Novel guanidine-Based inhibitors of inosine monophosphate dehydrogenase

Article abstract of DOI:10.1016/S0960-894X(02)00600-5

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

Full text of DOI:10.1016/S0960-894X(02)00600-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2931–2934
Novel Guanidine-Based Inhibitors of Inosine
Monophosphate Dehydrogenase
Edwin J. Iwanowicz,* Scott H. Watterson, Chunjian Liu, Henry H. Gu, Toomas Mitt,
Katerina Leftheris, Joel C. Barrish, Catherine A. Fleener, Katherine Rouleau,
N. Z. Sherbina and Diane L. Hollenbaugh
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 22 April 2002; accepted 10 July 2002
Abstract—A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was
explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immuno-
suppressive and antiviral therapy. The synthesis and the structure–activity relationships (SARs), derived from in vitro studies, for
this new series of inhibitors is given.
# 2002 Elsevier Science Ltd. All rights reserved.
The regulation of purine nucleotides in cells is governed
by two biochemical synthetic routes, the de novo
synthesis and the salvage pathways. Inosine monophos-
phate dehydrogenase (IMPDH), a key enzyme in the de
novo synthesis of guanosine nucleotides, catalyzes the
irreversible NAD-dependent oxidation of inosine-50-
monophosphate (IMP) to xanthosine-50-monopho-
sphate (XMP).1 Two distinct cDNA’s encoding
IMPDH have been identified and isolated. These tran-
scripts labeled type I and type II are of identical size
(514 amino acids).2ꢀ4 IMPDH II activity is markedly
upregulated in actively proliferating cell types including
cancers and activated peripheral blood lymphocytes.5 B-
and T-lymphocytes depend on the de novo, rather than
salvage pathway, to generate sufficient levels of nucleo-
tides necessary to initiate a proliferative response to
mitogen or antigen. Due to the B- and T-cell’s unique
reliance on the de novo pathway, IMPDH is an attrac-
tive target for selectively inhibiting a lymphocyte-medi-
ated immune response without undue inhibition of
other noncancerous proliferating cells. Mutations that
lead to a loss in function of wild-type p53 are the most
common genetic change associated a number of diverse
cancers.6 A role for IMPDH in cancer is supported by
the observation that upregulation of the enzyme pre-
vents p53-dependent growth inhibition.7 Inhibitors of
IMPDH have shown marked cytotoxicity against human
leukemia and several colon and ovarian cell lines.5,8ꢀ10
Mycophenolic acid (MPA) has been shown to be a
potent, uncompetitive, reversible inhibitor of human
IMPDH type I and type II.11,12 MPA has been demon-
strated to block the response of B-, and T-cells to mito-
gen or antigen. CellCept1, a prodrug of MPA, has
clinical use for the treatment of transplant rejection and
autoimmune disorders, such as psoriasis and rheuma-
toid arthritis.13 MPA has been shown to have potent in
vitro cytotoxicity, however marked in vivo antitumor
activity has not been observed. The metabolite of Tia-
zofurin (TAD), a nucleotide inhibitor of IMPDH, has
shown in vivo antitumor activity and efficacy in clinical
trials for the treatment of myelogenous leukemia.5 Tia-
zofurin is currently under investigation for the treat-
ment of ovarian cancer.14
Dose-limiting gastrointestinal (GI) toxicity is observed
in a clinical setting from oral administration of either
MPA or CellCept1 (mycophenolate mofetil, MMF).
MMF is rapidly and to a large extent completely
absorbed following oral administration whereby it is
converted to MPA.13,15 Three metabolites have been
identified in humans, the 7-O-glucuronide (MPAG),
7-O-glucoside conjugate (MPAG1s), and the acyl glu-
curonide (AcMPAG).16 MPAG, the major metabolite,
is inactive against IMPDH and is found in substantially
higher concentrations in the plasma of healthy subjects
*Corresponding author. Tel.: +1-609-252-5416; e-mail: iwanowie@
bms.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00600-5

2932
E. J. Iwanowicz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2931–2934
than MPA contributing to the high doses required for
efficacy. Significant MPAG is excreted in the bile and
converted into MPA by the action of intestinal flora
contributing to the gastrointestinal toxicity.
Structurally related analogues disclosed by Vertex and
Bristol-Myers Squibb, devoid of phenolic functionality,
are reputed to have an improved therapeutic window
with regard to dose limiting GI toxicity.17ꢀ19 A con-
temporary approach to IMPDH inhibition utilizing
mycophenolic adenine dinucelotide analogues has been
recently reported.20
Scheme 1. Synthesis of compounds 6a–l. Reagents and conditions: (a)
1,10thiocarbonyldi-2(1H)-pyridone, CH2Cl2, rt, 94%; (b) NaNHCN,
EtOH, rt, 76%; (c) substituted aniline, EDC, DMF, 50 ꢁC, 12–75%.
This report describes a new class of IMPDH inhibitors.
Our focus is the identification and development of
potent, selective inhibitors of IMPDH with improved
pharmacological properties. Recently we have disclosed
a new class of inhibitors, exemplified by 1 (Fig. 1).21
Through protein crystallographic studies, Vertex has
shown that analogues of 2 bind to IMPDH forming a
hydrogen bond between the urea NH and the carbox-
ylate of Asp 274.22 The biarylurea moiety of the Vertex
chemical series contributes to the poor aqueous solubi-
lity for compound 2. Our strategy was to develop a new
class of inhibitors utilizing the oxazole-functionalized
aniline residue, 3, and replacing the urea moiety with
guanidine-based functionality.23
Scheme 2. Synthesis of compounds 8a–c. Reagents and conditions: (a)
1-isothiocyanato-3-methyl-benzene, CH2Cl2, reflux 3.5 days, 70%; (b)
R3=Me: MeNH2, DCC, Hunig’s base (0.2 equiv), rt, 92%; (c) R3=H/
NH3, DCC, Hunig’s base (0.2 equiv), rt, 73%; (d) R3=OMe/
MeONH2, WSC, Hunig’s base (0.2 equiv), rt, 73%.
The biaryl cyanoguanidines were conveniently prepared
using established methodologies outlined in Scheme 1.24
The synthetic route employed aniline 3, whose prepara-
tion was previously described.21 Several other guani-
dines were prepared as outlined in Schemes 2–5.
Table 1 reports the in vitro inhibitor potencies against
IMPDH II for a range of inhibitors featuring a central
guanidine residue. Replacement of the urea linkage of 2
with a cyanoguanidine moiety, 6a, resulted in a 13-fold
loss in potency, (IMPDH II IC50 0.24 mM).25 Attempts
to enhance the potency through the use of more basic
residues, 8a–c, led to a further loss in inhibitory activity.
Moderating the guanidine basicity through acylation,
analogues 10a,b and 12, yielded inactive analogues. In
addition, removal of the tolyl residue of 6a resulted in
little change in potency. Likewise, the removal of the
methyl residue of 6b, gave little change in inhibitory
activity. Further investigation into the SAR for the
cyanoguanidine series showed that although a methyl
residue is tolerated in the meta position of the phenyl
Scheme 3. Synthesis of compounds 10a,b. Reagents and conditions:
(a) R4=Me: acetyl chloride, KSCN, acetone, rt, 49%; (b) R4=OEt:
EtOC(O)NCS, CH2Cl2, reflux, 2 h, 91%; (c) m-toluidine, WSC,
Hunig’s base, DMF, 65 ꢁC, 15 h, 82%.
Scheme 4. Synthesis of compound 12. Reagents and conditions: (a)
benzoyl isothiocyanate, CH2Cl2, rt, 3 h, 94%; (b) in a sealed tube:
NH3, WSC, Hunig’s base, DMF, 65 ꢁC, 16 h, 70%.
Figure 1. Chemical structures of MPA, diamide 1, and urea 2.

E. J. Iwanowicz et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2931–2934
Table 2. SAR of cyanoguanidine-based inhibitors of IMPDH
2933
Compd
R
IMPDH II
IC50, mM
6f
6g
6h
CH2OH
O(CH2)2OPh
C(O)NH(CH2)3Me
0.28
0.38
0.13
Scheme 5. Synthesis of compounds 14a,b. Reagents and conditions:
(a) (PhO)2CNCN, CH3CN, rt, 94%; (b) R5=H:NH3, EtOH, 100 ꢁC,
94%; (c) R5=CH2-m-MePh:MeCN, 80 ꢁC, 80%.
6i
0.34
residue (R2), substitution at ortho and para positions
yield poorly active inhibitors (6c,d). Extending the meta
tolyl pharmacophor by a methylene residue, 14b, also
had a negative effect on potency. Lastly, replacement of
the phenyl residue (R2) with either 2- or 3-pyridyl
enhanced the polarity of the molecule without a sig-
nificant change in inhibitory activity.
6j
0.11
0.18
6k
6l
0.016
An effort to explore and develop the SAR at the meta
position of the ‘right hand’ phenyl ring of 6a is sum-
marized in Table 2. Both polar and lyphophilic residues
are tolerated at this position. Notably, the carbonate
side chain utilized by Vertex in VX-497 only modestly
increases the potency in this series, as demonstrated by
6i. Structurally related side chains (6j,k) gave similar
results. Interestingly, replacement of the carbonate with
an amidinoether moiety, 6l, enhanced binding affinity
for IMPDH II, comparable to both MPA and 2
(IC50=0.016 mM). In addition, this analogue was a less
potent inhibitor of IMPDH I (IC50=0.42 mM).
We have identified a new series of novel guanidine-
based inhibitors of IMPDH II. Replacement of the
central urea linkage of 2 with a cyanoguanidine moiety
resulted in a 13-fold loss in potency. This lost potency may
be regained through the appropriate substitution on the
‘right hand’ phenyl ring, as observe with 6l. This com-
pound was examined in a T-Cell proliferation assay26 and
was found to have an IC50 of 0.410 mM (MPA
IC50=0.390mM). Studies to optimize this series of ana-
logues through further modifications of the central guani-
dine moiety and the ‘right hand’ functionality to achieve
oral activity in a T-cell mediated pharmacodynamic model
are ongoing. The objective of future studies is to evaluate
cyanoguanidine-based inhibitors in an in vivo setting to
establish the relationship between efficacy and toxicity.
Table 1. SAR of guanidine-based inhibitors of IMPDH
References and Notes
R1
R2
IMPDH II
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