Synthesis and discovery of (I-3,II-3)-biacacetin as a novel non-zinc binding inhibitor of MMP-2 and MMP-9

Article abstract of DOI:10.1016/j.bmc.2015.03.084

Eleven biflavones (7a-b and 9a-i) were synthesised by a simple and efficient protocol and screened for MMP-2 and MMP-9 inhibitory activities. Amongst them, a natural product-like analog, (I-3,II-3)-biacacetin (9h) was found to be the most potent inhibitor

Full text of DOI:10.1016/j.bmc.2015.03.084

Bioorganic & Medicinal Chemistry 23 (2015) 3781–3787
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Bioorganic & Medicinal Chemistry
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Synthesis and discovery of (I-3,II-3)-biacacetin as a novel non-zinc
binding inhibitor of MMP-2 and MMP-9
,à
à
Pandurangan Nanjan ,à, Jyotsna Nambiar , Bipin G. Nair , Asoke Banerji
⇑
Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Amrita University, Kollam, Kerala 690525, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven biflavones (7a–b and 9a–i) were synthesised by a simple and efficient protocol and screened for
MMP-2 and MMP-9 inhibitory activities. Amongst them, a natural product-like analog, (I-3,II-3)-bia-
cacetin (9h) was found to be the most potent inhibitor. Molecular docking studies suggest that unlike
most of the known inhibitors, 9h inhibits MMP-2 and MMP-9 through non-zinc binding interactions.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 15 January 2015
Revised 29 March 2015
Accepted 31 March 2015
Available online 9 April 2015
Keywords:
Cerium ammonium nitrate (CAN)
(I-3,II-3)-Biflavones
Biacacetin
Non-zinc binding inhibitor
MMP-2 and MMP-9
1. Introduction
could be a good option for dimerization of flavones through C–C
oxidative coupling by a single electron transfer mechanism.6
(I-3,II-3)-Biflavones form a small group of dimeric flavonoids.1
Due to limited occurrence and lack of convenient synthesis, this
group of flavonoids have not been subjected to detailed bio-evalua-
tion. Unlike synthetic strategies for C–O–C or C–C linked biflavones
(e.g., Ullmann coupling and Suzuki reactions),2 synthesis of (I-3,II-
3)-biflavones has not been studied in detail.3 During last three dec-
ades large numbers of inhibitors of matrix metalloproteinases
(MMPs) have been identified but they lack selectivity due to the
similarities in their active sites. Our earlier studies showed that
anacardic acid inhibits MMP-2 and MMP-9 by interacting with
the zinc in the catalytic site.4 Recently non-zinc binding inhibitors
are attracting attention due to their possible selectivity over
MMPs.5 With this in view, we screened eleven (I-3,II-3)-biflavones
(7a–b and 9a–i) for inhibition of MMP-2 and MMP-9 activities.
Oxidative dimerization of the flavones seems to be an attractive
possibility for the synthesis of (I-3,II-3)-biflavones. Synthesis of (I-
3,II-3)-hexamethyl biapigenin, 9e by oxidative coupling of tri-
methyl apigenin using alkaline K3[Fe(CN)6] has been described.3a
But the yields of the reactions are very low due to side-reactions
which makes the isolation process very cumbersome. Perusal of
literature suggests that the use of CAN (ceric ammonium nitrate)
However, reaction of flavone 4a with CAN under different reaction
conditions did not yield biflavone 2 (Scheme 1). A retrosynthetic
analysis (Fig. 1) suggests that a tetraketone intermediate (II,
Fig. 1) obtainable from appropriately placed o-OH functionality in
1,3-diketone (I, Fig. 1) provides a good option for the synthesis of
(I-3,II-3)-biflavones. Some examples of dimerization of 1,3-dike-
tones to tetraketones by using Ag2CO3,7 electrochemical8 and
CAN9 reactions have been reported. To our knowledge, dimeriza-
tion of diaryl-1,3-diketones have not been reported so far. The
present article describes, development of a one pot synthesis of
(I-3,II-3)-biflavones through the application of CAN mediated
dimerization of appropriately substituted diaryl-1,3-diketones.
2. Results and discussion
2.1. Chemistry
The diaryl-1,3-diketones were conveniently synthesized by the
Baker–Venkataraman transformation of appropriately substituted
benzoyloxy esters of 2-hydroxyacetophenones as described
earlier.10 1,3-Diketone 4 [1-(2-hydroxyphenyl)3-phenyl propane
1,3-dione)] did not undergo any change on treatment with CAN
under various reaction conditions, and the starting material was
recovered. We conjecture that this could be due to presence of
the free o-hydroxyl group in 4. Acetylation of 4 with acetic anhy-
dride/pyridine gave 5 which on reaction with CAN at ambient
⇑
Corresponding author. Tel.: +91 (476) 2801280; fax: +91 (476) 2899722.
E-mail address: banerjiasoke@gmail.com (A. Banerji).
Equally contributed to this article.
Tel.: +91 (476) 2801280; fax: +91 (476) 2899722.
à
http://dx.doi.org/10.1016/j.bmc.2015.03.084
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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P. Nanjan et al. / Bioorg. Med. Chem. 23 (2015) 3781–3787
O
Cl
O
OH
O
O
O
OH
CH3
O
O
BV
reaction
H+
CH3
2
KOH/ 50oC
Pyridine/rt
4
O
4a
O
1
3
AC2O/rt
O
O
CH3
CH3
B
O
C
O
O
O
CAN/-20oC
O
HCl/reflux
A
7
2
3
1
O
C
O
A
O
O
O
2
B
5
6
BV- Baker-Venkataraman,
CAN- Cerium Ammonium Nitrate
7
O
7b , B =
7a , B =
________________________________________________________________________________________________
R4
R3
R5
OCH3
R1
O
O
HO
O
O
O
CH3
R3
R4
O
O
R2
CAN/ -20oC
MeOH-HCl/Reflux.
R1
O
O
R5
R2
OH
10
R1
R2
O
R5
R3
9
8
R4
R1, R2 = OCH3 /or MOM
MOM = Methoxymethyl
Entry
Yield (%)
R1
R2
H
R3
R4
R5
OCH3
H
H
H
H
H
H
H
H
H
H
85
88
83
82
86
85
83
80
78
9a
9b
9c
9d
9e
9f
5'
OCH3
4'
3'
O
4
H
H
OCH3
H
H
6'
1'
H
H
OCH3
H
8
1
HO
O
I
OCH3
OCH3
OH
OCH3
OCH3
H
H
7
6
2
2'
3
OH
5
H
OCH3
H
3
4
5
H
6
II
OH
O
2'
3'
2
OH
OH
H
H
9g
9h
9i
1'
O
7
OH
1
8
OH
OH
H
OCH3`
OCH3
6'
4'
OH
OH
H
OCH3
H3CO
5'
9h
Scheme 1. Synthesis of (I-3,II-3)-biflavones.
OH
B
O
C
OH
A
O
O
O
O
O
O
O
O
O
OH
I
III
II
Figure 1. Retro-synthetic analysis of (I-3,II-3)-biflavones.
temperature gave the tetraketone 6 in 50% yield. The mass spec-
trum of 6 showed peak at m/z 563 [M+H]+ corresponding to
molecular formula, C34H26O8 indicating successful dimerization
of 5. MS/MS of m/z 563 showed successive loss of two 60 units sug-
gesting loss of two ketene (CH2@C@O) units and two water (H2O)
molecules to give ion at m/z 443. Since 6 is a symmetrical dimer,
only monomeric protons were observed in the 1H NMR spectrum
except that the methylene protons (2H) of 5 at d 4.54 were
replaced by the methine proton singlet (1H) at d 6.71 in 6, confirm-
ing that coupling reaction has occurred regiospecifically at 2-posi-
tion (in structure 5). Other features of MS, 1H and 13C NMR as well
as IR and UV spectra were in complete agreement with the
assigned structure 6. When 6 was refluxed with 10% methanolic
HCl (2 h), 7 was obtained in 78% yield. Biflavone 7 gave the
expected molecular mass of m/z 443 [M+H]+. MS/MS of m/z 443
ion showed successive loss of two fragments with mass unit 120,
each giving ion at m/z 203. The loss of two 20-hydroxy acetophe-
none moieties (2 ꢀ 120 units) arises from cleavage of two A rings.
The mass spectral fragmentation along with the absence of singlet
proton (d 6.43)10 at 3-position (corresponding to monomer, 10) in
1H NMR provide unequivocal evidence for the linkages of two fla-
vone units through I-3 and II-3 positions. It is known that the CAN
directed dimerization reactions are greatly influenced by the
experimental factors such as solvents, temperature, etc. Therefore
it was necessary to optimize the reaction conditions by conducting
experiments using different solvent media and temperature (see
Supplementary information). The best result for the dimerization
of 5–6 (80–92% yield) was obtained using a mixture of MeCN/

P. Nanjan et al. / Bioorg. Med. Chem. 23 (2015) 3781–3787
3783
MeOH/H2O (1:1:1, v/v), CAN (1.1 equiv), at ꢁ20 °C for 30 min.
Increasing the quantity of CAN did not improve the yield substan-
tially. During the optimization of the protocol, it was observed that
diketone 5 could be directly converted to biflavone 7 by adding
CAN (1.1 equiv) under similar conditions (MeCN/MeOH/H2O,
ꢁ20 °C) followed by refluxing (2 h) in methanolic HCl (10%) with-
out the isolation of tetraketone, 6. Biflavone 7 was obtained with
high yield (92%). Therefore, in subsequent experiments the dike-
tones were directly transformed into biflavones in a one pot reac-
tion. Scope of the reaction was further expanded by the
preparation of 7a (87%) and 7b (84%) (Scheme 1). The generality
of the protocol was established by taking differently substituted
diketones. The methoxy diketones
8 were transformed into
methoxybiflavones 9a–e in good yields (Scheme 1). For the synthe-
sis of polyhydroxy biflavones, it was necessary to protect the
hydroxyl groups. MOM (methoxymethyl) group was preferred
option for the protection of hydroxyl groups since it offers certain
advantages over the conventional groups (e.g., benzyl, benzoyl)
such as ease of preparation and regeneration of hydroxyl groups.
The protected diketones were prepared using 4,6-diMOM
phloroacetophenone, as described in our earlier publication.10
The treatment of the diketones 8 with CAN, followed by methano-
lic–HCl (10%, 3 h) gave polyhydroxy and regiospecific partial
methyl ethers of biflavones 9f–i in good yield (Scheme 1).
Deprotection of the MOM groups occurred during the final double
cyclodehydration step. Thus, it was inferred that MOM and acetyl
groups are compatible for the CAN mediated oxidative coupling.
Figure 2. Regulation of MMP-2 and -9 (gelatinases) activity by (I-3,II-3)-biflavones.
2.2. Biological evaluation
(A) Zymogram showing gelatinase activity of conditioned media from HT1080 cells
treated with 0.5% DMSO (lane 1), 10 lM 9f, 9g, 9h, 9i, 7a, 7b (lanes 2–7,
respectively). (B) A representative plot of percentage inhibition observed in the
zymogram. Each bar represents the Mean SE of triplicate determinations from
three independent experiments. ⁄⁄⁄P <0.001 (one-way analysis of variance with
Dunnett’s multiple-comparison post-test).
In the present investigation, eleven different biflavones (7a–b,
9a–i) were screened for their inhibition of MMP-2 and -9 activities
in fibrosarcoma cells (HT1080). Amongst them, 9h showed signifi-
cant inhibition (90%) of both MMP-2 and -9 activities at a concen-
tration of 10 lM (Fig. 2). Other compounds 9a–e showed much less
inhibition of gelatinases (data not shown). Dose-dependent inhibi-
tion of MMP-2 and -9 activities were observed when the cells were
treated with different concentrations of 9h (Fig. 3). These results
were further confirmed using a fluorescent-based gelatin degrada-
tion assay which clearly shows the reduction in degradation of the
gelatin at a concentration of 5 lM when compared to the control
(Fig. 4). Analysis of the cytotoxicity of 9h to HT1080 cells indicated
that there was no significant toxicity up to a concentration of
7.5
10 did not show inhibition of MMP-2 and MMP-9 at a concentra-
tion of 5 M (data not shown), whereas 9h inhibits MMP-2 and
lM (data not shown). The monomer of 9h, that is, acacetin
l
MMP-9 by 60% and 90%, respectively, at this same concentration.
This provides an evidence that the dimer 9h is a better inhibitor
of MMP-2 compared to its monomer. Since MMP-2 and MMP-9
are known to play a significant role in the migration of cancer cells,
the effect of 9h on cell migration was studied using transwell
migration assay. 9h showed reduction in migration of HT1080 cells
from a concentration of 5 lM (Fig. 5).
2.3. SAR studies
Eleven variously substituted biflavones having hydroxy-, meth-
oxy-, furano- and cinnamyl- moieties were evaluated for the
inhibition of MMP-2 and MMP-9. While biacacetin (9h) showed
maximum inhibition for both MMP-2 and MMP-9, other com-
pounds showed differential activities. For example, biflavones 7a,
9f and 9i showed significantly lower inhibitions for MMP-2 com-
pared to 9h (Fig. 2). Similarly methoxy derivatives (9a–9e) did
not show significant inhibition of MMP-2 and MMP-9 (data not
shown). The compounds which lack the hydroxyl in the position
Figure 3. Dose dependent study of 9h. (A) Zymogram showing gelatinase activity of
conditioned media from HT1080 cells treated with 0.5% DMSO (lane 1) and 2.5, 5,
7.5 and 10 lM 9h (lanes 2–5, respectively). (B) A representative plot of percentage
inhibition observed in the zymogram. Each bar represents the Mean SE of
triplicate determinations from three independent experiments. ⁄⁄⁄P <0.001 (one-
way analysis of variance with Dunnett’s multiple-comparison post-test).

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P. Nanjan et al. / Bioorg. Med. Chem. 23 (2015) 3781–3787
Figure 4. Regulation of gelatinase activity by 9h. Gelatin degradation assay showing the reduction in degradation of the fluorescent gelatin at 5 lM 9h when compared to
control.
R2 (as in 9f and 7b in Scheme 1) did not show significant inhibition
of MMP-2 and MMP-9.
inhibitory activity of this molecule is under further studied for
selectivity and specificity of the different MMPs.
2.4. Molecular docking
3. Conclusion
Active sites of most of the MMPs are conserved and result in
lack of selectivity in inhibition.5,11 Amongst the biflavones (7a–b
and 9a–i) tested, 9h was found to be the most potent inhibitor of
MMP-2 and -9. Molecular dynamic simulation studies indicate
that, while the S10 pocket in MMP-2 is narrow, that of MMP-9
has a relatively larger pocket-like subsite.5,11 To simulate the inter-
action of 9h with the target proteins, ‘Autodock’-based molecular
modeling programme was used. The parameters for docking were
fixed on the basis of our previously published work.4 9h forms four
hydrogen bonds with MMP-2 catalytic domain [(PDB code—1QIB)
(docking score; ꢁ7.6)]; one between 7-OH (I) and Thr229, while
the other three are between Arg119 and 5-OH (I), 4-(I) and 4(II)
carbonyls (Fig. 6A, Supplementary Fig. 1). 9h also forms hydropho-
bic interactions with Phe196, Phe232, Leu197 and Gln122. It is
noteworthy that unlike most of the MMP inhibitors such as anac-
ardic acid (Supplementary Fig. 2) and batimastat, 9h does not bind
to the zinc in the catalytic site. A probable explanation could be the
larger size and rigid nature of 9h that results in reduced accessibil-
ity to the active site of MMP-2 (deeper S10 tunnel). 9h binds in a
In conclusion, a new synthesis of (I-3,II-3)-biflavones through
oxidative dimerization of poly-substituted diaryl-1,3-diketones
with cerium ammonium nitrate followed by double cyclodehydra-
tion has been developed. A systematic screening of biflavones (7a–
b, 9a–i) for inhibition of MMP-2 and -9 was carried out; 9h was
found to be best inhibitor. In silico docking studies suggests that
(I-3,II-3)-biacacetin inhibits the gelatinases through non-zinc bind-
ing interactions. The non-zinc binding nature of (I-3,II-3)-bia-
cacetin could be an important factor for designing selective
molecules of therapeutic importance through inhibition of MMP-
2 and MMP-9.
4. Experimental
4.1. Chemistry
4.1.1. Preparation of tetraketone 6
To 5 (1 mmol) in methanol (4 ml) at 28 °C, Ceric ammonium
nitrate (1 mmol, in 1 ml methanol) was added and stirred for
12 h. (monitored by TLC; toluene 5: methanol 5: formic acid 2),
methanol was removed under reduced pressure, water (10 ml)
was added, then extracted with dichloromethane (10 ml ꢀ 2),
dried over anhydrous Na2SO4. Solvent removed and white solid
was obtained. After column chromatography, product 6 was
obtained. 6 were characterized by 1H NMR, 13C NMR, MASS, IR,
UV spectral and HPLC analysis.
region between
a
-helix and S10 specificity loop (indicated in
Fig. 6A). The MMP-9 catalytic domain [(PDB code—2OVX) (docking
score; ꢁ7.5)] was also subjected to docking study. 9h did not show
any hydrogen bonding with MMP-9; it exhibited only hydrophobic
interactions with the amino acid residues (Phe110, Leu187, His401,
His405, His411 and Pro421) (Supplementary Fig. 3). The lengthy
S10 sub-site in MMP-9 precludes the interaction of 9h with S10 site
and hence prevents interaction of 9h with the zinc (Fig. 6B). 9h was
also compared with its monomer 10 with respect to its binding
(data not shown). Although 10, exhibited interaction at the active
site (zinc) in both MMP-2 and MMP-9, inhibition of MMP-2 activity
was significantly reduced. We report here for the first time that
this natural product like analog, (I-3,II-3)-biacacetin is a novel
non-zinc binding inhibitor for MMP-2 and -9. The non-zinc-binding
4.1.2. Deprotection and cyclodehydration of 6 (preparation of 7)
To 6 (1 mmol), methanolic–HCl (20 ml, 10%) was added and
refluxed for 2 h. After monitoring by TLC, solvent was removed
under reduced pressure. Water added to the reaction mass,
extracted with ethyl acetate (10 ml ꢀ 2). Washed with water
(5 ml ꢀ 2), dried with anhydrous Na2SO4. Solvent removed and

P. Nanjan et al. / Bioorg. Med. Chem. 23 (2015) 3781–3787
3785
Figure 5. Effects of 9h on migration of HT1080 cells. (A) The cells were treated with different concentrations of 9h in serum-free medium and DMEM with 10% FBS was used
as the attractant. After 24 h of culture, the cells that migrated through the pores were stained and photographed under microscope. (B) The graph displays percentage of cells
migrated with respect to control in five random fields. Each bar represents the Mean SE of the number of cells obtained in the five random fields. ⁄⁄⁄P <0.001 (one-way
analysis of variance with Dunnett’s multiple-comparison post-test).
white solid was obtained. The overall conversion 5–7, via tetrake-
tone 6 was 39%. The (I-3, II-3)-biflavone crystallized with metha-
nol-chloroform mixture. Characterization of 7 is given below.
4.1.3.1. Characterization of 6.
colorless needles. Mp 150–151 °C. UV kmax (MeOH)/nm; 250. IR
max/cmꢁ1; 1761 (CO) and 1687 (CO). 1H NMR (400 MHz, DMSO-
Tetraketone 6 was obtained as
m
d6); d 7.94, m, 2H; 7.6, m, 1H; 7.53, m, 1H; 7.45, t, 2H; 7.32, t,
2H; 7.09, dd, 1H; 6.71, s, 1H; 2.26, s, 3H. 13C NMR (100 MHz,
DMSO-d6); 193.84, 193.68, 168.65, 148.11, 135.75, 134.03, 133.9,
130.01, 129.97, 128.71, 126.23, 124.06, 59.94 and 20.66. LC–MS;
Rt-16.133, Purity-97%, [M+1]+ = 563, MS/MS = 503, 443.
4.1.3. General procedure for the conversion of 1,3-diketones to
(I-3,II-3)-biflavones; one pot synthesis of (I-3,II-3)-biflavones (7–
7b and 9a–9j)
Acetate protected 1,3-diketones (1 mmol) were taken in MeCN/
MeOH (1:1, 8 ml). CAN (1.1 equiv in water 4 ml) was added to the
reaction mixture at ꢁ20 °C over 5 min. Then reaction mixture was
stirred for 30 min; after monitoring (TLC) methanolic–HCl (10%,
30 ml) was added and refluxed for 2–3 h. After that, solvent was
removed; water was added and extracted with ethyl acetate
(20 ml ꢀ 2). Washed well with water (5 ml ꢀ 2), dried over anhy-
drous Na2SO4. Fast column chromatography was performed using
SiO2. Biflavones were obtained in good yield: 78–92% directly
without isolating tetraketones. Characterizations of all biflavones
are given below.
4.1.3.2. Characterization of 7.
crystals. Mp 330–332 °C. UV kmax (MeOH)/nm; 254 and 310. IR
max/cmꢁ1; 1643 (CO). 1H NMR (400 MHz, DMSO-d6): d 8.12, m,
7 was obtained as colorless
m
1H; 7.9, m, 1H; 7.72, d (J = 8.0 Hz), 1H; 7.59, m, 1H; 7.42, m, 1H;
7.36, t, 2H; 7.22, t, 2H. 13C NMR (100 MHz, DMSO-d6): 176.01,
162.64, 155.59, 134.7, 132.2, 130.69, 128.44, 127.36, 125.87,
125.35, 121.92 and 118.47. LC–MS: Rt-15.848, Purity-95%
[M+1]+ = 443, MS/MS = 323, 203.

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P. Nanjan et al. / Bioorg. Med. Chem. 23 (2015) 3781–3787
(J = 8.8 Hz), 1H; 7.44, m, 1H; 7.34, m, 2H; 7.2, m, 3H; 7.13, dd,
1H; 3.91, s, 3H. 13C NMR (100 MHz, DMSO-d6): 175.38, 164.15,
162, 157.43, 132.28, 130.53, 128.35, 127.30, 126.78, 115.81,
115.73, 115.18, 100.71 and 56.16. LC–MS: Rt-6.722, Purity-96%,
[M+1]+ = 503, MS/MS = 488, 369, 235.
4.1.3.6. Characterization of 9b.
white crystals. Mp 257 °C (dec). UV kmax (MeOH)/nm; 243 and
308. IR
max/cmꢁ1; 1631 (CO). 1H NMR (400 MHz, DMSO-d6): d
9b was obtained as dull
m
8.16, m, 1H; 7.9, m, 1H; 7.83, m, 2H; 7.54, m, 1H; 7.4, m, 1H;
6.94, d, (J = 8.4 Hz), 1H: 6.81, m, 2H: 3.61, s, 3H. 13C NMR
(100 MHz, DMSO-d6): 175.94, 156.43, 155.66, 134.27, 131.9,
129.01, 125.47, 125.19, 122.23, 120.73, 119.87, 118.24, 118.06,
111.27 and 55.03. LC–MS: Rt-5.456, Purity-97%, [M+1]+ = 503,
MS/MS = 488, 383, 263.
4.1.3.7. Characterization of 9c.
crystals. Mp 248–249 °C. UV kmax (MeOH)/nm; 269 and 307. IR
max/cmꢁ1; 1685 (CO). 1H NMR (400 MHz, DMSO-d6): d 7.90, d
9c was obtained as yellow
m
(J = 8.8 Hz), 2H; 7.59, d (J = 2.4 Hz), 1H; 7.3, t, 1H; 7.2, t, 2H; 7.10,
d (J = 8.8 Hz), 2H; 7.02, dd, 1H; 3.82, s, 3H. 13C NMR (100 MHz,
DMSO-d6): 175.38, 166.95, 162.82, 132.28, 131.3, 122.96, 115.81,
115.72, 115.18, 100.71 and 55.4. LC–MS: Rt-6.551, Purity-96.5%,
[M+1]+ = 503, MS/MS = 488, 383, 263.
4.1.3.8. Characterization of 9d.
crystals. Mp 175–178 °C. UV kmax (MeOH)/nm; 269 and 303. IR
max/cmꢁ1; 1628 (CO). 1H NMR (400 MHz, DMSO-d6): d 8.05, dd,
9d was obtained as white
m
2H; 7.58, m, 3H; 6.88, d (J = 2.4), 1H; 6.52, d (J = 2.4), 1H. 3.90, s,
3H; 3.64, s, 3H. 13C NMR (100 MHz, DMSO-d6): 143.98, 139.26,
129.29, 123,116.1, 116.7, 116.8, 95.2, 80.25, 60.32 and 55.4.
LC–MS: Rt-16.563, Purity-98%, [M+1]+ = 563, MS/MS = 381,
199.
Figure 6. Predicted binding mode of 9h to MMP-2 and MMP-9. (A) Ligand 9h (stick
model, carbon-yellow, red-oxygen, hydrogen-white and methyl group-cyan color)
interaction with MMP-2 catalytic domain (violet sphere-zinc metal, Orange-S10
specificity loop, light blue-interaction of amino acid residues with the ligand). (B)
Surface structure of MMP-9 with 9h. Surface structure shows that 9h binds outside
the S10 active site.
4.1.3.9. Characterization of 9e.
crystals. Mp 190 °C (dec).3a UV kmax (MeOH)/nm; 265 and 310. IR
max/cmꢁ1; 1622 (CO). 1H NMR (400 MHz, DMSO-d6): d 7.22, d
9e was obtained as colorless
m
(J = 8.8 Hz), 2H; 6.89, d (J = 8.8 Hz), 2H; 6.72, s, 1H; 6.52, s, 1H;
3.88, s, 3H; 3.82, s, 3H; 3.74, s, 3H. 13C NMR (100 MHz, DMSO-
d6): 162.15, 128.88, 113.78, 111.72, 109.49, 103.84, 103.76, 56.61
and 55.13. LC–MS: Rt-17.362, Purity-96%, [M+1]+ = 623, MS/
MS = 608, 441, 259.
4.1.3.3. Characterization of 7a.
crystals. Mp 200–202 °C. UV kmax (MeOH)/nm; 247 and 314. IR
max/cmꢁ1; 1641 (CO). 1H NMR (400 MHz, DMSO-d6): d 8.11, m,
7a was obtained as yellow
m
1H; 8.02, m, 1H; 7.93, d (J = 8.0 Hz), 1H; 7.9, m, 1H; 7.85, d
(J = 16.0 Hz), 1H; 7.77, d, (J = 8.4 Hz), 1H: 7.61, d (J = 36.0 Hz),
7.59, m, 1H: 7.55, m, 1H: 7.06, m, 2H: 6.8, m, 1H. 13C NMR
(100 MHz, DMSO-d6): 176.09, 174.77, 155.24, 154.83, 154.45,
153.5, 148.56, 147.53, 145.01, 144.87, 134.96, 134.37, 126.03,
125.59, 125.3, 124.84, 123.57, 122.37, 118.36, 118.19, 117.12,
115.64, 115.39, 112.9, 109.08 and 104.56. LC–MS: Rt-15.491,
Purity-97.5%, [M+1]+ = 423, MS/MS = 303, 183.
4.1.3.10. Characterization of 9f.
low needles. Mp 220 °C (dec). UV kmax (MeOH)/nm; 249 and 308. IR
max/cmꢁ1; 1627 (CO). 1H NMR (400 MHz, DMSO-d6): d 10.8, s, 1H;
8.07, dd, 2H; 7.91, d (J = 8.8 Hz), 1H; 7.59, m, 3H; 7.01, d
(J = 2.4 Hz), 1H; 6.99, dd, 1H; 6.95, d (J = 2.4 Hz), 1H. 13C NMR
(100 MHz, DMSO-d6): 176.37, 162.75, 161.95, 157.5, 131.51, 131,
130, 129.06, 126.52, 126.15, 116.16, 115.06, 106.63 and 102.54.
LC–MS: Rt-4.765, Purity-97%, [M+1]+ = 475, MS/MS = 355, 219.
9f was obtained as pale yel-
m
4.1.3.4. Characterization of 7b.
crystals. Mp 269–272 °C. UV kmax (MeOH)/nm; 259 and 337. IR
max/cmꢁ1; 1622 (CO). 1H NMR (400 MHz, DMSO-d6): d 7.99, m,
7b was obtained as yellow
4.1.3.11. Characterization of 9g.
low solid. Mp 228–230 °C. UV kmax (MeOH)/nm; 269 and 306. IR
max/cmꢁ1; 1638 (CO). 1H NMR (400 MHz, DMSO-d6): d 7.27, d
9g was obtained as pale yel-
m
m
1H; 7.85, m, 1H; 7.7, m, 2H; 7.59, m, 2H; 7.45, m, 1H; 7.28, m,
3H: 6.94, d (J = 16.0 Hz). 13C NMR (100 MHz, DMSO-d6): 175.76,
159.93, 155.32, 137.49, 134.94, 134.24, 129.8, 128.81, 128.12,
125.21, 125.14, 122.8, 119.03, 118.21 and 114.17. LC–MS: Rt-
15.335, Purity-94.5%, [M+1]+ = 503, MS/MS = 383, 263.
(J = 8.8 Hz), 1H; 6.88, d, (J = 8.8 Hz), 1H: 6.38, d (J = 2.0 Hz), 1H:
6.19, d (J = 8.0 Hz), 1H. 13C NMR (100 MHz, DMSO-d6): 181.85,
164.41, 163.87, 161.45, 157.45, 132, 130.71, 129.12, 126.4,
105.18, 103.96, 99.01 and 94.11. LC–MS: Rt-4.701, Purity-94%,
[M+1]+ = 507, MS/MS = 354, 201.
4.1.3.5. Characterization of 9a.
crystals. Mp 238–241 °C. UV kmax (MeOH)/nm; 244 and 306. IR
max/cmꢁ1; 1635 (CO). 1H NMR (400 MHz, DMSO-d6): d 8.0, d
9a was obtained as colorless
4.1.3.12. Characterization of 9h.
yellow solid. Mp 305 °C (dec). UV kmax (MeOH)/nm; 270 and 333.
IR
max/cmꢁ1; 1649 (CO). 1H NMR (400 MHz, DMSO-d6): d 7.27, d
9h was obtained as pale
m
m

P. Nanjan et al. / Bioorg. Med. Chem. 23 (2015) 3781–3787
3787
(J = 8.8 Hz), 1H; 6.88, d, (J = 8.8 Hz), 1H: 6.38, d (J = 2.0 Hz), 1H:
4.3. Molecular docking
6.19, d (J = 8.0 Hz), 1H: 3.61, s, 3H. 13C NMR (100 MHz, DMSO-
d6): 180.72, 164.71, 161.43, 161.28, 157.26, 129.26, 123.91,
114.13, 111.82, 98.96, 93.72 and 55.37. LC–MS: Rt-3.477, Purity-
96%, [M+1]+ = 567, MS/MS = 552, 414, 261.
In order to identify the binding mode of the compound 9h,
AutoDockTools (http://mgltools.scripps.edu) was used. The crystal
structure of MMP-2 catalytic domain (PDB code 1QIB) and MMP-9
catalytic domain (PDB code 2OVX) were obtained from RCSB PDB
data bank. For docking studies, a grid box of 44 ꢀ 46 ꢀ 60 points
on 0.375-Å grid spacing, centered on the ligand-binding sites for
MMP-2 or MMP-9 identified by autoligand, were used to generate
affinity maps. The best docking pose of 9h was selected and ana-
lyzed. For validation, docking of 9h was compared with anacardic
acid.4
4.1.3.13. Characterization of 9i.
9i was obtained as yellow
solid. Mp 263 °C (dec). UV kmax (MeOH)/nm; 270 and 341. IR mmax
/
cmꢁ1; 1651 (CO). 1H NMR (400 MHz, DMSO-d6): d 12.91, s, 1H;
10.83, s, 1H; 7.69, dd, 1H: 7.56, d, (J = 2.0 Hz), 1H; 7.14, d
(J = 8.4 Hz), 1H: 6.53, d (J = 4.8 Hz), 1H; 6.21, d, (J = 2.4 Hz), 1H;
3.88, s, 3H; 3.84, s, 3H. 13C NMR (100 MHz, DMSO-d6): 181.8,
164.18, 161.41, 157.35, 152.15, 149.02, 122.91, 120.04, 111.72,
109.42, 103.84, 103.76, 98.86, 94.09 and 55.73. LC–MS: Rt-5.273,
Purity-98%, [M+1]+ = 597, MS/MS = 444, 291.
Acknowledgements
We would like to thank Mr. Muralidharan V (Amrita Agilent
Analytical Research Lab) for acquiring the mass spectrometry data,
Dr. Uday Singh (Sam Higginbottom Institute of Agriculture,
Technology & Sciences, Deemed University, Allahabad, India) for
his valuable suggestions on docking studies, Ms. Asha Vijayan
and Mr. Krishna Chaitanya Medini for helping with Corel DRAW
and MATLAB and University Grants Commission [F.2-7/2012(SA-
I)] for providing financial assistance to Ms. Jyotsna Nambiar.
4.2. Biological evaluation
4.2.1. Cell culture
HT1080 (human fibrosarcoma cells) were obtained from
National Centre for Cell Sciences, Pune, Maharashtra, India.
HT1080 cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (v/v), 1% peni-
cillin, 1% streptomycin, 0.1% amphotericin B (Sigma–Aldrich, St.
Louis, MO). Fetal bovine serum (FBS) was purchased from
Invitrogen (Carsbad, CA).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.03.084.
4.2.2. Gelatin Zymography
The zymography assay was performed according to the protocol
described by Ratnikov et al.12 Gelatin substrate gels were prepared
by incorporating gelatin (2 mg/ml) into 10% polyacrylamide gel
containing 0.4% SDS. Electrophoresis was carried out under non-re-
ducing conditions at 120 V for 150 min. The gels were washed in
2.5% Triton X-100 (v/v) for 30 min and then incubated overnight
at 37 °C in developing buffer containing 50 mM Tris–HCl, pH 7.6,
200 mM NaCl, 5 mM CaCl2 and 0.2% (v/v) Brij-35. Digestion bands
were quantified using BIORAD image analyzer systems.
References and notes
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4.2.3. Gelatin degradation assay
The assay was carried out according to the protocol described
previously.13 The cells were treated with 5
lM 9 h for 24 h at
37 °C. After treatment, the cells were fixed, mounted in ProLongÒ
Gold Antifade reagent (Molecular probes, P-36931) and observed
using IX71 Inverted microscope (Olympus).
10. Pandurangan, N. Lett. Org. Chem. 2014, 11, 225.
4.2.4. Cell migration assay
11. Nagase, H. In Cancer Drug Discovery and Development: Matrix Metalloproteinase
Inhibitors in Cancer Therapy; Neil, J. C., Krzysztof, A., Eds.; Humana Press:
Totowa, NJ, 2001; pp 33–66.
12. Mueller, S. C.; Chen, W. T. J. Cell Sci. 1991, 99, 213.
13. Ratnikov, B. I.; Deryugina, E. I.; Strongin, A. Y. Lab. Invest. 2002, 82, 1583.
HT1080 cells (8 ꢀ 104 cells) were treated with different concen-
trations of 9 h in 300 lL serum-free media and plated in the upper
chamber. The lower chamber contained 750 ll of 10% serum-con-
taining media. After 24 h of incubation, non-migratory cells in the
upper chamber were carefully removed with a cotton swab.
Migrated Cells were stained with crystal violet solution. The cell
numbers were randomly counted in five fields.