Design, docking studies and molecular iodine catalyzed synthesis of benzo[a]xanthen-one derivatives as hyaluronidase inhibitors

Article abstract of DOI:10.1007/s00044-016-1655-2

A series of novel benzo[a]xanthen-11(12H)-one derivatives 4a–m were designed and subjected to docking studies. The title compounds were synthesized from 3-aryl-4-formylsydnones 1a–m, β-naphthol and dimedone in presence of molecular iodine as a catalyst an

Full text of DOI:10.1007/s00044-016-1655-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1655-2
ORIGINAL RESEARCH
Design, docking studies and molecular iodine catalyzed synthesis of benzo
[a]xanthen-one derivatives as hyaluronidase inhibitors
Mahadev N. Kumbar1 Ravindra R. Kamble1 Atulkumar A. Kamble1
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Shrinivas D. Joshi2 Sheshagiri R. Dixit2 Joy Hoskeri3
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Received: 13 May 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract A series of novel benzo[a]xanthen-11(12H)-one
derivatives 4a–m were designed and subjected to docking
studies. The title compounds were synthesized from 3-aryl-
4-formylsydnones 1a–m, β-naphthol and dimedone in pre-
sence of molecular iodine as a catalyst and evaluated for
their in vitro inhibitory effects on the hyaluronidase.
1997, Cao et al., 2007, Djoufack et al., 2010, Niu et al.,
2012, Tao et al., 2008, Zelefack et al., 2009). Sydnones
(Browne and Harrity, 2010; Stewart, 1964; Baker and Ollis,
1957) are the most prominent five-member non benzenoid
hetero-aromatic compounds. They are the well-defined class
of mesoionic compounds and have oxadiazole skeleton
exhibiting aromaticity through a net separation of formal
positive and negative charges. Since their discovery in
1935, sydnones have attracted significant attention from a
wide variety of researchers due to the interesting structural
and physicochemical properties. Their derivatives have
been recognized to exhibit a wide spectrum of bioactivities
including antibacterial (Mohamed et al., 2004), anticancer
(Christopher and Charles, 2003) and anti-inflammatory
(Wagner and Hill, 1974) etc.
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●
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Keywords Sydnone Dimedone Molecular iodine
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Molecular docking Hyaluronidase Anti-inflammatory
activity
Introduction
During drug development of non-steroidal anti-inflammatory
drugs (NSAIDs) it was believed that serotonin was a pos-
sible mediator for inflammation. Consequently, serotonin
was used as a lead for anti-inflammatory agents and on
these bases the anti-inflammatory drug Indomethacin was
developed. So also sydnone appended to dihydropyr-
imidines (DHPMs) have been found to show anti-inflam-
matory properties (Tegginamath et al., 2013) (Fig. 1).
Structure activity relationship (SAR) for the above drug
development results and the structures of various class of
clinically established NSAID’s confirm to a broadly
accepted pharmacophore (Fig. 2) which suggests four
important structural requirements for anti-inflammatory
action on hyaluronidase enzyme viz., i) aromatic ring A and
naphthalene nucleus C (help in crossing biological mem-
brane) fused with six member heterocyclic unit i.e., pyran B
(required for bioactivity) ii) Pyran is further attached to a
nitrogen containing aromatic/heterocyclic unit D (pharma-
cophore). Based on the above observations, the lead
molecules were designed in this report (Fig. 3).
Xanthenes and benzoxanthenes are the most important
classes of biodynamic heterocycles and hence their synth-
esis has received much attention especially in the field of
medicinal and pharmaceutical chemistry (Lambert et al.,
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1655-2) contains supplementary material,
which is available to authorized users.
* Ravindra R. Kamble
ravichem@kud.ac.in
1
Department of Chemistry, Karnatak University, Dharwad 580003
Karnataka, India
2
Novel Drug Design and Discovery Laboratory, Department of
Pharmaceutical Chemistry, S.E.T.’s College of Pharmacy,
Sangolli Rayanna Nagar, Dharwad 580 002 Karnataka, India
3
Department of Biochemical Pharmacology, Institute of
Experimental Pharmacology and Toxicology, Slovak Academy of
Sciences, Bratislava, Slovak Republic

Med Chem Res
Fig. 1 DHPMs containing
sydnone
O
N
O
N
O
N
N
N
O
O
N
O
O
R
EtOOC
NH
R
R
NH
NH
H3C
N
X
X
N
X
H
O
H
X=O, S; R= H, CH3
Fraser, 1992). Therefore, hyaluronidase is one of the drug
targets to exhibit the anti-inflammatory property and hence
our efforts were concentrated to develop hyaluronidase
inhibitors to maintain the HA level which may lead to the
development of new class of therapeutics of NSAIDs.
All the newly designed molecules 4a–m were docked
within the “active site” of the hyaluronidase (hyaluronate
lyase PDB ID: 1W3Y) enzyme with different orientations.
The protein structure files and the ligand molecules were
drawn by using Surflex-Dock program interfaced with
Sybyl-X 2.0. The acting forces of this binding mode are
mainly hydrogen bonding, electrostatic forces, Vander
Waal’s forces, and hydrophobic interaction due to non-polar
residue interaction. Docking results were followed by in
vitro hyaluronidase inhibition analysis.
D
Pharmacophore
Linker
To cross
A
C
To cross
Biological
membrane
B
Biological
membrane
Bioactivity
Fig. 2 SAR of anti-inflammatory drug
N
O
N
Ar
O
O
Results and discussion
Chemistry
O
4a-m
One-pot three component synthetic route to target com-
pounds is outlined in Scheme 1. This involves the con-
densation of 3-aryl-4-formylsydnones 1a–m with 2-
naphthol and dimedone using molecular iodine as catalyst at
90 °C in ethanol to give 9,10-dihydro-12-(3-arylsydnon-4-
yl)-9,9-dimethyl-8H-benzo[a]xanthen-11(12H)-one deriva-
tives 4a–m. Scheme 2 depicts the plausible mechanism of
formation of title compounds 4a–m under the influence of
molecular iodine as catalyst.
Fig. 3 Newly designed xanthenes containing sydnone
After designing the molecules based on the SAR studies,
we planned to carry out the docking. Molecular docking
study is a well-established technique to determine the
interaction of two molecules and find the best orientation of
ligand that would form a complex with overall minimum
energy (Thangasamy et al., 2014). Hyaluronidase is one of
the most important enzymes in the development of many
diseases that degrade hyaluronic acid (HA). HA is ubiqui-
tous component of the extracellular matrix of vertebrates
which is a linear polysaccharide composed of a repeating
unit with the structure [D-Glucuronic acid (1-β-3) N-acetyl-
D-glucosamine (1-β-3)]. HA can form highly viscous solu-
tion and thereby influence the properties of this matrix. It
has been implicated in many biological processes such as
fertilization, embryonic development, cell migration and
differentiation, inflammation, growth including metastasis
of tumor cells. The activity of hyaluronidase is increased
during chronic inflammatory conditions including
inflammatory joint disease (Foschi, 1990; Laurent and
The structures of synthesized compounds 4a–m were
1
confirmed by infrared (IR), H & 13C NMR, MS and ele-
mental analyses. In case of IR spectral studies, the com-
pounds have shown strong adsorption band for carbonyl of
sydnone and xanthene rings at 1715–1739 and 1672–1689
cm−1 respectively. 1H NMR spectral studies exhibit a singlet
for six protons in the range 1.00–1.11 ppm. due to two
methyl groups and a singlet for one proton present on pyran
ring at 3.28–3.41 ppm. Further, two singlets for each two
protons in the range of 2.96–2.49 and 2.54–2.05 ppm. were
observed due to CH2 groups of xanthene. The aromatic
protons in all compounds appeared as multiplets in the range
6.76–7.99 ppm. In case of 13C NMR spectral study, the

Med Chem Res
Scheme 1 Synthetic route for the
title compounds 4a–m. Where a,
R = –C6H5; b, R = 4–CH3–C6H4;
c, R = 4–OCH3–C6H4; d, R =
4–Cl–C6H4; e, R = 4–Br–C6H4;
f, R = 4–NO2–C6H4; g, R =
3–CH3–C6H4; h, R =2
–CH3–C6H4; i, R = 3–Cl–C6H4;
j, R = 2–Cl–C6H4; k, R = 2–
OCH3–C6H4; l, R = 3–OCH3–
C6H4; m, R = –C–(CH3)3
N
O
O
R
O
N
O
R
CHO
O
OH
I2
N
EtOH
N
O
O
O
2
3
1a-m
4a-m
Scheme 2 Plausible mechanism
for the formation of
δ
δ
I
I
benzoxanthene derivatives
4a–m showing the catalytic role
of molecular iodine
R
R
N
O
R
N
N
N
O
N
H
I
O
OH
O
O
H
N
3
O
O
H
H
O
O
δ
- I2
+I2
O
O
O
1a-m
I
2
O
N
R
R
R
O
N
O
H
O
N
O
N
N
N
H
O
O
- H2O
- I2
O
OH
δ
O
O
δ
OH
O
I
I
O
4 a-m
numbers of signals are in consistent with number of mag-
netically nonequivalent carbon atoms in the molecule and in
mass spectra of all the synthesized compounds showed the
molecular ion peaks at their respective m/z values.
interactions, oxygen atom of sydnone interacts with
hydrogen of AGR336 (–O ----- H–ARG336; 1.95 & 1.92
Å), nitrogen atom of sydnone ring makes a hydrogen
bonding interaction with H atom of ARG336 (N ------ H–
ARG336; 2.69 Å), and oxygen atom present at fifth position
(carbonyl) makes the hydrogen bonding interaction with H
atom of ARG336 (C–O -----H–ARG336; 2.42 Å) and
remaining hydrogen bond interaction was raised from the
interaction of carbonyl oxygen group of xanthene ring
(pyran) with H atom of ASN349 (C=O -----H–ASN349;
2.10 Å). Another compound 4k also has shown three
hydrogen bonding interactions with the enzyme. The first
hydrogen bonding was due to the interaction of H atom of
ASN290 with nitrogen atom of sydnone (N -----H-ASN290;
2.50 Å). Remaining two interactions were due to oxygen
atom of xanthene carbonyl with H atoms of ARG462 (C=O
-----H-ARG462; 1.73 Å) and H atom of TYR408 (C=O
-----H– TYR408; 2.28 Å). The C score (consensus score)
values indicated the summary of all four interactions
between the inhibitors and the enzyme. Crash score
revealed the penetration into the binding site which was in
Docking studies
All the inhibitors were docked into the binding site of
hyaluronidase and the energy scores of the inhibitors are
shown in Table 1 where a precise correlation can be found
between docking studies and in vitro activity results.
A complete overview of receptor – inhibitor binding inter-
actions is presented in Figs. 4 and 5. They vividly represent
the interaction model of the most potent inhibitors 4f and 4k
with hyaluronidase (hyaluronate lyase), respectively in
which both the inhibitors are suitably situated at the enzyme
binding site and there are various interactions between them
and the binding region of the enzyme. Compound 4f
showed four hydrogen binding interactions at the active site
of the enzyme (PDB ID: 1W3Y) with the amino acids such
as ARG336, and ASN349. Among the four hydrogen bond

Med Chem Res
Chem scoreg
Table 1 Surflex docking score (kcal/mol) of the compounds 4a–m
Compounds
C scorea
Crash scoreb
Polar scorec
D scored
PMF scoree
G scoref
4a
4.73
4.85
5.36
4.91
5.44
6.39
5.32
4.07
6.11
3.99
5.65
5.14
4.86
4.29
−0.70
−0.73
−2.23
−1.06
−1.89
−1.06
−1.40
−0.51
−0.88
−1.28
−1.12
−1.64
−1.29
−0.84
1.71
1.26
0.68
1.10
0.01
1.62
1.84
0.99
1.38
0.54
1.93
0.96
1.46
2.09
−2674.24
−2664.21
−3119.85
−2908.13
−3744.14
−2819.74
−3046.88
−3638.24
−2535.01
−2326.51
−2691.52
−3636.93
−2644.67
−2010.66
−84.68
−90.07
−55.62
−97.61
−60.23
−102.47
−82.60
−70.60
−88.27
−103.34
−117.04
−110.64
−89.14
−56.00
−202.41
−212.33
−252.49
−255.69
−260.16
−229.90
−231.38
−188.08
−235.11
−232.72
−225.52
−261.43
−237.05
−145.66
−38.83
−38.41
−26.75
−37.74
−28.66
−37.97
−33.50
−30.63
−29.73
−30.95
−37.51
−37.92
−41.64
−26.98
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
Indomethacin
a
C score (Consensus score) integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a
receptor and reports the output of total score
b
Crash score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable. Negative numbers indicate
penetration
c Polar indicating the contribution of the polar interactions to the total score. The polar score may be useful for excluding docking results that make
no hydrogen bonds
d
D score for charge and Vander Waals interactions between the protein and the ligand
e
PMF score indicating the Helmholtz free energies of interactions for protein-ligand atom pairs (potential of mean force, PMF)
f
G score showing hydrogen bonding, complex (ligand–protein), and internal (ligand–ligand) energies
g
Chem score points for H-bonding, lipophilic contact, and rotational entropy, along with an intercept term
The bold values indicate that the results are impressive
favor of the compounds under analysis. The compounds 4f
and 4k showed better hydrogen bonding and favorable
internal energies than the other compounds including stan-
dard drug Indomethacin. These compounds are attributed to
the lipophilicity of the sydnone moiety fused with xanthene
(pyran) due to which better Vander Waal’s interaction was
achieved between ligand and the receptor. Molecular
docking study revealed that majority of the docked com-
pounds exhibited H-bonds and this fact prompted us to
carry out in vitro hyaluronidase inhibition analysis. (Fig. 6)
moieties have shown least activity. The standard deviation
for percentage inhibition of hyaluronidase enzyme at 10,
50, and 100 μg concentrations were found to be 32.15,
59.86, and 96.44 respectively (Fig. 7).
Conclusions
In summary, a series of novel 9,10-dihydro-12-(3-arylsyd-
non-4-yl)-9,9-dimethyl-8H-benzo[a]xanthen-11(12H)-one
derivatives 4a–m were designed and synthesized using
molecular iodine as a catalyst. Molecular docking results
against hyaluronidase (hyaluronate lyase) were sub-
stantiated by in vitro inhibition analysis. It is interesting to
note that synthesized compounds showed moderate to
potent inhibition. These results indicate that the compounds
reported in this paper are useful for further studies exhi-
biting the anti-inflammatory activity.
In vitro hyaluronidase inhibition (anti-inflammatory
activity) of title compounds
The docking results were further substantiated by in vitro
anti-inflammatory activity using inhibition of hyaluronidase
enzyme as a model. From Table 2 it can be concluded that
the synthesized molecules 4f and 4k have shown 86–88 %
growth inhibition which was almost equal to the standard
Indomethacin at 100 μg concentration. However, the com-
pounds 4a–b, 4h–i have exhibited 52–65 % growth inhi-
bition at the 100 μg concentration. The remaining
compounds 4c–e, 4g, 4l and 4m with large aromatic
Experimental protocols
Melting points were determined in open capillaries and are
uncorrected. Progress of the reaction checked by Aluchrosep

Med Chem Res
Fig. 4 Docked view of compound 4f at the active site of enzyme hyaluronate lyase
Fig. 5 Interaction of 4k at the binding site of enzyme hyaluronate lyase
Silica Gel 60/UV 254 thin layer chromatography (TLC) on
silica gel coated plates in ethyl acetate and hexane (3:7, 30
%) as eluent. IR spectra were recorded in a KBr disc matrix
General procedure for the preparation of title
compounds
1
using an IMPACT-410 Nicolet (USA). H and 13C NMR
3-Aryl-4-formylsydnone 1a–m was prepared by formyla-
tion of 3-arylsydnone using N-methylformanilide in POCl3
(Thoman et al., 1964). Iodine (0.2 mmol) in absolute etha-
nol (6 ml) was added to a mixture of 3-aryl-4-for-
spectra were recorded on Bruker Avance 300 MHz FT NMR
spectrometers with (DMSO-d6) and CDCl3 as solvent and
tetramethylsilane was used as internal standard. The mass
spectra were recorded on Shimadzu gas chromatography–
mass spectrometry instrument operating at 70 eV spectro-
meter and elemental analyses were carried out using Heraus
CHN rapid analyzer. All the solvents and reagents were of
analytical grade and are purchased from Sigma-Aldrich and
E-Merck used without further purification.
mylsydnone (1 mmol), β-naphthol
2 (1 mmol), and
dimedone 3 (1.2 mmol). The mixture was stirred at 90 °C
for 3–4 h. After completion (TLC), the reaction mixture was
quenched into crushed ice. The crude product formed was
filtered, washed and dried. Further recrystallized in ethanol
to get pure title compounds 4a–m.

Med Chem Res
Fig. 6 Docked image of standard drug Indomethacin with hyaluronate lyase
9,10-Dihydro-12-(3-phenylsydnon-4-yl)-9,9-dimethyl-8H-
Table 2 Results of in vitro hyaluronidase inhibition of title
compounds 4a–m
benzo[a]xanthen-11(12H)-one (4a)
Compounds
Concentration (µg)
Yellow solid, recrystallized from ethanol, yield: 82 %. m.p.:
10
50
100
196–197 °C; IR (KBr) cm−1: 1716 (sydnone C=O), 1669
1
4a
10.12
11.92
09.98
09.62
17.21
21.47
09.18
12.75
11.45
05.56
29.14
09.16
09.42
32.15
51.27
53.14
14.96
15.42
19.45
51.60
16.03
53.14
49.43
11.42
50.16
15.76
14.56
59.86
60.42
65.73
24.94
27.99
31.17
86.76
25.63
62.86
64.98
22.92
88.43
27.87
28.31
96.44
(xanthene C=O). H NMR (400 MHz, DMSO-d6): δ 7.86–
7.21 (11H, m, Ar–H), 4.25 (1H, s, pyran C4–H), 2.49 (2H,
s, CH2), 2.08 (2H, s, CH2), 1.01 (6H, s, -(CH3)2); 13C NMR
(100 MHz, CDCl3): δ 200.02 (xanthene C=O), 175.90
(sydnone C=O), 172.52 (C–O), 166.03, 160.02 (Ar–C),
155.22 (Ar–C), 146.02 (Ar–C), 140.01 (Ar–C), 134.54 (Ar–
C), 129.21 (Ar–C), 127.66 (Ar–C), 127.46 (Ar–C), 126.02
(Ar–C), 125.91 (Ar–C), 122.53 (Ar–C), 118.54 (Ar–C),
115.20 (Ar–C), 112.51 (Ar–C), 108.58, 106.02 (Ar–C),
102.54 (Ar–C), 100.08 (sydnone C4), 55.70, 34.61, 32.50
4b
4c
4d
4e
4f
4g
4h
4i
4j
(pyran
C4),
30.54,
27.02
(–CH3)2;
Anal.
4k
calcd. for C27H22N2O4: C, 74.02; H, 5.08; N, 6.45. Found
C, 73.96; H, 5.06; N, 6.39 %. MS (EI, 70 eV) m/z: 438.5
(M + 1).
4l
4m
Indomethacin
The bold values indicate that the results are impressive.
9,10-Dihydro-12-(-3-p-tolylsydnon-4-yl)-9,9-dimethyl-8H-
benzo[a]xanthen-11(12H)-one (4b)
122.90 (Ar–C), 118.75 (Ar–C), 115.46 (Ar–C), 114.51,
108.68 (Ar–C), 101.77 (sydnone C4), 55.09, 43.22, 32.88
(pyran C4), 27.90 (–CH3)2, 25.63 (–CH3); Anal. calcd. for
C28H24N2O4: C, 74.39; H, 5.41; N, 6.24. Found C, 74.32;
H, 5.35; N, 6.19 %. MS (EI, 70 eV) m/z: 452.5 (M + 1).
Brown color solid, recrystallized from ethanol, yield: 76 %.
m.p.: 172–173 °C; IR (KBr) cm−1: 1724 (sydnone C=O),
1677 (xanthene C=O). 1H NMR (400 MHz, CDCl3): δ 7.72
–7.01 (10H, m, Ar–H), 3.34 (1H, s, pyran C4–H), 2.77 (2H,
s, CH2), 2.51 (2H, s, CH2), 2.37 (3H, s, –CH3), 1.08 (6H, s,
–(CH3)2); 13C NMR (100 MHz, CDCl3): δ 200.45 (C=O),
177.42 (C=O), 175.27 (C–O), 162.94, 154.05 (Ar–C),
152.19 (Ar–C), 139.47 (Ar–C), 137.92 (Ar–C), 135.20 (Ar–
C), 134.18 (A–C), 131.18 (Ar–C), 129.25 (Ar–C), 128.31
(Ar–C), 126.67 (Ar–C), 125.35 (Ar–C), 123.24 (Ar–C),
9,10-Dihydro-12-(3-p-methoxyphenyl-sydnon-4-yl)-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4c)
Pale yellow color solid, recrystallized from ethanol. Yield:
65 %. m.p.: 156–157 °C; IR (KBr) cm−1: 1722 (sydnone

Med Chem Res
Fig. 7 Bar graph of in vitro
hyaluronidase inhibition of
compounds 4a–m
C=O), 1673 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.99–7.42 (6H, m, Ar–H), 7.21 (2H, d, Ar–H), 7.08 (2H,
d, Ar–H), 4.38 (1H, s, pyran C4–H), 3.29 (3H, s, OCH3),
2.49 (2H, s, CH2), 2.48 (2H, s, CH2), 1.50 (6H, s, –(CH3)2);
13C NMR (100 MHz, CDCl3): δ 200.53 (C=O), 177.00
(C=O), 174.80 (C–O), 163.01, 153.91 (Ar–C), 138.91
(Ar–C) 137.21 (Ar–C), 135.48 (Ar–C), 134.64 (A–C),
129.59 (Ar–C), 128.67 (Ar–C), 127.65 (Ar–C), 126.27
(Ar–C), 125.98 (Ar–C), 123.48 (Ar–C), 123.26 (Ar–C),
118.00 (Ar–C), 114.89, 109.39 (Ar–C), 101.31 (sydnone
C4), 64.37 (–OCH3), 55.82, 50.52, 42.92, 32.49 (pyran C4),
28.19 (–CH3)2, 27.64; Anal. calcd. for C28H24N2O5:
C, 71.84; H, 5.21; N, 6.07. Found C, 71.78; H, 5.16;
N, 5.98 %. MS (EI, 70 eV) m/z: 468.5 (M + 1).
12-(3-(p-Bromophenylsydnon-4-yl)-9,10-dihydro-9,
9-dimethyl-8H-benzo[a]xanthen-11(12H)-one (4e)
Dark brown color, recrystallized from ethanol. Yield: 72 %.
m.p.: 174–175 °C; IR (KBr) cm−1: 1715 (sydnone C=O),
1666 (xanthene C=O). 1H NMR (400 MHz, CDCl3): δ 7.75
(2H, d, Ar–H), 7.73 (2H, d, Ar-H), 7.72–7.23 (6H, m, Ar–
H), 3.28 (1H, s, pyran C4–H), 2.73 (2H, s, CH2), 2.49 (2H,
s, CH2), 1.07 (6H, s, –(CH3)2); 13C NMR (100 MHz,
CDCl3): δ 195.03 (C=O), 176.19 (C=O), 172.38 (C–O),
164.12, 162.88 (Ar–C), 159.40 (Ar–C), 154.45 (Ar–C),
141.12 (Ar–C), 140.49 (Ar–C), 134.52 (Ar–C), 129.59 (Ar–
C), 128.68 (Ar–C), 126.01 (Ar–C), 124.52 (Ar–C), 122.23
(Ar–C), 121.79 (Ar–C), 115.03, 113.74 (Ar–C), 111.23
(Ar–C), 107.12 (Ar–C), 101.23 (sydnone C4), 50.38, 40.61,
33.16 (pyran C4), 31.23, 28.20 (–CH3)2; Anal. calcd. for
C27H21N2BrO4: C, 62.76; H, 4.17; N, 5.55. Found C, 62.68;
H, 4.09; N, 5.41 %. MS (EI, 70 eV) m/z: 518.1 (M + 2),
516.1.
12-(3-(p-Chlorophenylsydnon-4-yl)-9,10-dihydro-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4d)
Yellow solid, recrystallized from ethanol, yield: 77 %. m.p.:
197–198 °C; IR (KBr) cm−1: 1729 (sydnone C=O), 1678
(xanthene C=O). 1H NMR (400 MHz, CDCl3): δ 7.74 (2H,
d, Ar–H), 7.70 (2H, d, Ar–H), 7.68–7.05 (6H, m, Ar–H),
3.32 (1H, s, pyran C4–H), 2.92 (2H, s, CH2), 2.54 (2H, s,
CH2), 1.11 (6H, s, –(CH3)2); 13C NMR (100 MHz, CDCl3):
δ 196.09 (C=O), 175.24 (C=O), 171.38 (C–O), 166.56,
161.24 (Ar–C), 157.31 (Ar–C), 155.63 (Ar–C), 140.92 (Ar–
C), 138.67 (Ar–C), 132.79 (Ar–C), 129.99 (Ar–C), 127.82
(Ar–C), 125.62 (Ar–C), 123.46 (Ar–C), 122.90 (Ar–C),
120.82 (Ar–C), 114.46, 113.67 (Ar–C), 110.34 (Ar–C),
109.29 (Ar–C), 100.37 (sydnone C4), 54.43, 42.75, 33.19
(pyran C4), 31.92, 28.2 (–CH3)2, 27.12; Anal. calcd. for
C27H21N2ClO4: C, 68.51; H, 4.53; N, 5.98. Found C, 68.57;
H, 4.48; N, 5.92 %. MS (EI, 70 eV) m/z: 474.1 (M + 2),
472.1 (M + 1).
9,10-Dihydro-12-(-3-(p-nitrophenylsydnon-4-yl)-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4f)
Pale yellow color solid, recrystallized from ethanol. Yield:
52 %. m.p: 132–133 °C; IR (KBr) cm−1: 1715 (sydnone
C=O), 1666 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.87 (2H, d Ar–H), 7.84 (2H, d, Ar–H), 7.46–7.03 (6H, m,
Ar–H), 3.37 (1H, s, pyran C4–H), 2.70 (2H, s, CH2), 2.55
(2H, s, CH2), 1.09 (6H, s, –(CH3)2); 13C NMR (100 MHz,
CDCl3): δ 198.62 (C=O), 176.56 (C=O), 174.19 (C–O),
161.87, 154.91 (Ar–C), 139.20 (Ar–C) 136.46 (Ar–C),
135.94 (Ar–C), 132.16 (A–C), 130.73 (Ar–C), 129.02 (Ar–
C), 128.34 (A–C), 126.98 (Ar–C), 124.43 (Ar–C), 122.61
(Ar–C), 121.65 (Ar–C), 119.00 (Ar–C), 113.76, 108.49

Med Chem Res
(Ar–C), 102.73 (sydnone C4), 66.51 (–OCH3), 57.52,
49.68, 31.76 (pyran C4), 30.91, 28.63 (–CH3)2, 27.94; Anal.
calcd. for C27H21N3O6: C, 67.09; H, 4.42; N, 8.77. Found
C, 67.07; H, 4.38; N, 8.69 %. MS (EI, 70 eV) m/z: 483.5
(M + 1).
(2H, s, CH2), 2.49 (2H, s, CH2), 1.07 (6H, s, –(CH3)2); 13
C
NMR (100 MHz, CDCl3): δ 195.64 (C=O), 176.64 (C=O),
172.61 (C–O), 164.16, 160.49 (Ar–C), 156.12 (Ar–C),
153.01 (Ar–C), 141.84 (Ar–C), 133.14 (Ar–C), 131.67 (Ar–
C), 130.13 (Ar–C), 126.62 (Ar–C), 124.13 (Ar–C), 124.68
(Ar–C), 121.29 (Ar–C), 120.72 (Ar–C), 115.79, 114.67
(Ar–C), 111.42 (Ar–C), 107.81 (Ar–C), 102.19 (sydnone
C4), 55.49, 40.57, 32.26 (pyran C4), 30.74, 28.34 (–CH3)2,
26.63; Anal. calcd. for C27H21N2ClO4: C, 68.62; H, 4.51;
N, 6.01. Found C, 68.57; H, 4.48; N, 5.92 %. MS (EI, 70
eV) m/z: 474.1 (M + 2), 472.1 (M+).
9,10-Dihydro-12-(3-m-tolylsydnon-4-yl)-9,9-dimethyl-8H-
benzo[a]xanthen-11(12H)-one (4g)
Pale brown color solid, recrystallized from ethanol, yield:
72 %. m.p.: 182–183 °C; IR (KBr) cm−1: 1727 (sydnone
C=O), 1682 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.49–7.03 (10H, m, Ar–H), 3.39 (1H, s, pyran C4–H),
2.96 (2H, s, CH2), 2.51 (2H, s, CH2), 2.42 (3H, s, –CH3),
1.02 (6H, s, –(CH3)2); 13C NMR (100 MHz, CDCl3): δ
197.72 (C=O), 177.58 (C=O), 174.62 (C–O), 160.21,
153.14 (Ar–C), 150.54 (Ar–C), 1742.67 (Ar–C), 139.69
(Ar–C), 136.82 (Ar–C), 133.49 (A–C), 130.82 (Ar–C),
129.61 (Ar–C), 127.73 (Ar–C), 127.02 (Ar–C), 124.87 (Ar–
C), 122.23 (Ar–C), 121.34 (Ar–C), 116.86 (Ar–C), 115.73
(Ar–C), 112.87, 110.24 (AR–C), 103.72 (sydnone C4),
53.51, 41.16, 31.97 (pyran C4), 28.64 (–CH3)2, 27.03 (–
CH3); Anal. calcd. for C28H24N2O4: C, 74.37; H, 5.41; N,
6.22. Found C, 74.32; H, 5.35; N, 6.19 %. MS (EI, 70 eV)
m/z: 452.2 (M + 1).
12-(3-(o-Chlorophenylsydnon-4-yl)-9,10-dihydro-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4j)
Yellow color solid, recrystallized from ethanol. Yield:
71 %. m.p.: 162–163 °C; IR (KBr) cm−1: 1729 (sydnone
C=O), 1672 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.74–7.05 (10H, m, Ar–H), 3.36 (1H, s, pyran C4–H),
2.79 (2H, s, CH2), 2.51 (2H, s, CH2), 1.11 (6H, s, –(CH3)2);
13C NMR (100 MHz, CDCl3): δ 196.77 (C=O), 174.56
(C=O), 172.94 (C–O), 162.19, 161.85 (Ar–C), 155.81 (Ar–
C), 152.07 (Ar–C), 140.46 (Ar–C), 132.87 (Ar–C), 131.98
(Ar–C), 130.02 (Ar–C), 125.31 (Ar–C), 124.74 (Ar–C),
124.08 (Ar–C), 122.34 (Ar–C), 120.97 (Ar–C), 114.80,
114.12 (Ar–C), 111.09 (Ar–C), 108.26 (Ar–C), 101.53
(sydnone C4), 56.34, 41.61, 31.71 (pyran C4), 30.82, 28.73
(–CH3)2, 26.93; Anal. calcd. for C27H21N2ClO4: C, 68.60;
H, 4.53; N, 5.98. Found C, 68.57; H, 4.48; N, 5.92 %. MS
(EI, 70 eV) m/z: 474.1 (M + 2), 472.1 (M+).
9,10-Dihydro-12-(3-o-tolylsydnon-4-yl)-9,9-dimethyl-8H-
benzo[a]xanthen-11(12H)-one (4h)
Pale brown color solid, recrystallized from ethanol. Yield:
72 %. m.p.: 144–145 °C; IR (KBr) cm−1: 1732 (sydnone
C=O), 1689 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.34–6.76 (10H, m, Ar–H), 3.41 (1H, s, pyran C4–H),
2.84 (2H, s, CH2), 2.66 (2H, s, CH2), 2.45 (3H, s, –CH3),
1.10 (6H, s, –(CH3)2); 13C NMR (100 MHz, CDCl3): δ
199.02 (C=O), 176.09 (C=O), 174.49 (C–O), 161.16,
154.81 (Ar–C), 151.31 (Ar–C), 140.57 (Ar–C), 138.39 (Ar–
C), 136.67 (Ar–C), 134.50 (A–C), 132.59 (Ar–C), 130.19
(Ar–C), 129.42 (Ar–C), 127.61 (Ar–C), 124.91 (Ar–C),
123.84 (Ar–C), 121.13 (Ar–C), 117.25 (Ar–C), 116.43 (Ar–
C), 113.64, 109.97 (AR–C), 106.04 (sydnone C4), 56.61,
40.19, 30.96 (pyran C4), 28.42 (–CH3)2, 26.32 (–CH3);
Anal. calcd. for C28H24N2O4: C, 74.39; H, 5.43; N, 6.27.
Found C, 74.32; H, 5.35; N, 6.19 %. MS (EI, 70 eV) m/z:
452.2 (M + 1).
9,10-Dihydro-12-(3-(o-methoxyphenylsydnon-4-yl)-9,
9-dimethyl-8H-benzo[a]xanthen-11(12H)-one (4k)
Dark yellow color solid, recrystallized from ethanol. Yield:
62 %. m.p.: 137–138 °C; IR (KBr) cm−1: 1734 (sydnone
C=O), 1674 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.93 (1H, d Ar–H), 7.88 (1H, d, Ar–H), 7.81 (1H, d, Ar–
H), 7.47 (1H, t, Ar–H), 7.39 (1H, t, Ar–H), 7.23 (1H, d, Ar–
H), 7.09 (1H, d, Ar–H), 7.06 (1H, d, Ar–H), 7.03 (1H, d,
Ar–H), 6.81 (1H, d, Ar–H), 4.29 (1H, s, pyran C4–H), 3.77
(3H, s, OCH3), 2.76 (2H, s, CH2), 2.51 (2H, s, CH2), 1.04
(6H, s, –(CH3)2); 13C NMR (100 MHz, CDCl3): δ 200.06
(C=O), 177.49 (C=O), 174.15 (C–O), 162.48, 151.81 (Ar–
C), 139.06 (Ar–C) 137.75 (Ar–C), 135.67 (Ar–C), 133.15
(A–C), 130.79 (Ar–C), 128.16 (Ar–C), 127.90 (Ar–C),
126.73 (Ar–C), 124.36 (Ar–C), 123.79 (Ar–C), 122.05 (Ar–
C), 117.86 ( Ar–C), 115.62, 108.46 (Ar–C), 102.94 (syd-
none C4), 65.86 (OCH3), 56.31, 49.79, 41.64, 31.32 (pyran
C4), 28.97 (–CH3)2, 26.95; Anal. calcd. for C28H24N2O5: C,
71.82; H, 5.21; N, 6.03. Found C, 71.78; H, 5.16; N, 5.98
%. MS (EI, 70 eV) m/z: 468.5 (M + 1).
12-(3-(m-Chlorophenylsydnon-4-yl)-9,10-dihydro-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4i)
Yellow solid, recrystallized from ethanol. Yield: 75 %.
m.p.: 177–178 °C; IR (KBr) cm−1: 1726 (sydnone C=O),
1678 (xanthene C=O). 1H NMR (400 MHz, CDCl3): δ
7.68–7.09 (10H, m, Ar–H), 3.39 (1H, s, pyran C4–H), 2.72

Med Chem Res
9,10-Dihydro-12-(3-(m-methoxyphenylsydnon-4-yl)-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4l)
adding polar hydrogen atom with Gasteiger-Hückel (Gas-
teiger and Marsili, 1980) charges and water molecules were
removed. The 3D structure of the ligands was generated by
the SKETCH module implemented in the SYBYL program
(Tripos Inc., St. Louis, USA) and its energy-minimized
conformation was obtained with the help of Tripos force
field using Gasteiger - Hückel charges and molecular
docking was performed with Surflex-Dock program that is
interfaced with Sybyl-X 2.0 (Tripos International, 2012).
Other miscellaneous parameters were assigned with the
default values given by the software. In the present work the
default parameters of Surflex Docking program were used
for the molecular docking of ligands. To find the correct
conformations of the ligands and to obtain minimum energy
structures ligands were allowed to be flexible. At the end of
docking, the best conformations of the ligands were ana-
lyzed for their binding interactions.
Yellow color solid, recrystallized from ethanol. Yield:
67 %. m.p: 155–156 °C; IR (KBr) cm−1: 1731 (sydnone
C=O), 1677 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.92 (1H, d, Ar–H), 7.83 (1H, d, Ar–H), 7.80 (1H, d, Ar–
H), 7.46 (1H, t, Ar–H), 7.39 (1H, t, Ar–H), 7.18 (1H, d, Ar–
H), 7.12 (1H, d, Ar–H), 7.07 (1H, d, Ar–H), 7.02 (1H, d,
Ar–H), 6.76 (1H, d, Ar–H), 4.32 (1H, s, pyran C4–H), 3.79
(3H, –OCH3, s), 2.78 (2H, s, CH2), 2.61 (2H, s, CH2), 1.07
(6H, s, –(CH3,)2); 13C NMR (100 MHz, CDCl3): δ 200.16
(C=O), 176.49 (C=O), 173.43 (C–O), 162.79, 153.47 (Ar–
C), 139.03 (Ar–C) 137.68 (Ar–C), 136.17 (Ar–C), 132.15
(A–C), 129.61 (Ar–C), 128.94 (Ar–C), 127.37 (Ar–C),
127.03 (Ar–C), 124.24 (Ar–C), 123.92 (Ar–C), 122.47 (Ar–
C), 119.84 (Ar–C), 113.76, 108.64 (Ar–C), 102.85 (syd-
none C4), 66.53 (–OCH3), 57.29, 51.37, 40.18, 30.96
(pyran C4), 28.71 (–CH3)2, 26.59; Anal. calcd. for
C28H24N2O5: C, 71.84; H, 5.19; N, 6.06. Found C, 71.78;
H, 5.16; N, 5.98 %. MS (EI, 70 eV) m/z: 468.5 (M + 1).
Hyaluronidase inhibition (anti-inflammatory) assay
(Ling et al., 2003)
The assay medium consisting of 3-5U hyaluronidase (from
Sigma–Aldrich, Bangalore) in 100 µl of 20 mM sodium
phosphate buffer (pH 7.0) with 77 mM sodium chloride,
0.01 % bovine serum albumin (BSA) was pre-incubated
with different concentrations (10, 50 and 100 μg) of the test
compound for 15 min at 37 °C. The assay was commenced
by adding HA (100 µl, Sigma–Aldrich, Bangalore; 0.03 %
in 300 mM sodium phosphate, pH 5.35) to the incubation
mixture and incubated for further 45 min at 37 °C. The
undigested hyaluronic acid was precipitated with acid
albumin solution (1 ml) made up of 0.1 % BSA in 24 mM
sodium acetate and 79 mM acetic acid, (pH 3.75). After
standing at room temperature for 10 min, the absorbance of
the reaction mixture was measured at 600 nm. The absor-
bance in absence of enzyme was used as reference value for
maximum inhibition. The inhibitory activity of test com-
pound was calculated as the percentage ratio of absorbance
in presence of test compound vs. absorbance in the absence
of enzyme. The enzyme activity was checked by control
experiment run simultaneously, in which the enzyme was
pre-incubated with DMSO (5 µl) followed by the assay
procedures described above. Samples were tested in the
range of 5 µg, 10 µg and 100 µg in the reaction mixture.
Indomethacin was used as reference standard.
12-(3-(p-t-Butylphenylsydnon-4-yl)-9,10-dihydro-9,9-
dimethyl-8H-benzo[a]xanthen-11(12H)-one (4m)
Dark yellow color solid, recrystallized from ethanol. Yield:
73 %. m.p.: 149–150 °C; IR (KBr) cm−1: 1739 (sydnone
C=O), 1679 (xanthene C=O). 1H NMR (400 MHz, CDCl3):
δ 7.69 (1H, d, Ar–H), 7.59 (1H, d, Ar–H), 7.57 (1H, d, Ar–
H), 7.48 (1H, d, Ar–H), 7.30 (1H, t, Ar–H), 7.21 (1H, t, Ar–
H), 7.16 (1H, d, Ar–H), 7.09 (1H, d, Ar–H), 7.05 (1H, d
Ar–H), 6.98 (1H, d Ar–H), 3.92 (1H, s, pyran C4–H), 2.95
(2H, s, CH2), 2.07 (2H, s, CH2), 1.38 (9H, s, –(CH3)3), 1.10
(6H, s, –(CH3)2); 13C NMR (100 MHz, CDCl3): δ 198.6
(C=O), 176.31 (C=O), 174.56 (C–O), 160.68, 154.78 (Ar–
C), 152.35 (Ar–C), 141.51 (Ar–C), 136.71 (Ar–C), 133.49
(Ar–C), 131.78 (Ar–C), 130.67 (Ar–C), 129.96 (Ar–C),
126.28 (Ar–C), 125.23 (Ar–C), 123.83 (Ar–C), 122.79 (Ar–
C), 120.46 (Ar–C), 118.58 (Ar–C), 114.32, 110.87 (Ar–C),
102.68 (sydnone C4), 52.87, 45.60, 41.79, 32.8 (–(CH3)3),
30.8, 28.3 (–CH3)2, 27.90 (pyran C4); Anal. calcd. for
C31H30N2O4: C, 71.82; H, 5.21; N, 6.03. Found C, 71.78;
H, 5.16; N, 5.98 %. MS (EI, 70 eV) m/z: 468.5 (M + 1).
Pharmacological evaluation
Molecular docking
Acknowledgments The authors are thankful to Dr. V. H. Kulkarni,
Professor and Principal, S.E.T.’s College of Pharmacy, Dharwad, India
for docking study. Also we are grateful to Biogenics Ltd, Hubballi,
India for in vitro anti-inflammatory analysis. We acknowledge Uni-
versity Scientific Instrumentation Centre (USIC), Karnataka Uni-
versity, Dharwad, (KUD) for IR and Mass and NMR Research Centre,
Indian Institute of Science (IISc), Bangalore, India for carrying out 1H
and 13C spectral analyses. The authors are also thankful to UGC, New
The crystal structure used was that of hyaluronidase (hya-
luronate lyase) in complex with palmitoyl-vitamin C (PDB
ID: 1W3Y) for the docking studies obtained from the Pro-
tein Data Bank. The enzyme for docking was prepared by

Med Chem Res
Delhi for providing financial assistance vide No. F 540/2/DSA
(SAP I), MNK also thanks UGC New Delhi for providing financial
assistance under UPE program.
Laurent TC, Fraser JRE (1992) Hyaluronan. FASEB J 6:2397–2404
Ling SK, Tanaka T, Kouno I (2003) Effects of iridoids on lipox-
ygenase and hyaluronidase activities and their activation by β
-glucosidase in the presence of amino acids. Biol Pharm Bull
26:352–356
Mohamed AM, Gineinah MN, Nasr WA, Bayoumi H (2004) novel
analogues of sydnone: synthesis, characterization and anti-
bacterial evaluation. Archiv der Pharmazie 337:427–433
Niu SL, Li ZL, Ji F, Liu GY, Zhao N, Liu XQ, Jing YK, Hua HM
(2012) Xanthones from the stem bark of Garcinia bracteata with
growth inhibitory effects against HL-60 cells. Phytochemistry
77:280–286
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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