Novel amide-based inhibitors of inosine 5′-monophosphate dehydrogenase

Article abstract of DOI:10.1016/S0960-894X(02)00601-7

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

Full text of DOI:10.1016/S0960-894X(02)00601-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2879–2882
0
Novel Amide-Based Inhibitors of Inosine 5 -Monophosphate
Dehydrogenase
Scott H. Watterson,* Chunjian Liu, T. G. Murali Dhar, Henry H. Gu, William J. Pitts,
Joel C. Barrish, Catherine A. Fleener, Katherine Rouleau, N. Z. Sherbina,
Diane L. Hollenbaugh and Edwin J. Iwanowicz
Bristol-Myers Squibb PRI, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 19 April 2002; accepted 15 July 2002
Abstract—A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was
explored. The synthesis and the structure–activity relationships (SARs) derived from in vitro studies are described.
#
2002 Elsevier Science Ltd. All rights reserved.
0
Inosine 5 -monophosphate dehydrogenase (IMPDH) is
a key enzyme in the regulation of cell proliferation and
differentiation. In mammals, nucleotides may be syn-
thesized through one of two pathways: a de novo
synthesis pathway or through a salvage pathway utiliz-
ing existing purines and their nucleotides and nucleo-
a proliferative response to mitogen or antigen. As a
result, IMPDH is an attractive target for selectively
inhibiting the immune system without also inhibiting
the proliferation of other cells. Inhibition of this enzyme
has implications in immunosuppressive, antiviral, and
oncology therapies.
1
sides. The extent of utilization of each pathway is
dependent on the cell type. IMPDH is a crucial enzyme
involved in the de novo synthesis of guanine nucleo-
tides. Specifically, IMPDH catalyzes the irreversible
Mycophenolic acid (MPA, Fig. 2) and some of its deri-
vatives have been shown to be potent, uncompetitive,
reversible inhibitors of human IMPDH type I and type
0
9,10
NAD dependent oxidation of inosine 5 -monopho-
0
II.
MPA has been demonstrated to block the
2
sphate (IMP) to xanthosine 5 -monophosphate (XMP).
In the enzymatic mechanism, an active site cysteine
residue plays a key role in the activation of IMP during
response of B- and T-cells to mitogen or antigen.
IMPDH inhibitors, such as the prodrug of MPA, Cell-
1
Cept (MMF), are useful drugs in the treatment of
transplant rejection and autoimmune disorders, such as
3
,4
enzymatic oxidation (Fig. 1). Two distinct cDNA’s
encoding IMPDH have been identified and isolated.
These transcripts labeled type I and type II possess 84%
1
1
psoriasis. In addition, various cancer cell lines are
sensitive to IMPDH inhibition, and clinical trials eval-
5
ꢀ7
sequence identity.
IMPDH type II activity is mark-
edly upregulated in actively proliferating cell types
including cancers and activated peripheral blood lym-
8
phocytes.
The de novo synthesis of guanine nucleotides, and thus
the activity of IMPDH, is particularly important in B-
and T-lymphocytes. These cells are dependent on the de
novo synthesis rather than the salvage pathway to gen-
erate sufficient levels of nucleotides necessary to initiate
*
6
Corresponding author. Tel.: +1-609-252-6778; fax: +1-609-252-
601; e-mail: scott.watterson@bms.com
Figure 1. The mechanistic conversion of IMP to XMP by IMPDH.
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00601-7

2880
S. H. Watterson et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2879–2882
of phenolic functionality, are reputed to have an
improved therapeutic window with regard to dose lim-
1
3ꢀ16
iting GI toxicity.
Our focus is on the identification and development of
potent inhibitors of IMPDH type II with improved
pharmacological properties. Of particular note, is the
dose limiting GI toxicity that largely restricts the clinical
use of MPA and MPA analogues to the treatment of
acute allograft rejection. Recently, we have disclosed
three new classes of inhibitors of IMPDH, exemplified
1
4,17,18
by structures 2 and 3 (Fig. 2).
In this report, we
have outlined the synthesis and biological evaluation of
a new class of inhibitors of IMPDH, represented by
structures 4 and 5.
Through protein crystallographic studies, Vertex has
shown that analogues of 1 bind to IMPDH forming a
hydrogen bond between the urea NH and the carbox-
3
,19
ylate of Asp 274.
Our strategy was to develop a new
class of inhibitors utilizing the oxazole-functionalized
aniline residue 10 in which the urea moiety of 1 is
replaced with amide-based functionality, as depicted in
structures 4 and 5 (Fig. 2).
Figure 2. Chemical structures of MPA, MMF, Vertex urea 1, diamide
, triazine 3, amide 4, and a,b-unsaturated amide 5.
2
uating IMPDH inhibitors for their antileukemic effects
8
The syntheses of analogues 4 and 5 are shown in
Scheme 1. The 3-methoxy-4-(5-oxazolyl)-aniline 10 was
prepared from 2-methoxy-4-nitro-toluene 6 on multi-
gram scale utilizing a synthetic procedure described by
Vertex. The reaction of aniline 10 with various com-
mercially available carboxylic acids in the presence of 1
equivalent of 1-ethyl-3-(3-dimethyl-aminopropyl)-car-
bodiimide HCl (EDC) in dimethyl formamide (DMF)
provided the amides of structure 4. Alternatively, com-
are currently in phase III. An alternative approach to
1
2
IMPDH inhibition has been described by Pankiewicz.
1
5
Dose-limiting gastrointestinal (GI) toxicity is observed
1
from oral administration of either MPA or CellCept
1
1
in a clinical setting. Structurally related analogues
disclosed by Vertex and Bristol-Myers Squibb, devoid
Table 1. SAR of amide-based inhibitors of structure 4
Compd
R1
IMPDH II IC50 (mM)
1
NA
Phenyl
Benzyl
0.019
0.63
0.97
0.26
0.33
0.63
0.11
0.13
>3
0.21
0.17
0.14
>1
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
3,4-Dimethylphenyl
3,4-Benzodioxolyl
5-Indolyl
2-Indolyl
2-(1-Methyl)indolyl
3-Indolyl
2-Benzofuranyl
2-Benzothiophenyl
2-Indolizino
3-(1H)-Indazolyl
2-Furanyl
2-(3-Methyl)furanyl
2-(4,5-Dimethyl)furanyl
2-(5-t-Butyl)furanyl
2-Thiophenyl
2-(5-Pyrido)thiophenyl
2-(5-Methyl)thiophenyl
2-(N-Methyl)pyrolyl
a
2.5
>1
0.54
0.13
0.81
0.87
0.11
0.89
Scheme 1. Reagents and conditions: (a) HOAc, Ac
2
O, concd H
2
SO
4
,
4r
4s
4t
ꢁ
CrO
MIC, K
95%); (e) R -CO
CH Cl /DMF.
3
, 0–10 C (51%); (b) concd HCl, dioxane, reflux (91%); (c) Tos-
2
CO
3
, MeOH, reflux (84%); (d) 40 psi H
2
, Pd/C, EtOH,
ꢁ
2 2
H, EDC, DMF, 50 C; (f) R -CO H, EDC, DMAP,
1
2
(
aIC50 >1.0 mM (IMPDH type I).
2
2

S. H. Watterson et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2879–2882
Table 2. SAR of amide-based inhibitors of structure 5
2881
Compd
R2
Bond orientation
IMPDH II IC50 (mM)
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
Phenyl
o-Methylphenyl
m-Methylphenyl
p-Methylphenyl
o-Fluorophenyl
m-Fluorophenyl
p-Fluorophenyl
2,6-Dimethylfluorophenyl
o-Chlorophenyl
m-Chlorophenyl
p-Chlorophenyl
o-Methoxyphenyl
o-Methoxyphenyl
2-Furanyl
trans
trans
trans
trans
trans
trans
trans
trans
trans
trans
trans
trans
cis
trans
trans
trans
trans
trans
trans
0.073
0.035
0.59
0.21
0.74
0.20
0.12
0.075
0.060
0.30
0.23
0.11
0.96
0.028
0.066
0.21
0.057
0.63
0.41
2-Thiophenyl
3-Pyrido
4-Pyrido
2-Quinolinyl
3-Indolyl
s
pounds of structure 5 were prepared by the reaction of
aniline 10 with various commercially available a,b-
unsaturated carboxylic acids in the presence of EDC
and dimethylaminopyridine in a mixture of dichloro-
methane (CH Cl ) and DMF.
and 5i–4k clearly demonstrated that ortho-substitution
of the phenyl ring is favored over the unsubstituted
inhibitor 5a. Substitution at the meta- and para-posti-
tions resulted in an overall decrease in potency relative
to compound 5a. Interestingly, potency was maintained
when the ortho-phenyl substituted compound 5c was
further elaborated with a methyl at the opposing ortho-
position (5h). Placement of a methoxy group at the
ortho-position (5l) led to a slight decrease in activitiy
relative to 5a. The cis-analogue, 5m, was prepared in
order to determine the necessity of the trans orientation
of the double bond of the amide. This configuration led
to an 8-fold loss in potency. Substitution of the a,b-
unsaturated amides with various heterocycles was also
explored. Furan substituted amide 5n was the most
potent compound is this series with an IC50 value of
0.028 mM.
2
2
The SARs for the inhibition of IMPDH type II catalytic
activity are summarized in Tables 1 and 2. In the amide
series 4, simple replacement of one of the NH groups in
the Vertex urea 1 with CH provided benzyl amide 4b,
2
which showed a 50-fold loss in potency against IMPDH
type II. Shortening the carbon tether, via elimination of
the methylene unit, (4a) resulted in a similar loss in
2
0
potency versus 1, (IC =0.63 mM). Further substitu-
5
0
tion on the phenyl ring, compounds 4c and 4d, provided
an increase in activity. Replacement of the phenyl ring
with various heterocycles was also explored. Bicyclic
heterocycles like 2-indolyl 4f, 5-indolyl 4e, 2-benzofur-
anyl 4i, 2-benzotiophenyl 4j, and 2-indolizino 4k all
proved to be well tolerated and provided enhanced
potency over 4a. Interestingly, activity was maintained
in the 2-(1-methyl)indolyl 4g derivative. Bicyclic hetero-
cycles 4h and 4l were not well tolerated. Simple unsub-
stituted heterocycles, 4m, 4q, and 4t, were less active
than 4a; however, further elaboration of both the furan
ring and the thiophene ring at the 5-postion resulted in
an increase in potency (4p, 0.13 mM and 4s, 0.11 mM,
respectively). The indole analogue 4f was evaluated
against IMPDH type I and was found to have an IC50
value >1 mM, demonstrating a selectivity preference for
IMPDH type II (IC =0.11 mM).
The compounds that showed potent inhibitory activity
against IMPDH type II were examined in a T-Cell pro-
2
1
liferation assay. In this assay, MPA inhibited T-cell
proliferation with an IC50 of 0.39 mM. None of the
amide-based inhibitors discussed in this paper inhibited
proliferation with an IC50 of less than 1 mM. For exam-
ple 4g, 4s, 5n, and 5q inhibited T-cell proliferation with
IC50 values of 7.7, 8.8, 6.6, and 8.7 mM, respectively.
We have identified two new series of novel amide-based
inhibitors of IMPDH Type II catalytic activity. These
compounds demonstrate that the urea or cyanoquani-
dine isosteres can be effectively replaced by an amide
moiety. Studies to optimize this series of analogues to
achieve oral activity in a T-cell mediated pharmacody-
namic model are ongoing. The emphasize of future
studies is to evaluate this amide-based series relative to
MPA and other IMPDH inhibitors, as well as to establish
in vivo the relationship between efficacy and toxicity.
5
0
A series of a,b-unsaturated amides of the structure 5
was also explored. Overall, this series proved to be more
potent then the amide series represented by 4. Phenyl
substituted amide 5a had an IC50 value of 0.073 mM
against IMPDH Type II enzyme. Compounds 5b–5g

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S. H. Watterson et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2879–2882
Zhen, Z.; Pitts, W. J.; Dhar, T. G. M.; Fleener, C. A.; Rou-
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1