Quinazolinethiones and quinazolinediones, novel inhibitors of inosine monophosphate dehydrogenase: Synthesis and initial structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2004.11.015

The development of a series of novel quinazolinethiones and quinazolinediones as inhibitors of inosine monophosphate dehydrogenase (IMPDH) is described. The synthesis, in vitro inhibitory values for IMPDH II and in vitro inhibitory value for PBMC proliferation are discussed.

Full text of DOI:10.1016/j.bmcl.2004.11.015

Bioorganic & Medicinal Chemistry Letters 15 (2005) 751–754
Quinazolinethiones and quinazolinediones, novel inhibitors of
inosine monophosphate dehydrogenase: synthesis and
initial structure–activity relationships
George M. Buckley, Natasha Davies, Hazel J. Dyke, Philip J. Gilbert,
Duncan R. Hannah, Alan F. Haughan,* Caroline A. Hunt, William R. Pitt,
Rachael H. Profit, Nicholas C. Ray, Marianna D. Richard, Andrew Sharpe,
Alicia J. Taylor, Justine M. Whitworth and Sophie C. Williams
Celltech R&D, Granta Park, Great Abington, Cambridge CB1 6GS, UK
Received 16 July 2004; revised 2 November 2004; accepted 4 November 2004
Available online 23 November 2004
Abstract—The development of a series of novel quinazolinethiones and quinazolinediones as inhibitors of inosine monophosphate
dehydrogenase (IMPDH) is described. The synthesis, in vitro inhibitory values for IMPDH II and in vitro inhibitory value for
PBMC proliferation are discussed.
ꢀ 2004 Elsevier Ltd. All rights reserved.
A key enzyme involved in the de novo synthesis of guano-
sine nucleotides is inosine monophosphate dehydroge-
nase (IMPDH) which catalyses the irreversible NAD
dependent oxidation of inosine-50-monophosphate
(IMP) to xanthosine-50-monophosphate (XMP).1 Two
isoforms of the enzyme have been identified and desig-
nated type I and type II.2,3 IMPDH II activity is mark-
edly up-regulated in actively proliferating cell types
including cancers and activated peripheral blood
lymphocytes.4 Thus, inhibition of IMPDH II results in
inhibition of cell proliferation in the immune system
via inhibition of guanosine nucleotide production. This
has led to a number of companies5 identifying IMPDH
as a potential target for medical intervention in diseases
such as systemic lupus erythematosus, psoriasis and
rheumatoid arthritis as well as organ transplant
rejection.
rejection.6 Other end points in autoimmune diseases
have been, and are currently being, sought but they
may well be restricted by reported dose limiting gastro-
intestinal toxicity of this compound, which is believed to
be due to glucuronidation and excretion of MPA via bile
and is not mechanism based.7
The search for alternative IMPDH inhibitors with an
improved therapeutic window has been led by Vertex
with VX-497 and VX-1488,9 as well as BMS with
BMS-33719710 and other structural classes.5 Many of
these have been designed using knowledge of binding
interactions aided by crystal structures (Fig. 1).11
At Celltech our focus has been on the identification and
development of potent, selective inhibitors of IMPDH II
with improved pharmacological properties. Our efforts
to find optimal replacements for the urea of VX-497
led us to the cyclised quinazolinethione (1) (Fig. 2).
Cellceptꢁ (mycophenolate mofetil, MMF), which is a
prodrug of mycophenolic acid (MPA) currently has clin-
ical utility, as an IMPDH inhibitor, in organ transplant
With this initial positive result in hand we developed and
utilised parallel solution phase chemistry to scope out
the SAR around the 3-position with 60 analogues
(Scheme 1). Known amino ester (2) was readily con-
verted to isothiocyanate (3), which was reacted with a
number of primary amines in parallel to give quinazoline-
thiones (4).
*
Corresponding author. Tel.: +44 1223 896497; fax: +44 1223
896400; e-mail: alan.haughan@celltechgroup.com
Present address: Argenta Discovery Ltd, 8/9 Spire Green Centre, Flex
Meadow, Harlow, CM19 5TR, UK.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.015

752
G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 15 (2005) 751–754
OH
Me
Me
MPA, R=H
O
OR
MMF, R=
N
O
O
O
OMe
H
N
O
O
N
O
O
O
VX-497
MeO
N
H
N
H
H
Me
N
O
CN
NC
O
O
VX-148
MeO
N
N
H
H
O
N
N
O
O
Me
N
N
BMS-337197
Figure 3. Quinazolinethione 4a docked into IMPDH.
O
MeO
N
H
Figure 1. Structures of MPA, MMF, VX-497, VX-148 and BMS-
337197.
From the initial set of compounds synthesised (Table 1)
it became obvious that branching close to the bicyclic
ring of our quinazolinethiones as exemplified by com-
pounds 4e, 4g and 4h gave us a fall in activity. This
was consistent with docking of these structures where
clashes with protein could be observed. Some of our
more potent compounds (e.g., 4c) pleasingly gave sub-
micromolar IC50s in our peripheral blood monocyte
(PBMC) proliferation assay.13
O
O
N
H
S
N
MeO
N
H
1
IMPDH II (IC50) = 30nM
Figure 2. Quinazolinethione 1.
Table 1. SAR of quinazolinethiones
O
O
N
O
R
S
O
C(S)Cl2
N
N
N
CO2Me
NH2
CO2Me
NCS
MeO
N
H
Et3N
CH2Cl2
MeO
MeO
4
2
3
Compd
R
IMPDH II,14 nM
PBMC,13 lM
RNH2
THF
heat
4a
Me
17
1.1
4b
4c
91
13
1.0
N
N
O
O
N
N
R
N
0.78
MeO
N
H
S
4d
4e
22
0.98
n.t.
N
4
1710
N
N
Scheme 1. Solution phase parallel synthesis of quinazolinethiones.
4f
100
4.1
N
Selection of groups for inclusion in this set was driven
by CADD as we not only desired diversity within the
set synthesised but also wanted to incorporate groups
likely to yield favourable physicochemical properties.
We used docked structures to guide our search for addi-
tional binding affinity and to trim out groups, which
would have putative negative interactions (Fig. 3).12
4g
4h
4i
821
n.t.
n.t.
2.76
i-Pr
27% @ 1lM
O
71
n.t. = not tested.

G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 15 (2005) 751–754
753
O
O
O
O
N
O
N
N
N
R
S
CO2H
NH2
N
N
OH
H
MeO
N
H
MeO
MeO
4
9
O
O
10
O
O
N
O
O
N
R
O
R
PyBOP
O
RNH2
O
N
N
MeO
MeO
N
H
6
O
O
5
N
DMF
N
R
N
NHR
NH
Figure 4. In vitro microsomal turnover of quinazolinethiones by rat
liver microsomes at a concentration of 1mg/mL.
125oC
MeO
MeO
N
H
O
O
O
6
11
Scheme 3. Solid phase synthesis of quinazolinediones.
However, many of the quinazolinethiones, with differing
3-substituents, were found to be rapidly turned over in
in vitro microsomes assays. Subsequent metabolite identi-
fication studies (Fig. 4) indicated that two major
metabolites were formed in each case, irrespective of
3-substituent. One route of metabolism resulted in the
formation of the corresponding quinazolinediones
(conversion of C@S to C@O). Once formed, the diones
(6), which also inhibited IMPDH II, appeared stable
and did not undergo further metabolism. The other
route of metabolism led to compounds in which the S
was replaced by H and these quinazolinones (5) were
inactive against IMPDH II. The identity of both of these
metabolites were confirmed by comparison with authe-
ntic samples. As a consequence of these results no
further thiones were produced and instead optimisation
of the diones was undertaken.
logues in parallel. The known aminoacid (9) was loaded
onto Merrifield resin as the carbamate.15 Amines were
coupled with PyBOP, then cyclisation and release of
product was achieved at 125ꢂC with DMF as solvent
to give diones (6) without the need for any purification
(Scheme 3).
The SAR of the quinazolinediones generally mirrored
that of the quinazolinethiones as one would envisage
from docking studies, but were intrinsically less potent
(Table 2). We postulate that this lower potency is due
to a weaker key H-bond interaction between the N–H
and Asp274 (Fig. 3).
The synthetic tractability of the quinazolinediones was
initially not as good as had been found with the quinazo-
linethiones. The corresponding isocyanate (7) was not
as stable as its isothiocyanate analogue (3) had been
and so was not isolable. This required us to prepare it
each time in situ. The intermediate ureas (8) also had
to be purified and so this lowered the throughput of tar-
get molecules (Scheme 2).
In conclusion the quinazolinediones seem to hit a pla-
teau of activity beyond which it was difficult to drive
activities lower. They routinely gave poor absorbance
in vivo and also in in vitro Caco-2 diffusion experiments
(data not shown).
We intend to publish further modifications of this tem-
plate, which resulted in compounds with improved PK
profiles and cellular potencies in due course.
We therefore developed and utilised a solid phase Ôcatch
and releaseÕ route, which allowed us to generate 80 ana-
Table 2. SAR of quinazolinediones
O
O
N
R
O
O
N
O
N
N
CO2Me
NH2
CO2Me
NCO
MeO
N
H
MeO
MeO
6
2
IMPDH II,14 nM
PBMC,13 lM
7
Compd
6a
R
Me
104
188
4
RNH2
THF
N
6b
1.2
H
O
O
O
N
N
6c
47
Inactive
N
CO2Me
DBN
R
N
N
O
DME
heat
MeO
N
H
MeO
N
H
O
6d
6e
86
2.4
N
O
N R
H
N
N
8
6
297
9.13
Scheme 2. Solution phase synthesis of quinazolinediones.

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G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 15 (2005) 751–754
12. Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245,
43–53.
References and notes
13. Inhibitors are dissolved in 100% DMSO at a concentration
of 20mM and an initial dilution of 1:71 into RPMI 1640
medium containing penicillin/streptomycin and glutamine
5% human serum. 1:10 serial dilutions are then prepared,
these inhibitors are added at T = 0. PBMCÕs are plated out
at a density of 2 · 106/mL in RPMI medium containing
5% human serum in the presence and absence of inhibitor
and 100lM guanosine. The plates are incubated at 37ꢂC/
5% CO2 for 48h. After 48h 0.5lCi of 3H thymidine is
added per well and the plates incubated for a further 18h.
The final assay volume is 150lL. The contents of the plate
are then transferred to a filter plate and the cells washed
with saline using the Millipore microtitre plate filtration
system. The plates are dried and 100lL of microscintill-
ation Ô0Õ fluid is added to each well and the plate counted
in the TopCount.
14. IMPDH activity was determined by monitoring the
production of the fluorescent product, NADH. Assays
were performed in a final volume of 200lL containing
IMPDH (0.4lg), NAD (100lM), IMP (100lM), 1%
DMSO, 30mM KCl and 100mM Tris/HCl, pH7.5.
Fluorescence at excitation 340nm/emmission 465nm
was read continuously at 25ꢂC for 30min. From this
data, initial rates (i.e., change in fluorescence intensity
per minute) were calculated. To determine the IC50
values, test compounds were prepared at an initial
concentration of 1.0mM in 100% DMSO, then diluted
in assay buffer to 0.2mM. Further dilutions were made in
assay buffer containing 20% DMSO, prior to diluting 20-
fold into the assay, to allow testing across the range 1nM
to 10lM.
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