mineral o~l, sorbitan ~onostearatR~ polyæorbate 60~ cetyl ~ster~ wax~
cot~aryl alcohol, 2-octyldodecanol, benzyl alco~ol and water.
~ opical application for the lower intestinal tract can be effected in a rectal suppository formu~ation (~e~
abov~) or in a ~uitable enama formulation.
DosagQ lev~l~ on the ord~r of O.Ol to lOO mg~kg per day of the ~ctive ~ngredient compound ar~ useful in th~
trQat~ent Or the a~ove conditions. The amount of activQ
ingredient that may be combined with the carrier materials to produce a ~ingl~ dosage form will vary depending upon th~ host treat~d and th~ particular mode of ndministration.
It is understood, however, that a ~pecific dose l~v~l for any particular patient will depend upon a ~ariety of ~actors, including the ac~i~ity of the specific oompound employed, th~ age, body weight, general h~alth, sex, diet, ti~e of ~dmini~tration, rate of excretion, drug combination and th~ everity of the particular disease being treated.
The compound can also be administered in combination with a steroid, such as methyl prednisalone acetate, ~or add~tional immunosuppressive effect. The steroid is administered orally, intravenously, rectally, topically or by inhalation. Dosage~ ~based upon methyl pre-dnisalone acetate) of 0.1-5 mglkg/day may b~ employed.
An initial loading do~e o~ 100-S00 mg may be employed.
Steroid do~es ~ay be decreased uith time ~rom the higher toward the lower doses as t~e clinical situation indicates.

W ~21313 2 1 0 2 1 7 8 PCT/US9~0439l ~

The compounds can be administerea ~ith other immuno-suppressant drugs, ~uch a~ rapamycin, azathioprine, 15-deoxy~pergu~lin, cyclo~por~n, FK-506 or combinations of thess, to increa~e the immunosuppress~ve effQct.
Admini~tration of cyclosporin ~nd FK-506 together ~hould be avoided duQ to contraindications reported resulting from coadmini~tration of these iYmunosuppres~ants. Th~
dosag~ level of other i~unosuppressant drUg8 will depend upon the factors previously ~tatcd and the immuno-supprQssive Q~fectivonQ~s of th~ drug combination.
ORT3, which i~ a murine monoclonal antibody to CD3 gurface antigen of human T lymphocytes, can also be :~
coadministered intra~nously with compound~ of the present invention~ for rescue and reversal of a¢ute allograft r~ections, particularly in renal transplan-tations.
Th~ inv~ntion will bs further illustrated by way of the following ~xamples, whi¢h are not intended to be ~ ting in any way.
, - EXAMPLES

General Proton nuclear magnetic resonance (lH NMR) ~pectra were recorded at 500 MHz on a Bruker AMX 500. Chemical 8hifts are reported in parts per million (~) relative to Me4Si (~ 0.0). Analytical high performancQ liquid chro~atography (HPLC) was performed on either a W~ters 600~ or ~ H~wlett P~ck~rd 1050 liquid chromatograph.

~9U~1313 2 1 0 2 1 7 8 PCT/US92/043~1 S~nthe~i~ of (S)-3-PhenylbutYl N-(4-methYlsulfonYl)-pi~ecolate (5) (S)-3-PhenYlbutyl Pipecolate (38) To a ~lurry of 5.0 g (17.9 ~ol) of th~ tartrate ~alt of ~S)-pipecolic aaid (Egbert~on, M. and 5.J.
Danishefsky, J. or. Chem. 54:11 (1989)) in 50 mL o~ dry bQnzenQ wa~ added 13.8 g ~89.5 ~ol) o~ ~-phenyl-l-bu-tanol (Aldrich Chem~cal Co.) and 3.76 g (19.8 mmol) o~
p-toluQnQsulfoni¢ acid monohydr~te and th~ resulting mixture was heaSed at reflux o~ernight under a Dean Stark trap. The resulting homogeneous solut~on was concentrated, dis~olved into 100 ~L of 4:1 ether:~thyl acetate and extracted with 0.5 N HCl. The acid~c aqueous layer wa~ wa~hea with 100 mL of 4:1 ether:ethyl acetate, b~l*i~d by th addit~on of 8.0 ~L o~ 30~ NR4OH and wa~
then extracted into ethyl acetate. Th~ combined organic , extracts wer~ washed with brine, dried over MgS04 and concentrated to giv~ 4.5 g of the free a~in~ (38) as an oil. ~ NM~ oonsist~nt with structure.

~S)-3-PhenYlbutYl N-(4-methylsulfonYl)pipecolate (S) To a ~olution of 30 mg ~0.12 mmol) of thQ free amine (38) ~n 0.5 mL of CH2C12 was added 22 ~L (Q.13 mmol) of diisopropylethylamine and 24 mg (0~13 mmol) of p-toluene~ul~onyl chloride and the resulting mixture was allow0d ~o stir at room tempQra~ure for 1 hr. The x~actlon mlxtur~ was concentratQd and flash Chro~atographQd (elution with a gradient of 5% ethyl acetate in hexane to 100% ethyl acetate) to give 29 mg of ~ ~2t21313 2 1 0 2 1 7 8 Pcr/US92/0439l the sulfonamid~ (5) as an oil. The compound i5 illu~trated balow. lH NMR (500 MHz CDC13) ~ 7~62 (d), 7.29-7.09 (m), 4.71 (br d), 4.02-3.g5 (m), 3.91-3.83 (m), 3.71 (br d), 3.21-3.12 (m), 2~58 (t), 2.46 ~s), 2010 (br d), 1.75-1.66 (m), 1.65-1.40 (m), 1.30-1.15 (m).

~, H
IJ~
,~

SYnthesis of (S)-1,7-DiphenYl-4-hePtanYl N-(4-methoxysulfonYl)piPecolate ~33) 4-PhenYl-l-butYraldehyde (39) r To a solution of 3.2 mL ~20.8 mmol) of 4-phenyl-1-butanol (Aldrich Chemical Co.) in 20 m~ o~
CH2C12 at oC was added 3.2 g of powdered 3 A molecular ~ieves and then 5.37 g (24.9 m~ol) of pyridinium chlorochromate (PCC). The resulting suspension was stirrea at O-C for 1 h at which tim~ an additional 2.16 g (10.0 mmol) o~ PCC was added and the reaction mixture was warmed to room temperature. ~fter stirring at ambient temperature for O.S h, the reaction mixtur~ was diluted ~9~21313 2 1 0 2 1 7 8 PCT/US92/04391 with ~ther and filtered through celite to give 2.5 g of the crud~ product. Flash chromatography ~elution with 5 ethyl acetatQ in hQxane) yielded 700 mg of the aldehyde (39). lH NMR ~onsistent with the productO

3-Phenyl-~-PropYlmagnesiu~ ~romide ~40) To a ~usp~nsion of 736 mg ~30.3 ~mol) o~ magnesium turn~ng~ in 50 ~L of THF at room temperatur~ wa~ added 50 o~ 1,2-dibromoQthanQ followed by the dropw~e addition o~ 5.5 g (2S.1 ~mol) Or 1-bromo-3-phenylpropane (Aldrich Chemical Co.). After stirring at room temperature for 0.5 h the supernatent wa~ transferred via cannula to a 100 m~ storage vessel and subsequently used aæ a 0.5 ~HF ~olut~on of the Gr~gnard reag~nt (40).

1,7-D~hen~1-4-he~tanol (41) To a ~olution Or 700 mg (4.7 mmol) of 4-pheny~
butanal (39) in 5.0 mL of TKF at 0C was added 10.0 mL
(5.0 mmol) o~ 3-phenyl-1-propylmagnesium bromide (40) and the resulting ~ixture W8~ stirred at 0C for O.S h. The mixture was then quenched by the dropwise addition of saturat~d NH4Cl and diluted with ether. The phases were ~eparated and the organic layer was wa~hed with watar and ~.
brine and then dried over MgS04. Concentration gave 1.12 g o~ the alcohol (41) a~ an o~l. The lH NMR spectrum of thi8 compound ~CDC13) was consi~tent with the structure.

~S)-Boc-Pipe¢olyl-1,7-diphenyl-4-h~Ptanyl ester (42L
To ~ ~olution of 164 ~g (0.72 mmol) (S)-Boc-~-pipecolic a¢id in 5~0 mL of CH2C12 at room temperature was ~dd~d 174 m~ (0.65 ~mol~ of alcohol 41, 140 mg (Q.72 ' PCI~/US92/043~1 ~213~3 2 1 0 21.7 8 D~ol) of 1-(3-dim~thylaminopropyl)-3-~thylcarbodiimlde hydrochloride (EDC) and a catalytic amoun~ of N,N-di~ethyl~minopyridine (DMAP). .The reaction mixture was ~tirred at ambient temperature for 0.5 h and then applied d~rectly to a silic~ gel column. Elution with lOS ethyl acetate in hexan~ afforded 76.2 mg of the ester (42) a~ ~n oil. ~ NMR consi~tent with proauct.

~S)-1,7-DiPhen~1-4-heptanylpiPecolat~ (43) To a ~olu~ion o~ 47 mg (0.10 ~mol) o~ (42) in 1.0 mh of CH2C12 at ~mbient temperature was added 1.0 m~ of trifluoracetic acid. ~fter stirring at room temperature ~or 0.5 h, the resulting ~olution was neutralized by the -~
dropwi~e addition of saturated g2co3 The layers were ~Qp~rated ana thQ organia pha~ was washed with water, dr~d over NgS04 and concentrated to yield 23 mg of the aminQ (43) a~ an oil. ~ NNR ¢on~i~tent with structure.

(S)-1,7-DiphenYl-4-he~tan N-(4-m~thoxYsulfonyl)PiPecolate ~33) To a ~olution of 12.1 mg (0.031 mmol) of the free `~
a~ine (43) ~n 1.0 mL of CH2C12 was added 6.0 ~L (0.035 mmol) of diisopropylethylamine and 7.1 ~g ~0.034 mmol) of 4-~Qthoxybenzenesulfonyl chloride and the resulting mixtur~ ~as ~llowed to stir at room temperature for 1 h.
The reaction ~ixture wa~ concentratQd and flash chro~tographed (elution with 5% ethyl ac~tate in hexane?
to gi~e~~O.S ~g o~ th~ sul~onamid~ (33) aB ~n oil. The structur~ of (33) is shown below. lH NMR ~S00 NHz CDC13) 7.43-7.33 (br d~, 7.09-6.97 ~m), 6.96-6.83 (m), 6.54 (br d), 4.61 (br d), 4.48 ~), 3.58-3.45 (~), 3.01-2.92 ~ 21313 PCT/US92/04391 o25--~br t)t 2.39-2.25 (br t), 1.88 ~br d), 1.59-1.47 (br d), 1.46-1~19 (m), 1.06-0.95 (~).

'''.

~,J, 33 EXAMPL2 3, Cell Sourc~ and Culture Fr~sh p~xipheral blood lymphocyte~ (PBhs) from LeukoPak cells or whole blood from random normal blood donors ~tested HIV-negative and hepatitis negative) are isolatea and separated by density centrifugation over Hi8topaque 1077 (Sigma Chemical Co., St. ~ouis, M0~. The ~ur~ne CTLL ~ytotoxic T cell l~ne and the human JurXa~ T
~811 line are from ATC~ ~CTLL-2 ATCC TIB214, JURK~T CLONE
E6-1 ATCC ~IB152). Th~ human allogeneic B c~ll lines u~ed for act~vation of the fr~h PBLs ar~ EBV-transformed ly~phocytes from normal healthy adult donors wit~ two completely diff~rent HLA haplotypes. All cell l~ne~ were routinely tested for the presence of MYcoplasma V 9~21313 2~ 1 . 8 contamination using the Gibco Mycotect test kit and are Mycoplasma-free. Culture medium consists of RPMI ~640 (Gibco, Grand Island, NY) containing penicillin (50 U/ml) and streptomycin (50 ~g/~l), L-gluta~in~ 2 mM, 2 mercaptoethanol (5 x 10 5), 10% heat-inactivated FCS and 10 mN HEPES.

Com~ound Solutions and Titrations All chemical stocks WerQ dissolved in DMS0.
Titrations of co~pounds were ~ade into the meaiu~ the individual assay was carried out in, i.e., complete RPMI
or HB 104 for final diluted concentrations, using ~ult~ple three-~old dilutions from 1 ~M or 10 ~M stock solution~.

NTT Assav The NTT assay is a colori~etric technique to deter~ine the toxicity of the compounds on growing ly~phoid ~nd non-lymphoid csll lines based on reduction o~ the tetrasol$u~ 8alt by ~ntact ~itochondria (Mossman, T., J. Immunol. Methods 65:55 tl983)). Cell viability in the presence or absence of different concentrations of test compounds in serum-free medium (HB 104, HANA
Biologic, Inc.) wa~ a~se~sed usinq MTT
(3-t4,5-d$methyl-thiasoyl-2-yl~2,5-diphenyl-tetrazolium bro~ide). At 4 h before th~ end of the 3-day toxicity assay culture period, 20 ~1 of NTT dy~ (g mg/ml in pH 7.2 PBS) wer~ ~dded to each ~icrotit~r well. At the end of the incubation t~m~, most of the culture media was carcfully aspirated out of each well. Then 100 ~1 of acidified isopropyl alcohol (0.04 N HCl) was added to "

W09~2~313 PCT/US92/04391 -27~ 1 ~217 8 solubillze the dye and optical density is read at 570 nm ~inus OD at 6~0 nm (Nolecular Devices Thermomax plate reader and Softmax ~oftware program, Nenlo Park, CA).
Results were compared with ~ean OD in controls (medium with no drug8) and dose~ causing 50% toxicity (TC50) were calculated.

Mitoqenesis Assay~ (~PMA~ and ~OXr3~) Th~ inh~bitory e~t~ct o~ test co~pound~ on the proliferation of hu~an PBLs in respon~e to mitogens (Waithe, W.X. and X. Hirschhorn, H~ndbook o~ ExPerimental _mmunoloqv, 3d Ed. Blackwell Scientific Publications, Oxford (1978); ~ishell, B.B. and S.M. Shiigi, Selected Method~ in Cellular ImmunoloqY W.H. Preeman and Co., San Fran¢i~co, CA (1980)) wa~ a~ses~ed by ~timulation of 5 x 104 cell~ wlth O~T3 ~10 4 dilution final) or PMA
(~Ong/~l) plu~ iono~ycin ~250 ng/ml) in the presenc~ or ab~ence of different concentratians o~ test compound~ and control drug~ (CsA, FK506, Pagamycin) in final volume of 200 ~1 per well in 96 well round bottomed plates. After 48 h incubation ~37C, ~S C02), cells were pulsed with 1 ~C$ of 3H-thymidine, harvested 24 h later with a ~om Tek cell harvester, and counted in LKB ~-scintillation counter. Compounds were evaluated on their ability to inhibit proliferation of human peripheral blood ly~phocytes. Results (cpm) were compared with controls witb mediu~ alone, and concentrations cau~ing 50t reduction in counts (IC50) were calculated.

~; .

~ 92/21313 PCT/US92/04391 2~ 2~ 0217~ ~-NLR Bioassays (~LB" and "~VM~) Antigen activated proliferation o* PBLs in a primary mlxed lymphocyte rea¢tion wa~ a6sessed in the presence or absence of different concentrations of tested compounds and ¢ontrol drug~. 5 x 104 fresh PB~s were stimulated with 5 x 103 of ~itomycin C treated-allogeneic EB~-transformed ~-lymphobla~toid cells, LB and JVM, in a final volu~e o~ 200 ~1 per well ~n 96-~ell roun~-bottomed plates ~ hell, B.B. ~nd S.N. Shiigi, S~lected Methods in Cellular Immunoloqy W.H. Freaman and Co., San ~:
Franci~co, CA (1980); Nelson, P.A. et al., Transplantation 50:286 ~1990)). Cultures were pulsed on day 6, harvested 24 h later and counted as in previous ~ection. The compound~ were evaluated on t~eir ability to inhibit proliferation in a mixed lymphocyte reaction. ~-IL-2 N~croassay (~CTLL~) To determine if test compounds inhib~t the later T
cQll activation process of cytokine utili~at~on, the proliferativa respons~ o~ the IL-2 dependent CTLL-20 murine T cell line (ATCC) was assessed ~Gillis, S. et al., J. Immunoloqy 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 proliferation o~ the CTLLs, and CsA and FK506 do not ~Dumont, F.J. et al., J. I~munoloqy l44:251 (~990)). 3 x 103 CTLLs were e~posed to di~ferent conc~ntra~ions of test compounds ~nd control drugs in th~ presence of l U/ml of human recombinant IL-2 ~Genzyme, rIL-2) for 24 h.
Four h after adding drugs, cells wer~ pulsed w~th 1 ~CI

W09~21313 PCT/USg2/04391 -2g~ 1 7 ~

o~ 3H-thymid~ne, incubated for an add~tional 20 h (37-C, 5~ C02), and then harveste~ and counted as previously described.
.

Results The result~ of the~e assay~ are tabulated in Table 3 bel~w.

TABLE 3: ASSAY RESULTS

No. Ki(nm) Xd(nm) PMA OKT3 ~B JVM CTLL

216,000 ND>10,000>10,000>10,000 >10,000 >10,000 32,500 ND~10,000~10,000~10,000 ~lO,OOQ ~10,000 41,500 1000~10,000>lo,OOO~lo,OOO>lo,OOO ~lo,OOo 5 130 ND5,000 3,500 ~10,000 >to,~oo 7,500 6 180 ND~10,000~10,000 9,000 4,500 l,QOO
7 200 ND~10,000 8,000 ~10,000 ~10,000 ~10,000 82,400 ND~10,00~>10,~00 ~0,000 ~10,000 ~10,000 9 100 90~10,000~10,000~10,000 >10,000 ~10,000 10200 ND~10,000>10,000>10,000 ~10,000 ~10,000 112,200 ND>10,000>10,000 8,000 >lO,OQO >10,000 128,000 ND>10,000>10,000~10,000 >10,000 >10,000 133,000 ND~10,000>10,000>10,000 >10,000 >10,000 ~14240 ND>10,000 8,000 ~10,000 ~10,000 >10,000 156,000 ND~10,000~10,000>10,000 ~10,000 >10,000 16900 ND>10,000>~O,QOO>10,000 >10,000 >lO,OQO
17>100,400 ND~10,000~10,000~10,000 ~10,000 ~10,000 182,000 . ND ~10,000 ~10,000 ~10,000 ~10,000 ~10,000 195,000 ND >10,000 >lO,OQO >10,000 ~10,000 >10,000 202,000 ND >10,000 >10,000 ~10,000 >10,000 >10,000 21500 ND >10,000 ~10,000 >10,000 >lQ,OOO >10,000 SUBSrITUTE SHEET

W ~2/2131~ PCI~/USg2~04391 TABIa~ 3: ASSAY RESULTS ~continued) No .Ki (nm2 Kd (nm) ~ OKT3 L~B Jy~lCTIL

22 ~100,000 ND>10,000>10~000 >10,000 >10,000>10,000 23 2,000 ND~10,000>10,000 ~10,000 >10,000>10,000 24 900 ND>10, 000>10, 000 4, 000 ~10, 000~10, OQ0 1,900 ND9,000 >10,000 >10,000 >10,000>10,000 26 2,000 ND~10,000>10,000 >10,000 ~10,000~10,000 27 12,000 ND>10,000>1~,000 >10,000 >10,000>10,000 28 1,500 ND>10,000>10,000 >10,000 >10,000>10,000 29 1,700 ~JD>10,000>10,000 ~10,000 >10,000~10,000 3,000 ND>10,000>10,000 >10,000 >10,0~>10,000 31 6,000 ND>10,000 8,000 >10,000 ~10,000>10,000 32 8,000 ND>10,000>10,000 ~10,000 ~10,000>10,000 33 5,000 ND7,500 9,500 7,500 >10,000~10,000 34 700 ~D>10,000~10,000 >10,000 >10,00~>10,000 2,500 IJD>10,000~10,000 ND >10,0009,000 36 400 ND>10,000>10,000 >10,000 ~10,000>10,000 Ki - inhibition of FXBP rotamase activity K - binding to FKBP
P ~ and OXT3 - mitogens used to stimulate proliferation of human peripheral blood lymphocytes tPB~). Compounds are evaluated on their ability to inhibit proliferation.
IB and JUN - human viral-transformed B lymphoblastoid cell lines stimulated to proliferate in a mixed ly~phocyte reaction (MIR). The compounds are evaluated on their abi~ity to inhibit this proliferation.
CTLL - inh~bition of proliferation of cytotoxic T cells stimulatsd by IL~2.
ND - not determined~
Eouivalents Those ~killed in the art will recognize, or be able to ascertain, using no more than routin~ experimentation, D ny eguivalents to the ~pecific embodiment~ of the invention described herein. Such equivalents are intended to be encompassed by the following claims:

SUBSI-ITUTE SHEET

Claims (19)

1. A compound represented by the formula:
and pharmaceutically acceptable salts thereof, wherein A is CH2, oxygen, NH or N-(C1-C4 alkyl);
wherein B and D are independently Ar, hydrogen, (C1-C6)-straight or branched alkyl, (C1-C6)-straight or branched alkenyl, (C1-C6)-straight or branched alkyl or alkenyl that is substituted with a (C5-C7)-cycloalkyl, (C1-C6)-straight or branched alkyl or alkenyl that is substituted with a (C5-C7)-cycloalkenyl, or Ar substituted (C1-C6)-straight or branched alkyl or alkenyl, wherein, in each case, one or two of the CH2 groups of the alkyl or alkenyl chains may contain 1-2 heteroatoms selected from the group consisting of oxygen, sulfur, SO and SO2 or provided that both B and D are not hydrogen;

wherein Q is hydrogen, (C1-C6)-straight or branched alkyl or (C1-C6)-straight or branched alkenyl;
wherein T is Ar or substituted 5-7 membered cycloalkyl with substituents at positions 3 and 4 which are independently selected from the group consisting of hydrogen, hydroxyl, O-(C1-C4)-alkyl, O-(C1-C4)-alkenyl and carbonyl;
wherein Ar is selected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, monocyclic and bicyclic heterocyclic ring systems with individual ring sizes being 5 or 6 whirh may contain in either or both rings a total of 1-4 heteroatoms independently selected from O, N and S; wherein Ar may contain one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-straight or branched alkyl, (C2-C6)-straight or branched alkenyl, O-(C1-C4)-straight or branched alkyl, O-(C2-C4)-straight or branched alkenyl, O-benzyl, O-phenyl, 1,2-methylenedioxy, amino, carboxyl and phenyl;
wherein E is (C1-C6)-straight or branched alkyl, (C1-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl, (C5-C7)-cycloalkenyl substituted with (C1-C4)-straight or branched alkyl or (C1-C4)-straight or branched alkenyl, [(C2-C4)-alkyl or (C2-C4)-alkenyl)]-Ar or Ar (Ar as described above);
wherein J and K are taken together to form a 5-7 membered heterocyclic ring which may contain an oxygen, sulfur, SO or SO2 substituent therein;
wherein n is 0 to 3; and wherein the stereochemistry at carbon position 1 and 2 are R or S.
2. The compound according to claim 1, said compound having an affinity for FK-506 binding protein.
3. The compound according to claim 1, said compound being capable of inhibiting the prolyl peptidyl cis-trans isomerase activity of the FK-506 binding protein.
4. The compound according to claim 1, said compound having a molecular weight below about 750 amu.
5. The compound according to claim 4, said compound having a molecular weight below about 500 amu.
6. The compound according to claim 1, wherein the stereochemistry at carbon position 1 is S.
7. The compound according to claim 1, wherein J
and K are taken together as represented by the formula:

wherein n is 1 or 2 and m is 0 to 1.
8. The compound according to claim 7, wherein B
is selected from the group consisting of hydrogen, benzyl, 2-phenylethyl and 3-phenylpropyl;
D is selected from the group consisting of phenyl, 3-phenylpropyl, 4-phenoxyphenyl and 4-phenoxyphenyl; and E is selected from the group consisting of phenyl, 4-methylphenyl, 4-methoxyphenyl, 2-thienyl, 2,4,6-triisopropylphenyl, 4-fluorophenyl, 3-methoxyphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, methyl, 1-naphthyl, 8-quinolyl, 1-(5-N,N-dimethylamino)-naphthyl, 4-iodophenyl, 2,4,6-trimethylphenyl, benzyl, 4-nitrophenyl, 2-nitrophenyl, 4-chlorophenyl and E-styrenyl.
9. A compound represented by any of the structures shown in Table 1, said compound having an affinity for FX-506 binding protein.
10. A composition for use in suppressing an immune response in a mammal, said composition comprising a compound according to claim 1 having an affinity for FK-506 binding protein and having a molecular weight below about 750 amu, in a physiologically acceptable vehicle.
11. The composition according to claim 10, wherein the immune response to be suppressed is an autoimmune response or an immune response associated with graft rejection.
12. The composition according to claim 10 or 11, wherein the immunosuppressant compound is represented by the structures shown in Table 1.
13. The composition according to any one of claims 10, 11, or 12, further comprising an immuno-suppressant selected from the group consisting of cyclosporin, rapamycin, FR506, 15-deoxyspergualin, OKT3 and azathioprine.
14. The composition according to any one of claims 10, 11, 12, or 13, further comprising a steroid.
15. The use of a compound according to claim 1 having an affinity for FK-506 binding protein and having a molecular weight below about 750 amu for the manufacture of a medicament for use in suppressing an immune response in a mammal.
16. The use according to claim 15, wherein the immune response to be suppressed in an autoimmune response or an immune response associated with graft rejection.
17. The use according to claim 15 or 16, wherein the immunosuppressant compound is represented by the structures shown in Table 1.
18. The use according to any one of claims 15, 16, or 17, wherein said compound is used in combination with an immunosuppressant selected from the group consisting of cyclosporin, rapamycin, FK506, 15-deoxyspergualin, OKT3 and azathioprine.
19. The use according to any one of claims 15, 16, 17, or 18, wherein said compound is used in combination with a steroid.
CA 2102178 1991-05-24 1992-05-26 Novel immunosuppressive compounds Abandoned CA2102178A1 (en)

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