Immunosuppressive compounds

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 (rotamass) 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.

DETAILED DESCRIPTION OF THE INVENTION 
This invention relates to a novel class of immunosuppressive compounds 
represented by the formula I: 
##STR1## 
and pharmaceutically acceptable salts thereof, 
wherein A is O, 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 
##STR2## 
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 independently 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 consisting of 1-naphthyl, 2-naphthyl, 
2-fury1, 3-furyl, 2-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl and phenyl 
having one to three substituents which are independently selected from the 
group consisting of hydrogen, halo, hydroxyl, nitro, CF.sub.3, 
(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 its 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 or 
(C1-C4)-straight or branched alkenyl, 2-indolyl, 3-indolyl, [(C1-C4)-alkyl 
or (C1-C4)-alkenyl)]-Ar or Ar (Ar as described above); 
wherein J is hydrogen or C1 or C2 alkyl or benzyl; K is (C1-C4)-straight or 
branched alkyl, benzyl or cyclohexyhethyl; or wherein J and K may be taken 
together to form a 5-7 membered heterocyclic ring which may contain an 
oxygen (O), sulfur (S) , SO or SO.sub.2 substituent therein. 
The stereochemistry at position 1 (Formula I) is (R) or (S), with (S) 
preferred. 
The compounds of the present invention can be used in the form of salts 
derived from inorganic or organic acids and bases. Included among such 
acid salts are the following: acetate, adipate, alginate, aspartate, 
benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, 
camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, 
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, 
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 
2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, 
persulfate, 3-phenylpropionate, pictate, pivalate, propionate, succinate, 
tartrate, thiocyanate, rosylate and undecanoate. Base salts include 
ammonium salts, alkali metal salts such as sodium and potassium salts, 
alkaline earth metal salts such as calcium and magnesium salts, salt with 
organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and 
salts with amino acids such as arginine, lysine, and so forth. Also, the 
basic nitrogen-containing groups can be quaternized with such agents as 
lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, 
bromides and iodides dialkyl sulfates like dimethyl, diethyl, dibutyl and 
diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and 
stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and 
phenethyl bromides and others. Water or oil-soluble or dispersible 
products are thereby obtained. 
Preferably, the compounds will have a molecular weight below about 750 
atomic mass units (a.m.u.) and most preferably below about 500 a.m.u. 
Examples of compounds in which the J and K substituents are taken together 
to form a heterocyclic ring are shown in Tables 1 and 2. Examples of other 
preferred compounds of this invention are listed in Tables 3 and 4. 
TABLE 1 
__________________________________________________________________________ 
##STR3## 
No. 
B D n Ki K.sub.d 
__________________________________________________________________________ 
2 Benzyl Phenyl 1 25 .mu.M 
&gt;5.0 
.mu.M 
3 Benzyl Phenyl 2 1.5 .mu.M 
&gt;2.0 
.mu.M 
4 Allyl Phenyl 2 8 .mu.M 
ND 
5 1-Naphthyl Phenyl 2 0.9 .mu.M 
ND 
6 2-Naphthyl Pehnyl 2 7.0 .mu.M 
1.0 .mu.M 
7 Benzyl 2-Methylpropyl 
2 0.9 .mu.M 
ND 
8 Benzyl 2-Methoxyphenyl 
2 17 .mu.M 
&gt;75 .mu.M 
9 Benzyl 3-Methoxyphenyl 
2 0.3 .mu.M 
&gt;1.3 
.mu.M 
10 Benzyl 4-Methoxyphenyl 
2 5.0 .mu.M 
5.0 .mu.M 
11 Benzyl 3,5-Dimethoxyphenyl 
2 2.0 .mu.M 
0.6 .mu.M 
12 Benzyl 2,6-Dimethoxyphenyl 
2 50 .mu.M 
25 .mu.M 
13 Benzyl 3,4,5-Trimethoxy- 
2 0.1 .mu.M 
2 .mu.M 
phenyl 
14 Benzyl 4-Fluorophenyl 
2 4.0 .mu.M 
10 .mu.M 
15 Benzyl 3-Nitrophenyl 
2 160 .mu.M 
&gt;75 .mu.M 
16 Benzyl 4-Nitrophenyl 
2 160 .mu.M 
100 .mu.M 
17 Benzyl 2-Pyridyl 2 130 .mu.M 
&gt;1000 
.mu.M 
18 Benzyl 2-Pyridyl-N-oxide 
2 &gt;500 
.mu.M 
10 .mu.M 
19 tert-Butyl 2-Furyl 1 200 .mu.M 
&gt;500 
.mu.M 
20 Benzyl 2-Furyl 2 3 .mu.M 
&gt;12 .mu.M 
21 Benzyl 3-Indolyl 2 25 .mu.M 
20 .mu.M 
22 Benzyl 2-Thiophenyl 2 0.8 .mu.M 
4 .mu.M 
23 E-3-Phenyl-2- Phenyl 2 1.5 .mu.M 
ND 
methyl-prop- 
2-enyl 
24 E-3-(4-Hydroxy- 
Phenyl 2 6 .mu.M 
ND 
phenyl)-2- 
methyl-prop- 
2-enyl 
25 E-3-[cis-(4- Phenyl 2 0.6 .mu.M 
ND 
hydroxycyclo- 
hexyl)]-2- 
methyl-prop-2- 
enyl 
26 E-3-[trans-(4- Phenyl 2 0.5 .mu.M 
ND 
Hydroxycyclo- 
hexyl)]-2-methyl- 
prop-2-enyl 
27 Benzyl 2-Nitrobenzyl 
1 26 .mu.M 
&gt;25 .mu.M 
28 Hydrogen Methoxy 2 ND ND 
29 tert-Butyl Methoxy 1 600 .mu.M 
&gt;500 
.mu.M 
30 Allyl Methoxy 2 190 .mu.M 
&gt;25 .mu.M 
31 Benzyl Methoxy 2 80 .mu.M 
&gt;50 .mu.M 
32 2-Cyclohexylethyl 
Methoxy 2 45 .mu.M 
&gt;40 .mu.M 
33 3-Cyclohexylpropyl 
Methoxy 2 20 .mu.M 
12 .mu.M 
34 4-Cyclohexylbutyl 
Methoxy 2 6 .mu.M 
2-3 .mu.M 
35 Cyclopentyl- Methoxy 2 35 .mu.M 
ND 
propyl 
36 E-3-(4-Methoxy- 
Methoxy 2 40 .mu.M 
&gt;30 .mu.M 
phenyl)-2-methyl- 
prop-2-enyl 
37 E-3-(3,4-Dime- Methoxy 2 10 .mu.M 
ND 
thoxyphenyl-2- 
methyl-prop-2-enyl 
38 E-3-(4-Hydroxy- 
Methoxy 2 60 .mu.M 
ND 
phenyl)-2-methyl- 
prop-2-enyl 
39 E-3-[cis-(4- Methoxy 2 70 .mu.M 
&gt;20 .mu.M 
Hydroxycyclo- 
hexyl)]-2-methyl- 
prop-2-enyl 
40 Benzyl Cyclohexyl 2 1.3 .mu.M 
3 .mu.M 
41 Benzyl Ethyl 1 400 .mu.M 
&gt;300 
.mu.M 
42 Benzyl 3-Methoxyphenyl 
1 5 .mu.M 
80 .mu.M 
43 Benzyl 2-Pyridyl 1 300 .mu.M 
ND 
44 Benzyl 3,4-Difluorophenyl 
2 3 .mu.M 
ND 
45 Benzyl (E)-2-(4-Methoxyphenyl)- 
2 1 .mu.M 
ND 
ethenyl 
46 Benzyl 1-Hydroxy-1-cyclohexyl 
2 1 .mu.M 
2 .mu.M 
47 Benzyl 2-Naphthyl 2 1.5 .mu.M 
0.3 .mu.M 
48 Benzyl Naphthyl 2 1 .mu.M 
2 .mu.M 
49 (S)-.alpha.-Methylbenzyl 
Phenyl 2 0.5 .mu.M 
0.6 .mu.M 
50 Benzyl 2-Hydroxy-2- 2 12 .mu. M 
0.35 
.mu.M 
tetrahydropyranyl 
51 (R)-.alpha.-Methylbenzyl 
Phenyl 2 1.5 .mu.M 
1 .mu.M 
52 Benzyl 3-Trifluoromethylphenyl 
2 1.5 .mu.M 
1.3 .mu.M 
53 Benzyl 3-Benzyloxyphenyl 
2 0.5 .mu.M 
0.2 .mu.M 
54 Benzyl (E)-2-tert-Butylethenyl 
2 9 .mu.M 
3 .mu.M 
55 Benzyl 2-Trifluoromethylphenyl 
2 5 .mu.M 
ND 
56 4-Cyclohexylbutyl 
Phenyl 2 0.4 .mu.M 
ND 
57 4-Cyclohexylbutyl 
3,4,5-Trimethoxyphenyl 
2 0.04 
.mu.M 
0.1 .mu.M 
58 4-Phenylbenzyl Phenyl 2 5 .mu.M 
ND 
59 4-Phenylbenzyl 3,4,5-Trimethoxyphenyl 
2 2 .mu.M 
ND 
60 Benzyl 3-Ethoxyphenyl 
2 0.56 
.mu.M 
ND 
61 3-Phenoxybenzyl 
3,4,5-Trimethoxyphenyl 
2 0.018 
.mu.M 
0.035 
.mu.M 
62 3-Phenoxybenzyl 
Phenyl 2 0.09 
.mu.M 
0.15 
.mu.M 
63 4-Phenylbutyl 3,4,5-Trimethoxyphenyl 
2 0.019 
.mu.M 
0.1 .mu.M 
64 4-Phenylbutyl Phenyl 2 0.35 
.mu.M 
ND 
65 Benzyl 3-(3-Propenyloxy)phenyl 
2 1 .mu.M 
ND 
66 Benzyl 3-(2-Propoxy)phenyl 
2 0.5 .mu.M 
ND 
67 Benzyl 1-Methylpropyl 
2 1 .mu.M 
ND 
68 2-Phenylethyl Phenyl 2 1.1 .mu.M 
ND 
69 6-Phenylhexyl Phenyl 2 0.5 .mu.M 
ND 
70 5-Phenylpentyl 3,4,5-Trimethoxyphenyl 
2 0.07 
.mu.M 
ND 
71 6-Phenylhexyl 3,4,5-Trimethoxyphenyl 
2 0.1 .mu.M 
0.05 
.mu.M 
72 6-Cyclohexylhexyl 
3,4,5-Trimethoxyphenyl 
2 0.05 
.mu.M 
0.5 .mu.M 
73 4-Phenoxybenzyl 
3,4,5-Trimethoxyphenyl 
2 0.8 .mu.M 
ND 
74 5-Cyclohexylpentyl 
3,4,5-Trimethoxyphenyl 
2 0.09 
.mu.M 
0.08 
.mu.M 
75 Benzyl 3-(1-Butoxy)phenyl 
2 0.36 
.mu.M 
ND 
76 4-Phenylbutyl 3-(2-Propoxy)phenyl 
2 0.1 .mu.M 
ND 
77 4-(4-Iodophenyl)butyl 
3,4,5-Trimethoxyphenyl 
2 0.016 
.mu.M 
0.06 
.mu.M 
78 4-Iodobenzyl 3,4,5-Trimethoxyphenyl 
2 1.4 .mu.M 
ND 
79 2-(2-Naphthyl)ethyl 
3,4,5-Trimethoxyphenyl 
2 0.22 
.mu.M 
ND 
80 2-(1-Naphthyl)ethyl 
3,4,5-Trimethoxyphenyl 
2 0.5 .mu.M 
ND 
81 4-Phenylbutyl 4-Iodophenyl 2 0.8 .mu.M 
0.25 
.mu.M 
82 4-Phenylbutyl 3-Iodophenyl 2 0.13 
.mu.M 
0.2 .mu.M 
83 3-Phenylpropyl 3,4,5-Trimethoxyphenyl 
2 0.11 
.mu.M 
ND 
84 3-(3-Indolyl)propyl 
3,4,5-Trimethoxyphenyl 
2 0.017 
.mu.M 
0.054 
.mu.M 
85 4-(4-Methoxyphenyl)butyl 
3,4,5-Trimethoxyphenyl 
2 0.013 
.mu.M 
0.049 
.mu.M 
86 4-Phenylbut-2-enyl 
3,4,5-Trimethoxyphenyl 
2 0.8 .mu.M 
ND 
87 4-Phenylbut-3-enyl 
3,4,5-Trimethoxyphenyl 
2 0.5 .mu.M 
ND 
88 4-(4-Allocaminophenyl)propyl 
3,4,5-Trimethoxyphenyl 
2 0.011 
.mu.M 
0.07 
.mu.M 
89 4-Phenylpropyl 1-Cyclohexenyl 
2 0.78 
.mu.M 
ND 
90 4-(4-Methoxyphenyl)but-3-enyl 
3,4,5-Trimethoxyphenyl 
2 0.011 
.mu.M 
0.60 
.mu.M 
91 4-Phenylpropyl 1-Fluoro-1-cyclohexyl 
2 0.54 
.mu.M 
ND 
92 4-Phenylpropyl 3-Butoxyphenyl 
2 1.4 .mu.M 
ND 
93 3-[3-(N-Formylindolyl)]propyl 
3,4,5-Trimethoxyphenyl 
2 0.015 
.mu.M 
0.06 
.mu.M 
94 4-(3-indolyl)butyl 
3,4,5-Trimethoxyphenyl 
2 0.016 
.mu.M 
0.05 
.mu.M 
95 4-Phenylbutyl Benzyl 2 0.35 
.mu.M 
ND 
96 4-Phenylbutyl 3-Biphenyl 2 0.04 
.mu.M 
0.033 
.mu.M 
97 4-Phenylbutyl 4-tert-Butylphenyl 
2 0.6 .mu.M 
ND 
98 4-Phenylbutyl Cyclohexyl 2 0.08 
.mu.M 
0.18 
.mu.M 
99 4-Phenylbutyl Cyclohexylmethyl 
2 0.12 
.mu.M 
ND 
100 
4-Phenylbutyl 3,4-Methylenedioxyphenyl 
2 0.25 
.mu.M 
ND 
101 
4-Phenylbutyl 4-Tetrahydropyranyl 
2 0.44 
.mu.M 
ND 
102 
4-Phenylbutyl 3-Cyclohexyl-4-methoxy- 
2 14 .mu.M 
ND 
phenyl 
103 
4-Phenylbutyl 4-(4-Methoxybenzyloxy- 
2 0.7 .mu.M 
ND 
methyl)-2-furyl 
104 
4-Phenylbutyl tert-Butyl 2 0.18 
.mu.M 
ND 
105 
4-Phenylbutyl Ethyl 2 1.6 .mu.M 
ND 
106 
3-(N-Benzimidazolyl)propyl 
3,4,5-Trimethoxyphenyl 
2 0.11 
.mu.M 
ND 
107 
3-(N-Purinyl)propyl 
3,4,5-Trimethoxyphenyl 
2 0.13 
.mu.M 
ND 
108 
(S,S)-2-Methyl-3-hydroxy-4- 
3,4,5-Trimethoxyphenyl 
2 0.25 
.mu.M 
ND 
phenylpropyl 
__________________________________________________________________________ 
ND indicates not determined. 
TABLE 2 
______________________________________ 
##STR4## 
No. B U n Ki K.sub.d 
______________________________________ 
109 Benzyl 3,4-Methylene- 
1 3 .mu.M 
&gt;15 .mu.M 
dioxyphenyl 
110 Benzyl 3,4-Methylene- 
2 3 .mu.M 
&gt;4 .mu.M 
dioxyphenyl 
111 Benzyl 4-Methoxyphenyl 
1 6 .mu.M 
&gt;30 .mu.M 
112 Benzyl 4-Methoxyphenyl 
2 4 .mu.M 
&gt;8 .mu.M 
113 Benzyl 2,5-Dimethoxy- 
1 10 .mu.M 
ND 
phenyl 
114 Benzyl 2,4,5-Trimethoxy- 
1 25 .mu.M 
ND 
phenyl 
115 Benzyl 3,4,5-Trimethoxy- 
1 450 .mu.M 
&gt;25 .mu.M 
phenyl 
116 Benzyl 4-Dimethylamino- 
2 20 .mu.M 
&gt;5 .mu.M 
phenyl 
117 Benzyl 4-Nitrophenyl 2 14 .mu.M 
&gt;5 .mu.M 
118 Benzyl 2-Furyl 2 2.5 .mu.M 
ND 
119 Benzyl 3-Furyl 2 2.5 .mu.M 
ND 
120 Benzyl 3-Indolyl 2 &gt; 60 .mu.M 
&gt;8 .mu.M 
121 Benzyl 3-Pyridyl 2 25 .mu.M 
ND 
122 Benzyl Hydrogen 2 300 .mu.M 
ND 
123 Benzyl Phenyl 2 11 .mu.M 
ND 
______________________________________ 
TABLE 3 
__________________________________________________________________________ 
##STR5## 
No B D J K Ki K.sub.d 
__________________________________________________________________________ 
124 Benzyl 
Methoxy 
Methyl 
Hydrogen 
1000 
.mu.M 
&gt;200 
.mu.M 
125 Benzyl 
Methoxy 
Methyl 
S-Methyl 
400 .mu.M 
&gt;200 
.mu.M 
126 Benzyl 
Methoxy 
Methyl 
S-Isopropyl 
170 .mu.M 
&gt;200 
.mu.M 
127 Ethyl 
Methoxy 
Benzyl 
Hydrogen 
&gt;1200 
.mu.M 
&gt;300 
.mu.M 
128 tert- 
Methoxy 
Ethyl 
S-Methyl 
&gt;400 
.mu.M 
&gt;500 
.mu.M 
Butyl 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
##STR6## 
No B U J K Ki K.sub.d 
__________________________________________________________________________ 
129 
Benzyl 
4-Methoxy- 
Methyl 
S-Methyl 
80 .mu.M 
&gt;150 
.mu.M 
phenyl 
130 
Benzyl 
4-Methoxy- 
Methyl 
S-Iso- 
30 .mu.M 
&gt;20 .mu.M 
phenyl propyl 
131 
Benzyl 
3,4-Methylene- 
Methyl 
S-Methyl 
50 .mu.M 
ND 
dioxyphenyl 
132 
Benzyl 
3,4-Methylene- 
Hydrogen 
S-Methyl 
60 .mu.M 
ND 
dioxyphenyl 
__________________________________________________________________________ 
The immunosuppressive compounds of this invention have an affinity for the 
FK-506 binding protein which is located in the cytosol of lymphocytes, 
particularly T lymphocytes. When the immunosuppressive compounds are bound 
to the FKBP, they act to inhibit the prolylpeptidyl cis-trans isomerase 
activity of the binding protein and inhibit lymphocyte activation mediated 
by FKBP. One particular FK-50G binding protein has been identified by 
Harding, M. W. et al., Nature 341:758-760 (1989) and can be used as the 
standard by which to evaluate binding affinity of the compounds for FKBP. 
Compounds of this invention, however, may have an affinity for other 
FK-50G binding proteins. Inhibition of the prolyl peptidyl cis-trans 
isomerase may further be indicative of binding to an FK-506 binding 
protein. 
Human FK-506 binding protein can be obtained as described by Harding, M. W. 
et al., Nature 341:758-760 (1989). Values for the apparent K.sub.d can be 
determined from a competitive Za-20 binding assay performed as described 
by Harding et al., using 32-[1-.sup.14 C]-benzoyl FK-506 as a reporting 
ligand; or using [3H]dihydro-FK-506, as described by Siekierka, J. J. et 
al., Nature 341:755-757 (1989). The binding affinities for several 
compounds of this invention for the FKBP are reported in Tables 1-4. The 
data was obtained using the latter method, where the ability of an 
unlabeled compound to compete with the binding of [.sup.3 H]dihydro-FK-506 
to FK-506 binding protein was measured. 
The inhibition of the PPIEse (rotamase) enzyme activity of the FKBP 
(apparent "Ki" values) can 81so be measured according to the methods 
described by either Harding, M. W. et al., Nature 341:758-760 (1989) or 
Siekierka, J. J. et al., Nature 341:755-757 (1989). The cis-trans 
isomerization of the proline-alanine peptide bond in a model substrate, 
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, is monitored 
spectrophotometrically in a coupled assay with chymotrypsin, which 
releases 4-nitroanilide from the trans form of the substrate. Fischer, G. 
et al., Nature 337:476-478 (1989). The inhibitory effect of the addition 
of different concentrations of inhibitor on the extent of the reaction is 
determined, and analysis of the change in first order rate constant as a 
function of inhibitor concentration yields an estimate of the apparent Ki 
value. The extent of enzyme inhibition (K.sub.i) of some preferred 
compounds is shown in Tables 1-4. 
The compounds of the present invention can be further characterized in 
cellular biological experiments in vitro where their resemblance in 
function and use to cyclosporin A and to FK-506 is apparent. (See Tables 5 
and 6). 
TABLE 5 
______________________________________ 
Assays and 
IC.sub.50 Value Cyclosporin 
for Drugs A Rapamycin FK-506 
______________________________________ 
1) Human PBL + OKT3 
&lt;1 &lt;1 &lt;1 
.mu.g/ml .mu.g/ml 
.mu.g/ml 
2) T-Cell Hybridoma + 
&lt;1 &lt;1 &lt;1 
TCR/CD2 .mu.g/ml .mu.g/ml 
.mu.g/ml 
3) Apoptosis Blocks Inactive 
Blocks 
at at at 
1 1 1 
.mu.g/ml .mu.g/ml 
.mu.g/ml 
4) CTLL Proliferation + 
&gt;&gt;1 .perspectiveto.0.01 
&gt;&gt;1 
IL-2 .mu.g/ml .mu.g/ml 
.mu.g/ml 
______________________________________ 
TABLE 6 
______________________________________ 
Cellular Assay Results 
PMA OKT3 LB JVM CTLL 
No. (.mu.M) (.mu.M) (.mu.M) 
(.mu.M) (.mu.M) 
______________________________________ 
2 ND ND ND ND ND 
3 7.6 4.6 &gt;10 &gt;10 &gt;8.5 
4 ND ND ND ND ND 
5 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
6 ND ND ND ND ND 
7 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
8 ND ND ND ND ND 
9 &gt;10 6.5 &gt;10 &gt;10 &gt;10 
10 ND ND ND ND ND 
11 ND ND ND ND ND 
12 ND ND ND ND ND 
13 &gt;10 5.9 &gt;10 &gt;10 &gt;10 
14 ND ND ND ND ND 
15 ND ND ND ND ND 
16 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
17 ND ND ND ND ND 
18 ND ND ND ND ND 
19 ND ND ND ND ND 
20 ND ND ND ND ND 
21 ND ND ND ND ND 
22 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
23 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
24 ND ND ND ND ND 
25 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
26 &gt;10 6.5 &gt;10 &gt;10 &gt;10 
27 ND ND ND ND ND 
28 ND ND ND ND ND 
29 ND ND ND ND ND 
30 ND ND ND ND ND 
31 ND ND ND ND ND 
32 ND ND ND ND ND 
33 ND ND ND ND ND 
34 ND ND ND ND ND 
35 ND ND ND ND ND 
36 ND ND ND ND ND 
37 ND ND ND ND ND 
38 ND ND ND ND ND 
39 ND ND ND ND ND 
40 7.0 1.0 &gt;10 &gt;10 &gt;10 
41 ND ND ND ND ND 
42 ND ND ND ND ND 
43 ND ND ND ND ND 
44 ND ND ND ND ND 
45 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
46 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
47 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
48 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
49 &gt;10 6.2 &gt;10 &gt;10 &gt;10 
50 ND ND ND ND ND 
51 ND ND ND ND ND 
52 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
53 &gt;10 8.0 &gt;10 &gt;10 8.0 
54 ND ND ND ND ND 
55 ND ND ND ND ND 
56 &gt;10 &gt;10 &gt;10 6.5 5.0 
57 4.0 4.5 &gt;10 8.0 6.0 
58 ND ND ND ND ND 
59 ND ND ND ND ND 
60 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
61 4.0 8.0 &gt;10 &gt;10 3.2 
62 6.5 &gt;10 &gt;10 &gt;10 &gt;10 
63 6.0 3.1 10 8.5 3.8 
64 10 6.0 &gt;10 &gt;10 &gt;10 
65 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
66 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
67 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
68 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
69 6.1 &gt;10 &gt;10 &gt;10 &gt;10 
70 7.0 4.5 &gt;10 9.0 4.2 
71 5.0 5.5 7.5 8.0 3.8 
72 9.0 &gt;10 &gt;10 &gt;10 5.5 
73 8.0 &gt;10 4.5 6.0 7.0 
74 8.0 9.0 10 10 5.0 
75 8.0 &gt;10 9.0 &gt;10 4.5 
76 5.0 10 9.0 &gt;10 6.0 
77 4.5 8.0 6.0 7.0 2.1 
78 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
79 10 2.5 &gt;10 &gt;10 8.0 
80 3.0 4.0 10 10 6.0 
81 7.0 &gt;10 &gt;10 &gt;10 &gt;10 
82 10 &gt;10 &gt;10 &gt;10 &gt;10 
83 5.5 5.5 8.5 7.5 5.0 
84 4.5 6.0 6.0 &gt;10 2.0 
85 4.5 4.5 7.0 10 1.5 
86 9.0 &gt;10 &gt;10 &gt;10 &gt;10 
87 7.0 &gt;10 &gt;10 &gt;10 &gt;10 
88 2.2 2.2 2.5 4.5 4.0 
89 8.0 &gt;10 &gt;10 &gt;10 &gt;10 
90 8.0 &gt;10 &gt;10 &gt;10 7.0 
91 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
92 9.0 &gt;10 &gt;10 &gt;10 &gt;10 
93 6.0 7.0 10 8.7 3.7 
94 5.0 5.5 &gt;10 7.0 4.0 
95 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
96 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
97 &gt;10 &gt;10 &gt;10 &gt;10 6.0 
98 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
99 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
100 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
101 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
102 &gt;10 &gt;10 10 10 &gt;10 
103 7.0 10 10 &gt;10 10 
104 4.0 &gt;10 &gt;10 &gt;10 &gt;10 
105 &gt;10 &gt;10 &gt;10 &gt;10 &gt;10 
106 7.0 &gt;10 &gt;10 &gt;10 3.0 
107 6.5 &gt;10 &gt;10 &gt;10 &gt;10 
108 &gt;10 &gt;10 8.0 &gt;10 &gt;10 
109 ND ND ND ND ND 
110 ND ND ND ND ND 
111 ND ND ND ND ND 
112 ND ND ND ND ND 
113 ND ND ND ND ND 
114 ND ND ND ND ND 
115 ND ND ND ND ND 
116 ND ND ND ND ND 
117 ND ND ND ND ND 
118 ND ND ND ND ND 
119 ND ND ND ND ND 
120 ND ND ND ND ND 
121 ND ND ND ND ND 
122 ND ND ND ND ND 
123 ND ND ND ND ND 
124 ND ND ND ND ND 
125 ND ND ND ND ND 
126 ND ND ND ND ND 
127 ND ND ND ND ND 
128 ND ND ND ND ND 
129 ND ND ND ND ND 
130 ND ND ND ND ND 
131 ND ND ND ND ND 
132 ND ND ND ND ND 
______________________________________ 
All of the compounds in Table 6 showed toxicity at higher concentrations 
that their immunosuppresive activity and were typically concentrations &gt; 
10 .mu.M. 
PMA and OKT3 mitogens used to stimulate proliferation of human peripheral 
blood lymphocytes (PBC). Compounds are evaluated on their ability to 
inhibit proliferation. 
LB and JVM human viraltransformed B lymphoblastoid cell lines stimulated 
to proliferate in a mixed lymphocyte reaction (MLR). The compounds are 
evaluated on their ability to inhibit this proliferation. 
CTLL inhibition of proliferation of cytotoxiv T cells stimulated by IL2. 
1) Assay similar to Yoshimura, N. et al., Transplantation 47:356-359 
(1989). Assay uses fresh human peripheral blood lymphocytes isolated by 
Ficoll-Hypaque density centrifugation, stimulated by the OKT3 antibody 
(anti-CD3) which stimulates via interaction with CD3. Stimulation is 
measured by incorporation of radioactive thymidine [(.sup.3 H)TdR] into 
proliferating cells, with an uninhibited control signal of 48,000-75,000 
cpm. IC.sub.50 values are estimated from inhibitions of proliferation 
observed at various drug concentrations. 
2) Assay similar to above, but using T-cell clone stimulated with antibody 
to the T-cell receptor (TCR) and antibody to CD2. Stimulation is measured 
by incorporation of radioactive thymidine [(.sup.3 H)TdR] into 
proliferating cells, with an uninhibited control signal of 23,000 cpm. 
IC.sub.50 values are estimated from inhibitions of proliferation observed 
at various drug concentrations. 
3) Assay according to Shi, Y. et el., Nature 339:625-626 (1989). The assay 
uses a T-cell hybridoma similar to that described. The assay measures 
activation-induced (anti-CD3) cell death (evaluated by counting viable 
cells after staining as described) in a T-cell hybridoma that mimics the 
effect known to occur in immature thymocytes. The ability of cyclosporin A 
and FK-506 to inhibit this cell death is herein used as a sensitive 
indication of compounds with cyclosporin-like and/or FK-506-like mechanism 
of action. Note that the chemically related, but mechanistically distinct, 
immunosuppressant rapamycin is inactive in this assay. 
4) Assay according to DuMont, F. et al., J. Immunol. 144:251-258 (1990). 
The assay measures the stimulation of CTLL cells in response to IL-2. 
Proliferation is measured by Incorporation of (.sup.3 H)TdR. 
Immunosuppressants which work by a similar Mechanism to cyclosporin A and 
FK-506 will not inhibit in this IL-2 driven process, since they function 
by the inhibition of production of endogenous IL-2. In this assay, 
exogenous IL-2 is provided to overcome this block. Note that the 
chemically related, but mechanistically distinct immunosuppressant, 
rapamycin, is active in this assay. 
These assays can be used to profile the cellular activity of the compounds 
of the present invention. Thus, it is clear from these results that the 
compounds resemble both cyclosporin A and FK-506 in its cellular activity, 
including immunosuppression, in contrast to the mechanistically dissimilar 
immunosuppressant agent rapamycin. Furthermore, the observed cellular 
activity is consistent quantitatively with the activity observed for FKBP 
binding and inhibition of PPIase (rotamase) activity shown in Table 1. 
Thus, the compounds can be used as immunosuppressants for prophylaxis of 
organ rejection or treatment of chronic graft rejection and for the 
treatment of autoimmune diseases. 
The immunosuppressive compounds of this invention can be periodically 
administered to a patient undergoing bone marrow or organ transplantation 
or for another reason in which it is desirable to substantially reduce or 
suppress a patient's immune response, such as in various autoimmune 
diseases. The compounds of this invention can also be administered to 
mammals other than humans for treatment of various mammalian autoimmune 
diseases. 
The novel compounds of the present invention possess an excellent degree of 
activity in suppression of antigen-stimulated growth and clonal expansion 
of T-cells, especially those T-cells characterized as "helper" T-cells. 
This activity is useful in the primary prevention of organ transplant 
rejection, in the rescue of transplanted organs during a rejection 
episode, and in the treatment of any of several autoimmune diseases known 
to be associated with inappropriate autoimmune responses. These autoimmune 
diseases include: dyeiris, Behcet's disease, Graves ophthahopathy, 
psoriasis, acute dermatomyositis, 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 glomerulonephritis, rheumatoid arthritis 
and insulin-dependent diabetes mellitus. In 811 of the above-listed 
autoimmune diseases, treatment is effective to reduce the symptoms and 
slow progression of the disease. In the case of insulin-dependent diabetes 
mellitus, treatment as described below is most effective when instituted 
before the complete cessation of natural insulin production and transition 
to complete dependence on external insulin. 
For these purposes the compounds of the present invention may be 
administered orally, parenterally, by inhalation spray, topically, 
rectally, nasally, buccally, vaginally or via an implanted reservoir in 
dosage formulations containing conventional non-toxic 
pharmaceutically-acceptable carriers, adjuvants and vehicles. The term 
parenteral as used herein includes subcutaneous, intravenous, 
intramuscular, intrasternal and intracranial injection or infusion 
techniques. 
The pharmaceutical compositions may be in the form of a sterile injectable 
preparation, for example as a sterile injectible aqueous or oleagenous 
suspension. This suspension may be formulated according to techniques 
known in the art using suitable dispersing or wetting agents and 
suspending agents. The sterile injectable preparation may also be a 
sterile injectable solution or suspension in a non-toxic 
parenterally-acceptable diluent or solvent, for example as a solution in 
1,3-butanediol. Among the acceptable vehicles and solvents that may be 
employed are water, Ringer's solution and isotonic sodium chloride 
solution. In addition, sterile, fixed oils are conventionally employed as 
a solvent or suspending medium. For this purpose any bland fixed oil may 
be employed including synthetic mono- or di-glycerides. Fatty acids such 
as oleic acid and its glyceride derivatives find use in the preparation of 
injectables, as do natural pharmaceutically-acceptable oils, such as olive 
oil or castor oil, especially in their polyoxyethylated versions. These 
oil solutions or suspensions may also contain a long-chain alcohol diluent 
or dispersant such as Ph. Helv or similar alcohol. 
The compounds may be administered orally, in the form of capsules or 
tablets, for example, or as an aqueous suspension or solution. In the case 
of tablets for oral use, carriers which are commonly used include lactose 
and corn starch. Lubricating agents, such as magnesium stearate, are also 
typically added. For oral administration in a capsule form, useful 
diluents include lactose and dried corn starch. When aqueous suspensions 
are required for oral use, the active ingredient is combined with 
emulsifying and suspending agents. If desired, certain sweetening and/or 
flavoring and/or coloring agents may be added. 
The compounds of this invention may also be administered in the form of 
suppositories for rectal administration of the drug. These compositions 
can be prepared by mixing the drug with a suitable non-irritating 
excipient which is solid at room temperature but liquid at the rectal 
temperature and therefore will melt in the rectum to release the drug. 
Such materials include cocoa butter, beeswax and polyethylene glycols. 
The compounds of this invention may also be administered topically, 
especially when the conditions addressed for treatment involve areas or 
organs readily accessible by topical application, including autoimmune 
diseases of the eye, the skin, or the lower intestinal tract. Suitable 
topical formulations are readily prepared for each of these areas. 
For ophthalmic use, the compounds can be formulated as micronized 
suspensions in isotonic, pH adjusted sterile saline, or, preferably, as 
solutions in isotonic, pH adjusted sterile saline, either with or without 
a preservative such as benzylalkonium chloride. Alternatively for the 
ophthahic uses, the compounds may be formulated in an ointment such as 
petrolatum. 
For application topically to the skin, the compounds can be formulated in a 
suitable ointment containing the compound suspended or dissolved in, for 
example, a mixture with one or more of the following: mineral oil, liquid 
petrolatum, white petrolatum, propylene glycol, polyoxyethylene 
polyoxypropylene compound, emulsifying wax and water. Alternatively, the 
compounds can be formulated in a suitable lotion or cream containing the 
active compound suspended or dissolved in, for example, a mixture of one 
or more of the following: mineral oil, sorbitan monostearate, polysorbate 
60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol 
and water. 
Topical application for the lower intestinal tract can be effected in a 
rectal suppository formulation (see above) or in a suitable enema 
formulation. 
Dosage levels on the order of 0.01 to 100 mg/kg per day of the active 
ingredient compound are useful in the treatment of the above conditions. 
The amount of active ingredient that may be combined with the carrier 
materials to produce a single dosage form will vary depending upon the 
host treated and the particular mode of administration. 
It is understood, however, that a specific dose level for any particular 
patient will depend upon a variety of factors, including the activity of 
the specific compound employed, the age, body weight, general health, sex, 
diet, time of administration, rate of excretion, drug combination and the 
severity of the particular disease being treated. 
The compound can also be administered in combination with a steroid, such 
as methyl prednisalone acetate, for additional immunosuppressive effect. 
The steroid is administered orally, intravenously, rectally, topically or 
by inhalation. Dosages (based upon methyl prednisalone acetate) of 0.1-5 
mg/kg/day may be employed. An initial loading dose of 100-500 mg may be 
employed. Steroid doses may be decreased with time from the higher toward 
the lower doses as the clinical situation indicates. 
The compounds can be administered with other immunosuppressant drugs, such 
as rapamycin, azathioprine, 15-deoxyspergualin, cyclosporin, FK-506 or 
combinations of these, to increase the immunosuppressive effect. 
Administration of cyclosporin and FK-506 together should be avoided due to 
contraindications reported resulting from coadministration of these 
immunosuppressants. The dosage level of other immunosuppressant drugs will 
depend upon the factors previously stated and the immunosuppressive 
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 transplantations. 
The invention will be further illustrated by way of the following examples, 
which are not intended to be limiting in any way. 
EXAMPLES 
General 
Proton nuclear magnetic resonance (.sup.1 H NMR) spectra were recorded at 
300 MHz on a Bruker AC 300 or at 500 MHz on a Bruker AMX 500. Chemical 
shifts for proton resonances are reported in parts per million (.delta.) 
relative to Me.sub.4 Si (.delta. 0.0). Analytical high performance liquid 
chromatography (HPLC) was performed on either a Waters 600 E or a Hewlett 
Packard 1050 liquid chromatograph. HPLC assessments of compounds were run 
on a Waters Associates Delta Pak 5 micron, 15 cm. column at a flow rate of 
1.5 mL per minute. The solvent system used was: A=0.1% H.sub.3 PO.sub.4 
/H.sub.2 O; B=0.1% H.sub.3 PO.sub.4 /CH.sub.3 CN. A linear gradient of 95% 
A/5% B to 100% B over 15 minutes followed by 1.5 minutes at 100% B was 
used, with detection at 214 nM. 
The compounds described below are illustrated in FIG. 1. 
EXAMPLE 1 
Synthesis of (E)-3-[cis-4-(hydroxycyclohexyl)]-2- methylprop-2-enyl 
N-(phenylgloxyl)-pipecolate (25) 
1. (S)-Benzyl Pipecolate (133) 
To a slurry of 7.3 g (26.14 mmol) of the tartrate salt of (S)-pipecolic 
acid (Egbertson M. and S. J. Danishefsky, J. Org. Chem. 54:11 (1989) in 75 
mL of dry benzene was added 13.5 mL (0.13 mol) of benzyl alcohol and 5.48 g 
(28.8 mmol) of p-toluenesulfonic acid monohydrate. The reaction mixture was 
heated at reflux under a Dean-Stark trap for 2 h and then cooled to room 
temperature. The solution was then diluted with 400 mL of ether and 
stirred overnight at 4.degree. 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-toluenesulfonic acid salt of benzyl pipecolate (134). .sup.1 HNMR 
(300 MHz, D.sub.2 O) .delta. 7.63 (d), 7.41 (s), 7.28 (d), 5.26 (ABq), 4.8 
(s), 4.03 (dd), 3.92 (dd), 3.51-3.39 (m), 3.15-2.93 (s), 2.40 (s), 
2.36-2.24 (m), 1.98-1.53 (m). 
Benzyl pipecolate was routinely generated by treating an ethyl acetate 
suspension of this salt with saturated sodium bicarbonate until 
dissolution of the organic material. The aqueous layer was extracted twice 
with ethyl acetate and the combined organic extracts were dried with 
MgSO.sub.4 and evaporated to yield (S)-Benzyl pipecolate (133) as a pale 
yellow oil. 
2. (S)-N-(Phenylglyoxyl)pipecolic Acid (135) 
To a solution of 4.95 g (17.72 mmol) of the tartrate salt of 
L-(S)-pipecolic acid in 18.0 mL of methylene chloride at 0.degree. C. was 
added 20.4 mL (117.10 mmol) of diisopropylethylamine followed by 12.4 mL 
(97.7 mmol) of chlorotrimethylsilane and the resulting solution was 
stirred at 0.degree. C. for 15 minutes. To this mixture was added 17.72 
mmol of benzoylformyl chloride, which was freshly prepared in a separate 
reaction flask at ambient temperature from 2.66 g (17.72 mmol) of 
benzoylformic acid and 2.3 mL (26.37 mmol) of oxalyl chloride in 18.0 mL 
of methylene chloride containing a catalytic amount of dimethylformamide. 
The reaction mixture was stirred at 25.degree. C. overnight at which time 
the mixture was poured into 1.0 N HCl. The aqueous layer was discarded and 
the organic layer was washed twice with saturated sodium bicarbonate. The 
combined aqueous layers were washed with methylene chloride, acidified to 
pH 2.0 with concentrated HCl, and then extracted repeatedly with ether. 
Flash chromatography (Still, W. C. et al., J. Org. Chem. 43:2923 (1978)) 
(slutton 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. .sup.1 H NMR (500 MHz, CDCl.sub.3) .delta. 11.4-11.1 (br s) , 
8.02 (d), 7.98 (d), 7.65 (t), 7.58-7.43 (m), 5.45 (d), 4.64 (dd), 4.43 
(d), 3.52 (dd), 3.25 (ddd), 3.01 (ddd), 2.42 (d), 2.24 (d), 1.86-1.78 (m), 
1.68-1.38 (m). 
3. cis-and trans-4-(tert.-butyldimethylsilyloxy)cyclohexane-1-ol (136) and 
(137) 
To a solution of 3.43 g (21.7 mmol) of cis- and trans-methyl 
4-hydroxycyclohexane carboxylate (Noyce, D. S. and D. B. Denney, J. Am. 
Chem. Soc. 74:5912 (1952)) in 45 mL of methylene chloride at 0.degree. C. 
was added 3.0 mL (26.0 mmol) of 2,6-butidine followed by 5.5 mL (23.8) 
mmol of tert-butyldimethylsilyl trifluoromethanesulfonate. The ice bath 
was removed and the reaction mixture was allowed to stir at 25.degree. C. 
for 2 h at which time the solution was poured into saturated sodium 
bicarbonate. The layers were partitioned and the organic layer was washed 
with saturated copper sulfate and water and then dried over MgSO.sub.4 to 
give 5.9 g of the crude methyl esters. A solution of 5.72 g (21.0 mmol) of 
this mixture in 45 mL of anhydrous THF was treated with 400 mg (10.5 mmol) 
of lithium aluminum hydride. The reaction mixture was stirred at 
25.degree. C. for 0.5 h and was then quenched by the slow addition of a 
saturated solution of Rochelle's salt. The mixture was diluted with ether, 
the layers were partitioned and the aqueous layer was washed twice with 
ethyl acetate. The combined organic extracts were dried over MgSO.sub.4 
and concentrated to give 4.9 g of the diastereomeric alcohols. Flash 
chromatography (elution with 1: 5 ethyl acetate-hexane ) gave 650 mg of 
(136), 1.10 g of (137) and 2.40 g of a mixture of the two. Data for (136): 
.sup.1 H NMR (300 MHz, CDCl.sub.3) .delta. 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): .sup.1 H 
NMR (300 MHz, CDCl.sub.3) .delta. 3.47 (dddd), 3.38 (d), 1.86-1.67 (m), 
1.47-1.16 (m), 1.05-0.77 (m), 0.72 (s), -0.02 (s). 
4. (E)-Ethyl 
3-[cis-(4-tert-Butyldimethylsilyloxycyclohexyl)]-2-methylprop-2-enoate 
(138). 
To a -78.degree. C. solution of oxalyl chloride (465 .mu.L, 5.33 mmol) in 
5.0 mL of methylene chloride was added dimethylsulfoxide (755 .mu.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 -78.degree. C. for 45 min at which time 
2.2 mL (16.0 mmol) of triethylamine was added and the solution was allowed 
to warm to ambient temperature. The reaction was quenched with 1.0 N HCl 
and the aqueous layer was extracted with three portions of methylene 
chloride. The combined organic extracts were dried over MgSO.sub.4 and 
evaporated to dryness to give 620 mg of the intermediate aldehyde which 
was treated directly with 1.22 g (3.36 mmol) of 
(carbethoxyethylidine)triphenylphosphorane in 5.0 mL of methylene 
chloride. The resulting reaction mixture was stirred at ambient 
temperature overnight and was then poured into water. The layers were 
partitioned and the aqueous layer was extracted twice with methylene 
chloride. The combined organic layers were dried over MgSO.sub.4 and 
concentrated 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-[Cis-(4-tert-Butyldimethylsilyloxycyclohexyl)]-2-methylprop-2-en-1-o 
l (139) 
To a solution of 300 mg (0.95 mmol) of enoate (138) in 2.0 mL of anhydrous 
tetrahydrofuran at 25.degree. C. was added 18 mg (0.43 mmol) of lithium 
aluminum hydride and the resulting mixture was allowed to stir for 30 min. 
The reaction was quenched by the slow addition of saturated Rochelle's salt 
and diluted with ethyl acetate. The layers were separated and the aqueous 
layer was extracted with two portions of ethyl acetate. The combined 
organic extracts were washed with water and brine and then dried over 
MgSO.sub.4. Evaporation and flash chromatography (elution with 1:10 ethyl 
acetate-hexane) gave 220 mg of the allyic alcohol (139). .sup.1 H NMR (300 
MHz, CDCl.sub.3) .delta. 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), 0.0 (s). 
6. (E)-S-[cis-(4-tert-Butyldimethylsilyloxycyclohexyl)]-2-methylprop-2-enyl 
N-(phenylglyoxyl)pipecolate (140) 
To a solution of 68 mg (0.24 mmol) of allylic alcohol (139), 42.3 mg (0.16 
retool) of-acid (135) and 39.8 mg (0.20 retool) of 
1-(3-dimethylamino-propyl)-3-ethylcarbodiimide 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 partitioned and the aqueous layer was extracted twice with 
methylene chloride. The combined organic extracts were dried over 
MgSO.sub.4 and concentrated to yield a yellow oil. Flash chromatography 
(slution with 15% ethyl acetate in hexane) gave 9.2 mg of the ester (140) 
as a rotameric mixture. .sup.1 H NMR (500 MHz, CDCl.sub.3) .delta. 8.01 
(d) , 7.94 (d) , 7.59-7.52 (m), 7.46-7.39 (m), 7.19 (d), 7.12 (d), 6.82 
(d), 6.51 (s), 6.38 (s), 5.43 (d), 4.78 (ABq), 4.62 (dd), 4.58 (s), 4.41 
(d), 3.51 (dd), 3.23 (ddd), 3.01 (ddd), 2.41 (d), 2.24 (d), 1.91 (s), 
1.84-1.76 (m), 1.65-1.48 (m), 0.96 (s), 0.18 (s). 
7. (E)-3-[cis-4-(hydroxycyclohexyl)]-2-methylprop-2-enyl 
N-(phenylglyoxyl)-pipecolate (25) 
To a solution of 9.2 mg (0.02 mmol) of ester (140) in 1.0 mL of 
acetonitrile at 25.degree. C. was added dropwise a solution consisting of 
a 95:5 mixture of acetonitrile: 48% hydrofluoric acid and the reaction was 
stirred until thin layer chromatography (TLC) indicated the disappearance 
of starting material. The reaction was quenched by the addition of 
saturated potassium carbonate. The reaction mixture was extracted with 
three portions of ethyl acetate, dried over MgSO.sub.4 and concentrated. 
Flash chromatography (slution with 35% ethyl acetate in hexane ) yielded 
6.1 mg(82% ) of the ester (25 ) as a rotameric mixture. .sup.1 H NMR (500 
MHz, CDCl.sub.3 ) .delta. 8.02 (dd), 7.97 (dd), 7.67-7.59 (m), 7.56-7.48 
(m), 5.49 (d), 5.44 (d), 4.61 (ABq), 4.44 (s), 4.41 (d), 3.98-3.91 (m), 
3.5 (br d), 3.26 (ddd), 3.11 (ddd), 2.43-2.18 (m), 1.86-1.37 (m), 1.72 
(d), R.sub.f 0.57 (3:1 ethyl acetate-hexane). 
EXAMPLE 2 
Synthesis of (S)-Benzyl N-(phenylglyoxyl)pipecolate (3) 
To a solution of 43 mg (0.19 mmol) of freshly generated (S)-Benzyl 
pipecolate (133) (described in Example 1) in 2.0 mL of anhydrous methylene 
chloride was added 44 mg (0.29 retool) of benzoylformic acid and 56 mg 
(0.29 mmol) of 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide 
hydrochloride (EDC) and the resulting mixture was stirred overnight at 
room temperature. The reaction mixture was then poured into water, the 
layers partitioned and the aqueous layer was extracted twice with 
methylene chloride. The combined organic extracts were dried over 
MgSO.sub.4 and concentrated to yield a yellow oil. Flash chromatography 
(slution with 1:3 ether-hexane) gave 49 mg (72%) of the keto-amide (3) as 
a rotameric mixture. .sup.1 H NMR (500 MHz, CDCl.sub.3) .delta. 7.98 (d) , 
7.91 (d) , 7.58 (t) , 7.41-7.30 (m), 5.45 (d), 5.21 (ABq), 5.06 (ABq), 4.61 
(dd), 4.42 (d), 3.48 (dd), 3.19 (ddd), 2.96 (ddd), 2.40 (d), 2.21 (d), 
1.83-1.72 (m), 1.61-1.49 (m), 1.46-1.33 (m), R.sub.f 0.55 (1:1 
ether-hexane). 
EXAMPLE 3 
Synthesis of (S) -Benzyl N-[(3-methoxyphenyl)glyoxyl)]-pipecolate (9) 
The keto-amide (9) was prepared from 45 mg (0.205 mmol) of (S)-Benzyl 
pipecolate (133) (described in Example 1 ) and 55 mg(0.306 mmol) of 
3-methoxybenzoylformic acid (Barnish, T. et al., J. Med. Chem. 24:339 
(1981)) as described in Example 2. Flash chromatography (elution with 1:4 
ether-hexane) gave 73 mg (93%) of (9) as a rotameric mixture. .sup.1 H NMR 
(300 MHz, CDCl.sub.3 ) .delta. 7.59-7.10 (m), 5.42 (d), 5.23 (ABq), 5.09 
(ABq), 4.59 (dd), 4.38 (d), 3.82 (s), 3.81 (s), 3.48-3.40 (m), 3.20 (ddd), 
2.98 (ddd), 2.39 (d), 2.21 (d), 1.82-1.70 (m), 1.61-1.22 (m), R.sub.f 0.45 
(1:1 ether-hexane). 
EXAMPLE 4 
Synthesis of (S)-Benzyl N-(2-furylglyoxyl)pipecolate (20) 
To a 0.degree. C. solution of 412 mg (1.03 mmol) of (S)-Benzyl pipecolate 
salt (134) (described in Example 1) in 40 mL of acetonitrile was added 198 
.mu.L (1.14 retool) of diisopropylethylamine, 174 mg (1.24 retool) of 
.alpha.-oxo-2furanacetic acid, 579 mg (1.24 mmol) of 
benzotriazol-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate 
and then 216 .mu.L (1.24 mmol) of diisopropylethylamine.. The resulting 
reaction mixture was stirred at ambient temperature for 14 h and then 
evaporated to dryness. The residue was dissolved into 150 mL of ethyl 
acetate, washed sequentially with 50 mL of 0.5 N HCl, 50 mL of saturated 
NaHCO.sub.3, 50 mL of brine and was then dried over MgSO.sub.4 and 
concentrated. Flash chromatography (elution with 2% ether in methylene 
chloride) provided 163 mg (46%) of the keto-amide (20) as an oil. The 
.sup.1 H NMR spectrum of this compound (300 MHz, CDCl.sub.3) was 
consistent with the product as a mixture of rotamers. R.sub.f 0.2 (2% 
ether in methylene chloride). HPLC, Rt=12.83 min. 
EXAMPLE 5 
Synthesis of (S)-Benzyl N-(4-Methoxycinnamoyl)pipecolate (112) 
To a solution of 145 mg (0.37 mmol) of (S)-Benzyl pipecolate salt (134) 
(described in Example 1) in 8.0 mL of methylene chloride was added 102 mg 
(0.57 mmol) of 4-methoxycinnamic acid, 107 mg (0.55 mmol) of 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 130 
.mu.L (0.74 mmol) of diisopropylethylamine. The resulting solution was 
stirred at ambient temperature for 12 h and was then concentrated under 
reduced pressure. Flash chromatography (slution with 1:1 ethyl 
acetate-hexane) gave 91 mg (65%) of the amide (112) as a colorless oil. 
.sup.1 H NMR (300 MHz, CDCl.sub.3) .delta. 7.66 (d), 7.50 (d), 7.35 (m), 
6.90 (d), 6.82 (d), 6.63 (d), 5.55 (d), 5.20 (br s), 4.86 (br s), 4.67 (br 
d) 4.03 (br d), 3.83 (s), 3.37 (dr), 2.78 (dt) 2.33 (br d), 1.74 (m), 1.43 
(m). R.sub.f 0.40 (1:1 ether-hexane). 
EXAMPLE 6 
Synthesis of (S)-3-(3,4-Methylenedioxyphenyl)-prop-2enoylproline benzyl 
ester (109) and (S)-3-(3,4-Methylene-dioxyphenyl)-prop-2-enoylalanine 
benzyl ester (132) 
A solution of 192 mg (1.0 mmol) of 3,4-methylenedioxycinnamic acid, 121 mg 
(0.5 mmol) of proline benzyl ester hydrochloride and 108 mg (0.5 mmol) of 
alanins benzyl ester hydrochloride in 6 mL of acetonitrile was treated 
sequentially with 0.35 mL (2.0 mmol) of diisopropylethylamine and 443 mg 
(1.0 mmol) of benzotriazol-1-yloxytris(dimethylamino)phosphonium 
hexafluorophosphate. The mixture was stirred for 16 hrs then concentrated 
in vacuo. The residue was dissolved in 10 mL of dichloromethane and poured 
into 3 volumes of diethyl ether. The mixture was washed sequentially with 
water, 10% potassium bisulfate solution, water, saturated sodium 
bicarbonate solution, water and saturated sodium chloride solution. The 
organic layer was dried over magnesium sulfate, filtered and concentrated 
in vacuo. The residue was purified by silica gel column chromatography, 
using a stepwise gradient of 30%, 35%, 40%, and 45% ethyl acetate in 
hexane as eluant. Ester (109) (142 mg) was obtained as a colorless oil, 
R.sub.f 0.4 (40% ethyl acetate/hexane); HPLC, R.sub.t =12.26; 1H NMR (300 
MHz) consistent with structure. Ester (132) (162 mg) was obtained as a 
colorless oil; TLC, R.sub.f =0.22 (40% ethyl acetate/hexane); HPLC, 
R.sub.t =11.84 min; .sup.1 H NMR (300 MHz) consistent with structure. 
EXAMPLE 7 
Synthesis of (S)-N-3-(4-methoxyphenyl)-prop-2-enoyl-N-methylalanine benzyl 
ester (129) 
1. (S)-N-Methylalanine benzyl ester p-toluene sulfonic acid salt (141) 
A suspension of 1.55 g (15.0 mmol) of (S)-N-methylalanins and 9.31 (90.0 
mmol) of benzyl alcohol in 30 mL of toluene was treated with 3.00 g (15.8 
mmol) of p-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 into 200 mL of ether, 
precipitating a yellow oil. The solvents were decanted and the residue was 
taken up into ethyl acetate and concentrated to yield a viscous, light 
yellow oil (141); TLC: R.sub.f =0.34, 95:5:0.5 CH.sub.2 Cl.sub.2 
/MeOH/concentrated ammonium hydroxide; .sup.1 H NMR (300 MHz) consistent 
with structure. 
2. (S)-N-3-(4-methoxyphenyl)-prop-2-enoyl-N-methylalanine benzyl ester 
(129) 
A suspension of 96 mg (0.5 mmol) of 4-methoxycinnamic acid and 121 mg (0.5 
mmol) of (141) in 6 mL of dichloromethane was cooled in an ice/water bath 
under nitrogen. The mixture was treated with 0.26 mL (1.5 mmol) of 
diisopropylethylamine and 135 mg (0.53 mmol) of 
N,N-bis-(2-oxo-3-oxazolidinyl) phosphinic chloride and then stirred for 16 
hours, warming slowly to ambient temperature. The mixture was poured into 
three volumes of diethyl ether and washed sequentially with water, 10% 
potassium bisulfate solution, water, saturated sodium bicarbonate 
solution, water, and saturated sodium chloride solution. The organic layer 
was dried over magnesium sulfate, filtered, and concentrated in vacuo. The 
residue was purified by preparative thick layer silica gel chromatography 
using 40% ethyl acetate/hexane as eluant. Ester (129) (40 mg) was obtained 
as a yellow oil : TLC, R.sub.f =0.24 (35% ethyl acetate/hexane); HPLC, 
R.sub.t =13.34 min; .sup.1 H NMR (300 MHz) consistent with structure. 
EXAMPLE 8 
Synthesis of (S)-N-Methyloxalyl-N-ethylalanine benzyl ester (127) 
1. (S)-N-9-Fluorenylmethoxycarbonyl-N-ethylalanine benzylester (142) 
A suspension of 848 mg (2.5 mmol) of (S)-N-Fmoc-N-ethylalanine in 10 mL of 
dichloromethane was treated with 436 .mu.L (5.0 mmol) of oxalyl chloride 
followed by a catalytic amount (1 drop) of dimethylformamide. The mixture 
was stirred for one hour, then concentrated in vacuo. The yellow, oily 
residue was treated with 10 mL of toluene followed by 517 mg (0.5 mmol) of 
benzyl alcohol and 669 mg (5.0mmol) of silver cyanide. The mixture was 
heated in an 80.degree. C oil bath with vigorous stirring for 20 minutes, 
then cooled and filtered through a pad of diatomaceous earth. The filtrate 
was concentrated, and the residue was purified by silica gel column 
chromatography, using 10% acetone in hexane as eluant. Ester (142) (810 
mg) was obtained as a colorless oil; TLC: R.sub.f =0.28, 15% 
acetone/hexane; .sup.1 H NMR (300 MHz) consistent with structure. 
2. (S)-N-Methyloxalyl-N-ethylalanine benzyl ester (177) 
A solution of 0.25 g (0.58 mmol) of (142) in 3 mL of acetonitrile was 
treated with 3 mL of diethylamine and the mixture was allowed to stand for 
10 min. The mixture was concentrated in vacuo and the residue was taken up 
in 10mL of acetonitrile and again evaporated. After repeating this 
process, the residue was dissolved in 4 mL of dichloromethane, cooled in 
an ice/water bath under nitrogen, and treated with 111 .mu.L (0.64 mmol) 
of diisopropylethylamine 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 sequentially with water, 10% potassium 
bisulfate solution, water, saturated sodium bicarbonate solution, water, 
and saturated sodium chloride solution. The organic layer was dried over 
magnesium sulfate, filtered, and concentrated in vacuo. The residue was 
purified by silica gel column chromatography using 1:7 acetone: hexane as 
eluant. Ester (127) (147 mg) was obtained as a colorless oil, R.sub.f 
=0.36, 35% acetone/hexane; HPLC, R.sub.t =12.19 min; .sup.1 H NMR (300 
MHz) consistent with structure. 
EXAMPLE 9 
Synthesis of (S)-3-cyclopentylpropyl N-(2-Methyloxalyl)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 (S)-N-(Phenylglyoxyl)pipecolic acid (135). 
Thus, 3.16 g (11.32 retool) of the tartrate salt of (S)-pipecolic acid and 
1.19 mL (12.45 mmol) of methyl oxalyl chloride gave 1.25 g (51%) of the 
acid (143) as a tan solid. 1M NMR (300 MHz, CDCl.sub.3) a 5.31 (d), 4.62 
(d), 4.49 (br d), 3.61 (br d), 3.90 (s), 3.88 (s), 3.46 (dr), 2.97 (dt), 
2.40-1.40 (m). 
2. (S)-3-cyclopentylpropyl N-(2-Methyloxalyl)pipecolate (35) 
The ester (35) was prepared from 3-cyclopentylpropan-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 .sup.1 H NMR spectrum of this compound (300 MHz, CDCl.sub.3) was 
consistent with the product as rotamers. R.sub.f 0.56 (10% ether in 
methylene chloride). HPLC, R.sub.t =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 MIr-negative and hepatitis 
negative) are isolated and separated by density centrifugation over 
Mistopaque 1077 (Sigma Chemical Co., St. Louis, MO). The murine CTLL 
cytotoxic T cell line and the human Jurkat T cell line are from ATCC 
(CTLL-2 ATCC TIB214, JURKAT CLONE E6-1 ATCC TIB152). The human allogeneic 
B cell lines used for activation of the fresh PBLs are EBV-transformed 
lymphocytes from normal healthy adult donors with two completely different 
HLA haplotypes. All cell lines were routinely tested for the presence of 
Mycoplasma contamination using the Gibco Mycotect test kit and are 
Mycoplasma-free. Culture medium consists of RPMI 1640 (Gibco, Grand 
Island, N.Y.) containing penicillin (50 U/ml) and streptomycin (50 
.mu.g/ml), L-glutamine 2 mM, 2 mercaptoethanol (5.times.10.sup.-5), 10% 
heat-inactivated FCS and 10 mM HEPES. 
Compound Solutions and Titrations 
All chemical stocks were dissolved in DMSO. 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, using multiple 
three-fold dilutions from 1 .mu.M or 10 .mu.M stock solutions. 
MTT Assay 
The MTT assay is a colorimetric technique to determine the toxicity of the 
compounds on growing lymphold and non-lymphoid cell lines based on 
reduction of the tetrazolium salt by intact mitochondria (Mossman, T., J. 
Immunol. Methods 65:55 (1983)). Cell viability in the presence or absence 
of different concentrations of test compounds in serum-free medium (HB 
104, HANA Biologic, Inc.) was assessed using MTT 
(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 .mu.l of MTT 
dye (5 mg/ml in pH 7.2 PBS) were added to each microtiter well. At the end 
of the incubation time, most of the culture media was carefully aspirated 
out of each well. Then 100 .mu.l of acidified isopropyl alcohol (0.04 N 
HCl). was added to solubilize the dye and optical density is read at 570 
nm minus OD at 630 nm (Molecular Devices Thermomax plate reader and 
Softmax software program, Menlo Park, Calif.). Results were compared with 
mean OD in controls (medium with no drugs) and doses causing 50% toxicity 
(TO.sub.50) were calculated. 
Mitogenesis Assays ("PMA" and "OKT3") 
The inhibitory effect of test compounds on the proliferation of human PBLs 
in response to mitogens (Waithe, W. K. and K. Hirschhorn, Handbook of 
Experimental Immunology, 3d Ed. Blackwell Scientific Publications, Oxford 
(1978); Mishell, B. B. and S. M. Shiigi, Selected Methods in Cellular 
Immunology W. H. Freeman and Co., San Francisco, Calif. (1980)) was 
assessed by stimulation of 5.times.10.sup.4 cells with OKT3 (10.sup.-4 
dilution final) or PMA (10 ng/ml) plus ionomycin (250 ng/ml) in the 
presence or absence of different concentrations of test compounds and 
control drugs (CsA, FK506, Pagamycin) in final volume of 200 .mu.l per 
well in 96 well round bottomed plates. After 48 h incubation (37.degree. 
C., 5% CO.sub.2), cells were pulsed with 1 .mu.Ci of .sup.3 H-thymidine, 
harvested 24 h later with a Tom Tek cell harvester, and counted in LKB 
.beta.-scintillation counter. Results (cpm) were compared with controls 
with medium alone, and concentrations causing 50 % reduction in counts 
(IC.sub.50) were calculated. 
MLR Bioassays ("LB" and "JVM") 
Antigen activated proliferation of PBLs in a primary mixed lymphocyte 
reaction was assessed in the presence or absence of different 
concentrations of tested compounds and control drugs. 5.times.10.sup.4 
fresh PBLs were stimulated with 5.times.10.sup.3 of Mitomycin C 
treated-allogeneic EBV-transformed .beta.-lymphoglastoid cells, LB and 
JVM, in a final volume of 200 .mu.l per well in 96-well round-bottomed 
plates (Mishell, B. B. and S. M. Shiigi, Selected Methods in Cellular 
Immunology W. H. Freeman and Co., San Francisco, Calif. (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 section. 
IL-2 Microassay ("CTLU") 
To determine if test compounds inhibit the later T cell activation process 
of cytokine utilization, the proliferative response of the IL-2 dependent 
CTLL-20 murine T cell line (ATCC) was assessed (Gillis, S. et al., J. 
Immunology 120:2027 (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 of the CTLLs, 
and CsA and FK506 do not (Dumont, F. J. et al., J. Immunology 144:251 
(1990)). 3.times.10.sup.3 CTLLs were exposed to different concentrations 
of test compounds and control drugs in the presence of I U/ml of human 
recombinant IL-2 (Genzyme, rIL-2) for 24 h. Four h after adding drugs, 
cells were pulsed with 1 .mu.CI of 3H-thymidine, incubated for an 
additional 20 h (37.degree. C., 5% CO.sub.2), and then harvested and 
counted as previously described. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain, using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.