Identification of novel isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual screening hits

Article abstract of DOI:10.1016/j.bmc.2012.03.019

In recent years, xanthine oxidase has emerged as an important target not only for gout but also for cardiovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical trials with uricosurics have demonstrated that

Full text of DOI:10.1016/j.bmc.2012.03.019

Bioorganic & Medicinal Chemistry 20 (2012) 2930–2939
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
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Identification of novel isocytosine derivatives as xanthine oxidase
inhibitors from a set of virtual screening hits
Chandrika B-Rao a, , Asha Kulkarni-Almeida b, Kamlesh V. Katkar b, Smriti Khanna a, Usha Ghosh c,
⇑
Ashish Keche c, Pranay Shah c, Ankita Srivastava b, Vaidehi Korde a, Kumar V.S. Nemmani d,
Nitin J. Deshmukh d, Amol Dixit d, Manoja K. Brahma d, Umakant Bahirat d, Lalit Doshi d, Rajiv Sharma c,
H. Sivaramakrishnan c
a Discovery Informatics, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
b Department of High Throughput Screening and Biotechnology, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
c Department of Medicinal Chemistry, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
d Department of Pharmacology, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In recent years, xanthine oxidase has emerged as an important target not only for gout but also for car-
diovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical
trials with uricosurics have demonstrated that enhanced excretion of uric acid is, by itself, not adequate
to treat hyperuricemia; simultaneous inhibition of production of uric acid by inhibition of xanthine oxi-
dase is also important. Virtual screening of in-house synthetic library followed by in vitro and in vivo
testing led to the identification of a novel scaffold for xanthine oxidase inhibition. In vitro activity results
corroborated the results from molecular docking studies of the virtual screening hits. The isocytosine
scaffold maintains key hydrogen bonding and pi-stacking interactions in the deep end of the xanthine-
binding pocket, which anchors it in an appropriate pose to inhibit binding of xanthine and shows promise
for further lead optimization using structure-based drug design approach.
Received 6 January 2012
Revised 6 March 2012
Accepted 6 March 2012
Available online 14 March 2012
Keywords:
Xanthine oxidase inhibitors
Isocytosine
Virtual screening
Docking
Ó 2012 Elsevier Ltd. All rights reserved.
Hit identification
1. Introduction
tion stimulants (e.g., probenecid).3 Some of the small molecules
currently in advanced clinical trials are topiroxostat (FYX-051, an
Xanthine oxidase (XO) is an important enzyme that catalyzes
the transformation of physiological substrates such as hypoxan-
thine to xanthine and xanthine to uric acid which is excreted by
kidneys. Excessive production and/or inadequate excretion of uric
acid results in hyperuricemia. Hyperuricemia is associated with
conditions like gout, cardiovascular mortality and metabolic syn-
drome including hyperinsulinemia and hypertriglyceridemia. Alle-
viating hyperuricemia, therefore, has therapeutic significance, and
XO is a key target towards this end.1
Gout is a state of inflammation caused by deposition of mono-
sodium urate crystals in joints, and is preceded by hyperuricemia.2
Therapies include symptomatic treatments (e.g., nonsteroidal anti-
inflammatory drugs, corticosteroids, IL1-traps and colchicine), and
mechanism-based treatments like urate production inhibitors (XO
inhibitors or XOIs, e.g., allopurinol and febuxostat) and urate excre-
XOI) and RDEA-594 (a URAT-1 transport inhibitor, which facilitates
excretion of uric acid).4,5 Pegloticase, a pegylated porcine urate oxi-
dase, which mops up the excess uric acid and is effective in rever-
sal of chronic tophaceous gout, has recently been approved by US
FDA for advanced cases of treatment-resistant gout.4,6
It is commonly believed that 90% of the cases of hyperuricemia
are due to under-excretion rather than overproduction of uric
acid.7 However, recent clinical trials of RDEA-594 demonstrated
that stimulation of uric acid excretion was successful in reducing
uric acid levels in only about 67% of the patients, while combina-
tion therapy with an XOI (allopurinol or febuxostat) could give
up to 100% response in patients.8 Thus, XOIs continue to be impor-
tant for treatment of hyperuricemia.
All the currently available treatments come with their own sets
of adverse effects. A common side-effect of all cause-based treat-
ments (XOIs and uric acid excretion stimulants) seems to be an in-
crease in the occurrence of gout flares in the early stages of
treatment, which usually subside as the uric acid levels in the body
decrease. Although allopurinol has been widely used all over the
world for over 50 years, in about 5% of the cases, it is associated
with hypersensitivity reactions which have sometimes even been
Abbreviations: XO, xanthine oxidase; XOI, xanthine oxidase inhibitor; po, per
oral; ip, intraperitoneal; Mo-Pt, molybdopterin; PASS, Prediction of Activity Spectra
of Substances; AUC, area under curve; XDH, xanthine dehydrogenase.
⇑
Corresponding author. Tel.: +91 22 30818714; fax: +91 22 30818036.
E-mail address: chandrika.rao@piramal.com (Chandrika B-Rao).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.019

Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
2931
fatal. Moreover, it is contraindicated in patients with renal impair-
ment as it is metabolized by kidney and excreted by renal route.9
Febuxostat is metabolized in the liver and may avoid the renal side
effects shown by allopurinol, but a somewhat higher incidence of
hepatotoxicity was observed in clinical trials.10 With pegloticase,
the main concerns identified are related to infusion reactions, car-
diovascular events, allergic reactions and immunogenicity6 and
applicability to a limited patient population with severe topha-
cious gout. Hence the search for better drugs having fewer adverse
effects continues.
ships of over 250,000 compounds. Virtual screening by PASS was
resorted to in order to eliminate, at the outset, compounds that
lack the features of known XOIs. Thirty-two compounds with prob-
ability of activity, Pa, greater than 0.3, were selected after in silico
screening of 2648 compounds from in-house synthetic library
using PASS, as detailed in Section 5. Twenty-one of these were also
predicted for Gout Treatment with net probability of activity over
inactivity, Pa–Pi, greater than 0.3, which adds to the evidence in fa-
vor of their being active.
Our work was focused on finding a novel class of XOIs which may
avoid some of the adverse effects of drugs currently in the market. It
is known that many natural product derivatives like flavonoids,11,12
folates13 and lithospermic acid14 demonstrate xanthine oxidase
inhibitory activity; however, none of these is potent enough to show
clinical efficacy. Among the many small molecule scaffolds reported
by various researchers15 to inhibit XO are triazoles,16,17 pyra-
zoles,18,19 isoxazoles,20 thiadiazolopyrimidinones,21 chalcones,22
pteridines,23 purine derivatives,24 salicylic acid derivatives,25 curcu-
min analogs,26 oxopropylamides27 and dihydropyrazoles.28
Although allopurinol and febuxostat are both XOIs which bind to
the xanthine-binding site of XO, their exact molecular mechanisms
of action differ. While allopurinol acts as a substrate that is metab-
olized to oxypurinol by the molybdopterin (Mo-Pt) unit,29 which
further inhibits the binding of xanthine by co-ordinating with Mo-
Pt, febuxostat binds tightly in the active site and blocks the binding
of xanthine, without interacting with Mo-Pt.30 Among the other
known XOIs, FYX-05131 interacts with Mo-Pt while salicylic acid,32
lumazine,33 piraxostat19 and BOF-427234 do not (Fig. 1).
2.2. Molecular docking studies
Twenty-four compounds (Table 1) predicted by PASS were phys-
ically available in the library. These virtual hits were docked to xan-
thine-binding site of xanthine dehydrogenase (XDH) co-crystallized
with piraxostat (PDB code 1VDV)19 using Glide XP docking protocol
available in Schrodinger Suite of programs.36 Glide docking scores
were used to assess the docking results. Of the 24 docked com-
pounds, 3 scored better than allopurinol (compounds 1, 2, 4) and
3 scored within 2 kcal/mol (compounds 5, 6, 7) less than allopurinol
(Table 1). Based on the dock scores, only the best docked com-
pounds were expected to show binding affinity in the in vitro assay,
but all the virtual screening hits were submitted for in vitro screen-
ing as a cross-check of PASS and docking predictions.
The docked pose of compound 1 in enol form (grey) shows a good
geometric fit and interactions similar to those of piraxostat with the
protein (Fig. 2A). Differences were seen in the docked poses of 1, 2, 3,
4 on the one hand and 5, 6, 7 on the other (Fig. 2). Key H-bonds, pi-
stacking interactions (face-to-face with Phe914 and face-to-edge
with Phe1009) and other hydrophobic interactions found for
piraxostat are maintained by 1, 2, 3 and 4 (Fig. 2A and B). Com-
pounds 5, 6 and 7 do not have H-bonds with Thr1010 and Arg880
and either lack or have weaker pi-stacking interactions with
Phe1009 and Phe914. Hydrophobic interactions at the mouth of
the pocket with Val1011, Leu1014, Leu648, Phe649 and Phe1013
seem to be responsible for their high dock scores (Fig. 2C).
Using computational methods, we have identified a novel scaf-
fold containing isocytosine ring as an inhibitor of XO, which has
features common to both allopurinol and febuxostat. Although
the substituted pyrimidine ring shows similar non-bonded interac-
tions as purine substrates (xanthine), the mechanism of action is
akin to febuxostat as our scaffold is a pure inhibitor and not a sub-
strate-inhibitor.
2.3. In vitro xanthine oxidase inhibition
2. Results
Xanthine oxidase inhibition assay was carried out on a high-
throughput system after standardization as described in the Section
5. XO was added to the test compounds or vehicle control and reaction
was initiated by addition of xanthine. The UV absorbance at 310 nm,
indicating the formation of uric acid was measured at 30 min at ambi-
ent temperature. The IC50 values were calculated for test compounds.
Allopurinol was used as positive control. Three compounds, 1, 2 and 3
exhibited xanthine oxidase inhibition in enzymatic assay. Compound
2.1. Virtual screening using Prediction of Activity Spectra of
Substances (PASS)
The PASS35 software gives prediction of potential biological
activities of a compound based on a comparison of its chemical
structure with a database of available structure–activity relation-
HOOC
HOOC
3 showed 98% inhibition at 100 lM followed by compound 2 with
OH
N
S
79% and compound 1 with 76% inhibition (Table 1). Compounds 4, 5,
6 and 7 were inactive. In view of its good dock score, pose and interac-
tions with protein, it was decided to test a fresh sample of 4 although it
was found to be inactive.
N
N
N
N
N
N
H
O
O
CN
CN
Febuxostat
Launched
2.4. Synthesis of compounds 1, 2, 3, 4 and confirmation of
in vitro activity
Piraxostat
Allopurinol
Launched
Discontinued
O-Na+
N
N
Since the tested compounds were taken from a chemical library,
it was necessary to synthesize them and confirm their in vitro
activities. The synthesis of in vitro hits (compounds 1, 2 and 3)
and inactive compound 4 was carried out as per Scheme 1.
The synthesized compounds were tested for xanthine oxidase
inhibition using the same enzymatic assay protocol used for the li-
brary compounds. All the three active compounds showed good
IC50 values (Fig. 3) on re-testing. Compound 3, with an IC50 value
N
N
N
N
H
N
N
N
CN
O
S
FYX-051
Phase II
BOF-4272
Phase I
O
of 1.4
l
M, was better than allopurinol (5.7
lM) followed by 1
Figure 1. Structures of some important xanthine oxidase inhibitors of clinical
significance.
(9.4 M) and 2 (30.2 l
l
M). Although 3 was the most active com-

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Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
Table 1
Compounds screened using PASS, docked and tested in in vitro xanthine oxidase inhibition assay
Compound ID
XOIa
Pab
Gout treatment
Dock scores
kcal/mol
In vitro screening
% inhibition (100 lM)
Pa–Pic
0.293
Pa
Pa–Pi
HO
N
O
1
0.314
0.307
0.401
0.313
0.386
À12.6
76.25
79.63
N
H2N
O
HO
N
2
0.284
0.443
À12.99
O
O
N
H2N
HS
N
O
3
4
0.427
0.318
0.42
NPd
NP
À7.61
98.3
N
H2N
O
NH
O
HN
O
0.298
0.457
0.407
À11.06
0
O
O
5
0.303
0.278
0.522
0.497
À9.58
0
N
N
N
O
N+ O-
-O
O
6
7
0.304
0.381
0.28
0.451
0.739
0.398
0.734
À8.94
À8.82
0
0
N
N
O
O-
0.371
O
O
O
O
O
8
O
0.689
0.686
0.920
0.918
À8.32
0
NH
N
O
O-
N+
O
-O
9
0.335
0.311
0.318
0.289
0.595
0.456
0.583
0.406
À7.57
À7.39
0
0
N
-O
O
NH2
N+
10
O
N
11
12
0.367
0.316
0.355
0.295
0.497
NP
0.464
NP
À7.36
À7.25
0
0
NH
O
N
O
N
N
Cl
O
-O N+
O
N+
O-
13
0.299
0.273
NP
NP
À7.25
0
O
O

Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
2933
Table 1 (continued)
Compound ID
XOIa
Pab
Gout treatment
Dock scores
kcal/mol
In vitro screening
% inhibition (100 lM)
Pa–Pic
0.289
Pa
Pa–Pi
Cl
O
O
O
14
0.311
NP
NP
À7.15
0
N
O
O
O
NH
-O
15
16
0.333
0.314
0.316
0.293
0.48
0.441
0.516
À6.97
À6.85
0
0
N+
O
O
O
N+
N
0.538
-O
O
N
O
O
N
17
18
0.3
0.275
0.353
NP
NP
À6.78
À6.74
0
0
O
NH
H2N
O
N
NH
0.365
0.629
0.621
OH
S
N
N
19
0.463
0.457
0.727
0.722
À6.62
0
N
N
O
Cl
Cl
20
21
0.326
0.352
0.307
0.338
0.441
0.382
À6.48
À5.81
0
0
O
N
O
S
H
N
N
N
H
Cl
O
O
S
O
O
NP
NP
HN
H2N
O
Cl
-O
N+
O
N
22
23
0.303
0.318
0.279
0.298
NP
NP
À5.75
À5.14
0
0
O
O
O
NH
0.532
0.509
O
N
NH2
HN
O
O
O
24
0.376
0.365
NP
NP
À4.46
0
HN
O
O
Allopurinole
Febuxostat
À10.58
À14.97
100
Not tested
a
XOI: xanthine oxidase inhibition.
Pa: probability of being active.
Pi: probability of being inactive.
NP: not predicted.
b
c
d
e
Tested at 10 lM concentration.

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Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
rats. Test compounds were administered 1 h later and percent
reduction in serum uric acid level was computed for treated animals
with respect to vehicle controls as described in Section 5. All 4 treat-
ment groups (compounds 1, 2, allopurinol and febuxostat) had low-
er serum uric acid levels than the vehicle control group (Fig. 4A).
However, the percent reductions in AUC shown by compounds 1
at a dose of 100 mg/kg po and 2 at a dose of 50 mg/kg. ip (84% and
57% respectively, Fig. 4B) were less than those shown by allopurinol
and febuxostat (184% and 209% respectively at 10 mg/kg po). Uric
acid levels in allopurinol and febuxostat group were below basal
levels, hence percent reduction was more than 100 for these com-
pounds. These results suggested that potently active compounds
could be derived by chemical modifications of compound 1 for
improving enzymatic activity.
2.6. Synthesis, in vitro results and docking of compound 1
analogs
Compounds 1 and 2, which exhibited some activity in vivo, share
the same scaffold. A few analogs of 1, varying at R1 and R2 positions
(Table 2), were synthesized (Scheme 1). Methoxyphenyl pyrimidine
substructure search was done on Pubchem and a few compounds
(1e, 1f, 1g) with different substitutions at R1 and R2 on pyrimidine
were purchased from commercial vendors. All these compounds
failed to show in vitro enzymatic activity (Table 2). These com-
pounds were docked to XO to find their binding modes. Most of
the compounds attained very poor dock scores and did not maintain
the docked pose of 1. Removal of –OH group at R1 or its substitution
with a polar group such as –NH2 resulted in tremendous decrease in
dock scores due to loss of H-bond interactions with either Arg880 or
Thr1010 or both. Hydrophobic groups (–CF3, –Cl) or bulky substitu-
ents (–NHSO2CH3, –OCH3, –CN) at R1 position could not be accom-
modated well in the active site and the compounds adopted a
flipped pose after docking. Similar flipped poses were obtained
when –Cl, or –NHNH2 groups were attempted at R2 position. Struc-
ture activity relationship based on this small set of molecules clearly
shows that steric bulk is not tolerated at R1 and R2 positions be-
cause very little space is available at the deep end of the active site.
3. Discussion
In vitro screening of 24 virtual screening hits gave 3 active com-
pounds, 1, 2, and 3, with IC50 similar to or better than that of allo-
purinol. While compounds 1 and 2 were not predicted to be the
most probable inhibitors of XO in the set of compounds assessed
by PASS, they had the best dock scores. However, compound 3,
which was ranked 3rd by PASS, had a poor dock score and best
in vitro activity. Its high in vitro activity could be an artifact caused
by aggregation through the –SH group present in the compound.39
Furthermore, the reactive –SH group can interact non-specifically
with SH-containing compounds and proteins resulting in adverse
reactions in vivo.38 On the whole, while PASS helped in quickly
identifying a set of potential actives, results of docking were much
more reliable. Poorly docked compounds, with either low dock
scores or poor docked poses, were found to be inactive.
Compound 4, which attained a good dock score (À11.06 kcal/
mol) and pose but turned out to be inactive in the enzymatic assay,
was perhaps exceptional. It bears –OH groups at both R1 and R2
positions that can exist in tautomeric keto or enol forms. In docked
pose, R2 is directed towards the oxygen atoms of Mo-Pt, and in ro-
tated pose, R1 would face Mo-Pt. This may result in electrostatic
repulsions which would prevent the molecule from adopting a
pose which has pyrimidine at the deep end of the active site, but
the docking program did not reflect it. It is observed that the nat-
ural substrate, hypoxanthine, flips after getting hydroxylated to
Figure 2. Docking of virtual hits in the active site of XDH. H-Bonds are depicted by
blue dashed lines. Residues are colored according to the type of interaction with
ligand—dark blue for pi-stack, magenta for other hydrophobic and cyan for polar
interactions. (A) Docked pose of compound 1 (grey) and co-crystallized ligand
piraxostat (lime green) show similar interactions with protein. (B) Poses of virtual
hits, 2 (yellow), 3 (spring green) and 4 (brown) also show key interactions. (C) Poses
of some PASS hits with good dock scores (5 in grey, 6 in spring green and 7 in
yellow) but lacking interactions at the deep end of the pocket. These figures have
been generated in Pymol.37
pound, it was dropped due to potential toxicity issues.38 Com-
pounds 1 and 2 were taken forward for in vivo tests. Compound
4, which was earlier inactive in the library screen, was again found
to be inactive despite good interactions and dock score.
2.5. Effect of compounds 1 and 2 on serum uric acid in
potassium oxonate-induced hyperuricemic rats
Hyperuricemia was induced by administering potassium oxo-
nate at a dose of 250 mg/kg intraperitoneally to overnight fasted

Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
2935
H2N
NH
OH
N
SH
N
.HCl
Lawesson's
Reagent
O
O
NH2
N
N
EtO
Base
H2N
H2N
O
O
O
25
R
R
R
1 : R=H
3 : R=OMe
2
: R=OMe
H2N
OMe
NH2
O
N
POCl3
OH
N
Cl
H2N
N
N
N
NaOMe
O
HO
H2N
N
R
O
O
1d : R=H
4 : R=H
R
R
26
MeSO2NH2
NHSO2Me
POCl3
N
aq. NH3 / EtOH
H2N
N
Cl
N
O
NH2
R
1c : R=H
N
aq. NH3 / EtOH
Cl
N
H2N
N
O
O
R
1a : R=H
R
1b : R=H
Scheme 1. Synthetic scheme for compounds 1, 2, 3, 4 (Table 1) and analogs of 1 (Table 2).
Figure 3. Dose–response curves for in vitro hits. Synthesized compounds 1, 2 and 3 show response comparable to that of allopurinol.
xanthine and presents a different carbon atom to Mo-Pt. When this
carbon also gets hydroxylated to give uric acid, the compound flips
in the active site such that none of the 3 –OH groups faces Mo-Pt,
and it is then ejected from the active site. The drug, allopurinol,
also flips on getting hydroxylated to oxypurinol, such that both
the –OH groups stay away from Mo-Pt.29 It is also reported in

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Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
Figure 4. Antihyperuricemic effect of compounds 1, 2, allopurinol and febuxostat in hyperuricemic rat model. (A) Time course of serum uric acid levels for 4 h after
treatment. (B) AUC of serum uric acid levels. Values in parenthesis are percent decrease in serum uric acid levels with respect to vehicle.
Table 2
the literature that when dihydroxy substitution was made on pur-
Analogs of compound 1, synthesized or purchased and tested for in vitro activity
ine derivatives known to be XOIs, it resulted in an inactive com-
R1
pound.24 All these examples support our view that the dihydroxy
N
compound cannot bind stably in the pose obtained by docking,
and this explains why the high dock score did not translate into
in vitro activity.
R2
N
O
Further SAR at R1 and R2 positions and subsequent docking
clearly show that hydrophobic and bulky substituents were not
tolerated. On the whole, docking results were more reliable indica-
tors of activity than PASS predictions. –OH group at R1 and –NH2 at
R2 seem to be the most appropriate choices of functional groups
for this scaffold. Further improvement in the enzymatic activity
may be achieved through structure-based drug design at other
sites on the ligand, as outlined in section 3.1.
In the in vivo potassium oxonate-induced hyperuricemic rat
model, compounds 1 and 2 showed reduction in uric acid levels
but not to the same extent as the standard compounds, allopurinol
and febuxostat. Compounds 1 and 2 were also the best docked
molecules with dock scores of À12.6 and À12.99 kcal/mol, better
than allopurinol (À10.58 kcal/mol) but not as good as febuxostat
(À14.97 kcal/mol). These results suggest that more potent enzy-
matic activity and better oral exposure may provide improved
in vivo efficacy.
Compound
ID
R1
R2
Dock scores In vitro
(kcal/mol)
Source
IC50 (lM)
4
1a
1b
1c
1d
1e
1f
OH
Cl
NH2
OH
Cl
NH2
À11.06
À6.46
À7.87
À6.71
À5.77
À6.96
À5.93
À5.21
>100
>100
>100
>100
>100
>100
>100
>100
Synthesized
Synthesized
Synthesized
Synthesized
Synthesized
Purchased
NHSO2CH3 NH2
OCH3
H
CN
CF3
NH2
NH2
NHNH2
NH2
Purchased
Purchased
1g
which seems to play the role of anchoring the molecule in appro-
priate pose in the active site. None of the other substitutions at-
tempted at R1 and R2 positions, be it hydrophobic or polar,
showed these H-bonds, nor did they have any in vitro activity.
The limited SAR at R1 and R2 positions indicates that –OH at R1 po-
sition and –NH2 at R2 position are important for activity. One
nitrogen of the pyrimidine ring H-bonds to Glu802, similar to pyr-
azole ring nitrogen in piraxostat.
The mechanism of metabolism of substrate by XO requires an
electrophilic carbon next to a ring nitrogen of the substrate to be
positioned adjacent to Mo-Pt, with nitrogen towards Glu1261.
Glu1261 acts as a general base and abstracts a proton from Mo-Pt
hydroxyl group. The ionized Mo-Pt facilitates nucleophilic attack
on the electrophilic carbon center. This type of motif is seen in the
substrate inhibitors, allopurinol and FYX-051. Febuxostat and
piraxostat do not possess this motif and do not get metabolized
by Mo-Pt. Our hit has a nitrogen in the desired position, but the car-
bon is substituted with –NH2, and is not available for attack by Mo-
Pt. Hence we say that compounds 1 and 2 are pure inhibitors and not
substrate inhibitors.
3.1. Some directions from structure-based design approach
The co-crystallized ligand, piraxostat, which is a low nanomolar
inhibitor of XO (IC50 = 5nM), shows several interactions with the
active site residues of the protein (Fig. 2A). The carboxyl group is
involved in electrostatic interactions with guanidinium group of
Arg880 and H-bonds to Thr1010 as well. The pyrazole ring nitrogen
is involved in H-bond interaction with Glu802. Asn768 forms an-
other crucial H-bond with the cyano group of the ligand. Besides
these polar interactions, a number of hydrophobic interactions
are observed as well. The pyrazole ring is pi-stacked between
Phe914 and Phe1009. The phenyl ring has hydrophobic interac-
tions with Leu873, Val1011 and Leu1014. The alkoxy side chain ex-
tends towards the solvent accessible region and is engaged in
hydrophobic interactions with various residues at the entrance of
the pocket such as Leu648, Phe649 and Phe1013.19
Similar interactions were observed by docking the isocytosine
series of compounds. Highly polar groups such as –OH (compounds
1, 2) and weakly polar ones such as –SH (compound 3) at R1 posi-
tion correspond to carboxylate of piraxostat and retain H-bonds
with Arg880 and Thr1010. The –NH2 at R2 H-bonds to Glu802,
The terminal methoxy group exhibits weak hydrophobic inter-
actions. Longer aliphatic chains and more hydrophobic groups
could enhance these interactions and lead to further improvement
in activity. Suitable groups may also be tried at meta position on

Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
2937
phenyl ring to obtain an additional H-bond with Asn768. Scaffold
modifications in place of pyrimidine ring could be attempted as
the nitrogen atom next to –OH group is not directly involved in
any obvious interactions except possibly through water molecules,
if present. Successful implementation of these modifications will
be described in follow-on publications on this scaffold (in
preparation).
by Protein Preparation Wizard using OPLS-2005 force field. Active
site for docking was defined as grid box of dimensions
a
25 Â 25 Â 25 Å3 around the centroid of the ligand assuming that
the ligands to be docked are of similar size as the co-crystallized
ligand. Docking of molecules was done using Glide XP module with
Epik penalties for different ionizations and tautomeric states. Ten
docked poses per ligand were generated and analyzed for interpre-
tation of final results.
4. Conclusions
5.3. Chemistry
The isocytosine scaffold has been identified from a virtual
screening exercise followed by screening in uric acid production
inhibition assay as a novel scaffold for xanthine oxidase inhibition.
Two molecules of the scaffold exhibited in vitro xanthine oxidase
inhibitory activity comparable to the standard compound allopuri-
nol. They were tested in in vivo hyperuricemic rat model and
showed reduction in uric acid levels as compared to vehicle control.
Molecular docking studies of these compounds provided important
insights into their binding mode and opened up the prospect of
using structure-based design for improving in vitro and in vivo po-
tency of the compounds.
Compounds listed in Tables 1 and 2 are either commercially
available (Interbioscreen, Maybridge) or were synthesized as de-
scribed in Scheme 1. All chemicals and solvents used were of re-
agent grade and were purified and dried by standard methods
before use. All air-sensitive reactions were run under nitrogen
atmosphere. All the reactions were monitored by thin layer chro-
matography (TLC) on pre-coated silica gel G plates at 254 nm un-
der UV lamp using ethyl acetate/petroleum ether as eluent. Flash
chromatography separations were obtained on silica gel (100–
200 mesh). 1H NMR spectra were recorded on Bruker Advance
spectrometer (300 MHz) using tetramethylsilane as internal stan-
dard; J values are in Hertz. Chemical shifts are reported in ppm
(d) relative to the solvent peak. Mass spectra were recorded on
either GCMS (focus GC with TSQ II mass analyzer and thermoelec-
tron) with auto sampler/direct injection (EI/CI) or LCMS (APCI/ESI;
Bruker daltanoics Micro TOFQ). All chromatographic purifications
were done on silica gel (100–200 mesh). Melting points were mea-
sured on a MR-VIS visual melting range apparatus.
The syntheses of hits (Table 1; compounds 1, 2 and 3) and ana-
logs (Table 2; 4, 1a, 1b, 1c and 1d) were carried out as per Scheme
1 and experimental procedure below. 2-Amino-6-aryl-substituted-
pyrimidin-4-ol (1) was prepared from corresponding ethyl-3-aryl-
substituted-3-oxopropanoate (25) and guanidine and subse-
quently converted to thiol derivative (3) by Lawesson’s reagent
and to 4-chloro-6-aryl-substituted-pyrimidin-2-amine (26) by
treatment with POCl3. Compound 26 was later converted to its
amine (1b), methanesulfonamide (1c) and methoxy (1d) deriva-
tives by treatment with ammonia, methanesulfonamide and so-
dium methoxide respectively. Compound 1b was also prepared
by ammonia reaction of 2,4-dichloro-6-(4-methoxyphenyl)pyrimi-
dine (1a) as shown in Scheme 1.
5. Materials and methods
5.1. Bioactivity predictions using PASS
PASS was developed as a tool for evaluating biological activity
potential of a molecule of interest.35 Predictions are made using
2D QSAR-type models based on a large training set which includes
over 250,000 substances. PASS predicts about 4000 different kinds
of biological activities with a mean prediction accuracy of 85%. The
list of predictable activities include main pharmacological effects
(e.g., Gout Treatment, Uric Acid Excretion Stimulant), mechanisms
of action (e.g. Xanthine Oxidase Inhibitor, Collagenase Inhibitor)
and other effects of interest like toxicity, metabolism etc. The bio-
logical activity spectrum of the query compound is estimated from
the structure–activity relationship knowledgebase (SARBase) and
output in the form of probabilities of the compound being ‘active’
(Pa) or ‘inactive’ (Pi) for a biological activity. These probabilities are
obtained by combining contributions made by groups of atoms in
the compound, which favor or disfavor the particular activity as
seen from a large structure–activity database at the backend of
the software. Higher the values obtained for Pa–Pi, more the
chances of the compound showing activity on a scale of 0–1. The
net probability for activity of a compound can be estimated as
the difference Pa–Pi. In the current study, the in silico-filtered in-
house synthetic chemistry database, containing 2648 compounds
which violate not more than 1 of Lipinski’s Rule of 5, was screened
using PASS and 646 compounds were found to have Pa–Pi >0 for
Gout Treatment or Xanthine Oxidase Inhibition. Thirty-two com-
pounds with Pa P 0.3 for Xanthine Oxidase Inhibition were se-
lected as ‘hits’; all of them had Pa–Pi P 0.27.
The structures of commercially purchased compounds (Table 2;
1e, 1f and 1g) (Interbioscreen, Maybridge) were confirmed by
melting point and spectral data.
5.3.1. Preparation of 2-amino-6-(4-methoxyphenyl)pyrimidin-
4-ol (1)
To a stirred solution of 25 (1.0 g, 4.49 mmol) and guanidine
hydrochloride (0.64 g, 6.75 mmol) in DMF (10 ml), sodium acetate
(0.55 g, 6.75 mmol) was added and heated at 100 °C for 48 h. Reac-
tion mixture was cooled to rt, diluted with water (5 ml) and pH
was adjusted to 6.5. The precipitated solid was filtered and washed
with ethyl acetate (3 Â 5 ml). Solid was dried under reduced pres-
sure to give 1 as off-white solid (0.14 g, 14.2%). Mp 245–247 °C; 1H
NMR (DMSO-d6, 300 MHz): d 3.19 (s, 3H, OCH3), 6.15 (s, 1H, Ar),
7.03 (d, 2H, J = 8.4 Hz, Ar), 7.35 (broad s, 2H, NH2), 7.90 (d, 2H,
J = 8.4 Hz, Ar), 11.68 (s, 1H, Ar-OH); MS (ESI+) m/z 218 (M+H)+.
5.2. Molecular docking studies
Molecular docking studies were done using GLIDE 5.6 module of
Schrodinger Suite of programs (2010).36,40 Co-crystal structure of
xanthine dehydrogenase (XDH) with piraxostat (PDB code 1VDV)
was selected based on superior crystal structure parameters com-
pared to other XO or XDH structures. No difference was seen in the
xanthine binding-sites of XO and XDH crystal structures; hence we
could use either of them for docking. The ligands were built within
Maestro BUILD and prepared by LIGPREP module. All tautomeric
forms generated at physiological pH including keto and enol forms
were docked. The protein was prepared, optimized and minimized
5.3.2. Preparation of 2-amino-6-(3,4-dimethoxyphenyl)pyrimidin-
4-ol (2)
To a stirred solution of 25 (0.2 g, 0.79 mmol) and guanidine
hydrochloride (0.11 g, 1.18 mmol) in ethanol (10 ml), potassium
carbonate (0.16 g, 1.18 mmol) was added and refluxed for 48 h.
Reaction mixture was cooled to rt, ethanol removed under vacuum,
diluted with water (5 ml) and pH was adjusted to 6.5. The precip-

2938
Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
itated solid was filtered and washed with ethyl acetate (3 Â 5 ml).
Solid was dried under reduced pressure to give 2 as off-white solid
(0.043 g, 22%). Mp 288–291 °C; 1H NMR (CDCl3, 300 MHz): d 3.81
(s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.16 (s, 1H, Ar), 6.95 (broad s,
2H, NH2), 7.01 (d, 1H, J = 8.4 Hz, Ar), 7.50 (s, 1H, Ar), 7.54 (dd,
1H, J = 4.5 and 8.4 Hz, Ar); MS (ESI+) m/z 248 (M+H).
tion. Organic layer was washed with brine solution (2 Â 50 ml)
and the solvent was distilled under vacuum to get crude product
as yellow liquid oil. Crude compound was purified by silica gel col-
umn chromatography using methanol/chloroform as eluent to give
26 (1.65 g, 76.3%). Mp 201–203 °C; 1H NMR (DMSO-d6, 300 MHz): d
3.82 (s, 3H, OCH3), 7.01 (broad s, 2H, NH2), 7.05 (d, 2H, J = 7.8 Hz,
Ar), 7.19 (s, 1H, Ar), 8.06 (d, 2H, J = 7.8 Hz, Ar); MS (ESI+) m/z 236
(M+H)+.
5.3.3. Preparation of 2-amino-6-(3,4-dimethoxyphenyl)pyrimidin-
4-thiol (3)
To a suspension of 2 (0.15 g, 0.61 mmol) in toluene (5 ml) was
added Lawesson’s reagent (0.98 ml, 2.44 mmol) and heated at
110 °C for 8 h. Toluene was distilled out from the reaction mixture
under vacuum and treated with ice (5 g) followed by sodium bicar-
bonate solution (5 ml). The resulting mixture was extracted with
chloroform (2 Â 50 ml) and washed with brine (2 Â 20 ml). Solvent
was removed under reduced pressure and crude compound was
purified by silica gel column using methanol/chloroform as eluent.
Solid dried under reduced pressure to give 3 as off-white solid
(0.056 g, 35%). Mp 243–245 °C; 1H NMR (CDCl3, 300 MHz): d 3.81
(s, 3H, OCH3), 4.01 (s, 3H, OCH3), 6.94 (broad s, 2H, NH2), 7.01 (s,
1H, Ar), 7.03 (d, 1H, J = 8.4 Hz, Ar), 7.57 (d, 1H, J = 2.1 Hz, Ar),
7.65 (dd, 1H, J = 2.1 and 8.4 Hz, Ar); MS (ESI+) m/z 264 (M+H)+.
5.3.8. Preparation of N-(2-amino-6-(4-methoxyphenyl)pyrimidin-
4-yl) methanesulfonamide (1c)
A suspension of 26 (200 mg, 0.848 mmol), methane sulfon-
amide (161.4 mg, 1.69 mmol), potassium carbonate (234.4 mg,
1.69 mmol) in dimethyl sulfoxide (5 ml) was heated to 170 °C for
3–4 h. Reaction was monitored by TLC. The resulting mixture con-
centrated to dryness under reduced pressure. Cold water was
added to reaction mixture and pH was neutralized to 6–7 by add-
ing dil HCl solution. Crude product was filtered off and purified by
silica gel column chromatography using methanol/chloroform elu-
ent to give 1c (82 mg, 32.8%). Mp 242–244 °C; 1H NMR (acetone-d6,
300 MHz): d 3.33 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 6.23 (broad s, H,
NH2), 6.78 (s, 1H, Ar), 7.01 (d, 2H, J = 8.1 Hz, Ar), 7.98 (d, 2H,
J = 8.1 Hz, Ar); MS (ESI+) m/z 295 (M+H)+; HRMS calcd for
5.3.4. Preparation of 6-(4-methoxyphenyl)pyrimidine-2,4-diol (4)
The suspension of urea (500 mg, 8.32 mmol) in 25 (4 ml) was
heated at 180–190 °C for 5–6 h. Reaction was monitored by TLC.
Reaction mixture was cooled to rt, diluted with ethyl acetate
(10 ml). The solid was filtered, washed with ethyl acetate
(2 Â 5 ml) to give 4 (437 mg, 24.06%). Mp 286–288 °C; 1H NMR
(DMSO-d6, 300 MHz): d 3.82 (s, 3H, OCH3), 5.77 (s, 1H, Ar), 7.03
(d, 2H, J = 9.0 Hz, Ar), 7.70 (d, 2H, J = 9.0 Hz, Ar), 11.02 (s, 1H, Ar-
OH), 11.07 (s, 1H, Ar-OH); MS (ESI+) m/z 219 (M+H)+.
C12H15N4O3S, 295.0859; found, 295.0848.
5.3.9. Preparation of 4-methoxy-6-(4-methoxyphenyl)pyrimidin-
2-amine (1d)
Na metal (100 mg, 4.34 mmol) was dissolved in dry methanol
(5 ml). 26 (100 mg, 4.24 mmol) was added and reaction mass
was heated to reflux for 3–4 h. Reaction was monitored by TLC. Sol-
vent was distilled under reduced pressure. Water was added to the
reaction mixture and pH was adjusted to 6–7 by dil HCl solution.
Product was filtered off and dried to give 1d (62 mg, 63.26%). Mp
159–161 °C; 1H NMR (DMSO-d6, 300 MHz): d 3.81 (s, 6H, OCH3),
6.49 (s, 1H, Ar), 6.57 (broad s, 2H, NH2), 7.04 (d, 2H, J = 5.4 Hz,
Ar), 8.01 (d, 2H, J = 5.4 Hz, Ar); MS (ESI+) m/z 231(M+H)+.
5.3.5. Preparation of 2,4-dichloro-6-(4-methoxyphenyl)pyrimidine
(1a)
The suspension of 4 (200 mg, 0.91 mmol) in POCl3 (4 ml) was
heated at 90 °C for 1 h. Reaction was monitored by TLC. After com-
pletion, excess POCl3 was distilled off under vacuum. Ice and water
were added to the residue, and the yellow product was filtered and
washed with water till it reached neutral pH. Crude product was
purified by silica gel column chromatography using chloroform
as eluent to give 1a (126.6 mg, 53.80%). Mp 103–105 °C; 1H NMR
(CDCl3, 300 MHz): d 3.91 (s, 3H, OCH3), 7.03 (d, 2H, J = 9.0 Hz, Ar),
7.60 (s, 1H, Ar), 8.06 (d, 2H, J = 9.0 Hz, Ar); MS (ESI+) m/z 255
(M+H)+.
5.4. High throughput in vitro assay
The assay was standardized based on previous protocols re-
ported by Okamoto et al. and Sathisha et al.21,30 Previous studies
have reported uric acid absorption to be maximum at 290 nm
which was reproduced by us on the Jasco spectrophotometer. Since
the objective was to establish a high throughput assay, the assay
was standardized using the Tecan system with the integrated Sa-
fire 2 reader. For this purpose, we used 96 well UV transparent
plates from Corning Life Sciences, USA, instead of the quartz cuv-
ettes used in the Jasco spectrophotometer. In this set up, the
absorption peak of uric acid was found at 310 nm. At this wave-
length a linearly increasing trend was observed for absorbance
with increasing concentrations of uric acid. All further xanthine
oxidase activity was monitored spectrophotometrically on the
HTS system following the absorbance of uric acid at 310 nm under
aerobic condition.
5.3.6. Preparation of 6-(4-methoxyphenyl)pyrimidine-2,4-diamine
(1b)
In a sealed tube, suspension of 1a (60 mg, 0.235 mmol) in liq.
ammonia (9 ml) and ethanol (1 ml) was heated to 110 °C for 4–
5 h. Reaction was monitored by TLC. Solvent was distilled off under
vacuum to give crude product. Crude product was purified by silica
gel column chromatography using methanol/chloroform as eluent
to give 1b (12 mg, 23.57%). Mp 210–212 °C; 1H NMR (DMSO-d6,
300 MHz): d 3.82 (s, 3H, OCH3), 6.20 (s, 1H, Ar), 6.55 (broad s,
2H, NH2), 6.98 (broad s, 2H, NH2), 7.04 d, 2H, J = 8.7 Hz, Ar), 7.81
(d, 2H, J = 8.7 Hz, Ar); MS (ESI+) m/z 217 (M+H)+.
The XO activity was observed in presence of varied concentra-
tions of bovine XO (Calbiochem) and xanthine (Sigma). The con-
centrations at which the formation of uric acid stabilized were
found to be 200 mU XO and 400 lM xanthine at 30 min. These
5.3.7. Preparation of 4-chloro-6-(4-methoxyphenyl)pyrimidin-
2-amine (26)
parameters were used for further screening. The enzyme solution
of XO prepared in water was added to the test compounds or vehi-
cle control (0.5% DMSO). The reaction was initiated by addition of
xanthine in 50 mM potassium phosphate buffer (pH 7.5) as the
substrate to the above assay mixture. The absorbance at 310 nm,
indicating the formation of uric acid was measured at 30 min at
ambient temperature. Duplicate assays were repeated three times.
Allopurinol was used as positive control. The inhibitory activity of
A stirred mixture of 1 (2.0 g, 9.21 mmol) and POCl3 (50 ml) was
heated to 120–125 °C for 2 h under dry nitrogen atmosphere. Once
reaction mixture became clear, it was monitored by TLC for com-
pletion. After the completion of reaction, POCl3 was distilled out
under vacuum. Chloroform (100 ml) was added to crude mass
and pH was adjusted to 6–7 by adding sodium bicarbonate solu-

Chandrika B-Rao et al. / Bioorg. Med. Chem. 20 (2012) 2930–2939
2939
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.019.
An animated Interactive 3D Complement (I3DC) is available in
Proteopedia at http://proteopedia.org/w/Journal:BMC:3.
References and notes
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