Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid

Article abstract of DOI:10.1021/jm9603633

Structure-activity relationships in the region of the phthalide ring of the inosine monophosphate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the lactone ring with other cyclic moieties resulted in loss of potency, especia

Full text of DOI:10.1021/jm9603633

J . Med. Chem. 1996, 39, 4181-4196
4181
Str u ctu r e-Activity Rela tion sh ip s for In h ibition of In osin e Mon op h osp h a te
Deh yd r ogen a se by Nu clea r Va r ia n ts of Mycop h en olic Acid 1
Peter H. Nelson,† Stephen F. Carr,‡ Bruce H. Devens,§ Elsie M. Eugui,§ Fidencio Franco,| Carlos Gonzalez,|
Ronald C. Hawley,† David G. Loughhead,*,† David J . Milan,† Eva Papp,‡ J ohn W. Patterson,† Sussan Rouhafza,§
Eric B. Sjogren,† David B. Smith,† Rebecca A. Stephenson,† Francisco X. Talamas,| Ann-Marie Waltos,†
Robert J . Weikert,† and J ohn C. Wu‡
Institutes of Organic Chemistry, Immunology and Biological Sciences, and Biochemistry and Cell Biology, Syntex Research,
3401 Hillview Avenue, Palo Alto, California 94304
Received May 20, 1996X
Structure-activity relationships in the region of the phthalide ring of the inosine monophos-
phate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the
lactone ring with other cyclic moieties resulted in loss of potency, especially for larger groups.
Replacement of the ring by acyclic substituents also indicated a strong sensitivity to steric
bulk. A phenolic hydroxyl group, with an adjacent hydrogen bond acceptor, was found to be
essential for high potency. The aromatic methyl group was essential for activity; the methoxyl
group could be replaced by ethyl to give a compound with 2-4 times the potency of mycophenolic
acid in vitro and in vivo.
Mycophenolic acid (MPA) (1a ) is produced by fermen-
tation of several penicillium species.2 It has diverse in
vitro and in vivo biological activities, including antifun-
gal,3 antibacterial,4 antiviral,5 and immunosuppressive6
properties. Mycophenolic acid itself has been tested
clinically against various tumors without success7 and
has been found to be effective as an antipsoriatic.8 The
compound is an inhibitor of inosine-5′-monophosphate
dehydrogenase (IMPDH, EC 1.1.1.205), and this inhibi-
tion is believed to mediate the above-described biological
properties.9 IMPDH catalyzes the NAD-dependent
conversion of inosine-5′-phosphate to xanthosine-5′-
phosphate and is thus a key enzyme in the de novo
synthesis of guanine nucleotides. IMPDH exists in type
I and type II isoforms;10 the type II isoform is selectively
upregulated during cell proliferation.11 Since, unlike
F igu r e 1.
many other cell types, lymphocytes are dependent on
de novo purine biosynthesis,12 inhibitors of IMPDH
ships (SAR) associated with changes in the nucleus of
would be expected to be immunosuppressive without
MPA. The goal of the work has been to identify
exhibiting general cytotoxicity. Thus we have sought
compounds with increased in vitro and in vivo immu-
MPA analogs or derivatives with high IMPDH inhibi-
nosuppressive potency, preferably with simpler struc-
tory potency for the potential therapy of autoimmune
tures more amenable to total synthesis than MPA itself.
diseases and for the prevention of allograft rejection.
We have also prepared compounds designed to increase
Mycophenolate Mofetil (RS-61443),13 a prodrug of MPA,
the in vivo potency by alteration of the metabolism.
has been found to be clinically effective in rheumatoid
Although MPA has an IC50 of ca. 20 nM against IMPDH,
arthritis14 and, following clinical trials for the reversal
effective immunosuppressive doses in both animals17
and prevention of kidney and heart transplant rejec-
and man7 are in the range of 10-100 mg/kg. MPA is
tion,15 was approved in the United States in 1995 for
well absorbed after oral administration, but it is rapidly
use by renal transplant patients.
converted into the biologically inactive phenolic glucu-
We have previously attempted to increase the inhibi-
ronide in several species which have been examined,
tory potency of mycophenolic acid against IMPDH by
including man,18 and subsequently excreted as such.
modification of the side-chain moiety, particularly by
Since removal of this mode of inactivation and excretion
replacement of the double bond by other groups.16
might result in compounds with higher in vivo potencies,
Inhibitory potency was found to be very sensitive to
we have prepared a number of compounds in which the
small changes in the side-chain structure. In the
phenolic hydroxyl has been replaced by groups not
present paper we report on structure-activity relation-
susceptible to glucuronidation. Other workers have
described a limited number of MPA analogs in which
* Send correspondence to this author at Roche Bioscience, 3401
the phthalide nucleus has been either replaced or
Hillview Ave., Palo Alto, CA 94304.
modified, though often without biological data.19 The
somewhat fragmentary published data suggest that
changes in the nucleus, as in the side chain, result in
considerable loss of potency.
† Institute of Organic Chemistry.
‡ Institute of Biochemistry and Cell Biology.
§ Institute of Immunology and Biological Sciences.
| Divisio´n de Investigacio´n, Syntex S.A. de C.V., Cuernavaca, Me´xico.
X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0022-2623(96)00363-9 CCC: $12.00 © 1996 American Chemical Society

4182 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nelson et al.
Sch em e 1
Sch em e 2
Sch em e 3
products (1v,w ) were prepared from the ester of 7-bromo
compound 1t (Scheme 7). Synthesis of most of the
monocyclic phenols 2 was achieved by preparation of
the appropriately substituted phenolic precursor and
then attachment of the side chain as in Scheme 3.
Other compounds of this type were made from MPA,
which upon prolonged treatment with strong aqueous
base afforded the decarboxylated product 27a (Scheme
8). Following reductive deoxygenation to give 2a , vari-
ous substituents X (Table 2) were introduced.
The non-phenols 3a -s were synthesized analogously
to Scheme 3, except that the phenolic oxygen atom in
the Claisen product (12) became part of the methoxyl
group in the final product. For the amines 4, another
variant of the ortho-ester Claisen methodology, shown
in Scheme 9, was employed. In this route, the side
chain was derived from an o-methylaniline (28). Con-
version to the t-BOC derivative 29 facilitated metalation
with tert-butyllithium,21 and reaction of the resultant
benzylic anion with methacrolein then afforded the
carbinols 30 which underwent ortho-ester Claisen rear-
rangement to give the products 31.
Ch em istr y
The compounds synthesized can be divided into four
groups: structures 1, in which the lactone ring has been
replaced by other cyclic moieties or in which the
aromatic 6-methoxy and 7-methyl groups have been
varied; monocyclic phenols 2; monocyclic non-phenols
3; and monocyclic amines 4. The majority of the analogs
were made by total synthesis and some by degradation/
modification of mycophenolic acid. The bicyclic com-
pounds 1 were made by both approaches: Trimethyl-
aluminum-mediated aminolysis of the protected MPA
derivative 5a gave the ring-opened benzyl alcohol
intermediate 6a (Scheme 1). Conversion of the alcohol
to the mesylate 6b, followed by displacement with
nucleophiles, then allowed construction of the new
heterocycles 1b,e,f (Table 1).
Biologica l Resu lts
Alternatively, modified nuclei were made by Diels-
Alder reactions (Scheme 2) to afford the dihydroaro-
matic products 9, which were aromatized to the phenols
10. The side chain was then introduced by a previously
published route, in which the critical step is conversion
of the allylic carbinol 14 to esters 15 using the ortho-
ester Claisen rearrangement (Scheme 3).20 Most of the
6-substituted compounds 1h -q were made from differ-
entially protected intermediates such as 16d (Scheme
4), from which the triflate group could be displaced by
nucleophiles or replaced by coupling with organostan-
nanes under palladium catalysis to introduce carbon
substituents (vinyl, cyclopropyl, phenyl). The 6-methyl
(1m ) and 7-ethyl (1s) analogs were made by total
syntheses (Schemes 5 and 6). Two of the 7-substituted
Compounds were tested as inhibitors of human re-
combinant type II IMPDH.22 Results (Tables 1-4) are
expressed as micromolar IC50 concentrations. Most of
the compounds were also assayed as inhibitors of
mitogen-induced human peripheral lymphocyte prolif-
eration,16 an assay which is an in vitro model for in vivo
immunosuppression but independent of absorption,
metabolism, and excretion parameters which affect in
vivo potencies. In general, results from the two assays
correlate, although potencies in the cell proliferation
assay were always somewhat lower than for enzyme
inhibition. A number of the more potent compounds
were also tested in vivo for immunosuppressant activity.
The bicyclic analogs 1b-g were all less potent inhibi-
tors of IMPDH than the parent 1a . Replacement of the

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4183
Sch em e 4
Sch em e 5
Sch em e 7
Sch em e 8
Sch em e 9
Sch em e 6
which was more than 400-fold less potent than the NH
compound 1e, a result which suggests a strong sensitiv-
ity to steric bulk in this area of the molecule. The
N-methyl group occupies a volume which is not explored
in other analogs, and its low inhibitory potency suggests
that it cannot be accommodated at the IMPDH active
site. Increasing the size of the lactone ring in 1a to 6
(1d ) diminished the potency 25-fold. Addition of a
methyl substituent to the phthalide methylene (1g) also
resulted in a marked decrease in potency attributable
to a steric effect. More encouraging results were
lactone oxygen with sulfur (1b), CH2 (1c), or NH (1e)
resulted in a 5-10-fold reduction in potency. Replace-
ment with the larger NCH3 group gave a compound (1f)

4184 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Ta ble 1. Bicyclic Compounds
Nelson et al.
compd
A
B
C
D
IMPDH IC50 (µM)
ASEa
lc prolifn IC50 (µM)
0.058 ( 0.003
1a
O
S
H
CH3
OCH3
0.0251b (31)c
0.0248d (50)
0.121d (3)
0.140b
0.0092
0.00044
0.069
0.042
0.231
1b
1c
1d e
1e
1f
1g
1h g
1ig
1j
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OC2H5
H
CHdCH2
C2H5
1.7
0.62, 0.83
8.6
CH2
OCH2
NH
NCH3
O
0.644d
0.244d
0.244
1.50
>100d,f
>10f
0.60
0.420d
0.139
0.0105
0.0273
0.624
0.0036
0.0025
0.0018
0.0036
0.018
2.93
0.043
0.137
0.050
0.152
0.0097
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
0.0877d (2)
0.0898d (2)
6.96d
>10f
0.43, 0.43
NT
1k
1l
0.00851b
0.0126b (2)
0.0186d (2)
0.0507b
0.093
0.057, 0.028
0.25, 0.11
0.34
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
CH3
cyclopropyl
C6H5
0.130b
2.8
CN
15.5b
>10f
>10f
NT
CONH2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
0.231b
0.515d
C2H5
Br
OCH3
CN
0.410d
3.0
4.0
0.975d
0.126d (2)
>100d,f (2)
>100d,f (2)
0.13
>10f
CONH2
>10f
a
b
d
Asymptotic standard error for IC50
.
Measured at pH 7.4. c Number of determinations if >1. Measured at pH 8.0. e Number system
for compound 1d :
f Compounds were tested to 100 and 10 µM, respectively, in the IMPDH and lc prolifn assays. If no inhibition was observed, potency is
g
designated as >100 and >10. Otherwise IC50 values are reported. See ref 19b.
obtained by variations at the 6 (methoxyl)-position.
Replacement of the methoxyl with vinyl, ethyl, or
methyl (1k -m ) gave products with equal or higher
inhibitory potencies to MPA. Larger groups such as
ethoxy (1i),19b,23 cyclopropyl (1n ), or phenyl (1o) or a
smaller one such as hydrogen (1j) were much less
potent. The 6-phenyl compound 1o was less potent in
the lymphocyte proliferation assay than would be
expected from the IMPDH potency, an effect which may
be due to less facile penetration through the cell
membrane by this more lipophilic compound. The high
potency of the 6-ethyl compound 1l suggests that the
6-substituent does not interact with IMPDH by hydro-
gen bonding and, in view of the low potency of the
6-unsubstituted compound 1j, suggests that a major
function of the 6-substituent is to influence the orienta-
tion of the adjacent side chain. The lower potency of
the 6-cyano (1p ) and 6-carbamoyl (1q) compounds can
be ascribed to the undesirability of dipolar character at
this locus and/or to an unfavorable reduction in electron
density at the lactone carbonyl group. Variations at the
7-position indicated that the methyl group present in
MPA was the optimum. Larger (ethyl, 1s; methoxyl,
1u ) or smaller (H, 1r ) groups resulted in a ca. 10-50-
fold drop in potency. Electron-withdrawing substituents
(CN, CONH2) gave inactive compounds, either through
a local effect on binding or due to increased acidity of
the phenol moiety.
the MPA phthalide. In most cases, the aromatic methyl
and methoxyl groups present in MPA were retained so
as to allow the effect of single structural changes to be
assessed. The results for the monocyclic phenols (Table
2) also suggest sensitivity to steric bulk, though other
factors are also relevant. Compounds 2a -l differ only
in the substituent adjacent to the phenolic hydroxyl
group, yet the IMPDH inhibitory potencies range over
more than 3 orders of magnitude. The unsubstituted
phenol 2a is of low potency; introduction of a methyl
group into the ortho position (2b) results in a 60-fold
increase. Yet isosteric24 replacement of the methyl
group with chloro (2d ) causes a further 1 order of
magnitude increase in potency, indicating a major effect
not related to steric bulk. The chlorophenol 2d is the
most potent non-phthalide analog of MPA yet reported,
showing 0.7 times the potency of MPA in inhibition of
IMPDH. Rationalization of the significant differences
in potency between the analogs is difficult, though some
trends are discernible. Two criteria for high potency
are that the substituent X can function as a hydrogen
bond acceptor and it must be neither too large (iodo,
2f) nor too small (fluoro, 2c). Based on these criteria,
the formyl-substituted compound 2i was expected to be
among the most potent analogs, yet it was about 30-
fold less potent than the almost isosteric chloro com-
pound 2d . We hypothesize that the intramolecular
H-bond in 2i is strong enough to greatly diminish,
relative to 1a , participation by the phenol hydroxyl
group in H-bond donation to the enzyme. Table 2 also
contains some compounds in which the aromatic meth-
Since replacement of the lactone ring with signifi-
cantly larger groups was not fruitful, we sought mono-
cyclic replacements whose bulk would not be larger than

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4185
Ta ble 4. Amino Compounds
Ta ble 2. Monocyclic Phenols
IMPDH
IC50 (µM)a
0.0248 (50)c 0.00044 0.058 ( 0.003
lc prolifn
IMPDH
lc prolifn
IC50 (µM)
compd
X
Y
ASEb
IC50 (µM)
compd
X
Y
IC50 (µM)a
ASEb
0.0248 (50)c 0.00044 0.058 ( 0.003
90.8
0.763
1a
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
N/A
H
CH3
F
Cl
Br
N/A
1a
4a
4b
4c
4d
4e
4f
N/A N/A
H
Br
Br
OCH3 51.8 (2)
OCH3 0.959 (2)
OCH3 1.08 (2)
OCH3 0.0335 (3)
OCH3 0.0367 (3)
OCH3 0.592
OCH3 0.184
OCH3 0.534
OCH3 1.12 (3)
OCH3 11.9
68.0
NT
3.7
4.1
0.20, 0.33
0.28, 0.26
4.9
NT
1.7
CH3
CH3
414
NT
NT
4.1, 2.3, 1.7
2.8
4.1
0.214
0.114
0.0065
0.0384
0.119
0.083
0.344
0.789
1.27
0.089
0.129
0.093
2.75
OCH3 0.462 (3)
NO2 OCH3 0.465
CN
Cl
OCH3 14.8
OCH3 0.489 (2)
I
0.236
4.1, 3.4, 1.4
NO2
CN
CHO
a
b
Measured at pH 8.0. Asymptotic standard error for IC50
.
c Number of determinations if >1.
10.0
CHdNOH
>10d
>10d
NT
CHdNOCH3 OCH3 52.8
4.35
1.27
comparable potencies of about 1 µM. As in previous
series, larger substituents such as methoxyl (3f) and
methylthio (3h ) or smaller ones such as H (3n ) or F (3k )
were not tolerated in the Y position. With a chloro at
Y, compounds of about equal potency were obtained by
replacing the hydroxyl with H (3a ), F (3m ), Cl (3l), or
CH3 (3s). In the phenol series, Cl (2d ) and CH3 (2b)
were greatly superior to F (2c) and H (2a ) as adjacent
substituents. Thus the phenolic hydroxyl group, whose
removal obviates the predicted principal route of me-
tabolism and excretion for the compounds, is essential
for high inhibitory potency.
CH3O
H
OCH3 11.9
CH3
CH3
CH3
CH3
>100d
NT
0.36, 0.87
1.65
Cl
Br
NO2
0.0693
0.620 (2)
0.289
0.0087
0.174
0.251
3.2
a
b
Measured at pH 8.0. Asymptotic standard error for IC50
.
d
c Number of determinations if >1. Compounds were tested to 100
and 10 µM, respectively, in the IMPDH and lc prolifn assays. If
no inhibition was observed, potency is designated as >100 and
>10. Otherwise IC50 values are reported.
Ta ble 3. Non-phenols
A small series of compounds in which the phenolic
hydroxyl group had been replaced by amino was pre-
pared (Table 4). The amines were 3-30-fold less potent
than the corresponding phenols where direct compari-
sons could be made (e.g., 4a vs 2m , 4b vs 2o, 4c vs 2e),
and the SAR appeared to be similar. Replacement of
the MPA phenolic hydroxyl with amino results in a
similar decrease in inhibitory potency.25,26
In summary, we have found that the MPA hydroxyl
group cannot be replaced without major loss in potency.
The adjacent substituent must be a hydrogen bond
acceptor, with chloro the optimum in size and/or H-bond
acceptor ability. These criteria suggest that the inhibi-
tors bind to IMPDH via H-bond donation at the hydroxyl
locus and by H-bond acceptance at the adjacent position.
In MPA itself, the lactone carbonyl group serves as the
H-bond acceptor. A small (>H) lipophilic substituent
is required at both the 6- and 7-positions. The role of
the 6-substituent may be to influence the conformation
of the side chain at C-5.
IMPDH
lc prolifn
compd
X
Y
IC50 (µM)a
ASEb
IC50 (µM)
1a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
N/A
H
H
H
H
H
H
H
H
N/A
0.0248 (50)c
0.537
0.795 (4)
1.22
0.00044
0.371
0.192
0.136
1.11
0.058 ( 0.003
6.7
Cl
Br
>10d
9.5
NT
NT
NT
NO2
NH2
OH
CH3O
CN
CH3S
CH3SO
F
17.0
>100d
50.0
11.1
0.366
3.62
1.60
NT
52.3
>10d
>10d
>10d
>10d
>10d
7.5
H
>100d
1.38
F
0.271
1.20
0.141
0.208
9.84
0.185
0.910
0.0837
0.357
0.957
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
CH3
F
Cl
Cl
H
11.8
0.557
0.437 (6)
18.8
1.80
2.88
0.681
1.04
1.70
>10d
>10d
>10d
>10d
>10d
>10d
NO2
OH
CN
Br
3s
Cl
Representative compounds were tested in vivo, in the
mouse J erne plaque assay.27 Animals were immunized
by intraperitoneal injection of sheep red blood cells
(SRBC). Oral administration of immunosuppressant
compounds for the 4 subsequent days reduced the in
vitro response of the spleen cells to SRBC in a dose-
dependent manner. The results are shown in Table 5.
The most potent compound was 1l in which the aromatic
methoxyl was replaced by ethyl. This compound is 2-4
times as potent as 1a in vitro and in vivo. The
corresponding methyl-substituted compound 1m was
considerably less potent, despite having in vitro poten-
cies comparable to 1l. In contrast, the 7-methoxyl
analog 1u showed higher in vivo potency than would
be predicted from its in vitro results. Monocyclic
compounds, whether phenols (2d ,e), amines (4c,f), or
a
b
Measured at pH 8.0. Asymptotic standard error for IC50
.
c Number of determinations if >1. Compounds were tested to 100
and 10 µM, respectively, in the IMPDH and lc prolifn assays. If
no inhibition was observed, potency is designated as >100 and
>10. Otherwise IC50 values are reported.
d
oxyl group has been replaced by methyl (2m -p ). These
compounds, which are more easily accessible, are 2-10-
fold less potent than the methoxyl analogs. In contrast,
replacement of the methoxyl group in MPA itself by
methyl (1m ) had no effect on inhibitory potency.
Replacement of the phenolic hydroxyl group with H
resulted in a 3-20-fold decrease in potency, depending
on the adjacent substituent (Table 3). Substituent
effects among the non-phenols were less pronounced
than for the phenols, however, and 10 compounds had

4186 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Ta ble 5. Inhibition of the Murine PFC Response
Nelson et al.
% inhibition, PFC/106 WBC @ daily dose (mg/kg)
compd
potency relative to 1a
100
75
50
25
12.5
6.25
ED50 (mg/kg)
1a
1l
1.0
3.6
0.5
0.5
0.3
0.3
0.3
0.3
0.3
0
81 ( 2
36 ( 4
20 ( 4
16 ( 4
25
33
9
93
73
1m
1u
2d
3b
4c
4f
3l
2e
3m
45
49
25
83, 44
0
0
36
30
42
32, 40, 28
5
0
0
0
4. Or th o-Ester Cla isen Rea r r a n gem en t. The carbinol
14 was dissolved in freshly distilled28 trimethyl orthoformate
(20 mL/g). Pivalic acid (0.1 mol equiv) was added, and the
solution was heated in a 90 °C bath. When the reaction was
complete (TLC, 2-8 h), the cooled solution was added to ether
and water. The organic phase was dried and evaporated and
the residue chromatographed to give the silyl ether/methyl
ester product 15. Yields were 40-70%.
compounds in which the phenolic hydroxyl had been
replaced by H (3b), Cl (3l), or F (3m ), had low in vivo
potencies. In general, in vivo potencies correlated with
the potencies in the lymphocyte proliferation assay. It
is apparent that the possible increase in plasma levels
and half-lives which might occur for the non-phenols is
not sufficient to overcome the considerable loss of
inhibitory potency caused by the replacement of the
phenolic group.
5. Desilyla tion .29
The silyl ether was dissolved in THF
(10 mL/g) at 0 °C, and 1.0 M tetrabutylammonium fluoride in
THF (1.0 mol equiv) was added. After 10 min the solution
was added to water and extracted with EtOAc. The extract
was dried and evaporated to give the free phenol. Yields were
>90%.
6. Ester Hyd r olysis. The methyl ester was dissolved in
dimethoxyethane or MeOH (30 mL/g), and a solution of
LiOH‚H2O (3 mol equiv) in an equal volume of water was
added. When hydrolysis was complete (0.5-2 h), the solution
was added to water and washed with ether. The aqueous
phase was acidifed with 2 N HCl and extracted with EtOAc.
The extract was dried and evaporated, and the residue was
recrystallized. Yields were 80-100%.
(E)-6-(4-H yd r oxy-6-m et h oxy-7-m et h yl-3-oxo-1,3-d ih y-
dr oben zo[c]th ioph en -5-yl)-4-m eth yl-4-h exen oic Acid (1b).
(a ) ter t-Bu t yl (E)-6-[6-Met h oxy-4-[(m et h oxyet h oxy)-
m et h oxy]-7-m et h yl-3-oxo-1,3-d ih yd r oisob en zofu r a n -5-
yl]-4-m eth yl-4-h exen oa te (5a ). To a 0 °C solution of methyl
mycophenolate (26.2 g, 78.3 mmol) in CH2Cl2 (350 mL) were
added diisopropylethylamine (19 mL, 109 mmol) and MEM
chloride (11.7 g, 94 mmol). After 3 d at room temperature the
solution was washed with water and brine, dried (MgSO4), and
concentrated to an oil. This material was dissolved in MeOH
(400 mL) and treated with 4 N aqueous KOH (80 mL). After
2 d the bulk of the MeOH was removed under reduced pressure
and the residue partitioned between EtOAc and cold 0.5 M
NaHSO4. The organic layer was washed with brine, dried
(Na2SO4), and concentrated to give the MEM ether of 1a (22
g, 69%), mp 90-91 °C (t-BuOMe). This material (10.5 g, 25.7
mmol) in EtOAc was treated with oxalyl chloride (4 mL, 46
mmol) and a trace of DMF. After gas evolution was complete
(ca. 1 h), the solution was concentrated, redissolved in ethyl
acetate (100 mL), and concentrated again. The residue was
dissolved in CH2Cl2 (30 mL) and added dropwise to a 0 °C
solution of tert-butyl alcohol (50 mL) and DMAP (4.7 g, 38.5
mmol) in CH2Cl2 (75 mL). The solution was allowed to warm
to room temperature and stir for 18 h. The reaction mixture
was partitioned between EtOAc and cold 1 M aqueous NaH-
SO4. The organic phase was dried and concentrated and the
residue chromatographed on silica gel to give 6.0 g of 5a as
an oil: NMR δ 1.37 (s, 9H), 1.79 (s, 3H), 3.37 (s, 3H), 3.46 (d,
J ) 6.9 Hz, 2H), 3.57 (m, 2H), 3.76 (s, 3H), 3.94 (m, 2H), 5.12
(s, 2H), 5.22 (br t, J ) 6 Hz, 1H), 5.43 (s, 3H).
Exp er im en ta l Section
Melting points are uncorrected. Flash chromatography was
performed with silica gel A 230-400. 1H NMR spectra were
obtained at 300 MHz in CDCl3 unless otherwise stated and
are reported in ppm downfield of TMS. Microanalyses were
within (0.4% of theory unless otherwise stated.
Gen er a l P r oced u r e for th e P r ep a r a tion a n d Cla isen
Rea r r a n gem en t of P h en olic Allyl Eth er s. The phenol (1
mol), K2CO3 (1.5 mol), and allyl bromide (1.5 mol) were stirred
in DMF (10 vol) at room temperature for 1-8 h. Water and
ether were added, and the organic phase was dried and
evaporated. Traces of DMF were removed by percolation
through silica gel. Yields of the ethers 11 were ca. 90%. The
allyl ether was dissolved in N,N-diethylaniline (30 mL/g), and
the solution was heated in a 200 °C oil bath under N2 until
reaction was complete (TLC, usually about 4 h). The cooled
solution was added to 2 N HCl and EtOAc. The organic phase
was washed with 2 N HCl and water, dried, and evaporated.
The residue was chromatographed on silica gel if necessary.
Yields of the rearrangement products 12 were 60-80%.
Gen er a l P r oced u r e for Con ver sion of a n Allyl Su b-
st it u en t in t o t h e MP A Sid e Ch a in . 1. Silyla t ion of
P h en ols. The phenol 12 (1 mol) was dissolved in DMF (10
mL/g), and imidazole (2.0 mol equiv) and tert-butylchlorodi-
methylsilane (1.5 mol equiv) were added. After 8-24 h, water
and ether were added. The organic phase was washed with 2
N HCl, dried, and evaporated. The silyl ether was usually
used without purification.
2. Ozon olysis of th e Allyl Gr ou p to th e Cor r esp on d in g
Ar yla ceta ld eh yd e. The allyl compound was dissolved in 1:1
CH2Cl2:MeOH (50 mL/g) containing pyridine (0.25%). The
solution was cooled to -78 °C, and ozonized O2 was passed
through until a blue color was present. N2 was then bubbled
through to discharge the blue color. Me2S (3 mol) was added.
The reaction mixture was allowed to warm and left overnight.
If a starch KI test was negative, the solution was washed with
2 N HCl and aqueous NaHCO3 and then dried and evaporated.
The residue was chromatographed to obtain the arylacetal-
dehyde 13. Yields were 70-90%.
3. Gr ign a r d Rea ction To P r od u ce Ca r bin ols 14. The
arylacetaldehyde 13 (1.0 mol) was dissolved in THF (20 mL/
g), and the solution was cooled to -78 °C. Isopropenyl MgBr
(0.7 M in THF, 1.25 mol) was then added. In some cases, an
additional 0.25 mol of Grignard reagent was added after 1 h,
if appreciable aldehyde was still present. Saturated aqueous
NH4Cl was added, and the cooling bath was removed. Water
and ether were added, and the organic phase was dried and
evaporated. Chromatography then yielded 14. Yields were
50-80%.
(b) ter t-Bu tyl (E)-6-[4-[(Acetylth io)m eth yl]-2-m eth oxy-
6-[(2-m eth oxyeth oxy)m eth oxy]-3-m eth yl-5-(p yr r olid in -1-
ylca r bon yl)p h en yl]-4-m eth yl-4-h exen oa te (6c). A solution
of pyrrolidine (1.4 mL, 16.8 mmol) in THF (30 mL) was treated
with 2 M Me3Al in toluene (8.4 mL, 16.8 mmol). After 30 min,
a solution of 5a (1.96 g, 4.21 mmol) was added and the solution
stirred overnight and then heated at 50 °C for 24 h to complete
the reaction. After cooling, the reaction mixture was cau-

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4187
tiously poured into a slurry of ice and 1 N HCl and then
extracted with EtOAc. The organic phase was washed with
brine and, aqueous NaHCO3, dried, and stripped to give 6a
as an oil. This material was immediately redissolved in
CH2Cl2 (35 mL), cooled to -10 °C, and treated sequentially
with triethylamine (1.8 mL, 13 mmol) and mesyl chloride (0.8
mL, 10 mmol). After 1 h, ice was added and the mixture
partitioned between EtOAc and aqueous 1 M NaHSO4. The
organic layer was washed with brine, dried, and concentrated
to give 6b. Thiolacetic acid (0.46 mL, 6.4 mmol) was added to
a stirred suspension of Cs2CO3 (1.13 g, 3.47 mmol) in DMF (5
mL). After 1 h, a solution of 6b (ca. 2 g) in DMF (5 mL) was
added. After 2 h the reaction mixture was partitioned between
EtOAc and aqueous NaHCO3. The organic layer was dried,
concentrated, and chromatographed (7:3 EtOAc/hexane) to give
1.44 g of 6c as an oil: NMR δ 1.40 (s, 9H), 1.75 (s, 3H), 1.8-
2.0 (m, 4H), 2.2 (s, 3H), 2.23-2.30 (m, 4H), 2.31 (s, 3H), 3.0-
3.1 (m, 1H), 3.25-3.4 (m, 3H), 3.37 (s, 3H), 3.54-3.65 (m, 4H),
3.65 (s, 3H), 3.72-3.9 (m, 2H), 4.1 and 4.3 (2d of AB, J ) 13.3
Hz, 2H), 5.04 (AB, J ) 5.2 Hz, 2H), 5.17 (br t, J ) 6 Hz, 1H).
(c) 6c (1.44 g) was dissolved in N2-flushed MeOH (10 mL).
Ammonia was bubbled through the solution for several
minutes and the reaction mixture stirred at room temperature
until starting material was consumed (ca. 3 h). The reaction
was quenched with AcOH and the mixture partitioned between
EtOAc and water. The organic layer was dried, concentrated,
and then redissolved in EtOAc-AcOH (1:1). After 3 h, the
solution was partitioned between EtOAc and water and the
organic layer dried and concentrated. Chromatography (7:3
hexane/EtOAc) afforded the MEM ether/t-Bu ester of 1b (0.88
g) as an oil. The MEM group was removed (with concomitant
ester exchange) by heating in MeOH (30 mL) containing
p-TsOH (0.23 g) at reflux for 12 h. Workup and base
hydrolysis of the resultant methyl ester gave 1b (0.16 g): mp
164.5-168 °C (hexane/EtOAc); NMR30 δ 1.8 (s, 2H), 2.20 (s,
3H), 2.27-2.47 (m, 4H), 3.38 (d, J ) 6.8 Hz, 2H), 3.74 (s, 3H),
4.25 (s, 2H), 5.25 (br t, J ) 6 Hz, 1H), 9.53 (s, 1H). Anal.
(C17H20O5S) C, H.
Hz, 2H), 3.50 (t, J ) 6.7 Hz, 2H), 5.15 (br t, J ) 7.0 Hz, 1H).
Anal. (C18H22O6‚1.25H2O) C, H.
(E)-6-(4-Hydr oxy-6-m eth oxy-7-m eth yl-3-oxo-2,3-dih ydr o-
1H-isoin d ol-5-yl)-4-m eth yl-4-h exen oic Acid (1e). 6b (ca.
2 g) was dissolved in DMF (5 mL) and treated with NaN3 (0.8
g, 12.3 mmol). After 5 h, the mixture was partitioned between
hexane-EtOAc (1:1) and water. The organic phase was
washed with brine, dried, and concentrated to an oil which
was chromatographed (7:3 EtOAc/hexane) to give 6d (1.38 g)
as an oil. This material was dissolved in THF (5 mL) and
treated with PPh3 (0.93 g, 3.5 mmol). After 5 h, water (0.5
mL) and NH4OH (0.2 mL) were added, and the solution was
heated at 50 °C for 4 d to effect hydrolysis of the phosphine
imine and lactam formation. The solvents were removed, and
the residue was dissolved in MeOH (5 mL) and treated with
p-TsOH (250 mg). After 1 d the reaction mixture was
partitioned between EtOAc and water; the organic layer was
washed with aqueous NaHCO3 and brine, dried, and concen-
trated. Chromatography of the residue (7:3 EtOAc/hexane)
gave the tert-butyl ester of 1e (0.73 g) which upon hydrolysis
afforded 1e: mp 179-184 °C (aqueous EtOH); NMR30 (DMSO-
d6) δ 1.74 (s, 3H), 2.10 (s, 3H), 2.17-2.25 (m, 4H), 3.31 (d, J )
8.1 Hz, 2H), 3.67 (s, 3H), 4.27 (s, 2H), 5.17 (br t, J ) 6 Hz,
1H), 8.55 (s, 1H), 8.84 (s, 1H). Anal. (C17H21NO5) C, H, N.
(E)-6-(4-Hyd r oxy-6-m eth oxy-2,7-d im eth yl-3-oxo-2,3-d i-
h yd r o-1H-isoin d ol-5-yl)-4-m eth yl-4-h exen oic Acid (1f). To
a solution of 1e (0.042 g, 0.132 mmol) in DMF (1.3 mL) was
added NaH (0.052 g of a 60% dispersion in oil, 1.3 mmol). The
mixture was stirred at room temperature for 30 min and then
recooled to 0 °C and treated with CH3I (0.022 g, 0.15 mmol).
After stirring at 10 °C for 1 h an additional 0.011 g of CH3I
was added and the mixture stirred for an additional 30 min.
The reaction was quenched with 1 M aqueous NaHSO4 and
the mixture extracted with EtOAc. The organic phase was
dried and concentrated to an oil. Chromatography (97:2:1
CH2Cl2/MeOH/AcOH) afforded 0.037 g (84%) of 1f: mp 160-
162 °C (EtOAc/hexane); NMR30 δ 1.80 (s, 3H), 2.13 (s, 3H),
2.28-2.44 (m, 4H), 3.13 (s, 3H), 3.37 (d, J ) 6.8 Hz, 2H), 3.71
(s, 3H), 4.22 (s, 3H), 5.28 (br t, J ) 6 Hz, 1H). Anal. (C18H23
NO5) C, H, N.
-
(E)-6-(4-H yd r oxy-6-m et h oxy-7-m et h yl-3-oxoin d a n -5-
yl)-4-m eth yl-4-h exen oic Acid (1c). 2-(Phenylselenyl)cyclo-
pentenone (7a )31 (15.0 g, 0.063 mol) and 1,3-dimethoxy-1-
[(trimethylsilyl)oxy]penta-1,3-diene (8)32 (34.0 g, 0.157 mol)
were dissolved in toluene (220 mL). After 24 h at room
temperature and 5 h at reflux, the solvent was evaporated and
the residue chromatographed (1:4 hexane/EtOAc and then
EtOAc) to give 9a as an oil (5.5g, 46%).33 This product was
dissolved in toluene (180 mL), and DDQ (9.6 g, 1.5 mol equiv)
was added in portions over 1 h. The red solution was then
evaporated, and the residue was chromatographed (EtOAc/
CH2Cl2, 1:9) to afford 7-hydroxy-5-methoxy-4-methylindan-1-
one (10a ) (3.55 g, 65%), mp 138-139 °C (EtOAc-hexane).
Using procedures described above, 10a was converted into
1c: mp 148-149 °C (EtOAc/hexane); NMR30 δ 1.79 (s, 3H),
2.16 (s, 3H), 2.2-2.36 (m, 2H), 2.36-2.50 (m, 2H), 2.9-3.0 (m,
2H), 3.34 (d, J ) 7 Hz, 2H), 3.76 (s, 3H), 5.26 (t, J ) 7 Hz,
1H), 9.15 (br, 1H). Anal. (C18H22O5) C, H.
(E)-6-(4-Hyd r oxy-6-m eth oxy-1,7-d im eth yl-3-oxo-1,3-d i-
h yd r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1g).
5b (225 mg, 0.5 mmol) was dissolved in dry DMF (10 mL),
and 50% NaH in oil (288 mg, 6.0 mmol) was added. After 15
min MeI (0.3 mL, 4.8 mmol) was added. The reaction mixture
was heated to 60 °C for 24 h and then cooled and poured into
water. The product was extracted with EtOAc and chromato-
graphed (hexane/EtOAc, 4:1) to give the methyl ester of 1g
(110 mg, 47%) as an oil, NMR30 δ 0.27 (s, 6H), 1.05 (s, 9H),
1.60 (d, J ) 6.8 Hz, 3H), 1.78 (s, 3H), 2.22 (s, 3H), 2.20-2.51
(m, 4H), 3.41 (d, J ) 6.2 Hz, 2H), 3.64 (s, 3H), 3.76 (s, 3H),
5.20 (t, J ) 6.2 Hz, 1H), 5.40 (q, J ) 6.5 Hz, 1H). Basic
hydrolysis then gave 1g: mp 82-84 °C (ether/hexane); NMR30
δ 1.65 (d, J ) 6 Hz, 3H), 1.82 (s, 3H), 2.20 (s, 3H), 2.30-2.50
(m, 4H), 3.39 (d, J ) 6.9 Hz, 2H), 3.77 (s, 3H), 5.22 (t, J ) 6.5
Hz, 1H), 5.54 (q, J ) 6.5 Hz, 1H), 7.84 (s, 1H). Anal.
(C18H22O6) C, H.
(E)-6-(8-Hydr oxy-6-m eth oxy-5-m eth yl-1-oxoisoch r om an -
7-yl)-4-m eth yl-4-h exen oic Acid (1d ). A solution of the
lactone 7b34 (7.0 g, 0.033 mol) and 1,3-dimethoxy-1-[(trimeth-
ylsilyl)oxy]penta-1,3-diene (8)32 (10.5 g, 0.048 mol) in toluene
(150 mL) was refluxed for 18 h. The reaction mixture was
brought to dryness, and the crude product was chromato-
graphed (hexane/EtOAc, 3:7) to give 9b (2.0 g, 18%) as a
mixture of isomers. This compound (2.0 g, 6.31 mmol) was
dissolved in dichloromethane (250 mL) at 0 °C. m-CPBA (1.41
g, 8.17 mmol) was added in portions. After 1 h the solution
was added to 20% aqueous NaHSO3; the organic phase was
separated, washed with saturated NaHCO3, dried over Na2SO4,
and evaporated. The residue was chromatographed (hexane/
EtOAc, 7:3) to obtain 8-hydroxy-6-methoxy-5-methylisochro-
man-1-one (10b) (472 mg, 36%), mp 164-165 °C (EtOAc/
hexane). Anal. (C11H12O4) C, H. Using the procedures
described above, 10b was converted into 1d : mp 160-161 °C
(MeOH-ether); NMR30 (DMSO-d6) δ 1.73 (s, 3H), 2.15 (s, 3H),
2.23-2.05 (m, 4H), 3.00 (t, J ) 7.3 Hz, 2H), 3.26 (d, J ) 7.0
(E)-6-(4,6-Dih ydr oxy-7-m eth yl-3-oxo-1,3-dih ydr oisoben -
zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1h ) a n d (E)-6-
(6-Eth oxy-4-h yd r oxy-7-m eth yl-3-oxo-1,3-d ih yd r oisoben -
zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1i). These were
prepared as previously described.19b
(E)-6-(4-Hyd r oxy-7-m eth yl-3-oxo-1,3-d ih yd r oisoben zo-
fu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1j). The methyl
ester of 1h (500 mg, 1.56 mmol) was dissolved in CH2Cl2 (10
mL) at -10 °C, and 2,6-lutidine (1.32 g, 4.68 mmol) and then
triflic anhydride (668 mg, 6.2 mmol) were added. After 15 min
the mixture was added to water/EtOAc. The organic solution
was washed with 2 N NaHSO4 and then dried and evaporated
to give 16a as a gum. This material was stirred in MeOH (15
mL) containing K2CO3 (645 mg, 4.68 mmol) for 1 h. The
mixture was added to dilute HCl and extracted with EtOAc.
The extract was dried and evaporated, and the residue was
chromatographed to give 16b which was converted into the
MEM ether 16c as described above. The latter compound (465
mg, 0.88 mmol) was dissolved in DMF (8 mL) to which Et3N

4188 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nelson et al.
(267 mg, 2.6 mmol), formic acid (81 mg, 1.67 mmol), and Pd-
(1,1′-bis(diphenylphosphino)ferrocene)Cl235 (37 mg, 0.044 mmol)
were added. The reaction mixture was heated at 60 °C for 48
h and then added to water and extracted with EtOAc. The
extract was washed with aqueous NaHSO4, dried, and evapo-
rated. The residue was chromatographed (1:1 EtOAc/hexane)
to give 17a (150 mg, 43%), which after removal of the MEM
ether and basic hydrolysis gave 1j: mp 135-139 °C (EtOAc/
hexane); NMR30 (DMSO-d6) δ 1.70 (s, 3H), 2.16 (s, 3H), 2.15-
2.35 (m, 4H), 3.35 (d, J ) 6 Hz, 2H), 5.25 (s, 2H), 5.35 (t, J )
7 Hz, 1H), 7.34 (s, 1H). Anal. (C16H18O5) C, H.
(E)-6-(4-H yd r oxy-7-m et h yl-3-oxo-6-vin yl-1,3-d ih yd r o-
isoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1k ). (a )
Met h yl (E)-4-Met h yl-6-[7-m et h yl-3-oxo-4-[(p -t olylsu lfo-
n yl)oxy]-6-[[(t r iflu or om e t h yl)su lfon yl]oxy]-1,3-d ih y-
d r oisoben zofu r a n -5-yl]-4-h exen oa te (16d ). A mixture of
mycophenolic acid (201 g, 0.627 mol), TsOH (3 g, 16 mmol),
and MeOH (1.8 L) was stirred at 25 °C for 18 h. The reaction
mixture was cooled on ice, and the product 16e (189.4 g, 90%)
was isolated by filtration. A solution of 16e (70.0 g, 0.209 mol)
and TsCl (45.4 g, 0.238 mol) in CH2Cl2 (400 mL) was cooled
on ice and treated with Et3N (37.8 mL, 0.27 mol) and DMAP
(1.2 g). The reaction mixture was stirred for 1.5 h at 0 °C and
then poured into ice water. Extraction with CH2Cl2 gave the
tosylate 16f which was used without further purification. The
compound was dissolved in collidine (500 mL), LiI (102 g, 0.76
mol) was added, and the reaction mixture was heated at 70
°C with mechanical stirring for 17 h. The mixture was cooled
and poured into ice water and concentrated HCl (350 mL).
Extraction with EtOAc (6 × 500 mL) gave the phenolic acid
16g. Esterification as above gave the phenolic ester 16h (33.7
g). A solution of 16h (33.7 g, 71 mmol) in CH2Cl2 (600 mL)
and pyridine (14.6 mL) was cooled to 0 °C and treated with
triflic anhydride (13.8 mL, 82 mmol). After 15 min the reaction
mixture was poured into 1 N aqueous NaHSO4. Extraction
with CH2Cl2, drying, and evaporation gave a residue which
was recrystallized to give 16d (39.47 g, 92%): mp 135.7-136.7
°C (EtOAc/hexane). Anal. (C25H25F3O10S2) C, H.
(b) Meth yl (E)-4-Meth yl-6-[7-m eth yl-3-oxo-4-[(p-tolyl-
su lfon yl)oxy]-6-vin yl-1,3-d ih yd r oisoben zofu r a n -5-yl]-4-
h exen oa te (17b). 16d (40.0 g, 65.9 mmol), LiCl (7.74 g, 180
mmol), Ph3As (2.00 g, 6.5 mmol), tributylvinyltin (21.5 mL,
73.6 mmol), and tris(dibenzylideneacetone)dipalladium(0)-
chloroform adduct (0.90 g, 0.98 mmol) were heated in N-
methylpyrrolidinone (300 mL) at 55 °C for 3 h. The reaction
mixture was cooled and poured into a mixture of ice, KF (24
g), water, and EtOAc. After stirring for 1 h at 25 °C this
mixture was filtered through Celite and extracted with EtOAc.
Concentration of these extracts followed by recrystallization
from t-BuOMe/EtOAc mixtures gave 17b (30.52 g, 95%): mp
105.0-106.2 °C. Anal. (C26H28O7S) C, H.
(c) Basic hydrolysis, as described below for 1l, gave 1k : mp
148.4-148.7 °C (t-BuOMe); NMR30 δ 1.76 (s, 3H), 2.14 (s, 3H),
2.25-2.5 (m, 4H), 3.41 (d, J ) 6.7 Hz, 2H), 5.13 (t, J ) 6.7 Hz,
1H), 5.22 (s, 2H), 5.26 (dd, J ) 18, 1.7 Hz, 1H), 5.64 (dd, J )
11.6, 1.7 Hz, 1H), 6.66 (dd, J ) 18.1, 11.6 Hz, 1H). Anal.
(C18H20O5) C, H.
(E)-6-(4-Hydr oxy-6,7-dim eth yl-3-oxo-1,3-dih ydr oisoben -
zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1m ). (a ) Meth yl
4,5-Dim eth yl-2-(p r op -2-en yloxy)ben zoa te (18a ). A mix-
ture of 2-hydroxy-4,5-dimethylbenzoic acid36 (5.00 g, 30 mmol),
Li2CO3 (5.57 g, 75 mmol), MeI (4.67 mL, 75 mmol), and DMF
(50 mL) was stirred at 50 °C for 2 h. After cooling to 25 °C
the reaction mixture was poured into ice water, extracted with
EtOAc, dried, and evaporated to give methyl 2-hydroxy-4,5-
dimethylbenzoate. This ester was added over 30 min to a
mixture of NaH (1.48 g, 60%, 37 mmol) and allyl bromide (5.2
mL, 60 mmol) in DMF (30 mL) at 0 °C. After 30 min the
reaction mixture was warmed to 25 °C and poured into water.
Ether extraction, drying, evaporation, and chromatography
(10% EtOAc/hexane) gave 18a (4.511 g, 68% overall): bp 90
°C/0.10 mmHg. Anal. (C13H16O3) C, H.
(b) N,N-Dieth yl-4,5-d im eth yl-2-(p r op -2-en yloxy)ben z-
a m id e (18b). Trimethylaluminum (40 mL, 8.0 mmol, 2 M in
toluene) was added to a solution of diethylamine (8.3 mL, 160
mmol) in benzene (80 mL) at 0 °C. The reaction mixture was
stirred for 1 h at 25 °C, treated with ester 18a (8.50 g, 38.6
mmol), and heated at 80 °C for 24 h. The mixture was cooled
and poured cautiously into ice water containing HCl (30 mL,
concentrated). Isolation by EtOAc extraction and Kugelrohr
distillation gave 18b (9.25 g, 92%): bp 105 °C/0.09 mmHg; mp
57.3-58.2 °C (t-BuOMe/hexane). Anal. (C16
H23NO2) C, H, N.
(c) N,N-Dieth yl-2-h ydr oxy-4,5-dim eth yl-3-(pr op-2-en yl)-
ben za m id e (19a ). This was produced by Claisen rearrange-
ment of 18b as described above: yield 71%; bp 115 °C/0.08
mmHg. Anal. (C16H23NO2) H, N; C: calcd, 73.52; found, 72.79.
(d ) 4-[(ter t-Bu tyld im eth ylsilyl)oxy]-6,7-d im eth yl-3-oxo-
5-(p r op -2-en yl)-1,3-d ih yd r oisoben zofu r a n (20a ). Silyla-
tion of 19a gave 19b (5.66 g, 15.1 mmol) which, in THF (8
mL), was added to a solution of 1.7 M t-BuLi in pentane (20
mL, 34 mmol) in THF (40 mL) and TMEDA (4.52 mL) at -90
°C over 30 min. After stirring for 40 min at -90 °C, DMF (9
mL) was added; the reaction mixture was warmed to -15 °C
and then poured into ice water. Isolation by EtOAc extraction
and chromatography (EtOAc/hexane) gave the aldehyde 19c
(4.35 g, 71%). This compound (4.35 g, 10.8 mmol) was
dissolved in EtOH (50 mL) and cooled to 5 °C. Sodium
borohydride (225 mg, 6 mmol) was added to the reaction
mixture which was stirred at 0 °C for 1 h. The reaction
mixture was diluted with ice water, and the carbinol 19d was
isolated by EtOAc extraction and evaporation. This crude
product was dissolved in EtOAc (12 mL) and HOAc (3 mL)
and heated to 50 °C for 40 min. The reaction mixture was
partitioned between EtOAc and aqueous NaHCO3. Extraction
with EtOAc followed by recrystallization gave 20a (2.639 g,
53%): mp 81.1-81.5 °C (t-BuOMe/hexane). Anal. (C19H28O3-
Si) C, H.
(e) Ozonolysis then gave [4-[(tert-butyldimethylsilyl)oxy]-
6,7-dimethyl-3-oxo-1,3-dihydroisobenzofuran-5-yl]acetalde-
hyde (20b), 82%, mp 107.8-109.2 °C (t-BuOMe/hexane). Anal.
(C18
H26O4Si) C, H. 20b was converted into 1m : mp 167.1-
169.7 °C (EtOAc); NMR30 δ 1.81 (s, 3H), 2.13 (s, 3H), 2.25 (s,
3H), 2.30 (m, 2H), 2.45 (m, 2H), 3.44 (d, J ) 6.7 Hz, 2H), 5.09
(t, J ) 6.7 Hz, 1H), 5.21 (s, 2H). Anal. (C17H20O5) C, H.
(E)-6-(6-Cyclop r op yl-4-h yd r oxy-7-m eth yl-3-oxo-1,3-d i-
h yd r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1n ).
A mixture of 16d (1.20 g, 1.98 mmol), Ph3As (65 mg, 0.21
mmol), LiCl (270 mg, 6.4 mmol), tris(dibenzylideneacetone)-
dipalladium(0)-chloroform adduct (40 mg, 0.039 mmol), tribu-
tylcyclopropyltin (0.80 mL, 2.45 mmol), and N-methylpyrro-
lidinone (6 mL) was heated at 95 °C for 3 h. An aqueous KF
workup and chromatography (as described above for 1k ) gave
the ester 17d (134 mg, 14%). Base hydrolysis then gave 1n :
mp 172.0-173.6 °C (EtOAc/t-BuOMe); NMR30 (DMSO-d6) δ
0.51 (m, 2H), 1.06 (m, 2H), 1.73 (s, 3H), 2.22 (s, 3H), 2.1-2.3
(m, 4H), 3.58 (d, J ) 6.4 Hz, 2H), 5.09 (t, J ) 6.4 Hz, 1H),
5.23 (s, 2H). Anal. (C19H22O5) C, H.
(E)-6-(6-E t h yl-4-h yd r oxy-7-m et h yl-3-oxo-1,3-d ih yd r o-
isoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1l).
A
solution of the styrene 17b (29.31 g, 60.5 mmol) in EtOAc (300
mL) and benzene (300 mL) containing tris(triphenylphos-
phine)rhodium(I) chloride (2.00 g, 2.2 mmol) was hydrogenated
at 1 atm for 5 h. The solvent was removed in vacuo and the
residue filtered through a short silica gel column (EtOAc).
Recrystallization from t-BuOMe/EtOAc gave 17c (28.41 g,
96%), mp 101.3-102.8 °C. Anal. (C26H30O7S) C, H. A mixture
of this compound (3.142 g, 6.47 mmol), MeOH (20 mL), water
(20 mL), and LiOH (1.2 g, 28.6 mmol) was heated at 62 °C for
17 h. The MeOH was evaporated, and the resulting aqueous
solution was poured into 1 N aqueous NaHSO4. Extraction
with EtOAc followed by chromatography (EtOAc/hexane with
1% HOAc) gave 1l (1.417 g, 68%): mp 145.2-145.6 °C (EtOAc/
t-BuOMe); NMR30 δ 1.12 (t, J ) 7.5 Hz, 3H), 1.81 (s, 3H), 2.16
(s, 3H), 5.21 (s, 2H), 2.30 (m, 2H), 2.45 (m, 2H), 2.68 (q, J )
7.5 Hz, 2H), 3.42 (d, J ) 6.3 Hz, 2H), 5.11 (t, J ) 6.6 Hz, 1H).
Anal. (C18H22O5) C, H.
(E )-6-(4-H yd r oxy-7-m e t h yl-3-oxo-6-p h e n yl-1,3-d ih y-
d r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1o). In
a procedure analogous to that described for the preparation
of 1n , the triflate 16d (0.688 g, 1.13 mmol) on reaction with
tributylphenyltin gave the 6-phenyl compound 17e (0.109 g,
18%). Hydrolysis then gave 1o (0.050 g, 67%): mp 156.0-

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4189
156.5 °C (EtOAc/t-BuOMe); NMR30 δ 1.27 (s, 3H), 1.83 (s, 3H),
2.1-2.4 (m, 4H), 3.12 (d, J ) 7.0 Hz, 2H), 5.01 (t, J ) 7.0 Hz,
1H), 5.25 (s, 2H), 7.0-7.1 (m, 2H), 7.3-7.5 (m, 3H). Anal.
(C22H22O5) C, H.
Anal. (C16H20O6) C, H. Claisen rearrangement then gave 24a
as an oil. Anal. (C16H20O6) C, H.
(c) 7-Eth yl-4-h yd r oxy-6-m eth oxy-3-oxo-5-(2-p r op en yl)-
1,3-d ih yd r oisoben zofu r a n (25a ). 24a (2.80 g, 9.1 mmol),
LiOH (2.1 g, 50 mmol), water (25 mL), and MeOH (17 mL)
were heated at 53 °C for 4 h. The reaction mixture was cooled
on ice and acidified with HCl. Extraction with EtOAc and
concentration gave the diacid 24b. This compound, in HOAc
(4 mL), was heated to 90 °C and treated with zinc dust (1.3 g
in three portions) over 1.5 h while a solution of HCl (concen-
trated, 2 mL) in HOAc (2 mL) was added dropwise. The
reaction mixture was cooled, diluted with water, and extracted
with EtOAc to give 25a (0.711 g, 31% from 24a ): mp 94.0-
95.1 °C (t-BuOMe/hexane). Anal. (C14H16O4) C, H.
(d ) Silylation followed by side-chain elaboration then gave
1s: mp 142-145 °C (t-BuOMe/hexane); NMR30 δ 1.20 (t, J )
7.6 Hz, 3H), 1.82 (s, 3H), 2.25-2.5 (m, 4H), 2.59 (q, J ) 7.6
Hz, 2H), 3.41 (d, J ) 6.7 Hz, 2H), 3.79 (s, 3H), 5.27 (s, 2H),
5.28 (t, J ) 6.7 Hz, 1H). Anal. (C18H22O6) C, H.
(E)-6-(7-Br om o-4-h yd r oxy-6-m et h oxy-3-oxo-1,3-d ih y-
d r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1t).39
Br2 (1.81 mL, 3.64 mmol) in CH2Cl2 (7 mL) was added to 25b40
(200 mg, 0.91 mmol) in CH2Cl2 (25 mL). After 1 h the solvent
was removed under vacuum, and the residue 26 was dissolved
in HOAc (30 mL). Zn dust (250 mg, 325 mesh) was added.
After stirring for 36 h the reaction mixture was added to water
and extracted with CH2Cl2. The extract was washed, dried,
and evaporated, and the residue was chromatographed (90:
10:1 hexane/EtOAc/HOAc) to give 25c, 200 mg, 74%; mp 57-
58 °C (ether). Anal. (C12H11BrO4) C, H. 25c was converted
into 1t: mp 154-156 °C (ether) (lit.39 mp 165-166 °C); NMR30
(DMSO-d6) δ 1.71 (s, 3H), 2.16-2.28 (m, 4H), 3.37 (d, J ) 7
Hz, 2H), 3.79 (s, 3H), 5.12 (t, J ) 7 Hz, 1H), 5.19 (s, 2H). Anal.
(C16H17BrO6) C, H.
(E )-6-(4-H y d r o x y -6,7-d i m e t h o x y -3-o x o -1,3-d i h y -
d r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1u ). A
solution of 4,6,7-trimethoxy-3-oxo-1,3-dihydroisobenzofuran41
(1.74 g, 7.76 mmol) in CH2Cl2 (150 mL) was added to a
suspension of AlCl3 (2.2 g, 16.3 mmol) in CH2Cl2 (150 mL).
After 26 h at reflux, 10% aqueous HCl (25 mL) was added.
After 45 min the organic layer was separated, dried, and
evaporated to give 4-hydroxy-6,7-dimethoxy-3-oxo-1,3-dihy-
droisobenzofuran42 (1.0 g, 61%). This compound was then
converted as described above, except that a modified silylation
procedure was required,43 into 1u : mp 129-132 °C (ether);
NMR30 δ 1.80 (s, 3H), 2.30-2.46 (m, 4H), 3.37 (d, J ) 7.1 Hz,
2H), 3.84 (s, 3H), 3.87 (s, 3H), 5.23 (t, J ) 7 Hz, 1H), 5.32 (s,
2H), 7.26 (s, 1H). Anal. (C17H20O7) C, H.
(E)-6-(7-Cya n o-4-h yd r oxy-6-m et h oxy-3-oxo-1,3-d ih y-
d r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1v)
a n d (E)-6-(7-Ca r ba m oyl-4-h yd r oxy-6-m eth oxy-3-oxo-1,3-
d ih yd r oisob en zofu r a n -5-yl)-4-m et h yl-4-h exen oic Acid
(1w ). The methyl ester of 1t (1.2 g, 3.01 mmol) was dissolved
in pyridine (20 mL), and Ac2O (0.71 mL, 7.5 mmol) was added.
After 36 h the solution was added to water and extracted with
EtOAc. The extract was washed with dilute HCl, dried, and
evaporated to give 850 mg (75%) of the phenolic acetate. This
material (400 mg, 0.91 mmol) was dissolved in dry dioxane
(40 mL), and KCN (106 mg, 1.63 mmol) and Pd(PPh3)4 (208
mg, 0.2 mmol) were added. After 8 h at 95 °C, additional KCN
(100 mg) and Pd(PPh3)4 (100 mg) were added. After another
12 h at 95 °C, the mixture was added to water. The solution
was extracted with EtOAc (discarded) and then acidified with
dilute HCl and extracted with EtOAc. The extract was dried
and evaporated to give the methyl ester of 1v44 (150 mg, 48%).
This compound (75 mg, 0.22 mmol) was dissolved in MeOH (5
mL) and water (1 mL), and LiOH‚H2O (19 mg, 0.45 mmol) was
added. After 2 h water was added, and the solution was
acidified with dilute HCl and then extracted with EtOAc. The
extract was dried and evaporated, and the residue was
chromatographed (70:30:0.1 hexane/EtOAc/HOAc) to afford
1v: 35 mg, 45%, mp 125-130 °C (ether/hexane); NMR30
(DMSO-d6) δ 1.73 (s, 3H), 2.15-2.25 (m, 4H), 3.29 (d, J ) 7
Hz, 2H), 4.03 (s, 3H), 5.07 (t, J ) 7 Hz, 1H), 5.41 (s, 2H), 12.0
(br s, 1H). Anal. (C17H17NO6) C, H, N. Also, 1w : 15 mg, 20%;
mp 194-196 °C (ether/hexane); NMR30 (DMSO-d6) δ 1.77 (s,
(E )-6-(6-Cya n o-4-h yd r oxy-7-m e t h yl-3-oxo-1,3-d ih y-
d r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1p ).
To a solution of 16d (2.8 g, 4.9 mmol) in dioxane (60 mL) was
added KCN (0.644 g, 10 mmol) and Pd(PPh3)4 (0.80 g, 0.63
mmol). After 5 d at reflux, the mixture was added to water
and extracted with EtOAc. The extract was dried and
evaporated, and the residue was chromatographed (1:1 EtOAc/
hexane) to afford methyl (E)-6-(6-cyano-4-hydroxy-7-methyl-
3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-4-hexenoate (17f)
(510 mg, 32%), mp 139-140 °C (EtOAc/hexane); NMR δ 1.84
(s, 3H), 2.4-2.5 (m, 4H), 2.43 (s, 3H), 3.63 (d, J ) 7.6 Hz, 2H),
3.64 (s, 3H), 5.23 (s, 2H), 5.23 (t, 1H), 5.28 (s, 2H), 7.90 (s,
1H). Anal. (C18H19NO5) C, H, N. Basic hydrolysis as de-
scribed above then gave 1p , mp 142-145.5 °C (EtOAc/hexane);
NMR30 δ 1.84 (s, 3H), 2.3-2.5 (m, 4H), 2.43 (s, 3H), 3.63 (s,
3H), 5.25 (2, J ) 7 Hz, 1H), 5.28 (s, 2H), 7.26 (s, 1H). Anal.
(C17H17NO5) C, H, N.
(E)-6-(6-Ca r b a m oyl-4-h yd r oxy-7-m et h yl-3-oxo-1,3-d i-
h yd r oisoben zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1q).
17f (200 mg, 0.6 mmol) was refluxed in 0.7 M aqueous NaOH
(7 mL) for 48 h. The solution was then acidified with 2 N HCl
and extracted with EtOAc. The extract was dried and
evaporated, and the residue was recrystallized from EtOAc/
cyclohexane to give 1q (80 mg, 40%): mp 200.6-202.4 °C;
NMR30 δ 1.69 (s, 3H), 2.09 (s, 3H), 2.12-2.28 (m, 4H), 3.31 (d,
J ) 10 Hz, 2H), 5.15 (t, J ) 6.7 Hz, 1H), 5.28 (s, 2H), 7.67 (d,
J ) 15 Hz, 1H), 7.86 (d, J ) 15 Hz, 1H), 9.4 (s, 1H), 11.99 (s,
1H). Anal. (C17H19NO6) H, N; C: calcd, 61.25; found, 60.59.
(E)-6-(4-Hyd r oxy-6-m eth oxy-3-oxo-1,3-d ih yd r oisoben -
zofu r a n -5-yl)-4-m eth yl-4-h exen oic Acid (1r ). 4-Hydroxy-
6-methoxy-3-oxo-1,3-dihydroisobenzofuran37 was converted into
1r using the reactions of Scheme 3: mp 145-147 °C (MeOH);
NMR30 (DMSO-d6) δ 1.71 (s, 3H), 2.13-2.26 (m, 4H), 3.25 (d,
J ) 6.9 Hz, 2H), 3.85 (s, 3H), 5.11 (t, J ) 7 Hz, 1H), 5.23 (s,
2H), 7.64 (s, 1H). Anal. (C16H18O6‚0.25H2O) C, H.
(E )-6-(7-E t h yl-4-h yd r oxy-6-m e t h oxy-3-oxo-1,3-d ih y-
d r oisob en zofu r a n -5-yl)-4-m et h yl-4-h exen oic Acid (1s).
(a ) 6-Eth yl-3-(2-p r op en yloxy)-2-cycloh exen on e (21b). A
solution of i-Pr2NH (16.8 mL, 0.12 mol) in THF (100 mL) was
cooled to 0 °C and treated with 1.6 M n-BuLi (75 mL, 0.12
mol). The reaction mixture was cooled to -70 °C, and a
solution of 3-(2-propenyloxy)-2-cyclohexenone38 (21a ) (17.4 g,
0.115 mol) in THF (5 mL) was added over 20 min. EtI (25
mL) and HMPA (15 mL) were added to the reaction mixture.
After 17 h at -22 °C, AcOH (10 mL) and water (20 mL) were
added and the THF was evaporated. The residue was parti-
tioned between EtOAc and water. Extraction and evaporation
gave a residue which was chromatographed to give 21b (15.2
g, 73%): bp 100 °C/0.11 mmHg. Anal. (C11H16O2) C, H.
(b) Dim eth yl 6-Eth yl-3-h yd r oxy-5-m eth oxy-4-(2-p r o-
p en yl)p h th a la te (24a ). A solution of LDA (90 mmol) pre-
pared from i-Pr2NH (12.61 mL, 90 mmol) and 1.6 N n-BuLi
(56.2 mL, 90 mmol) in THF (120 mL) was cooled to -65 °C
and treated with TMSCl (14 mL, 110 mmol). 21b (15.2 g, 84.4
mmol) in THF (5 mL) was added to the reaction mixture over
15 min. Et3N (15 mL) was added to the reaction mixture
which was poured into hexane/ice water. Separation followed
by drying and evaporation gave the silyl enol ether 22 which
was diluted with xylene (50 mL), cooled to -50 °C, and treated
with dimethylacetylene dicarboxylate (14 mL, 114 mmol) and
NaH (100 mg). The reaction mixture was heated at 50 °C for
90 min and at 120 °C for 2 h. The xylene was removed in
vacuo, and following an EtOAc/water workup, the crude
product was chromatographed on silica gel (EtOAc/hexane) to
give phenol 23a (6.62 g, 36% from 21b). 23a (6.62 g, 22.5
mmol) in DMF (40 mL) was treated with K2CO3 (6.9 g, 50
mmol) and MeI (4.36 mL, 70 mmol) at 25 °C for 15 h. The
reaction mixture was diluted with ice water and extracted with
ether. After concentration the residue was recrystallized to
give dimethyl 3-ethyl-4-methoxy-6-(2-propenyloxy)phthalate
(23b) (6.132 g, 88% from 23a ), mp 73.4-74.6 °C (t-BuOMe).

4190 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nelson et al.
3H), 2.14-2.32 (m, 4H), 3.35 (d, J ) 8.6 Hz, 2H), 3.79 (s, 3H),
5.16 (t, J ) 8.4 Hz, 1H), 5.43 (s, 2H), 7.61 (br s, 2H). Anal.
(C17H19NO7‚0.25H2O) C, H, N.
(E)-6-(6-H yd r oxy-2-m et h oxy-3,4-d im et h ylp h en yl)-4-
m et h yl-4-h exen oic Acid (2a ). 1a (5.0 g, 15.6 mmol) and
LiOH‚H2O (2.61 g, 62.4 mmol) were refluxed in water (100
mL) for 48 h; then a further 6.0 g (103 mmol) of LiOH was
added. After a total of 96 h reflux, the mixture was acidified
with dilute HCl and extracted with EtOAc. The extract was
dried and evaporated to give the decarboxylated product 27a
(3.55 g, 82%).45 This material was dissolved in MeOH (50 mL),
and p-TsOH (90 mg) was added. After 16 h the solution was
added to water and extracted with EtOAc. The extract was
dried and evaporated and the residue chromatographed (EtOAc/
hexane, 1:1) to give the methyl ester 27b (3.53 g, 95%). The
product (3.0 g, 9.7 mmol) was dissolved in acetone (50 mL) at
0 °C, and Cs2CO3 (3.16 g, 9.7 mmol) and Ac2O (0.99 g, 9.7
mmol) were added. After 3 h the mixture was poured into
water and extracted with EtOAc. The extract was dried and
evaporated and the residue chromatographed (EtOAc/hexane,
1:1) to give the acetate 27c (2.7 g, 79%). Using the procedures
described in the preparation of 1b, this compound was
transformed via the benzylic mesylate to the thioacetate 27d .
This material was added to a refluxing suspension of Raney
nickel catalyst (23 g of 50% aqueous suspension) in acetone
(250 mL). After 4 h reflux the reaction mixture was filtered
through Celite and then evaporated. The residue was parti-
tioned between EtOAc and water. The organic phase was
dried and evaporated to give 27e as an oil. Basic hydrolysis,
as described above, then afforded 2a : mp 92.5-93 °C (hexane/
EtOAc); NMR30 δ 1.82 (s, 3H), 2.09 (s, 3H), 2.16 (s, 3H), 2.28-
2.48 (m, 4H), 3.47 (d, J ) 7 Hz, 2H), 3.63 (s, 3H), 5.26 (t, J )
7 Hz, 1H), 6.44 (s, 1H). Anal. (C16H22O4) C, H.
(E)-6-(2-Hyd r oxy-6-m eth oxy-3,4,5-tr im eth ylp h en yl)-4-
m eth yl-4-h exen oic Acid (2b). A mixture of 2i methyl ester
(0.36 g, 1.13 mmol) and Zn powder (1.5 g, 23 mmol) in AcOH
(10 mL) was heated at 60 °C for 1 h. The solids were filtered
off and washed with EtOAc, and the filtrate was washed with
water (2×), aqueous NaHCO3, and brine. The organic phase
was dried and concentrated, and the residue was chromato-
graphed (85:15 hexane/EtOAc) to give the methyl ester of 2b
(0.3 g), which upon basic hydrolysis afforded 2b: mp 99-100
°C (hexane); NMR30 δ 1.79 (s, 3H), 2.12-2.13 (br s, 9H), 2.2-
2.33 (m, 4H), 3.32 (d, J ) 6.8 Hz, 2H), 3.58 (s, 3H), 5.21 (br t,
J ) 6 Hz, 1H), 7.9 (br s, 1H). Anal. (C17H24O4) C, H.
(E )-6-(3-F lu o r o -2-h y d r o x y -6-m e t h o x y -4,5-d im e t h -
ylp h en yl)-4-m eth yl-4-h exen oic Acid (2c). (a ) 2-F lu or o-
5-m eth oxy-3,4-d im eth ylp h en ol (32a ). Methyl 6-hydroxy-
4-methoxy-2,3-dimethylbenzoate46 (25.0 g) was refluxed in
AcOH (75 mL) and concentrated HCl (75 mL) for 40 min. The
mixture was added to water and extracted with EtOAc. The
extract was dried and evaporated and the residue crystallized
from hexane to afford 3-methoxy-4,5-dimethylphenol (32b)
(14.6 g, 81%), mp 71-73 °C. Anal. (C9H12O3) C, H. The
product (10.0 g, 0.066 mmol) and N-fluoropyridinium pyridine
heptafluorodiborate (23.4 g, 0.072 mol) were dissolved in
acetonitrile (100 mL). After 12 days the solution was added
to water and extracted with ether. The extract was washed
with 2 N aqueous HCl, dried, and evaporated. The residue
was chromatographed (3:1 hexane/ether) to afford 32a (1.84
g, 15%): mp 57-60 °C (hexane). Anal. (C9H11FO2) C, H.
(b ) 2-F lu or o-5-m et h oxy-3,4-d im et h yl-6-(2-p r op en yl)-
p h en ol (32c).47 32a (1.8 g, 10.6 mmol) was dissolved in
CH2Cl2 (50 mL) and MeOH (25 mL) at 0 °C. Tetrabutylam-
monium tribromide (5.1 g, 10.6 mmol) was added. After 6 h
the solution was added to ether/water. The organic solution
was dried and evaporated to give 2-bromo-6-fluoro-3-methoxy-
4,5-dimethylphenol (32d ) (2.6 g, 96%), mp 120-123 °C (ether/
hexane). Anal. (C9H10BrFO2) C, H. This material (2.4 g,
0.0096 mol) was dissolved in pyridine (25 mL), and acetic
anhydride (4 mL) was added. After 1 h the solution was added
to water and extracted with ether. The ethereal solution was
washed with 2 N aqueous HCl, dried, and evaporated to afford
2-bromo-6-fluoro-3-methoxy-4,5-dimethylphenyl acetate (32e)
(2.88 g, 100%), mp 66-68 °C (hexane). Anal. (C11H12BrFO3)
C, H. This material (1.97 g, 0.0068 mol) was dissolved in
F igu r e 2.
toluene, and allyltributyltin (2.47 g, 0.0075 mol) and Pd(PPh3)4
(0.371 g, 0.000 32 mol) were added. The mixture was heated
at 90 °C for 60 h. Two further additions of 371 mg of Pd
catalyst were then made at 24 h intervals. The mixture was
then added to water, and the organic solution was dried and
evaporated. The residue was chromatographed (100:100:4
hexane/toluene/ether) to afford 2-fluoro-5-methoxy-3,4-di-
methyl-6-(2-propenyl)phenyl acetate (794 mg, 43%) as an oil.
This material was dissolved in MeOH (10 mL) and water (1.5
mL), to which saturated aqueous Na2CO3 (2 mL) was added.
The mixture was heated at 50 °C for 4 h and then added to
water and extracted with ether. The extract was dried and
evaporated to give 32c (658 mg, 91%) as an oil.
(c) Using the procedures described above, this material was
converted in 21% overall yield into 2c: mp 96-98 °C (ether/
hexane); NMR30 δ 1.81 (s, 3H), 2.13 (s, 3H), 2.14 (d, J ) 2 Hz,
3H), 2.3-2.5 (m, 4H), 3.36 (d, J ) 7 Hz, 2H), 3.64 (s, 3H), 5.26
(t, J ) 7 Hz, 1H). Anal. (C16H21FO4) C, H.
(E )-6-(3-Ch lor o-2-h yd r oxy-6-m e t h oxy-4,5-d im e t h yl-
p h en yl)-4-m eth yl-4-h exen oic Acid (2d ). 32b (14.6 g, 0.096
mol) and N-chlorosuccinimide (12.8 g, 0.096 mol) were dis-
solved in DMF (100 mL). After 24 h the solution was added
to water and extracted with ether. The extract was dried and
evaporated, and the residue was chromatographed (4:1 hexane/
ether) to afford 2-chloro-5-methoxy-3,4-dimethylphenol (32f)
(12.8 g, 72%), mp 99-100 °C (hexane/ether). Anal. (C9H11
-
ClO2) C, H. O-Allylation then gave 2-chloro-5-methoxy-3,4-
dimethyl-1-(2-propenyloxy)benzene (32g), mp 59-61 °C (MeOH).
Anal. (C12H15ClO2) C, H. Claisen rearrangement and side-
chain elaboration afforded 2d in an overall yield of 12%: mp
114-117 °C (aqueous MeOH); NMR30 δ 1.83 (s, 3H), 2.19 (s,
3H), 2.32 (s, 3H), 2.3-2.5 (m, 4H), 3.41 (d, J ) 6 Hz, 2H), 3.67
(s, 3H), 5.30 (t, J ) 6 Hz, 1H). Anal. (C16H21ClO4) C, H.
(E )-6-(3-Br om o-2-h yd r oxy-6-m e t h oxy-4,5-d im e t h yl-
p h en yl)-4-m eth yl-4-h exen oic Acid (2e). 27e (1.99 g, 5.7
mmol) was dissolved in MeOH (30 mL), and K2CO3 (3.9 g, 28.5
mmol) was added. After 3 h reflux, the reaction was added to
1 N aqueous NaHSO4 and extracted with EtOAc. The extract
was dried and evaporated and the residue chromatographed
(4:1 hexane/EtOAc) to give 27f as an oil. This compound (0.5
g, 1.7 mmol) in CH2Cl2 (3 mL) was added to a solution of Br2
(0.27 g, 1.7 mmol) in a mixture of PhMe (10 mL) and t-BuNH2
(0.25 g, 3.4 mmol) at -78 °C. After 3 h the reaction mixture
was added to EtOAc/aqueous NaHSO4 containing a few drops
of aqueous Na2SO3. The organic phase was dried and evapo-
rated, and the residue was subjected to basic hydrolysis to give
2e: mp 99.5-103.5 °C (hexane/EtOAc); NMR30 δ 1.80 (s, 3H),
2.19 (s, 3H), 2.34 (s, 3H), 2.2-2.45 (m, 4H), 3.40 (d, J ) 6 Hz,
2H), 3.66 (s, 3H), 5.27 (t, J ) 7 Hz, 1H). Anal. (C16H21BrO4)
C, H.
(E)-6-(2-Hydr oxy-3-iodo-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (2f). A solution of 27f (0.64 g,
2.2 mmol) in CH2Cl2 (1 mL) was added to a solution of t-BuNH2

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4191
(0.32 g, 4.4 mmol) and I2 (0.556 g, 2.2 mmol) in PhMe (12 mL)
at 0 °C. After 1 h, the reaction mixture was worked up as for
2e to give the methyl ester of 2f as an oil (0.9 g, 98%). Basic
hydrolysis then afforded 2f: mp 89-91 °C (t-BuOMe/hexane);
NMR30 δ 1.80 (s, 3H), 2.23 (s, 3H), 2.44 (s, 3H), 2.3-2.5 (m,
4H), 3.44 (d, J ) 6 Hz, 2H), 3.67 (s, 3H), 5.28 (t, J ) 7 Hz,
1H). Anal. (C16H21IO4) C, H.
was dried and concentrated to an oil. Chromatography (4:1
hexane/EtOAc) gave 0.19 g of the methyl ester of 2j. Basic
hydrolysis afforded 2j: mp 165-166 °C (hexane/EtOAc);
NMR30 (DMSO-d6) δ 1.80 (s, 3H), 2.15 (s, 3H), 2.15-2.35 (m,
4H), 2.30 (s, 3H), 3.32 (d, J ) 6.8 Hz, 2H), 3.67 (s, 3H), 5.23
(br t, J ) 6 Hz, 1H), 8.63 (s, 1H), 11.1 (s, 1H), 11.5 (s, 1H).
Anal. (C17H23NO5) C, H; N: calcd, 7.21; found, 7.86.
(E)-6-[2-Hydr oxy-6-m eth oxy-3-[(m eth oxyim in o)m eth yl]-
4,5-d im eth ylp h en yl]-4-m eth yl-4-h exen oic Acid (2k ). This
compound was prepared as above (2j) using methoxyamine
hydrochloride: mp 112-113 °C (hexane); NMR30 δ 1.84 (s, 3H),
2.18 (s, 3H), 2.27 (s, 3H), 2.28-2.48 (m, 4H), 3.41 (d, J ) 6.6
Hz, 2H), 3.68 (s, 3H), 4.0 (s, 3H), 5.32 (br t, J ) 6 Hz, 1H),
8.57 (s, 1H), 10.61 (br s, 1H). Anal. (C18H25NO5) C, H, N.
(E)-6-(2-Hyd r oxy-3,6-d im eth oxy-4,5-d im eth ylp h en yl)-
4-m eth yl-4-h exen oic Acid (2l). This was prepared from 2,5-
dimethoxy-3,4-dimethylphenol50 in an overall yield of 13%: mp
83-86 °C (hexanes); NMR30 δ 1.80 (s, 3H), 2.11 (s, 3H), 2.17
(s, 3H), 2.35 (m, 2H), 2.44 (m, 2H), 3.36 (d, J ) 7 Hz, 2H),
3.64 (s, 3H), 3.78 (s, 3H), 5.31 (t, J ) 7 Hz, 1H). Anal.
(C17H24O5) C, H.
(E)-6-(6-H yd r oxy-2,3,4-t r im et h ylp h en yl)-4-m et h yl-4-
h exen oic Acid (2m ). This was prepared from 3,4,5-
trimethylphenol: mp 109.5-111.5 °C (EtOAc/hexane); NMR30
δ 1.82 (s, 3H), 2.13 (s, 3H), 2.20 (s, 3H), 2.23 (s, 3H), 2.25-
2.50 (m, 4H), 3.40 (d, J ) 6 Hz, 2H), 5.15 (t, J ) 6 Hz, 1H),
6.50 (s, 1H). Anal. (C16H22O3) C, H.
(E )-6-(2-H y d r o x y -6-m e t h o x y -4,5-d im e t h y l-3-n it r o -
p h en yl)-4-m eth yl-4-h exen oic Acid (2g). 27f (0.75 g, 2.6
mmol) was dissolved in pyridine (8.4 mL), and a solution of
tetranitromethane (0.60 g, 3.1 mmol) in EtOH (4.8 mL) was
added. After 16 h, the mixture was partitioned between EtOAc
and aqueous NaHSO4. The organic layer was dried and
evaporated and the residue chromatographed (9:1 hexane/
EtOAc) to give the methyl ester of 2g (0.167 g, 19%) as a yellow
oil. Basic hydrolysis then gave 2g: mp 84-86 °C (t-BuOMe/
hexane); NMR30 δ 1.79 (s, 3H), 2.18 (s, 3H), 2.41 (s, 3H), 2.25-
2.50 (m, 4H), 3.42 (d, J ) 6 Hz, 2H), 3.70 (s, 3H), 5.23 (t, J )
7 Hz, 1H). Anal. (C16H21NO6) H, N; C: calcd, 59.43; found,
59.98.
(E )-6-(3-Cya n o-2-h yd r oxy-6-m e t h oxy-4,5-d im e t h yl-
p h en yl)-4-m eth yl-4-h exen oic Acid (2h ). Ethyl cyanoac-
etate (56.5 g, 0.5 mol) was added to 1 M ethanolic sodium
ethoxide (500 mL, 0.5 mol), and the mixture was stirred for
30 min. Mesityl oxide (49 g, 0.5 mol) was added, and the
mixture was heated at reflux for 1 h. The mixture was poured
in water and extracted with Et2O. The aqueous solution was
acidified (HCl) and extracted with EtOAc. The extract was
washed with 1 N HCl and water, dried, and evaporated, and
the residue was crystallized twice from EtOAc/hexane to afford
4-cyano-5,5-dimethyl-1,3-cyclohexanedione (33a ) (49 g, 60%),
mp 129-132 °C. Anal. (C9H11NO2) C, H, N. This material
(10 g, 60.5 mmol) was dissolved in MeOH (100 mL), and
trimethyl orthoformate (6.4 g, 60.5 mmol) and p-TsOH (57 mg,
0.3 mmol) were added. Two additional portions of trimethyl
orthoformate (6.4 g, 60.5 mmol each) were added at 24 h
intervals. After 7 d the mixture was added to water and
extracted with EtOAc. The extract was washed with H2O and
brine, dried, and evaporated. The residue was chromato-
graphed (CH2Cl2/hexane/acetone, 5:5:1) to give 6-cyano-3-
methoxy-5,5-dimethylcyclohex-2-enone (33b) (3.7 g, 34%), mp
78-80 °C. Anal. (C10H12NO2) C, H, N. The enone (1.9 g, 10.5
mmol) was cooled to 0 °C and cold trifluoroacetic anhydride
(20 mL) added. Sulfuric acid (0.64 mL, 11.6 mmol) was added
dropwise and the mixture stirred at room temperature for 18
h. The volatiles were evaporated. Ice and solid Na2CO3 were
added to pH 8. The mixture was reacidified (2 N HCl) and
extracted with EtOAc. The extract was dried and evaporated.
The residue was chromatographed (EtOAc/hexane, 1:1) to give
2-cyano-5-methoxy-3,4-dimethylphenol48 (32h ) (1.2 g, 64%), mp
232-238 °C. Anal. (C10H11NO2‚1/8EtOAc) C, H, N. Using the
procedures described above, the latter was converted into 2h :
mp 112-115 °C (EtOAc/cyclohexane); NMR30 δ 1.83 (s, 3H),
2.14 (s, 3H), 2.3-2.5 (m, 4H), 2.40 (s, 3H), 3.40 (d, J ) 7 Hz,
2H), 3.68 (s, 3H), 5.32 (t, J ) 7 Hz, 1H). Anal. (C17H21NO4‚
1/4H2O) C, H, N.
(E)-6-(3-Ch lor o-2-h yd r oxy-4,5,6-t r im et h ylp h en yl)-4-
m eth yl-4-h exen oic Acid (2n ). N-Chlorosuccinimide (8.5 g,
63.5 mmol) in DMF (400 mL) was added dropwise over 0.5 h
to a cooled (0 °C) solution of 3,4,5-trimethylphenol (5.6 g, 31.8
mmol) in DMF (300 mL). The mixture was allowed to warm
to room temperature, and after 48 h the reaction was quenched
with water and the mixture extracted with ether. The ether
layer was stirred with a slurry of zinc and saturated am-
monium chloride. The organic layer was dried and evaporated
to an orange oil which was chromatographed (50:1 hexanes/
EtOAc) to give 1.96 g (29%) of 2-chloro-3,4,5-trimethylphenol
as a yellow oil. After O-allylation, Claisen rearrangement, and
silylation, ozonolysis gave a mixture so an alternative oxida-
tion was employed. To a solution of 1-[(tert-butyldimethylsi-
lyl)oxy]-2-chloro-3,4,5-trimethyl-6-(2-propenyl)benzene (910 mg,
2.8 mmol) in THF/water (1:1) was added OsO4 (2.5 wt % in
2-methyl-2-propanol, 0.5 mL). After 5 min of stirring, sodium
periodate (1.26 g, 5.88 mmol) was added. After 5 h the reaction
mixture was diluted with ether and washed with saturated
aqueous NaHCO3 and brine. The organic layer was dried
(MgSO4), filtered, and stripped to a brown oil which was
chromatographed (30:1 hexanes/EtOAc) to afford [2-[(tert-
b u t y l d i m e t h y l s i l y l ) o x y ] - 3 - c h l o r o - 4 , 5 , 6 - t r i -
methylphenyl]acetaldehyde (34) (600 mg, 65%), mp 44-45 °C.
Anal. (C17H27ClO2Si) C, H, Cl. This compound was then
transformed into 2n : mp 148-150 °C (EtOAc/hexane); NMR30
δ 1.8 (s, 3H), 2.15 (s, 6H), 2.32 (s, 3H), 2.25-2.4 (m, 4H), 3.4
(d, J ) 6.7 Hz, 2H), 5.11 (br t, J ) 6.7 Hz, 1H). Anal. (C16H21
ClO3) C, H, Cl.
-
(E)-6-(3-F or m yl-2-h yd r oxy-6-m et h oxy-4,5-d im et h yl-
p h en yl)-4-m eth yl-4-h exen oic Acid (2i). A mixture of the
methyl ester of 2a (2.29 g, 7.84 mmol) and hexamethylene-
tetramine (1.35 g, 9.63 mmol) in AcOH (20 mL) was heated at
60 °C for 24 h.49 Water (25 mL) was added, and the mixture
was stirred at 50 °C for 8 h. The mixture was partitioned
between EtOAc and water; the organic phase was washed with
brine, dried, and concentrated to an oil. Chromatography (85:
15 hexane/EtOAc) afforded the methyl ester of 2i (1.02 g, 57%)
as a low-melting solid. Basic hydrolysis gave 2i: mp 109-
110 °C (hexane/EtOAc); NMR30 δ 1.80 (s, 3H), 2.15 (s, 3H),
2.26-2.50 (m, 4H), 2.47 (s, 3H), 3.35 (d, J ) 7.6 Hz, 2H), 3.7
(s, 3H), 5.26 (br t, J ) 6 Hz, 1H), 10.29 (s, 1H), 12.43 (s, 1H).
Anal. (C17H22O5) C, H.
(E)-6-(3-Br om o-2-h yd r oxy-4,5,6-t r im et h ylp h en yl)-4-
m eth yl-4-h exen oic Acid (2o). t-BuNH2 (0.277 g, 3.8 mmol)
was added to PhMe (88 mL) at -25 °C. After 15 min the
solution was cooled to -78 °C, and Br2 (0.276 g, 1.72 mmol)
was added. The ethyl ester of 2m (0.5 g, 1.72 mmol) in CH2
Cl2
(1 mL) was added. After 1 h the reaction mixture was warmed
to room temperature and partitioned between EtOAc and
aqueous Na2SO3. The organic phase was dried and evaporated
and the residue chromatographed to give the ethyl ester of 2o
(0.38 g, 60%). Hydrolysis then afforded 2o: mp 140-141.5
°C; NMR30
δ 1.80 (s, 3H), 2.16 (s, 3H), 2.19 (s, 3H), 2.25-2.50
(m, 4H), 2.38 (s, 3H), 3.46 (d, J ) 6 Hz, 2H), 5.13 (t, J ) 6 Hz,
1H). Anal. (C16H21BrO3) C, H.
(E)-6-(2-Hydr oxy-4,5,6-tr im eth yl-3-n itr oph en yl)-4-m eth -
yl-4-h exen oic Acid (2p ). Nitration of the ethyl ester of 2m ,
as described above (2g), gave 33% of the ethyl ester of 2p as
a yellow oil. Hydrolysis then afforded 2p : mp 114-117.5 °C
(t-BuMe ether/hexane); NMR30 δ 1.80 (s, 3H), 2.19 (s, 3H), 2.22
(s, 3H), 2.25-2.50 (m, 4H), 3.34 (d, J ) 6 Hz, 2H), 5.08 (t, J )
6 Hz, 1H). Anal. (C16H21NO5) C, H, N.
(E)-6-[2-Hydr oxy-3-[(h ydr oxyim in o)m eth yl]-6-m eth oxy-
4,5-d im eth ylp h en yl]-4-m eth yl-4-h exen oic Acid (2j).
A
mixture of the methyl ester of 2i (0.19 g, 0.59 mmol) and NH2-
OH‚HCl (0.065 g, 0.94 mmol) in pyridine (1 mL) was stirred
at room temperature for 5 h. The mixture was partitioned
between EtOAc and aqueous 1 M NaHSO4; the organic layer

4192 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nelson et al.
(E)-6-(5-Ch lor o-2-m eth oxy-3,4-dim eth ylph en yl)-4-m eth -
yl-4-h exen oic Acid (3a ). Using procedures described above,
4-chloro-2,3-dimethylphenol was converted into methyl (E)-6-
(5-chloro-2-hydroxy-3,4-dimethylphenyl)-4-methyl-4-hex-
enoate as an oil. This material (200 mg, 0.7 mmol) was
dissolved in acetone (25 mL), and K2CO3 (2.2 g, 16 mmol) and
MeI (0.5 mL, 0.8 mmol) were added. After stirring for 20 h,
the mixture was added to water (100 mL) and extracted with
EtOAc. The organic layer was washed, dried, and evaporated
to afford the methyl ester of 3a (130 mg, 62%) as a clear oil
which was hydrolyzed to give 3a (oil, 77%): NMR30 δ 1.73 (s,
3H), 2.22 (s, 3H), 2.32 (s, 3H), 2.3-2.5 (m, 4H), 3.31 (d, J ) 7
Hz, 2H), 3.66 (s, 3H), 5.31 (t, J ) 7 Hz, 1H), 6.99 (s, 1H).
Anal. (C16H21ClO3) C, H.
The organic layer was washed, dried, and evaporated to afford
the methyl ester of 3f (oil, 86%). Basic hydrolysis then gave
3f (85%): mp 39-40 °C (ether); NMR30 δ 1.76 (s, 3H), 2.11 (s,
3H), 2.20 (s, 3H), 2.3-2.5 (m, 4H), 3.55 (d, J ) 7 Hz, 2H), 3.64
(s, 3H), 3.77 (s, 3H), 5.0 (s, 1H), 5.36 (t, J ) 7 Hz, 1H). Anal.
(C17H24O4‚0.25H2O) C, H.
(E)-6-(5-Cya n o-2-m eth oxy-3,4-dim eth ylp h en yl)-4-m eth -
yl-4-h exen oic Acid (3g). The methyl ester of 3e was
converted via the triflate to the nitrile (see 1p ), which upon
basic hydrolysis gave 3g (60%, foam): MS m/z 287 (M+), 269,
227, 174; NMR30 δ 1.73 (s, 3H), 2.22 (s, 3H), 2.43 (s, 3H), 2.4-
2.5 (m, 4H), 3.34 (d, J ) 7 Hz, 2H), 3.71 (s, 3H), 5.30 (t, J )
7 Hz, 1H).
(E)-6-[2-Meth oxy-3,4-d im eth yl-5-(m eth ylth io)p h en yl]-
4-m eth yl-4-h exen oic Acid (3h ).51 35a (2.5 g, 6.0 mmol) was
dissolved in THF (40 mL), purged with N2, and cooled to -70
°C. n-BuLi (1.6 M in hexane, 5.3 mL, 8.5 mmol) was added
dropwise. After 15 min, dimethyl disulfide (0.65 mL, 8.9
mmol) was added dropwise. After 30 min, the reaction was
quenched with aqueous NH4Cl and the mixture partitioned
between EtOAc and H2O. The organic layer was washed,
dried, and evaporated. Chromatography (3:1 hexane/toluene)
then gave 35b as an oil (1.6 g, 70%). Desilylation, ortho-ester
Claisen rearrangement, and basic hydrolysis then gave 3h
(50%): mp 47-49 °C (hexane); NMR30 δ 1.75 (s, 3H), 2.22 (s,
3H), 2.29 (s, 3H), 2.35-2.5 (m, 4H), 3.35 (d, J ) 7 Hz, 2H),
3.66 (s, 3H), 5.35 (t, J ) 7 Hz, 1H). Anal. (C17H24O3S) C, H.
(E )-6-[5-(Me t h y ls u lfin y l)-2-m e t h o x y -3,4-d im e t h y l-
p h en yl]-4-m eth yl-4-h exen oic Acid (3i). To the methyl
ester of 3h (200 mg, 0.62 mmol) in CH2Cl2 (8 mL) were added
wet alumina52 (620 mg) and Oxone (382 mg, 0.62 mmol), the
mixture was heated to reflux for 20 h, cooled, and filtered, and
the solvent was removed. Chromatography (3:1 EtOAc/hex-
ane) gave the methyl ester of 3i (140 mg, 67%) as an oil. Basic
hydrolysis then gave 3i (92%): mp 83-85 °C (hexane); NMR30
δ 1.73 (s, 3H), 2.22 (s, 3H), 2.25 (s, 3H), 2.4-2.55 (m, 4H), 2.83
(s, 3H), 3.43 (d, J ) 7 Hz, 2H), 3.73 (s, 3H), 5.47 (t, J ) 7 Hz,
1H), 7.62 (s, 1H). Anal. (C17H24O4S) C, H.
(E)-6-(2,3-Diflu or o-6-m et h oxy-4,5-d im et h ylp h en yl)-4-
m eth yl-4-h exen oic Acid (3j). A solution of 5-amino-2,3-
dimethylphenol53 (10.1 g, 73.6 mmol) in ether (380 mL) and
48% fluoboric acid (74 mL) was chilled with an ice bath while
74 g of 4A molecular sieves was added. After stirring for 1 h
at room temperature, it was filtered through glass wool and
chilled to 0 °C, and isoamyl nitrite (10.9 mL, 81.1 mmol) was
added. After 1 h, the mixture was warmed to room temper-
ature during 0.5 h and then refluxed for 3 h. The reaction
mixture was diluted with ether (500 mL) and washed with
aqueous NaHCO3 until the washes were alkaline. The ether
solution was then dried and evaporated. Chromatography
(93:7 hexane/ether) then gave 5-fluoro-2,3-dimethylphenol
(3.73 g, 36.2%), mp 71.9-72.8 °C. Anal. (C8H9FO) H; C: calcd,
68.56; found, 69.34. This compound (1.52 g, 10.8 mmol) and
1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis-
(tetrafluoroborate)54 (3.89 g, 11.9 mmol) were refluxed in
MeOH (55 mL) for 2.5 h. The reaction mixture was partitioned
between EtOAc and brine. The EtOAc solution was dried and
evaporated. Chromatography (93:7 hexane/ether) then gave
4,5-difluoro-2,3-dimethylphenol (246 mg, 14%), slightly more
polar than the starting material. Using the procedures
described above, this compound was converted into 3j: mp
78.4-78.7 °C (hexane); NMR30 δ 1.78 (s, 3H), 2.16 (m, 6H),
2.30 (m, 2H), 2.43 (m, 2H), 3.36 (br d, J ) 7 Hz, 2H), 3.65 (s,
3H), 5.23 (br t, J ) 7 Hz, 1H). Anal. (C16H20F2O3) C, H.
(E)-6-(2-Ch lor o-3-flu or o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (3k ). 5-Chloro-2,3-dimethylphe-
nol46 was fluorinated as described above to give 5-chloro-4-
fluoro-2,3-dimethylphenol, 13.3%; mp 84.4-100.6 °C. Anal.
(C8H8ClFO) C, H. This was converted into 3k : mp 96.5-97.1
°C (hexane); NMR30 δ 1.81 (br s, 3H), 2.18 (m, 6H), 2.31 (m,
2H), 2.44 (m, 2H), 3.47 (br d, J ) 7 Hz, 2H), 3.64 (s, 3H), 5.18
(br t, J ) 7 Hz, 1H). Anal. (C16H20ClFO3) C, H.
(E)-6-(5-Br om o-2-m eth oxy-3,4-dim eth ylph en yl)-4-m eth -
yl-4-h exen oic Acid (3b). 2,3-Dimethylphenol was converted,
as described above, into 2,3-dimethyl-6-(2-propenyl)phenol
(67%, oil). Tribromination/reductive debromination, as de-
scribed above (1t), gave 4-bromo-2,3-dimethyl-6-(2-propenyl)-
phenol as a yellow oil (5.1 g, 61% overall) which was O-meth-
ylated to give 4-bromo-2,3-dimethyl-6-(2-propenyl)anisole (89%),
mp 55-56 °C (hexane). This compound was then transformed
into 3b: mp 62-64 °C (EtOH); NMR30 δ 1.73 (s, 3H), 2.25 (s,
3H), 2.32 (s, 3H), 2.4-2.5 (m, 4H), 3.31 (d, J ) 7 Hz, 2H), 3.74
(s, 3H), 5.31 (t, J ) 7 Hz, 1H), 7.18 (s, 1H). Anal. (C16H21
BrO3‚0.25H2O) C, H.
-
(E)-6-(2-Meth oxy-3,4-d im eth yl-5-n itr op h en yl)-4-m eth -
yl-4-h exen oic Acid (3c). To cold TFA (0-5 °C) was added
2,3-dimethyl-6-(2-propenyl)phenol (see preparation of 3b) (6.7
g, 0.038 mol) followed by dropwise addition of nitric acid (1.7
mL, 0.042 mol). After 15 min, the reaction mixture was poured
into H2O and extracted with EtOAc, and the organic layer was
washed, dried, and evaporated to a red oil. Chromatography
(19:1 hexane/EtOAc) afforded 2,3-dimethyl-4-nitro-6-(2-pro-
penyl)phenol as a red liquid (2.9 g, 35%). Methylation, as
described above, gave 2,3-dimethyl-4-nitro-6-(2-propenyl)ani-
sole which was converted into 3c: mp 64-65 °C (ether); NMR30
δ 1.75 (s, 3H), 2.29 (s, 3H), 2.38 (s, 3H), 2.4-2.6 (m, 4H), 3.39
(d, J ) 7 Hz, 2H), 3.74 (s, 3H), 5.33 (t, J ) 7 Hz, 1H), 7.49 (s,
1H). Anal. (C16H21NO5) C, H, N.
(E)-6-(5-Am in o-2-m eth oxy-3,4-dim eth ylph en yl)-4-m eth -
yl-4-h exen oic Acid (3d ). 3c (150 mg, 0.49 mmol) was
dissolved in DMF (3 mL), NaHCO3 (123 mg, 1.46 mmol) and
NaS2O4 (255 mg, 1.46 mmol) were added, and the mixture was
stirred at 60 °C for 4 h. Water was added, and the reaction
mixture was acidified with HOAc and extracted with EtOAc
and then CH2Cl2. Combined organic layers were washed,
dried, and evaporated. Chromatography (1:1 EtOAc/hexane
0.1% HOAc) then gave 3d (35 mg, 26%): mp 130-132 °C
(acetone); NMR30 (DMSO-d6) δ 1.67 (s, 3H), 1.91 (s, 3H), 2.06
(s, 3H), 2.2-2.3 (m, 4H), 3.14 (d, J ) 7 Hz, 2H), 3.48 (s, 3H),
5.20 (t, J ) 7 Hz, 1H), 6.27 (s, 1H). Anal. (C16H23NO3‚0.25H2O)
C, H, N.
(E)-6-(5-H yd r oxy-2-m et h oxy-3,4-d im et h ylp h en yl)-4-
m eth yl-4-h exen oic Acid (3e). 2,3-Dimethylbenzene-1,4-diol
was converted into 4-hydroxy-2,3-dimethylphenyl allyl ether,
35%, mp 96-97 °C (ether), by alkylation with 1 mol of allyl
bromide. Silylation and Claisen rearrangement gave 4-[(tert-
butyldimethylsilyl)oxy]-2,3-dimethyl-6-(2-propenyl)phenol (61%,
oil). The phenol (2.0 g, 6.77 mmol) was dissolved in DMF (60
mL) and cooled to 5 °C. To this were successively added NaOH
(325 mg, 8.12 mmol) dissolved in H2O (3 mL) and dimethyl
sulfate (1.4 mL, 14.9 mmol). After 1 h the reaction mixture
was partitioned between EtOAc and 10% HCl. The organic
layer was dried and evaporated and the residue chromato-
graphed (97:3 hexane/acetone) to give 4-[(tert-butyldimethyl-
silyl)oxy]-2,3-dimethyl-6-(2-propenyl)anisole (oil, 86%) which
was converted into 3e (36%): mp 127-130 °C (EtOAc); NMR30
(DMSO-d6) δ 1.67 (s, 3H), 1.98 (s, 3H), 2.07 (s, 3H), 2.2-2.35
(m, 4H), 3.17 (d, J ) 7 Hz, 2H), 3.52 (s, 3H), 5.21 (t, J ) 7 Hz,
1H), 8.77 (s, 1H). Anal. (C16H22O4) C, H.
(E)-6-(2,5-Dim eth oxy-3,4-d im eth ylp h en yl)-4-m eth yl-4-
h exen oic Acid (3f). The methyl ester of 3e (100 mg, 0.34
mmol) was dissolved in DMF (5 mL), and K2CO3 (66 mg, 0.48
mmol) and MeI (26 µL, 0.4 mmol) were added. After 20 h,
the reaction mixture was partitioned between H2O and EtOAc.
(E)-6-(2,3-Dich lor o-6-m eth oxy-4,5-d im eth ylp h en yl)-4-
m et h yl-4-h exen oic Acid (3l). (a ) 4,5-Dich lor o-2,3-d i-
m eth ylp h en ol. 5-Chloro-2,3-dimethylphenol46 (8.98 g, 57.3
mmol) and N-chlorosuccinimide (8.42 g, 63.1 mmol) in DMF

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4193
(180 mL) were warmed in a 60 °C oil bath for 3 h. The reaction
mixture was partitioned between ether and water. After
washing with 250 mL of 10% aqueous Na2SO3, the ether layer
was dried (Na2SO4) and concentrated. Chromatography (95:5
and then 90:10 hexane/acetone) gave the title compound55 (6.35
g, 58.0%): mp 90-92 °C. Anal. (C8H8Cl2O) C, H; Cl: calcd,
37.11; found, 37.79.
(b) Using procedures described above, 4,5-dichloro-2,3-
dimethylphenol was converted to 3l: mp 94-95 °C (hexane);
NMR30 δ 1.81 (br s, 3H), 2.23 (s, 3H), 2.26-2.36 (m, 2H), 2.35
(s, 3H), 2.43 (m, 2H), 3.51 (br d, J ) 7 Hz, 2H), 3.65 (s, 3H),
5.18 (br t, J ) 6 Hz, 1H). Anal. (C16H20Cl2O3) C, H.
(E)-6-(3-Ch lor o-2-flu or o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (3m ). 5-Fluoro-2,3-dimethylphe-
nol was chlorinated as described above to give 4-chloro-5-
fluoro-2,3-dimethylphenol,55 71.2%; mp 74-77 °C. Anal.
(C8H8ClFO) H; C: calcd, 55.03; found, 55.74. This was
converted into 3m : mp 82-83 °C (hexane); NMR30 δ 1.78 (br
s, 3H), 2.19 (s, 3H), 2.28-2.35 (m, 2H), 2.31 (s, 3H), 2.43 (m,
2H), 3.37 (br d, J ) 7 Hz, 2H), 3.66 (s, 3H), 5.23 (br t, J ) 7
Hz, 1H). Anal. (C16H20ClFO3) C, H.
(E)-6-(6-Ch lor o-2-m eth oxy-3,4-dim eth ylph en yl)-4-m eth -
yl-4-h exen oic Acid (3n ). This was prepared from 5-chloro-
2,3-dimethylphenol:46 mp 81-83 °C (hexane); NMR30 δ 1.80
(br s, 3H), 2.15 (s, 3H), 2.20 (s, 3H), 2.30 (m, 2H), 2.43 (m,
2H), 3.45 (br d, J ) 7 Hz, 2H), 3.66 (s, 3H), 5.20 (br t, J ) 7
Hz, 1H), 6.96 (s, 1H). Anal. (C16H21ClO3) C, H, Cl.
(E)-6-(2-Ch lor o-6-m eth oxy-4,5-dim eth yl-3-n itr oph en yl)-
4-m eth yl-4-h exen oic Acid (3o). A solution of 5-chloro-2,3-
dimethyl-6-(2-propenyl)phenol (1.13 g, 5.75 mmol) in AcOH
(51.8 mL) and water (5.7 mL) was chilled to 0 °C, and 70%
HNO3 (0.40 mL, 6.3 mmol) was added. After 1.25 h at 0 °C,
it was diluted with EtOAc (200 mL) and washed with water,
aqueous NaHCO3 until the washes were alkaline, and then
brine. After drying the EtOAc solution was evaporated.
Chromatography (90:10 hexane/EtOAc) then gave 5-chloro-2,3-
dimethyl-4-nitro-6-(2-propenyl)phenol (0.962 g, 69.2%) which
was converted into 3o: mp 101.2-101.9 °C (acetone/hexane);
NMR30 δ 1.80 (br s, 3H), 2.17 (s, 3H), 2.23 (s, 3H), 2.31 (m,
2H), 2.45 (m, 2H), 3.50 (br d, J ) 7 Hz, 2H), 3.69 (s, 3H), 5.15
(br t, J ) 7 Hz, 1H). Anal. (C16H20ClNO5) C, H, N.
(E )-6-(2-Ch lor o-3-h yd r oxy-6-m e t h oxy-4,5-d im e t h yl-
p h en yl)-4-m eth yl-4-h exen oic Acid (3p ). To 2,3-dimethyl-
benzene-1,4-diol (5.0 g, 36.2 mmol) in DMF (40 mL) was added
NCS (5.3 g, 39.8 mmol) in one portion. The reaction mixture
was warmed to 60 °C for 3 h and then allowed to cool. The
reaction mixture was poured over H2O (1.5 L) and extracted
with EtOAc, and the combined acetate layers were washed,
dried, and evaporated. Chromatography (9:1 hexane/EtOAc)
then afforded 2.9 g (39%) of 5-chloro-2,3-dimethylbenzene-1,4-
diol as an oil. To this material (3.8 g, 22 mmol) in DMF (40
mL) was added DBU (3.9 mL, 26.4 mmol) followed by t-BuMe2-
SiCl (3.3 g, 22 mmol), and the reaction mixture was allowed
to stir at room temperature for 24 h. Reaction was quenched
with H2O (1 L), the mixture was extracted with EtOAc (4 ×
100 mL), and the combined acetate layers were washed, dried,
and evaporated. Flash chromatography (hexane) afforded 2.0
g (32%) of 4-[(tert-butyldimethylsilyl)oxy]-6-chloro-2,3-dimeth-
ylphenol as a clear liquid. To a solution of the phenol (2.0 g,
6.97 mmol) in pyridine (20 mL) was added Ac2O (1.7 mL, 17
mmol) in one portion. After 36 h, the reaction was quenched
with H2O and the mixture extracted with Et2O (3 × 50 mL).
The combined ether layers were washed, dried, and evaporated
to afford 4-[(tert-butyldimethylsilyl)oxy]-6-chloro-2,3-dimeth-
ylphenyl acetate (2.2 g, 96%) as a clear oil. Desilylation
(TBAF) then gave 6-chloro-4-hydroxy-2,3-dimethylphenyl ac-
etate which was converted into 2-chloro-4-hydroxy-5,6-di-
methyl-3-(2-propenyl)phenyl acetate by allylation/Claisen re-
arrangement. To a solution of the latter compound (1.4 g, 5.5
mmol) in acetone (40 mL) was added K2CO3 (3.0 g, 21.98 mmol)
followed by MeI (0.5 mL, 7.7 mmol), and the solution was
allowed to stir for 7 h; then the reaction was quenched with
H2O (500 mL), the mixture was extracted with EtOAc (3 × 50
mL), and the combined acetate layers were washed, dried, and
evaporated. Chromatography (20:1 hexane/acetone) afforded
2-chloro-4-methoxy-5,6-dimethyl-3-(2-propenyl)phenyl acetate
(1.4 g, 95%) as an oil. To this compound (1.2 g, 4.47 mmol) in
MeOH (20 mL) and H2O (8 mL) was added K2CO3 (0.92 g, 6.7
mmol). After 20 h the MeOH was removed under vacuum,
the residue was diluted with H2O and extracted with EtOAc,
and the combined acetate layers were washed, dried, and
evaporated to give 2-chloro-4-methoxy-5,6-dimethyl-3-(2-pro-
penyl)phenol as a light brown oil. Using the previously
described procedures, this material was converted into 3p : mp
60-62 °C (2-propanol); NMR30 δ 1.80 (s, 3H), 2.18 (s, 6H),
2.28-2.46 (m, 4H), 3.45 (d, J ) 6.7 Hz, 2H), 3.62 (s, 3H), 5.17
(t, J ) 7.8 Hz, 1H). Anal. (C16H21ClO4) C, H.
(E)-6-(2-Ch lor o-3-cyan o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (3q). Using the procedures de-
scribed above (1k ,p ), the methyl ester of 3p was converted into
the phenolic triflate which was displaced with cyanide using
Pd catalysis to give, after ester hydrolysis, 3q: mp 116-118
°C (ether); NMR30 δ 1.80 (s, 3H), 2.20 (s, 3H), 2.29-2.45 (m,
4H), 2.47 (s, 3H), 3.48 (d, J ) 6.6 Hz, 2H), 3.70 (s, 3H), 5.14
(m, 1H). Anal. (C17H20ClNO3) C, H, N.
(E)-6-(3-Br om o-2-ch lor o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (3r ). 3-Chloro-5,6-dimethyl-2-(2-
propenyl)phenol was subjected to bromination/reductive de-
bromination (Zn/AcOH) as described above to give 4-bromo-
3-chloro-5,6-dimethyl-2-(2-propenyl)phenol, oil. Anal. (C11H12
-
BrClO) C, H. This was converted into 3r , mp 90-93 °C
(hexane); NMR30 δ 1.80 (br s, 3H), 2.25 (s, 3H), 2.31 (m, 2H),
2.41 (s, 3H), 2.44 (m, 2H), 3.54 (br d, J ) 7 Hz, 2H), 3.65 (s,
3H), 5.18 (br t, J ) 6 Hz, 1H). Anal. (C16H20BrClO3) C, H.
(E)-6-(3-Ch lor o-6-m et h oxy-2,4,5-t r im et h ylp h en yl)-4-
m eth yl-4-h exen oic Acid (3s). 4-Chloro-2,3,5-trimethylphe-
nol was converted into 4-chloro-2,3,5-trimethyl-6-(2-propenyl)-
phenol, mp 53-54 °C (EtOH). Anal. (C12H15ClO‚0.125H2O)
C, H. This was transformed into 3s: mp 105-107 °C (hexane);
NMR30 δ 1.77 (s, 3H), 2.21 (s, 3H), 2.26 (s, 3H), 2.30 (s, 3H),
2.28-2.42 (m, 4H), 3.39 (d, J ) 6.4 Hz, 2H), 3.61 (s, 3H), 5.07
(m, 1H). Anal. (C17H23ClO3) C, H.
(E)-6-(6-Am in o-2,3,4-tr im eth ylp h en yl)-4-m eth yl-4-h ex-
en oic Acid (4a ). To a solution of 2,3,4,5-tetramethylaniline56
(2.61 g, 17.5 mmol) in THF (15 mL) was added di-tert-butyl
dicarbonate (3.9 g, 17.87 mmol). After 24 h the solvent was
removed and the residue recrystallized from hexane to give
3.1 g (71%) of N-(tert-butoxycarbonyl)-2,3,4,5-tetramethyl-
aniline, mp 108-109 °C. To a -78 °C solution of this
compound (3.02 g, 12.11 mmol) in THF (40 mL) was added
t-BuLi21 (16 mL of a 1.6 M solution in pentane, 25.6 mmol).
The yellow solution was allowed to warm to -20 °C and then
recooled to -78 °C. Methacrolein (1.8 mL, 21.7 mmol, freshly
distilled and dried over Na2SO4) was added, the solution was
stirred for 5 min, and then the reaction was quenched with
aqueous NH4Cl. The mixture was partitioned between EtOAc
and water; the organic phase was washed with brine, dried,
and concentrated to give 30 (R ) 2,3,4-trimethyl), 1.48 g, 38%;
mp 142-143 °C (EtOAc/hexane). Claisen rearrangement of
this compound (1.36 g, 4.25 mmol) gave 1.63 g of crude product.
This material was dissolved in MeOH (40 mL) and treated with
p-TsOH‚H2O (1.29 g, 6.78 mmol). The reaction mixture was
heated at 50 °C for 12 h and then partitioned between EtOAc
and aqueous NaHCO3. The organic phase was dried and
concentrated to an oil. Chromatography (4:1 hexane/EtOAc)
gave 1.04 g (88%) of the methyl ester of 4a as an oil. Basic
hydrolysis afforded 4a : mp 176-179 °C (EtOAc); NMR30
(DMSO-d6) δ 1.73 (s, 3H), 1.98 (s, 3H), 2.04 (s, 3H), 2.08 (s,
3H), 2.1-2.3 (m, 4H), 3.13 (d, J ) 6.4 Hz, 2H), 3.35 (br s, ca.
3 H), 4.93 (br t, J ) 6 Hz, 1H), 6.35 (s, 1H). Anal. (C16H23
NO2) C, H, N.
-
(E)-6-(2-Am in o-3-br om o-4,5,6-tr im eth ylph en yl)-4-m eth -
yl-4-h exen oic Acid (4b). To 4a methyl ester (0.58 g, 2.1
mmol) in DMF (5 mL) at 0 °C was added a solution of NBS
(0.37 g, 2.1 mmol) in DMF (5 mL). The reaction mixture was
stirred an additional 15 min; then the reaction was quenched
with aqueous Na2SO3 and the mixture partitioned between
EtOAc and water. The organic phase was dried and concen-
trated to an oil. Chromatography gave the methyl ester of 4b
(0.29 g), which upon basic hydrolysis afforded 4b (0.21 g): mp
139-141 °C (hexane/EtOAc); NMR30 δ 1.75 (s, 3H), 2.07 (s,

4194 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nelson et al.
3H), 2.1-2.35 (m, 4H), 2.30 (s, 3H), 3.26 (d, J ) 6.4 Hz, 2H),
4.53 (br s, 2H), 4.93 (br t, J ) 6 Hz, 1H). Anal. (C16H22BrNO2)
C, H, N.
between EtOAc and H2O and the organic phase washed with
water, filtered through Celite, and then further washed with
aqueous NaHCO3, dried, and concentrated. The residue was
suspended in aqueous 1 N HCl and heated at reflux for 24 h.
After cooling the reaction mixture was basified with NH4OH
and extracted with hexane/EtOAc (4:1). The organic phase
was dried and concentrated. Chromatography (hexane/EtOAc,
9:1) gave 2-chloro-5-methoxy-3,4,6-trimethylaniline (2.3 g). A
-78 °C solution of this material (2.08 g, 11.45 mmol) and di-
tert-butyl dicarbonate (2.9 g, 13.28 mmol) was treated with
NaHMDS (25 mL of a 1.0 M solution in THF). After 10 min
the reaction mixture was poured into saturated aqueous NH4Cl
and extracted with EtOAc. The organic phase was dried,
concentrated, and chromatographed (hexane/t-BuOMe). The
product fractions were concentrated, redissolved in hexane (20
mL), and allowed to crystallize to yield 1.42 g of N-(tert-
butoxycarbonyl)-2-chloro-5-methoxy-3,4,6-trimethylaniline, mp
95 °C (hexane). Side-chain elaboration, N-deprotection, and
ester hydrolysis then gave 4f: mp 92-96 °C (hexane/EtOAc);
NMR30 δ 1.83 (s, 3H), 2.16 (s, 3H), 2.29 (s, 3H), 2.3-2.5 (m,
4H), 3.35 (d, J ) 6.4 Hz, 2H), 3.62 (s, 3H), 5.12 (br t, J ) 6
Hz, 1H). Anal. (C16H22ClNO3) C, H, N.
In h ibition of IMP DH. The assay for IMPDH activity
measures the formation of NADH (λmax ) 340 nm, ꢀ340 ) 6220
M-1 cm-1) as IMP is converted to XMP by human type II IMP
dehydrogenase. Two different buffers and pH conditions were
employed. One set of reactions was performed at pH 7.4, in
0.1 M potassium phosphate, 0.5 M KCl, 3 mM EDTA, and 10
µg/mL bovine serum albumin (BSA). The second set of
reactions was performed at pH 8.0, in 0.1 M Tris-HCl, 0.1 M
KCl, 3 mM EDTA, and 100 µg/mL BSA. The concentrations
of the substrates IMP and NAD were 50 and 100 µM (∼2 ×
Km), respectively. The kinetics of inhibition of the compounds
did not differ significantly in the two buffer conditions.
Compounds were dissolved and diluted in DMSO or water and
assayed for inhibitory activity. DMSO at concentrations above
10% of the total reaction volume significantly inhibited the
reaction (∼40%); thus, DMSO concentrations were maintained
at or below 10% when it was used. The assays contained 0.5-
1.0 mL total volume and were initiated by addition of 12.5-
25 pmol of recombinant human type II IMPDH (0.0008-0.0016
units).22 One unit of enzyme catalyzes the formation of 1 µmol
of NADH/min at 40 °C at saturating substrate concentrations
(200 µM IMP and 400 µM NAD). The reactions were per-
formed in disposable methacrylic plastic microcuvettes (UV
transparent, 1 cm path length, 1.5 mL capacity). Enzymatic
activity was monitored at 340 nm in a UV/vis spectrophotom-
eter fitted with a water-jacketed multicell transporter main-
tained at 40 °C by a recirculating bath. The 50% inhibitory
value (IC50) for each compound was determined by computer
using nonlinear regression fit according to the following
equation:
(E)-6-(2-Am in o-3-br om o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (4c). To 2,3,6-trimethylphenol
(22.4 g, 164 mmol) in CH2Cl2/MeOH (3:2; 500 mL) at 0 °C was
added tetrabutylammonium tribromide (77 g, 162 mmol).57
After 10 min the reaction was quenched with aqueous NaH-
SO3. The reaction mixture was partitioned between water and
hexane and the organic layer dried and concentrated to give
29.5 g (84%) of 4-bromo-2,3,6 trimethylphenol. A solution of
this material (24.1 g, 112 mmol) in CH2Cl2 (100 mL) was
treated with trifluoroacetic anhydride and DMAP (0.3 g). After
3 h the reaction mixture was concentrated and the residue
redissolved in trifluoroacetic acid (50 mL). The solution was
cooled to 0 °C and treated dropwise with 90% HNO3 (10 mL).
The reaction mixture was stirred at 0 °C for 1 h, warmed to
20 °C over 1 h, and then poured onto ice. The mixture was
extracted with EtOAc and the organic phase washed with
water and aqueous NaHCO3, dried, and concentrated. The
residue was dissolved in MeOH and treated with excess NH4-
OH. After 30 min the reaction mixture was acidified with
aqueous HCl and partitioned between EtOAc and H2O. The
combined organic phases were washed, dried, and concen-
trated. The residue was chromatographed (85:15 hexane/
EtOAc) to give 4-bromo-2,3,6-trimethyl-5-nitrophenol (6.2 g,
21%), mp 143-144 °C (hexane/toluene). Methylation (MeI/
K2CO3/DMF) gave 4-bromo-5-nitro-2,3,6-trimethylanisole, mp
85-86 °C (hexane). This material (6.13 g) was hydrogenated
in EtOH containing NaOAc (2 g) over 5% Pd/C (2.5 g). After
filtration of the catalyst, the solvent was evaporated and the
residue was partitioned between ether and aqueous 1 N
NaOH. The organic layer was dried and concentrated to give
3.5 g of 3-methoxy-2,4,5-trimethylaniline, mp 85-86 °C (hex-
ane). Using the methods described above for side-chain
elaboration (see 4a ), the aniline was converted into 31 (R )
2-methoxy-3,4-dimethyl, R1 ) CH3). Bromination, as described
above for 4b, gave 4c: mp 91-94 °C (hexane/EtOAc); NMR30
δ 1.85 (s, 3H), 2.22 (s, 3H), 2.27-2.51 (m, 4H), 2.37 (s, 3H),
3.45 (d, J ) 6.3 Hz, 2H), 3.65 (s, 3H), 5.15 (br t, J ) 6 Hz,
1H). Anal. (C16H22BrNO3) C, H; N: calcd, 3.93; found, 3.48.
(E)-6-(2-Am in o-6-m eth oxy-4,5-dim eth yl-3-n itr oph en yl)-
4-m eth yl-4-h exen oic Acid (4d ). 31 (R ) 2-methoxy-3,4-
dimethyl, R1 ) CH3) (0.54 g, 1.85 mmol) and 2,3-dimethyl-2-
butene (0.23 g) in pyridine (8 mL) at 0 °C were treated with a
solution of tetranitromethane (0.4 g, 2.0 mmol). After 30 min
the reaction mixture was partitioned between aqueous 1 M
NaHSO4 and EtOAc. The organic phase was washed with
brine, dried, and concentrated to an oil. Chromatography (4:1
hexane/EtOAc) afforded the methyl ester of 4d (0.15 g) which
gave 4d upon base hydrolysis: mp 112-113 °C (hexane/
EtOAc); NMR30 δ 1.86 (s, 3H), 2.17 (s, 3H), 2.25 (s, 3H), 2.35-
2.54 (m, 4H), 3.40 (d, J ) 6.1 Hz, 2H), 3.67 (s, 3H), 5.12 (br t,
J ) 6 Hz, 1H), 6.7 (v br s, 1H). Anal. (C16H22N2O5) C, H; N:
calcd, 8.69; found, 8.22.
fractional activity ) V0/[(I/IC50)n + 1]
(E)-6-(2-Am in o-3-cyan o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m eth yl-4-h exen oic Acid (4e). The methyl ester of 4c
(0.165 g, 0.45 mmol), KCN (0.087 g, 1.3 mmol), and Pd(Ph3P)4
(0.05 g, 0.04 mmol) in dioxane (2 mL) were heated at 100 °C
for 18 h. The reaction mixture was partitioned between EtOAc
and H2O and the organic layer dried and concentrated.
Chromatography then gave the methyl ester of 4e (0.06 g,
42%). Basic hydrolysis then gave 4e: mp 111-112 °C (hex-
ane); NMR30 δ 1.84 (s, 3H), 2.12 (s, 3H), 2.35-2.53 (m, 4H),
2.38 (s, 3H), 3.33 (d, J ) 6.2 Hz, 2H), 3.67 (s, 3H), 5.10 (br t,
J ) 6 Hz, 1H). Anal. (C17H22N2O3) C, N; H: calcd, 7.33; found,
8.02.
(E)-6-(2-Am in o-3-ch lor o-6-m eth oxy-4,5-dim eth ylph en yl)-
4-m et h yl-4-h exen oic Acid (4f). Chlorination of 2,3,6-tri-
methylphenol (NCS/DMF) gave the 4-chloro product, which
after nitration using tetranitromethane, followed by O-meth-
ylation, afforded 4-chloro-2,3,6-trimethyl-5-nitroanisole. This
compound (3.28 g, 14.28 mmol), Pd/C (5%, 0.3 g), and Et3N
(25 mL) were heated to 90 °C and treated with formic acid
portionwise over 48 h until starting material was consumed
(ca. 10 mL total of formic acid).58 The mixture was partitioned
where V0 is the maximum rate, I is the concentration of the
compound, and n is the Hill coefficient.
In h ibition of Hu m a n Lym p h ocyte P r olifer a tion .59 Hu-
man peripheral blood mononuclear cells (PBMC) were sepa-
rated from heparinized whole blood by density-gradient cen-
trifugation in Ficoll-paque (Pharmacia). After washing, 2 ×
105 cells/well were cultured in microtiter plates with RPMI-
1640 (Gibco) supplemented with 5% fetal calf serum, penicillin,
and streptomycin. Phytohemagglutinin (PHA; Sigma) was
used as a T-cell mitogen at a final concentration of 10 µg/mL.
Compounds were tested at four to six different concentrations
between 0.001 and 20 µM, by addition to the culture at time
0. Compounds were dissolved in DMSO at 10-2 M, and further
dilutions were made with RPMI-1640 medium. Cultures were
set up in quadruplicate and incubated at 37 °C in an
atmosphere of air with 7% CO2 and 100% humidity for 72 h.
A pulse of 0.5 µCi/well [3H]thymidine was added for the last 6
h. Cells were collected on glass fiber filters with an automatic
harvester, and radioactivity was measured by standard scintil-
lation procedures. A mean of quadruplicate determinations

SAR for Inhibition of IMPDH
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4195
(6) Mitsui, A.; Suzuki, S. Immunosuppressive Effects of Mycophe-
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was calculated for each compound concentration, and the 50%
inhibitory concentration (IC50) for mitogenic stimulation was
determined by interpolation, using the cubic spline determi-
nation. (Curfit, Interactive Microwave Inc., State College, PA).
The results are shown in Tables 1-4.
In Vivo Im m u n osu p p r essive Assa y. A modification of
the J erne hemolytic plaque (PFC) assay60 was used. Groups
of 5-7 adult C3H or CD-1 female mice (J ackson Laboratories,
Bar Harbour, ME, or Charles River, Portage, MI), 10-12
weeks of age, were immunized on day 0 by the ip administra-
tion of 1.25 × 108 sheep red blood cells (SRBC) in 0.25 mL of
Hanks buffered salt solution (HBSS). Commencing on day 0,
each treatment group (n ) 5) received four consecutive daily
doses of the appropriate compound. Test materials were
prepared as suspensions in an aqueous vehicle containing 0.9%
NaCl, 0.5% sodium carboxymethylcellulose, 0.4% polysorbate
80, 0.9% benzyl alcohol, and 97% distilled water and were
delivered in a volume of 0.25 mL. Control animals received
aqueous vehicle. On day 4, the mice were euthanized and the
spleens were removed and gently dispersed in HBSS; 1 mL of
this cell suspension was transferred to a 15 mL tube and
diluted to 10 mL with HBSS. The cells were centrifuged for
10 min at 300g and resuspended in 10 mL of HBSS. SRBC
were washed twice with saline by centrifugation, and the final
pellet was resuspended at a final concentration of 20% in
HBSS. Guinea pig complement (Gibco), diluted 1:4 in HBSS,
was mixed with the SRBC suspension in a 1:1 ratio. A gel
consisting of 0.5% agar (Difco) in HBSS was melted in a boiling
water bath and then maintained at 47-48 °C. Aliquots (0.7
mL) of the agar solution were dispensed into warmed tubes,
and spleen cell suspensions (0.2 mL) and SRBC/complement
mixtures (0.1 mL) were added and mixed. Aliquots (0.1 mL)
of this mixture were dropped into four separate quadrants of
a warmed 47-48 °C Petri dish, and glass coverslips were
placed on each. Incubation was carried out for 2-2.5 h at 37
°C in an atmosphere of 5% CO2 in air. Areas of hemolysis
surrounding the plaque-forming cells (PFC) were enumerated
with the aid of a dissecting microscope. The data of the four
replicates were averaged for each spleen cell suspension, and
these averages were used for the calculation of the mean for
each experimental group. Total white blood cells/spleen,
plaque-forming cells/spleen, and PFC/108 WBC were calculated
for each spleen. The latter results are shown in Table 5. ED50
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Ack n ow led gm en t. We would like to thank David
J . Morgans, J r. (Institute of Organic Chemistry, Syntex
Research), for helpful discussions and suggestions, Ms.
Lilia Kurz and Ms. J anis Nelson (Institute of Analytical
Research, Syntex Research) for extensive NMR work
leading to the assignment of substitution patterns of the
polysubstituted benzenes described herein, and Ms.
Sandra J . McNabb for assistance in preparing this
manuscript.
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ment.
J M9603633