Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

Article abstract of DOI:10.1016/S0960-894X(02)00351-7

A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

Full text of DOI:10.1016/S0960-894X(02)00351-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2137–2140
Rapid Synthesis of Triazine Inhibitors of Inosine
Monophosphate Dehydrogenase
William J. Pitts,* Junqing Guo, T. G. Murali Dhar, Zhongqi Shen, Henry H. Gu,
Scott H. Watterson, Mark S. Bednarz, Bang-Chi Chen, Joel C. Barrish,
Donna Bassolino, Daniel Cheney, Catherine A. Fleener, Katherine A. Rouleau,
Diane L. Hollenbaugh andEdwin J. Iwanowicz
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received21 March 2002; accepted6 May 2002
Abstract—A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared.
The synthesis and the structure–activity relationships (SAR) derived from in vitro studies are described. # 2002 Elsevier Science
Ltd. All rights reserved.
Inosine monophosphate dehydrogenase (IMPDH) is an
enzyme in the de novo synthetic pathway of guanosine
nucleotides. This enzyme catalyzes the irreversible
NAD-dependent oxidation of inosine-50-monophos-
phate (IMP) to xanthosine-50-monophosphate (XMP).1
Two distinct cDNA’s encoding IMPDH have been
identified and isolated. These transcripts labeled type I
andtype II, possess 84% sequence identity, with one
conservative change in the active site binding pocket.2ꢀ4
B and T-lymphocytes depend on the de novo, rather
than salvage pathway, to generate sufficient levels of
nucleotides necessary to initiate a proliferative response
to mitogen or antigen. A therapeutic window for the
anti-metabolite effects of IMPDH inhibition depends
upon the B andT cell’s unique reliance on the de novo
pathway relative to other proliferating cells in the body.
rejection and autoimmune disorders, such as psoriasis.9
Dose-limiting gastrointestinal (GI) toxicity is observed
from oral administration of either MPA or MMF in a
clinical setting. MPA undergoes extensive glucuroni-
dation and biliary excretion. High levels of MPA in the
GI tract has been postulatedto be a cause of GI side
effects.10 Vertex has shown that biarylureas, exemplified
by VX-497, are highly potent inhibitors of IMPDH
catalytic activity (Fig. 1) and these compounds do not
have an obvious site of glucuronidation.11 This com-
poundis currently unedr evaluation in the clinic in
combination with interferon alpha for the treatment of
hepatitis C.12
Our initial approach was to examine urea isosteres in an
effort to define the optimal linkage between the two
halves of the Vertex ureas.13 The synthesis andIMPDH
SAR of a cyanoguanidine urea isostere II (Fig. 2) will be
Mycophenolic acid(MPA), andsome of its derivatives
have been shown to be potent, uncompetitive, reversible
5,6
inhibitors of human IMPDH type I andtype II.
The
enzymatic activity of human IMPDH II was measured
using a procedure similar to reported methods.7,8 MPA
has been demonstrated to block the response of B- and
T-cells to mitogen or antigen. IMPDH inhibitors, such
as the prodrug of MPA, mycophenolate mofetil
(MMF), are useful drugs in the treatment of transplant
*Corresponding author. Tel.: +1-609-252-5252; fax: +1-609-252-
7410; e-mail: william.pitts@bms.com
Figure 1. Chemical structures of MPA, MMF andVX-497.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00351-7

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W. J. Pitts et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2137–2140
14
18
reportedshortly. It was realizedthat reaction of phe-
nylhydrazine in place of simple amines, with thiourea I
wouldprovide a heterocyclic analogue. Triazole III was
foundto be a potent inhibitor of IMPDH (IC 50=150
nM), andwas sufficiently idfferent from the urea
isosteres to warrant further investigation.
bedby Vertex.
Reaction of cyanuric chloride 2, with
phenylmagnesium bromide produced triazine 3. Stirring
a solution of aniline 1 with triazine 3 at room tempera-
ture resultedin the precipitation of intermediate 4 in
high yieldandpurity. Intermediate
4 was subjectedto
nucleophilic substitution with a variety of amines to
produce triazine analogues 5a–o. Treatment of triazine
4 with hydroxide or alkoxides provided analogues 5p–r.
In a similar synthetic sequence, cyanuric chloride 2 was
reactedwith aniline 1 at 0 ꢁC to produce the mono
addition product, which was followed by addition of
methylamine at room temperature to produce inter-
mediate 7. Reaction of intermediate 6 with aryltin
reagents produced analogues 7a–d in moderate yield.
Reaction of 6 with a variety of amines produced the
analogues 7e–i. Approximately 180 compounds of
structure 5 and 7 were preparedin two separate libra-
ries. Purity for all compounds was >85% as determined
by LCMS. The syntheses of triazine 11 (Scheme 2) was
accomplishedby condensation of phenylpyruvate 9 and
methyl hydrazinocarboximidothioate 10. Oxidation to
the sulfone 12, proceeded quantitatively, however this
material was unstable (hydrolysis) and best used the
same day it was prepared. Nucleophilic displacement of
the sulfone did not proceed at an appreciable rate with
aniline 1, but occurred rapidly with the anion derived
Analysis of this triazole (III) suggestedthat rapid
diversification might be possible if we could substitute a
triazine (IV) for the leadtriazole scaffold.
The crystal structure of MPA boundto IMPDH has
been reported.15 Based on this data a model of the tria-
zine (5a) docked into the active site of IMPDH could be
prepared(Fig. 3). The amine of the triazine ( 6a), which
wouldsupport projection of appeneddgroups either
towardmethionine 420, glutamine 441, serine 276 or
solvent, was usedas a point of attachment for reagent
docking.16 Compounds that scored well in the docking
run were pre-screenedfor violation of Lipinski’s rule of
517 before final selection. Additional compounds were
prepared to provide a more complete SAR study.
The synthesis of analogues 6 and 8 is shown in Scheme 1.
The 3-methoxy-4-(5-oxazolyl)-aniline 1 was preparedon
multigram scale utilizing a synthetic procedure descri-
Figure 3. Triazine (5a) docked into IMPDH. The amine (blue) was
usedas the point of attachment for reagent docking.
Figure 2. Chemical structures of triazole III andtriazine IV. Reagents
andconditions: (a) PhNHNH 2, EDC, DMF 60 ꢁC.
Scheme 1. Reagents andconditions: (a) PhMgBr, toluene 0 ꢁC (60%); (b) 1 and 3, THF, rt (99%); (c) HNR3R4, dioxane 80 ꢁC, (70–90%); (d)
HOR1, NaH, dioxane 60 ꢁC (70–85%); (e) 1 and 2, acetone, K2CO3, 0 ꢁC, 2 h (63%); (f) NH2Me, THF, (92%); (g) ArSnBu3, Pd(OAc)2, DMF,
80 ꢁC, (15–35%).

W. J. Pitts et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2137–2140
2139
from formamide 8. Acidic hydrolysis of the formamide
group, provided triazine 13.
the initial hypothesis that the triazine couldeffectively
function as a replacement for the triazole. The most
potent compound, 5p (IC50=18 nM), was comparable
in potency to MPA (IC50=15 nM). Representative
compounds were chosen from this compound library to
illustrate several SAR points.
The structure–activity relationships (SARs) for the
inhibition of IMPDH type II catalytic activity are sum-
marizedin Table 1. Several chemical filters were
employed during the library design phase. 82% of the
compounds prepared did not break any Lipinski rule,
andas such we hopedto bias this library towardinhi-
bitors of IMPDH II with improveddrug-like character.
Triazine 5a was 3-foldmore potent at inhibiting
IMPDH II than the leadtriazole III, which validated
Primary andsecondary amines 5a and 5b were relatively
potent inhibitors of IMPDH II with IC50 values of 0.054
and0.076 mM, respectively. Tertiary amines such as 5c,
5j, 5k, and 5l, are generally less potent. Tertiary amines
5m and 5n appear to be an exception to the general
Table 1. SAR of 5a–r and 7a–i with respect to inhibition of IMPDH II
1
1
CompdR
IMPDH II
IC50, mM
CompdR
IMPDH II
IC50, mM
CompdR
IMPDH II
IC50, mM
1
5a
–NH2
0.054
5j
0.19
7a
0.31
5b
5c
5d
–NHMe
–NMe2
0.076
0.27
0.25
5k
5l
0.33
0.16
0.078
7b
7c
7d
0.13
0.17
0.057
5m
5e
5f
0.13
5n
5o
0.072
0.29
7e
7f
0.72
0.13
0.078
5g
5h
5i
>1.0
0.41
5p
5q
5r
–OH
0.018
0.041
0.032
7g
7h
7i
0.39
0.16
0.62
–OCH3
0.058
–OCH2Ph
–NHMe
Scheme 2. Reagents andconditions: (a) HCO 2H, 150 ꢁC, 2 h (99%); (b) 9 and 10, NaHCO3, EtOH, reflux 1 h (98%); (c) H2O2/AcOH, Na2WO4,
(quant); (d) 8 and 12, NaH, DMF 0 ꢁC, 0.5 h then 4 N HCl, 70 ꢁC, 0.5 h (70%).

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W. J. Pitts et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2137–2140
trend, and this may be due to a positive interaction with
the enzyme from the pendant hydroxyl group. The ser-
ies of acidic compounds 5d, 5e, and 5f provide some
insight into the binding of these compounds to IMPDH.
The most potent carboxylate 5f, is can be overlapped
effectively with the carboxylate of MPA in its enzyme
boundconformation. The crystal structure of MPA
with IMPDH shows a bidentate interaction of the car-
boxylate of MPA with Serine 276. Oxygen substituted
triazines 5p–r were generally more potent inhibitors
than their corresponding Nitrogen analogues 5a, 5b,
and 5h. Basedon the SAR of the series of compounds,
5, it appearedthat a substitutent at R 1, was not required
for activity. To test this hypothesis, the isomeric triazine
13 was prepared, and found to also be a potent inhibitor
of IMPDH II with IC50 value of 0.045 mM. Compounds
of structure 7 represent an effort to findalternatives to
the phenyl ring present in structure 5. Compound 7i was
foundto be relatively less potent than 5b, suggesting
that the methylamine group is not a satisfactory replace-
ment for the phenyl ring. The methylamine substituent
was therefore selectedto remain constant, in an attempt
to simplify interpretation of the results. Methyl group
placement (7a–c) was toleratedat the 3- or 4-position.
The furyl compound 7d, was comparable in potency to
the corresponding phenyl analogue 5b. Interestingly
analogue 7f was a potent inhibitor of IMPDH II while
the enantiomer 7e, andthe methyl ether 7g, were not.
The structural basis for the activity of alcohol 7f is not
clear.
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12. Information presentedon company website: http://
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19. CEM proliferation assay: The human T-lymphoblast
CEM cell line (ATCC) was culturedin RPMI 1640 (Gibco)
containing 10% heat inactivatedFBS and100 units/mL of
penicillin andstreptomycin. Cells were seeeddin 96-well
Costar flat-bottom tissue culture plates at a concentration of
3000 cells/well in the presence of 0.5% DMSO. Test
compounds were added in triplicate at a final concentration of
10 uM with 3-foldserial dilutions. Cell cultures were main-
Compounds that were potent inhibitors of IMPDH II
19
were examinedin a T cell proliferation assay.
inhibitedT cell proliferation with an IC
MPA
of 0.39 mM.
50
None of the triazines discussed in this paper inhibited T
cell proliferation with an IC50 of less than 1 mM. For
example 5p, 5i, and 13 inhibitedT cell proliferation with
an IC50 of 5.9, 2.4, and3.5 mM, respectively.
In summary, we have identified several series of novel
triazine inhibitors of the enzyme IMPDH II. These
compounds demonstrate that the urea or diamide iso-
steres can be effectively replacedby heterocycles. The
SAR of other heterocyclic replacements for triazines
will be the subject of a separate paper.20 Studies to opti-
mize this series of analogues to achieve oral activity in a
T-cell mediated pharmacodynamic model are ongoing.
tainedin a 5% CO humidified atmosphere for 72 h. Cell via-
2
bility was measuredafter a final 5 h incubation with 10% (v/v)
Alamar Blue dye. The fluorometric conversion of Alamar Blue
was readon a Cytoflour II multi-well plate reader with exci-
tation/emission settings of 530/590 nm, respectively.
20. Dhar, T. G. M.; Pitts, W. J.; Watterson, S. H.; Liu, C.;
Guo, J.; Barrish, J. C.; Fleener, C. A.; Rouleau, K.; Sherbina,
N. Z.; Hollenbaugh, D.; Iwanowicz, E. J. Bioorg. Med. Chem.
Lett. Submittedfor publication.
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