Identification of novel and potent isoquinoline aminooxazole-based IMPDH inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00107-0

Screening of our in-house compound collection led to the discovery of 5-bromo-6-amino-2-isoquinoline 1 as a weak inhibitor of IMPDH. Subsequent optimization of 1 afforded a series of novel 2-isoquinolinoaminooxazole-based inhibitors, represented by 17, with single-digit nanomolar potency against the enzyme.

Full text of DOI:10.1016/S0960-894X(03)00107-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1345–1348
Identification of Novel and Potent Isoquinoline
Aminooxazole-Based IMPDH Inhibitors
Ping Chen,a,* Derek Norris,a Kristin D. Haslow,a T. G. Murali Dhar,a William J. Pitts,a
Scott H. Watterson,a Daniel L. Cheney,b Donna A. Bassolino,b Catherine A. Fleener,c
Katherine A. Rouleau,c Diane L. Hollenbaugh,c Robert M. Townsend,c Joel C. Barrisha
and Edwin J. Iwanowicza
aDiscovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543, USA
bComputer Aided Drug Design, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543, USA
cImmunology, Inflammation and Pulmonary Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 4000, Princeton, NJ 08543, USA
Received 28 October 2002; accepted 7 January 2003
Abstract—Screening of our in-house compound collection led to the discovery of 5-bromo-6-amino-2-isoquinoline 1 as a weak
inhibitor of IMPDH. Subsequent optimization of 1 afforded a series of novel 2-isoquinolinoaminooxazole-based inhibitors, repre-
sented by 17, with single-digit nanomolar potency against the enzyme.
# 2003 Elsevier Science Ltd. All rights reserved.
As a rate-limiting key enzyme in the de novo biosyn-
thetic pathway of guanine nucleotides, inosine
50-monophosphate dehydrogenase (IMPDH) catalyzes
the irreversible NAD-dependent oxidation of inosine
50-monophosphate (IMP) to xanthosine 50-mono-phos-
phate (XMP). This ultimately leads to the formation of
guanosine phosphate precursors of the nucleic acid
pool.1 Studies have shown that there is a strong corre-
lation between IMPDH activity and cell proliferation, a
reflection of the dependence of growing cells on de novo
purine synthesis.2 Due to its intimate involvement in the
DNA synthesis, IMPDH has long been recognized as a
possible oncology target.1,3 In addition, it was found
that inhibition of this enzyme displayed a profound
anti-proliferative effect on B and T lymphocytes owing
to their dependence on the de novo pathway for the
production of guanine nucleotides. Consequently,
IMPDH has recently emerged as an immunology target
for treatment of transplant rejection and other auto-
immune disorders4 in addition to its role as an oncology
target for human leukemia and several colon and ovar-
ian cell lines.5À8
We have been interested in developing small molecule
IMPDH inhibitors for the treatment of B and T lym-
phocyte-mediated immunological diseases, such as rheu-
matoid arthritis (RA) and psoriasis. We report herein the
identification of aminoisoquinoline oxazoles, repre-
sented by 17, as a new class of IMPDH inhibitors that
display excellent in vitro potency against the enzyme.
Screening of our in-house compound collection identified
1, 5-bromo-6-aminoisoquinoline, as a relatively small
molecule possessing modest activity (IC50=0.55 mM)
against IMPDH-II (Fig. 1). Our initial focus was to
establish the structural elements of 1 required for potency
and to identify sites for further chemical modification.
The synthetic route to analogues 1–14 is shown in Scheme
1. Briefly, compounds 1 and 3 were obtained from the
treatment of 2 (prepared from 5-bromoiso-quinoline9)
with N-bromosuccinimide and N-chloro-succinimide,
respectively. Alkylation of 1 with tetra-alkyltin in the
presence of bis(triphenylphosphine) palladium dichlor-
ide afforded compounds 4–6. Acetyllation of 1–3,
respectively, gave the corresponding analogues 9–11.
Reduction of 6 under hydrogenation conditions gener-
ated 7. Compound 8 was obtained from 5-aminoquino-
line in a five-step sequence via 12.10 Finally, compound
*Corresponding author. Tel.: +1-609-252-5809; fax: +1-609-252-
6601; e-mail: ping.chen@bms.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00107-0

1346
P. Chen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1345–1348
Scheme 2.
Figure 1. IMPDH inhibitors.
14 was obtained from 13, which in turn was prepared
from p-anisaldehyde.11
Further investigation of the SAR of the aminoquinoline
lead was extended to analogues containing urea and
amino oxazole linkages, two known scaffolds found in
other IMPDH inhibitors.12 The synthetic route to the
diaryl urea analogues 15a–k is straightforward, as exem-
plified by the preparation of analogue 15a in Scheme 2.
Thus, the reaction of 5-bromo-6-amino-isoquinoline 1
with commercially available phenyl isocyanate afforded
the desired compound 15a in quantitative yield.
Scheme 3.
activity (2 vs 1). Exchanging the 2-bromo substituent of
1 with 2-chloro group led to a 2-fold loss in activity (3
vs 1). In addition, replacement of the 5-bromo group of
1 with small alkyl groups, such as methyl (5), was not
tolerated and resulted in a compound with >5-fold
reduction in activity. This trend is continued with other
alkyl or amino analogues (6–8), with the exception of
the vinyl analogue 5 (IC50=0.82 mM). Other efforts to
improve the potency by simple replacement of the
6-amino group of 1 with acetoamino or methoxy sub-
stituents again resulted in large losses (>10-fold) in
activity (9–11 and 14).
Synthesis of oxazole analogues 17 and 18 were achieved
by a two-step sequence, as exemplified by preparation of
17. Reaction of 6-aminoisoquinoline 2 with 1,10-thio-
carbonyldi-2(1H)-pyridone provided thio-isocyanate
intermediate 16. Subsequent treatment with a-azidoace-
tophenone (prepared from commercially available acet-
ophenone) in the presence of triphenyl phosphine gave
desired 17 in good overall yield. (Scheme 3).
The analogues prepared in this study were evaluated for
their inhibitory activity against IMPDH type II enzyme
and are expressed as IC50 values.13 As shown in Table 1,
deletion of the 5-bromo-substituent from 1 was detri-
mental to the potency, resulting in a>10-fold loss in
We next turned our attention to modifications around
the 6-amino functionality of 1. Previous work16 in our
lab as well as in others17 has demonstrated that a biaryl
urea isostere possess potent IMPDH inhibitory activity.
Scheme 1.

P. Chen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1345–1348
1347
Table 1. IMPDH Inhibition (IC50 values) for compounds 1–11 and
14
The significant differences in potency displayed by the
regioisomers of 16d, 16e and 16f are noteworthy: the
4-methylphenyl urea analogue 16f is >13-fold less
potent than 16e, whereas the 2-methylphenyl urea 16d is
>200-fold less potent than 16e, indicating a strong
preference for the meta-substitution. In addition, the
benzyl urea 16g was roughly 2-fold more potent than
the phenyl urea 16c.
Compd
R1
R2
IC50 (mM)13
1
2
3
NH2
NH2
NH2
Br
H
Cl
0.55
>5
1.2
We also examined the SAR of other bicycloheteroaryl
ureas in order to elucidate the effect of the isoquinoline
nitrogen on the binding affinity. The 3-isoquinoline
phenyl urea 16h, a regioisomer of 16c, was completely
devoid of activity (IC50 >5 mM), implying a disruption
of a possible hydrogen-bond interaction between the
isoquinoline nitrogen and the backbone of the enzyme.
In addition, the 1,2-cinnoline urea 16i was significantly
less active whereas its regioisomer, the 2,3-phthalazine
urea 16j was inactive (IC50>5 mM). Interestingly, the 2,4-
quinazoline urea 16k displayed enhanced enzyme activity
(IC50=150 nM), although it is still 8-fold less potent than
the 2-isoquinoline 16e. We speculated that the improved
activity of 16k may reflect a partially restored hydrogen-
bond interaction with the enzyme, although the basicity
of the N2-nitrogen of 16k may not be optimal.
4
5
6
7
8
9
10
11
14
NH2
NH2
NH2
NH2
NH2
NHAc
NHAc
NHAc
OMe
Me
CH¼CH2
>5
0.82
3.0
>5
>5
>5
>5
>5
>5
CH2CH¼CH2
CH2CH2CH3
NH2
Br
Cl
H
Br
It was reported and confirmed by X-ray crystallographic
studies that biaryl ureas can form a key hydrogen bond
interaction between one of the urea NH and the
carboxylate of Asp 274 in the active site of the IMPDH
enzyme.18 In addition, the second aryl moiety of the
biaryl urea occupies space in a large hydrophobic pocket.
We envisioned that the extension of 1 through a rigid
urea linkage would place the second aryl group in the
proximity of the same hydrophobic pocket, thus allow-
ing for further improvements in the binding affinity of 1.
Encouraged by the significant improvement in binding
affinity of biaryl urea 16e relative to the original lead 1
(30-fold), we set out to examine various con-
formationally constrained urea linkages. Initial efforts
to mimic the conformation of the urea led to the pre-
paration of 6-aminoisoquinoline oxazole 17 and 7-ami-
noquinazoline oxazole 18.12c To our delight, both
compounds displayed further enhanced potency against
the enzyme (IC50’s=5 and 60 nM, respectively) over their
corresponding urea counterparts 16e and 16k (Fig. 2).
As shown in Table 2, biaryl urea 16a displayed com-
parable activity to 1. Replacement of the 5-bromo
group of 16a with a 5-chloro group (16b) resulted in a
2-fold loss in potency. However, deletion of the
5-chlorine regained activity (16c).
Interestingly, the most drastic change in activity
observed in this series was derived from the substitution
pattern of the phenyl ring. 3-Methylphenyl urea 16e
showed an IC50 of 18 nM, a >40-fold increase in
potency from its parent compound 16c!
In summary, the isoquinoline aminooxazole scaffold,
represented by 17, was identified as a novel structural
array of highly potent IMPDH type II inhibitors from
our initial screen lead, 6-amino-2-isoquinoline 1, with
over a 100-fold improvement in enzyme inhibition over
1. The results of follow up SAR studies as well as the
Table 2. IMPDH Inhibition (IC50 values) for urea analogues 16a–k
Compd
A, B, C, D
R1
R2
IC50 (mM)13
1
0.55
0.74
1.66
0.74
3.8
0.018
0.247
0.4
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
A=C=D=CH, B=N
A=C=D=CH, B=N
A=C=D=CH, B=N
A=C=D=CH, B=N
A=C=D=CH, B=N
A=C=D=CH, B=N
A=C=D=CH, B=N
A=B=D=CH, C=N
A=B=N, C=D=CH
A=D=CH, B=C=N
A=C=CH, B=D=N
Br
Cl
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
CH2Ph
Ph
3-Me-Ph
3-Me-Ph
3-Me-Ph
>5
>1.7
>5
0.15

1348
P. Chen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1345–1348
W. Synth. Commun. 1999, 29, 1617. Compound 8 was pre-
pared based on a modified procedure of this reference.
11. Kumar, P.; Agarwa, S. L.; Bhakuni, D. S. Indian J. Chem.,
Sect. B 1992, 31, 177.
12. (a) Armistead, D. M.; Badia, M. C.; Bemis, G. W.;
Bethiel, R. S.; Frank, C. A.; Novak, P. M.; Ronkin, S. M.;
Saunders, J. O. PCT Int. Appl. 1997 ; WO 9740028. (b) Dhar,
M. T. G.; Watterson, S. H.; Liu, C.; Barrish, J. C.; Fleener, C.
A.; Rouleau, K. A.; Sherbina, N. Z.; Hollenbaugh, D. L.;
Iwanowicz, E. J. Bioorg. Med. Chem. Lett. 2002, 12, 3305. (c)
Dhar, M. T. G.; Shen, Z.; Guo, J.-Q.; Lin, C.; Watterson,
S. H.; Gu, H. H.; Pitts, W. J.; Fleener, C. A.; Rouleau, K. A.;
Sherbina, Z. N.; McIntyre, K. W.; Witmer, M.; Tredup, J. A.;
Chen, B.-C.; Zhao, R.; Bednarz, M. S.; Cheney, D. L.; Mac-
Master, J. F.; Miller, L. M.; Berry, K. K.; Harper, T. W.;
Barrish, J. C.; Hollenbaugh, D. L.; Iwanowicz, E. J. J. Med.
Chem. 2002, 45, 2127.
Figure 2.
optimization of the physical and chemical properties of
17 and 18 through various substitutions around the
phenyl ring will be reported in due course.
13. The enzymatic activity of human IMPDH II was quanti-
fied using a procedure similar to reported methods.14,15 The
conversion of NAD+ to NADH was followed spectro-
photometrically at 340 nM. A reaction mixture containing
0.1 M Tris, 0.1 M KCl, 3 mM EDTA pH 8.0, 400 mM IMP, 2
mM DTT and 40 nM IMPDH II was added to the wells of flat
bottom UV-transparent 96-well plates (Costar 3635). To test
inhibitors, compounds resuspended in DMSO were diluted in
the reaction to give a final DMSO concentration of 2.5%.
IMPDH II used in these assays was purified from Escherichia
coli expressing the gene for the human Type II enzyme. The
reaction was initiated by addition of NAD to a final concen-
tration of 400 mM. After a 2 h incubation at 25 ꢀC, readings
were taken at 340 nM. The concentrations of compound
required to inhibit NADH accumulation by 50% (IC50) were
calculated using a four-parameter logistic plot.
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