Synthesis and evaluation of naphthoflavones as a new class of non purine xanthine oxidase inhibitors This Letter is dedicated to Dr. K. L. Dhar on the occasion of his 78th birthday.

Article abstract of DOI:10.1016/j.bmcl.2014.07.041

In view of reported xanthine oxidase inhibitory potential of naphthopyrans and flavones, naphthoflavones as hybrids of the two were designed, synthesized and evaluated for in vitro xanthine oxidase inhibitory activity in the present study. The results of the assay revealed that the naphthoflavones possess promising inhibitory potential against the enzyme with IC₅₀ values ranging from 0.62 to 41.2 μM. Structure activity relationship indicated that the nature and placement of substituents on the phenyl ring at 2nd position remarkably influences the inhibitory activity. Substitution of halo and nitro groups at ortho and para position of the phenyl ring (2nd position) remarkably favored the activity. NF-4 with p-fluoro phenyl ring was the most potent inhibitor with IC₅₀ value of 0.62 μM. Enzyme kinetics study was also performed to investigate the inhibition mechanism and it was found that the naphthoflavones displayed mixed type inhibition. The basis of significant inhibition of xanthine oxidase by NF-4 was rationalized by molecular modeling studies.

Full text of DOI:10.1016/j.bmcl.2014.07.041

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of naphthoflavones as a new class of non
purine xanthine oxidase inhibitors
a
Harbinder Singh a, Sahil Sharma a, Ritu Ojha a, Manish K. Gupta b,c, Kunal Nepali a, , P. M. S. Bedi
⇑
a Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005, India
b Department of pharmaceutical sciences, ISF College of pharmacy, Punjab, India
c Lloyd Institute of Management and Technology, Greater Noida (UP), India
a r t i c l e i n f o
a b s t r a c t
Article history:
In view of reported xanthine oxidase inhibitory potential of naphthopyrans and flavones, naphthoflav-
ones as hybrids of the two were designed, synthesized and evaluated for in vitro xanthine oxidase inhib-
itory activity in the present study. The results of the assay revealed that the naphthoflavones possess
Received 24 March 2014
Revised 10 July 2014
Accepted 15 July 2014
Available online xxxx
promising inhibitory potential against the enzyme with IC50 values ranging from 0.62 to 41.2 lM. Struc-
ture activity relationship indicated that the nature and placement of substituents on the phenyl ring at
2nd position remarkably influences the inhibitory activity. Substitution of halo and nitro groups at ortho
and para position of the phenyl ring (2nd position) remarkably favored the activity. NF-4 with p-fluoro
This Letter is dedicated to Dr. K. L. Dhar on
the occasion of his 78th birthday.
phenyl ring was the most potent inhibitor with IC50 value of 0.62 lM. Enzyme kinetics study was also
performed to investigate the inhibition mechanism and it was found that the naphthoflavones displayed
mixed type inhibition. The basis of significant inhibition of xanthine oxidase by NF-4 was rationalized by
molecular modeling studies.
Keywords:
Xanthine oxidase
Naphthopyran
Flavone
Ó 2014 Elsevier Ltd. All rights reserved.
Assay
Kinetics
Inhibitory
Oxidative hydroxylation of hypoxanthine and xanthine cata-
lyzed by xanthine oxidase to produce uric acid and reactive oxygen
species leads to many diseases like gout and at least symptoms of
diseases like oxidative damage to the tissue.1–3 Therefore the selec-
tive inhibition of XO may result in broad spectrum chemothera-
peutic for gout, cancer, inflammation and oxidative damage.2–4
Allopurinol,3,4 2-alkyl hypoxanthines,5 pterin and 6-formylpterin6
represents the class of purine based xanthine oxidase inhibitors.
All these inhibitors have been successfully utilized and have
proved their inhibitory potential towards the enzyme. However
these purine based inhibitors have been reported to be associated
with Steven Johnson syndrome and worsening of renal function
induced in some of the patients.2–4 Keeping in view these side
effects, we have been actively involved in the design of some non
purine xanthine oxidase inhibitors in the recent past such as n-
acetyl pyrazolines,7 b-acetamido compounds,8 azaflavones9, naph-
thopyrans10 and 4,6-diaryl/heteroarylpyrimidin-2(1H)-ones.11
Flavones represents the class of secondary metabolites possess-
ing diverse array of biological activities.7,12–16 Among the reported
biological attributes, there are numerous reports on the xanthine
oxidase inhibitory potential of flavones.9,14–16 The structure activ-
ity relationship of flavones for xanthine oxidase inhibition has
been extensively explored. SAR study revealed that a substituent
free C-3 and overall planarity are two of the essential structural
features required for xanthine oxidase inhibition of flavones.14–16
Keeping these structural features intact, azaflavones were earlier
designed via bioisosteric replacement of benzopyrans with quino-
lones by our research group.9 The most potent azaflavone was
found to possess significant inhibitory potential against the
enzyme with an IC50 value of 6.24 lM.
Recently we synthesised and evaluated a series of naphthopy-
rans for in vitro xanthine oxidase inhibition in view of some of
the potent non purine xanthine oxidase inhibitors possessing
benzopyran skeleton. The results of the study proved that naphthyl
moiety is an effective surrogate for the hydroxy substituted fused
benzene ring in benzopyrans. The potent inhibitory potential of
some naphthopyrans was attributed to the interactions of naphthyl
ring as indicated by molecular modeling studies.10
With this background, naphthoflavones as hybrids of the naph-
thopyrans and flavones were designed keeping intact the two most
important structural requisites for flavones, that is, a substituent
free C-3 and overall planarity. Figure 1 represents the strategy
for the design of naphthoflavones as xanthine oxidase inhibitors.
The hybrids were synthesized via Scheme 1.
subjected to fries rearrangement as per the method reported by
a-Naphthol was
⇑
Corresponding author. Tel.: +91 8146096564.
E-mail address: kunal_8855@rediffmail.com (K. Nepali).
http://dx.doi.org/10.1016/j.bmcl.2014.07.041
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Singh, H.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.041

2
H. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
NAPHTHOPYRAN SKELETON
R
MOLECULAR
O
HYBRIDIZATION
R
O
O
O
R
NAPHTHOFLAVONES
HYBRIDS OF NAPHTHOPYRAN
AND FLAVONE
FLAVONE FRAMEWORK
Figure 1. Design Strategy.
Table 1 (continued)
R
OH
O
OH
Code
Structure
IC50 (lM)
b
a
CH3
O
O
O
F
CH3
O
NF-4
NF-5
0.62
O
c
Cl
OH
O
O
OH OH
O
O
O
27.5
C
R
R
H
d
Cl
Cl
O
NF-6
NF-7
NF-8
41.2
16.8
31.2
R
O
O
O
Br
O
Scheme 1. Reagents and conditions: (a) MW, ZnCl2, CH3COOH, 20 min; (b) benzoyl
chloride, pyridine, stirring rt, 1 h; (c) KOH, pyridine, warm, 30 min; (d) a drop of
conc. H2SO4, CH3COOH, reflux, 1 h.
O
Br
Table 1
O
Structures of naphthoflavones along with IC50 values
Code
Structure
IC50 (lM)
O
Br
O
O
O
NF-9
8.93
NF-1
32.5
O
O
I
F
O
NF-10
19.4
25.6
NF-2
NF-3
4.94
O
O
NO2
F
O
O
O
23.6
NF-11
O
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H. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
appeared as a merged signal in a multiplet at 7.26–7.36 ppm).
The rearranged product was then cyclized by treatment with sulfu-
ric acid to yield the desired naphthoflavones. All the reactions pro-
ceeded smoothly with diverse benzoylchlorides and products were
obtained in good yields. No Retro-Diels fragmentation was
observed for naphthoflavones in the mass spectrum.
The structures of the synthesized compounds were elucidated
by 1H NMR, 13C NMR. All spectral data were in accordance with
assumed structures.18
In vitro screening of the naphthoflavones using bovine milk
xanthine oxidase (grade 1, ammonium sulfate suspension) enzy-
matic assay was performed as described in the literature.19 Apige-
nin (one of the most potent flavone as xanthine oxidase inhibitor)2
and allopurinol20 were employed as reference inhibitor. The results
of the in vitro assay (Table 1) indicated that naphthoflavones pos-
sessed significant xanthine oxidase inhibitory activity with IC50
Table 1 (continued)
Code
Structure
IC50 (lM)
NO2
O
NF-12
NF-13
1.95
O
O
NO2
37.9
NO2
O
O
OCH3
NF-14
NF-15
18.9
21.3
value ranging from 0.62 to 41.2
NF-12 were found to be endowed with significant activity with
IC50 <5 M. NF-4 was the most potent of the series displaying an
IC50 value of 0.62 M followed by NF-2 and NF-12 with an IC50
value of 4.94 and 1.95 M. The in vitro screening of the naphthof-
lM. Naphthoflavones NF-2, NF-4,
O
O
CH3
l
l
l
O
lavones revealed some interesting aspects about the structure
inhibitory relationship. The inhibitory potential of the compounds
was sensitive towards the nature as well as positioning of the sub-
stituents on the phenyl ring at 2nd position. Compounds possess-
ing substituted phenyl rings (any substitution whether electron
withdrawing and electron donating) were more active than the
compounds with an unsubstituted phenyl ring (Compare NF-1
with NF-2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 14, 15). However only the
naphthoflavones with disubstituted phenyl ring were less potent
than the inhibitors with unsubstituted phenyl ring (compare NF-
1 with NF-6 and 13). Overall the preference order of substituents
(monosubstitution) is as follows: halo and nitro > methoxy >
methyl > hydrogen. Inhibitors with fluoro and nitro (ortho and
para) substituted phenyl rings were the most active (NF-2, 4, 12)
Apigenin
Allopurinol
1.11
8.69
Table 2
Inhibition of xanthine oxidase by potent compounds at 5 different concentrations
Code
1
l
M
5
l
M
10
l
M
25
l
M
50
l
M
IC50 (lM)
NF-2
NF-4
NF-9
NF-12
43.87
49.39
41.98
47.76
49.21
52.10
41.56
50.13
57.27
61.67
52.21
61.18
71.87
78.51
72.30
73.83
92.61
99.47
90.23
94.27
4.94
0.62
8.93
1.95
as they displayed IC50 value of <5 lM. Weak inhibitory potential
Naeimi et al.17 to yield 2-acetyl naphthol (1) (characterized by the
appearance of a singlet for D2O exchangeable proton at 14.01 ppm)
which was further benzoylated using various substituted benzoyl-
chlorides. The benzoylated product was then subjected to Baker
Venkataraman rearrangement.18 The Baker Venkataraman rear-
ranged product existed in enol form (confirmed by the appearance
of singlets for two D2O exchangeable protons at 15.2 and
was displayed by compounds with meta substituted phenyl rings
as compared to compounds with ortho and para substituted phenyl
rings. The decline in activity by the placement of meta substituted
phenyl rings was evident from the IC50 values of NF-3 (with m-flu-
oro phenyl, IC50 = 23.6
IC50 = 0.62 M) and NF-2 (with o-fluoro phenyl, IC50 = 4.94) as well
as from the the IC50 values of NF-8 (with m-bromo phenyl,
IC50 = 31.2 M), NF-7 (with o-bromo phenyl, IC50 = 16.8 M) and
lM), NF-4 (with p-fluoro phenyl,
l
13.68 ppm along with the vinylic proton
a to carbonyl which
l
l
Figure 2. % Age inhibition of potent naphthoflavones.
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4
H. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 3. Plot of V versus [I] for NF-4.
Figure 4. Lineweaver Burk plots.
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H. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
NF-9 (with p-bromo phenyl, IC50 = 8.93
l
M). NF-4 was almost 36
mode of inhibition of NF-2, NF-4 and NF-12 is mixed-type but it
seems that they have a strong competitive component.
folds and NF-2 was 5 folds more active than NF-3. Similar pattern
was observed for NF-9 and NF-7 which was around 3.5 folds and 2
folds more active than NF-8. Overall the preference order for posi-
tioning of the substituents follows the order: para > ortho > meta. It
was also observed that the nature of substituents at p-position
influenced the activity as substituents with a stronger ÀI (Induc-
tive) effect such as fluoro, nitro and bromo remarkably favoring
the inhibitory potential in comparison to the substituents with
stronger +R (Resonance) (OCH3) and hyper conjugation effect
(CH3) (Compare NF-2, 4, 12 with NF-14, 15). The remarkable inhib-
itory potential of NF-4 possessing fluoro substituted (para) phenyl
ring could be attributed to the presence of fluorophilic environ-
ments in the cavity of enzyme. In general, F substituents on ligands
prefer to orient toward electropositive regions of receptor sites21
and this has led to the increased use of this element to enhance
the binding affinity to the target protein.22 Moreover the ability
of fluorine to get involved in hydrogen bonding/dipolar interac-
tions have also been evidenced through number of reports.22,23
Thus fluorine effect could be the probable reason for the significant
inhibition of xanthine oxidase by NF-4.
Table 2 represents the inhibitory potential of NF-2, 4, 9, 12 (dis-
playing IC50 values <10 lM) at five different concentration.
Figure 2 represents the % age inhibition of potent naphthoflav-
ones against xanthine oxidase.
Figure 3 represents the plot of V versus [I] for the most potent
inhibitor, that is, NF-4.
Enzyme kinetic study was also studied for the potent com-
pounds. The Lineweaver–Burk plot (Fig. 4) revealed that com-
pounds NF-2, NF-4, NF-12 were mixed-type XO inhibitors. The
pattern of graph shows that it is a form of mixed inhibition sce-
nario. The Km, Vmax and slope are all affected by the inhibitor.
The inhibitors have increased the Km and slope (Km/Vmax) while
decreasing the Vmax. Moreover carefully observing the Figure 4, 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
In order to understand the binding conformation of NF-4 (most
potent XO inhibitor), it was docked using the GOLD software.25 into
the salicylic acid binding site of XO.26
To validate the docking procedure, the salicylic acid was
extracted from the original X-ray structure of XO (1FIQ)26 and
docked using GOLD. The highest scoring conformation was
selected and compared with original X-ray structure conformation.
The docked conformation of salicylic acid was found to be the sim-
ilar with the original X-ray structure. The root mean square devia-
tion (RMSD) between the best scored conformation from docking
and X-ray structure was found to be 0.21 Å.
The best fit conformation of NF-4 was selected on the basis of
Chemplp scoring function and visual inspection. The Figure 5
shows the binding conformation of the NF-4 at the binding site
of XO. The binding site residues and overall binding mode was
found to be similar to those observed with salicylic acid26 and fab-
uxostat.27 The NF-4 gets stabilized by various electrostatic and
hydrophobic interactions. In docking pose, the naphthyl ring (ring
A and B) of NF-4 was found sandwiched between Phe914 and
Phe1009. The Phe914 and Phe1009 are involved in ‘face-to-face’
and ‘face-to-edge’ pai–pai stacking interactions, respectively with
NF-4. This arrangement of energetically favorable arene/arene
interactions28 is also present in the co-crystal structure of XO with
salicylate23 and fabuxostat.27 Its conservation argues for an impor-
tant role in stabilizing the binding positions of aromatic inhibitors
and also represent one of the key features of substrate recognition
(Fig. 5).7 Two crucial H-bond interactions were observed between
NF-4 and binding residues of XO. Here, first H-bond was formed
between hydroxyl group of Ser876 and carbonyl group of ring C
(d = 1.91 Å; h = 145.9°). Also, the fluorine atom at ring D is involved
in H-bond interaction with side chain N–H of Asn768 (d = 2.24 Å;
h = 101.1°). This can be suggested that these H-bonds may contrib-
ute significantly towards the binding free energy between NF-4
and XO. The ring D of NF-4 face towards the side chain of
Leu648 and showed the hydrophobic interaction (Fig. 5).
a
(a constant that defines the degree to which inhibitor binding
affects the affinity of the enzyme for substrate) is greater than
1.24 This confirms that the inhibitor preferentially binds to the free
enzyme and not the enzyme substrate complex. Therefore, the
The present study employs molecular hybridization technique
for the design of naphthoflavones as hybrids of naphthopyrans
and flavones (two previously reported classes of xanthine oxidase
Figure 5. Binding interactions of NF-4 with the amino acid residues.
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6
H. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
7. Nepali, K.; Singh, G.; Turan, A.; Agarwal, A.; Sapra, S.; Kumar, R.; Banerjee, U. C.;
Verma, P. K.; Satti, N. K.; Gupta, M. K.; Suri, O. P.; Dhar, K. L. Bioorg. Med. Chem.
2011, 19, 1950.
8. Nepali, K.; Agarwal, A.; Sapra, S.; Mittal, V.; Kumar, R.; Banerjee, U. C.; Gupta, M.
K.; Satti, N. K.; Suri, O. P.; Dhar, K. L. Bioorg. Med. Chem. 2011, 19, 5569.
9. Dhiman, R.; Sharma, S.; Singh, G.; Nepali, K.; Bedi, P. M. S. Arch. Pharm. Chem.
Life Sci. 2012, 346, 7.
10. Sharma, S.; Sharma, K.; Ojha, R.; Kumar, D.; Singh, G.; Nepali, K.; Bedi, P. M. S.
Bioorg. Med. Chem. Lett. 2014, 24, 495.
11. Shukla, S.; Nepali, K.; Bedi, P. M. S.; Ojha, R.; Kumar, D.; Gupta, M. K. Arch.
Pharm. Chem. Life Sci. 2014, 347, 486.
12. Fernandez, S. P.; Wasowski, C.; Loscalzo, L. M.; Granger, R. E.; Johnston, G. A. R.;
Paladini, A. C.; Marder, M. Eur. J. Pharmacol. 2006, 539, 168.
13. Harborne, J. B.; Williams, C. A. Phytochemistry 2000, 55, 481.
14. Cos, P.; Ying, L.; Calomme, M.; Hu, J. P.; Cimanga, K.; Van-Poel, B.; Pieters, L.;
Vlietinck, A. J.; Berghe, D. V. J. Nat. Prod. 1998, 61, 71.
15. Da-Silva, S. L.; Da-Silva, A.; Honorio, K. M.; Marangoni, S.; Toyama, M. H.; Da-
Silva, A. B. F. J. Mol. Struct. Theochem 2004, 684, 1.
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inhibitors). The designed hybrids have been synthesized and eval-
uated for xanthine oxidase inhibition for the first time. Structural
features such as substituent free C-3 and overall planarity were
kept intact as revealed by the well established structure activity
relationship of flavones. Naphthopyran was employed as a surro-
gate for the hydroxy substituted benzopyran in flavone framework.
In vitro xanthine oxidase inhibitory assay indicated the promising
inhibitory profile of the naphthoflavones as the most potent napht-
hoflavones, that is, NF-4 and NF-12 displayed strong inhibition at
0.62 lM and 1.95 lM. Structure activity relationship indicated that
halo and nitro at para position of phenyl ring (2nd position) were
the preferred substituents for potent inhibition. Naphthoflavones
were found to be mixed type inhibitors as both the Km and Vmax
were changed with the most potent naphthoflavones and the inter-
section points of the plot indicated strong competitive component
of the inhibitors. Molecular modeling study confirmed naphthopy-
ran as a potential surrogate owing to its interactions with the
amino acid residues. NF-4 displayed interesting interactions with
the amino acid residues in the substrate binding site further sup-
porting the competitive aspect of the mixed type inhibitor. Thus
NF-4 and NF-12 appears to the good hits among the series of
naphthoflavones and displays promising attributes for detailed
investigation.
17. Naeimi, H.; Moradi, L. J. Mol. Catal. A: Chem. 2006, 256, 242.
18. 2-(2-Fluorophenyl)-4H-benzo[h]chromen-4-one (NF-2): Yield: 71%; mp: 118–
120 °C; 1H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.55 (1H, m), 8.17 (1H, d,
J = 8.7 Hz), 7.92–8.05 (2H, m), 7.69–7.79 (3H, m), 7.55 (1H, m), 7.24–7.41 (2H,
m), 6.91 (1H, s); 13C NMR (CDCl3, 75 MHz, d, 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, 162.2, 178.2. Anal. Calcd for
C19H11FO2: C, 78.61; H, 3.82; F, 6.54. Found: C, 78.73; H, 3.54. 2-(4-
Fluorophenyl)-4H-benzo[h]chromen-4-one (NF-4): Yield: 78%; mp: 154–156 °C;
1H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.55 (1H, br s), 8.14 (1H, d, J = 8.7 Mz),
7.92–8.07 (3H, m), 7.77 (1H, d, J = 8.7 Hz), 7.64–7.74 (2H, m), 7.23–7.29 (2H,
m), 6.91 (1H, s); 13C NMR (CDCl3, 75 MHz, d, 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. Anal. Calcd for C19H11FO2: C, 78.61; H, 3.82; F, 6.54. Found:
C, 79.00; H, 3.53. 2-(4-Nitrophenyl)-4H-benzo[h]chromen-4-one (NF-12): Yield:
76%; mp: 73–75 °C; 1H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.60 (1H, m), 8.45
(2H, d, J = 9.00 Hz), 8.18 (3H, m), 8.01 (1H, m), 7.67–7.86 (3H, m), 7.08 (1H, s);
13C NMR (CDCl3, 75 MHz, d, 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.
Acknowledgments
The authors are grateful to the Dr. K.A. Suri, Ex-director grade
scientist, IIIM (C.S.I.R), Jammu, India for providing us pure samples
of Apigenin employed in the study as a reference inhibitor of
xanthine oxidase.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.
041.
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Please cite this article in press as: Singh, H.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.041