Discovery of N-[2-[2-[[3-methoxy-4-(5-oxazolyl)phenyl]-amino]-5-oxazolyl]phenyl]-N- methyl-4-morpholinea0cetamide as a novel and potent inhibitor of inosine monophosphate dehydrogenase with excellent in vivo activity

Article abstract of DOI:10.1021/jm0105777

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this che

Full text of DOI:10.1021/jm0105777

J . Med. Chem. 2002, 45, 2127-2130
2127
Discover y of
N-[2-[2-[[3-Meth oxy-4-(5-oxa zolyl)p h en yl]-
a m in o]-5-oxa zolyl]p h en yl]-N-m eth yl-4-
m or p h olin ea ceta m id e a s a Novel a n d
P oten t In h ibitor of In osin e
Mon op h osp h a te Deh yd r ogen a se w ith
Excellen t in Vivo Activity
T. G. Murali Dhar,* Zhongqi Shen, J unqing Guo,
Chunjian Liu, Scott H. Watterson, Henry H. Gu,
William J . Pitts, Catherine A. Fleener,
F igu r e 1. Mechanism of IMPDH inhibition.
Katherine A. Rouleau, N. Z. Sherbina,
Kim W. McIntyre, Mark R. Witmer,
J effrey A. Tredup, Bang-Chi Chen, Rulin Zhao,
Mark S. Bednarz, Daniel L. Cheney,
J ohn F. MacMaster, Laura M. Miller,
Karen K. Berry, Timothy W. Harper, J oel C. Barrish,
Diane L. Hollenbaugh, and Edwin J . Iwanowicz*
Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New J ersey 08543-4000
F igu r e 2. IMPDH inhibitors.
Received December 21, 2001
tor. The reaction involves nucleophilic addition of a
cysteine residue (Cys 331 in human type II) to the
2-position of IMP. The resulting enzyme-IMP interme-
diate (E-IMP) transfers a hydride to NAD, result-
ing in an enzyme-XMP* (E-XMP*) intermediate,
which hydrolyzes to XMP.8,9 Recent isotope labeling
studies suggest that the substrates (IMP and NAD)
bind randomly to the enzyme with an ordered release
of products (NADH release preceding hydrolysis of
E-XMP*).10 Crystal structures of apo IMPDH and
IMPDH complexes with inhibitors bound have been
disclosed recently.11
Mycophenolate mofetil (MMF or Cellcept, Figure 2),
the morpholino ester prodrug of mycophenolic acid
(MPA), is an uncompetitive inhibitor of IMPDH and has
been approved for the treatment of kidney and heart
transplantation in combination with cyclosporin A and
corticosteroids.12 Recently the U.S. FDA has approved
Cellcept for use in preventing rejection of organ trans-
plantation in allogeneic liver transplant recipients.
Despite the clinical efficacy of Cellcept, its clinical utility
is limited because of an unfavorable gastrointestinal
(GI) tolerability profile. This has been attributed to the
high concentrations of free MPA in the GI tract resulting
from enterohepatic circulation of the phenolic glucoro-
nide.13 Mizoribine and ribavarin, unlike MPA, are
competitive inhibitors of IMPDH. Mizoribine is mar-
keted in J apan for lupus nephritis, nephrotic syndrome,
rheumatoid arthritis, and the prevention of renal trans-
plant rejection. However, its use is also limited by GI
toxicity. Ribavarin (Virazole, Rebetol) has been ap-
proved in the U.S. for the treatment of respiratory
syncytial virus and in combination with interferon-R for
treatment of hepatitis C. The use of ribavarin is limited
because of its side effects, which include hemolytic
anemia.
Abstr a ct: Inosine monophosphate dehydrogenase (IMPDH)
is a key enzyme that is involved in the de novo synthesis of
purine nucleotides. Novel 2-aminooxazoles were synthesized
and tested for inhibition of IMPDH catalytic activity. Multiple
analogues based on this chemotype were found to inhibit
IMPDH with low nanomolar potency. One of the analogues
(compound 23) showed excellent in vivo activity in the inhibi-
tion of antibody production in mice and in the adjuvant
induced arthritis model in rats.
In tr od u ction . Inosine monophosphate dehydroge-
nase (IMPDH) is an enzyme that catalyzes the nicotin-
amide adenosine dinucleotide (NAD) dependent conver-
sion of inosine 5′-monophosphate (IMP) to xanthosine
5′-monophosphate (XMP).1 The reaction is irreversible
and is the first step in the de novo synthesis of guanine
nucleotides. Rapidly proliferating cells such as lympho-
cytes are dependent on the availability of the nucleotide
pool to meet their metabolic requirement, and it is
known that the activity of IMPDH is higher in prolif-
erating cells.2 Because of these cell requirements, IM-
PDH is an attractive target for immunosuppressive,
anticancer, and antiviral therapies.3
Two isoforms of the IMPDH enzyme are known to
exist: type I and type II. Human types I and II IMPDH
cDNAs encode the same-size proteins (514 amino acids)
and show 84% sequence identity.4 It was initially
thought that because type II expression is upregulated
in neoplastic and replicating cells, it is this isoform that
is responsible for cell differentiation and neoplastic
transformation.5 However, more recent work has indi-
cated that induction of types I and II isoforms contrib-
utes significantly to the T-cell proliferation response.6
The mechanism of IMPDH reaction has been studied
in detail (Figure 1).7 The oxidation of IMP to XMP is
an irreversible reaction and utilizes NAD as the cofac-
To overcome the limitations of current IMPDH inhibi-
tors, Vertex, using a combination of high-throughput
screening and structure-based drug design, identified
* To whom correspondence should be addressed. Phone: 609-252-
4158 or 609-252-5416. Fax: 609 252-6804. E-mail for T.G.M.D.:
dharm@bms.com. E-mail for E.J .I.: edwin.iwanowicz@bms.com.
10.1021/jm0105777 CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/20/2002

2128 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Letters
Ta ble 1. In Vitro Activity of 5-Phenyl-2-amino-1,3-oxazole
Derivatives
Sch em e 1. General Method for the Synthesis of
2-Amino-1,3-oxazoles
IC50(IMPDH II), nM IC50(CEM), µM
n ) 3 n ) 3
compd
R
R′
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
H
H
o-Me
m-Me
p-Me
20 ( 4
25 ( 3
70 ( 23
85 ( 17
23 ( 4
97 ( 19
7 ( 3
H
H
H
H
H
H
H
H
H
H
Me
Et
H
H
>10
>10
>10
7 ( 2
>10
9 ( 1
>10
VX-497 (merimempodib) (Figure 2) as an orally bio-
available inhibitor of IMPDH.14,15 VX-497 is currently
in phase IIb trials for the treatment of hepatitis C in
combination with interferon-R.
o-OMe
m-OMe
p-OMe
o-CO2Et
o-CO2H
o-CONMe2
o-CONHMe 104 ( 7
H
H
115 ( 56
127 ( 10
11% inhib at 20 µM
141 ( 34
A program to identify novel IMPDH inhibitors was
initiated with the goal of identifying compounds with
improved physicochemical and pharmacological proper-
ties. Our strategy was to retain the aniline moiety of
VX-497 and to develop the SAR for a new series
featuring various heterocycles as replacements for the
central urea linkage.16 In this communication, we
describe the synthesis and SAR of a novel series of
2-aminooxazoles resulting in the identification of com-
pound 23 as a potent IMPDH inhibitor with excellent
9 ( 1
8 ( 3
9 ( 0.4
>10
1 ( 0.7
0.73 ( 0.2
27 ( 8
86 ( 47
-NHCOMe 30 ( 5
-NMeCOMe 18 ( 9
In Vitr o SAR. Table 1 summarizes the structure-
activity relationships for the inhibition of IMPDH II
activity in the 2-amino-5-phenyloxazole series. The
parent phenyl oxazole (1) is a potent inhibitor of IMPDH
II with an IC50 of 20 nM. However, in a secondary T-cell
(CEM) proliferation assay, the compound had a potency
of only 5 µM. Hypothesizing that the poor cell activity
might be due to poor cell permeability and/or solubility
of the compound, we decided to incorporate solubilizing
residues on the phenyl ring to improve cell potency
while retaining enzyme activity. The ortho position of
the phenyl ring appeared to be optimal for the introduc-
tion of solubilizing residues based on the data for
compounds 2 and 5. Compound 2 with an ortho methyl
substituent is approximately 2- to 3-fold more potent
than the corresponding meta 3 and para-substituted 4
derivatives. Introduction of polar residues 8-15 led to
mixed results. While ester 8, acid 9, and carboxamides
10 and 11 led to a 5- to 6-fold decrease in potency for
the enzyme and consequently no improvement in cell
activity, the acetamide derivative 14 maintained en-
zyme potency and inhibited T-cell proliferation with an
IC50 of 1 µM. On the other hand, the N-methylacetamide
15 is a potent inhibitor of IMPDH II (IC50 ) 26 nM)
and was the first compound in this series that exhibited
activity in a mouse model for inhibition of antibody
production and in a rat adjuvant induced arthritis
model.
Ch em istr y. The 2-aminooxazoles were synthesized
utilizing a modified17 iminophosphorane/heterocume-
lene mediated approach18 as outlined in Scheme 1. In
general, reaction of a isothiocyanate with an acyl azide
and triphenylphosphine in dioxane at 95-100 °C or in
dichloromethane at room temperature yielded the cor-
responding 2-aminooxazoles.19 The acyl azides can
either be obtained from the corresponding acylbromides
or the treatment of an enol ether with N-bromosucci-
namide and water. As a representative example, the
synthesis of compound 23 is outlined in Scheme 2.
Sch em e 2. Synthesis of Compound 23a
a
(a) ClCOCH2OAc, pyridine, CH2Cl2, 2 h; (b) NaH, MeI, DMF, 1 h; (c) Pd(PPh3)2Cl2, CH2C(OEt)(Bu3Sn), dioxane, 100 °C, 18 h; (d)
NBS, H2O, THF, 50 °C, 10 min; (e) NaN3, acetone, H2O, room temp, 30 min; (f) PPh3, dioxane, 90 °C, 15 min; (g) LiOH, MeOH, H2O, room
temp, 30 min; (h) MsCl, Et3N, THF, 1 h; then morpholine, Et3N, DMF, room. See Supporting Information for full experimental details.

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2129
Ta ble 2. In Vitro Activity of 5-Acetamido-2-amino-1,3-oxazole
Derivatives
F igu r e 3. Compound 23 suppressing a humoral immune
response to KLH. BALB/c mice were immunized with KLH
on day 0 and then were treated by oral gavage, as indicated
on days 0-4. Serum titers were measured on day 10 by ELISA.
Ta ble 3. PK Profile of Compound 23 in Rats (n ) 3)
bio-
avail,
%
dose,
µmol/kg
Cmax
µM
,
t1/2
h
,
Cl,
mL/min/kg
Vss,
L/kg
AUCo-inf
,
µM min
F igu r e 4. Treatment of adjuvant induced arthritis in rats
with compound 23 (p < 0.05 vs vehicle, Student’s t test). The
graph depicts SEM for n ) 8 rats per treatment.
IA
PO
10
20
5.9 ( 1.1
1.5 ( 0.5 2.5 (0.8
71 ( 8
2.1 ( 0.3 142 ( 15
72 ( 13 25
hemocyanin (KLH) as the immunogen.20 In this model,
BALB/c mice are immunized by intraperitoneal injection
of KLH and then treated with compound 23 or control
compounds for 4 days. Antibodies to KLH in the serum
are measured by ELISA on day 10 (Figure 3). Compound
23 was equally efficacious as CellCept when dosed twice
daily. Further studies indicated that twice daily dosing
was not necessary to maintain efficacy in this model
(data not shown).
Adjuvant induced arthritis in Lewis rats is a widely
utilized model of human rheumatoid arthritis.21 This
model has been used to evaluate the antiarthritic
potential of agents such as methotrexate.22 Here, we
demonstrate the efficacy of the IMPDH inhibitor com-
pound 23 in rat adjuvant induced arthritis.
Arthritis was induced by the subcutaneous injection
of Freund’s complete adjuvant into the base of the tail
of male Lewis rats. Compound 23 (5 or 100 mg/kg) or
mycophenolate mofetil (MMF, 25 mg/kg) was adminis-
tered to rats by daily oral gavage beginning on the day
of adjuvant injection. Baseline measurements of hind
paw volume were obtained by plethysmometry. Ad-
ditional paw volume measurements were performed
over the next 3 weeks, and the increases in paw volume
above baseline were calculated. Hind paws (hocks) were
collected at necropsy and were submitted for histo-
pathological examination.
submicromolar potency (IC50 ) 530 nM) in inhibiting
T-cell proliferation.
To further explore the SAR around acetamide 15 and
to address potential formulation issues, compounds
having a basic residue were examined (Table 2). As is
evident from Table 2, most of the compounds examined
maintained excellent enzyme and cell potency. From
this set of compounds, compound 23 was identified for
further in vivo evaluation. Compound 23 is a potent
uncompetitive inhibitor of IMPDH II enzyme (IC50
)
16 nM, Ki ) 3.2 nM, IMPDH I IC50 ) 120 nM) and has
an IC50 of 520 nM in inhibiting the proliferation of the
CEM T-cell line. It inhibited the proliferation of normal
human PBMCs stimulated with anti-CD3 MAB and
anti-CD28 MAB, with an IC50 of 130 nM. More impor-
tantly, the hydrochloride salt of compound 23 is highly
water-soluble, thus enabling it to be dosed using an
aqueous vehicle for in vivo studies.
P K P r ofile of Com p ou n d 23. Before the compound
was subjected to in vivo efficacy studies, pharmakoki-
netic characteristics of compound 23 were evaluated in
rats. Results are outlined in Table 3. Compound 23 has
an estimated oral bioavailability of 25% and an appar-
ent elimination half-life of 5.9 h, suggesting the com-
pound was suitable for further evaluation in the inhi-
bition of antibody production in mice and in the adjuvant
induced arthritis model in rats.
As shown in Figure 4, compound 23 prevented disease
onset and progression. At 100 mg/kg daily, it almost
completely suppressed disease development, signifi-
cantly (p < 0.05 vs water vehicle, Student’s t test)
In Vivo Eva lu a tion of Com p ou n d 23. The ability
of the compound to inhibit an antibody response to a
soluble antigen was examined using keyhole limpet

2130 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Letters
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(16) The synthesis and SAR of the various heterocycles that were
used to replace the central urea linkage will be the subject of a
separate paper.
inhibiting paw swelling from day 13 onward. These rats
showed no signs of compound-related toxicity, and they
continued to gain weight during the course of the study
(data not shown). Specifically we did not see GI toxicity
by either gross examination or histopathology. At a dose
level of 5 mg/kg daily, compound 23 also delayed the
progression of paw swelling compared to vehicle-treated
rats, although the reduction in swelling was statistically
significant in only 2 days. MMF, when dosed to rats at
25 mg/kg, was highly efficacious in preventing arthritic
paw swelling. Higher doses of Cellcept could not be
tolerated because of significant GI toxicity. These data
demonstrate the efficacy of the novel, orally active
2-amino-1,3-oxazole, compound 23, in adjuvant arthri-
tis, a rat model of human rheumatoid arthritis.
Con clu sion . In summary, novel 2-aminooxazoles
were synthesized and tested for inhibition of IMPDH.
Multiple analogues based on this chemotype were found
to inhibit IMPDH with low nanomolar potency. One of
the analogues, compound 23, showed excellent in vivo
activity in the inhibition of antibody production in mice
and in the adjuvant induced arthritis model in rats.
Further exploration of the 2-amino-1,3-oxazole series
and evaluation of compound 23 are underway and will
be reported.
Su p p or tin g In for m a tion Ava ila ble: Full experimental
details for the preparation of compound 23 and the protocols
used for rat PK, the enzyme (IMPDH) inhibition assay,
inhibition of proliferation of the CEM T-cell line, and PBMCs.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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