Novel indole-based inhibitors of IMPDH: Introduction of hydrogen bond acceptors at indole C-3

Article abstract of DOI:10.1016/S0960-894X(03)00109-4

The development of a series of novel indole-based inhibitors of 5′-inosine monophosphate dehydrogenase (IMPDH) is described. Various hydrogen bond acceptors at C-3 of the indole were explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are outlined.

Full text of DOI:10.1016/S0960-894X(03)00109-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1273–1276
Novel Indole-Based Inhibitors of IMPDH: Introduction of
Hydrogen Bond Acceptors at Indole C-3
Scott H. Watterson,* T. G. Murali Dhar, Shelley K. Ballentine, Zhongqi Shen,
Joel C. Barrish, Daniel Cheney, Catherine A. Fleener, Katherine A. Rouleau,
Robert Townsend, Diane L. Hollenbaugh and Edwin J. Iwanowicz
Bristol-Myers Squibb PRI, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 31 October 2002; accepted 21 January 2003
Abstract—The development of a series of novel indole-based inhibitors of 50-inosine monophosphate dehydrogenase (IMPDH) is
described. Various hydrogen bond acceptors at C-3 of the indole were explored. The synthesis and the structure–activity relation-
ships (SARs) derived from in vitro studies are outlined.
# 2003 Elsevier Science Ltd. All rights reserved.
Maintaining adequate nucleotide levels is essential for
normal cellular function, including the synthesis of RNA
and DNA. In mammals, nucleotides may be synthesized
through one of two pathways: a de novo synthesis path-
way or through a salvage pathway which utilizes existing
purines and their nucleotides and nucleosides.1 The
extent to which each pathway is utilized is dependent on
the cell type. The de novo synthesis of guanine nucleo-
tides is particularly important in B- and T-lymphocytes.
These cells are dependent on the de novo synthesis to
generate sufficient levels of nucleotides necessary to
initiate a proliferative response to mitogen or antigen.
Mycophenolic acid (MPA, Fig. 1) and some of its deri-
vatives have been shown to be potent, uncompetitive,
reversible inhibitors of human IMPDH type I and type
II.10,11 CellCept1 (mycophenolate mofetil, MMF), a
prodrug of MPA, has clinical utility, based on its inhi-
bition of IMPDH, for the treatment of transplant
rejection. Dose-limiting gastrointestinal (GI) toxicity
exhibited from administration of either CellCept1 or
Inosine 50-monophosphate 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 xan-
thosine 50-monophosphate (XMP).2ꢀ4 Two distinct
cDNA’s encoding IMPDH have been identified and
isolated. These transcripts, labeled type I and type II,
possess 84% sequence identity.5ꢀ7 IMPDH type II
activity is markedly up-regulated in actively proliferat-
ing cell types including cancers and activated peripheral
blood lymphocytes.8,9 As a result, IMPDH has emerged
as an attractive target for inhibiting the immune system
without also inhibiting the proliferation of other cells.
*Corresponding author. Tel.: +1-609-252-6778; fax: +1-609-252-
6601; e-mail: scott.watterson@bms.com
Figure 1. Chemical structures of MPA, MMF, VX-497, quinolone 1,
and amino-oxazole 2.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00109-4

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S. H. Watterson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1273–1276
MPA limits its potential for the treatment of other
autoimmune disorders, such as psoriasis and rheuma-
toid arthritis.12
complexed with Chinese hamster IMPDH type II.3 The
key binding interactions for VX-497 are depicted in
Figure 3. The phenyl oxazole moiety of VX-497 binds in
the NAD pocket forming hydrogen bonds between the
oxazole nitrogen and oxygen and Gly 326 and Thr 333,
respectively. Additionally, the methoxy residue binds in
a hydrophobic pocket defined by the side chains of Asn
303 and Arg 322. The hydrogen bond interaction
between the urea NH and the carboxylate of Asp 274 is
a significant contributor to the biological activity of
VX-497.3
Glucuronidation at either the phenolic or carboxylic
acid residues of MPA is implicated as a contributing
factor in the poor therapeutic index observed for Cell-
Cept1.13,14 Recent, novel inhibitiors of IMPDH,
including VX-497 and BMS-337197, are devoid of phe-
nolic and acid functionalities and are reputed to have an
improved therapeutic window with regard to dose lim-
iting GI toxicity.15ꢀ17
In the quinolone series, we believe that the interactions
observed for the methoxy oxazole aniline portion are
conserved, including the critical hydrogen bond inter-
action between the quinolone NH and the carboxylate
of Asp 274. More importantly, we believe that the qui-
nolone carbonyl serves as a hydrogen bond acceptor
that engages in an interaction with Gln 441 (Fig. 3),
contributing to the high potency observed for quinolone
1 (IC50=0.008 mM).22 For the indole chemotype (2), we
anticipated that the incorporation of an appropriate
hydrogen bond acceptor at the 3-position of the indole
ring could potentially improve activity through estab-
lishing the key interaction with Gln 441.
Our focus is on the identification and development of
potent, selective inhibitors of IMPDH type II with
improved pharmacological properties.18ꢀ23 In an effort
to find optimal replacements for the urea isostere of
VX-497, including amides18 and diamides,20 we became
increasingly more concerned about the potential libera-
tion of the aniline moiety in vivo. We have recently dis-
closed an approach which alleviates this concern.22 This
was accomplished through appending a vinylogous
amide to the 3-methoxy-5-oxazolylaniline residue to
form a quinolone scaffold (1). In this paper, we outline
an indole scaffold as an alternative bicyclic ring system
for the quinolone chemotype (1).
The first objective in developing this chemotype was to
prepare indoles (3) with varying hydrogen bond accep-
tors at the 3-position of the indole ring. The synthetic
pathways utilized in the preparation of these indoles (3)
are outlined in Schemes 1–4. The synthesis of aniline 4
has been previously described.22 A Sonogashira24 palla-
dium-catalyzed coupling of aniline 4 with various sub-
stituted acetylenes provided acetylenic anilines 5, as
depicted in Scheme 1. Subsequent heating of anilines 5
in N-methylpyrrolidinone in the presence of potas-
sium t-butoxide gave indoles 6 in good yield.25 This
sequence was used for the preparation of indoles 2, 3f,
and 3i. The acetylenes were either commercially available
Indole 2 was prepared (Scheme 1, Fig. 2) and was found
to have only modest potency against IMPDH type II
relative to quinolone 1 (IC50=0.50 mM). Vertex has
reported the crystallographic studies on VX-497
Figure 2. Indoles 2 and 3.
Figure 3. Proposed binding model for the Vertex urea VX-497, and a
potential binding model for quinolone 1 and indole 3.
Scheme 1.

S. H. Watterson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1273–1276
Table 1. SAR of quinolone-based inhibitors of structure 3
1275
Scheme 2.
Compd
R1
R2
H
IMPDH II IC50 (mM)
2
0.50
3a
3b
3c
3d
3e
CO2Et
CHO
CN
>1
0.23
0.13
0.15
0.29
Scheme 3.
or were prepared through a Sonogashira coupling.26
Additionally, indoles 7 were substituted with a nitrile at
C-3 by treatment with chlorosulfonyl isocyanate. Indoles
containing an aldehyde at C-3 (8) were prepared utilizing
a Vilsmeier–Haack reaction as outlined in Scheme 1. The
preparation of indoles substituted with an ester group is
shown in Scheme 2 and involves a one-pot coupling/
cyclization sequence with aniline 4 to give indole 3a. An
alternative approach to 3-formyl-indoles is outlined in
Scheme 3. This sequence was used for the preparation of
indole 3j.
3f
H
0.60
0.36
0.22
3g
3h
CHO
CN
The preparation of cyclic-ketone indoles 3d and 3e is
outlined in Scheme 4 and relies on a novel intramole-
cular Heck Reaction for the indole ring construction.28
3i
H
0.50
The in vitro inhibitory activity against IMPDH type II
for this series of indoles is summarized in Table 1. As
mentioned earlier, indole 2 was only a modestly potent
inhibitor of IMPDH type II with an IC50 value of
0.5 mM.26 As depicted in Figure 3, we anticipated that
the addition of a carbonyl residue at the 3-position of
the indole would direct the carbonyl oxygen proximal to
Gln 441. In order to establish the viability of our
hypothesis, we choose to explore both acyclic and cyclic
3j
CHO
CN
0.030
0.064
3k
indoles. Substituting the 3-position of the indole core
with an ethyl ester (3a) resulted in a loss in activity.
Substitution with a smaller hydrogen bond acceptor, a
formyl group (3b), provided a 2-fold improvement in
binding affinity. Interestingly, when the 3-position was
appended with a nitrile moiety (3c), a 3–4-fold increase
in activity was observed (IC50=0.13 mM). In an effort to
decrease the entropy of the core by increasing the rigid-
ity of the R2 substituent, cyclic indoles 3d and 3e were
examined. The 6-5-6 tricyclic indole 3d was considerably
more active than indole 2 with an IC50 value of 0.15 mM.
Contracting the ring to a 6-5-5 ring system (3e) resulted
in a 2-fold decrease in activity, relative to 3d. This data
Scheme 4.

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S. H. Watterson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1273–1276
supports our hypothesis that a hydrogen bond acceptor
at the 3-position of the indole can interact with Gln 441
in a similar fashion to quinolone 1.
8. Jayaram, H. N.; Grusch, M.; Cooney, D. A.; Krupitza, G.
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mann, P. D.; Oellerich, M.; Armstrong, V. W. Transplant.
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15. Dhar, T. G. M.; Shen, Z.; Guo, J.; Liu, C.; Watterson,
S. H.; Gu, H. H.; Pitts, W. J.; Fleener, C. A.; Rouleau, K. A.;
Sherbina, N. Z.; McIntyre, K. W.; Witmer, M. R.; Tredup,
J. A.; Chen, B.-C.; Zhao, R.; Bednarz, M. S.; Cheney, D. L.;
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Pitts, W. J.; Barrish, J. C.; Fleener, C. A.; Rouleau, K.; Sher-
bina, N. Z.; Hollenbaugh, D.; Iwanowicz, E. J. Bioorg. Med.
Chem. Lett. 2002, 12, 2879.
19. Iwanowicz, E. J.; Watterson, S. H.; Liu, C.; Gu, H. H.;
Barrish, J. C.; Fleener, C. A.; Rouleau, K.; Sherbina, N. Z.;
Hollenbaugh, D. Bioorg. Med. Chem. Lett. 2002, 12, 2931.
20. Gu, H. H.; Iwanowicz, E. J.; Guo, J.; Watterson, S. H.;
Zhen, Z.; Pitts, W. J.; Dhar, T. G. M.; Fleener, C. A.; Rou-
leau, K.; Sherbina, N. Z.; Witmer, M.; Tredup, J.; Hollen-
baugh, D. Bioorg. Med. Chem. Lett. 2002, 12, 1323.
21. Pitts, W. J.; Guo, J.; Dhar, T. G. M.; Shen, Z.; Gu, H. H.;
Watterson, S. H.; Bednarz, M. S.; Chen, B.-C.; Barrish, J. C.;
Bassolino, D.; Cheney, D.; Fleener, C. A.; Rouleau, K. A.;
Hollenbaugh, D. L.; Iwanowicz, E. J. Bioorg. Med. Chem.
Lett. 2002, 12, 2137.
22. Watterson, S. H.; Carlsen, C.; Dhar, T. G. M.; Shen, Z.;
Pitts, W. J.; Guo, J.; Gu, H. H.; Norris, D.; Chorba, J.; Chen,
P.; Cheney, D.; Witmer, M.; Fleener, C. A.; Rouleau, K.;
Townsend, R.; Hollenbaugh, D.; Iwanowicz, E. J. Bioorg.
Med. Chem. Lett. 2003, 13, 543.
With improvement in activity with the addition of a
hydrogen bond acceptor moiety at the 3-position, we
next focused our attention on improving the trajectory
of the appended R1 phenyl group. The structural over-
lap of indole 3 and quinolone 1 in Figure 3 indicated
that the orientation of the indole phenyl ring needed to
be optimized to fully mimic the spatial arrangement of
the quinolone core. Our first approach in addressing
this issue was through a benzyl substituent. Indole 3f
with a benzyl substituent did not offer any improvement
over indole 2. Consequently, the appendage of a formyl
or nitrile substituent at the 3-position of the indole core
(3g and 3h) did not improve potency relative to 3b and
3c, respectfully. In an effort to lower the entropy of the
benzyl group and further optimize the orientation rela-
tive to the phenyl group of quinolone 1, a benzothio-
phene substituent was explored. Although the core
indole 3i was equivalent to indole 2 in the inhibition of
IMPDH type II (IC50=0.50 mM), the addition of a
hydrogen bond acceptor at the 3-position provided a
significant enhancement in binding affinity. The formyl-
substituted indole (3j) and the nitrile-substitituted indole
(3k) had IC50 values against IMPDH type II of 0.030 and
0.064 mM, respectfully. Indole 3j also showed good selec-
tivity versus IMPDH type I (IC50=0.160mM).26 Fur-
thermore, analogue 3j had an IC50 value of 0.55mM in a
T-cell proliferation assay (CEM).29
In summary, we have developed a novel indole-based
series of potent inhibitors of IMPDH type II. Through
the incorporation of a hydrogen bond acceptor at the 3-
position of indole 3, we have demonstrated that we can
significantly improve binding affinity, as seen in indole
3j, which has an IC50 value of 30 nM. Studies to opti-
mize this series of analogues to achieve oral activity in a
T-cell mediated pharmacodynamic model are ongoing.
The emphasis of future studies is to evaluate this series
relative to MPA and other IMPDH inhibitors, as well
as to establish in vivo the relationship between efficacy
and toxicity.
23. Dhar, T. G. M.; Watterson, S. H.;; Chen, P.; Shen, Z.; Gu,
H. H.; Norris, D.; Carlsen, M.; Haslow, K.; Pitts, W. J.; Guo, J.;
Chorba, J.; Fleener, C. A.; Rouleau, K.; Townsend, R.; Hollen-
baugh, D.; Iwanowicz, E. J. Bioorg. Med. Chem. Lett. 2003, 13,
547.
References and Notes
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28. The IMPDH enzymatic inhibition protocol is outlined in
ref 22 (ref 27).
29. The CEM proliferation protocol is outlined in ref 18
(ref 21).