Isocytosine-based inhibitors of xanthine oxidase: Design, synthesis, SAR, PK and in vivo efficacy in rat model of hyperuricemia

Article abstract of DOI:10.1016/j.bmcl.2012.10.029

Structure-activity relationship studies were carried out for lead generation following structure-guided design approach from an isocytosine scaffold identified earlier for xanthine oxidase inhibition. A 470-fold improvement in in vitro IC₅₀ was obtained in the process. Five most potent compounds with nanomolar IC₅₀ values were selected for pharmacokinetics and in vivo experiments. The best compound showed good in vivo activity when administered intraperitoneally but was not active by oral route. The results suggest that improvement in oral exposure could improve the in vivo efficacy of this series.

Full text of DOI:10.1016/j.bmcl.2012.10.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7543–7546
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isocytosine-based inhibitors of xanthine oxidase: Design, synthesis, SAR, PK
and in vivo efficacy in rat model of hyperuricemia
Smriti Khanna a, Sandeep Burudkar b, Komal Bajaj b, Pranay Shah b, Ashish Keche b, Usha Ghosh b,
Avani Desai c, Ankita Srivastava c, Asha Kulkarni-Almeida c, Nitin J. Deshmukh d, Amol Dixit d,
Manoja K. Brahma d, Umakant Bahirat d, Lalit Doshi d, Kumar V. S. Nemmani d, Prashant Tannu e,
b
b
Anagha Damre e, Chandrika B-Rao a, , Rajiv Sharma , H. Sivaramakrishnan
⇑
a Discovery Informatics, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
b Department of Medicinal Chemistry, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
c Department of High Throughput Screening and Biotechnology, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
d Department of Pharmacology, Piramal Healthcare Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
e Department of Drug Metabolism and Pharmacokinetics, 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:
Structure–activity relationship studies were carried out for lead generation following structure-guided
design approach from an isocytosine scaffold identified earlier for xanthine oxidase inhibition. A 470-fold
improvement in in vitro IC50 was obtained in the process. Five most potent compounds with nanomolar
IC50 values were selected for pharmacokinetics and in vivo experiments. The best compound showed
good in vivo activity when administered intraperitoneally but was not active by oral route. The results
suggest that improvement in oral exposure could improve the in vivo efficacy of this series.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 15 March 2012
Revised 28 September 2012
Accepted 5 October 2012
Available online 13 October 2012
Keywords:
Xanthine oxidase inhibitors
Isocytosine
Structure–activity relationship
Docking
Lead optimization
Hyperuricemia is a metabolic disorder characterized by ele-
vated levels of serum uric acid (sUA). It is often associated with
diabetes, hypertension, and cardiovascular problems and is consid-
ered as a high risk factor for gout and oxidative stress.1
tual screening and testing in enzymatic assay.8a It showed an IC50
of 9.4 M in XO inhibition assay and 84% reduction in sUA level in
l
potassium oxonate-induced hyperuricemic rat model. Docking to
XO showed that keto/enol group in isocytosine formed H-bonds
with Arg880 and Thr1010 and –NH2 with Glu802. Limited struc-
ture–activity relationship (SAR) suggested that these are essential
interactions, which probably serve the role of anchoring the mole-
cules in the active site. Docking studies suggested possible direc-
tions for modifications that could improve in vitro potency of the
series.8a Here, we present our hit-to-lead efforts by repeated cycles
of structure-guided design, synthesis and in vitro screening in XO
inhibition assay,8b resulting in compounds with nanomolar activ-
ity. Selected active compounds were tested in vivo in hyperurice-
mic rat model,8c revealing 2 compounds with good efficacy.
Analysis of docking (PDB code 1VDV)9 of the novel hit revealed
that R points towards the solvent accessible region at the entrance
of the pocket, which is lined by many hydrophobic and a few polar
residues. SAR was obtained by diverse substitutions at R (Table 1),
docked using GLIDE10,11 synthesized (Scheme 1) and tested
in vitro.8b
Hyperuricemia is caused by dietary, environmental and genetic
factors2 with two main lines of treatment3 being reducing produc-
tion and increasing excretion of uric acid (UA). The former is
achieved by administration of xanthine oxidase (XO) inhibitors
(XOIs) and the latter by uricosuric agents. XO is the key enzyme
in purine metabolism to UA in the last two steps of the pathway.4
Currently used XOIs for treatment of hyperuricemia are allopuri-
nol5 and febuxostat6,7 (Fig. 1). They are associated with an adverse
event profile that includes gastrointestinal, hepatic, renal and
hematological toxicities, skin problems and allergic reactions.
We recently identified compound 1 (Fig. 1), a novel isocytosine
based scaffold from our in-house corporate collection through vir-
Abbreviations: XO, xanthine oxidase; XOI, xanthine oxidase inhibitor; PK,
pharmacokinetics; AUC, area under curve; po, per oral; ip, intraperitoneal; SAR,
structure–activity relationship; UA, uric acid; sUA, serum UA.
The observed in vitro activities corresponded well with predic-
tions made by docking studies (Table 1) using docking scores,
⇑
Corresponding author. Tel.: +91 22 30818714; fax: +91 22 30818036.
E-mail address: chandrika.rao@piramal.com (C. B-Rao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.029

7544
S. Khanna et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7543–7546
good score, but lost hydrophobic interactions with nonpolar resi-
dues at the entrance of the pocket and was inactive. Branched ali-
phatic chain (3) showed good activity. Branched and linear chains
with thioether or ether linker (4, 5, 6, 7, 8) had similar scores and
gave significant improvement in activity, probably due to im-
proved electrostatic interactions with Asn768 and Lys771. Isobut-
oxy group (7, Fig. 2) resulted in one of the most potent compounds
from this set with an IC50 value of 0.6 lM. Saturated cyclic rings
with polarity such as morpholine (9) docked well but did not im-
prove IC50 values, perhaps due to badly placed Ns in hydrophobic
region of protein. However, aromatic phenyl ring (10) boosted
the activity by 30-fold from 9.4
lM to 0.3 lM. This may be attrib-
uted to -stacking interactions of the terminal aromatic ring with
p
Figure 1. Structures of hypoxanthine, allopurinol, febuxostat and compound 1.
Potential sites of modifications on the phenylisocytosine scaffold are also shown.
Phe649 and Phe1013 at the entrance to the pocket. Introducing a
linker between the two phenyl rings improved dock scores but
did not translate to improvement in potency (11, 12).
Plasma pharmacokinetics (PK) of 7 was studied in Wistar/Spra-
gue Dawley rats at a single 100 mg/kg dose by oral route (po) over
docked poses and essential interactions with active site residues.
Small and polar –OH group (2) docked in the correct pose with
Table 1
Analogs of compound 1 with modifications at R
Compound ID
R
Yields (%)
49.18
Docking scores kcal/mol
In vitro IC50
>30
lM
2
3
–OH
À9.77
54.90
À10.80
4.95 0.19
4
5
6
7
32.27
87.66
46.38
49.94
À10.75
À10.51
À10.15
À9.84
1.63 0.08
2.44 0.20
2.10 0.19
0.60 0.07
S
S
O
O
8
19.55
7.66
À10.04
À10.25
À9.08
9.24 0.17
16.52 3.05
0.31 0.08
O
OH
N
O
9
10
39.80
11
34.70
40.89
À11.18
3.65 0.79
O
12
À10.70
2.47 1.43
Febuxostat
Allopurinol
À12.05
À8.47
0.03 0.00
4.19 52
Scheme 1. Synthetic scheme for compounds in Tables 1 and 3: Reagents and conditions: (a) Pd(dppf) Cl2.CH2Cl2, dioxan, bis-pinacolato diborane, potassium acetate,120 °C,
8 h. (b) Pd(PPh3)4, DMF, Na2CO3, microwave, 140 °C, 1–2 h. (c) Raney-Ni /H2, 40 psi, DMF. (d) NaOH, MeOH, 140 °C, 12 h.

S. Khanna et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7543–7546
7545
Table 3
Analogs of compound 7 with modifications at R1
Compound
ID
R1
Yields
(%)
Docking scores kcal/
mol
In vitro IC50
lM
7a
7b
7c
7d
7e
7f
OCF3
NH2
CONH2
OCH3
NO2
20.90
50.00
2.57
35.87
35.88
19.11
À9.96
>20
À10.33
À11.35
À10.53
À10.33
À10.98
À12.05
À8.47
6.72 0.09
1.92 0.7
0.95 0.65
0.14 0.03
0.02 0.00
0.03 0.00
5.77 0.42
CN
Febuxostat
Allopurinol
Fig. 2. Docked poses of compounds 7 (green) and 7f (brown). Compound 7f forms
additional H-bond with Asn768 which adds to its potency. This figure has been
generated in Pymol.12
Table 4
Pharmacokinetic parameters of compounds 7d, 7e and 7f in rats
PK parameters
7d(50 mg/
7e(50 mg/
7f(10 mg/kg 7f(2 mg/
kg ip)
kg ip)
po)
kg iv)
Table 2
Tmax (h)
2
2
0.19
0.07
0.23
0.2
0.03
1.19
4.19
0.47
0.48
Pharmacokinetic parameters of compounds 1, 7 and 10
Cmax
Cmax
(
(
l
l
g/ml)
M)
9.91
34.27
27.6
2.92
9.59
13.98
17.55
PK Parameters
1 (100 mg/
kg, po)
7 (100 mg/
kg, po)
10 (100 mg/
kg, po)
10 (50 mg/
kg, ip)
AUClast (h⁄
lg/ml)
AUC0-infinity (h⁄
l
g/ 27.72
0.31
ml)
Tmax (h)
Cmax
Cmax
0.25
5.1
23.48
4.08
0.5
0.88
0.21
0.81
0.6
3.33
1.88
7.13
Half-life (h)
%F (AUClast
%F (AUC0-infinity
CO (ng/ml)
Vd (mg/kg)
CL (ml/h/kg)
0.85
—
—
—
—
12.25
—
—
—
—
11.45
8.6
12.7
NA
NA
NA
0.63
(
(
l
l
g/ml)
M)
1.31
5.16
4.39
)
NAa
)
NA
AUClast (h⁄
lg/
21.92
1360.78
3825.58
4279.22
ml)
AUC0-infinity
(h⁄ g/ml)
Half-life (h)
4.11
6.59
5.68
3.42
0.89
2.85
23.14
5.52
—
—
l
a
NA: not applicable.
Figure 3. Plasma concentration-time profiles of compounds (A) 1, 7 and 10 (B) 7d, 7e and 7f in rats.
Figure 4. Time course of response of sUA levels for different treatments in hyperuricemic rats. Antihyperuricemic effect of (A) orally administered 7 (B) intraperitoneally
administered 10 and 7d.

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S. Khanna et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7543–7546
Figure 5. Time course of response of sUA levels for different treatments in hyperuricemic rats. Antihyperuricemic effect of (A) orally and intraperitoneally administered 7e
(B) intraperitoneally administered 7f and (C) orally administered 7f.
24 h (Table 2; Fig. 3A shows plot up to 6 h). Due to low oral expo-
sure, efficacy was tested at a high dose of 100 mg/kg po. The reduc-
tion in sUA level for 7 in hyperuricemic rat model8c was only 23.7%
as against 130% for allopurinol (Fig. 4A). This translates to 18% of the
efficacy of allopurinol as against a corresponding value of 44% for
the original hit, 1,8a in spite of 16-fold improvement in in vitro
activity. This is perhaps due to the much poorer oral exposure of
7 (Table 2). PK of another compound, 10, with better in vitro po-
tency, was studied by both po and ip routes (Fig. 3A). As oral expo-
sure of 10 (Table 2) was worse than that of 7, its in vivo efficacy was
tested by ip route at 100 mg/kg. It showed a reduction of 77% in sUA
levels as against 177% and 181% for reference standards, allopurinol
and febuxostat, respectively, in the same experiment (Fig. 4B).
R1 position on 7 was explored next (Table 3, Scheme 1). Func-
tional groups such as –OCH3 (7d), –NO2 (7e), –CN (7f) gave im-
proved IC50 values but –OCF3 (7a), –NH2 (7b) and –CONH2 (7c)
showed low activity.
first pass metabolism, indicating that the compounds are active
against the molecular target but unable to reach it in vivo.
In summary, a lead compound (IC50 = 20 nM) that was 470
times more potent in enzymatic assay than the original hit
(IC50 = 9.4 lM) was obtained by hit-to-lead efforts. Although it
exhibited considerable reduction in sUA levels by ip route as mea-
sured by AUC for UA level over 5 h post administration, it did not
show oral efficacy equiv to saturation doses of allopurinol and feb-
uxostat. Further efforts at lead optimization to improve PK profile
could result in better oral efficacy of this series of compounds.
Acknowledgment
We are grateful to Dr. Somesh Sharma for his continuous sup-
port to this structure-guided drug discovery project. We would like
to thank Prof Krishna Iyer for his inputs on PK studies.
Comparison of PK results of the three potent compounds, 7d, 7e
and 7f, (Fig 3B, Table 4) shows that ip administration generally
gave reasonable Cmax values. About 5-fold higher Cmax was ob-
tained for 7d compared to 10 administered i.p. and it was also
tested for efficacy by ip route. The 55% reduction in UA level seen
for 7d (Fig. 4B) was better than that for 7 (23.7%) (Fig. 4A), but not
as much as for 10 (77%). This may be due to the fact that 10 is more
potent than 7d. It was therefore felt that further improvement in
in vitro potency was required, which was attained by 7e and 7f.
7e and 7f were tested in animal models by both po and ip routes
of administration (Fig. 5). 7e showed reduction of 93% and 133% in
sUA levels after po and ip dosing respectively as against allopurinol
with 139% reduction (Fig. 5A). Higher enzymatic potency of 7e
seemed to compensate for reduced exposure as compared to 7d.
7f also showed reduction of 124% in sUA levels as against febuxo-
stat with 180% reduction (Fig. 5B). However, by po route, it was
essentially inactive (Fig. 5C). This may be explained by the PK re-
sults (Table 4). A moderate volume of distribution and very high
clearance for 7f resulted in an absolute oral bioavailability (%F) of
only 8.6%. Poor oral efficacy could be due to poor absorption, which
may be due to both poor dissolution and poor permeability.
In general, greater reduction in sUA levels is achieved by ip than
by po route, where the compound bypasses oral absorption and
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