Design of dual MMP-2/HDAC-8 inhibitors by pharmacophore mapping, molecular docking, synthesis and biological activity

Article abstract of DOI:10.1039/c5ra12606a

Recent analyses have highlighted the promotion of cancer migration and invasion, mediated through HDAC via MMP-2 and MMP-9. Since both class 1 HDACs and MMP-2/9 are involved in the migration and invasion of cancer, an attempt has been taken to design dual MMP-2/HDAC-8 inhibitors by pharmacophore mapping and molecular docking approaches. The designed molecules were synthesized and showed a range of inhibitory activity against different MMP subtypes. Most of these designed compounds were selective towards MMP-2 but less potent against anti-targets like MMP-8, -12, etc. The highly active MMP-2 inhibitors were also found to be active towards HDAC-8 but less potent against other class 1 HDACs (HDAC-1 and -2). Molecular dynamics simulations revealed that the designed compounds may be acting through a distinct mechanism of action in the 'acetate ion channel' of HDAC-8. Some potent dual MMP-2/HDAC-8 inhibitors were further explored for in vitro cellular assays against human lung carcinoma cell line A549. These analyses revealed that some of these dual inhibitors have considerable anti-migratory and anti-invasive properties. The work may help to obtain some useful dual inhibitors.

Full text of DOI:10.1039/c5ra12606a

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Design of dual MMP-2/HDAC-8 inhibitors by
pharmacophore mapping, molecular docking,
Cite this: RSC Adv., 2015, 5, 72373
synthesis and biological activity†
Amit K. Halder,a Sumana Mallick,b Deep Shikha,a Achintya Saha,c Krishna D. Sahab
a
*
and Tarun Jha
Recent analyses have highlighted the promotion of cancer migration and invasion, mediated through HDAC
via MMP-2 and MMP-9. Since both class 1 HDACs and MMP-2/9 are involved in the migration and invasion
of cancer, an attempt has been taken to design dual MMP-2/HDAC-8 inhibitors by pharmacophore
mapping and molecular docking approaches. The designed molecules were synthesized and showed a
range of inhibitory activity against different MMP subtypes. Most of these designed compounds were
selective towards MMP-2 but less potent against anti-targets like MMP-8, -12, etc. The highly active
MMP-2 inhibitors were also found to be active towards HDAC-8 but less potent against other class
1 HDACs (HDAC-1 and -2). Molecular dynamics simulations revealed that the designed compounds may
be acting through a distinct mechanism of action in the ‘acetate ion channel’ of HDAC-8. Some potent
dual MMP-2/HDAC-8 inhibitors were further explored for in vitro cellular assays against human lung
carcinoma cell line A549. These analyses revealed that some of these dual inhibitors have considerable
anti-migratory and anti-invasive properties. The work may help to obtain some useful dual inhibitors.
Received 29th June 2015
Accepted 17th August 2015
DOI: 10.1039/c5ra12606a
www.rsc.org/advances
class of metalloenzymes that catalyse deacetylation of
N-terminal lysine residues of various proteins including nucle-
osomal histones.12,13 The HDACs are involved in the chromatin
1. Introduction
Matrix metalloproteinase is an important class of zinc-
dependent endopeptidase involved in the degradation of
extracellular matrix (ECM) and remodelling of tissues.1,2 So far,
28 different MMPs are known and classied into six subgroups.
The overexpression of MMPs is related to various pathological
conditions such as cardiovascular, neurological, periodontal,
central nervous system, emphysema, angiogenesis, metastases
as well as cancer.3–5 Among all MMP subtypes, MMP-2 (or
gelatinase A) is considered the most important target of cancer
metastasis because of its strong correlation with cancer
progression.6–8 In last few decades, several attempts were made
to design potential MMP-2 inhibitors as anticancer agents
though none of these was successful in clinical trials. One of the
major reasons for this failure is undesirable adverse effects
caused by broad spectrum MMP inhibition.8–11 On the other
hand, histone deacetylases (HDACs) are another important
assembly and remodelling, down-regulation of various gene
expressions as well as DNA repair and re-combinations. Addi-
tionally, HDACs participate in various cellular processes by
interacting with several non-histone proteins like transcription
factors, molecular chaperones, cytoskeletal proteins, etc.13 Due
to these epigenetic and cellular functions, deregulation of
HDACs leads to several pathological conditions including
cancer.12,14,15 Eighteen different isotypes of HDACs are identied
and classied into four subgroups – class I (HDAC1-3 and 8),
class II (HDAC4–7, 9 and 10), class III (sirtuins 1–7) and class IV
(HDAC11). Among these, over-expression of class 1 HDACs is
associated with various human cancers. Several attempts were
made in last few years to develop class I HDAC inhibitors. Two
of these inhibitors, suberoylanilide hydroxamic acid (SAHA or
vorinostat) and the FK228 (romidepsin) have already been
approved by US food and drug administration (US FDA) for the
treatment of cutaneous T cell lymphoma whereas some other
inhibitors are under clinical trial.16–18 Most of these HDAC
inhibitors are pan-inhibitors (non-selective towards specic
isoform). Selectivity towards specic HDAC isoform is prefer-
able but not essential criteria for HDAC inhibitor design.14,19
Both HDACs and MMPs are related to cancer migration,
invasion and metastases.20–22 Moreover, HDAC inhibitors
suppress MMP-2 activation and lung cancer cell invasion via
RECK protein upregulation.23 Similar suppression of migration
aNatural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry,
Department of Pharmaceutical Technology, Jadavpur University, P.O. Box 17020,
Kolkata 700032, India. E-mail: tjupharm@yahoo.com; Tel: +91 33 2457 2495, +91
33 2438 3814, +91 9433187443
bCancer Biology & Inammatory Disorder Division, CSIR-Indian Institute of Chemical
Biology, Kolkata 700032, India
cDepartment of Chemical Technology, University of Calcutta, 92, APC Ray Road,
Kolkata 700009, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra12606a
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and invasion was found in breast carcinoma cell lines where the ESI (Text S1†). Briey, a dataset30–34 containing 111 reported
HDAC inhibition led to the downregulation of both MMP-2 and HDAC-8 inhibitors (Table S1, ESI†) was selected and structurally
MMP-9.24 Furthermore, HDAC-1, -6 and -8 have been found to diverse 24 molecules (Fig. S2†) of this dataset were used to build
suppress MMP-9 to inhibit breast cancer cell invasion.22 the ligand-based pharmacophore models. The remaining 87
Therefore, dual MMP-2/HDAC-8 inhibitors may be potential compounds were used as the test set to validate the developed
candidates to impart the higher anti-migratory and anti- models. For development of the structure-based pharmaco-
invasive properties of cancer cells than the selective MMP or phore, X-ray crystal structure of HDAC-8 (PDB: 1VKG)35,36 was
the selective HDAC inhibitor. Such designing of dual inhibitor used. The best ligand-based pharmacophore (LigPharm01, Text
is complicated since both these metalloenzymes have several S1, ESI†) was generated with acceptor (A), hydrophobic
isoforms, and broad spectrum inhibition (especially MMPs) aromatic (HAr) and zinc binding (Z) features (Fig. 1(a)), whereas
may give rise to undesirable adverse effects. Ideally, these the best structure-based pharmacophore (StrPharm02, Text S1,
inhibitors should specically block the targets responsible for ESI†) comprises of two ring aromatic (Ra), one hydrophobic (H)
invasion and metastases (such as MMP-2, MMP-9) and spare and one zinc binding features (Fig. 1(b)). These pharmacophore
anti-targets (like MMP-8, -12, etc.). Design of dual MMP- models of MMP-2 and HDAC-8 were used for the design of their
2/HDAC-8 inhibitors has earlier been explored by Cheng et al. dual inhibitors.
though the cellular effect or isoform selectivity of these inhibi-
tors was not described.25 In the current study, we attempted to
design novel dual MMP-2/HDAC-8 inhibitors by pharmaco-
2.2. Design of dual MMP-2 and HDAC-8 inhibitors
phore mapping techniques, with an aim to investigate the Previously, the molecular modeling studies of MMP-2 inhibitors
potency of these inhibitors on lung cancer cell migration and were reported.11 In that MMP-2 inhibitor dataset,11 some
invasion. Due to structurally distinct active site of HDAC-8, most compounds were designed from antineoplaston (A-10) metab-
broad spectrum HDAC inhibitors are least potent against this olite N2-phenylacetyl L-isoglutamine (AS2-5), and those deriva-
enzyme.14 Therefore, the HDAC-8 remains as the least investi- tives showed variable MMP-2 inhibitory activities.37,38 The AS2-5
gated class 1 HDAC isoform as far as its biological functions are derived MMP-2 inhibitors contained either the benzoyl37 or the
concerned. The HDAC-8 knockdown was earlier reported to phenylacryloyl38 moieties conjugated with L(+)-isoglutamine.
suppress cell proliferation in lung, cervical, colon and neuro- The best active molecules of these two series37,38 were considered
blastoma cell lines. Moreover, invasion of HDAC-8 transfected for designing a hypothetical molecule (to retain the structural
cells was found to be higher than HDAC-1 transfected cells.22 information of MMP-2 inhibition) that would map all features of
Presently, more than 20 human ligand-bound HDAC-8 protein the StrPharm02 (so that HDAC-8 inhibitory activity of this
structures are available that may serve as good starting points hypothetical molecule may be ensured). The design and structure
for the generation of the structure-based pharmacophore of the hypothetical molecule (H1, 4(S)-5-(benzylamino)-4-(2-(4-
models.26 In the current investigation, the designed dual methoxyphenyl)acetamido)-5-oxopentanoic acid) is shown in
MMP-2/HDAC-8 inhibitors were synthesized, screened against Fig. 2. The H1 was found to map all features of StrPharm02
different MMP and HDAC isoforms to understand their potency [Fig. 1(b)]. Furthermore, this compound showed the predicted
and selectivity as well as tested against human lung cancer cell HDAC-8 inhibitory activity (IC50) of 3.5 mM when mapped with
line A549 for cytotoxicity. Finally, migration and invasion assays LigPharm01 [Fig. 1(a)]. Both these mappings indicated that the
were performed for the active compounds against A549 human H1 may be a potential inhibitor of HDAC-8 enzyme. Furthermore,
lung carcinoma cell line which highly expresses both MMP-2 the mapping of the H1 in the best ligand-based pharmacophore
and HDAC-8. Immunouorescence assay was also done for of MMP-2 (Hypo2)11 provides a predicted activity (IC50) of 40.5 mM
cellular MMP-2 expression followed by molecular dynamics (pIC50 ¼ 4.392) [Fig. 2]. Therefore, the pharmacophore mapping
(MD) studies.
analyses indicated that H1 may be considered as a virtual hit for
dual MMP-2/HDAC-8 inhibition. To conrm this hypothesis, H1
was synthesized and its activity was determined against both
MMP-2 and HDAC-8. The IC50 activities against MMP-2 and
HDAC-8 were found to be 83.7 mM (pIC50 ¼ 4.077) and 3.8 mM,
2. Results and discussion
2.1. Development of pharmacophore model
Pharmacophore mapping is one of the most essential tools for respectively. Since the MMP-2 inhibitory activity of the molecule
development of anticancer agents.27–29 For the design of the dual was not found to be satisfactory, H1 was subjected to tailoring
MMP-2/HDAC-8 inhibitors, pharmacophore models were and a series of derivatives and analogs (compounds D1–D35),
developed and validated for both these enzymes separately. The structurally similar to H1 were synthesized for lead modication
development and validation of pharmacophore models for purpose. The activity of these derivatives and analogs was rst
MMP-2 inhibitors were reported earlier,11 where the best ligand- screened against MMP-2 enzyme. The best active compounds
based pharmacophore (Hypo 2) was generated with one were identied and were further evaluated with other MMPs
hydrophobic (H), one hydrogen bond acceptor (A) and two (MMP-1, -8, -9, -12, -14). The most suitable derivatives were tested
hydrogen bond acceptor lipid (AL) features (Fig. S1, ESI†).
against HDAC-8 enzyme as well as the nuclear extract of HELA
In the current study, ligand- and structure-based pharma- cells containing HDACs (rich in HDAC-1 and -2).
cophore models for HDAC-8 inhibitors were developed. The
The design of the modied leads (D1–D35) was done on the
details of model development and validation are discussed in basis of the molecular docking prediction. The H1 was docked
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Fig. 1 Interfeature distance constraints of (a) LigPharm01 and (b) StrPharm02.
in the protein structure of MMP-2 (PDB: 1HOV)39 using these compounds depends largely on the charge transfer (pi–pi
quantum polarized ligand docking (QPLD) tool.40,41 The best and pi–anion) interactions. Therefore, the substitution of this
docked pose (Dockscore: ꢀ8.907) is illustrated in Fig. 3. It is phenyl moiety should be more favourable for the charge
found that the methoxyphenyl moiety of H1 interacts with transfer reactions with residual amino acids. Since both pi–pi
His85 and Asp72 by pi–pi and pi–anion interactions respectively and pi–anion interactions may be possible, the charge transfer
whereas the benzyl moiety is inserted into the hydrophobic S10 interactions are difficult to predict in this case. Therefore, the
cavity to form pi–pi interaction with His120 and pi–charge R1 groups of the designed molecule were selected in a way so
interactions with the catalytic zinc atom. Interestingly, no that the variable electron densities of the adjacent phenyl
interaction is observed with the methoxy group of H1. These residue may be achieved to alter these charge transfer interac-
interactions are consistent with the docking interactions tions. Since the other unsubstituted benzyl residue of H1 is
observed by Li et al.38 Since the methoxyphenyl residue of H1 is involved in the charge transfer interactions at the S10 cavity,
exposed to the hydrophilic atmosphere, the higher activity of increase in the hydrophobic as well as charge transfer
Fig. 2 Design of dual MMP-2/HDAC-8 inhibitors from pharmacophore mapping analyses. A: hydrogen bond acceptor, H: hydrophobic, Ra: ring
aromatic, AL: hydrogen bond acceptor lipid, Z: zinc binding feature.
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Fig. 3 Best docking pose of compound H1; left side: three-dimensional (3D) representation with hydrophobic contour map, hydrophobic
contour map is shown as higher to lower as brown to blue (
follows ( charged (negative), charged (positive), hydrophobic,
salt bridge, solvent exposure).
); right side: 2D representation of the docking interactions, colours are as
metal, polar, hydrogen bond (backbone), pi–pi interaction,
interactions should alter the biological activity. To understand
It is evident from Table 1 that the 4-chloro- and the 4-bro-
the important interactions for the higher biological activity, the mophenyl and the naphthalene derivatives are the most suit-
R2 group of the designed molecule was substituted with able among other substitutions. The long straight aliphatic
different aliphatic and aromatic residues. It is worth mentioning chains at the R2 position are mostly favourable than both the
that the binding interactions of the S10 pocket have been thor- short and the branched aliphatic chains. Although the benzyl
oughly investigated earlier. Devel et al.9,10 reported that the bulky substitution at the R2 position shows the higher activities but
aromatic substitutions in this cavity increase MMP-2 inhibitory the phenyl substitution at the same position is unfavourable.
activity in several folds at the loss of isoform selectivity towards The benzyl and n-hexyl moieties at the R2 position are more
MMP-2. Moreover, such substitutions may increase the activity active than other analogs. Initially, compounds D1–D29 were
of these molecules towards MMP-8 and MMP-12 which are synthesized to understand these SARs. Aer conrming D11 as
considered as anti-targets for cancer treatment. Therefore, such the most active MMP-2 inhibitor of this series (D1–D29), the R2
bulky substitutions were avoided during the design of H1 and its position was substituted with different benzyl residues to
modied leads (D1–D35). In addition, Lipinski42 and Veber43 obtain D30–D35 in order to further improve the MMP-2 inhib-
rules were also taken into consideration during this design. itory activity. This decision was monitored by the docking
Although Veber rules were relaxed in some cases, Lipinski rule interaction of D11, provided in the ESI (Fig. S3†). The docking
(except molecular weight <500) was strictly followed.
pose (Dockscore: ꢀ9.681) suggested that the hydrophobic and
the charge transfer interactions at the S10 pocket have high
impact on the biological activity of these compounds.
Compounds D30–D35 showed variable MMP-2 inhibitory
activity. Substitution of the R2 position with the 4-nitrobenzyl
(D33) and the 2-chlorobenzyl (D35) derivatives showed the
higher MMP-2 inhibition than D11. The other analogs exhibited
the similar or the lower activity comparing to that of D11. The
2.3. Syntheses and enzymatic assay of the designed
compounds
The designed compounds (D1–D35) were synthesized through
the route outlined in Scheme 1.
The synthesized compounds (D1–D35) are listed in Table 1.
The MMP-2 inhibitory activity of these derivatives was tested
and the IC50 value is also depicted in Table 1.
D33 was found to be the highest active MMP-2 inhibitor (IC50
6.40 mM) in the current series.
¼
Scheme 1 Reagents and conditions: (a) SOCl2 in benzene; (b) L(+)-glutamic acid, 2 N NaOH (pH 7.5–8.5), then 1 N HCl; (c) DCC, CHCl3; (d) RNH2,
1 N Na2CO3, 1 N HCl.
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Table 1 Structures and MMP-2 inhibitory activity of the designed molecules
Cpd
R1
R2
MMP-2 (IC50) (mM)
D1
D2
D3
D4
D5/H1
D6
D7
D8
D9
4Cl
4Cl
4Cl
4Cl
4OCH3
4OCH3
4NO2
4NO2
4Br
4Br
4Br
4Br
2Cl
2Cl
2Cl
2Cl
2Br
2Br
2Br
2Br
2F
2F
2F
2,4-diCl
2,4-diCl
—
—
—
—
4Br
4Br
4Br
4Br
4Br
4Br
CH2C6H5
i-C4H9
n-C4H9
n-C6H13
CH2C6H5
i-C4H9
n-C5H11
t-C4H9
i-C3H7
n-C5H11
CH2C6H5
n-C6H13
i-C4H9
CH2C6H5
i-C3H7
n-C5H11
i-C3H7
i-C4H9
n-C5H11
C6H5
n-C4H9
n-C5H11
C6H5
CH2C6H5
n-C6H13
t-C4H9
i-C3H7
CH2C6H5
n-C6H13
CH2C6H4 4-Cl
CH2C6H4 4 F
CH2C6H3 3,4Cl2
CH2C6H4 4-NO2
CH2C6H4 4-OCH3
CH2C6H4 2-Cl
37.60
199.53
141.25
44.60
83.75
81.03
57.90
101.60
105.40
73.15
19.60
39.40
78.60
66.60
76.40
86.90
177.83
84.30
110.60
162.18
140.60
80.40
151.36
71.50
23.10
88.00
91.42
43.40
6.50
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
23.30
42.40
49.70
6.40
38.30
7.70
Fourteen compounds of the current series (D1, D4, D11, D12, gelatinase-B is closely associated with cancer progression,
D24–D25, D28–D35) were selected for further investigations. inhibition of this enzyme may develop adverse effects in
These compounds were evaluated for their inhibitory activities advanced stage of disease.6,21 Noticeably, the activity of the
towards MMP-1, -8, -9, -12 and ꢀ14 to understand the selectivity designed compounds towards MMP-9 varied to a considerable
proles. The inhibitory potentials of these compounds towards extent. Although these compounds showed a range of activities
HDAC-8 and the nuclear HDACs were also determined. These for different MMPs, the HDAC-8 inhibitory activity of these
enzyme inhibitory activities are presented in Table 2.
derivatives was almost similar. The IC50 of HDAC-8 of all
It is observed that the most of these compounds are non- compounds except D29 (52.56 mM) is lower than 10 mM. Inter-
selective towards other MMPs like MMP-1, MMP-8, MMP-12 estingly, all these compounds were found to be selective
and MMP-14. The MMP-8 is considered as an antitarget for towards HDAC-8 as compared to HELA nuclear extract of
cancer treatment and the inhibition of it may give rise to HDACs (rich in HDAC1 and 2). On the basis of these enzymatic
undesirable adverse effects.7,8 The MMP-12 has also been expressions, four compounds (D11, D29, D33 and D35) were
reported to be an antitarget since its inhibition may increase nally selected for cellular assays. These compounds were
angiogenesis and lung metastases. The exact disease context of tested for their anti-proliferative, anti-migratory and anti-
MMP-14 is unknown but inhibition of it may lower tumour invasive properties against A549 cells.
growth, angiogenesis and metastases.8 Though the MMP-9 or
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Table 2 MMPs and HDACs inhibitory activities of the designed compounds
IC50 (mM)
Cpd
MMP-1
MMP-2a
MMP-8
MMP-9
MMP-12
MMP-14
Nuclear HDACs
HDAC-8
D1
D4
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
37.6
44.6
19.6
39.4
71.5
23.1
43.4
6.5
23.3
42.4
49.7
6.4
207.67
169.53
201.05
240.32
101.53
168.23
230.4
183.89
205.75
244.53
208.02
205.89
180.83
181.63
5.2
57.82
8.9
238
>250
94.52
199.73
>250
>250
>250
>250
168.98
132.31
>250
138.32
50.56
68.78
45.83
73.92
23.56
100.32
85.6
85.79
90.19
98.69
91.99
83.16
193.35
56.33
99.29
102.67
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.33
5.02
3.03
4.21
6.07
8.6
6.05
52.56
4.09
4.27
4.45
2.89
5.09
6.29
D11
D12
D24
D25
D28
D29
D30
D31
D32
D33
D34
D35
74.57
63.06
83.1
74.57
81.36
64.82
70.42
>250
4.83
38.3
7.7
5.64
86.01
a
For comparison.
Fig. 4 (A) Antiproliferative activity of higher active MMP-2/HDAC-8 inhibitors. The results of annexin-V/PI binding assay through flow cytometry
for (B) untreated control, (C) D11 (50 mM), (D) D11 (100 mM), (E) D29 (50 mM), (F) D29 (100 mM).
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considerably; the most prominent anti-migratory effect was
noted for D33. The antimigratory effect of D11 was lower than
D33 but higher than other two derivatives, D29 and D35.
2.4. Cytotoxicity and ow cytometry apoptotic assays
Compounds D11, D29, D33 and D35 were tested for cytotoxicity
against
A549
cells
by
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The cytotoxicity
assay [Fig. 4(A)] depicted that none of these dual inhibitors are
highly cytotoxic in nature. These showed negligible cytotoxicity
(5–25%) at the lower concentrations (50–100 mM). Only at the
higher concentrations (300 mM), these showed perceptible
changes in cell viabilities (35–44%). To check the involvement of
apoptosis for anti-proliferative effects, annexin V-PI ow cytom-
etry analyses were conducted with two derivatives, D11 and D29.
Two lower dose concentrations (50 and 100 mM) were selected for
these cytometry analyses. The results are presented in Fig. 4(B)–
(F). It demonstrates that these compounds show minimal
apoptotic as well as necrotic effects on the cells. The purpose of
these cytotoxicity and ow cytometry analyses was to select an
appropriate non-cytotoxic dose for the migration and invasion
assays. Both these assays are performed in non-cytotoxic doses
where the migration and invasion properties may not be affected
by the cytotoxicity of these compounds.21 From these analyses, 50
mM dose was selected for both the migration and invasion assays.
2.6. Invasion assay
Inhibition of A549 cell invasion by these derivatives (D11, D29,
D33 and D35) was estimated through uorimetric QCM
ECMatrix cell invasion assay (ECM 555, Millipore). The
percentages of invaded cells are graphically presented in
Fig. 5(B). The inhibition was the most prominent for D33 which
showed 38–42% reduction in cell invasion. The D11 reduced the
invasion upto 33–34%. The other two compounds failed to show
such inhibition. Compounds D35 and D29 showed inhibition of
16–18% and 10–13%, respectively. Thus, from both the migra-
tion and the invasion assays, it may be conrmed that D33 and
D11 are two most promising candidates in the current series.
Interestingly, both these compounds showed comparatively
higher activity against MMP-9 enzyme. These results indicate
that the higher MMP-9 inhibition along with MMP-2 and HDAC-
8 are proportional to the higher anti-migration and anti-
invasion properties of these compounds. These results may
validate the ndings of previously reported investigations22,24
that highlighted HDAC mediated upregulation of both MMP-2
and MMP-9 in the increase of cancer cell invasion.
2.5. Migration assay
The anti-migratory properties of the selected derivatives
(compounds D11, D29, D33 and D35) were tested by the wound
healing assay. The A549 cells were treated with 50 mM of these
compounds (except the control) for 48 hours aer a scratch was
2.7. Immunouorescence assay for cellular MMP-2
expression
made on the monolayer of cells. The observations are illustrated To understand the potency of the designed compounds for
in Fig. 5(A). It demonstrates extensive migration of the lowering the cellular MMP-2 expression, immunouorescence
untreated cells (control) in previously created denuded area. microscopy technique was used to analyze the cellular expres-
The migration of the compound treated cells varied sions of MMP-2 enzyme aer the compound treatment. For
Fig. 5 Results of (A) wound healing migration assay and (B) Matrigel invasion assay of active designed molecules.
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nucleus counterstaining, 40-6-diamidino-2-phenylindole (DAPI) are provided in the ESI (Fig. S4†). These observations demon-
was used. The expressions of MMP-2 antibodies in untreated strate stabilities of these complexes during 10 ns simulation.
control and treated cells are represented in Fig. 6 along with The ligand–protein contacts of these two complexes are illus-
phase contrast, DAPI and merged MMP-2/DAPI images. The trated in Fig. 7. In both MD simulation runs, the carboxyl group
intensity of the MMP-2 antibody expressions was measured by of D33 was found to interact strongly with the catalytic zinc
ImageJ tool.44 It is observed that the expression of the cellular atom of both of HDAC-8 and MMP-2 enzymes. For MMP-2
MMP-2 is reduced by both D33 and D11; compound D33 complex, His120 made a strong pi–pi interaction with the
reduced the expression of MMP-2 up to 40%, whereas D11 nitrophenyl residue of D33. Noticeably, all amino groups of D33
showed 30% reduction aer 24 hours comparing with that of were involved in the polar interactions with the catalytic amino
the control.
acid residues. This compound made hydrogen bond interac-
tions with Leu83 and Gly81 with the occurrences of 65% and
71%, respectively. Water bridged interaction with Pro140
residue was also observed with a low percentage (27%). The
lower number of interactions in the S10 pocket may explain the
higher selectivity towards MMP-2 enzyme as well as the varia-
tions in the MMP inhibitory activities of the designed
compounds. On the other hand, D33 produced multiple
hydrogen bond interactions with Cys153, His143 and Tyr306
residues of HDAC-8 protein. The 4-bromophenyl residue of D33
made pi–pi interactions with Trp141 and Arg37. Interestingly,
Tpr141 is non-conserved amino acid in HDAC-8 and the
2.8. Molecular dynamics (MD) simulation analysis
In vitro analyses conrmed that D33 was active against both
HDAC-8 and MMP-2. To get insights into its mechanisms of
action, D33 was docked at the binding sites of MMP-2 (PDB:
1HOV)39 and HDAC-8 (PDB: 1VKG)35 enzymes separately. The
best docked poses were selected (Dockscore, MMP-2: ꢀ10.026
and HDAC-8: ꢀ7.899), and the docked complexes (MMP-2: D33
and HDAC-8: D33) were subjected to MD simulations at 10 ns
separately. The root mean square deviation (RMSD) and root
mean square uctuation (RMSF) diagrams of both complexes
Fig. 6 Confocal microscopy images of cellular MMP-2 expressions in untreated, compounds D11 and D33 treated A549 cell lines.
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Fig. 7 The percentage occurrence of interactions observed in 10 ns MD simulations of D33 complex of (A) MMP-2 and (B) HDAC-8.
tryptophan residue is replaced by leucine residue in HDAC1- (HDAC-8) but less active against other MMPs (i.e., MMP-8,
3.45,46 The selective inhibition of HDAC-8 is possible through MMP-12, MMP-1, etc.) that are considered as anti-targets of
interactions with the amino acid residues present in the cancer treatment.7,8 Since the scopes of MMP-2 (ref. 3–5) and
internal ‘acetate release channel’ of this enzyme.47 This ‘acetate HDAC-8 inhibitors16 are not limited to cancer, the designed
release channel’ is smaller in HDAC-8 than other HDACs. This molecules may be further studied to understand the aspects of
work also showed that the released acetate ion (aer reaction of these molecules for other diseases. Moreover, the active
HDAC-8 with lysine) is stabilized by Trp141 and Arg37 and this HDAC-8 inhibitors may be studied for anti-proliferative activity
mechanism is unique in HDAC-8. Therefore, interactions with against T-lymphocyte cells.48 Since, both MMP-2 and HDACs are
one of these amino acids may lead to the selective inhibition of involved in the cancer cell migration and invasion, the inhibi-
HDAC-8 enzyme. Such interactions are found in the X-ray crystal tory potentials of the dual inhibitors towards A549 lung carci-
structures of the HDAC-8 (PDB IDs: 3SFF and 3SFH)47 [ESI noma cell migration and invasion were explored. The designed
(Fig. S5†)], where HDAC-8 selective inhibitors interact strongly compounds provide a range of MMP-2 inhibitory activities
with Trp141 by pi–pi interactions. The D33, on the other hand, whereas the HDAC-8 inhibition is almost constant for the
was found to interact with both Trp141 and Arg37, and these higher active dual inhibitors. Molecular dynamics simulation
interactions may be responsible for its isoform specicity revealed that the designed compounds may be acting through a
towards HDAC-8. The histogram and timeline interaction distinct mechanism of action through the ‘acetate ion channel’
diagrams of HDAC-8:D33 complex are provided in ESI (Fig. S6†). of HDAC-8. These provide a scope to understand the role of
These diagrams reveal that D33 made several interactions with different MMPs and HDACs for migration and invasion of
the amino acid residues (Ile34, His143, Phe152, Cys153 and cancer cell. It was also observed that compounds simulta-
Tyr306) present in the ‘acetate release channel’ of HDAC-8. neously inhibiting MMP-2, MMP-9 and HDAC-8 were more
These interactions may also contribute to the higher isoform active than compounds having activity against one or two of
specicity of D33.
these enzymes. This nding partially validates the mechanism
of HDAC-8 mediated progress of cancer cell invasion via
MMP-9. These analyses may be taken into consideration to
design the potential dual inhibitors in future.
3. Conclusion
In the current report, the design, syntheses and biological
activities of dual MMP-2/HDAC-8 inhibitors have been pre-
sented. Pharmacophore-based technique is adopted for the
design of these dual inhibitors. The pharmacophore models
correctly predicted the activity of the designed molecules. The
4. Materials and methods
4.1. Computational chemistry
4.1.1. Ligand-based pharmacophore mapping. All ligand
models may be used for development of the higher active structures were prepared by Prepare ligand for QSAR tool of
molecules for these targets. Apart from the observed activity Discovery Studio (DS)49 where duplicate structures of these
against MMP-2 and HDAC-8, the selectivity of the designed compounds were removed and the pH of these molecules was
molecules against other MMPs and HDACs were taken into set to 7.4. These structures were subsequently minimized by
consideration. It was observed that the best active molecules Smart Minimizer algorithm (max steps: 10 000, RMS gradient:
were selective towards specic MMP (i.e., MMP-2) and HDAC 0.01) and distance dependent dielectric implicit solvent method
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using CHARMm input forceeld. Hypogen algorithm50 was used Model G1316C (Agilent Technologies), Wellplate Sampler
to develop 3D-QSAR pharmacophore models using DS49 1290-ALS, Model G4226A; Binary pump 1290-Bin Pump, Model
considering zinc binding feature (Z) as a mandatory feature for G4220A] coupled to electrospray ion (ESI) mass spectrometer
the hypothesis generation. The detail procedure is similar (MS QQQ) of multimode system, controlled through Mass
described in previous work.51
Hunter Quantitative Analysis soware. The 1H nuclear magnetic
4.1.2. Structure-based pharmacophore mapping. The resonance (NMR) spectra were measured on a AC Bruker
pharmacophore hypotheses generation was carried out on 300 MHz FT-NMR machine with TMS as the internal standards
different PDB structures of human HDAC-8 complexes following aer dissolving these compounds in dimethyl sulphoxide-d6
the protocols outlined in previous work (except maximum (DMSO-d6) solvent (Chembridge Isotope Laboratories Inc.,
charge distance that was set at 5.6).51 Two validation procedures USA). Infrared spectroscopy (IR) analyses were performed in a
were implemented – (1) selectivity score based ranking approach Bruker alpha 11960095 FT-IR instrument. Splitting patterns are
and (2) the decoy set validation. The active and confusing decoy designated as s (singlet), d (doublet) and m (multiplet). The
set compounds were collected from DUD-E database (http:// HMBC analysis was performed with a Bruker Avance DPX-500
dude.docking.org)51,52 and the rest of the validation proce- spectrometer using DMSO-d6 solvent. Optical rotation of
dures were similar to previously reported methods.51
these compounds was observed by Perkin-Elmer Type 141
4.1.3. Quantum polarized ligand docking (QPLD). Since polarimeter. Melting point of all synthesized compounds was
both MMPs and HDACs are the metalloenzymes having catalytic measured on Mel-Temp electrothermal apparatus and a capil-
zinc metal, accurate docking estimation requires quantum lary melting point apparatus and was further veried in
mechanics (QM) level treatment. Therefore, quantum polarized CTRONICS-a digital melting point apparatus. Reactions were
ligand docking (QPLD) technique53 was used to dock the monitored by the analytical thin layer chromatography (TLC)
designed inhibitors into the active site of these metal- performed on silica gel G plates (TLC silica gel 60 F254, Merck,
loenzymes. The NMR structure of MMP-2 enzyme (PDB ID: Germany). Spots were visualized by keeping the TLC plates in
1HOV)39 and X-ray crystal structure of HDAC-8 (PDB ID: 1VKG)35 ultraviolet (UV) light (254 nm) or placing the plates in iodine
were obtained from the PDB.36 The receptor structures were vapour. The column chromatography was performed using
described using OPLS force eld parameters. The partial 200–300 mesh silica gel with appropriate solvent systems. All
charges and the protonation states of the receptor molecules solvents were reagent grade and when necessary were puried
were described using Epik module and the catalytic zinc atom or dried by standard methods.
was assigned with +2 atomic charge. The restrained minimiza-
4.2.2. Syntheses. The designed compounds were synthe-
tion of hydrogen atoms of these protein molecules was per- sized by following the methods earlier proposed by Li et al.37,38
formed by the protein preparation wizard using OPLS2005 force with appropriate modications. The general synthetic tech-
˚
˚
˚
eld. A receptor grid box (size 15 A ꢁ 15 A ꢁ 15 A) was generated nique was outlined in Scheme 1.
considering the bound ligands of the respective protein as a
Syntheses of substituted phenylacetyl chlorides/napthylacetyl
centre of the box.54 The initial docking of the prepared ligands chloride (I1–I9). Substituted phenyl acetic acid/naphthyl acetic
was performed using the standard precision (SP) protocol to acid (0.1 mol) (S1–S9) and benzene (50–60 ml) were taken
generate the preliminary ligand poses. The single point calcu- separately in 250 ml round bottomed ask. The ask was tted
lation of the ligand poses using the density functional theory with a reux condenser attached with a calcium chloride drying
(DFT) based Becke–Lee–Yang–Parr (BLYP)/6-31G* model was tube and a dropping funnel. Thionyl chloride (0.4–0.5 mol) was
done to calculate the polarizable charges of these inhibitors. added drop-wise from the dropping funnel to the round
These charges were assigned for the inhibitors by electrostatic bottomed ask under anhydrous condition. The reaction
potential (ESP) tting. Finally, these inhibitors with new partial mixture was reuxed for 3–4 h until HCl gas evaluation stops.
charges were re-docked into the enzyme active site to obtain the The evolution of HCl gas was checked by congo red paper. The
most energetically favourable ligand poses. The best pose of the excess of thionyl chloride was removed by distillation with three
ligands was selected from the highest Dockscore value.
50 ml portions of dry benzene to obtain the products (I1–I9)
4.1.4. Molecular dynamic (MD) simulation. The MD simu- which were used immediately in the next reaction without
lation of the protein-ligand complexes was carried out through further purications.
Desmond/Maestro.54 The detailed protocols of MD simulation
Syntheses of 2-N-(substituted phenylacetyl)-L(+)-isoglutamic
were described previously.51 The protein complexes were simu- acids (I10–I17) and 2-N-napthylacetyl-L(+)-isoglutamic acid (I18).
lated for 10 ns with a step of 2 fs NPT ensemble. The Nose–Hoover L(+)-Glutamic acid (0.1 mol) was taken in a 250 ml conical ask.
chain thermostat and Martyna–Tobias–Klein barostat were used Sodium hydroxide solution (2 N) was added slowly to the conical
to maintain temperature and pressure at 300 K and 1.013 bar ask till all glutamic acid dissolved and the mixture become
respectively. The atomic coordinate data and system energies slightly alkaline (pH 7.5–8.0). The reaction mixture was stirred
were measured every 10 ps recording the trajectory at 4.8 ps.51,55 on a magnetic stirrer with the temperature maintained at
0–5 ꢂC. Maintaining the alkalinity, the substituted phenylacetyl
chlorides/napthylacetyl chlorides (I1–I9) were added individu-
ally in small portions to the reaction mixture with constant
4.2. Organic chemistry
4.2.1. General. The mass spectroscopy (MS) analysis was stirring, and sodium hydroxide (2 N) was added time-to-time to
performed through a LC MS/MS [the LC, Agilent 1290-column, keep the reaction mixture alkaline. The reaction end points
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were monitored by TLC. Aer the completion of the reaction,
the reaction mixture was washed with 10–15 ml of benzene or
diethyl ether. The aqueous solutions were then acidied with
6 N HCl in ice cold condition to precipitate the solid products
at the bottom and the solution was kept cooled for about 1/2–1
h to allow more precipitations. The precipitated products were
ltered, washed several time with distilled water and dried as
much as possible under vacuum. These were again washed
with 15–20 ml of benzene and dried to obtain the solid
products (I10–I18). All these intermediate products were
puried by recrystallization with warm water. Physical data of
intermediate compounds (I10–I18) are shown in Table S2 in
the ESI.†
4.3. Biology
4.3.1. Matrix metalloproteinase inhibition assay. The
MMPs inhibition assays were carried out using MMP inhibitor
proling kits, purchased from Enzo Life Science International,
Inc., Plymouth Meeting, PA, USA, following the manufacturer's
protocol. The concentration of chromogenic substrates was
detected by its absorbance value at 410 nm in a microplate
photometer (Thermo Scientic Multiscan FC, USA). Enzyme
reactions were performed at 37 ꢂC for 1 h in a 100 ml nal
volume of solutions with at least six concentrations of inhibi-
tors (except the control) in triplicate. Aer addition of the
substrate, the increase of absorbance was recorded in 1 min
time intervals for 30 minutes (except for MMP-12, 60 min) at 410
nm with the help of a microplate photometer (Thermo Scientic
Multiscan FC, USA). An inhibitor, N-isobutyl-N-(4-methoxy-
phenylsulfonyl)glycyl hydroxamic acid (NNGH) was included as
a prototype control inhibitor. The concentration of compounds
that provide 50% inhibition of enzymatic activity (IC50) was
determined by semi-logarithmic dose–response plots (Graph
Pad Prism 5.0 for Windows, Graph Pad Soware Inc., San Diego,
California, USA, 2007).
4.3.2. HDAC-8 inhibition assay. The HDAC-8 inhibitory
activities of these designed compounds were determined by
using the Fluor-de-lys HDAC-8 activity assay kit (Enzo Life
Sciences International, Inc., Plymouth Meeting, PA, USA)
according to the manufacturer's protocol. The uorescence was
measured with a multi-mode microplate reader at 380 nm
excitation and 460 nm emission (SpectraMax, Molecular
Devices, USA) spectra. Inhibition of HDAC-8 activity was
monitored by a decrease in uorescence signal.
4.3.3. HDAC-1/2 inhibition assay. The activity of the
designed compounds towards other class 1 HDACs was esti-
mated using Color-de-lys HDAC activity assay kit (Enzo Life
Sciences International, Inc., Plymouth Meeting, PA, U.S.A.) that
contain the nuclear extract of HELA cells. This nuclear extract is
rich in HDAC1 and HDAC2. The HDAC-8 is found in this extract
in a very low to negligible amount. The assay was performed
according to manufacturer's protocol. The absorbance was
measured at 410 nm by a microplate photometer (Thermo
Scientic Multiscan FC, USA).
4.3.4. Cytotoxicity assay. The lung carcinoma cell line A549
was maintained as monolayer cultures in Dulbecco's Modied
Eagle Medium (DMEM) (Gibco) supplemented with 10% heat-
inactivated FBS (Invitrogen, USA) and Penicillin–Streptomycin
(100 IU mlꢀ1 to 100 mg mlꢀ1). The anti-proliferative activity of
the compound was evaluated by MTT assay. Briey, cells were
seeded into 96-well plates at 5000 cells per well, and allowed to
adhere for 18–24 h prior to addition of these compounds
separately. The designed synthesized compounds (diluted with
serum free DMEM media) were added in different concentra-
tions and kept for 48 h. 10 ml of MTT solution (10 mg mlꢀ1) was
added to each well before 4 h at the end of incubation. Aer 4 h
of incubation, the formazan crystals were solubilised in 100 ml
of DMSO. The absorbance was measured at 570 nm using a
microplate photometer (Thermo Scientic Multiscan FC, USA).
2(S)-5-(Benzylamino)-4-(2-(4-chlorophenyl)acetamido)-5-oxopen-
tanoic acid (D1). A mixture of 2(S)-2-(2-(4-chlorophenyl)acet-
amido)pentanedioic acid (I10) and 50–60 ml of dry chloroform
was taken in a 250 ml at bottom ask. The reaction mixture
was stirred on a magnetic stirrer with the temperature main-
ꢂ
tained at 0–5 C under anhydrous condition. The N,N-dicyclo-
hexylcarbidiimide (DCC) (0.01 mol) was dissolved in 20 ml dry
chloroform and it was added slowly and gradually to the reac-
tion mixture with continuous stirring. Aer 1 h of stirring,
benzylamine (0.02 mol) was added to the reaction mixture and it
was continuously stirred for 5–6 h. Aer the reaction was over,
the reaction mixture was kept in the refrigerator for overnight.
The solution was ltered and both the solid part [dicyclohexy-
lurea (DCU)] and the liquid (chloroform) layer were extracted
with cold 1 N sodium carbonate (Na2CO3) solution. The aqueous
part was then acidied to pH 2 with 1 N HCl in cold condition to
obtain solid precipitations of these products separately. These
solid products were extracted with ethyl acetate (3 ꢁ 30 ml) and
were dried with anhydrous Na2SO4 overnight. The solvents
were subsequently removed under reduced pressure and the
resulting crude residues were recrystallized from ethanol and
dried to obtain the target compounds (D1) as white solid.
+
ꢂ
Yield: 47.8%. MP 143–145 C. MS (ESI positive) m/z [M + Na ]
407.21.1H NMR (DMSO-d6, 300 MHz, ppm) d 12.09 (s, 1H,
COOH), d 8.40 (m, 1H, CONH), d 8.17 (d, 1H, CONH, J ¼ 8.31), d
6.80 (d, 9H, benzene); d 4.26 (m, 1H, CH), d 4.24 (m, 2H, CH2), d
3.70 (s, 3H, OCH3); d 3.38 (s, 2H, CH2), d 2.15 (m, 2H, CH2), d
1.86 and 1.74 (m, 2H, CH2). IR (KBr, cmꢀ1):3319 (N–H str of
CONH), 3065 (aromatic ¼ C–H str), 2960 (assym. aliphatic –C–
H str), 2858 (sym. aliphatic –C–H str), 1723 (C]O str COOH),
1647 (C]O str of CONH), 1619 (aromatic C]C str), 1541 (N–H
deformation), 1435 (aliphatic –C–H deformation), 1414, 1266
(C–O str and O–H bending of COOH). The NMR spectra of I10
and D1 are provided in the ESI (Fig. S7 and S8†). The HMBC
spectra of D1 are also provided in the ESI (Fig. S9†). For D1,
cross peaks were found between N5–H (8.43) and C4 (171.22),
N3–H (4.24) and C4 (171.22) as well as C7–H (2.15) and C8
(173.87). However, no cross-peak was found between N5–H
(8.43) and C8 (173.87). The current results are consistent with
the observation of Li et al.38
The other nal products were synthesized according to the
general procedure as described for the syntheses of D1. The
details of spectral analyses are provided in ESI (Text S2†).
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The viability was calculated by considering the control con- Laboratories, USA). Cells were observed under an Andor spin-
taining the solvent control (0.1% DMSO) to be 100% viable. ning disk confocal microscope and images were acquired. The
4.3.5. Flow cytometry apoptosis assay. The apoptosis assay corrected total cell uorescence (CTCF) was measured by
was done by using an annexin V-FITC apoptosis detection kit ImageJ tool.44 All integrated density of each cells (presented in
(Calbiochem, Germany). Briey, cells were treated with different these images) and at least six background areas were measured
concentrations of compounds and were stained with propidium and CTCF value was determined as per eqn (1).
iodide (PI) and annexin V-FITC according to the manufacturer's
CTCF ¼ [integrated desnity ꢀ (area of selected cell
instructions. The percentage of the live, apoptotic and necrotic
ꢁ mean fluorescence of background readings)]
(1)
cells were analyzed by BD LSR Fortessa cell analyzer (Becton
Dickinson, USA). Data from 106 cells were analyzed for each
sample.
4.3.6. Would healing migration assay. The A549 cells were
plated in 24 well plate at a density of 5 ꢁ 104 cells per well with
the complete media (DMEM + 10% FBS). Aer 60–70% conu-
ence, these cells were starved overnight. A straight scratch was
made in the cell monolayer by 20–200 ml pipette tip. The
detached as well as loosely adhered cells were washed thrice
with PBS. These cells were then treated with different concen-
trations of the designed compounds for 48 h and then
photographed.
Abbreviations
DS
Discovery studio soware
FT-IR
HDAC
HMBC
MMP
MP
Fourier transform infrared spectroscopy
Histone deacetylase
Heteronuclear multiple bond correlation
Matrix metalloproteanse
Melting point
4.3.7. Invasion assay. The anti-invasiveness of the synthe-
sized compounds was evaluated by a uorimetric QCM ECMa-
trix Cell Invasion Assay kit (Millipore, USA). The assay was
performed in a 96-well invasion plate based on Boyden chamber
principle. The A549 cells were starved overnight in FBS-free
DMEM, followed by harvesting and placing 1 ꢁ 105 cells
(resuspended in FBS-free medium with 5% BSA) in each well of
the insert. The lower portion of the chamber is called the feeder
tray that contained the medium with 5% FBS as a chemo-
attractant. The synthesized compounds were added to the cell
suspensions in triplicates. These cells were incubated in the
absence (untreated) as well as in the presence of these
compounds at xed concentrations (50 mM) for 24 h. Invasive
cells are able to invade through a basement layer of matrix
membrane solution and cross the pores of polycarbonate
membranes to adhere at the bottom of the inserts. The adhered
cells were dissociated with cell detachment solution and the
detached cells were detected by CyQuantGR dye. The uores-
cence was measured with a multi-mode microplate reader at
480 nm excitation and 520 nm emission (SpectraMax, Molecular
Devices, USA) spectra.
4.3.8. Immunouorescence assay. Anti-MMP-2 antibody
(sc-10736) and CFL-tagged secondary antibody (sc-362252,
IgG-CFL 488) were purchased from Santa Cruz Biotechnology,
Inc, USA. The A549 cells were seeded into glass cover slips at a
density of 1 ꢁ 106 cells per cm2 and treated with these
compounds (except the control). Aer 24 h, these cells were
xed with 4% paraformaldehyde in PBS for 15 min, per-
meabilized with 0.1% Triton X-100 in PBS for 20 min and
exposed to the blocking solution (PBS containing 5% bovine
serum albumin and 0.2% Tween 20) for 1 h at room tempera-
ture. The xed cells were then incubated overnight with anti-
MMP-2 primary antibody (1 : 250 dilutions), washed with PBS
and subsequently treated with secondary antibody (1 : 250
dilutions) for 1 h. For nuclear localization, the culture was then
treated with 10 mg mlꢀ1 of DAPI solution for 1 min. Samples
were mounted in Vectashield hard set mounting media (Vector
MS
Mass spectrometry
NMR
QPLD
QSAR
RMSD
RMSF
Nuclear magnetic resonance
Quantum polarized ligand docking
Quantitative structure activity relationship
Root mean square deviation
Root mean square uctuation
Acknowledgements
Authors are thankful to the All India Council for Technical
Education (AICTE), New Delhi, Council of Scientic and
Industrial Research (CSIR), New Delhi and University Grants
Commission (UGC), New Delhi for providing nancial support.
One author (AKH) is grateful to CSIR, New Delhi for providing
Senior Research Fellowships. Authors are also thankful to
Department of Chemistry, Jadavpur University for providing
facility for NMR spectroscopy and also to the authorities of
Jadavpur University, University of Calcutta and Indian Institute
of Chemical Biology for providing facilities required for the
work.
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