Synthesis and xanthine oxidase inhibitory activity of 5,6-dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives

Article abstract of DOI:10.1016/j.bioorg.2014.08.007

Some 5,6-dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives were rationally designed, synthesized and evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Some notions about structure activity relationships are presented. Th

Full text of DOI:10.1016/j.bioorg.2014.08.007

Bioorganic Chemistry 57 (2014) 57–64
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and xanthine oxidase inhibitory activity
of 5,6-dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives
Deependra Kumar a, Gagandeep Kaur a, Arvind Negi a, Sanjeev Kumar b, Sandeep Singh c, Raj Kumar a, ,1
⇑
a Laboratory for Drug Design and Synthesis, Centre for Chemical and Pharmaceutical Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda 151
001, India
b Centre for Biosciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda 151 001, India
c Centre for Genetic Diseases and Molecular Medicine, Central University of Punjab, Bathinda 151 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 May 2014
Available online 30 August 2014
Some 5,6-dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives were rationally designed, synthesized
and evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Some notions about struc-
ture activity relationships are presented. The compounds 6g, 6h and 6e were found to be significantly
active against XO. The compound 6g emerged as the most potent XO inhibitor as compared to allopurinol
and free radical scavenger. The molecular docking of 6g into the XO active site highlighted its mode of
Keywords:
Xanthine oxidase
5,6-Dihydropyrazolo/pyrazolo[1,5-
c]quinazoline
Synthesis Molecular modeling
Antioxidant
binding and important interactions such as hydrogen bonding,
p–p stacking with amino acid residues
like Ser876, Thr1010, Phen914, Phe1009 and Phe649 and its close proximity to dioxothiomolybdenum
(MOS).
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
febuxostat [17] (2) having lesser and non-life threatening side
effects as compared to allopurinol [18–20].
Xanthine oxidase (XO; EC 1.17.3.2) is well characterized
druggable target [1–3] for the treatment and management of dis-
ease conditions involving high uric acid level; hyperuricemia and
gout [4,5] due its significant role in catalytic oxidative hydroxyl-
ation of hypoxanthine and xanthine to produce uric acid. Further
XO is also associated and unregulated in pathological conditions
involving inflammation, metabolic disorders, cellular aging, reper-
fusion damage, atherosclerosis, hypertension [6,7] and carcinogen-
esis as it generates superoxide anions, H2O2 and highly reactive
species (ROS) during the catalytic process [2]. Thus the selective
inhibition of XO may result in broad spectrum therapeutics for
gout, cancer, inflammation and oxidative damage [2–4].
In general XO inhibitors have been classified into purine and
non-purines based on their scaffolds mimicking to naturally occur-
ring purines (Fig. 1). Allopurinol (1) was the first purine based XO
inhibitor approved by the US FDA in 1966 for treatment of gout and
hyperuricemia [8]. Due to the reported hypersensitivity (Stevens–
Johnsons) syndrome induced by allopurinol use [9,10], researchers
initiated their search for new XO inhibitors derived non-purine
skeleton [4,11–16] and a great success has been achieved in this
direction which is evident by a recent US FDA approval of
In continuation to our earlier successful attempts made towards
the search for new xanthine oxidase inhibitors based on N-(1,3-
diaryl-3-oxopropyl)amides [11] (3) and N-acetyl pyrazolines [12]
(4), we thought of designing and synthesizing 5,6-dihydro pyrazol-
o[1,5-c]quinazolines (5 and 6) via structural modification of 3, 4
and FYX-051 keeping in mind the following considerations
(Fig. 2): (i) two aryl/heteroaryl rings (preferably joined by three
atoms) oriented in such a way so that favorable arene–arene inter-
actions with Phe914 and Phe1009 amino acid residues may take
place; (ii) a site for hydroxylation near molybdenum metal and
(iii) bioisosteric replacement of free C@O with C@N/CANH fol-
lowed by ring cyclization might enhance activity and target speci-
ficity through hydrogen bond formation with Ser876 and/or
Thr1010 amino acid residues of XO active site.
Pyrazolo[1,5-c]quinazolines have previously been reported to
possess bioactivities such as benzodiazepine binding ligands [21],
Gly/NMDA receptor, excitatory amino acid antagonists [22,23],
IKK [24], phosphodiesterase 10A [25] inhibitors, AMPA and kainate
receptor antagonists [26] and adenosine receptor antagonists [27].
2. Materials and methods
2.1. Chemistry
⇑
Corresponding author. Fax: +91 164 2240555.
E-mail addresses: raj.khunger@gmail.com, rajcps@cup.ac.in (R. Kumar).
CUPB Library Communication Number: P128/14.
All the reagents were of AR/GR quality and were purchased
from Sigma–Aldrich, Loba-Chemie Pt. Ltd., SD Fine Chemicals, Sisco
1
http://dx.doi.org/10.1016/j.bioorg.2014.08.007
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

58
D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
Fig. 1. Some reported xanthine oxidase inhibitors from our laboratory and others.
Fig. 2. Design of target compounds 5 and 6 as xanthine oxidase inhibitors.
Research Laboratory and HiMedia Laboratories Ltd. and were used
without further purification. Sartorius analytical balance
(BSA224S-CW) was used for the weighing purposes. JSGW heating
mantle, Tarson spinot digital, ILMVAC ROdist digital rotary evapo-
rator, digital hop top and NSW oven/vacuum oven were used dur-
ing the course of reaction. The progress of the reaction was
monitored by TLC, using hexane/ethyl acetate and chloroform/
methanol as the mobile phase on pre-coated Merck TLC plates in
JSGW UV/fluorescent analysis cabinet and/or iodine chamber.
Melting points were recorded on Stuart melting point apparatus
(SMP-30) with open glass capillary tubed and was uncorrected.
Infrared (IR) spectra of compounds were recorded with KBr on a
Bruker FT-IR spectrophotometer. 1H and 13C Nuclear magnetic res-
onance (NMR) spectra was obtained in CDCl3/d6-DMSO on a Bruker
Avance II (400 MHz) NMR spectrometer using TMS (d = 0) as
internal standard.
2.1.1. Representative procedure for the synthesis of 7
2.1.1.1.
(E)-3-(2-nitrophenyl)-1-phenylprop-2-en-1-one
(7a). A
mixture of 2-nitrobenzaldehyde (4 g, 26.50 mmol) and acetophe-
none (3.18 g, 26.50 mmol) in glacial acetic acid (10 mL) (q.s.) was
stirred in 100 mL round bottomed flask (RBF). Conc. Sulfuric acid

D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
59
(4–5 drops) was added to the reaction mixture in ice cold condition
and further stirred at rt for 24 h. After the completion of the
reaction (TLC), the reaction mixture was poured on ice cold water
and filtered. The solid was washed with water, dried and was
recrystallized from methanol to afford the pure compound. Yield:
85%; Creamy white solid; MP: 85–87 °C; IR (KBr, cmꢀ1): 1666
(C@O), 1610 (C@C), 1510 (N@O), 1342 (N@O).
(ESI): m/z = 236.1 [M+1]+. All the remaining reactions were carried
out using this general procedure [28].
2.1.1.5. Representative procedure for the synthesis of 2-phenylpyraz-
olo[1,5-c]quinazoline (5a). To a solution of 10 (1 mmol, 1.0 equiv)
in MeCN (2 mL) was added CH(OEt)3 (1.1 equiv) and was kept in
a vial, sealed and put in Biotage Initiator Microwave synthesizer.
The reaction mixture was then heated at 100 °C for 10 min (TLC)
under MW. After the completion of the reaction, the reaction mix-
ture was left to cool. The precipitates were filtered, dried and
washed with n-hexane (5 mL ꢁ 3) to afford the pure product 5a.
Yield: 91%, Creamy solid: Mp 135–136 °C; IR (KBr, cmꢀ1): 3019
(CH), 1620 (C@N), 1539 (C@C), 1215 (CAN); 1H NMR (DMSO-d6,
600 MHz, d with TMS = 0): 9.42 (1H, s), 8.30 (1H, d, J = 7.8 Hz),
8.09 (2H, d, J = 8.4 Hz), 7.93 (1H, m), 7.87 (1H, s), 7.75 (2H, m),
7.6 (2H, m), 7.5 (1H, m); 13C NMR (DMSO-d6, 150 MHz, d with
TMS = 0): 155.27, 140.34, 139.96, 139.67, 132.35, 130.57, 129.81,
129.54, 128.85, 128.73, 126.81, 124.21, 119.98, 96.65; MS (ESI):
m/z = 246 [M+1]+. All the remaining reactions were carried out
using this general procedure [28].
1H NMR (CDCl3, 300 MHz, d with TMS = 0): 8.12 (1H, d,
J = 15.9 Hz), 8.02 (3H, bs), 7.69–7.74 (2H, m), 7.50–7.57 (4H, m),
7.34 (1H, d, J = 15.9 Hz); 13C NMR (CDCl3, 75 MHz,
d with
TMS = 0): 190.22, 148.34, 140.03, 137.20, 133.56, 133.09, 131.08,
130.31, 129.13, 128.62, 126.97, 124.86. Similar protocol was
utilized for the synthesis of (E)-1-(4-chlorophenyl)-3-(2-nitro-
phenyl)prop-2-en-1-one (7b). The above compounds were used
for next steps.
2.1.1.2. 4,5-Dihydro-5-(2-nitrophenyl)-3-phenyl-1H-pyrazole (8a). A
mixture of 7a (3.3 g, 13.04 mmol) and hydrazine hydrate
(0.63 mL, 13.04 mmol) was refluxed under methanol for 2 h. After
completion of the reaction (TLC), the reaction mixture was cooled
to rt and the precipitates (8a) so obtained were collected, dried and
used for the next step for further reaction. Yield: 97%; Orange crys-
talline solid; MP: 140–142 °C; IR (KBr, cmꢀ1): 3310 (NAH), 1518
(N@O), 1333 (N@O), 1299 (C@N); 1H NMR (CDCl3, 400 MHz, d with
TMS = 0): 7.96 (2H, t, J = 8.4 Hz), 7.65 (3H, m), 7.41 (4H, m), 6.05
(D2O exchangeable NH, 1H, s), 5.42 (1H, t, J = 13.2 Hz), 3.80, (1H,
q, J = 14.2 Hz), 3.01 (1H, q, J = 13.2 Hz). 13C NMR (CDCl3, 100 MHz,
d with TMS = 0): 151.12, 148.54, 138.04, 133.87, 132.43, 129.06,
128.59, 128.53, 128.36, 126.04, 124.65, 59.83, 41.46. Similar proto-
col was utilized for the synthesis of 3-(4-chlorophenyl)-5-(2-nitro-
phenyl)-4,5-dihydro-1H-pyrazole (8b).
2.1.1.6. Representative procedure for the synthesis of 5-(2,5-dime-
thoxyphenyl)-2-phenyl-5,6-dihydropyrazolo
[1,5-c]quinazoline
(6a). A mixture of 10 (235 mg, 1 mmol, 1.0 equiv) and 2,5-dime-
thoxy benzaldehyde (1.0 equiv) in water (2 mL) was taken in a vial,
sealed and kept in Biotage Initiator Microwave synthesizer. The
reaction mixture was then heated at 100 °C for 15 min (TLC) under
MW. After the completion of the reaction, the reaction mixture was
left to cool. The precipitates were filtered, dried and washed with
n-hexane (10 mL ꢁ 3) to afford the pure product 6a. Yield: 80%,
Light yellow solid, Mp 230–232 °C; IR (KBr, cmꢀ1): 1644 (C@N),
1587 (C@C), 1220 (C-N), 1123 (C-O); 1H NMR (CDCl3, 400 MHz, d
with TMS = 0): 15.60 (D2O exchangeable NH, 1H, bs), 8.76 (1H, d,
J = 8.0 Hz), 8.37 (1H, d, J = 8 Hz), 8.04 (2H, m), 7.55 (2H, d,
J = 8.0 Hz), 7.19 (5H, m), 7.11 (3H, m), 7.27 (1H, d, J = 8.27 Hz),
3.70 (3H, s), 3.28 (3H, s); 13C NMR (CDCl3, 100 MHz, d with
TMS = 0): 153.51, 152.15, 149.94, 139.68, 139.25, 133.39, 129.38,
128.60, 127.85, 125.83, 124.06, 123.01, 119.52, 115.31, 113.80,
113.16, 112.70, 111.30, 96.62, 67.99, 56.05, 55.43; HRMS (TOF-
ESI) Calcd for C24H21N3O2, 383.1634 (M)+; observed: 384.1638
(M+H)+. The remaining reactions were carried out using this
general procedure [28].
2.1.1.3. Synthesis of 5-(2-nitrophenyl)-3-phenyl-1H-pyrazole (9a). 8a
(3.1 g, 11.70 mmol) was refluxed with catalytic amount of molecu-
lar iodine in DMSO at 130–140 °C for 2 h. After the completion of
reaction (TLC), the reaction mixture was poured in ice cold water
and was extracted using ethyl acetate (20 mL ꢁ 3). Organics were
washed with brine (10 mL ꢁ 3), dried over sodium sulfate and
evaporated under reduced pressure using rotary evaporator to
obtain the product (9a) which was used for next step. Yield; 92%;
Brownish color solid; MP: 194–196 °C; IR (KBr, cmꢀ1): 3456
(NAH), 1664 (C@N), 1524 & 1348 (N@O), 1217 (CAN); 1H NMR
(CDCl3, 400 MHz, d with TMS = 0): 7.67–7.76 (3H, m), 7.56–7.63
(3H. m), 7.33–7.51 (5H, m); 13C NMR (CDCl3, 100 MHz, d with
TMS = 0): 149.02, 146.37, 146.16, 132.09, 130.97, 129.04, 128.74,
128.04, 126.47, 125.62, 124.56, 123.82, 102.59. Similar protocol
was utilized for the synthesis of 5-(2-nitrophenyl)-3-phenyl-1H-
pyrazole (9b).
We have recently reported the synthesis and the physical data
of 10–10a, target compounds 5a–5b and 6b–6m [28].
2.2. Biological evaluation
Xanthine oxidase enzyme as lyophilized powder was purchased
from SRL Pt. Ltd. and stored at temperature below 4 °C. Allopurinol
was purchased from SRL Pt. Ltd. Potassium phosphate monobasic
(KH2PO4) and dibasic (K2HPO4), DMSO of AR grade were purchased
from Loba Chemie Pt. Ltd. Methanol was purchased from SDFCL.
Analytical balance (JB1603-c/FACT) and pH meter (Mettler Toledo)
were used for the purpose of weighing and measuring the pH,
respectively. REMI laboratory refrigerator and ice flaking machine
(MSW-136) by Macro scientific works were used for storing solu-
tions. Double-beam UV–Visible spectrophotometer (Shimadzu)
for measuring absorbance was used.
2.1.1.4. Representative procedure for the synthesis of 2-(3-Phenyl-1H-
pyrazol-5-yl)aniline (10). 9a (2.75 g, 10.38 mmol) was dissolved in
methanol and refluxed with 3 equivalent of stannous chloride
dihydrate for 2 h. After the completion of the reaction (TLC), meth-
anol was evaporated under reduced pressure using rotary evapora-
tor followed by neutralization of reaction mixture using 5% NaOH
solution. Solid product was than extracted with ethyl acetate
(10 mL ꢁ 3). Organics were dried over anhydrous sodium sulfate
and evaporated under reduced pressure using rotary evaporator
to obtain the product 10a which was used for next step. IR (KBr,
cmꢀ1): 3360 (NAH), 3285 (NAH), 1613 (C@N), 1579 (C@C), 1248
(CAN); 1H NMR (CDCl3, 400 MHz, d with TMS = 0): 13.23 (D2O
exchangeable NH, 1H, bs), 7.81 (1H, s), 7.79 (1H, s), 7.31–7.54
(4H, m), 7.00 (2H, s), 6.75 (1H, d, J = 7.91 Hz), 6.61 (1H, t,
J = 7.32 Hz), 6.22 (D2O exchangeable NH2, 2H, bs); 13C NMR (CDCl3,
100 MHz, d with TMS = 0): 149.11, 148.13, 145.37, 133.05, 129.13,
129.10, 128.57, 127.77, 125.14, 119.23, 115.63, 115.29, 99.58. MS
2.2.1. Evaluation of XO inhibitory activity
2.2.1.1. Principle of the XO assay. Xanthine oxidase assay is an enzy-
matic reaction in which xanthine oxidase produces uric acid during
oxidation of xanthine. Uric acid can be easily analyzed with an
absorption wavelength of 292 nm quantifying xanthine oxidase
activity [29].

60
D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
2.2.1.2. Preparation of reagents.
517 nm against the blank. Blank was prepared without the addi-
ꢂ Phosphate buffer (50 mM)
tion of DPPH. BHT was used as standard.
For the preparation of 50 mM phosphate buffer at pH 7.5,
9.4 mL (1 M) KH2PO4 (Potassium phosphate monobasic) and
40.6 mL (1 M) K2HPO4 (Potassium phosphate dibasic) was diluted
up to 1000 mL with deionised water.
2.2.2.3. Calculation of inhibition. The ability to scavenge DPPH rad-
ical was calculated by the following equation:
DPPH radical scavenging activityð%Þ ¼ ½ðAc ꢀ AsÞ=Acꢃ ꢁ 100
ꢂ Xanthine solution (0.10 mM)
where Ac = Control (Absorbance of DPPH radical + methanol),
As = Sample (Absorbance of DPPH radical + compound).
Stock solution of 0.1 (M) xanthine was prepared by dissolving
15.2 mg of xanthine in 1 mL of (1 M) NaOH solution. Working solu-
tion was made in accordance with 1
pH was maintained with 1 N HCl.
lL/mL in deionised water and
2.2.3. Molecular docking
The coordinates of the bovine milk XO complexed with salicylic
acid were obtained from the protein data bank (PDB entry: 1FIQ)
[32]. The ligands were drawn in ChemDraw and subjected to
energy minimization via MM2 force field, integrated in Chem3D
Ultra. The ligands were docked into the active sites of xanthine
oxidase using the Autodock 4.2.5.1, performs Lamarckian genetic
algorithm based ligand docking to optimize the conformation of
ligand at the receptor binding site. We utilized the grid size
60 ꢁ 60 ꢁ 60 with a centroid of 26.782 ꢁ 10.121 ꢁ 112.992 and
spacing 0.375. It utilizes energy score fitness function to evaluate
the various conformation of ligand at the binding site. This is com-
prised of various components such as binding energy, ligand effi-
ꢂ XO enzyme solutions (0.1–0.2 unit/mL)
Stock solution of 16U/mL was prepared by dissolving 1 mg of
lyophilised powder of xanthine oxidase enzyme in 1 mL of cold
phosphate buffer reagent. For working solution stock solution
was diluted up to required volume.
2.2.1.3. Procedure. The inhibitory effect on XO was measured spec-
trophotometrically at 292 nm under aerobic condition. Allopurinol
was used as a positive control for the inhibition test. The reaction
mixture consisted of 1.5 mL of 50 mM potassium phosphate buffer
(pH 7.5), 1 mL test sample solution (5, 10, 25, 50, and 100 lM) was
ciency, inhibition constant (lM), inter molecular energy, van der
dissolved in DMSO, 0.5 mL of freshly prepared XO enzyme solution
(0.2 units/mL of xanthine oxidase in phosphate buffer). The assay
mixture was pre-incubated at room temperature for 15 min. Then,
1 mL of substrate solution (0.10 mM of xanthine) was added into
the mixture. The mixture was incubated at room temperature for
30 min. Next, the reaction was stopped with the addition of 1 mL
of 1 M HCl. The absorbance was measured using UV–VIS spectro-
photometer against a blank prepared in the same way but the
xanthine was replaced with the phosphate buffer. Readings were
taken in triplicate.
Waals desolvation energy, electrostatic energy, total energy, tor-
sional energy, unbound energy. Each docking experiment was set
with 50 different runs with termination after 25,000 energy evalu-
ation. Population size was set out 100. The pose was ranked
according to its total energy Score fitness function.
3. Results and discussion
3.1. Chemistry
For the synthesis of target compounds, we followed the route as
depicted in Scheme 1. Briefly, the 2-nitrobenzaldehyde was con-
densed with aryl ketones to afford nitro substituted 1,3-diaryl
propenones (7) under acid catalyzed conditions. Introduction of
pyrazoline ring system (8) was made feasible by refluxing 7 with
hydrazine hydrate in methanol [33]. The structure of 8 was con-
firmed by the characteristic ABX pattern with three double dou-
blets in 1H NMR spectrum. Dehydrogenation of 8 to afford
pyrazole structure (9) was carried out by refluxing with iodine
and DMSO [34]. 1H NMR showed the disappearance of ABX pattern
due to aromatization and appearance of 13C NMR signal to 100–
103 ppm due to C-1 further confirmed the formation of 9. Reduc-
tion of 9 using SnCl2ꢄ2H2O in methanol under refluxing afforded
10. [35] IR spectra confirmed the formation of 10 as a sharp char-
acteristic peak of primary amine (NH2) at 3350–3380 cmꢀ1 was
obtained. The 10 was eventually cyclocondensed with triethyl
orthoformate and different aldehydes under microwave irradia-
tions to yield target compounds 5 and 6, respectively [28]. A gen-
eral observation on appearance of chemical shift values in 13C
NMR spectra ranging from 66–72 d and 140–145 d was noticed
for C-5 in case of 6 and 5, respectively (Fig. 3). All the final products
were fully characterized by mp, IR, NMR and HRMS spectral data.
2.2.1.4. Calculation of inhibition. The degree of XO inhibitory
activity was calculated by following formula
% Inhibition ¼ ½ðAc ꢀ AsÞ=Acꢃ ꢁ 100
where Ac = Absorbance of control (Sample in absence of XO inhibi-
tors), As = Absorbance of sample (Sample with potential XO
inhibitors).
2.2.2. Evaluation of antioxidant activity
Reactive oxygen species can harm various biomolecules includ-
ing DNA and consequences are oxidative stress in the biological
systems. Antioxidants are known to act via reducing the oxidative
stress by scavenging of the free radicals and therefore, prevent
many diseases including cancer. Hence, we performed DPPH assay
to study the antioxidant activity of our compounds in vitro. DPPH
assay was used to determine the total antioxidant activity by scav-
enging of the stable DPPH free radical. Antioxidant activity was
observed to escalate in the concentration dependent manner
[30,31].
2.2.2.1. Principle of DPPH assay. The principle of the assay is the use
of the stable free-radical for estimating antioxidant activity 2,2-
diphenyl-1-picrylhydrazyl (DPPH) which gets reduced and thus
stabilized in presence of a hydrogen donating substance [30].
3.2. Biological evaluation
3.2.1. XO inhibitory activity
In order to evaluate the biological activities of synthesized com-
pounds, in vitro biochemical screening of the compounds was car-
ried out using xanthine oxidase enzyme with UV–Visible
spectrophotometer at 292 nm. Four different concentrations of
compounds were used for inhibiting the xanthine oxidase enzyme
2.2.2.2. Procedure. 2 mL of the different concentrations of com-
pounds in methanol were added to 2 mL of 0.135 mM methanolic
solution of DPPH. The mixture was shaken vigorously and was left
to stand for 30 min in the dark. Absorbance was recorded at

D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
61
Scheme 1. Reagents and conditions: (i) Acetic acid, H2SO4 (4–5 drops), rt, 24 h; (ii) NH2NH2ꢄ2H2O, reflux, 2 h; (iii) I2/DMSO, reflux, 2 h; (iv) SnCl2ꢄ2H2O, methanol, reflux, 2 h;
(v) CH(OEt)3, acetonitrile, MW, 100 °C, 10–12 min; (f) RCHO, water, MW, 100 °C, 5–15 min.
All the synthesized target compounds 5a–5b, 6c–6m along with
the uncyclized compounds 10 and 10a were tested against the XO
enzyme as described in the literature [29] except compounds 6a
and 6c which were not soluble in the medium under given
conditions.
Each compound was tested in triplicate. Among the series
compounds tested, 10, 6b, 6e, 6f, 6g and 6h showed significant
xanthine oxidase inhibition and reduction in uric acid formation
(Fig. 4). Most of the compounds exhibited the good inhibitory
potential mainly at concentrations of 25
6g which was observed to exhibit the XO inhibitory activity at
M concentration. The compounds that showed the highest
lM and 50 lM except
5
l
in vitro XO inhibitory activity are collected in Table 1. Two
compounds 6g and 6h were found to be most active against XO
with IC50s 10.96 lM and 20.89 lM, respectively (Fig. 5 and Table 1)
in comparison to allopurinol (IC50 = 31.62
lM), standard inhibitor
of XO under tested conditions.
Fig. 3. Numbering to the target compounds 5 and 6.
i.e., uric acid formation as described in experimental section. XO
activity i.e. uric acid formation was started by addition of xanthine
substrate in different concentration of compounds (5, 10, 25 and
3.2.2. Structure activity relationship (SAR)
Some general notions about structure activity relationships
emerged from these studies (Fig. 3 and Table 1): (a) uncyclized
compounds 10 with no halogen substitution on ring A was found
to possess better XO inhibitory activity as compared to 10a,
whereas their products 5a and 5b, respectively obtained upon
cyclization with CH(OEt)3 were observed to be practically inactive
50 lM). The uric acid absorbance was measured spectrophotomet-
rically at 292 nm after an incubation of 30 min. The more potent
compounds tend to bind more strongly with XO and decrease the
uric acid formation.

62
D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
Fig. 4. Percent inhibition of XO with synthesized compounds at concentrations of 5 lM, 10 lM, 25 lM and 50 lM Data is expressed as mean values SD of three independent
experiments.
Table 1
activity (compare 6f, 6g and 6h with 6m). These observations
can be depicted as shown in below:
IC50 (half maximal inhibitory concentration) values of some selected compounds.
Compounds
% Inhibitiona
10
IC50 (lM)
xanthine oxidaseb
5
l
M
lM
25
l
M
50 lM
10
6b
6e
6g
9.84
8.06
4.07
35.99
17.95
33.95
9.63
13.96
7.39
47.96
23.98
42.03
25.08
19.86
11.46
69.38
57.32
46.53
60.74
45.53
62.59
73.76
87.78
59.23
41.69
NDc
43.65
10.96
20.89
31.62
6h
Allopurinol
a
b
c
Values are average means of three readings.
Values determined from the midpoint (i.e., 50% inhibition) of the semilog plot.
Not determined.
3.2.3. Antioxidant Activity
In order to confirm the antioxidant potential of best XO inhibi-
tory compounds i.e. 6g and 6h were tested for their antioxidant
potential of which both of them were observed to possess very
good antioxidant activity (6g, 99% and 6h, 96%) as they showed
significant decrease in the level of DPPH free radical (Table 2).
Butylated hydroxyltoluene (BHT) was used as positive control.
3.2.4. Molecular docking
For understanding the recognition process of R- and S-enantio-
mers of the most potent identified XO inhibitor 6g (Fig. 5) docking
experiments using Autodock software [36] were performed assum-
ing that it gets accommodated into the salicylic acid XO active site
(Fig. 6A) [32]. The docking study showed that R-isomer of 6g (6gR)
fits well into the XO active site (Fig. 6). The binding mode of 6gR
was found to be similar to those observed in with salicylic acid
[32], febuxostat [37] and curcumin [14]. The major interactions
of 6gR with XO include arene/arene interactions with Phe914,
Phe1009 and Phe649, three hydrogen bonds with Ser876 and
Fig. 5. Semilog plot of concentration vs % inhibition of xanthine oxidase (XO)
enzyme. The IC50 is identified from the midpoint (i.e., 50% inhibition) of the semilog
plot.
against XO, (b) Cyclization of 10 and 10a with aromatic aldehydes
also resulted in decrease or no change in the XO inhibitory activity
(compare 10 with 6b, 6d, 6e, 6f and 6i and compare 10a with 6j, 6k
and 6l) except in two cases where inhibitory activity was signifi-
cantly enhanced (compare 10 with 6g and 6h), (c) nitro group at
m-position on Ph at ring C was found to be more tolerable than
at o-position (compare 6f with 6h) whereas not such correlation
was noticed in case of activating groups and finally (d) in general,
change of Ph on ring C with 3-indolyl resulted in decrease in the
Table 2
Percentage inhibition of various compounds against DPPH.
S.no.
Compound
Absorbance
% Inhibition
1.
2.
3.
6g (4 mM)
6h (4 mM)
BHT (5 mM)
0.137
0.217
0.298
99
96
95

D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
63
Fig. 6. (A) Docking poses of R-isomer of 6g at the salicylic acid XO binding site (R-isomer: green color; salicylic acid: blue color) and, (B) Binding interactions of R-isomer of 6g
with the amino acid residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Thr1010 (Fig. 6B). The other amino acids such as Glu802, Asn768
and Lys771 also lie in close proximity of the compound. The phenyl
rings of Phe914 and Phe1009 lay parallel and perpendicular,
respectively to the plane of phenyl ring (ring C) of 6gR
(d = 4.59 Å and d = 3.62 Å). The phenyl residue of Phe649 lies per-
pendicular to phenyl ring (ring B) of 6gR (d = 3.67 Å). This kind of
arrangement of arene–arene interactions has been previously
observed in the co-crystal structures of XO with salicylic acid
[32] and febuxostat [37]. This conservation plays an important role
in stabilizing the binding positions of aromatic substrates and
might well represent one of the key features of the substrate recog-
nition. Three hydrogen bonds, one between NH of the inhibitor and
free OH group of Ser876, second between OH of the inhibitor and
OH of Ser876 and third between OH of the inhibitor and OH of
Thr1010 were observed and provided the rationale for binding of
inhibitor.
The ring C of the inhibitor gets positioned towards the dioxoth-
iomolybdenum (MOS) moiety at a distance of 4.70 Å indicating
that it might block the activity of the enzyme from binding to sub-
strate via hydroxylation. The docked conformation of S-enantiomer
was differing from the R-enantiomer. The favorable binding
conformation and higher interaction energy of R-enantiomer
suggests its dominant role in XO inhibitory activity.
4. Conclusions
We have rationally designed, synthesized and evaluated the XO
inhibitory activity of some dihydropyrazolo/pyrazolo[1,5-c]quina-
zolines first time. The SAR study disclosed that: (i) nature of rings
A, B and C, (ii) nature of substituents on the rings in particular on
ring C, and (iii) unsaturation i.e. 5,6-dihydropyrazolo[1,5-c]quina-
zolines were found to affect the XO inhibitory activity. Compound

64
D. Kumar et al. / Bioorganic Chemistry 57 (2014) 57–64
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and capable of scavenge the free radicals in DPPH free radical
assay. In silico studies highlighted the role of amino acid residues
involved in interactions with 6g and the results are in compliance
with the previous reported studies. Further lead optimization on
6g such as synthesis of compounds with diverse permutation and
combinations on ring A, B and C is under progress and will be pub-
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