Benzoflavone derivatives as potent antihyperuricemic agents

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RESEARCH ARTICLE

Benzoflavone derivatives as potent

antihyperuricemic agents†

Cite this: DOI: 10.1039/c8md00512e

Jatinder V. Singh,^aGurbachan Mal,^aGurleen Kaur,^aManish K. Gupta,^b

a

Amritpal Singh,^aKunal Nepali,^aHarbinder Singh,

*

a

*

Sahil Sharma and Preet Mohinder S. Bedi

Two series of benzoflavone derivatives were rationally designed, synthesized and evaluated for their xan-

thine oxidase (XO) inhibitory potential. Among both series, eight compounds (NF-2, NF-4, NF-9, NF-12,

NF-16, NF-25, NF-28, and NF-32) were found to exert significant XO inhibition with IC₅₀values lower than

10 μM. Enzyme kinetic studies revealed that the most potent benzoflavone derivatives (NF-4 and NF-28)

are mixed type inhibitors of the XO enzyme. Molecular modeling studies were also performed to investi-

gate the binding interactions of these molecules (NF-4 and NF-28) with the amino acid residues present in

the active site of the enzyme. Docking results confirmed that their favorable binding conformations in the

active site of XO can completely block the catalytic activity of the enzyme. Benzoflavone derivatives

exhibiting potent XO enzyme inhibition also showed promising results in a hyperuricemic mice model

when tested in vivo.

Received 10th October 2018,

Accepted 5th December 2018

DOI: 10.1039/c8md00512e

rsc.li/medchemcomm

significant effect on the activities of purine and pyrimidine

metabolizing enzymes have diverted researchers to develop

1. Introduction

The key role of the versatile molybdoflavoprotein xanthine ox-

idase (XO) has been longer familiar, today. There is a well-

known relationship between xanthine oxidase and hyperurice-

mia. The metabolism of purines, the oxidative hydroxylation

of hypoxanthine and xanthine to generate uric acid, and the

production of reactive oxygen species within the human body

are the primary functions of the XO enzyme.¹The potential

increase in serum uric acid level (hyperuricemia) leads to de-

position of sodium urate crystals in joints which results in in-

flammation and pain in joints. In this regard, xanthine oxi-

dase is an important and selective target for sustaining

broad-spectrum chemotherapy in hyperuricemic patients.^1,2

In the recent past, due to the success of allopurinol (a pu-

rine based xanthine oxidase inhibitor) in the treatment of hy-

peruricemia, several purine analogs like pterin and

6-formylpterin,³2-alkylhypoxanthines,^3,4and 2-substituted 7H-

pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]-pyrimidines^5–8were devel-

oped as potent xanthine oxidase inhibitors. However, the side

effects associated with these purine-based analogs and their

non-purine based xanthine oxidase inhibitors (Fig. 1).^9–11

Several non-purine based XO inhibitors have been devel-

oped so far. Among them, BOF-4272 is one of the potent XO

inhibitors but the variation in its efficacy from person to per-

son due to the difference in hepatic metabolism limits its

clinical use (Fig. 1).¹²In 2009, febuxostat got approved by the

USFDA as the first non-purine based XO inhibitor for the

treatment of hyperuricemia with prominent efficacy and lon-

ger lasting hypouricemic effect than allopurinol.^13–15But it

has certain adverse effects like liver function abnormalities,

headache, diarrhea, dizziness, and nausea similar to

allopurinol.^16–19Furthermore, a safety clinical trial carried

out by the FDA on febuxostat demonstrated that its use can

increase the risk of heart-related complications.²⁰

Working on similar lines, another notable compound,

pyranostat, was developed with potent in vivo efficacy but has

poor pharmacokinetic profile.²¹Topiroxostat, another non-

purine based XO inhibitor, was approved for use in Japan in

2013. A Japanese Phase III study revealed that the reduction

of serum uric acid by topiroxostat 120 mg per day was com-

parable to that of 200 mg per day of allopurinol but has ad-

verse effects similar to allopurinol (Fig. 1).²²

^aDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar,

Punjab 143005, India. E-mail: singh.harbinder40@gmail.com,

ss.gq2009@gmail.com, bedi_preet@yahoo.com

^bTERI-Deakin Nanobiotechnology Centre, The Energy and Resources Institute,

TERI Gram, Gual Pahari, Gurugram, Haryana 122001, India

† Electronic supplementary information (ESI) available. See DOI: 10.1039/

c8md00512e

These adverse events associated with both purine and non-

purine based xanthine oxidase inhibitors prompted re-

searchers working in this field to develop alternate scaffolds

as potent XO inhibitors that can be used for the treatment of

hyperuricemia.

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Fig. 1 Drugs used clinically for the treatment of hyperuricemia by inhibiting the xanthine oxidase enzyme.

Fig. 2 Various flavonoids as potent xanthine oxidase inhibitors.

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Among the various natural products, flavones have

attracted much attention due to their significant range of

pharmacological activities such as antioxidant, anti-

mutagenic, antibacterial, antiangiogenic, anti-inflammatory,

antiallergic, modulators of enzymatic activities and anti-

cancer activity.^23–26There are numerous reports too on the

xanthine oxidase inhibitory potential of flavones. Various

ical scavenging assay was performed to examine the antioxi-

dant potential for both the series of compounds (7,8- and

5,6-benzoflavones). Furthermore, the serum uric acid lower-

ing ability of synthesized benzoflavones exhibiting potent XO

enzyme inhibition was also investigated by using a potassium

oxonate (PO) induced hyperuricemic mice model.

reported flavones exerting considerable inhibitory potential 2. Results and discussion

against the xanthine oxidase enzyme are depicted in

2.1. Synthesis

Fig. 2.^27–33

Benzoflavone derivatives were synthesized via Scheme 1. α-

For the last five years, we have been actively involved in

the development of non-purine based xanthine oxidase

inhibitors.^34–40By keeping in view the significant XO inhibi-

tory potential of various flavonoids, we have previously

reported a series of 7,8-benzoflavone derivatives endowed

with significant XO inhibitory potential. The design was

based upon natural flavones exhibiting potent XO inhibition

taking apigenin as the lead molecule. The established struc-

ture–activity relationship for flavones reveals that their XO in-

hibitory potential is attributed to the presence of hydroxyl

groups on the benzopyran nucleus. We explored the role of

the naphthyl ring as a surrogate for the hydroxyl groups pres-

ent on the benzopyran moiety which actually enhanced the

arene–arene and hydrophobic interactions within the active

site of the xanthine oxidase enzyme.⁴¹Among all the deriva-

tives reported by our research group, this series was found to

be a prominent class exhibiting IC₅₀values ranging from

0.62–41.2 μM against the XO enzyme, which inspired us to

design a new series of compounds (i.e. 5,6-benzoflavones) by

changing the orientation of ring-A from trans- to cis-

configuration with respect to the carbonyl group of ring-C

(Fig. 3). Molecular modeling studies provided a strong ratio-

nale behind the design of this new series of molecules as 5,6-

benzoflavones found to be occupied well by the amino acid

residues present in the active site of the XO enzyme with

closer proximity as compared to the molecules of reported

7,8-benzoflavone series. Therefore, it can be suggested that

the cis-orientation of ring-A with respect to the carbonyl

group of ring-C is well tolerable for XO inhibitory potential.

In the present study, all the designed compounds were

synthesized and evaluated in vitro against the xanthine oxi-

dase enzyme by using spectrophotometric assay.^42,43The

mode of inhibition of the most potent compounds and their

binding interactions with the amino acid residues present in

the active site of the XO enzyme were also analyzed. Free rad-

and β-naphthol were subjected to Fries rearrangement. Fries

rearranged products (1a and 1b) were then benzoylated using

variously substituted benzoyl chlorides to obtain benzoylated

products (2a and 2b) which were then subjected to Baker

Venkataraman

rearrangement.

Baker

Venkataraman

rearranged products (3a & 3b) existing in the enol form (con-

firmed by the appearance of a singlet for two D₂O exchange-

able protons above 11 ppm along with the vinylic proton to

carbonyl which appeared as a merged signal in a multiplet)

were then cyclized by treatment with sulphuric acid to yield

the desired benzoflavones. All the reactions proceeded

smoothly with diverse benzoyl chlorides and products were

obtained in good yields. No retro-Diels fragmentation was ob-

served for derivatives in the mass spectrum. The structures of

all the synthesized compounds were elucidated by ¹H NMR,

¹³C NMR, MASS and elemental analysis. Spectral data were in

accordance with the assumed structures.

2.2. In vitro xanthine oxidase assay

The xanthine oxidase inhibitory potential of all the synthetics

was elucidated (Table 1) by using spectrophotometric assay

as described in the literature. Allopurinol, febuxostat, and

apigenin were used as reference compounds.^42,43Among the

previously reported 7,8-benzoflavones, compounds NF-2, NF-

4, and NF-12 exhibited IC₅₀values below 5 μM and NF-4 was

found to be the most potent XO inhibitor with an IC₅₀value

of 0.62 μM.⁴¹The established structure–activity relationship

for 7,8-benzoflavones revealed that the phenyl ring (ring-D) is

very sensitive to nature as well as the positioning of the sub-

stituent present. The substituted compounds were more ac-

tive than the unsubstituted ones. The overall preference or-

der for the substituent was found to be halo and nitro >

methoxy > methyl > hydrogen. Simultaneously, ortho and

para positioning of the substituents on ring-D is favorable for

Fig. 3 Design strategy for 5,6-benzoflavone derivatives.

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Scheme 1 Synthesis of benzoflavone derivatives. Reagents and conditions: (a) MW, ZnCl₂, CH₃COOH, 20 min; (b) various substituted benzoyl

chlorides, pyridine, stirring, rt, 1 h; (c) KOH, pyridine, warm, 15 min; (d) a drop of conc. H₂SO₄, CH₃COOH, reflux, 30 min.

XO inhibitory activity as compared to meta substitution.

Among the series of newly designed compounds (5,6-

benzoflavones), NF-28 exhibited the most potent XO inhibi-

tory activity with an IC₅₀value of 5.21 μM. Compounds with

ortho and para substituted phenyl rings were more potent

than meta-substituted compounds (see the IC₅₀values of NF-

19, NF-21, NF-24, and NF-27). It has been observed that the

stronger inductive effect at the para position favors the xan-

thine oxidase inhibitory activity (see the IC₅₀values of NF-25,

NF-28, and NF-32). The similar effect had also been seen in

the previously reported results for 7,8-benzoflavones, whereas,

the overall potency of 5,6-benzoflavones was lower than that

of 7,8-benzoflavones, which remains unjustified. The overall

results showed eight compounds (NF-2, NF-4, NF-9, NF-12,

NF-16, NF-25, NF-28, and NF-32) with IC₅₀values comparable

to that of allopurinol (IC₅₀< 10 μM) which were further se-

lected to perform in vivo experiments to check their serum

uric acid lowering ability by using a hyperuricemic mice

model. Moreover, the type of inhibition for the most potent

compounds has been analyzed by using enzyme kinetic

studies.

2.3. Enzyme kinetics

Compound NF-28 was further investigated for the type of in-

hibition by performing enzyme kinetic studies. The

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Table 1 IC₅₀values of benzoflavones

Code

Structure

Xanthine oxidase IC₅₀(μM + SD)

>20

Antioxidant IC₅₀(μM + SD)

NF-1

15.55 1.78

NF-2

NF-3

4.94 0.45

4.73 1.23

>20

10.13 1.98

NF-4

NF-5

0.62 0.10

0.83 0.14

>20

7.75 1.55

NF-6

>20

10.90 1.89

NF-7

NF-8

16.80 1.78

9.90 1.67

>20

5.98 1.45

NF-9

8.93 0.67

0.35 0.09

NF-10

19.40 1.89

7.33 1.54

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Table 1 (continued)

Code

Structure

Xanthine oxidase IC₅₀(μM + SD)

>20

Antioxidant IC₅₀(μM + SD)

NF-11

8.24 1.56

NF-12

NF-13

1.95 0.18

0.62 0.11

>20

5.55 1.55

NF-14

NF-15

NF-16

18.90 1.81

4.27 1.26

6.50 1.34

3.14 1.22

>20

6.95 0.62

NF-17

NF-18

>20

14.67 1.58

NF-19

>20

NF-20

12.44 1.34

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Table 1 (continued)

Code

Structure

Xanthine oxidase IC₅₀(μM + SD)

Antioxidant IC₅₀(μM + SD)

NF-21

NF-22

>20

18.58 1.23

NF-23

NF-24

17.07 1.63

>20

NF-25

NF-26

NF-27

8.90 0.56

>20

NF-28

5.21 0.65

>20

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Table 1 (continued)

Code

Structure

Xanthine oxidase IC₅₀(μM + SD)

>20

Antioxidant IC₅₀(μM + SD)

>20

NF-29

NF-30

NF-31

NF-32

>20

7.67 0.79

Allopurinol

Febuxostat

Apigenin

8.69

0.03

1.11^a

—

Quercetin

28.00^b

Literature value.^{41 b}Literature value.⁵²

a

Lineweaver–Burk plot (Fig. 4) observed for NF-28 was very

similar to that for NF-4.⁴¹The pattern of the graph shows

The software used can reproduce the co-crystallized confor-

mation of febuxostat with an RMSD value of 0.792 Å (Fig. 5),

which ensures the acceptability of the selected docking proto-

col. Several docked conformations of NF-28 were generated

and ranked according to their in silico binding affinity. The

conformation with the highest binding affinity (−11.1 kcal

mol⁻¹) was selected for discussion (Fig. 6).

The binding site residues and overall binding mode of NF-

28 are like those observed with febuxostat,⁴⁵salicylic acid⁴⁶

and curcumin.⁴⁷NF-28 gets stabilized at the binding cavity by

several types of electrostatic interactions. The major interac-

tions between NF-28 and XO include π–π stacking with Phe914

and Phe1009 and two hydrogen bonds with Ser876 and Arg880

(Fig. 6). Rings A and B get positioned in a cavity formed by hy-

drophobic residues Leu648, Phe649, Leu873, Leu1014, and

Pro1076. The carbonyl function at ring C has shown H-bond

interaction with the hydroxyl group of Ser876 (H-bond donor;

d = 2.254 Å). Another H-bond was observed between the N⁺–O⁻

function at ring D and terminal –NH₂of Arg880 (H-bond do-

nor; d = 2.251 Å). The phenyl rings of Phe914 and Phe1009 are

involved in face-to-face and edge-to-face π–π stacking interac-

tions, respectively, with ring D (d = 3.372 Å and 3.560 Å). This

that it is a form of mixed inhibition scenario where K_m, V_max

,

and the slope are all affected by the inhibitor. The inhibitor

has increased the K_mand slope (K_m/V_max) while decreasing

the V_max. Moreover, on careful observation, it was found that

intersecting lines on the graph converge to the left of the

y-axis and above the x-axis which indicates that the value of

α (a constant that defines the degree to which inhibitor bind-

ing affects the affinity of the enzyme for the substrate) is

greater than 1.⁴⁴This confirms that the inhibitor preferen-

tially binds to the free enzyme and not the enzyme–substrate

complex. Therefore, the mode of inhibition of NF-28 is

mixed-type but it seems that it has a strong competitive

component.

2.4. Docking study

To understand the binding conformation of inhibitors, the

potent XO inhibitors NF-4 and NF-28 were docked into the

febuxostat binding site. The docking procedure was validated

by reproducing a co-crystallized conformation of febuxostat.

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Fig. 4 Lineweaver–Burk plot of NF-28.

arrangement of energetically favorable π–π stacking interac-

tions was also seen in the co-crystal structure of XO with

febuxostat and salicylate.^45,46Its conservation argues for an

important role in stabilizing the binding positions of aromatic

substrates and it might well represent one of the key features

of substrate recognition.

We also compared the docked conformations of the previ-

ously reported most potent compounds NF-4 and NF-28 in

the febuxostat binding site using AutoDock Vina for a clear

and deep perception behind the potency against XO (Fig. 7).

The binding affinity of NF-4 for XO was found to be −9.5 kcal

mol⁻¹, which is approximately 14% lower than that of NF-28.

The docked conformation of both inhibitors (NF-4 and NF-

28) showed that, in both cases, ring-A and ring-B get posi-

tioned in a cavity formed by hydrophobic residues Leu648,

Phe649, Leu873, Leu1014, and Pro1076. The major differ-

ences in the binding modes of NF-4 and NF-28 include the

opposite orientation of ring-C and the higher distances be-

tween NF-4 and important binding site residues of XO

(Table 2, Fig. 7). The carbonyl function at ring-C adopted an

opposite orientation to that of NF-28 and is involved in

H-bonding interaction with the terminal –NH₂group of

Asn768 (d = 2.855 Å; 0.601 Å higher than that of NF-28 where

the carbonyl function makes a H-bond with Ser876). Also, the

distances between ring-D and Phe914 (face-to-face π–π stack-

ing interactions) and the 4-fluoro group at ring-D and Arg880

were also observed to be higher than those of NF-28

(Table 2). Therefore, according to computational studies, this

can suggest that ring-A in cis orientation with the carbonyl

function at ring-C is more favorable for binding than its trans

orientation and thus a series of 5,6-benzoflavones could be

more active than the reported 7,8-benzoflavone series which

was the rationale behind the synthesis of the current series

of molecules. But according to in vitro results, the molecules

of the previous series were found to be more active than the

molecules of the new series.

2.5. Antioxidant activity (DPPH assay)

During the oxidative hydroxylation of hypoxanthine and xan-

thine to produce uric acid, the reduction of oxygen at the fla-

vin center simultaneously generated a superoxide anion radi-

cal or hydrogen peroxide.¹Therefore, it would be beneficial if

the compounds with XO inhibitory potential can possess

antioxidant potential as well. 1,1-Diphenyl-2-picrylhydrazyl

(DPPH) radical scavenging assay was used to determine the

free radical scavenging ability of all the synthesized com-

pounds. The results of DPPH assay (Table 1) revealed that

some of the compounds have significant antioxidant poten-

tial. Among the active compounds, NF-9 was found to be the

most potent antioxidant with an IC₅₀value of 0.35 μM,

followed by NF-4 and NF-12 with IC₅₀values of 0.83 μM and

0.62 μM, respectively. Surprisingly, 5,6-benzoflavones were

found to be less potent as compared to 7,8-benzoflavones

with IC₅₀values greater than 20 μM for all the compounds.

Compounds with para substitution on the phenyl ring (ring

D) are more active than the ortho and meta-substituted

Fig. 5 Superimposition of co-crystalized (green) and docked confor-

mations (magenta) of febuxostat (RMSD: 0.792 Å).

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Fig. 6 Docking conformation of NF-28 at the febuxostat binding site (hydrogen atoms which are involved in H-bond interactions are shown).

compounds whereas the unsubstituted compound (NF-1)

showed moderate antioxidant activity.

the synthesized compounds cannot alter the normal activity of neu-

ronal cells. The basicity and lipophilicity of all the synthesized com-

pounds were determined with ChemAxon software MarvinSketch

and the results are shown in Table 4 which defines the compliance

of all the synthesized compounds with the Lipinski rule of five.

Tabular values indicate that a) all the molecules have a molecular

weight in the range of 286–408 which lies in the limit of 180–500;

b) compounds have no H-bond donating property; c) compounds

follow the H-bond acceptor criteria (<10); d) molar refractivities

are consistent and in the range of 83.64–98.07 which lies well in

the permitted range of 40–130 and e) log P of all the compounds is

lower than 5.6 which indicates that the compounds are not very li-

pophilic. These results suggest that all the compounds follow the

Lipinski rule of five and ADME properties which makes them phar-

macologically efficient for use in the animal model. Therefore,

2.6. In silico study

Furthermore, in silico physicochemical properties like absorption,

distribution, metabolism, and excretion (ADME) of the synthesized

compounds were determined by http://www.chemaxon.com/) and

PreADMET (http://preadmet.bmdrc.org/). Particularly, Caco-2 cell,

MDCK cell, blood brain barrier (BBB) & skin permeabilities, hu-

man intestinal absorption and plasma protein binding affinities

were predicted by the above-mentioned applications and are sum-

marized in Table 3. The results indicate that the compounds are

predicted to have lower blood–brain barrier permeation which is

less likely to cause neurotoxicity. In other words, we can say that

Fig. 7 Superimposition of the docked conformations of NF-4 (magenta) and NF-28 (green).

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Table 2 Differences in bond distances between XO and docked inhibi-

tors NF-4 and NF-28

fore, it is expected that these potent compounds can act as

clinical candidates for the treatment of hyperuricemia.

Distance^aÅ

S.

Difference^b

0.601^c

2.8. Acute toxicity study

no.

Residues

NF-4

NF-28

Acute toxicity studies were performed for the two highly po-

tent compounds (NF-2 and NF-32), according to OECD guide-

lines 2001.⁴⁸No gross behavioral abnormality was found in

any of the three groups of animals within 24 h after the ad-

ministration of test compounds.

1

2

3

4

5

Asn768

Ser876

Arg880

Phe914

Phe1009

2.855

—

2.619

4.306

3.421

—

2.254

2.251

3.732

3.560

0.368

0.574

−0.139

a

b

Refer to Fig. 7 for bond details. Difference between NF-4 and NF-

c

28. Difference in H-bond distance involving Asn768 and Ser876.

3. Conclusion

The present study is a follow-up to a previous report on xan-

thine oxidase inhibitors, 7,8-benzoflavones (naphthoflavones),

in which 5,6-benzoflavone derivatives were rationally

designed and synthesized. All the synthetics were evaluated

in vitro to check their inhibitory potential against xanthine

oxidase by using spectrophotometric assay. Many compounds

exhibited significant XO inhibition among which eight com-

pounds exhibited remarkable XO inhibitory potential. The

biological assay elucidated some interesting aspects about

the structure–activity relationship which stated that the sub-

stituents on the phenyl ring (ring-D) remarkably influence

the XO inhibitory activity. The para position on ring-D is the

most reliable one for the inhibitory activity of the com-

pounds. The mode of inhibition was also analyzed by using a

Lineweaver–Burk plot which showed a mixed type inhibition

pattern for the most potent compounds against XO enzyme.

The docking study suggests that the compounds NF-4 and

NF-28 fit well in the active site of the XO enzyme and

completely block its catalytic assembly. The compounds were

also found to exhibit antioxidant potential when evaluated by

DPPH assay. Moreover, in silico parameters revealed that

compounds with improved XO inhibitory potential can be

used for in vivo analysis. Eight potent compounds were tested

for their hypouricemic effect in a potassium oxonate-induced

hyperuricemic mice model. The results of the in vivo experi-

ment showed that the compounds can considerably reduce

the serum uric acid comparable to allopurinol and

febuxostat. It was found noteworthy that no sign of toxicity

was observed when the mice were administered with a 2000

mg kg⁻¹dose of benzoflavones. Thus, the overall study distin-

guished three compounds NF-2, NF-25 and NF-32 with a pro-

found pharmacological profile in both in vitro and in vivo

screening which can act as clinical candidates for the treat-

ment of hyperuricemia.

eight potent xanthine oxidase inhibitors were chosen for in vivo ex-

periments to evaluate their serum uric acid lowering capacity.

2.7. In vivo antihyperuricemic activity

Owing to their XO inhibitory potential, eight compounds

were subjected for the evaluation of antihyperuricemic effects

by using a potassium oxonate-induced hyperuricemic mice

model. The mean serum uric acid level of normal mice was

found to be 1.8 0.2 mg dL⁻¹. A significant enhancement

was observed in the serum uric acid level (5.9 0.42 mg dL⁻¹

)

with the administration of potassium oxonate (PO) and xan-

thine (Xn). Allopurinol and febuxostat were used as standard

drugs for comparison, which reduced the serum uric acid

level of potassium oxonate-induced hyperuricemic mice to

1.8 and 1.5 mg dL⁻¹, respectively. The serum uric acid levels

of potassium oxonate-induced hyperuricemic mice treated

with the synthesized compounds exhibited a dose-dependent

hypouricemic effect. All the compounds showed a significant

reduction in serum uric acid when tested at three dose levels

(1 mg kg⁻¹, 5 mg kg⁻¹& 10 mg kg⁻¹). Compounds NF-2, NF-9,

NF-16, NF-25 and NF-32 decreased the uric acid level ranging

from 1.8–2.8 mg dL⁻¹even at 1 mg kg⁻¹which is comparable

to standard drugs. These five compounds were further evalu-

ated at a dose level of 0.5 mg kg⁻¹but a significant decrease

in their hypouricemic effect was observed at this concentra-

tion. Two compounds NF-25 and NF-32 were found to exhibit

a hypouricemic effect comparable to febuxostat at a dose

level of 5 mg kg⁻¹. Compound NF-28 was found to be less po-

tent in vivo and showed a significant hypouricemic effect only

at a dose level of 10 mg kg⁻¹whereas compounds NF-4 and

NF-12 were not able to reduce serum uric acid considerably

even at a dose level of 10 mg kg⁻¹. The overall study revealed

that six compounds (NF-2, NF-9, NF-16, NF-25, NF-28, and

NF-32 except for NF-4 & NF-12) can reduce the serum uric

acid to its normal level. Careful examination of Fig. 8 and 9

shows that there is no significant difference between the

hypouricemic effect of standard drugs used and compounds

NF-2, NF-9, NF-16, NF-25 and NF-32 at 5 and 10 mg kg⁻¹.

Thus, the overall potency order of the compounds in compar-

ison with febuxostat (at 5 mg kg⁻¹) is as follows: NF-32 > NF-

25 > NF-2 > NF-9 > NF-16 > NF-28 > NF-12 > NF-4. There-

4. Future perspective

In the recent past, the search for purine and non-purine

based xanthine oxidase inhibitors for the treatment of hyper-

uricemia has been successfully carried out. But various side

effects associated with both purine and non-purine based

scaffolds limit their clinical use. The development of poten-

tial xanthine oxidase inhibitors based on natural moieties

like flavonoids is the future perspective in this area of

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Table 3 In silico ADME properties of benzoflavone derivatives

Absorption

Distribution

Human intestinal

absorption

(HIA)%

In vitro Caco-2 cell

In vitro MDCK cell

In vitro skin

permeability

(log K_p) cm h⁻¹

In vitro plasma

protein binding

(%)

In vivo blood brain

barrier penetration

(C. brain/C. blood)

permeability

Compound

(nm s⁻¹

)

(nm s⁻¹

)

NF-1

NF-2

NF-3

NF-4

NF-5

NF-6

NF-7

NF-8

100

56.74

54.20

54.21

55.39

47.71

49.57

46.32

46.46

46.45

46.34

18.77

6.12

17.99

57.27

56.23

52.39

56.73

54.71

54.75

55.39

47.91

50.14

46.44

46.57

46.46

46.45

21.43

15.17

20.38

57.28

56.22

73.77

35.60

21.03

29.51

11.79

26.74

29.92

0.19

0.14

0.08

0.26

1.74

−2.64

−2.95

−2.97

−2.93

−2.71

−2.62

−2.56

−2.58

−2.62

−3.67

−3.72

−2.79

−2.55

−3.11

−2.65

−2.91

−2.94

−2.70

−2.62

−2.57

−2.58

−2.62

−2.86

−2.83

−2.80

−2.56

−3.12

94.25

95.32

98.77

98.84

95.46

99.82

100

99.96

100

99.48

99.80

100

93.78

93.85

100

95.97

98.40

100

2.89

2.63

2.05

0.23

0.96

0.98

1.89

0.85

0.45

2.07

3.33

2.10

2.17

0.07

0.60

1.11

2.75

2.17

0.28

0.24

0.42

0.80

1.27

0.45

0.46

1.07

0.01

0.04

0.08

0.36

0.30

NF-9

100

NF-10

NF-11

NF-12

NF-13

NF-14

NF-15

NF-16

NF-17

NF-18

NF-19

NF-20

NF-21

NF-22

NF-23

NF-24

NF-25

NF-26

NF-27

NF-28

NF-29

NF-30

NF-31

NF-32

91.92

75.68

98.80

100

0.20

0.09

9.21

15.97

19.45

50.02

20.11

27.06

3.48

26.61

20.56

0.16

0.12

0.01

0.23

0.42

100

96.47

100

98.49

96.03

98.80

100

95.25

96.00

94.55

95.48

95.75

100

0.33

0.06

2.31

5.38

100

10.53

research. As observed, the modification of the flavone moiety

to benzoflavone was found to result in an improved and sig-

nificant xanthine oxidase inhibitory activity. Their potent

antioxidant potential as well as their ability to reduce the

high serum uric acid level in mice makes them a more effec-

tive and resourceful medication for hyperuricemia. By further

optimization of these promising hit molecules by pharmaco-

kinetic and chronic toxicity studies, we can conclude whether

the benzoflavones are fit for clinical use or not.

singlet, d – doublet, m – multiplet) and coupling constants

(J) in Hz (Hertz) in the solvent indicated. HRMS was recorded

on

a micrOTOF-QII Bruker Daltonik LC-MS/MS high-

resolution mass spectrometer. Melting points were deter-

mined in open capillaries and were uncorrected.

5.1.1. Procedure for the synthesis of 1-(1-hydroxy-

naphthalen-2-yl)ethanone (1a).⁴¹α-Naphthol (1 mmol) was

treated with glacial acetic acid (1.2 mmol) in the presence of

ZnCl₂(0.41 mmol) under microwave irradiation for 20 min at

200 °C. The crude mixture was dissolved in methanol and

adsorbed on silica (60–120 #). The desired product was puri-

fied by column chromatography with an increasing percent-

age of ethyl acetate in hexane as an eluting solvent. The char-

acterization data for 1-(1-hydroxynaphthalen-2-yl)ethanone are

as follows: yield: 74%, ¹H NMR (CDCl₃, 300 MHz, δ, TMS =

0): 14.01 (1H, s, OH), 8.45 (1H, d, J = 8.4 Hz, ArH), 7.75 (1H,

d, J = 8.1 Hz, ArH), 7.60–7.65 (2H, m, ArH), 7.52 (1H, m,

ArH), 7.26 (1H, d, J = 8.7 Hz, ArH), 2.69 (3H, s, COCH₃). Anal.

calcd. for C₁₂H₁₀O₂: C, 77.40; H, 5.41; O, 17.18; found: C,

77.71; H, 5.32.

5. Experimental

5.1. Materials and measurements

The reagents were procured from Sigma Aldrich, CDH and

Loba, India and used without further purification. All yields

refer to isolated products after purification. Products were

characterized by comparison with authentic samples and by

spectroscopic methods (¹H, ¹³C NMR and MASS). ¹H NMR

and ¹³C NMR spectra were recorded on a JEOL AL 300 NMR

spectrometer. The spectra were measured in CDCl₃relative to

1

TMS (0.00 ppm). In H NMR chemical shifts were reported in

5.1.2. Procedure for the synthesis of 1-(2-hydroxy-

naphthalen-1-yl)ethanone (1b). β-Naphthol (1 mmol) was

treated with glacial acetic acid (1.2 mmol) in the presence of

δ values using tetramethylsilane as an internal standard with

the number of protons, multiplicities (s – singlet, bs – broad

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Table 4 Physicochemical parameters of benzoflavone derivatives

Compound

Molecular weight

No. of H-bond donors

No. of H-bond acceptors

Molar refractivity

log P

No. of Lipinski violation

NF-1

NF-2

NF-3

NF-4

NF-5

NF-6

NF-7

NF-8

272

290

306

341

351

398

317

362

302

286

308

272

290

306

341

351

398

317

362

302

286

308

0

2

4

6

3

2

4

6

3

2

83.42

83.64

88.23

93.03

91.04

96.78

90.75

98.07

89.88

88.46

83.85

83.42

83.64

88.23

93.03

91.04

96.78

90.75

98.07

89.88

88.46

83.85

3.96

4.10

4.56

5.16

4.73

4.89

3.90

3.84

3.80

4.47

4.24

3.90

4.10

4.56

5.16

4.73

4.89

3.90

3.84

3.80

4.47

4.24

0

NF-9

NF-10

NF-11

NF-12

NF-13

NF-14

NF-15

NF-16

NF-17

NF-18

NF-19

NF-20

NF-21

NF-22

NF-23

NF-24

NF-25

NF-26

NF-27

NF-28

NF-29

NF-30

NF-31

NF-32

Fig. 8 Effect of compounds on the serum uric acid levels in a potassium oxonate-induced hyperuricemic mice model. The values are shown as

the mean S.E.M (n = 6). ^ap < 0.05 compared with the control (vehicle), ^bp < 0.05 compared with the hyperuricemic control.

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Fig. 9 Percent reduction in serum uric acid (SUA) level in a hyperuricemic mice model [the SUA level in the control (1.8 mg dL⁻¹) is 70% lower

than the SUA level in hyperuricemic mice (5.9 mg dL⁻¹)].

ZnCl₂(0.41 mmol) under microwave irradiation for 20 min at

200 °C. The crude mixture was dissolved in methanol and

adsorbed on silica (60–120 #). The desired product was puri-

fied by column chromatography with an increasing percent-

age of ethyl acetate in hexane as an eluting solvent. The char-

acterization data for 1-(2-hydroxynaphthalen-1-yl)ethanone are

as follows: yield: 60%, ¹H NMR (CDCl₃, 300 MHz, δ, TMS =

0): 13.48 (1H, s, OH), 8.09 (1H, d, J = 8.4 Hz, ArH), 7.89 (1H,

d, J = 9.0 Hz, ArH), 7.78 (1H, d, J = 8.1 Hz, ArH), 7.55–7.60

(1H, m, ArH), 7.37–7.42 (1H, m, ArH), 7.14 (1H, d, J = 9.0 Hz,

ArH), 2.87 (3H, s, COCH₃). Anal. calcd. For C₁₂H₁₀O₂: C,

77.40; H, 5.41; O, 17.18; found: C, 77.32; H, 5.55.

7.55 (4H, m, ArH), 7.36–7.40 (1H, m, ArH), 2.62 (3H, s,

COCH₃); anal. calcd. for C₁₉H₁₄O₃: C, 78.61; H, 4.86; found:

C, 78.93; H, 4.61.

5.1.5. Procedure for the synthesis of 1-(1-hydroxy-

naphthalen-2-yl)-3-phenylpropane-1,3-dione (3a).⁴¹A mixture

of 2-acetylnaphthalen-1-yl benzoate (0.01 mmol), KOH (1

mmol) and pyridine (2 ml) was warmed on a water bath for

30 min. Acetic acid solution (10%, 1.3 ml) was added to the

cooled mixture and the solid intermediate was collected by

filtration and dried. The characterization data for

1-(1-hydroxynaphthalen-2-yl)-3-(3-bromophenyl)propane-1,3-di-

1

one are as follows: yield: 72%; H NMR (CDCl₃, 300 MHz, δ,

5.1.3. Procedure for the synthesis of 2-acetylnaphthalen-1-

yl benzoate (2a).⁴¹To a solution of 1-(1-hydroxynaphthalen-2-

yl)ethanone (0.01 mol) in pyridine (5 ml), benzoylchloride

(0.01 mol) was added and stirred for 1 h at room tempera-

ture. The mixture was poured on ice and the precipitated

solid was collected and dried. The characterization data for

2-acetylnaphthalen-1-yl benzoate are as follows: yield: 80%,

¹H NMR (CDCl₃, 300 MHz, δ, TMS = 0): 8.34 (2H, d, J = 7.8

Hz, ArH), 8.00 (1H, d, J = 8.4 Hz, ArH), 7.81–7.93 (3H, m,

ArH), 7.71 (1H, m, ArH), 7.51–7.61 (4H, m, ArH), 2.63 (3H, s,

COCH₃); anal. calcd. for C₁₉H₁₄O₃: C, 78.61; H, 4.86; found:

C, 78.93; H, 4.61.

5.1.4. Procedure for the synthesis of 1-acetylnaphthalen-2-

yl benzoate (2b). To a solution of 1-(1-hydroxynaphthalen-2-

yl)ethanone (0.01 mol) in pyridine (5 ml), benzoylchloride

(0.01 mol) was added and stirred for 1 h at room tempera-

ture. The mixture was poured on ice and the precipitated

solid was collected and dried. The characterization data for

1-acetylnaphthalen-2-yl benzoate are as follows: yield: 84%,

¹H NMR (CDCl₃, 300 MHz, δ, TMS = 0): 8.19–8.21 (2H, m,

ArH), 7.81–7.96 (3H, m, ArH), 7.64–7.67 (1H, m, ArH), 7.53–

TMS = 0): 15. 2 (1H, s, D₂O exchangeable proton), 13.68 (1H,

s, D₂O exchangeable proton), 8.47 (1H, d, J = 8.4 Hz, ArH),

7.60–7.72 (3H, m, ArH), 7.69–7.77 (3H, m, ArH), 7.54 (1H, m,

ArH), 7.43 (1H, m, ArH), 7.26–7.36 (3H, m, ArH). Anal. calcd.

for C₁₉H₁₄O₃: C, 78.61; H, 4.86; O, 16.53; found: C, 78.91; H,

4.62.

5.1.6. Procedure for the synthesis of 1-(2-hydroxy-

naphthalen-1-yl)-3-phenylpropane-1,3-dione (3b). A mixture

of 1-acetylnaphthalen-2-yl benzoate (0.01 mmol), KOH (1

mmol) and pyridine (2 ml) was warmed on a water bath

for 30 min. Acetic acid solution (10%, 1.3 ml) was added

to the cooled mixture and the solid intermediate was col-

lected by filtration and dried. The characterization data for

1-(2-hydroxynaphthalen-1-yl)-3-(2-bromophenyl)propane-1,3-di-

one are as follows: yield: 65%; ¹H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 11.35 (1H, s, D₂O exchangeable proton), 8.27–

8.30 (1H, d, J = 8.4 Hz, ArH), 7.90 (1H, s, ArH), 7.67–7.70

(1H, m, ArH), 7.39–7.42 (1H, m, ArH), 7.15–7.26 (5H, m,

ArH), 6.66 (1H, s, –CH–), 1.29 (2H, s, COCH₂). Anal. calcd.

for C₁₉H₁₄O₃: C, 78.61; H, 4.86; O, 16.53; found: C, 78.72;

H, 4.73.

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5.1.7. Procedure for the synthesis of benzoflavones (NF-1

and NF-32). To a solution of phenylpropane-1,3-diones (1

mmol) in acetic acid (5 ml) a drop of concentrated sulfuric

acid was added and the mixture was refluxed for 1 hour. The

cooled mixture was poured into ice and the product was col-

lected by simple filtration. The characterization data for all

5.1.7.6. 2-(2,3-Dichlorophenyl)-4H-benzoĳh]chromen-4-one

(NF-6).⁴¹Yield: 83%; mp: 65–72 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 8.31 (1H, d, J = 8.1 Hz, ArH), 8.18 (1H, d, J

= 8.4 Hz, ArH), 7.97 (1H, d, J = 8.1 Hz, ArH), 7.82 (1H, d, J =

8.7 Hz, ArH), 7.64–7.75 (3H, m, ArH), 7.59 (1H, d, J = 7.8 Hz,

ArH), 7.41 (1H, m, ArH), 6.79 (1H, s, –CH–); ¹³C NMR (CDCl₃,

75 MHz, δ, TMS = 0): 114.1, 120.5, 122.5, 125.7, 127.3, 127.8,

128.1, 129.2, 129.6, 132.7, 161.8. MS: m/z: 342 (M⁺+ 1); anal.

calcd. for C₁₉H₁₀Cl₂O₂: C, 66.89; H, 2.95; Cl, 20.78; found: C,

67.10; H, 2.77; Cl, 20.98.

the synthetics are given below:

41

5.1.7.1. 2-Phenyl-4H-benzoĳh]chromen-4-one (NF-1).

Yield:

72%; mp: 167–169 °C; ¹H NMR (CDCl₃, 300 MHz, δ, TMS =

0): 8.56 (1H, bs, ArH), 8.13 (1H, d, J = 8.4 Hz, ArH), 7.91–7.99

(3H, m, ArH), 7.69–7.76 (6H, m, ArH), 6.98 (1H, s, –CH–); ¹³C

NMR (CDCl₃, 75 MHz, δ, TMS = 0): 108.6, 120.1, 120.6, 122.3,

124.0, 125.4, 126.2, 127.2, 128.2, 129.2, 129.3, 131.6, 131.8,

136.0, 153.6, 162.8, 178.3. MS: m/z: 273 (M⁺+ 1); anal. calcd.

for C₁₉H₁₂O₂: C, 83.81; H, 4.44; found: C, 83.53; H, 4.68.

5.1.7.2. 2-(2-Fluorophenyl)-4H-benzoĳh]chromen-4-one (NF-

5.1.7.7. 2-(2-Bromophenyl)-4H-benzoĳh]chromen-4-one (NF-7). ⁴¹

1

Yield: 67%; mp: 143–145 °C; H NMR (CDCl₃, 300 MHz, δ, TMS

= 0): 8.57 (1H, d, J = 8.1 Hz, ArH), 8.19 (1H, d, J = 8.1 Hz), 7.94

(1H, d, J = 7.8 Hz, ArH), 7.65–7.82 (5H, m, ArH), 7.40–7.53 (2H,

m, ArH), 6.76 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS

= 0): 113.9, 120.1, 120.6, 121.8, 122.6, 124.1, 125.5, 127.2, 127.8,

128.1, 129.4, 131.2, 132.0, 134.1, 134.1, 136.0, 154.0, 163.3,

178.0. MS: m/z: 352 (M⁺+ 1); anal. calcd. for C₁₉H₁₁BrO₂: C,

64.98; H, 3.16; Br, 22.75; found: C, 65.13; H, 3.36.

41

1

2). Yield: 71%; mp: 118–120 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.55 (1H, m, ArH), 8.17 (1H, d, J = 8.7 Hz, ArH),

7.92–8.05 (2H, m, ArH), 7.69–7.79 (3H, m, ArH), 7.55 (1H, m,

ArH), 7.24–7.41 (2H, m, ArH), 6.91 (1H, s, –CH–); ¹³C NMR

(CDCl₃, 75 MHz, δ, TMS = 0): 113.2, 113.4, 117.0, 117.3,

120.1, 120.4, 120.7, 122.4, 124.0, 124.4, 124.8, 125.4, 127.2,

128.2, 129.0, 129.3, 132.8, 133.0, 136.0, 153.7, 158.3, 158.8,

5.1.7.8. 2-(3-Bromophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

1

8). Yield: 74%; mp: 130–132 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.48 (1H, m, ArH), 8.09 (2H, m, ArH), 7.69–7.89

(6H, m, ArH), 7.41 (1H, m, ArH), 6.93 (1H, s, –CH–); ¹³C NMR

(CDCl₃, 75 MHz, δ, TMS = 0): 109.2, 120.0, 120.5, 122.2,

123.3, 123.8, 124.8, 125.6, 127.3, 128.2, 129.1, 129.4, 130.0,

130.7, 133.7, 134.4, 136.0, 153.5, 161.0, 178.0. MS: m/z: 352

(M⁺+ 1); anal. calcd. for C₁₉H₁₁BrO₂: C, 64.98; H, 3.16; Br,

22.75; found: C, 65.24; H, 2.98.

162.2, 178.2. MS: m/z: 291 (M⁺

+ 1); anal. calcd. for

C₁₉H₁₁FO₂: C, 78.61; H, 3.82; F, 6.54; found: C, 78.73; H, 3.54.

5.1.7.3. 2-(3-Fluorophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

1

3). Yield: 71%; mp: 143–145 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.57 (1H, bs, ArH), 8.15 (1H, d, J = 8.7 Hz, ArH),

7.95 (1H, m, ArH), 7.78 (2H, d, J = 8.4 Hz, ArH), 7.71–7.74

(3H, m, ArH), 7.55 (1H, m, ArH), 7.55 (1H, m, ArH), 7.28 (1H,

d, J = 8.4 Hz, ArH), 6.95 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75

MHz, δ, TMS = 0): 108.3, 112.1, 112.4, 117.3, 117.6, 119.2,

119.6, 120.9, 121.2, 123.0, 124.6, 126.3, 127.3, 128.4, 129.8,

129.9, 133.0, 133.1, 135.0, 152.5, 160.2, 160.4, 163.7, 177.104.

MS: m/z: 291 (M⁺+ 1); anal. calcd. for C₁₉H₁₁FO₂: C, 78.61; H,

3.82; F, 6.54; found: C, 78.83; H, 3.64.

5.1.7.9. 2-(4-Bromophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

1

9). Yield: 77% mp: 150–152 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.57 (1H, m, ArH), 8.18 (1H, d, J = 8.4 Hz, ArH),

7.96 (1H, m, ArH), 7.89 (2H, d, J = 8.4 Hz, ArH), 7.79 (1H, d, J

= 8.7 Hz, ArH), 7.70–7.73 (4H, m, ArH), 6.96 (1H, s, –CH–);

¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 108.9, 120.2, 120.7,

122.2, 125.6, 126.3, 127.3, 127.6, 128.3, 129.4, 130.8, 132.5,

136.1, 161.7, 178.2. MS: m/z: 350 (M⁺); anal. calcd. for

C₁₉H₁₁BrO₂: C, 64.98; H, 3.16; Br, 22.75; found: C, 64.67; H,

2.96.

5.1.7.4. 2-(4-Fluorophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

1

4). Yield: 78%; mp: 154–156 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.55 (1H, bs, ArH), 8.14 (1H, d, J = 8.7 Hz, ArH),

7.92–8.07 (3H, m, ArH), 7.77 (1H, d, J = 8.7 Hz, ArH), 7.64–

7.74 (2H, m, ArH), 7.23–7.29 (2H, m, ArH), 6.91 (1H, s, –CH

–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 108.5, 116.3, 116.6,

120.0, 120.6, 122.2, 124.0, 125.5, 127.2, 128.0, 128.3, 128.4,

128.5, 129.3, 136.0, 153.5, 161.8, 178.1. MS: m/z: 291 (M⁺+ 1);

anal. calcd. for C₁₉H₁₁FO₂: C, 78.61; H, 3.82; F, 6.54; found:

C, 79.00; H, 3.53.

5.1.7.10. 2-(2-Iodophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

10).

Yield: 69%; mp: 140–141 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 8.61 (1H, m, ArH), 8.18 (1H, m, ArH), 8.06

(1H, m, ArH), 7.93 (1H, m, ArH), 7.78–7.92 (1H, m, ArH),

7.55–7.70, (4H, m, ArH), 7.24 (1H, m, ArH), 6.68 (1H, s, –CH

–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 95.3, 113.6, 120.2,

120.6, 122.8, 124.1, 125.6, 127.2, 128.1, 128.5, 129.4, 130.8,

131.9, 136.0, 138.0, 140.5, 154.0, 165.2, 178.2. MS: m/z: 399

(M⁺+ 1); anal. calcd. for C₁₉H₁₁IO₂: C, 57.31; H, 2.78; I, 31.87;

found: C, 57.62; H, 2.94.

5.1.7.5. 2-(3-Chlorophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

1

5). Yield: 76%; mp: 140–142 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.57 (1H, bs, ArH), 8.15 (1H, d, J = 8.7 Hz, ArH),

7.99 (1H, s, ArH), 7.93 (1H, m, ArH), 7.87 (1H, d, J = 6.9 Hz,

ArH), 7.71–7.80 (3H, m, ArH), 7.48–7.56 (2H, m, ArH), 6.94

(1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 109.4,

120.2, 120.6, 122.3, 124.0, 124.3, 125.6, 126.3, 127.3, 128.3,

129.4, 130.5, 131.5, 133.7, 135.4, 136.1, 153.5, 161.1, 178.0.

MS: m/z: 307 (M⁺+ 1); anal. calcd. for C₁₉H₁₁ClO₂: C, 74.40;

H, 3.61; Cl, 11.56; found: C, 74.45; H, 3.31.

5.1.7.11. 2-(3-Nitrophenyl)-4H-benzoĳh]chromen-4-one (NF-

41

11).

Yield: 79%; mp: 139–141 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 8.89 (1H, s), 8.59 (1H, d, J = 8.4 Hz, ArH),

8.43 (1H, d, J = 7.6 Hz, ArH), 8.30 (1H, d, J = 7.8 Hz, ArH),

8.16 (1H, d, J = 8.7 Hz, ArH), 7.95 (1H, m, ArH), 7.71–7.83

(4H, m, ArH), 7.04 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz,

δ, TMS = 0): 110.1, 120.3, 120.5, 121.1, 122.2, 123.9, 125.8,

125.9, 127.6, 128.4, 129.6, 130.4, 131.7, 133.7, 136.2, 148.8,

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153.5, 159.9, 177.8. MS: m/z: 318 (M⁺+ 1); anal. calcd. for

C₁₉H₁₁NO₄: C, 71.92; H, 3.49; N, 4.41; found: C, 72.13; H,

3.10; N, 4.24.

(5H, m, ArH), 6.93 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz,

δ, TMS = 0): 108.6, 119.1, 120.4, 122.5, 124.4, 125.3, 126.4,

127.5, 128.3, 129.3, 129.2, 131,6, 131.34, 136.6, 153.3, 162.2,

178.3. MS: m/z: 273 (M⁺+ 1); anal. calcd for C₁₉H₁₂O₂: C,

83.81; H, 4.44; found: C, 83.59; H, 4.54.

5.1.7.12. 2-(4-Nitrophenyl)-4H-benzoĳh]chromen-4-one (NF-

1

12). ⁴¹Yield: 76%; mp: 73–75 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 8.60 (1H, m, ArH), 8.45 (2H, d, J = 9.00 Hz, ArH),

8.18 (3H, m, ArH), 8.01 (1H, m, ArH), 7.67–7.86 (3H, m, ArH),

7.08 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0):

110.8, 120.3, 120.5, 122.2, 124.4, 126.0, 127.1, 127.5, 128.4,

129.7, 137.7, 160.0, 177.9. MS: m/z: 318 (M⁺+ 1); anal. calcd.

for C₁₉H₁₁NO₄: C, 71.92; H, 3.49; N, 4.41; found: C, 71.65; H,

3.15; N, 4.75.

5.1.7.18. 3-(2-Fluorophenyl)-1H-benzoĳf ]chromen-1-one (NF-

18). Yield: 76%; mp: 105–109 °C; H NMR (CDCl₃, 300 MHz,

1

δ, TMS = 0): 10.06 (1H, d, J = 8.4 Hz, ArH), 8.13 (1H, d, J = 9.0

Hz, ArH), 7.91–7.99 (2H, m, ArH), 7.75–7.81 (1H, m, ArH),

7.35–7.66 (5H, m, ArH), 7.13 (1H, s, –CH–); ¹³C NMR (CDCl₃,

75 MHz, δ, TMS = 0): 115.1, 115.2, 116.9, 117.0, 117.5, 123.7,

124.3, 124.6, 126.7, 127.2, 128.1, 128.9, 129.3, 129.5, 130.4,

130.6, 132.7, 132.7, 135.6, 137.4, 156.4, 157.5, 180.2. MS: m/z:

291 (M⁺+ 1); anal. calcd. for C₁₉H₁₁FO₂: C, 78.61; H, 3.82; F,

6.54; found: C, 78.55; H, 3.91; F, 6.44.

5.1.7.13. 2-(3,5-Dinitrophenyl)-4H-benzoĳh]chromen-4-one

(NF-13). ⁴¹Yield: 81%; mp: 65–67 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 9.27 (1H, s, ArH), 9.20 (2H, s, ArH), 8.51

(1H, m, ArH), 8.10 (1H, m, ArH), 7.97 (1H, d, J = 8.7 Hz, ArH),

7.84 (1H, d, J = 8.1 Hz, ArH), 7.74–7.77 (2H, m, ArH), 6.98

(1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 26.8,

111.2, 118.3, 120.4, 122.0, 124.4, 124.9, 125.8, 127.3, 127.9,

5.1.7.19. 3-(3-Fluorophenyl)-1H-benzoĳf ]chromen-1-one (NF-

1

19). Yield: 78%; mp: 123–128 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 10.03 (1H, d, J = 8.6 Hz, ArH), 8.12–8.14 (1H, m,

ArH), 7.92–7.95 (2H, m, ArH), 7.75–7.79 (1H, m, ArH), 7.62–

7.66 (3H, m, ArH), 7.34–7.36 (2H, m, ArH), 7.14 (1H, s, –CH

–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 114.1, 115.4, 116.9,

117.5, 117.5, 123.7, 124.3, 124.6, 126.7, 127.2, 128.1, 128.9,

129.3, 129.8, 130.4, 130.3, 132.7, 132.4, 135.6, 137.4, 156.4,

128.5, 130.0. MS: m/z: 363 (M⁺

+ 1); anal. calcd. for

C₁₉H₁₀N₂O₆: C, 62.76; H, 2.93; N, 7.35; found: C, 62.59; H,

3.13; N, 7.29.

5.1.7.14. 2-(4-Methoxyphenyl)-4H-benzoĳh]chromen-4-one

(NF-14). ⁴¹Yield: 73%; mp: 57–58 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 8.62 (1H, m, ArH), 8.15 (2H, d, J = 8.4 Hz,

ArH), 8.07 (1H, m, ArH), 7.97 (1H, d, J = 8.7 Hz, ArH), 7.86

(1H, d, J = 8.1 Hz, ArH), 7.74–7.77 (2H, m, ArH), 7.13 (2H, d, J

= 8.7 Hz, ArH), 6.98 (1H, s, –CH–), 3.95 (3H, s, OCH₃); ¹³C

NMR (CDCl₃, 75 MHz, δ, TMS = 0): 55.9, 107.0, 115.1, 119.9,

120.4, 122.6, 123.7, 123.9, 125.7, 128.1, 128.6, 129.9, 135.8,

153.0, 162.5, 177.2. MS: m/z: 303 (M⁺+ 1); anal. calcd. for

C₂₀H₁₄O₃: C, 79.46; H, 4.67; found: C, 79.34; H, 4.97.

157.5, 180.4. MS: m/z: 291 (M⁺

+ 1); anal. calcd. for

C₁₉H₁₁FO₂: C, 78.61; H, 3.82; F, 6.54; found: C, 78.74; H, 3.75;

F, 6.55.

5.1.7.20. 3-(4-Fluorophenyl)-1H-benzoĳf ]chromen-1-one (NF-

1

20). Yield: 77%; mp: 130–136 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 10.04 (1H, d, J = 8.5 Hz, ArH), 8.13–8.15 (1H, m,

ArH), 7.91–7.94 (2H, m, ArH), 7.75–7.79 (2H, m, ArH), 7.62–

7.66 (2H, m, ArH), 7.22 (2H, d, J = 8.9 Hz, ArH), 7.13 (1H, s,

–CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 114.1, 115.4,

116.9, 117.5, 117.5, 123.7, 124.3, 124.6, 126.7, 127.2, 128.1,

128.9, 129.3, 129.8, 130.4, 130.3, 132.7, 132.4, 135.6, 137.4,

156.4, 157.5, 180.4. MS: m/z: 291 (M⁺+ 1); anal. calcd. for

C₁₉H₁₁FO₂: C, 78.61; H, 3.82; F, 6.54; found: C, 78.72; H, 3.64;

F, 6.59.

41

5.1.7.15. 2-p-Tolyl-4H-benzoĳh]chromen-4-one (NF-15).

1

Yield: 72%; mp: 68–70 °C; H NMR (CDCl₃, 300 MHz, δ, TMS

= 0): 8.63 (1H, d, J = 9.3 Hz, ArH), 8.18 (1H, d, J = 8.4 Hz,

ArH), 7.81–7.95 (3H, m, ArH), 7.71–7.81 (3H, m, ArH), 7.39

(2H, d, J = 8.4 Hz, ArH), 6.97 (1H, s, –CH–), 2.40 (3H, s, CH₃);

¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 21.5, 107.9, 120.0,

120.6, 122.3, 124.0, 125.3, 126.1, 127.1, 128.2, 128.9, 129.2,

129.9, 135.9, 142.3, 153.5, 163.0, 178.3. MS: m/z: 287 (M⁺+ 1);

anal. calcd. for C₂₀H₁₄O₂: C, 83.90; H, 4.93; found: C, 84.13;

H, 4.73.

5.1.7.16. 2-(3,5-Difluorophenyl)-4H-benzoĳh]chromen-4-one

(NF-16). Yield: 76%; mp: 124–139 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 8.55–8.58 (1H, m, ArH), 8.15 (1H, d, J = 8.7

Hz, ArH), 7.95–7.98 (1H, m, ArH), 7.79–7.82 (1H, m, ArH),

7.73–7.77 (2H, m, ArH), 7.52–7.55 (2H, m, ArH), 7.01–7.06

(1H, m, ArH), 6.92 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz,

δ, TMS = 0): 113.2, 115.4, 115.4, 115.8, 116.1, 126.8, 128.5,

130.1, 132.1, 133.7, 134.6, 135.8, 142.4, 159.8, 171.5, 184.2.

MS: m/z: 309 (M⁺+ 1); anal. calcd. for C₁₉H₁₀F₂O₂: C, 74.03;

H, 3.27; F, 12.33; found: C, 73.93; H, 3.17; F, 12.64.

5.1.7.21. 3-(3-Chlorophenyl)-1H-benzoĳf ]chromen-1-one (NF-

1

21). Yield: 78%; mp: 160–166 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 10.07 (1H, m, ArH), 8.11–8.17 (3H, m, ArH),

8.00–8.04 (2H, m, ArH), 7.66 (1H, s, ArH), 7.23–7.30 (3H, m,

ArH), 6.96 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS =

0): 115.4, 117.4, 117.6, 126.2, 127.6, 128.3, 129.0, 130.3, 131.6,

132.3, 133.3, 134.3, 135.4, 136.5, 153.5, 161.1, 180.0. MS: m/z:

307 (M⁺+ 1); anal. calcd. for C₁₉H₁₁ClO₂: C, 74.40; H, 3.61;

Cl, 11.56; found: C, 74.50; H, 3.59; Cl, 11.60.

5.1.7.22. 3-(2,3-Dichlorophenyl)-1H-benzoĳf ]chromen-1-one

(NF-22). Yield: 84%; mp: 160–165 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 10.08 (1H, m, ArH), 8.15–8.19 (3H, m,

ArH), 7.98–8.03 (2H, m, ArH), 7.32–7.36 (3H, m, ArH), 6.81

(1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0):

115.9, 117.2, 117.5, 126.8, 127.1, 127.6, 128.2, 129.0, 129.4,

130.4, 130.6, 131.6, 132.5, 133.7, 134.6, 135.8, 157.7, 159.7,

179.7. MS: m/z: 341 (M⁺+ 1); anal. calcd. for C₁₉H₁₀Cl₂O₂:

C, 66.89; H, 2.95; Cl, 20.78; found: C, 66.99; H, 2.85; Cl,

20.91.

5.1.7.17. 3-Phenyl-1H-benzoĳf ]chromen-1-one (NF-17). Yield:

91%; mp: 157–162 °C; ¹H NMR (CDCl₃, 300 MHz, δ, TMS =

0): 9.78 (1H, d, J = 9.0 Hz, ArH), 7.77–7.79 (1H, m, ArH),

7.73–7.74 (1H, m, ArH), 7.56–7.61 (3H, m, ArH), 7.36–7.47

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5.1.7.23. 3-(2-Bromophenyl)-1H-benzoĳf ]chromen-1-one (NF-

23). Yield: 75%; mp: 153–159 °C; H NMR (CDCl₃, 300 MHz,

5.1.7.29. 3-(3,5-Dinitrophenyl)-1H-benzoĳf ]chromen-1-one

(NF-29). Yield: 81%; mp: 101–106 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 10.15 (1H, s, ArH), 9.08 (1H, s, ArH), 8.65–

8.70 (2H, m, ArH), 8.11 (1H, m, ArH), 7.97–7.99 (1H, m, ArH),

7.77–7.84 (3H, m, ArH), 7.07 (1H, s, –CH–); ¹³C NMR (CDCl₃,

75 MHz, δ, TMS = 0): 112.4, 115.9, 117.3, 124.2, 124.3, 126.7,

126.9, 127.1, 128.2, 128.3, 129.3, 129.6, 136.1, 136.1, 137.4,

157.4, 158.1, 180.7. MS: m/z: 363 (M⁺+ 1); anal. calcd. for

C₁₉H₁₀N₂O₆: C, 62.76; H, 2.93; N, 7.35; found: C, 62.66; H,

3.03; N, 7.30.

5.1.7.30. 3-(4-Methoxyphenyl)-1H-benzoĳf ]chromen-1-one

(NF-30). Yield: 88%; mp: 72–78 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 10.11 (1H, m, ArH), 8.13–8.17 (3H, m,

ArH), 8.07 (1H, m, ArH), 7.97 (1H, m, ArH), 7.41 (2H, d, J =

8.5 Hz, ArH), 7.01–7.03 (2H, m, ArH), 6.95 (1H, s, –CH–), 3.96

(3H, s, OCH₃); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 56.9,

108.2, 114.1, 118.4, 119.5, 121.6, 123.4, 123.9, 126.7, 127.1,

128.3, 130.1, 130.6, 135.7, 153.3, 162.5, 177.7. MS: m/z: 303

(M⁺+ 1); anal. calcd for C₂₀H₁₄O₃: C, 79.46; H, 4.67; found: C,

79.57; H, 4.42.

1

δ, TMS = 0): 10.08 (1H, d, J = 8.7 Hz, ArH), 8.13 (1H, d, J = 9.0

Hz, ArH), 7.94 (1H, d, J = 7.5 Hz, ArH), 7.40–7.80 (7H, m,

ArH), 6.78 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS =

0): 115.1, 116.8, 117.0, 117.1, 117.5, 119.9, 124.6, 124.6, 126.7,

127.1, 128.1, 128.9, 129.3, 130.6, 132.7, 132.7, 135.6, 157.5,

180.3. MS: m/z: 352 (M⁺+ 1); anal. calcd. for C₁₉H₁₁BrO₂: C,

64.98; H, 3.16; Br, 22.75; found: C, 65.05; H, 3.03; Br, 22.88.

5.1.7.24. 3-(3-Bromophenyl)-1H-benzoĳf ]chromen-1-one (NF-

1

24). Yield: 78%; mp: 168–171 °C; H NMR (CDCl₃, 300 MHz, δ,

TMS = 0): 10.04 (1H, d, ArH), 8.15 (1H, d, J = 9.0 Hz, ArH), 7.95

(1H, m, ArH), 7.77–7.83 (3H, m, ArH), 7.63 (1H, s, ArH), 7.23–

7.33 (3H, m, ArH), 6.98 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75

MHz, δ, TMS = 0): 110.5, 117.2, 117.4, 126.0, 126.7, 127.1, 127.4,

128.1, 129.3, 130.3, 130.4, 130.6, 132.3, 135.6, 157.3, 159.7,

179.9. MS: m/z: 350 (M⁺+ 1); anal. calcd. for C₁₉H₁₁BrO₂: C,

64.98; H, 3.16; Br, 22.75; found: C, 65.02; H, 3.09; Br, 22.79.

5.1.7.25. 3-(4-Bromophenyl)-1H-benzoĳf ]chromen-1-one (NF-

25). Yield: 80%; mp: 220–225 °C; ¹H NMR (CDCl₃, 300 MHz, δ,

TMS = 0): 10.06 (1H, d, J = 8.7 Hz, ArH), 8.14 (1H, d, J = 9.0

Hz, ArH), 7.93 (1H, d, J = 8.1 Hz, ArH), 7.78–7.85 (3H, m, ArH),

7.61–7.70 (4H, m, ArH), 6.97 (1H, s, –CH–); ¹³C NMR (CDCl₃,

75 MHz, δ, TMS = 0): 110.5, 117.2, 117.4, 126.0, 126.7, 127.1,

127.4, 128.1, 129.3, 130.3, 130.4, 130.6, 132.3, 135.6, 157.3,

159.7, 180.0. MS: m/z: 351 (M⁺); anal. calcd. for C₁₉H₁₁BrO₂: C,

64.98; H, 3.16; Br, 22.75; found: C, 64.78; H, 3.00; Br, 22.65.

5.1.7.26. 3-(2-Iodophenyl)-1H-benzoĳf ]chromen-1-one (NF-

5.1.7.31. 3-p-Tolyl-1H-benzoĳf ]chromen-1-one (NF-31). Yield:

1

85%; mp: 96–102 °C; H NMR (CDCl₃, 300 MHz, δ, TMS = 0):

10.04 (1H, m, ArH), 8.12–8.18 (3H, m, ArH), 8.01–8.06 (2H,

m, ArH), 7.33 (2H, d, J = 8.4 Hz, ArH), 7.11–7.15 (2H, m,

ArH), 7.01 (1H, s, –CH–), 2.45 (3H, s, CH₃); ¹³C NMR (CDCl₃,

75 MHz, δ, TMS = 0): 26.4, 110.2, 119.9, 122.6, 123.7, 123.9,

126.3, 127.4, 128.1, 128.6, 129.0, 129.9, 130.4, 135.3, 138.5,

153.0, 162.2, 180.2. MS: m/z: 287 (M⁺+ 1); anal. calcd. for

C₂₀H₁₄O₂: C, 83.90; H, 4.93; found: C, 83.80; H, 4.98.

1

26). Yield: 69%; mp: 141–146 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 10.05 (1H, m, ArH), 8.19–8.25 (3H, m, ArH),

8.11–8.13 (2H, m, ArH), 7.82–7.84 (1H, m, ArH), 7.31–7.36

(3H, m, ArH), 6.96 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz,

δ, TMS = 0): 110.3, 113.6, 119.2, 120.6, 122.8, 124.1, 125.6,

127.2, 128.1, 128.5, 129.4, 130.8, 131.9, 136.0, 138.0, 140.5,

154.0, 165.2, 180.1. MS: m/z: 398 (M⁺+ 1); anal. calcd. for

C₁₉H₁₁IO₂: C, 57.31; H, 2.78; I, 31.87; found: C, 57.42; H,

2.88; I, 31.93.

5.1.7.32. 3-(3,5-Difluorophenyl)-1H-benzoĳf ]chromen-1-one

(NF-32). Yield: 78%; mp: 124–139 °C; ¹H NMR (CDCl₃, 300

MHz, δ, TMS = 0): 9.80 (1H, d, J = 8.4 Hz, ArH), 7.97 (1H, d, J =

9.0 Hz, ArH), 7.75 (1H, d, J = 8.1 Hz, ArH), 7.42–7.58 (4H, m,

ArH), 7.29–7.33 (2H, m, ArH), 6.76–6.85 (1H, m, –CH–); ¹³C

NMR (CDCl₃, 75 MHz, δ, TMS = 0): 114.5, 115.4, 116.9, 117.5,

117.5, 123.7, 128.1, 128.6, 129.9, 130.4, 135.3, 153.0, 158.4,

162.2, 179.9. MS: m/z: 309 (M⁺+ 1); anal. calcd. for C₁₉H₁₀F₂O₂:

C, 74.03; H, 3.27; F, 12.33; found: C, 73.99; H, 3.34; F, 12.15.

5.1.7.27. 3-(3-Nitrophenyl)-1H-benzoĳf ]chromen-1-one (NF-

1

27). Yield: 79%; mp: 139–141 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 10.15 (1H, m, ArH), 8.42 (1H, m, ArH), 8.11–8.14

(3H, m, ArH), 7.78–7.85 (2H, m, ArH), 7.62–7.65 (2H, m,

ArH), 7.07 (1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS =

0): 112.4, 115.9, 117.3, 124.2, 124.3, 126.7, 126.9, 127.1, 128.2,

128.3, 129.3, 129.6, 136.1, 136.1, 137.4, 157.4, 158.1, 179.7.

MS: m/z: 318 (M⁺+ 1); anal. calcd. for C₁₉H₁₁NO₄: C, 71.92; H,

3.49; N, 4.41; found: C, 72.00; H, 3.33; N, 4.34.

5.2. In vitro xanthine oxidase assay

All the synthesized compounds were evaluated against the

xanthine oxidase enzyme. Bovine milk xanthine oxidase

(grade 1, ammonium sulfate suspension, Sigma-Aldrich) ac-

tivity was assayed spectrophotometrically by measuring the

uric acid formation at 293 nm using a Hitachi U-3010 UV-

visible spectrophotometer at 25 °C. The reaction mixture con-

tains 50 mM potassium phosphate buffer (pH 7.6), 75 μM

xanthine and 0.08 units of xanthine oxidase. Inhibition of

xanthine oxidase activity of the synthetics at different concen-

trations (1, 5, 10, 25, 50 μM) was measured by monitoring

the decrease in uric acid formation at 293 nm at 25 °C. The

enzyme was pre-incubated for 5 min with a test compound

and dissolved in DMSO (1% v/v), and the reaction was started

by the addition of xanthine. The final concentration of DMSO

(1% v/v) did not interfere with the enzyme activity. All the

5.1.7.28. 3-(4-Nitrophenyl)-1H-benzoĳf ]chromen-1-one (NF-

1

28). Yield: 76%; mp: 102–106 °C; H NMR (CDCl₃, 300 MHz,

δ, TMS = 0): 10.14 (1H, d, J = 8.4 Hz, ArH), 8.41 (1H, d, J = 8.7

Hz, ArH), 8.14–8.19 (3H, m, ArH), 7.95 (1H, d, J = 8.4 Hz,

ArH), 7.78–7.80 (1H, m, ArH), 7.65–7.68 (2H, m, ArH), 7.08

(1H, s, –CH–); ¹³C NMR (CDCl₃, 75 MHz, δ, TMS = 0): 112.4,

115.9, 117.3, 124.2, 124.3, 126.7, 126.9, 127.1, 128.2, 128.3,

129.3, 129.6, 136.1, 136.1, 137.4, 157.4, 158.1, 179.7. MS: m/z:

318 (M⁺+ 1); anal. calcd. for C₁₉H₁₁NO₄: C, 71.92; H, 3.49; N,

4.41; found: C, 71.85; H, 3.35; N, 4.65.

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experiments were performed in triplicate and values were

expressed as the mean of three experiments.^42,43

domly divided into twelve experimental groups (n = 6). Each

group was kept in a plastic cage and allowed one week to

adapt to the environmental conditions before starting the ex-

periments. The animals were housed on a 12 h/12 h light/

dark cycle at a maintained temperature of 24 2 °C. They

were given standard animal feed and RO water during the

study. All experiments were carried out in accordance with

CPSCEA guidelines and animals were approved by the respec-

tive university committees for animal experiments with pro-

posal no. 226/CPCSEA-2016/17.

5.3. Enzyme kinetics study

Potent XO enzyme inhibitors were further investigated for the

type of inhibition and an enzyme kinetics study was carried out.

The Lineweaver–Burk plot was established from which we could

calculate the K_m, V_maxof the slope of the inhibitor and the value

of α (a constant that defines the degree to which inhibitor bind-

ing affects the affinity of the enzyme for the substrate).⁴⁴

5.6.2. Animal model of hyperuricemia. Potassium oxonate

(PO) (uricase inhibitor) and xanthine (Xn) were purchased

from Sigma-Aldrich and used to induce hyperuricemia. The

experiment was performed according to the reported method

by Qin et al. with some modifications.⁵¹Twelve experimental

groups (n = 6) were divided as follows: 1st “vehicle control

group”, 2nd “PO + Xn”, 3rd “PO + Xn + febuxostat, 4th “PO +

Xn + allopurinol”, and groups 5 to 12 “PO + Xn + synthetics”

with different doses. The animals were kept on 6 h fasting

(only the feed was withdrawn) before starting the experi-

ments. The mice were administered orally with xanthine (500

mg kg⁻¹in 0.5% CMC) followed by intraperitoneal injection

of potassium oxonate (300 mg kg⁻¹in 0.5% CMC) after 30 mi-

nutes to induce uric acid. After 1 h of potassium oxonate in-

jection, the mice were injected orally with different dose

levels of test compounds. Febuxostat and allopurinol drugs

were administered at an oral dose of 5 and 10 mg kg⁻¹re-

spectively. The mice in the “control” group were adminis-

trated 0.5% CMC only. Two hours after PO + Xn administra-

tion, blood samples were collected from the mice by retro-

orbital puncture. The collected blood was allowed to clot for

30 minutes at ambient temperature and then centrifuged

(REMI-Model no. C-24PLUS) at 10 000 rpm for 5 minutes at

−2 °C to obtain the serum. The fresh serum was used for fur-

ther uric acid detection by using an ERBA diagnostic kit.

5.6.3. Uric acid detection. The uric acid content of freshly

eluted serum was detected by using an ERBA Mannheim uric

acid detection kit. The principle is based upon the color forma-

tion during the enzymatic reaction between H₂O₂(produced by

the action of uricase on uric acid) and 4AAP + TBHB. Briefly,

the uric acid produced during an experiment in all samples of

the reaction mixture is oxidized to yield allantoin and hydrogen

peroxide in the presence of the uricase enzyme. The enzyme

peroxidase catalyzes the oxidative coupling of 4-AAP with phenol

to yield a colored quinone imine complex with absorbance pro-

portional to the concentration of uric acid in the sample. The

whole mechanism of the reaction is depicted below:

5.4. Docking study

The three-dimensional structure of bovine milk xanthine dehy-

drogenase (XDH) complexed with febuxostat (a potent XO inhib-

itor) was retrieved from the RCSB protein data bank (PDB code

1N5X).⁴⁵For docking simulations, hydrogen atoms were added

to the protein structure using MGL Tools – 1.5.6. The 2D struc-

ture of the ligands was drawn in ChemDraw and subjected to

energy minimization using the MM2 force field as implemented

in Chem 3D Ultra software (ChemDraw Ultra 6.0 and Chem3D

Ultra). Docking was performed using the AutoDock Vina soft-

ware.⁴⁹For the docking performance, a grid box was generated

by fixing the number of points in the x, y and z directions to 40,

each. The spacing was adjusted to 1.00 Å. The center of the grid

box was fixed to the point of 96, 52 and 38.

5.5. Antioxidant activity (DPPH assay)

All the synthesized compounds were tested for their antioxi-

dant potential by using 2,2-diphenyl-1-picrylhydrazyl (DPPH)

assay.⁵⁰Different concentrations of test compounds and

0.004% w/v DPPH solution were prepared in methanol. 1.5

mL DPPH solution and 1 mL methanol was taken as a con-

trol while methanol alone was taken as a blank. The reaction

mixture contained 1.5 mL of 0.004% w/v DPPH solution and

1 mL of test compounds in a test tube. The reaction mixture

was shaken at 37 °C for 1 min and placed in the dark for 30

min. Absorption measurements were then recorded using a

spectrophotometer (SYSTRONICS-Serial no. 2231 at 517 nm).

The ability to scavenge the DPPH radical (% inhibition) was

calculated using the following equation:

Absorbance of controlAbsorbance of sample

%inhibition 

100

Absorbance of control

The concentration of antioxidant substance causing a 50%

(IC₅₀) decrease in the concentration of free radicals was also

determined. All experiments were performed in triplicate and

values were then expressed as the mean.

Uric acid  O₂ H₂O ^U^rica^seAllantoin  CO₂ H₂O₂

H₂O₂ 4-AAP  TBHB ^P^erox^idas^eQuinoneimine  H₂O

4-AAP  4-Aminoantipyrine

TBHB  2,4,6-Tribromo-3-hydroxy benzoic acid

5.6. In vivo antihyperuricemic activity

5.6.1. Animals. Three-week-old male mice (Swiss albino

25–30 gm) were purchased from Central Research Institute,

Kasauli, Himachal Pradesh, India. The animals were ran-

The reaction mixture contained 30 μL of serum (sample)

and 1.5 mL of enzymatic reagent. This reaction mixture was

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mixed and incubated for 5 minutes at 37 °C. The absorbance

was recorded at 505 nm by using a colorimeter and further

used to calculate the concentration of uric acid by following

the given formula:

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Absorbance of test sample

Uric Acid mg dL^1







Absorbance of Standard

Concentration of Standard mg dL^1





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employed for acute toxicity studies for potent compounds.

The first group was treated with the vehicle and served as the

vehicle control. The remaining groups were treated with a

single higher dose (2000 mg kg⁻¹) of compounds. All the

treatments were administered immediately after 4 h of

fasting. The animals were observed continuously for any

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Conflicts of interest

18 H. R. J. Schumacher, M. A. Becker, R. L. Wortmann, P. A.

Macdonald, B. Hunt, J. Streit, C. Lademacher and N. Joseph-

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The authors confirm that they have no conflicts of interest.

19 M. Becker, H. Schumacher and P. A. MacDonald, Arthritis

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Acknowledgements

20 https://www.fda.gov/Drugs/DrugSafety/ucm584702.htm

retrieved on 2-july-2018.

The authors are grateful to the University Grants Commission

for providing funds under the Rajiv Gandhi National Fellow-

ship (RGNF) and they are thankful to Guru Nanak Dev Univer-

sity, Amritsar for providing various facilities to carry out the

research work.

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Article Doi

Benzoflavone derivatives as potent antihyperuricemic agents

DOI: 10.1039/c8md00512e

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Full text of DOI:10.1039/c8md00512e

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