Acridone-based inhibitors of inosine 5′-monophosphate dehydrogenase: Discovery and SAR leading to the identification of N-(2-(6-(4-ethylpiperazin-1- yl)pyridin-3-yl)propan-2-yl)-2-fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (BMS-566419)

Article abstract of DOI:10.1021/jm070299x

Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5′- monophosphate to xanthosine-5′-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model.

Full text of DOI:10.1021/jm070299x

3730
J. Med. Chem. 2007, 50, 3730-3742
Acridone-Based Inhibitors of Inosine 5′-Monophosphate Dehydrogenase: Discovery and SAR
Leading to the Identification of N-(2-(6-(4-Ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2-
fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (BMS-566419)
Scott H. Watterson,* Ping Chen, Yufen Zhao, Henry H. Gu, T. G. Murali Dhar, Zili Xiao, Shelley K. Ballentine, Zhongqi Shen,
Catherine A. Fleener, Katherine A. Rouleau, Mary Obermeier, Zheng Yang, Kim W. McIntyre, David J. Shuster, Mark Witmer,
Donna Dambach, Sam Chao, Arvind Mathur, Bang-Chi Chen, Joel C. Barrish, Jeffrey A. Robl, Robert Townsend, and
Edwin J. Iwanowicz
Bristol-Myers Squibb Pharmaceutical Research Institute, Post Office Box 4000, Princeton, New Jersey 08543
ReceiVed March 15, 2007
Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine
nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-
5′-monophosphate to xanthosine-5′-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of myco-
phenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH.
The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-
limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus,
development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may
reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery
and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability
when administered orally in a rat adjuvant arthritis model.
Introduction
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 synthetic pathway or through a salvage
pathway that utilizes existing purines and pyrimidines and their
nucleotides and nucleosides.1 The extent to which each pathway
is utilized is dependent on the cell type. B- and T-lymphocytes
are dependent on the de novo synthesis to generate sufficient
levels of nucleotides necessary to initiate a proliferative response
to mitogen or antigen.
Inosine 5′-monophosphate dehydrogenase (IMPDHa), a key
enzyme in the de novo synthesis of guanosine nucleotides,
catalyzes the irreversible nicotinamide-adenine dinucleotide
(NAD) dependent oxidation of inosine 5′-monophosphate (IMP)
to xanthosine 5′-monophosphate (XMP).2 Two distinct cDNAs
encoding IMPDH have been identified and isolated. These
transcripts, labeled type I and type II, possess 84% sequence
identity.2a,3 IMPDH type II is expressed at very low levels in
most resting cell types but is markedly up-regulated in actively
proliferating cell types, including cancer cells and activated
peripheral blood lymphocytes.2a,4 As a result, IMPDH type II
has emerged as an attractive target for selectively modulating
the immune response without also inhibiting the proliferation
of other cells.
Mycophenolic acid (MPA) has been shown to be a potent,
uncompetitive, reversible inhibitor of human IMPDH type I and
type II.5 Mycophenolate mofetil (MMF; Figure 1), a prodrug
of MPA, has clinical utility for the treatment of transplant
rejection based on its inhibition of IMPDH. Unfortunately, dose-
Figure 1.
limiting gastrointestinal (GI) toxicity is observed in a clinical
setting from the oral administration of either MMF or MPA.6
MMF is rapidly and, to a large extent, completely absorbed
following oral administration, whereby it is converted to MPA.6
Three metabolites have been identified in humans: the 7-O-
glucuronide (MPAG), 7-O-glucoside conjugate (MPAG1s), and
the acyl glucuronide (AcMPAG).6 MPAG, the major metabolite,
is inactive against IMPDH and is found in substantially higher
concentrations in the plasma of healthy subjects than MPA,
contributing to the high doses required to achieve the desired
plasma concentrations necessary for efficacy. Additionally,
significant amounts of MPAG are excreted in the bile and
* To whom correspondence should be addressed. Phone: 609-252-6778.
Fax: 609-252-7410. E-mail: scott.watterson@bms.com.
a Abbreviations: IMPDH, inosine 5′-monophosphate dehydrogenase;
MPA, mycophenolic acid; MMF, mycophenolate mofetil; HTS, high-
throughput screen; CEM, human T-lymphoblast cell line; PBMC, human
peripheral blood mononuclear cells; hERG, human ether-a-go-go-related
gene; GI, gastrointestinal.
10.1021/jm070299x CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/22/2007

Inhibitors of Inosine 5′-Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3731
Table 1. In Vitro Potency of Acridone Analogues (8)a
a IC50 values are shown as single determinations. b Inhibition of T-cell
proliferation (see Experimental Section for details).
Figure 2. aInhibition of T-cell proliferation in CEM lymphoblast cells
(see Experimental Section for details).
Table 2. In Vitro Potency of Fluoro-acridone Analogues (4) vs
Acridone Analogues (8)a
converted to MPA by the action of the intestinal flora.6 Coupled
with the high doses required for efficacy, this reintroduction of
MPA to the gut is likely a contributing factor to the clinically
observed GI toxicity.
In spite of the commercial success of MMF, dose limiting
GI toxicity has restricted its use primarily to the treatment of
transplant rejection, even though the inhibition of IMPDH may
provide a clinical benefit for other immune-mediated diseases
such as rheumatoid arthritis. There is a continuing effort to
identify and develop new inhibitors of IMPDH which are
differentiated from MMF with improved GI tolerability. Al-
though the GI toxicity associated with MPA is likely due to
the inhibition of intestinal epithelial cell proliferation, the toxicity
may be exacerbated by the unique pharmacokinetic profile (PK)
of MPA, resulting from glucoronidation, excretion to the GI
tract, and subsequent enterohepatic recirculation.6 Two strategies
have emerged in the literature to address the GI toxicity
associated with MPA. The first is to develop inhibitors with a
different PK profile that may minimize the exposure of the cells
of the gut to an inhibitor, thereby reducing the likelihood of GI
toxicity and allowing for increased exposure and greater
efficacy. This may be accomplished by identifying inhibitors,
which exclude the acid and phenolic chemical moieties impli-
cated in the glucuronidation and subsequent enterohepatic
recirculation. Several groups,2b,7-9 including our own,9 have
focused on this approach and have identified a number of new
classes of inhibitors. A second approach is to appropriately
formulate MPA to reduce upper GI toxicity and improve
absorption.10 With favorable clinical data,11 Novartis launched
an enteric-coated formulation of mycophenolate sodium (ERL-
80) in 2002.
IMPDH II
IC50 (nM)
CEMc
IC50 (nM)
PBMCc
IC50 (nM)
cmpd
R1
R2
8a
Ph
H
H
H
H
F
19
53
42
53
11 ( 5.9
29
11 ( 0.8
2.1
2.2
2.2
2.3
0.83 ( 0.69
1.0
0.42 ( 0.07
0.73
8fa
8ga
8ha
4ab
4ba
4cb
2-pyridyl
3-pyridyl
4-pyridyl
Ph
2-pyridyl
3-pyridyl
0.59
0.63
0.22 ( 0.07
F
F
0.14 ( 0.04
a IC50 values are shown as single determinations. b IC50 values are shown
as mean values of three determinations. c Inhibition of T-cell proliferation
(see Experimental Section for details).
the development of a novel acridone series and the SAR leading
to the discovery of 4m (BMS-566419;12 Figure 2).
Chemistry
The synthetic pathways utilized in the preparation of the
acridone inhibitors discussed in Tables 1-3 are outlined in
Schemes 1-9. Acridones 8a-h were prepared as shown in
Scheme 1. A Buchwald-Hartwig13 coupling of dimethyl
2-bromoterephthatate with aniline in the presence of Pd(OAC)2,
(S)-BINAP, and Cs2CO3 gave biphenylamine 6. Hydrolysis of
the esters with LiOH in MeOH/THF and subsequent cyclization
with polyphosphoric acid at 165 °C afforded the acridone
carboxylic acid 7 in high yield. Intermediate 7 was coupled with
various commercially available amines using BOP-Cl and Et3N
in DMF to give acridones 8a-e. The pyridyl-substituted tertiary-
alkyl amines used for acridone derivatives 8f-h were derived
from commercially available nitriles via dimethylation using
MeCeCl2 in THF,14 as outlined in Scheme 3.
In an effort to supplement our previously reported endeavors
to develop IMPDH inhibitors,9 we initiated a high-throughput
screening (HTS) approach of our compound deck to find novel
chemotypes. Acridone 1 (Figure 2) was identified as a potential
lead with an IC50 of 0.726 µM against IMPDH II. Further
analysis of the sample indicated the presence of an ∼12%
impurity. Subsequent purification revealed that the source of
the activity was a regio-isomeric acridone analogue (3), with
an IC50 of 0.059 µM against IMPDH II. In this article, we outline
Fluoro-acridones 4a-p were prepared as exemplified in the
preparation of analogue 4m depicted in Scheme 2. The biphenyl
amine intermediate 11 was prepared via a Buchwald-Hartwig13
coupling of dimethyl 2-fluoroterephthatate with aniline in the

3732 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Table 3. In Vitro Potency of Fluoro-acridone Analoguesa
Watterson et al.
Scheme 2a
a Reagents and conditions: (a) HNO3, H2SO4, 95%; (b) H2, Pd/C, EtOAc,
90%; (c) PhBr, Pd(OAc)2, (S)-BINAP, Cs2CO3, toluene, 80%; (d) LiOH-
H2O, H2O, THF, MeOH, 96%; (e) PPA, 165 °C, 98%; (f) 14, BOP-Cl,
Et3N, DMF, 75%.
Scheme 3a
a Reagents and conditions: (a) N-ethylpiperazine, EtOH, reflux, 95%;
(b) CeCl3, MeLi, THF, -78 °C-rt, 90%.
Scheme 4a
a Reagents and conditions: (a) 2-dimethylaminoethylchloride hydro-
chloride, K2CO3, DMF, 33%; (b) CeCl3, MeLi, THF, -78 °C-rt, 79%.
a IC50 values are shown as mean values of three determinations.
b Inhibition of T-cell proliferation (see Experimental Section for details).
Scheme 5a
Scheme 1a
a Reagents and conditions: (a) PhNH2, Pd(OAc)2, (S)-BINAP, Cs2CO3,
toluene, 97%; (b) LiOH, MeOH, THF, H2O, 96%; (c) PPA, 165 °C, 98%;
(d) RNH2, BOP-Cl, Et3N, DMF.
a Reagents and conditions: (a) CeCl3, MeLi, THF, -78 °C-rt; (b) 12,
BOP-Cl, Et3N, DMF, 59%; (c) mCPBA, DCM, MeOH, 56%.
presence of Pd(OAC)2, (S)-BINAP, and Cs2CO3. Hydrolysis of
the esters with LiOH in MeOH/THF followed by cyclization
with polyphosporic acid at 165 °C gave fluoro-acridone acid
12 in 98% yield. Acridone acid 12 was coupled with various
amines using BOP-Cl and Et3N in DMF to give fluoro-acridones
4a-p. The tertiary-alkyl amines used in the final couplings were
synthesized in good yield from the corresponding nitriles via
dimethylation using MeCeCl2 in THF,14 as shown in Schemes
3-9. Example 4l was prepared utilizing a similar route as
outlined in Scheme 3 starting with 2-chloro-4-cyanopyridine
and dimethylamine.

Inhibitors of Inosine 5′-Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3733
Scheme 6a
enantiomer 8d had an IC50 of 95 nM against IMPDH II, while
the (S)-enantiomer’s (8e) inhibitory potency was 4-5-fold less,
with an IC50 of 431 nM. With 8b as a viable lead, we turned
our focus toward improving the cell potency (T-cell proliferation
in CEM lymphoblastoid cells and in human peripheral blood
mononuclear cells (PBMCs); see Experimental Section for
details) and the physio-chemical properties of the series.
a Reagents and conditions: (a) dimethylamine, DCM, 0 °C, 95%; (b)
CeCl3, MeLi, THF, -78 °C-rt.
In exploring the structure-activity relationships (SARs)
around this lead, we rationalized that a small group substituted
at R2 in structure 4 (Table 2) might provide enhancements in
the in vitro potency, in particular, cell activity and physio-
chemical properties, through restricted rotation of the acridone-
amide carbonyl bond, forcing the carbonyl to orient 180 degrees
away from the group. As anticipated, incorporation of a fluoro
adjacent to the amide (4a; Table 2) provided a >3-fold
improvement in the inhibition of T-cell proliferation in human
PBMCs (see Experimental Section for details), with an IC50 of
0.22 µM. Interestingly, the effects were more pronounced when
the amide was substituted with a 3-pryidyl (8g vs 4c), resulting
in a 4-5-fold improvement in the inhibition of T-cell prolifera-
tion (CEM lymphoblastoid cells and PBMCs) and the inhibition
of IMPDH II. In the des-fluoro-acridone pyridyl analogues (8f-
h), the positioning of the nitrogen had very little impact on either
the inhibition of the enzyme or on T-cell proliferation. However,
in the fluoro-acridone series, the 3-pryridyl analogue 4b was
2-3-fold more active than the 2-pyridyl analogue 4c. With the
small but consistent improvement in in vitro potency, the fluoro-
acridones became the primary focus of our effort to optimize
this series of inhibitors.
Scheme 7a
a Reagents and conditions: (a) Ac2O, pyr, 1 M aq NaOH, 89%; (b) 1,2-
dibromoethane, K2CO3, acetone, water, reflux, 99%; (c) NBS, DMF, 75
°C, 63%; (d) Pd2(dba)3, DPPF, Zn, ZnCN2, DMA, 150 °C, 80%; (e) CeCl3,
MeLi, THF, -78 °C-rt, 95%.
Scheme 8a
With the fluoro-acridone core in hand, the optimal region
for further enhancing activity and physio-chemical character-
istics was the tertiary-alkyl amide substitution. We reasoned
that by introducing various polar residues to the phenyl
substituent of 4a (cLogP 3.3) or to the pyridyl substituent of
4c (cLogP 1.7) we could modulate the LogP and, consequently,
modulate the potency and the physio-chemical properties of the
series. As outlined in Table 3, a wide range of phenyl and
pyridyl substitution was well tolerated and provided several
potent analogues. The minor variations observed in the in vitro
activities suggest that this region of the molecule is likely
directed toward solvent. Examples 4d-h represent substituted
phenyl rings with sulfonamide 4g exhibiting highly potent in
a Reagents and conditions: (a) TMS-CHN2, MeOH, 99%; (b) morpholine,
125 °C, 88%; (c) NBS, DMF, 56%; (d) Pd2(dba)3, DPPF, Zn, ZnCN2, DMA,
150 °C, 87%; (e) CeCl3, MeLi, THF, -78 °C-rt, 99%.
Scheme 9a
vitro inhibitory activity (IMPDH IC50 ) 10 nM, CEM IC50
)
0.66 µM, PBMC IC50 ) 0.20 µM). As mentioned previously,
the 3-pyridyl substituted analogue 4c was also highly potent
with an IMPDH IC50 of 11 nM and an IC50 of 0.14 µM in the
PBMC cell assay. To decrease any potential metabolic liability
attributed to the 3-pyridyl nitrogen, substitution at the para-
position was explored. As depicted by examples 4i-k and 4m-
p, the substituted pyridyl analogues ranging from a simple
methyl to the more elaborate methyl substituted morpholinopy-
ridine (4p) provided significant in vitro potency with little
differentiation in activity. Interestingly, the 4-pyridyl analogue
4l exhibited equivalent, if not slightly better, activity overall
relative to the 3-pyridyl analogue 4k; however, the 4-pyridyl
derivatives proved to be less desirable in both advanced in vitro
profiling assays (e.g., CYP inhibition, hERG, metabolic stability)
and pharmacokentic studies.
a Reagents and conditions: (a) morpholine, 140 °C, 99%; (b) Pd2(dba)3,
DPPF, Zn, ZnCN2, DMA, 150 °C, 77%; (c) CeCl3, MeLi, THF, -78 °C-
rt, 95%.
Results and Discussion
Our preliminary library work around ester 3 led to the
identification of acridones 8a and 8b as potential leads with
the phenyl substituted tertiary-alkyl amide 8b demonstrating a
3-fold improvement in IMPDH II inhibitory activity over the
simple t-butyl 8a, as seen in Table 1. Further investigation of
8b revealed that quaternary substitution adjacent to the amide
nitrogen was optimal, with the benzyl amide (8c) having
significantly reduced inhibitory activity (IMPDH II IC50 ) 2.2
µM) relative to 8b (IMPDH II IC50 ) 0.019 µM). Additionally,
both monomethyl enatiomers were prepared and evaluated (8d
and 8e), and although they showed significant improvement over
8c, they were less potent than 8b (5-23-fold). The (R)-
Although the phenyl series provided many highly potent
analogues, substituted pyridyl acridones demonstrated optimal
in vivo efficacy with improved pharmacokinetic and in vitro
profiling properties. Based on this analysis, analogue 4m was
selected as our lead candidate. Steady-state enzyme kinetic
studies determined that 4m was a reversible and uncompetitive

3734 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Table 4. Partial In Vitro Profiling Data for Compound 4ma
Watterson et al.
parameter
result
62 ( 5.0 nM
92% in human serum
ames negative
IMPDH type I12 (IC50)
protein binding
mutagenicity
hERG (patch clamp) (IC50)
25 µM
CYPa,b inhibition (IC50)
>20 µM 1A2, 2C9, 2D6;
19 µM 2C19; 13 µM 3A4 BFC
2.5
LogP
Caco2 permeability
320 nm/s
a CYP ) cytochrome P450. b CYP inhibition assays were performed
against isolated CYP enzymes.
Table 5. Pharmacokinetic Parameters for Compound 4ma
parameter
rata
cynoa
10b
po dose (mg/kg)
iv dose (mg/kg)
10b
2
2
C
T
max (µM), PO
max ((µM), PO
AUC ((µM‚h), PO
1/2 (h), iv
0.41 ( 0.21
4.0 ( 0.0
5.2 ( 2.7
3.6 ( 1.8
4.4 ( 1.7
29 ( 3.0
7.4 ( 2.2
43
26 ( 24
2.9 ( 2.0
102 ( 32
4.0 ( 0.33
5.5 ( 0.45
3.4 ( 0.64
1.1 ( 0.19
88
T
MRT (h), iv
Cl (mL/min/kg), iv
V
F
ss (L/kg), iv
po (%)
a Average of three animals with associated standard deviation. b Vehicle:
60% PEG400/40% HCl at pH 3.
Figure 3. (A) Efficacy of 4m (25 and 50 mg/kg) and MMF (15 mg/
kg) vs vehicle in a rat adjuvant arthritis model. (B) Efficacy of 4m (5
and 10 mg/kg) vs vehicle in a rat adjuvant arthritis model. Animals
per group ) 8; 4m and MMF were administered PO/QD starting on
day 0. Vehicle was 60% PEG400/40% 0.01 N HCl; *p value < 0.05
compared to vehicle treatment group.
inhibitor of IMPDH II with a Ki of 25 ( 3 nM with respect to
IMP and 20 ( 4 nM with respect to NAD.9j A partial list of
profiling data is presented in Table 4. In pharmocokenetic (PK)
studies (Table 5), 4m had a bioavailability of 43% in rats with
a half-life of 3.6 h and a bioavailability of 88% in cynologous
monkey with a half-life of 4.0 h. Through these studies, 4m
was found to be metabolically stable, and in the absence of a
phenolic residue, 4m is not expected to exhibit the corresponding
enterohepatic recirculation associated with MMF.
Acridone 4m was evaluated in the fully preventative Lewis
rat adjuvant arthritis (AA rat) model, a widely utilized preclinical
model of human rheumatoid arthritis,15 to establish the effect
of compound treatment on paw swelling post-inoculation with
complete Freund’s adjuvant.16 The rat was chosen because of
its sensitivity to the immunosuppressive effects of IMPDH
inhibitors and its predisposition to the analogous mechanism-
based toxicities such as anemia and GI toxicity observed
clinically with MMF.
Arthritis was induced by the subcutaneous injection of
Freund’s complete adjuvant into the base of the tail of male
Lewis rats. Acridone 4m was administered at doses of 50 and
25 mg/kg PO/QD in the first experiment, along with MMF at
15 mg/kg (Figure 3A), and doses of 10 and 5 mg/kg PO/QD in
a second experiment (Figure 3B) daily for 21 days starting at
day 0. Baseline measurements of hind paw volume were
obtained by plethysmometry. Additional paw volume measure-
ments were performed over the next three weeks, and the
increases in paw volume above baseline were calculated.
Significant inhibition of paw swelling was seen at doses of 10,
25, and 50 mg/kg PO/QD (p < 0.05 vs water vehicle, Student’s
t test, Figures 3). Additionally, the rats showed no signs of GI
toxicity at any dose by either gross examination or histopathol-
ogy. MMF demonstrated significant inhibition at 15 mg/kg
(Figure 3A) and only modest (∼30%) but not statistically
significant inhibition of paw swelling at a dose of 10 mg/kg
PO/QD (data not shown), also showing no signs of GI toxicity
at these doses. This study clearly demonstrated the efficacy of
this novel, orally active acridone inhibitor (4m) in the rat
adjuvant arthritis model.
a
GI tolerability was assessed for both acridone 4m and MMF
in Lewis rats in the context of the rat AA model. To determine
the maximum tolerated dose with respect to GI toxicity, the
rats were given escalating doses of either MMF or 4m for up
to 14 days. The rats were then monitored for the development
of diarrhea and weight loss. Rats receiving either a daily dose
of 50 mg/kg of MMF or 125 mg/kg of 4m survived the two
week study with only mild transient weight loss and occasional
soft stools observed for some rats. Higher doses of MMF and
4m (e.g., 75 mg/kg and 150 mg/kg, respectively) resulted in
moderate to severe diarrhea, confirmed by histopathology (crypt
cell necrosis), leading to morbidity. As a result of these findings,
doses of 50 mg/kg for MMF and 125 mg/kg for 4m were
selected as the maximum tolerated doses for GI toxicity. The
therapeutic index (TI) of 4m and MMF for GI toxicity was
established based on exposure (AUC) in these rat studies. Based
on the AUCs for 4m at the lowest efficacious and maximally
tolerated doses, the TI for GI toxicity in the rat AA model was
determined to be 19-fold. Comparatively, the TI for MMF was
determined to be 6.9-fold.
Conclusion
In summary, a novel series of acridone-based inhibitors of
IMPDH II has been identified. Structure-activity relationship
studies led to multiple highly potent analogues exhibiting
significant IMPDH inhibition as well as the inhibition of the
proliferation of CEM lymphoblastoid cells and human PBMCs.
Based on its in vitro potency, in vivo activity, and pharmaco-
kinetic and safety profiles, 4m was selected as our lead drug
candidate. Compound 4m was efficacious at 10 mg/kg in the
rat adjuvant arthritis model, a preclinical model of human
rheumatoid arthritis. This compound has a different in vitro and
in vivo pharmacokinetic and metabolic profile than MMF and,
as a result, 4m was evaluated in rat to establish in vivo the

Inhibitors of Inosine 5′-Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3735
min) was employed on a Zorbax Rapid Res 3.5µ 4.6 × 50 mm
column. Flow rate was 2.5 mL/min and UV detection was set to
254 nm. The LC column was maintained at ambient temperature.
Method H. A linear gradient using 10% methanol, 90% water,
and 0.1% TFA (solvent A) and 90% acetonitrile, 10% water, 0.1%
TFA (solvent B); t ) 0 min, 0% B, t ) 4 min, 100% B (5 min)
was employed on a Chromolith SpeedRod 4.6 × 50 mm column.
Flow rate was 4 mL/min and UV detection was set to 254 nm. The
LC column was maintained at ambient temperature.
relationship between efficacy and toxicity and was found to have
an approximately 3-fold improvement over MMF in its thera-
peutic index with respect to GI toxicity. Overall, preclinical data
suggest that acridone 4m may have an improved safety window
in humans over MMF with respect to GI adverse events.
Experimental Section
Proton magnetic resonance (1H) spectra were recorded on either
a Bruker Avance 400 or a JEOL Eclipse 500 spectrometer and are
reported in ppm relative to the reference solvent of the sample in
which they were run. HPLC and LCMS analyses were conducted
using a Shimadzu SCL-10A liquid chromatograph and a SPD UV-
vis detector at 220 or 254 nm with the MS detection performed
with either a Micromass Platform LC spectrometer or a Waters
Micromass ZQ spectrometer. Preparative reverse-phase HPLC
purifications were performed using the following conditions:
Ballistic YMC S5 ODS 20 × 100 mm column with a binary solvent
system, where solvent A ) 10% methanol, 90% water, 0.1%
trifluoroacetic acid and solvent B ) 90% methanol, 10% water,
and 0.1% trifluoroacetic acid; flow rate ) 20 mL/min; linear
gradient time ) 10 min; start %B ) 20; final %B ) 100. Fractions
containing the product were concentrated in vacuo to remove the
methanol and were neutralized with aqueous sodium bicarbonate.
The products were collected by vacuum filtration or were extracted
with organic solvents and concentrated under reduced pressure. All
flash column chromatography was performed on EM Science silica
gel 60 (particle size of 40-60 µm). All reagents were purchased
from commercial sources and used without further purification
unless otherwise noted. All reactions were performed under an inert
atmosphere.
HPLC analyses were performed using the following conditions:
Method A. A linear gradient using 5% acetonitrile, 95% water,
and 0.05% TFA (solvent A) and 95% acetonitrile, 5% water, and
0.05% TFA (solvent B); t ) 0 min, 10% B, t ) 15 min, 100% B
(20 min) was employed on a SunFire C18 3.5u 4.6 × 150 mm
column. Flow rate was 1 mL/min and UV detection was set to 254
nm. The LC column was maintained at ambient temperature.
Method B. A linear gradient using 5% acetonitrile, 95% water,
and 0.05% TFA (solvent A) and 95% acetonitrile, 5% water, and
0.05% TFA (solvent B); t ) 0 min, 10% B, t ) 15 min, 100% B
(20 min) was employed on a XBridge Ph 3.5µ 4.6 × 150 mm
column. Flow rate was 1 mL/min and UV detection was set to 254
nm. The LC column was maintained at ambient temperature.
Method C. A linear gradient using 5% acetonitrile, 95% water,
and 0.05% TFA (solvent A) and 95% acetonitrile, 5% water, and
0.05% TFA (solvent B); t ) 0 min, 10% B, t ) 10 min, 50% B
(20 min) was employed on a SunFire C18 3.5µ 4.6 × 150 mm
column. Flow rate was 1 mL/min and UV detection was set to 254
nm. The LC column was maintained at ambient temperature.
Method D. A linear gradient using 5% acetonitrile, 95% water,
and 0.05% TFA (solvent A) and 95% acetonitrile, 5% water, and
0.05% TFA (solvent B); t ) 0 min, 10% B, t ) 10 min, 50% B
(20 min) was employed on a XBridge Ph 3.5µ 4.6 × 150 mm
column. Flow rate was 1 mL/min and UV detection was set to 254
nm. The LC column was maintained at ambient temperature.
Method E. A linear gradient using 10% methanol, 90% water,
and 0.1% TFA (solvent A) and 90% methanol, 10% water, and
0.1% TFA (solvent B); t ) 0 min, 0% B, t ) 4 min, 100% B (5
min) was employed on a YMC ODS 4.6 × 50 mm column. Flow
rate was 4 mL/min and UV detection was set to 220 or 254 nm.
The LC column was maintained at ambient temperature.
9-Oxo-9,10-dihydro-acridine-3-carboxylic Acid (7). To a
mixture of dimethyl bromoterephthalate (4.5 g, 16.0 mmol), aniline
(2.2 mL, 24.0 mmol), (S)-(-)-2,2′-bis(diphenylphosphino)-1,1′-
binaphthyl (922 mg, 1.48 mmol), and toluene (50 mL) was added
cesium carbonate (7.53 g, 23.1 mmol), followed by palladium(II)
acetate (0.22 g, 1.0 mmol). The mixture was heated to 100 °C for
24 h. HPLC indicated that the reaction was complete. The reaction
mixture was cooled to room temperature, diluted with diethyl ether,
and filtered under reduced pressure through a pad of Celite. The
filtrate was concentrated under reduced pressure and purified by
silica gel chromatography using a 1:10 mixture of ethyl acetate
and hexane to give 4.58 (97%) of 5-phenylamino-terephthalic acid
dimethyl ester as a bright yellow solid. HPLC tr ) 3.74 min
(condition E); LCMS (EI) m/z calcd for C16H15NO4 [M + H]+,
286.11; found, 286+. 1H NMR (400 MHz, CDCl3): δ 3.88 (s, 3H),
3.93 (s, 3H), 7.13 (m, 1H), 7.25 (m, 1H), 7.32-7.40 (m, 4H), 7.91
(d, J ) 1.50, 1H), 8.01 (d, J ) 8.30, 1H), 9.50 (bs, 1H).
A mixture of 5-phenylamino-terephthalic acid dimethyl ester
(4.56 g, 16.0 mmol) and lithium hydroxide monohydrate (2.0 g,
48.0 mmol) in methanol (32 mL), tetrahydrofuran (32 mL), and
water (16 mL) was heated to reflux for 0.5 h. HPLC indicated that
the reaction was complete. The organic solvents were removed
under reduced pressure, and the aqueous residue was diluted with
water. The pH was adjusted to 3.0 with 6 N aqueous hydrochloric
acid. The resulting precipitate was collected by vacuum filtration,
rinsed with water, and dried under reduced pressure to provide 3.95
g (96%) of 5-phenylamino-terephthalic acid (6) as a bright yellow
solid. HPLC tr ) 2.75 min (condition E); LCMS (EI) m/z calcd for
C14H11NO4 [M + H]+, 258.08; found, 258+.
To a round-bottom flask containing polyphosphoric acid (35 g)
at 165 °C was added finely ground 5-phenylamino-terephthalic acid
(6; 3.0 g, 1.17 mmol) over 10 min. After the addition was complete,
the reaction mixture was stirred for 30 min at 165 °C. HPLC
indicated that the reaction was complete. While at 165 °C, the
mixture was slowly added to a mixture of ice and sodium hydroxide.
The pH was adjusted to 3.0 with additional sodium hydroxide, and
the resulting mixture was filtered through a medium porosity fritted
funnel to give a yellow paste that was rinsed with methanol and
dichloromethane into a round-bottom flask. The organic solvents
were removed under reduced pressure, and the residue was
azeotroped several times with methanol and dichloromethane to
give 2.75 g (98%) of 9-oxo-9,10-dihydro-acridine-3-carboxylic acid
(7) as a yellow solid. HPLC tr ) 2.45 min (condition E); LCMS
1
(EI) m/z calcd for C14H9NO3 [M + H]+, 240.07; found, 240+. H
NMR (500 MHz, DMSO-d6) δ 7.30 (t, J ) 7.55 Hz, 1H), 7.55 (d,
J ) 8.20 Hz, 1H), 7.72 (d, J ) 8.42 Hz, 1H), 7.77 (t, J ) 7.55 Hz,
1H), 8.18 (s, 1H), 8.24 (d, J ) 8.20 Hz, 1H), 8.31 (d, J ) 8.42 Hz,
1H), 11.96 (s, 1H), 13.13 (br s, 1H).
2-Fluoro-9-oxo-9,10-dihydro-acridine-3-carboxylic Acid (12).
To a mixture of dimethyl 2-fluoroterephthalate (42.0 g, 0.198 mol)
in concentrated sulfuric acid (200 mL) at 0 °C was added 36 mL
of a 1:1 mixture of nitric acid and sulfuric acid dropwise. The
mixture was stirred at 0 °C for 15 min, the ice-bath was removed,
and the reaction mixture was stirred for an additional 45 min. HPLC
indicated that the starting material had been consumed. The reaction
mixture was poured over ice and extracted with ethyl acetate. The
organic layer was collected, washed with brine, and dried over
anhydrous sodium sulfate. Concentration under reduced pressure
followed by recrystallization from methanol afforded 48.5 g (95%)
of 2-fluoro-5-nitro-terephthalic acid dimethyl ester as a white solid.
Method F. A linear gradient using 10% methanol, 90% water,
and 0.2% H3PO4 (solvent A) and 90% acetonitrile, 10% water, and
0.2% H3PO4 (solvent B); t ) 0 min, 0% B, t ) 4 min, 100% B (5
min) was employed on a YMC ODS 4.6 × 50 mm column. Flow
rate was 4 mL/min and UV detection was set to 220 or 254 nm.
The LC column was maintained at ambient temperature.
Method G. A linear gradient using 10% methanol, 90% water,
and 0.2% H3PO4 (solvent A) and 90% acetonitrile, 10% water, and
0.2% H3PO4 (solvent B); t ) 0 min, 0% B, t ) 8 min, 100% B (11

3736 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Watterson et al.
1
HPLC purity >98%, tr ) 2.69 min (condition B). H NMR (500
bis[2-oxo-3-oxazolidinyl]phosphorodiamidic chloride (0.064 g,
0.251 mmol). The reaction mixture was stirred at room temperature
overnight, diluted with dichloromethane, washed with a 1.0 N
aqueous solution of sodium hydroxide (3×), and dried over
anhydrous sodium sulfate. Concentration under reduced pressure
afforded a yellow solid which was triturated with methanol with
sonication and filtered to give 9-oxo-N-(2-phenylpropan-2-yl)-9,-
10-dihydroacridine-3-carboxamide (8b, 0.054 g, 73%) as a pale
yellow solid (Alternatively, all compounds could be purified by
flash silica gel chromatography or preparative HPLC). HPLC purity
99.6%, tr ) 10.94 min (method A); 99.6%, tr ) 12.43 min (method
B); LCMS (EI) m/z calcd for C23H20N2O2 [M + H]+, 357.16; found,
mHz, CDCl3) δ 3.95 (s, 3H), 3.98 (s, 3H), 7.44 (d, 1H), 8.59 (d,
1H).
A mixture of 2-fluoro-5-nitro-terephthalic acid dimethyl ester
(48.5 g, 0.178 mol) and palladium/carbon (4.6 g) in ethyl acetate
(450 mL) was evacuated under reduced pressure and charged with
hydrogen (3×). The reaction mixture was subsequently stirred under
a steady stream of nitrogen for 1 h. The reaction mixture was filtered
through a pad of Celite, concentrated, and recrystallized from a
mixture of methanol and dichloromethane to provide 36.2 g (90%)
of 2-amino-5-fluoro-terephthalic acid dimethyl ester (10) as a bright
yellow solid. HPLC purity >98%, tr ) 2.68 min (condition B);
LCMS (EI) m/z calcd for C10H10FNO4 [M + H]+, 228.07; found,
1
357.14. H NMR (500 MHz, DMSO-d6) δ 1.69 (s, 6H), 7.18 (t, J
228+. H NMR (500 mHz, CDCl3) δ 3.89 (s, 3H), 3.92 (s, 3H),
) 7.42 Hz, 1H), 7.27-7.32 (m, 3H), 7.40 (s, 1H), 7.42 (s, 1H),
7.55 (d, J ) 8.80 Hz, 1H), 7.64 (d, J ) 7.15 Hz, 1H), 7.75 (t, J )
7.70 Hz, 1H), 7.91 (s, 1H), 8.23 (d, J ) 7.15 Hz, 1H), 8.27 (d, J
) 8.25 Hz, 1H), 8.73 (s, 1H), 11.90 (s, 1H).
N-tert-Butyl-9-oxo-9,10-dihydroacridine-3-carboxamide (8a).
Acridone 8a was prepared as a pale yellow solid following the
procedure described for 8b. HPLC purity 99.4%, tr ) 10.94 min
(method A); 99.5%, tr ) 12.94 min (method B); LCMS (EI) m/z
calcd for C18H18N2O2 [M + H]+, 295.14; Found, 295.17. 1H NMR
(500 MHz, DMSO-d6) δ 1.41 (s, 9H), 7.28 (m, 1H), 7.55-7.60
(m, 2H), 7.70 (m, 1H), 7.89 (s, 1H), 8.07 (s, 1H), 8.23-8.25 (m,
2H), 11.89 (s, 1H).
N-Benzyl-9-oxo-9,10-dihydroacridine-3-carboxamide (8c). Ac-
ridone 8c was prepared as a pale yellow solid following the
procedure described for 8b. HPLC purity 99.2%, tr ) 11.33 min
(method A); 99.1%, tr ) 13.58 min (method B); LCMS (EI) m/z
calcd for C21H16N2O2 [M + H]+, 329.13; found, 329.19. 1H NMR
(500 MHz, DMSO-d6) δ 4.52 (d, J ) 6.05 Hz, 2H), 7.23-7.30
(m, 2H), 7.32-7.37 (m, 4H), 7.55 (d, J ) 8.25 Hz, 1H), 7.69 (d,
J ) 8.25 Hz, 1H), 7.75 (t, J ) 7.70 Hz, 1H), 8.03 (s, 1H), 8.23 (d,
J ) 7.15 Hz, 1H), 8.28 (d, J ) 8.25 Hz, 1H), 9.31 (t, J ) 5.77 Hz,
1H), 11.91 (s, 1H).
1
7.20 (d, 1H), 7.61 (d, 1H).
To a mixture of 2-amino-5-fluoro-terephthalic acid dimethyl ester
(10; 10.0 g, 44.0 mmol), bromobenzene (4.6 mL, 44.0 mmol), (S)-
(-)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (2.06 g, 3.30 mmol),
and toluene (250 mL) was added cesium carbonate (20.0 g, 44.0
mmol) followed by palladium(II) acetate (0.494 g, 2.20 mmol). The
mixture was heated at 90 °C for 24 h. HPLC indicated that the
reaction was complete. The reaction mixture was cooled to room
temperature and filtered under reduced pressure through a pad of
Celite topped with a pad of silica gel, and the Celite/silica gel pads
were rinsed with ethyl acetate. The filtrate was concentrated under
reduced pressure and purified by silica gel chromatography using
a 1:10 mixture of EtOAc and hexane to give 10.7 g (80%) of
2-fluoro-5-phenylamino-terephthalic acid dimethyl ester as a pale
yellow solid. HPLC purity > 99%, tr ) 3.72 min (condition F). 1H
NMR (400 mHz, CDCl3) δ 3.89 (s, 3H), 3.93 (s, 3H), 7.12 (m,
1H), 7.22 (m, 2H), 7.37 (m, 2H), 7.74 (m, 2H), 9.22 (br s, 1H).
A mixture of 2-fluoro-5-phenylamino-terephthalic acid dimethyl
ester (30.0 g, 9.89 mmol) and lithium hydroxide monohydrate (12.5
g, 29.7 mmol) in MeOH (200 mL), tetrahydrofuran (200 mL), and
water (100 mL) was heated at 75 °C for 0.5 h. HPLC indicated
that the reaction was complete. The organic solvents were removed
under reduced pressure, and the aqueous residue was diluted with
water. The pH was adjusted to 3.0 with 6 N aqueous hydrochloric
acid, which resulted in a precipitate. The solid was collected by
vacuum filtration and dried under reduced pressure to provide a
quantitative yeild of compound 2-fluoro-5-phenylamino-terephthalic
acid (11) as an orange-yellow solid. HPLC purity 99%, tr ) 3.02
(R)-9-Oxo-N-(1-phenylethyl)-9,10-dihydroacridine-3-carboxa-
mide (8d). Acridone 8d was prepared as a pale yellow solid
following the procedure described for 8b. HPLC purity 98.8%, tr
) 11.83 min (method A); 99.0%, tr ) 14.10 min (method B);
LCMS (EI) m/z calcd for C22H18N2O2 [M + H]+, 343.14; found,
1
343.18. H NMR (500 MHz, DMSO-d6) δ 1.50 (d, J ) 7.15 Hz,
3H), 5.20 (qd, J ) 7.33, 7.15 Hz, 1H), 7.25 (ddd, J ) 18.01, 7.56,
7.42 Hz, 2H), 7.34 (t, J ) 7.70 Hz, 2H), 7.42 (d, J ) 7.70 Hz,
2H), 7.55 (d, J ) 8.25 Hz, 1H), 7.69 (d, J ) 7.15 Hz, 1H), 7.74 (t,
J ) 7.70 Hz, 1H), 7.98 (s, 1H), 8.23 (d, J ) 6.60 Hz, 1H), 8.28 (d,
J ) 8.25 Hz, 1H), 9.10 (d, J ) 7.70 Hz, 1H), 11.92 (s, 1H).
1
min (condition F). H NMR (400 mHz, DMSO) δ 7.11 (m, 1H),
7.25 (m, 2H), 7.39 (m, 2H), 7.60 (m, 1H), 7.68 (m, 1H), 9.32 (br
s, 1H), 13.55 (br s, 2H).
To a round-bottom flask containing polyphosphoric acid (254
g) at 165-172 °C was added finely ground 2-fluoro-5-phenylamino-
terephthalic acid (11; 15.0 g, 55.6 mmol) over 30 min. After the
addition was complete, the reaction mixture was stirred for 10 min
at 165 °C. HPLC indicated that the reaction was complete. While
at 165 °C, the reaction mixture was slowly added to a mixture of
ice and sodium bicarbonate. The pH was adjusted to 3.0 with
additional solid sodium bicarbonate, and the resulting mixture was
filtered through a medium porosity fritted funnel to give a greenish-
yellow paste that was washed with water (3×) and air-dried under
vacuum. The resulting paste was rinsed with methanol into a flask.
The paste/methanol mixture was then sonicated to disperse the paste.
Dichloromethane was added, and the mixture was concentrated
under reduced pressure. The azeotrope procedure was repeated 2×
to give 12.0 g (84%) of 2-fluoro-9-oxo-9,10-dihydro-acridine-3-
carboxylic acid (12) as a yellow solid. HPLC retention time ) 2.35
min (condition A); LCMS (EI) m/z calcd for C14H8FNO3 [M +
(S)-9-Oxo-N-(1-phenylethyl)-9,10-dihydroacridine-3-carboxa-
mide (8e). Acridone 8e was prepared as a pale yellow solid
following the procedure described for 8b. HPLC purity 98.2%, tr
) 11.86 min (method A); 98.4%, tr ) 14.16 min (method B);
LCMS (EI) m/z calcd for C22H18N2O2 [M + H]+, 343.14; found,
1
343.15. H NMR (500 MHz, DMSO-d6) δ 1.50 (d, J ) 7.15 Hz,
3H), 5.20 (qd, J ) 7.33, 7.15 Hz, 1H), 7.25 (dt, J ) 18.70, 7.42
Hz, 2H), 7.34 (t, J ) 7.70 Hz, 2H), 7.42 (d, J ) 7.70 Hz, 2H),
7.55 (d, J ) 8.25 Hz, 1H), 7.69 (d, J ) 9.90 Hz, 1H), 7.74 (t, J )
6.87 Hz, 1H), 7.98 (s, 1H), 8.23 (d, J ) 7.15 Hz, 1H), 8.28 (d, J
) 8.25 Hz, 1H), 9.10 (d, J ) 7.70 Hz, 1H), 11.91 (s, 1H).
9-Oxo-N-(2-(pyridin-2-yl)propan-2-yl)-9,10-dihydroacridine-
3-carboxamide (8f). Acridone 8f was prepared as a pale yellow
solid following the procedure described for 8b using acridone acid
(7) and 2-(pyridin-2-yl)propan-2-amine. The amine was prepared
from picolinonitrile as described in the preparation of 4i. HPLC
purity 98.2%, tr ) 6.97 min (method A); 99.1%, tr ) 10.56 min
(method B); LCMS (EI) m/z calcd for C22H19N3O2 [M + H]+,
358.16; found, 358.17. 1H NMR (500 MHz, DMSO-d6) δ 1.71 (s,
6H), 7.24 (d, J ) 6.60 Hz, 1H), 7.28 (t, J ) 7.15 Hz, 1H), 7.47 (d,
J ) 8.25 Hz, 1H), 7.55 (d, J ) 8.80 Hz, 1H), 7.66 (d, J ) 9.90
Hz, 1H), 7.73-7.77 (m, 2H), 7.94 (s, 1H), 8.23 (d, J ) 7.15 Hz,
1H), 8.28 (d, J ) 8.25 Hz, 1H), 8.52 (d, J ) 3.85 Hz, 1H), 8.91 (s,
1H), 11.91 (s, 1H).
H]+, 258.06; found, 258.03. H NMR (500 MHz, DMSO) δ 7.30
(t, J ) 7.6 Hz, 1H), 7.56 (d, J ) 7.6 Hz, 1H), 7.78 (t, J ) 7.6 Hz,
1H), 7.93 (d, J ) 11.0 Hz, 1H), 8.11 (d, J ) 6.1 Hz, 1H), 8.22 (d,
J ) 7.6 Hz, 1H), 12.06 (s, 1H), 13.69 (br s, 1H).
9-Oxo-N-(2-phenylpropan-2-yl)-9,10-dihydroacridine-3-car-
boxamide (8b). To a mixture of 9-oxo-9,10-dihydroacridine-3-
carboxylic acid (7, 0.050 g, 0.209 mmol), cumylamine (0.036 mL,
0.251 mmol), and triethylamine (0.087 mL, 0.627 mmol) in N,N-
dimethylformamide (5.0 mL) at room temperature was added N,N-
1

Inhibitors of Inosine 5′-Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3737
9-Oxo-N-(2-(pyridin-3-yl)propan-2-yl)-9,10-dihydroacridine-
3-carboxamide (8g). Acridone 8g was prepared as a pale yellow
solid following the procedure described for 8b using acridone acid
(7) and 2-(pyridin-3-yl)propan-2-amine. The amine was prepared
from nicotinonitrile as described in the preparation of 4i. HPLC
purity 97.3%, tr ) 7.02 min (method A); 98.0%, tr ) 10.59 min
(method B); LCMS (EI) m/z calcd for C22H19N3O2 [M + H]+,
358.16; found, 358.17. 1H NMR (500 MHz, DMSO-d6) δ 1.72 (s,
6H), 7.20-7.26 (m, 1H), 7.33 (dd, J ) 7.97, 4.67 Hz, 1H) 7.54 (d,
J ) 8.25 Hz, 1 H), 7.58-7.61 (m, 1H), 7.68-7.72 (m, 1H), 7.77
(d, J ) 7.70 Hz, 1H), 7.94 (s, 1 H), 8.22 (d, J ) 7.70 Hz, 1H),
8.26 (d, J ) 8.3 Hz, 1H), 8.40 (d, J ) 4.40 Hz, 1H), 8.64 (s, 1H),
8,81 (s, 1H), 11.95 (s, 1H).
analogous to that used for the preparation of compound 4i. HPLC
tr ) 0.203 min (condition E); LCMS (EI) m/z calcd for C13H22N2O
1
[M + H]+, 223.18; found, 223+. H NMR (400 MHz, CDCl3) δ
1.45 (s, 6H), 2.31 (s, 6H), 2.70 (t, J ) 5.72 Hz, 2H), 4.02 (t, J )
5.72 Hz, 2H), 6.85 (d, J ) 8.79 Hz, 2H), 7.39 (d, J ) 8.79 Hz,
2H).
Fluoro-acridone 4d was prepared as a yellow solid from 12 and
16 by a route analogous to that used for the preparation of 8b.
HPLC purity 96.9%, tr ) 8.96 min (method A); 96.7%, tr ) 9.76
min (method B); LCMS (EI) m/z calcd for C27H28FN3O3 [M +
H]+, 462.22; found, 462.16. 1H NMR (500 MHz, DMSO-d6) δ 1.65
(s, 6H), 2.37 (s, 6H), 2.83 (s, 2H), 4.10 (t, J ) 5.50 Hz, 2H), 6.91
(d, J ) 8.80 Hz, 2H), 7.29 (t, J ) 7.42 Hz, 1H), 7.35 (d, J ) 8.80
Hz, 2H), 7.55 (d, J ) 8.25 Hz, 1H), 7.68 (d, J ) 5.50 Hz, 1H),
7.76 (t, J ) 7.70 Hz, 1H), 7.91 (d, J ) 9.90 Hz, 1H), 8.21 (d, J )
8.25 Hz, 1H), 8.76 (s, 1H), 11.97 (s, 1H).
9-Oxo-N-(2-(pyridin-4-yl)propan-2-yl)-9,10-dihydroacridine-
3-carboxamide (8h). Acridone 8h was prepared as a pale yellow
solid following the procedure described for 8b using acridone acid
(7) and 2-(pyridin-4-yl)propan-2-amine. The amine was prepared
from isonicotinonitrile as described in the preparation of 4i. HPLC
purity 97.6%, tr ) 6.90 min (method A); 95.9%, tr ) 10.49 min
(method B); LCMS (EI) m/z calcd for C22H19N3O2 [M + H]+,
358.16; found, 358.17. 1H NMR (500 MHz, DMSO-d6) δ 1.67 (s,
6H), 7.27 (t, J ) 7.42 Hz, 1H), 7.37 (d, J ) 6.05 Hz, 2H), 7.55 (d,
J ) 8.25 Hz, 1H), 7.66 (d, J ) 8.25 Hz, 1H), 7.74 (t, J ) 7.70 Hz,
1H), 7.92 (s, 1H), 8.23 (d, J ) 8.25 Hz, 1H), 8.28 (d, J ) 8.80 Hz,
1H), 8.49 (d, J ) 6.05 Hz, 2H), 8.87 (s, 1H), 11.92 (s, 1H).
2-Fluoro-9-oxo-N-(2-phenylpropan-2-yl)-9,10-dihydroacridine-
3-carboxamide (4a). Acridone 4a was prepared as a bright yellow
solid following the procedure described for 8b starting with fluoro-
acridone acid 12. HPLC purity 99.1%, tr ) 13.39 min (method A);
98.7%, tr ) 15.38 min (method B); LCMS (EI) m/z calcd for C23H19-
2-Fluoro-N-(2-(4-(methylsulfonyl)phenyl)propan-2-yl)-9-oxo-
9,10-dihydroacridine-3-carboxamide (4e). Acridone 4e was pre-
pared as a bright yellow solid following the procedure described
for 4f, starting with 4-bromophenyl-methylsulfide. HPLC purity
98.0%, tr ) 11.34 min (method A); 98.0%, tr ) 13.77 min (method
B); LCMS (EI) m/z calcd for C24H21FN2O4S [M + H]+, 453.13;
1
found, 453.11. H NMR (500 MHz, DMSO-d6) δ 1.69 (s, 6H),
3.22 (s, 3H), 7.28 (t, J ) 7.42 Hz, 1H), 7.55 (d, J ) 8.25 Hz,1 H),
7.70 (d, J ) 8.25 Hz, 3H), 7.75 (t, J ) 7.42 Hz, 1H), 7.90 (d, J )
8.80 Hz, 2H), 7.93 (d, J ) 10.45 Hz, 1H), 8.21 (s, 1H) 9.03 (s,
1H), 11.97 (s, 1H).
2-Fluoro-N-(2-(3-(methylsulfonyl)phenyl)propan-2-yl)-9-oxo-
9,10-dihydroacridine-3-carboxamide (4f). To a solution of 3-bro-
mophenyl-methylsulfide (1.624 g, 8.0 mmol) in dimethylformamide
(30 mL) was added cuprous cyanide (0.788 g, 8.8 mmol). The
reaction mixture was heated at 150 °C overnight. The solvent was
removed under reduced pressure, and the residue was diluted wth
dichloromethane, washed with water, and dried over anhydrous
sodium sulfate. Concentration under reduced pressure followed by
purification by flash silica gel chromatography afforded 3-(meth-
1
FN2O2 [M + H]+, 375.15; found, 375.15. H NMR (500 MHz,
DMSO-d6) δ 1.67 (s, 6H), 7.21 (t, J ) 7.42 Hz, 1H), 7.29 (t, J )
7.42 Hz, 1H), 7.34 (t, J ) 7.70 Hz, 2H), 7.45 (d, J ) 7.15 Hz,
2H), 7.54 (d, J ) 8.25 Hz, 1H), 7.68 (d, J ) 5.50 Hz, 1H), 7.76 (t,
J ) 7.70 Hz, 1H), 7.92 (d, J ) 9.90 Hz, 1H), 8.22 (d, J ) 6.60
Hz, 1H), 8.85 (s, 1H), 11.94 (s, 1H).
1
ylthio)-benzonitrile as a light yellow solid. H NMR (400 MHz,
2-Fluoro-9-oxo-N-(2-(pyridin-2-yl)propan-2-yl)-9,10-dihy-
droacridine-3-carboxamide (4b). Acridone 4b was prepared as a
bright yellow solid following the procedure described for 8b using
fluro-acridone acid (12) and 2-(pyridin-2-yl)propan-2-amine. The
amine was prepared from picolinonitrile as described in the
preparation of 4i. HPLC purity 98.2%, tr ) 4.83 min (method G);
99%, tr ) 1.85 min (method H); LCMS (EI) m/z calcd for C22H18-
CDCl3) δ 2.53 (s, 3H), 7.44 (m, 4H).
1-Methyl-1-(3-methylthiophenyl)ethyl-amine (17) was prepared
from 3-bromophenyl methylsulfide as described in the preparation
of 4i. 1H NMR (400 MHz, CDCl3) δ 1.48 (s, 6H), 1.60 (br s, 2H),
2.50 (s, 3H), 7.12 (m, 1H), 7.27 (m, 2H), 7.44 (s, 1H).
2-Fluoro-N-(2-(3-(methylthio)phenyl)propan-2-yl)-9-oxo-9,10-di-
hydroacridine-3-carboxamide (18) was prepared as a bright yellow
solid following the procedure described for 8b using fluro-acridone
(12) and 1-methyl-1-(3-methylthiophenyl)ethyl-amine (17). HPLC
purity 99%, tr ) 3.28 min (method E); LCMS (EI) m/z calcd for
C24H21FN2O2S [M + H]+, 421.14; found, 421+.
1
FN3O2 [M + H]+, 376.15; found, 376.15. H NMR (500 MHz,
DMSO-d6) δ 1.70 (s, 6H), 7.24-7.31 (m, 2H), 7.54 (d, J ) 8.25
Hz, 2H), 7.73-7.83 (m, 3H), 7.94 (d, J ) 10.45 Hz, 1H), 8.22 (d,
J ) 7.70 Hz, 1H), 8.54 (d, J ) 3.85 Hz, 1H), 9.08 (d, J ) 2.20
Hz, 1H), 11.99 (s, 1H).
2-Fluoro-9-oxo-N-(2-(pyridin-3-yl)propan-2-yl)-9,10-dihy-
droacridine-3-carboxamide (4c). Acridone 4c was prepared as a
bright yellow solid following the procedure described for 8b using
fluro-acridone acid (12) and 2-(pyridin-3-yl)propan-2-amine. The
amine was prepared from nicotinonitrile as described in the
preparation of 4i. HPLC purity 96.4%, tr ) 3.86 min (method G);
99%, tr ) 1.78 min (method H); LCMS (EI) m/z calcd for C22H18-
2-Fluoro-N-(2-(3-(methylthio)phenyl)propan-2-yl)-9-oxo-9,10-di-
hydroacridine-3-carboxamide (18) was dissolved in a mixture of
dichloromethane and methanol. To the solution was added excess
mCPBA, and the reaction mixture was stirred overnight. The
solvents were removed under reduced pressure, and the resulting
residue was dissolved in dichloromethane, washed sequentially with
aqueous Na2S2O8 and aqueous sodium bicarbonate, and dried over
sodium sulfate. Concentration under reduced pressure followed by
purification by flash silica gel chromatography afforded (2-fluoro-
N-(2-(3-(methylsulfonyl)phenyl)propan-2-yl)-9-oxo-9,10-dihydroacri-
dine-3-carboxamide (4f; 56%) as a bright yellow solid. HPLC purity
98.0%, tr ) 11.37 min (method A); 98.2%, tr ) 13.92 min (method
B); LCMS (EI) m/z calcd for C24H21FN2O4S [M + H]+, 453.13;
1
FN3O2 [M + H]+, 376.15; found, 376.14. H NMR (500 MHz,
DMSO-d6) δ 1.70 (s, 6H), 7.29 (t, J ) 7.42 Hz, 1H), 7.33-7.39
(m, 1H), 7.54 (d, J ) 8.25 Hz, 1H), 7.69 (d, J ) 5.50 Hz, 1H),
7.76 (t, J ) 6.87 Hz, 1H), 7.81 (d, J ) 8.25 Hz, 1H), 7.93 (d, J )
9.90 Hz, 1H), 8.22 (d, J ) 8.25 Hz, 1H), 8.44 (d, J ) 6.05 Hz,
1H), 8.68 (d, J ) 2.20 Hz, 1H), 8.97 (s, 1H), 11.94 (s, 1H).
N-(2-(4-(2-(Dimethylamino)ethoxy)phenyl)propan-2-yl)-2-
fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (4d). A mix-
ture of 4-cyanophenol (1.19 g, 10 mmol), 2-dimethylaminoethyl-
chloride hydrochloride (2.16 g, 15 mmol), and potassium carbonate
(5.53 g, 40 mmol) in DMF (20 mL) was heated at 80 °C for 2 h
and then filtered. The filtrate was concentrated under reduced
pressure, and the resulting residue was purified by flash silica gel
chromatography to give 0.628 g of 4-(2-(dimethylamino)ethoxy)-
benzonitrile (15). 2-(4-(2-(Dimethylamino)-ethoxy)phenyl)propan-
2-amine (16) was then prepared as a colorless oil by a route
1
found, 453.15. H NMR (500 MHz, DMSO-d6) δ 1.71 (s, 6H),
3.22 (s, 3H), 7.29 (t, J ) 7.15 Hz, 1H), 7.55 (d, J ) 8.25 Hz, 1H),
7.62-7.68 (m, 2H), 7.75-7.82 (m, 3H), 7.92-7.96 (m, 2H), 8.22
(d, J ) 7.15 Hz, 1H), 9.06 (s, 1H), 11.97 (s, 1H).
N-(2-(4-(N,N-Dimethylsulfamoyl)phenyl)propan-2-yl)-2-fluoro-
9-oxo-9,10-dihydroacridine-3-carboxamide (4g). To a suspension
of 4-cyanobenzenesulfonyl chloride (0.402 g, 1.99 mmol) in
dichloromethane (3 mL) was added dimethylamine (3 mL, 2 M in
tetrahydrofuran). The reaction mixture was stirred for 30 min,
diluted with water, and extracted with dichloromethane. The organic

3738 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Watterson et al.
layer was collected, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give 0.409 g (98%) of
4-cyano-N,N-dimethylbenzenesulfonamide as a white solid. 4-(2-
Aminopropan-2-yl)-N,N-dimethylbenzene-sulfonamide (19) was
prepared from 4-cyano-N,N-dimethylbenzenesulfonamides, as de-
scribed in the preparation of 4i.
mmol), and zinc cyanide (0.080 g, 0.678 mmol) was flushed with
nitrogen. Dimethyl acetamide (4 mL) was added, and the resulting
mixture was heated at 150 °C overnight. HPLC indicated that the
reaction was complete. The reaction mixture was cooled to room
temperature, diluted with ethyl acetate, and washed with 2 N
aqueous ammonium hydroxide, washed with brine, and dried over
anhydrous sodium sulfate. Concentration under reduced pressure
followed by purification by flash silica gel chromatography afforded
3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-7-carbonitrile (21; 0.183
g, 80%) as an off-white solid. HPLC purity 98%, tr ) 0.997 min
(method F); LCMS (EI) m/z calcd for C8H7N3O [M + H]+, 162.07;
found, 162.03.
To a flame-dried flask under nitrogen was added cerium chloride
(0.918 g, 3.73 mmol) followed by anhydrous tetrahydrofuran (11
mL). The mixture was stirred vigorously for 45 min, during which
time the cerium chloride became suspended. The suspension was
cooled to -78 °C, and methyl lithium (2.66 mL, 3.73 mmol, 1.4
M in ether) was added dropwise. After the addition was complete,
the reaction mixture was stirred for 0.5 h at -78 °C. Compound
21 (0.120 g, 0.745 mmol) in tetrahydrofuran (4 mL) was added
via cannula to the -78 °C solution. The dry ice bath was removed,
and the reaction mixture was stirred at room temperature overnight.
The mixture was quenched with a few drops of a saturated aqueous
solution of ammonium chloride, and a 50% aqueous solution of
ammonium hydroxide was added dropwise until a precipitate
formed and settled to the bottom of the flask. The mixture was
filtered through Celite under reduced pressure, diluted with dichlo-
romethane, washed with water, and dried over anhydrous sodium
sulfate. Concentration under reduced pressure afforded 2-(3,4-
dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)propan-2-amine (22; 95%).
HPLC tr ) 0.197 min (method F); LCMS (EI) m/z calcd for
C10H15N3O [M + H]+, 194.13; found, 194.13. The amine was used
without any further purification.
Acridone 4g was prepared as a bright yellow solid following
the procedure described for 8b using fluro-acridone (12) and 4-(2-
aminopropan-2-yl)-N,N-dimethylbenzenesulfonamide (19). HPLC
purity 97.3%, tr ) 12.52 min (method A); 97.7%, tr ) 14.97 min
(method B); LCMS (EI) m/z calcd for C25H24FN3O4S [M + H]+,
482.15; found, 482.18. 1H NMR (500 MHz, DMSO-d6) δ 1.70 (s,
6H), 2.63 (s, 6H), 7.29 (t, J ) 7.42 Hz, 1H), 7.54 (d, J ) 8.25 Hz,
1H), 7.68-7.70 (m, 3H), 7.71-7.78 (m, 3H), 7.93 (d, J ) 10.45
Hz, 1H), 8.22 (d, J ) 8.25 Hz, 1H), 9.03 (s, 1H), 11.94 (s, 1H).
N-(2-(3-(N,N-Dimethylsulfamoyl)phenyl)propan-2-yl)-2-fluoro-
9-oxo-9,10-dihydroacridine-3-carboxamide (4h). Acirdone 4h
was prepared as a bright yellow solid following the procedure
described for 8b using fluro-acridone (12) and 3-(2-aminopropan-
2-yl)-N,N-dimethylbenzenesulfonamide. The amine was prepared
from 3-cyano-N,N-dimethylbenzene-sulfonamide as described in 4g.
HPLC purity 93.5%, tr ) 12.47 min (method A); 94.2%, tr ) 14.73
min (method B); LCMS (EI) m/z calcd for C25H24FN3O4S [M +
H]+, 482.15; found, 482.19. 1H NMR (500 MHz, DMSO-d6) δ 1.70
(s, 6H), 2.57 (s, 6H), 7.29 (t, J ) 7.97 Hz, 1H), 7.55 (d, J ) 8.25
Hz, 1H), 7.59-7.66 (m, 3H), 7.77 (q, J ) 7.15 Hz, 3H), 7.93 (d,
J ) 10.45 Hz, 1H), 8.22 (d, J ) 7.15 Hz, 1H), 9.07 (s, 1H), 11.98
(s, 1H).
N-(2-(3,4-Dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)propan-
2-yl)-2-fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (4i). A
mixture of 2-amino-3-hydroxypyridine (3.0 g, 27.2 mmol) and 2
mL of pyridine in 141 mL of acetic anhydride was heated at reflux
for 5 min, cooled to room temperature, and concentrated under
reduce pressure. The oily residue was then stirred in a 1 M aqueous
solution of sodium hydroxide (75 mL) until the mixture became
homogeneous. The solution was poured into a mixture of crushed
ice and 6 M aqueous hydrochloric acid, and the pH was adjusted
to ∼4.0. The aqueous mixture was extracted with dichloromethane,
washed with water, dried over anhydrous sodium sulfate, and
concentrated to give N-(3-hydroxypyridin-2-yl)acetamide (3.70 g,
89%) as an off-white solid. HPLC purity 99%, tr ) 0.193 min
(method F); LCMS (EI) m/z calcd for C7H8N2O2 [M + H]+, 153.07;
found, 153.07.
To a mixture of N-(3-hydroxypyridin-2-yl)acetamide (3.70 g, 24.3
mmol), 1,2-dibromoethane (8.4 mL, 97.2 mmol), water (30 mL),
and acetone (120 mL) was added potassium carbonate (13.4 g, 97.2
mmol. The reaction mixture was heated at reflux overnight and
concentrated. The residue was dissolved in dichloromethane, washed
with water, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The crude solid was dissolved in ether,
and the undissolved material was removed by vacuum filtration.
The filtrate was concentrated to give 4.28 g (99%) of 1-(2H-pyrido-
[3,2-b][1,4]oxazin-4(3H)-yl)ethanone (20) as an off-white solid.
HPLC tr ) 0.720 min (method F); LCMS (EI) m/z calcd for
C9H10N2O2 [M + H]+, 179.08; found, 179.10.
A mixture of 20 (0.438 g, 2.46 mmol) and N-bromosuccinimide
(0.438 g, 2.46 mmol) in 6 mL of dimethylformamide was heated
at 75 °C for 1 h. The solvent was removed under reduced pressure,
and the residue was dissolved in dichloromethane, washed with
water, and dried over anhydrous sodium sulfate. Concentration
under reduced pressure followed by purification by flash silica gel
chromatography afforded 0.315 g of 1-(7-bromo-2H-pyrido[3,2-b]-
[1,4]oxazin-4(3H)-yl)ethanone as a white solid. HPLC purity ∼95%,
tr ) 2.99 min (method F); LCMS (EI) m/z calcd for C9H9BrN2O2
[M + H]+, 256.99; found, 256.90 and 258.90. 1H NMR (500 MHz,
CDCl3) δ 2.58 (s, 3H), 4.04-4.07 (m, 2H), 4.25-4.27 (m, 2H),
7.37 (d, J ) 2.2 Hz, 1H), 7.51 (d, J ) 2.2 Hz, 1H).
To a mixture of 12 (0.020 g, 0.078 mmol), 22 (0.017 g, 0.086
mmol), and triethylamine (0.033 mL, 0.233 mmol) was added N,N-
bis[2-oxo-3-oxazolidinyl]phosphorodiamidic chloride (0.024 g,
0.093 mmol) in 4 mL of anhydrous dimethylformamide at room
temperature. The reaction mixture was stirred at room temperature
overnight. The mixture was diluted with dichloromethane, washed
with water, washed with 1 N aqueous sodium hydroxide (3×), and
dried over anhydrous sodium sulfate. Concentration under reduced
pressure followed by purification by preparative HPLC afforded
compound 4i (17.0 mg) as a bright yellow solid. HPLC purity
98.5%, tr ) 7.80 min (method A); 98.4% tr ) 11.80 min (method
B); LCMS (EI) m/z calcd for C24H21FN4O3 [M + H]+, 433.17;
1
found, 433.27. H NMR (500 MHz, DMSO-d6) δ 1.62 (s, 6H),
3.38 (br s, 2H), 4.11-4.12 (m, 2H), 6.54 (s, 1H), 6.98 (d, J ) 2.2
Hz, 1H), 7.29 (t, J ) 7.7 Hz, 1H), 7.53 (d, J ) 8.3 Hz, 1H), 7.65
(t, J ) 2.2 Hz, 2H), 7.76 (t, J ) 7.7 Hz, 1H), 7.91 (d, J ) 9.9 Hz,
1H), 8.21 (d, J ) 8.3 Hz, 1H), 8.72 (s, 1H), 11.94 (s, 1H).
2-Fluoro-N-(2-(6-methylpyridin-3-yl)propan-2-yl)-9-oxo-9,10-
dihydroacridine-3-carboxamide (4j). Acridone 4j was prepared
as a bright yellow solid following the procedure described for 8b
using fluro-acridone (12) and 2-(6-methylpyridin-3-yl)propan-2-
amine. The amine was prepared from 6-methylnicotinonitrile as
described in the preparation of 4i. HPLC purity 96%, tr ) 3.97
min (method G); 97%, tr ) 1.84 min (method H); LCMS (EI) m/z
calcd for C23H20FN3O2 [M + H]+, 390.16; found, 390.20. 1H NMR
(500 MHz, DMSO-d6) δ 1.67 (s, 6H), 2.44 (s, 3H), 7.21 (d, J )
7.70 Hz, 1H), 7.28 (t, J ) 7.15 Hz, 1H), 7.54 (d, J ) 8.25 Hz,
1H), 7.64-7.71 (m, 2H), 7.75 (t, J ) 6.87 Hz, 1H), 7.92 (d, J )
9.90 Hz, 1H), 8.21 (d, J ) 6.60 Hz, 1H), 8.53 (d, J ) 2.75 Hz,
1H), 8.92 (s, 1H), 11.95 (s, 1H).
N-(2-(6-(Dimethylamino)pyridin-3-yl)propan-2-yl)-2-fluoro-
9-oxo-9,10-dihydroacridine-3-carboxamide (4k). 6-(Dimethy-
lamino)nicotinonitrile was prepared from 6-chloronicotinonitrile and
dimethylamine as described for 4n. The nitrile was then dimethy-
lated as described for 4n to give 5-(2-aminopropan-2-yl)-N,N-
dimethylpyridin-2-amine. Acridone 4k was prepared as a bright
yellow solid from 12 and 5-(2-aminopropan-2-yl)-N,N-dimethylpy-
ridin-2-amine as described in the preparation of 8b. HPLC purity
A mixture of 1-(7-bromo-2H-pyrido[3,2-b][1,4]oxazin-4(3H)-yl)-
ethanone (0.290 g, 1.13 mmol), tris(dibenzylideneacetone)di-
palladium(0) (0.062 g, 0.068 mmol), 1,1′-bis(diphenylphosphino)-
ferrocene (0.075 g, 0.136 mmol), zinc powder (9.0 mg, 0.136

Inhibitors of Inosine 5′-Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3739
91.8%, tr ) 8.16 min (method A); 93.2%, tr ) 15.38 min (method
B); LCMS (EI) m/z calcd for C24H23FN4O2 [M + H]+, 419.19;
fluoro-9,10-dihydro-9-oxo-3-acridinecarboxamide (4m; 8.2 g, 56%)
as a bright yellow solid. HPLC purity 99.5%, tr ) 6.65 min (method
A); 99.7%, tr ) 10.53 min (method B); LCMS (EI) m/z calcd for
1
found, 419.18. H NMR (500 MHz, DMSO-d6) δ 1.64 (s, 6H),
1
3.00 (s, 6H), 6.61 (d, J ) 8.80 Hz, 1H), 7.26 (t, J ) 7.42 Hz, 1H),
7.52-7.57 (m, 2H), 7.66 (d, J ) 5.50 Hz, 1H), 7.74 (t, J ) 7.15
Hz, 1H), 7.90 (d, J ) 9.90 Hz, 1H), 8.17 (d, J ) 2.20 Hz, 1H),
8.21 (d, J ) 8.25 Hz, 1 H), 8.75 (s, 1H), 11.94 (s, 1H).
C28H30FN5O2 [M + H]+, 488.25; found, 488.22. H NMR (500
MHz, DMSO-d6) δ 1.03 (t, 3H, J ) 7.2 Hz), 1.66 (s, 6H), 2.36 (q,
J ) 14.3,7.2 Hz, 2H), 2.45 (m, 4H), 3.45 (m, 4H), 6.80 (d, J )
8.8, 1H), 7.29 (t, J ) 7.5 Hz, 1H), 7.53 (d, J ) 8.2 Hz, 1H), 7.57
(d, J ) 8.8 Hz, 1H), 7.66 (d, J ) 5.5 Hz, 1H), 7.76 (t, J ) 7.5 Hz,
1H), 7.91 (d, 9.9 Hz, 1H), 8.20 (s, 1H), 8.21 (d, J ) 8.2 Hz, 1H),
8.78 (s, 1H), 11.93 (s, 1H). Anal. Calcd for C28H30FN5O2, 1.1%
H2O: C, 68.22; H, 6.26; N, 14.21; F, 3.85. Found: C, 67.83; H,
6.21; N, 14.06; F, 3.76.
2-Fluoro-N-(2-(6-morpholinopyridin-3-yl)propan-2-yl)-9-oxo-
9,10-dihydroacridine-3-carboxamide (4n). A mixture of 6-chlo-
ronicotinonitrile (0.139 g, 1.00 mmol) and morpholine (0.35 mL,
4.00 mmol) in 2 mL of ethanol was heated to reflux for 1 h. Upon
cooling to room temperature, the solvent was removed under
reduced pressure and the residue was diluted with water. The
resulting white solid was collected by filtration, rinsed thoroughly
with water, and dried under high vacuum overnight to afford
6-morpholinonicotinonitrile as a white crystalline solid (36% yield).
HPLC tr ) 1.59 min (condition E); LCMS (EI) m/z calcd for
C10H11N3O [M + H]+, 190.10; found, 190+. 1H NMR (400 MHz,
DMSO-d6) δ 3.65 (t, J ) 4.92, 4H), 3.80 (t, J ) 4.92, 4H), 6.59
(d, J ) 8.74, 1H), 7.64 (dd, J ) 2.41, J ) 8.74, 1H), 8.42 (d, J )
2.41, 1H).
N-(2-(2-(Dimethylamino)pyridin-4-yl)propan-2-yl)-2-fluoro-
9-oxo-9,10-dihydroacridine-3-carboxamide (4l). 2-(Dimethylami-
no)isonicotinonitrile was prepared from 2-chloro-4-cyanopyridine
and dimethylamine as described for 4n. The nitrile was then
dimethylated as described for 4n to give 4-(2-aminopropan-2-yl)-
N,N-dimethylpyridin-2-amine. Acridone 4k was prepared as a bright
yellow solid from 12 and 4-(2-aminopropan-2-yl)-N,N-dimethylpy-
ridin-2-amine as described in the preparation of 8b. HPLC purity
95%, tr ) 4.06 min (method G); 98%, tr ) 2.01 min (method H);
LCMS (EI) m/z calcd for C24H23FN4O2 [M + H]+, 419.19; found,
1
419.23. H NMR (500 MHz, DMSO-d6) δ 1.62 (s, 6H), 3.02 (s,
6H), 6.61 (s, 1H), 6.63 (d, J ) 5.50 Hz, 1H), 7.29 (t, J ) 7.15 Hz,
1H), 7.55 (d, J ) 8.25 Hz, 1H), 7.67 (d, J ) 5.50 Hz, 1H), 7.76 (t,
J ) 6.87 Hz, 1H), 7.93 (d, J ) 10.45 Hz, 1H), 8.02 (d, J ) 5.50
Hz, 1H), 8.22 (d, J ) 8.25 Hz, 1H), 8.90 (s, 1H), 11.99 (s, 1H).
N-(2-(6-(4-Ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2-
fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (4m). To 2-chlo-
ropyridine-5-carbonitrile (100 g, 0.72 mol) in 400 mL of ethanol
was added N-ethylpiperazine (250 mL). The mixture was stirred at
80 °C for 3.5 h under nitrogen. The solvent was removed under
reduced pressure, and the resulting residue was cooled to room
temperature and diluted with 1 L of water. The aqueous mixture
was stirred overnight, and the resulting precipitate was collected
by vacuum filtration, washed with water (3×), and dried under
reduced pressure to give 6-(4-ethylpiperazin-1-yl)nicotinonitrile (13;
141 g, 91%) as an off-white solid. HPLC purity >99%, tr ) 0.580
min (method F); LCMS (EI) m/z calcd for C12H16N4 [M + H]+,
217.15; found, 217.12. 1H NMR (400 MHz, CDCl3) δ 1.13 (t, J )
7.2 Hz, 3H), 2.47 (q, J ) 14.5, 7.2 Hz, 2H), 2.52-2.54 (m, 4H),
3.69-3.71 (m, 4H), 6.59 (d, J ) 9.0 Hz, 1H), 7.60 (dd, J ) 9.0,
2.3 Hz, 1H), 8.40 (d, J ) 2.3 Hz, 1H).
2-(6-Morpholinopyridin-3-yl)propan-2-amine was prepared from
6-morpholinonicotinonitrile as described in the preparation of 4m
to give the product as a pale yellow solid. HPLC retention time )
0.203 min (condition E); LCMS (EI) m/z calcd for C12H19N3O [M
+ H]+, 222.16; found, 222+.
2-(6-Morpholinopyridin-3-yl)propan-2-amine was coupled with
12 following the procedure described in 4m to give 4n as a pale
yellow solid. HPLC purity 97.0%, tr ) 7.59 min (method A); 97.6%,
tr ) 11.65 min (method B); LCMS (EI) m/z calcd for C26H25FN4O3
1
[M + H]+, 461.20; found, 461.26. H NMR (400 MHz, DMSO-
d6) δ 1.65 (s, 6H), 3.40 (s, 4H), 3.69 (s, 4H), 6.81 (d, J ) 8.80 Hz,
1H), 7.28 (t, J ) 7.42 Hz, 1H), 7.53 (d, J ) 8.25 Hz, 1H), 7.61
(dd, J ) 8.80, 2.75 Hz, 1H), 7.66 (d, J ) 5.50 Hz, 1H), 7.76 (t, J
) 6.87 Hz, 1H), 7.91 (d, J ) 9.90 Hz, 1H), 8.20-8.23 (m, 2H),
8.80 (s, 1H), 11.94 (s, 1H).
To a flame dried flask under nitrogen was added cerium chloride
(Alfa Aesar) (68.4 g, 0.28 mol) and 450 mL of anhydrous
tetrahydrofuran. The mixture was stirred for 45 min at room
temperature and cooled to -78 °C. Methyl lithium (173 mL of a
1.6 M solution in ether, 0.28 mol) was added dropwise with stirring.
The reaction mixture was stirred for 1 h at -78 °C. 6-(4-
Ethylpiperazin-1-yl)nicotinonitrile (13; 20.0 g, 0.093 mol) in 50
mL of anhydrous tetrahydrofuran was added slowly via cannula at
-78 °C, and the mixture was stirred overnight at room temperature.
The reaction was quenched with a small volume of water (∼ 5
mL) at 0 °C, and then a 50% aqueous solution of ammonium
hydroxide was added until a precipitate formed and settled to the
bottom of the flask. The mixture was filtered through a pad of
Celite, concentrated, and purified by silica gel chromatography using
CH2Cl2/MeOH/TEA (100:2:1) to give 2-(6-(4-ethylpiperazin-1-yl)-
pyridin-3-yl)propan-2-amine (14; 16.5 g, 72%) as a pale yellow
solid. HPLC purity 86%, tr ) 0.183 min (method F); LCMS (EI)
m/z calcd for C14H24N4 [M + H]+, 249.21; found, 249.20. 1H NMR
(400 MHz, CDCl3) δ 1.13 (t, J ) 7.2 Hz, 3H), 1.47 (s, 6H), 2.47
(q, J ) 14.4, 7.2 Hz, 2H), 2.55-2.59 (m, 4H), 3.54-3.56 (m, 4H),
3.75 (m, 2H), 6.64 (d, J ) 8.8 Hz, 1H), 7.65 (dd, J ) 8.8, 2.6 Hz,
1H), 8.33 (d, J ) 2.6 Hz, 1H).
To a mixture of the F-acridone acid (7; 7.76 g, 30.2 mmol), 2-(6-
(4-ethylpiperazin-1-yl)pyridin-3-yl)propan-2-amine (14; 9.0 g, 36.2
mmol), and triethylamine (9.2 g, 90.6 mmol) in 160 mL of
anhydrous dimethylformamide was added N,N-bis[2-oxo-3-oxazo-
lidinyl]phosphorodiamidic chloride (9.2 g, 36.2 mmol). The reaction
mixture was stirred at room temperature overnight. To the mixture
was added 400 mL of a 5% aqueous solution of sodium bicarbonate,
and the mixture was stirred for 1 h. The resulting precipitate was
collected by vacuum filtration and washed with water (2×). The
wet solid was collected and crystallized from methanol to provide
pure N-[1-[6-(4-ethyl-1-piperazinyl)-3-pyridinyl]-1-methylethyl]-2-
2-Fluoro-N-(2-(5-methoxy-6-morpholinopyridin-3-yl)propan-
2-yl)-9-oxo-9,10-dihydroacridine-3-carboxamide (4o). To 2-bromo-
3-hydroxypyridine (8.20 g, 47.1 mmol) in 200 mL of dichlo-
romethane and 100 mL of methanol was added trimethylsilyl
diazomethane dropwise (2.0 M solution in hexane, 47.1 mL, 94.2
mmol). After 30 min at room temperature, an extra equivalent of
trimethylsilyl diazomethane was added, and the mixture was stirred
for 2.5 h. The solvent was removed under reduced pressure to give
a quantitative yield of 2-bromo-3-methoxypyridine as a light-brown
solid. HPLC purity 85%, tr ) 1.81 min (method F); LCMS (EI)
m/z calcd for C6H6BrNO [M + H]+, 187.97; found,187.93, 189.93.
A mixture of 2-bromo-3-methoxypyridine (8.50 g, 45.2 mmol)
and morpholine (60 mL) in a sealed tube was heated at 125 °C for
4.5 h. The reaction mixture was diluted with dichloromethane,
washed with water, and dried over anhydrous sodium sulfate.
Concentration under reduced pressure afforded 5.10 g (58%) of
4-(3-methoxypyridin-2-yl)morpholine (23) as a brownish oil. HPLC
tr ) 0.603 min (method F); LCMS (EI) m/z calcd for C10H14N2O2
[M + H]+, 195.11; found, 195.14. The compound was used without
any further purification.
A mixture of 23 (5.05 g, 26.0 mmol) and N-bromosuccinimide
(4.80 g, 27.0 mmol) in 60 mL of dimethylformamide was heated
at 85 °C for 3 h. The solvent was removed under reduced pressure,
and the residue was dissolved in dichloromethane, washed with
water, and dried over anhydrous sodium sulfate. Concentration
under reduced pressure followed by purification by flash silica gel
chromatography afforded 4.43 g (62%) of 4-(5-bromo-3-methoxy-
pyridin-2-yl)morpholine as a white solid. HPLC purity 99%, tr )
2.72 min (method F); LCMS (EI) m/z calcd for C10H13BrN2O2 [M
1
+ H]+, 273.02; found, 274.15. H NMR (500 MHz, CDCl3) δ

3740 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Watterson et al.
3.36-3.38 (m, 4H), 3.83-3.85 (m, 7H), 7.13-7.14 (m, 1 H), 7.90-
7.91 (m, 1H).
) 3.19 min (method F); LCMS (EI) m/z calcd for C6H5Br2N [M
+ H]+, 249.89; found, 249.89.
A mixture of the 2,5-dibromo-3-methylpyridine (3.3 g, 13.2
mmol) and morpholine (30 mL) in a sealed tube was heated at 140
°C for 6 h. The reaction mixture was cooled to room temperature,
diluted with water, and extracted with dichloromethane. The organic
layer was collected, washed with water, and dried over anhydrous
sodium sulfate. Concentration under reduced pressure afforded a
quantitative yield (3.67 g) of 4-(5-bromo-3-methylpyridin-2-yl)-
morpholine. HPLC tr ) 3.03 min (method F); LCMS (EI) m/z calcd
for C10H13BrN2O [M + H]+, 257.03; found, 257.05. The compound
was used without any further purification.
A mixture of 4-(5-bromo-3-methylpyridin-2-yl)morpholine (3.65
g, 14.2 mmol), tris-(dibenzylideneacetone)dipalladium(0) (0.78 g,
0.852 mmol), 1,1′-bis(diphenylphosphino)-ferrocene (0.945 g, 1.70
mmol), zinc powder (0.111 g, 1.70 mmol), and zinc cyanide (1.67
g, 14.2 mmol) was flushed with nitrogen. Dimethyl acetamide (50
mL) was added, and the resulting mixture was heated at 150 °C
overnight. HPLC indicated that the reaction was complete. The
reaction mixture was cooled to room temperature, diluted with ethyl
acetate, and washed with 2 N aqueous ammonium hydroxide,
washed with brine, and dried over anhydrous sodium sulfate.
Concentration under reduced pressure followed by purification by
flash silica gel chromatography afforded 5-methyl-6-morpholinoni-
cotinonitrile (26; 2.7 g, 94%) as an off-white solid. HPLC purity
>99%, tr ) 2.33 min (method F); LCMS (EI) m/z calcd for
C11H13N3O [M + H]+, 204.11; found, 204.09. 1H NMR (500 MHz,
CDCl3) δ 2.29 (s, 3H), 3.33-3.35 (m, 4H), 3.82-3.84 (m, 4H),
7.57 (s, 1H), 8.40 (s, 1H).
A mixture of 4-(5-bromo-3-methoxypyridin-2-yl)morpholine
(4.40 g, 16.0 mmol), tris(dibenzylideneacetone)dipalladium(0)
(0.885 g, 0.966 mmol), 1,1′-bis(diphenylphosphino)ferrocene (1.07
g, 1.93 mmol), zinc powder (0.124 g, 1.93 mmol), and zinc cyanide
(1.89 g, 16.0 mmol) was flushed with nitrogen. Dimethyl acetamide
(60 mL) was added, and the resulting mixture was heated at 150
°C overnight. HPLC indicated that the reaction was complete. The
reaction mixture was cooled to room temperature, diluted with ethyl
acetate, washed with 2 N aqueous ammonium hydroxide, washed
with brine, and dried over anhydrous sodium sulfate. Concentration
under reduced pressure followed by purification by flash silica gel
chromatography afforded 5-methoxy-6-morpholinonicotinonitrile
(24; 3.37 g, 96%) as an off-white solid. HPLC purity >99%, tr )
2.26 min (method F); LCMS (EI) m/z calcd for C11H13N3O2 [M +
H]+, 220.11; found, 220.05. 1H NMR (500 MHz, CDCl3) δ 3.65-
3.67 (m, 4H), 3.80-3.82 (m, 4H), 3.86 (s, 3H), 7.10 (s, 1H), 8.12
(s, 1H).
To a flame-dried flask under nitrogen was added cerium chloride
(18.8 g, 76.4 mmol) followed by 150 mL of anhydrous tetrahy-
drofuran. The mixture was stirred vigorously for 45 min, during
which time the cerium chloride became suspended. The suspension
was cooled to -78 °C, and methyl lithium (54.6 mL, 76.4 mmol,
1.4 M in ether) was added dropwise. After the addition was
complete, the reaction mixture was stirred for 0.5 h at -78 °C.
Compound 24 (3.35 g, 15.3 mmol) in tetrahydrofuran was added
via cannula to the -78 °C solution. The dry ice bath was removed,
and the reaction mixture was stirred at room temperature overnight.
The mixture was quenched with a few drops of a saturated aqueous
solution of ammonium chloride, and a 50% aqueous solution of
ammonium hydroxide was added dropwise until a precipitate
formed and settled to the bottom of the flask. The mixture was
filtered through Celite under reduced pressure, diluted with dichlo-
romethane, washed with water, and dried over anhydrous sodium
sulfate. Concentration under reduced pressure afforded 3.41 g (89%)
2-(5-methoxy-6-morpholinopyridin-3-yl)propan-2-amine (25) as a
yellowish-orange oil. HPLC purity 88%, tr ) 0.603 min (method
F); LCMS (EI) m/z calcd for C13H21N3O2 [M + H]+, 252.17; found,
252.20. The compound was used without any further purification.
To a flame-dried flask under nitrogen was added cerium chloride
(16.4 g, 66.4 mmol) followed by 150 mL of anhydrous tetrahy-
drofuran. The mixture was stirred vigorously for 45 min, during
which time the cerium chloride became suspended. The suspension
was cooled to -78 °C, and methyl lithium (47.4 mL, 66.4 mmol,
1.4 M in ether) was added dropwise. After the addition was
complete, the reaction mixture was stirred for 0.5 h at -78 °C.
Compound 26 (2.70 g, 13.3 mmol) in tetrahydrofuran was added
via cannula to the -78 °C solution. The dry ice bath was removed,
and the reaction mixture was stirred at room temperature overnight.
The mixture was quenched with a few drops of a saturated aqueous
solution of ammonium chloride, and a 50% aqueous solution of
ammonium hydroxide was added dropwise until a precipitate
formed and settled to the bottom of the flask. The mixture was
filtered through Celite under reduced pressure, diluted with dichlo-
romethane, washed with water, and dried over anhydrous sodium
sulfate. Concentration under reduced pressure afforded 3.10 g (99%)
of 2-(5-methyl-6-morpholinopyridin-3-yl)propan-2-amine (27) as
a yellowish-orange oil. HPLC purity 90%, tr ) 0.39 min (method
F); LCMS (EI) m/z calcd for C13H21N3O [M + H]+, 236.18; found,
236.21. The compound was used without any further purification.
To a mixture of 12 (2.67 g, 10.4 mmol), 27 (3.05 g, 13.0 mmol),
and triethylamine (5.44 mL, 39.0 mmol) was added N,N-bis[2-oxo-
3-oxazolidinyl]phosphorodiamidic chloride (3.30 g, 13.0 mmol) in
80 mL of anhydrous dimethylformamide at room temperature. The
reaction mixture was stirred at room temperature overnight. The
mixture was diluted with dichloromethane, washed with water,
washed with 1 N aqueous sodium hydroxide (2×), washed with
brine, and dried over anhydrous sodium sulfate. Concentration under
reduced pressure followed by recrystallization from methanol
afforded 3.31 g (67%) of 2-fluoro-N-(2-(5-methyl-6-morpholinopy-
ridin-3-yl)propan-2-yl)-9-oxo-9,10-dihydroacridine-3-carboxam-
ide (4p) as a bright yellow solid. HPLC purity 98.9%, tr ) 9.39
min (method C); 99.4%; tr ) 13.95 min (method D); LCMS (EI)
To a mixture of 12 (2.78 g, 10.8 mmol), 25 (3.40 g, 13.5 mmol),
and triethylamine (4.52 mL, 32.4 mmol) was added N,N-bis[2-oxo-
3-oxazolidinyl]phosphorodiamidic chloride (3.44 g, 13.5 mmol) in
90 mL of anhydrous dimethylformamide at room temperature. The
reaction mixture was stirred at room temperature overnight. The
mixture was diluted with dichloromethane, washed with water,
washed with 1 N aqueous sodium hydroxide (2×), washed with
brine (2×), and dried over anhydrous sodium sulfate. Concentration
under reduced pressure followed by recrystallization from methanol
afforded 4.42 g (83%) of 2-fluoro-N-(2-(5-methoxy-6-morpholi-
nopyridin-3-yl)propan-2-yl)-9-oxo-9,10-dihydroacridine-3-carboxa-
mide (4o) as a bright yellow solid. HPLC purity 99.4%, tr ) 9.61
min (method C); 99.3%, tr ) 14.10 min (method D); LCMS (EI)
1
m/z calcd for C27H27FN4O4 [M + H]+, 491.21; found, 491.30. H
NMR (400 MHz, DMSO-d6) δ 1.86 (s, 6H), 3.26-3.28 (m, 4H),
3.71-3.73 (m, 4H), 3.83 (s, 3H), 7.27 (d, J ) 2.0 Hz, 1H), 7.31 (t,
J ) 7.4 Hz, 1H), 7.56 (d, J ) 8.3 Hz, 1H), 7.68 (d, J ) 10.0 Hz,
1H), 7.77-7.79 (m, 1H), 7.90 (d, J ) 2.0 Hz, 1H), 7.94 (d, J ) 10
Hz, 1H), 8.23 (d, 7.4 Hz, 1H), 8.88 (s, 1H), 11.95 (s, 1H).
2-Fluoro-N-(2-(5-methyl-6-morpholinopyridin-3-yl)propan-2-
yl)-9-oxo-9,10-dihydroacridine-3-carboxamide (4p). To a het-
erogeneous suspension of 2-amino-5-bromo-3-methylpyridine (5.0
g, 26.7 mmol) in 17 mL of hydrobromic acid at 0 °C was added
bromine (4.25 mL). To this mixture was added sodium nitrite (5.25
g) in 7.5 mL of water slowly via pipet. The mixture was stirred for
15 min, and 11.3 g of sodium hydroxide in 29 mL of water was
slowly added. The resulting oil was extracted with ether, and the
organic layer was collected, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure to give 2,5-dibromo-3-
methylpyridine as a yellowish-orange solid. HPLC purity 99%, tr
1
m/z calcd for C27H27FN4O3 [M + H]+, 475.21; found, 475.54. H
NMR (500 MHz, DMSO-d6) δ 1.66 (s, 6H), 2.26 (s, 3H), 3.02-
3.04 (m, 4H), 3.72-3.73 (m, 4H), 7.28 (t, J ) 7.5 Hz, 1H), 7.53-
7.55 (m, 2H), 7.68 (d, J ) 5.5 Hz, 1H), 7.76 (t, J ) 7.5 Hz, 1H),
7.92 (d, J ) 10.4 Hz, 1H), 7.93 (s, 1H), 8.20-8.22 (m, 2H), 8.86
(s, 1H), and 11.94 (s, 1H).
IMPDH Type I and II Enzymatic Activity. The enzymatic
activity of human IMPDH type I or II was measured using a

Inhibitors of Inosine 5′-Monophosphate Dehydrogenase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3741
procedure similar to that previously reported.17 The conversion of
NAD+ to NADH was followed spectrophotometrically at 340 nm.
A reaction mixture containing 0.1 M Tris, 0.1 M KCl, 3 mM EDTA,
pH 8.0, 400 µM IMP, 2 mM DTT, and 40 nM of either IMPDH I
or II was added to the wells of flat bottom UV-transparent 96-well
plates (Costar 3635). To test inhibitors, 4m dissolved in DMSO
was diluted in the reaction to give a final DMSO concentration of
2.5%. IMPDH I and II used in these assays was purified from E.
coli expressing the gene for the human Type I or Type II enzyme,
respectively. The reaction was initiated by addition of NAD+ to a
final concentration of 400 µM. After a 2-hour 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.
(4) (a) Jayaram, H. N.; Grusch, M.; Cooney, D. A.; Krupitza, G.
Consequences of IMP dehydrogenase inhibition and its relationship
to cancer and apoptosis. Curr. Med. Chem. 1999, 6, 561-574. (b)
Carr, S. F.; Papp, E.; Wu, J. C.; Natsumeda, Y. Characterization of
human type I and type II IMP dehydrogenase. J. Biol. Chem. 1993,
268, 27286-27290.
(5) (a) Anderson, W. K.; Boehm, T. L.; Makara, G. M.; Swann, R. T.
Synthesis and modeling studies with monocyclic analogues of
mycophenolic acid. J. Med. Chem. 1996, 39, 46-55. (b) Nelson, P.
H.; Eugui, E.; Wang, C. C.; Allison, A. C. Synthesis and immuno-
suppressive activity of some side-chain variants of mycophenolic acid.
J. Med. Chem. 1990, 33, 833-838.
(6) (a) Shipkova, M.; Armstrong, V. W.; Oellerich, M.; Wieland, E.
Mycophenolate mofetil in organ transplantation: focus on metabo-
lism, safety, and tolerability. Expert Opin. Drug Metab. Toxicol. 2005,
1, 505-526. (b) Behrend, M. Adverse gastrointestinal effects of
mycophenolate mofetil: aetiology, incidence, and management. Drug
Safety 2001, 24, 645-663. (c) Bullingham, R. E. S.; Nicholls, A. J.;
Kamm, B. R. Clinical pharmacokinetics of mycophenolate mofetil.
Clin. Pharmacokinet. 1998, 34, 429-455.
Human T-Lymphoblast (CEM) Proliferation Inhibition As-
say. The human T-lymphoblast CEM cell line (ATCC) was cultured
in RPMI 1640 (Gibco) containing 10% heat inactivated FBS and
100 units/mL of penicillin and streptomycin. Cells were seeded in
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 µM with
3-fold serial dilutions. Cell cultures were maintained in a 5% CO2
humidified atmosphere for 72 h. Cell viability was measured after
a final 5 h incubation with 10% (v/v) Alamar Blue dye. The
fluorometric conversion of Alamar Blue was read on a Cytoflour
II multi-well plate reader with excitation/emission settings of 530/
590 nm, respectively. The IC50 values were calculated using a four-
parameter logistic plot.
(7) Review: Ratcliffe, A. J. Inosine 5′-monophosphate dehydrogenase
inhibitors for the treatment of autoimmune diseases. Curr. Opin. Drug
DiscoVery DeV. 2006, 9, 595-605.
(8) (a) Jain, J.; Almquist, S. J.; Shlyakhter, D.; Harding, M. W. VX-
497: A novel, selective IMPDH inhibitor and immunosuppressive
agent. J. Pharm. Sci. 2001, 90, 625-637. (b) Gummert, J. F.; Barten,
M. J.; Boeke, K.; Leon, E. J.;, Decker, C. J.; Nimmesgern, E.;
Billingham, M. E.; Morris, R. E. IMPDH inhibitor VX-497 is the
first rationally synthesized immunosuppressant to prolong allograft
survival. Transplantation 1999, 67, S62. (c) Tossing, G. Merime-
podib, Vertex IDrugs 2003, 6, 372-376. (d) Jain, J.; Almquist, S.
J.; Heiser, A. D.; Shlyakhter, D.; Leon, E.; Memmott, C.; Moody,
C. S.; Nimmesgen, E.; Decker, C. Characterization of pharmacologi-
cal efficacy of VX-148, a new, potent immunosuppressive inosine
5′-monophosphate dehydrogenase inhibitor. J. Pharmacol. Exp. Ther.
2002, 302, 1272-1277. (e) Beevers, R. E.; Buckley, G. M.; Davies,
N.; Fraser, J. L.; Galvin, F. C.; Hannah, D. R.; Haughan, A. F.;
Jenkins, K.; Mack, S. R.; Pitt, W. S.; Ratcliffe, A. J.; Richard, M.
D.; Sabin, V.; Sharpe, A.; Williams, S. C. Low molecular weight
indole fragments as IMPDH inhibitors. Bioorg. Med. Chem. Lett.
2006, 16, 2535-2538. (f) Beevers, R. E.; Buckley, G. M.; Davies,
N.; Fraser, J. L.; Galvin, F. C.; Hannah, D. R.; Haughan, A. F.;
Jenkins, K.; Mack, S. R.; Pitt, W. S.; Ratcliffe, A. J.; Richard, M.
D.; Sabin, V.; Sharpe, A.; Williams, S. C. Novel indole inhibitors of
IMPDH from fragments: synthesis and initial structure-activity
relationships. Bioorg. Med. Chem. Lett. 2006, 16, 2539-2542. (g)
Birch, H. L.; Buckley, G. M.; Davies, N.; Dyke, H. J.; Frost, E. J.;
Gilbert, P. J.; Hannah, D. R.; Haughan, A. F.; Madigan, M. J.;
Morgan, T.; Pitt, W. S.; Ratcliffe, A. J.; Ray, N. C.; Richard, M. D.;
Sharpe, A.; Taylor, A. J.; Whitworth, J. M.; Williams, S. C. Novel
7-methoxy-6-oxazol-5-yl-2,3-dihydro-1H-quinazolin-4-ones as IM-
PDH inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 5335-5339.
(h) Buckley, G. M.; Davies, N.; Dyke, H. J.; Gilbert, P. J.; Hannah,
D. R.; Haughan, A. F.; Hunt, C. A.; Pitt, W. S.; Profit, R. H.; Ray,
N. C.; Richard, M. D.; Sharpe, A.; Taylor, A. J.; Whitworth, J. M.;
Williams, S. C. Quinazolinethiones and quinazolinediones, novel
inhibitors of inosine 5′-monophosphate dehydrogenase: Synthesis
and initial structure-activity relationships. Bioorg. Med. Chem. Lett.
2005, 15, 751-754. (i) Pickett, S. D.; Sherborne, B. S.; Wilkinson,
T; Bennett, J.; Borkakoti, N.; Broadhurst, M.; Hurst, D.; Kilford, I.;
McKinnell, M.; Jones, P. S. Discovery of novel low molecular weight
inhibitors of IMPDH via virtual needle screening. Bioorg. Med. Chem.
Lett. 2003, 13, 1691-1694.
Human Peripheral Blood Mononuclear Cells Proliferation
Inhibition Assay. Human PBMCs were isolated from heparinized
whole blood by density gradient centrifugation according to ICN/
Cappel LSM specifications. PBMCs were maintained in RPMI 1640
medium containing 10% heat inactivated FBS and 100 units/mL
of penicillin and streptomycin. Directly following whole blood
isolation, 5 × 104 cells/well were plated into 96-well tissue culture
plates and stimulated with anti-CD3 mAb OKT3 at 200 ng/mL and
1 µg/mL soluble anti-CD28 antibody. Test compounds were added
in triplicate at a final starting concentration of 3.3 µM with 3-fold
serial dilutions. All culture wells included 0.5% DMSO. Cell
cultures were maintained in a 5% CO2 humidified atmosphere for
72 h followed by the addition of 1 µCi 3H-thymidine/well.
Radiolabeled thymidine [3H] (Amersham) incorporation was de-
termined after a 5 h incubation. The concentrations of compound
required to inhibit PBMC proliferation by 50% (IC50) were
calculated using a four-parameter logistic plot.
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