Design, synthesis, molecular docking, and biological studies of novel phytoestrogen-tanaproget hybrids

Article abstract of DOI:10.1080/00397911.2016.1144768

A diverse range of novel and highly functionalized flavonoid-based tanaproget hybrids were synthesized and evaluated in vitro for their antimicrobial and antiproliferative activities. Novel products were synthesized in good yields (81-95%) under Pd-cataly

Full text of DOI:10.1080/00397911.2016.1144768

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Design, Synthesis, Molecular Docking and
Biological Studies of Novel Phytoestrogen-
Tanaproget Hybrids
Sumit Kumar, Nishant Verma, Nikhil Kumar, Alok Patel, Partha Roy, Vikas
Pruthi & Naseem Ahmed
To cite this article: Sumit Kumar, Nishant Verma, Nikhil Kumar, Alok Patel, Partha Roy,
Vikas Pruthi & Naseem Ahmed (2016): Design, Synthesis, Molecular Docking and Biological
Studies of Novel Phytoestrogen-Tanaproget Hybrids, Synthetic Communications, DOI:
10.1080/00397911.2016.1144768
To link to this article: http://dx.doi.org/10.1080/00397911.2016.1144768
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Date: 02 March 2016, At: 07:37

Design, synthesis, molecular docking and biological studies of novel phytoestrogen-
tanaproget hybrids
1
1
2
2
2
2
Sumit Kumar , Nishant Verma , Nikhil Kumar , Alok Patel , Partha Roy , Vikas Pruthi ,
1
Naseem Ahmed
1
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee,
2
Uttarakhand, India Department of Biotechnology, Indian Institute of Technology
Roorkee, Roorkee, Uttarakhand, India
Corresponding author Sumit Kumar E-mail: sumitdcy@iitr.ac.in; Naseem Ahmed
E-mail: nasemfcy@iitr.ac.in
Supplemental data for this article can be accessed on the publisher’s website.
Abstract
A diverse range of novel and highly functionalized flavonoids based tanaproget hybrids
were synthesized and evaluated in vitro for their antimicrobial and anti-proliferative
activities. Novel products were synthesized in good yields (81-95%) under Pd-catalysed
0
reaction from bromo flavones and tanaproget boronic acids within 18-20 min at 60 C.
Bioassay results exhibited excellent activities against both hormone-dependent and
hormone-independent human breast cancer cells (MCF-7, MDA-MB-231, DU-145, PC-3
and HeLa). Among them, compounds 4e, 9a, 9c, 9e, 9g, 9h, 9m and 9n displayed
excellent activity. Compounds 4d, 4o and 9o were found equally potent against C.
albicans compared to fluconazole. Compound 5c showed better antibacterial activity
against S. aureus. Compounds 5a, 9i, 9o and 10c have shown admirable antibacterial
activity against E.coli.
1

KEYWORDS: Phytoestrogen-Tanaproget hybrids, Estrogen and progesterone receptors,
Anti-cancer activity, antimicrobial activity, Docking study
INTRODUCTION
Cancer is a generic term for a large group of diseases that can affect all parts of the body.
There are 32.6 million people living with cancers in 2012 worldwide and 14.1 million
new cases were reported every year in which 8.2 million led to cancer death. Therefore,
the search for anticancer agents that display broader spectrum of cytotoxicity to the tumor
cells is of great interest [1, 2]. Natural products obtained from plants, marines and
microbes are the major resources for the bioactive agents and play a vital role in the
discovery of lead molecules for new drugs. Flavonoids are the polyphenolic
phytochemicals that exist ubiquitously in the plant kingdom, which can be found in
several food sources such as fruits, vegetables, whole grains, and legumes [3, 4]. Earlier
studies have explored flavone and flavanone moeities as an extensive variety of
biological activities such as antioxidant [5], anti-inflammatory [6], antibacterial [7],
antifungal [8], anticancer activities [9], antiestrogenic [10], interaction with estrogen
receptors [11] and inhibitory activities against aromatase enzyme [12]. Due to their broad
spectrum of pharmacological activities, the flavonoids have been extensively studied as
2

therapeutic agents, mainly of relevance to our study as anticancer and antimicrobial
agents. In particular, due to their structural and functional resemblances to endogenous
estrogens, flavonoids have attracted noteworthy interest as alternative estrogens, termed
phytoestrogens and broadly studied for their potential role in many estrogen-dependent
diseases including breast cancer [13].
Tanaproget drug is used as non-steroidal progesterone receptor agonist with high affinity
and selectivity for the progesterone receptor [14] (Fig. 1). Generally due to the four-ring
structural motif found in natural steroid receptor ligands, steroidal progestins have higher
cross-reactivity with other steroid hormone receptors. Currently, available progestins
have side effects due to their lack of selectivity with other steroid hormone receptors.
Recently, the novel classes of non-steroidal progesterone receptor (PR) modulators with
improved receptor selectivity have been reported [15-18]. These new compounds exhibit
the potential for safer and wider pharmacological use as progestins.
Molecular hybridization is an effective tool to design and synthesize highly active novel
molecules by covalently bonding two or more pharmacophores into a single entity [19-
2
0] (Fig. 2). Moreover, the hybrid molecules may also minimize the undesirable side
effects and express synergic action [21]. These findings had encouraged us to investigate
the potential synergistic effect of phytoestrogen and tanaproget analogue scaffolds on
esterogen and progesterone receptors with respect to their parent core structures.
3

Herein, we report a series of hybrid compounds of these two pharmacophores with an
intention to synergize the anti-cancer activity against human cancer cell lines (MCF-7,
MDA-MB-231, DU-145, PC-3 and Hela). Our designed compounds have three parts,
namely flavonoid structure as a chief backbone, tanaproget-like moiety for enhancing
desired pharmacophore behaviour with drug like properties and non-polar butyl or pentyl
chain for lipophilicity control. The excellent cytotoxicity of various compounds for
harmone- dependent and harmone-independent breast cells pointed them as promising
lead molecules for anticancer drug design. Furthermore, molecular modelling of these
novel molecules was done to provide a comprehensive guide for further understanding of
their synergistic effects on estrogen and progesterone receptors.
2
. RESULTS AND DISCUSSION
2
.1. Chemistry
The general route for the synthesis of the target phytoestrogen-tanaproget hybrids (4a-r,
a-c, 6a-c, 9a-o, 10a-c) is depicted in Scheme 1 and 2. Briefly, the key starting bromo
5
flavonoids (1a-c, 2a-c, 5, 6, 7a, 7b, 8a-c, 10) and boronic acids (3a-c) were prepared by
the literature procedures [22, 23]. The Scheme 1 and 2 illustrates the routes for the
synthesis of the target hybrid compounds using bromo flavonoids, boronic acids of
0
tanaproget analogue, Pd-complex and K CO in ethanol at 60 C under microwave
2
3
irradiation. Generally, the products were obtained in high yields (81-95%) in 18-20 min.
Compounds 4a-r, 5a-c and 6a-c were prepared from 3-bromo flavone derivatives (1a-c,
2
a-c, 5 and 6) and the appropriate boronic acids of tanaproget analogues (3a-3c) using
4

Suzuki cross-coupling reaction which gave the products 4a-r, 5a-c and 6a-c in good to
excellent yields (Scheme 1).
Compounds 9a-o and 10a-c were synthesized from 3-butyl, 6-bromo flavone or
flavanone derivatives (7a, 7b, 8a-c and 10) and the appropriate boronic acids of
tanaproget analogs (3a-c) using Suzuki reaction which furnished the products 9a-o and
1
0a-c in 74-87% yields (Scheme 2).
The assigned structure of new products (4a-r, 5a-c, 6a-6c, 9a-o, 10a-c) were established
1
13
from their spectroscopic data (IR, H, C NMR, HRMS). For example, compound 4e
was obtained as a brown solid. IR spectrum of 4e showed absorptions at 3265, 1713,
-
1
1
652, 1621 and 1555 cm for –NH, >C=O, >C=C< (tanaproget analogue moeity), >C=O
and >C=C< (flavone moeity). HR-MS of 4e supported a molecular composition of
+
1
C H NNaO [M+Na] , representing 18 degrees of unsaturation. In the H-NMR spectra
29
25
6
of 4e, peak at δ 9.40 (s, D O exchangeable, 1H) confirms the presence of -NH group,
H
2
methyl group occurred at δ 1.68 (s, 3H), two methylene protons (-CH ) resonating at δ
H
H
2
2
.67-2.63 (m, 2H), two -OCH groups resonates at 3.93, three olefinic protons appears at
3
1
3
δ (m, 1H) and 5.14-5.09 (m, 2H). In C-NMR spectrum, δc 173.5 and 162.0 confirms
H
the presence of carbonyl group of flavone and tanaproget moiety respectively. Peaks at δc
8
4.6, 56.3, 56.2, 45.5 and 26.3 also confirm the presence of quaternary carbon, two –
OCH , methylene (CH ) and -CH groups.
3
2
3
2
.2. Biological Evaluations
5

2
.2.1. In Vitro Anticancer Activity
All compounds were screened for in vitro antiproliferative activities against the estrogen-
responsive breast cancer cell line (MCF-7), estrogen-independent breast cancer cell line
(MDA-MB-231), prostate cancer cell line (DU-145, PC-3) and cervix (HeLa) where 5-
FU, cyclophosphamide and cycloheximide were taken as positive control using MTT
assay. The results are reported in terms of % cell growth inhibition and IC values (Table
5
0
1
and 2). For a preliminary SAR evaluation, the initial series of compounds (4a-r, 5a-c,
a-c) was first evaluated against the above mentioned cell lines to investigate the effects
6
of different number of methoxy groups at different positions on the B-ring of oxa- and
aza-flavones core and vinyl, allyl or pentyl substitution on tanaproget scaffold. Among
this series, compound 4d, 4e and 6b with the methoxy groups at the C-3 and 4 positions
on ring-B and furan and allyl substitution showed the highest antiproliferative potency
against MCF-7, MDA-MB-231 and DU-145 cell lines. Among the tested cell lines, a
differential behaviour was detected when the cytotoxic effect of the active drugs was
comparatively studied in all cell lines; a higher cell growth inhibition was found in breast
cells (Table 1). Thus, whereas MCF-7, MDA-MB-231 and DU-145 cell proliferation was
inhibited ≥ 50% by compounds 4d, 4e, 9a, 9c, 9e, 9g, 9h, 9i, 9j, 9m and 9n, only
compounds 5b, 6a and 6c resulted to be cytotoxic in PC-3 cells. The synthesized
compounds were found to be weakly antiproliferative (inhibition ≤ 50%) for HeLa cells
except 6c and 9k. Compounds 4h, 4q, 4r against DU-145 cells and 4f, 4j, 4l, 4m, 5c, 6a,
9
a, 9n, 10c against HeLa cells failed to exhibit significant cytotoxicity.
6

In case of PC-3 cells, most of the compounds were not showing any considerable
cytotoxicity except compounds 5b, 6a, and 6c. Compound 9i exhibited excellent
cytotoxicity upto 94.29% with IC 19.51 ± 1.49 µM against MDA-MB-231 cells as
5
0
compared to the reference drugs. Compound 9h showed maximum cytotoxicity against
MDA-MB-231 and DU-145 cells (IC 13.49 ± 2.72 and 11.09 ± 1.32 µM) respectively.
5
0
In the structure –activity relationship (SAR), it was found that the introduction of
hydroxyl, methoxy or amino group in the aza-flavone core with the vinyl or allyl
substituent in tanaproget motif greatly enhanced their anticancer activities might be due
to some carbon type and π-alkyl type interaction of electron donating groups with the
amino acids which is supported by docking study also. Compounds (4d, 4e, 9a, 9c, 9e,
9
g, 9h, 9i, 9j, 9m and 9n) that inhibited ≥ 50% cell proliferation in at least two different
cell lines are shown in Fig. 3.
The anti-proliferative activities expressed as IC values are summarized in Table 2.
5
0
This table includes IC values of those compounds that at a 10 µM concentration caused
5
0
a reduction ≥ 50% in the growth of breast tumor cell lines. However, IC values
5
0
corresponding to compounds that inhibited tumour cell proliferation less than 50% at 10
µM were not evaluated (Fig. 4).
We also compared the antitumor activity of two related prostate cell lines, DU-145 and
PC-3 cells. A comparative analysis between these two cells showed that both cell lines
behaved differently. On the other hand, HeLa cells were less responsive to the
antiproliferative action of almost all the synthesized compounds except 4k, 4q, 4r, 5a, 6c,
7

9
c, 9f, 9k, 9l, 9o and 10a. Hence, the inspection of these results indicates the idea that
cancerous tumours generally contain cell sub-populations with different biological
properties.
2
.2.2. Antimicrobial Activity
Synthesized compounds (4a-r, 5a-c, 6a-c, 9a-o, 10a-c) were screened for their
antimicrobial activity. Yeast (Candida albicans), Gram +ve bacteria (S. aureus) and
Gram -ve bacteria (E. coli) were selected to evaluate the effectiveness of the test
compounds (details in Experimental section).
2
.2.2.1. Antibacterial Activity
All the hybrid compounds were preliminary screened for the antibacterial assay and some
of them were subjected to MIC (minimum inhibitory concentration) determination by
24
microdilution method and the results are given in Table 3. Reference drugs Cefadroxil
and Ampicillin were chosen as positive controls in antibacterial activity assay.
As seen in the Table 3, most of the compounds have shown lower activity than positive
control (Cefadroxil) while compounds 5c (MIC: 31.25 µg/ml) have shown excellent
antibacterial activity against S. aureus. Moreover, compounds 4r, 9e, 9o (MIC: 62.5
µg/ml) demonstrated equal potency against S. aureus than Cefadroxil. Compounds 4i, 4p,
5
a, 6c, 9h, 9i and 9n were found to be the least active (MIC: 250µg/ml) against S. aureus
compared to the reference drug. Compounds 5a, 9i, 9o and 10c exhibited comparable
antibacterial activity (MIC ≤ 62.5 µg/ml) than positive control against E. coli.
8

Compounds 4p, 4r, 5c, 6c, 9n were found to be the least potent (MIC: 250 µg/ml) against
E. coli. as compared to positive control Ampicillin (MIC: 62.5 µg/ml).
2
.2.2.2. Antifungal Activity.
Some previously screened compounds for the antifungal activity were subjected to MIC
determination by microdilution method [24] and the results are given in Table 3.
Reference drug Fluconazole was selected as positive control in antifungal activity assay.
Compounds 4d, 4o and 9o have shown equal potency (MIC: 31.25 µg/ml) as Fluconazole
against C. albicans. Compounds 4c, 4p, 9b and 9h exhibited moderate antifungal activity
(MIC: 62.5 µg/ml) while others displayed lower activity (MIC: 125-625 µg/ml).
2
.3. Molecular Docking Study
In order to examine the synergistic effect of the hybrid molecules on binding with
estrogen and progesterone receptors, an extensive docking study was done. Also, the
majority of the flavonoid-tanaproget hybrid compounds with significant antiproliferative
activities had shown distinct selectivity for the MCF-7 ER+ve breast cancer cell line that
expresses high levels of ER receptor. Therefore, to investigate whether the
antiproliferative activity of these synthesized derivatives (against the MCF-7 cell line) is
estrogen receptor-mediated, the binding interactions of the products with the ER receptor
were studied using molecular modelling (see supporting information). Genetic algorithm
was implemented using AutoDock4.2 and all the final results were visually inspected
using Discovery studio visualizer.
9

After the validation of the docking procedure (Fig. 5), the docking simulation suggested
that the binding affinity of 9a-i and 10a-c were higher than that of 4a-r, 5a-c and 6a-c.
Out of all the synthesized compounds 5b (PDB ID: 1ZUC) and 9h (PDB ID: 3EQM)
were found to be the most potent hit with AutoDock binding energy of -10.77 and -12.09
kcal/mol respectively which was significantly greater than the references used in the
present study (Table 4).
Visual analysis of docking pose indicated that compound 5b was fully buried inside the
active site of progesterone receptor (Fig. 6). The aromatic ring of tanaproget, flavone and
furan ring seemed to interact completely with hydrophobic residues likes Met759,
Leu763, Gly722, Leu718, Leu797 and Cys891 by π -alkyl interaction. The carbonyl and
sec-amine group in tanaproget moiety of compound 9h was found to be hydrogen bonded
with residue Arg145 and Arg435 of 3EQM. π -alkyl interaction between aromatic ring of
tanaproget and flavone moieties and residues likes Cys437, Phe430, Val369 and Met364
were also inspected and studied. π-sulphur interaction between the aromatic ring of
flavone and Met364 was also apparent by visualizers.
The present in-silico work clearly indicated that all the tested molecules displayed higher
affinity towards estrogen receptor (3EQM) than progesterone receptor (1ZUC). These
docking results are also sync with the outcome of in vitro antiproliferative activity against
a panel of five human cancer cell lines.
1
0

3
. CONCLUSION
We have reported a novel series of phytoestrogen-tanaproget hybrids which exhibited
excellent in vitro cytotoxicity against human cancer cell lines (MCF-7, MDA-MB-231,
DU-145, PC-3 and HeLa). Among them, compounds 4d, 4e, 9a, 9c, 9e, 9g, 9h, 9i, 9j, 9m
and 9n were found to exhibit excellent antiproliferative activities against the MCF-7,
MDA-MB-231 and DU-145 cells. Compounds 4d, 4o and 9o with MIC: 31.25µg/ml were
found equally potent against C. albicans compared to fluconazole. Compound 5c with
MIC 31.25 µg/ml showed better antibacterial activity compared to reference drug against
S. aureus (MIC: 62.5 µg/ml). Compounds 5a, 9i, 9o and 10c with MIC: 62.5µg/ml were
equally effective as standard drug (ampicillin) against E.coli. The molecular modelling
clearly indicates that all the hybrid compounds have higher affinity towards ER (3EQM)
than PR receptors (1ZUC). Compound 5b had improved binding affinity (Ki) at PR of
1
2.86 nM with docking score -10.77 kcal/mol compared to tanaproget. As a non-steroidal
PR antagonists, phytoestrogen-tanaproget hybrids (5a, 5b, 6a, 6b) were found more
potent as compared to tanaproget.
4
. EXPERIMENTAL PROTOCOLS
4
.1. Chemistry
All the required chemicals were purchased from Merck and Aldrich Chemical Company.
Pre-coated aluminium sheets (silica gel 60 F254, Merck) were used for thin-layer
chromatography (TLC) and spots were visualized under UV light. IR spectra were
1
13
recorded with KBr on Thermo Nicolet FT-IR spectrophotometer. H NMR and C NMR
1
1

spectra were recorded respectively on Bruker Spectrospin DPX 500 and Jeol Resonance
ECX 400II spectrometer using CDCl or DMSO as solvent and trimethylsilane (TMS) as
3
an internal standard. Spectra were processed using Bruker Topspin® 3.0.b.8. Melting
®
points were performed with Ambassador and Digital Melting point apparatus
(Nutronics), Popular India. Splitting patterns are designated as follows; s, singlet; d,
doublet; m, multiplet. Chemical shift (δ) values are given in ppm.
4
.2. Microwave Irradiation Experiment
All microwave experiments were carried out in a dedicated Anton Paar Monowave-300
reactor, operating at a frequency of 2.455 GHz with continuous irradiation power of 0 to
8
50 W. The reactions were performed in a G-10 Borosilicate glass vial sealed with
Teflon septum and placed in a microwave cavity. Before microwave heating, oxygen was
purged into the reaction vial. Initially, microwave of required power was used and
temperature was being ramped from room temperature to a desired temperature. Once
this temperature was attained, the process vial was held at this temperature for required
time. The reactions were continuously stirred. Temperature was measured by an IR
sensor. After the experiments a cooling jet cooled the reaction vessel to ambient
temperature.
4
.3. General Procedure For Synthesis Of Compounds (4a-R, 5a-C, 6a-C, 9a-O, 10a-
C)
To a G-4 process vial capped with Teflon septum was added 3-bromo flavone (0.5
mmol), boronic acid (0.6 mmol), Pd-complex C (0.2 mol%, 1.0 mg), TBAB (0.12 mmol),
1
2

K CO (1.2 mmol) and 6 ml of ethanol. After a pre-stirring for one minute, the vial was
2
3
subjected to microwave irradiation time of 18-20 minute at 60 °C. It was then cooled to
room temperature, diluted with water, and extracted with EtOAc for three times. The
organic phase was dried with Na SO and concentrated to yield the product. The crude
2
4
material was chromatographed on a silica gel column eluting with a mixture of hexane
and ethyl acetate (8:2) to afford the product in (81-95%) yield. The purified products
were identified by FTIR, NMR and HRMS spectra.
6
-(2-(4-Methoxyphenyl)-4-Oxo-4H-Chromen-3-Yl)-4-Methyl-4-Vinyl-1H-
Benzo[D][1,3] Oxazin-2(4H)-One (4a)
o
1
Brown solid, mp 160-168 C, Yield 95%. H NMR (500 MHz, CDCl ppm): δ 9.08 (s,
3
,
1
1
H, D O exchangeable), 8.27 (d, J = 8Hz, 1H), 7.80 (d, J = 9Hz, 1H), 7.70 (t, J = 8.5 Hz,
2
H), 7.48 (d, J = 8 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.37 (dd, J = 1.5, 8.5 Hz, 1H), 7.13
(d, J = 8Hz, 1H), 7.08-7.02 (m, 3H), 6.87 (d, J = 8Hz, 1H), 6.06-5.97 (m, 1H), 5.26-5.06
1
3
(
m, 2H), 3.90 (s, 3H), 1.82 (s, 3H). C NMR (125 MHz, CDCl , ppm): δ174.8, 164.5,
3
1
1
3
61.8, 155.9, 153.0, 139.4, 134.2, 132.3, 131.5, 129.4, 127.5, 127.3, 126.8, 125.9, 124.5,
-1
23.6, 120.0, 117.7, 115.7, 114.8, 113.8, 113.7, 87.7, 55.6, 25.7.FTIR (KBr, v = cm ):
256, 2955, 1709, 1655, 1615, 1587, 1505, 1463, 1345, 1333, 1306. HRMS (ESI+): m/z
+
calcd. for C H NO [M+H] : 440.1492 found : 440.1495.
2
7
22
5
4
4
4
.4. Biology
.4.1. In Vitro Antitumor Activity Assays
.4.1.1. Cell Culture
1
3

Human breast (MCF-7, MDA-MB-231), prostate (PC-3, DU-145) and cervical (HeLa)
cancer cell lines were all obtained from National Center for Cell Science (NCCS), Pune,
India. All cell culture reagents were from GIBCO (Invitrogen, USA). Penicillin,
streptomycin, MTT (3-(4,5-dimethyl-2-thiazolyl)2,5diphenyl-2H-tetrazoliumbromide),
cell culture grade dimethyl sulphoxide (DMSO), 5-fluorouracil (5-FU),
cyclophosphamide and actidione (cycloheximide) were from HiMedia (Mumbai, India).
All the cells were maintained in Dulbecco’s modified Eagle’s media (DMEM)
supplemented with 10% fetal bovine serum (heat inactivated) and 1% antibiotic
(100 U/ml of penicillin and 100 µg/ml streptomycin) (Himedia, Mumbai, India) mix
at 37 ºC in humidified atmosphere in a CO incubator.
2
4
.4.1.2. MTT Assay
3
MTT assay was carried out as described previously [25]. In brief, 5 × 10 cells in 200 µl
of medium were seeded in 96-well plates (Griener, Germany). After 24 h, the cells were
treated with various concentrations (0.1, 1, 10, 100 and 1000 μM) of test compound
dissolved in DMSO. The final DMSO concentration for all dilutions was 0.1% which was
used as vehicle control. The cultures were assayed after 24 h by the addition of 20 µl of 5
o
mg/ml MTT and incubating for 4 h at 37 C. The MTT-containing medium was then
aspirated and 200 µl of DMSO (Himedia, Mumbai, India) was added to lyse the cells and
solubilize the water insoluble formazone. The absorbances of the lysates were determined
on a Fluostar optima (BMG Labtech, Germany) microplate reader at 570 nm.
The percentage inhibition was calculated as
1
4

=
100 - [(Mean OD of treated cell X 100)/mean OD of vehicle treated cells (negative
control)]
The dose response curve and IC values were obtained by nonlinear regression analysis
5
0
[
non-linear regression (sigmoidal dose response with variable slope)] using Graph Pad
Prism, version 5.02 software (Graph Pad Software Inc., CA, USA).
4
4
.4.2. In Vitro Antimicrobial Assay/Studies
.4.2.1. Test Strain
The clinical isolates of C. albicans were obtained from the Department of Microbiology,
o
IITR, India. The isolate was sub-cultured on Sabouraud Dextrose Agar at 37 C for 48-
7
2h. Both Gram (+) bacteria (S. aureus) and Gram (-)bacteria (E. coli) were also obtained
from the Department of Microbiology, IITR, India, and used to evaluate the antibacterial
effectiveness of the test compounds.
4
.4.2.2. Microdilution Assay
Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial
compound that inhibits the visible growth of a microorganism. MIC values of the
compounds against bacterial and fungal isolates were determined on the basis of micro-
well dilution method following NCCLS recommendations [26]. In this method we made
stock of chemically synthesized compounds at a concentration of 10 mg/ml in DMSO,
which was converted to working solution of concentration 1 mg/ml solution in methanol.
Using a multichannel micropipette, 100 µl of media into all wells of pre-sterilized
1
5

microtitre plate was dispensed (experiment was done in triplicate). Two fold serial
dilutions were carried out from the well 1 to the well 10 and excess media (100 µl) was
discarded from the last well (No. 10). Liquid culture of test organisms were grown to a
suitable phase in corresponding medium (YPD; yeast extract peptone dextrose, for fungal
o
growth and LB; Luria Bertani Broth, for bacterial growth) for 12-18 h at 37 C. Then
optical density (OD) of liquid culture was determined at 600 nm and diluted in such a
4
7
way that each well received 10 cfu/100 µl of fungal suspension and 10 cfu/100 µl of
bacterial culture. Appropriate positive and negative control was also included in the
study. Positive control contained only microbial cells whereas negative control contained
only standard drug solution (Fluconazole for fungus and Ampicillin, Cifadroxil for
bacteria). All experimental procedures were performed under sterile condition using bio-
o
safety hood (ESCO, USA). The plates were incubated at 30 C (fungal culture) and 37
o
C (bacterial culture) for 12-16 h of growth phase.
4
.5. Docking Studies
AutoDock4.2 was used for docking simulation [27]. In AutoDock, we employed
Lamarckian genetic algorithm (LGA), which is the hybrid of genetic algorithm (GA) and
local search algorithm (LSA), for conformation searching. The RMSD between the
docked conformation, as generated by the docking algorithm and the native co-
crystallised ligand conformation which is well within the grid spacing used in the
docking procedure, showing that the docking method to be used was trustworthy and
valid. For protein preparation, the crystal structure of molecular and pharmacological
properties of a potent and selective novel non-steroidal Progesterone receptor agonist
1
6

tanaproget, at resolution 1.80 was retrieved from protein data bank (PDB code: 1ZUC)
and Structural basis for androgen specificity and oestrogen synthesis in human aromatase
(PDB code: 3EQM). The inhibitor, progesterone ligand and all water molecules were
removed, leaving only the residues of the receptor. Preparation of the target protein with
ADT involved the addition of polar hydrogens to the macromolecule, an essential step to
correct the calculation of partial charge. Finally, Gasteiger charges were calculated for
each atom of the macromolecule. The docking area was assigned visually around the
apparent active site. A grid of 80 Å × 80 Å × 80 Å with grid spacing of 0.375 Å was
positioned around the active site with all the ligand atom types using AutoGrid. All
parameters were set to defaults except ga_run (100). The docking results were clustered
on the basis of root mean square deviation (rmsd) and were ranked on the basis of free
energy of binding. All compounds were drawn and then converted to 3D by
ChemDraw(Cam-bridgesoft Inc.). Each structure was then energy minimized by AM1
force field. Discovery Studio Visualizer (DSV) was used for 3D visualization [28].
SUPPLEMENTAL MATERIAL
1
13
Characterization data H, and C NMR spectra associated with this article are available
on the publisher’s website.
ACKNOWLEDGEMENTS
The authors wish to express their gratitude to the BRNS, India for financial support,
DST, New Delhi for providing HRMS facility and CSIR, New Delhi for awarding SRF to
S.K.
1
7

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2
2
1

Table 1. In vitro anti-proliferative activity (% growth inhibition) at a concentration of 10
a
µM
S. No. Compounds MCF-7 MDA-MB-231 DU-145 PC-3 HeLa
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
4a
4b
4c
4d
4e
4f
-
19.66
28.48
22.55
75.95
68.05
6.91
21.41
-
15.62
31.23
25.41
67.86
51.12
25.48
32.52
9.63
6.32 21.45
5.21
17.28
20.22
59.66
47.89
28.16
12.72
-
9
14.53 22.36
35.66 27.64
20.01 13.25
30.02 4.82
14.57 15.27
18.34 26.49
36.25 19.84
4g
4h
4i
15.66
16.83
18.11
-
-
16.33
32.10
42.35
25.34
24
0
1
2
3
4
5
6
7
8
9
0
4j
37.79
34.41
28.59
31.45
24.88
41.16
16.47
38.86
-
41
0.82
4k
4l
29.65 39.45
18.09 7
4m
4n
4o
4p
4q
4r
5a
5b
14
24.05 0.67
18.5
14.39
-
30.21
19.25
23.94
7.62
15
10.20 30.64
25.73
33.57
8
-
3.53 41.01
5.34 36.26
-
4.28
25.44
9.94
35.97
48
45.28
35.37
19
37.27
56.08 11.24
2
2

2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
5c
19.22
27.27
37.11
22.55
48.72
24.22
42.22
19.11
66.16
-
16.68
42.36
70.13
31.95
77.73
40.5
22.57
44.38
55.32
42.78
61.62
45.32
63.35
28.16
51.24
21.54
71.52
79.29
86.75
75.19
29.38
39.65
61.45
56.87
19.64
26.34
38.40
37.51
10.32
26.54 5.69
51.23 4.61
32.59 14.60
44.64 55.04
34.58 7.09
6a
6b
6c
9a
9b
9c
15
34.49
75.22
20.54
86.43
19.61
81.06
86.43
94.29
73.06
35.06
43.88
70.31
74.43
27.86
33.59
36.52
15.95
20.22
11.54 36.26
33.5 29.52
23.47 18.5
9d
9e
9f
41
35.76
9g
9h
9i
63.05
66.16
47.77
62.77
14.55
19.05
40.94
45.5
39.06 16
33.85 12.38
31.28 26.58
36.87 23.47
0.95 54.72
20.28 42.85
24.67 15.27
17.61 2.54
9.27 47
9j
9k
9l
9m
9n
9o
10a
10b
10c
5-FU
17.38
16.33
19.05
25.5
13.27 38.28
0.89 23
38.41 5.68
30.43 13.00
15.54
2
3

4
4
4
5
CYC-PHO
13.17
15.84
17.96
12.10
14.30
25.02 9.19
CYC-HEXI 15.22
33.25 11.11
Bold values represent compounds showing good antiproliferative activity 5-FU 5-
a
fluorouracil, CYC-PHO cyclophosphamide, CYC-HEXI cycloheximide, Compounds
tested in triplicate, data expressed as mean value of three independent experiments.
2
4

a,b
Table 2. IC values of in vitro antiproliferative activity of active compounds.
5
0
S.
Compound MCF-7
MDA-MB-
231
DU-145
PC-3
HeLa
No.
IC in
5
0
µM
1
2
3
4
5
6
7
8
9
4d
4e
9a
9c
9e
9g
9h
9i
27.35 ±
.91
18.18 ±
.41
17.42
1.85
14.71 ±
.65
26.38 ±
.74
19.24 ±
.24
31.69 ±
.13
23.03 ±
.76
29.59 ±
.48
23.30 ± 2.83 45.00 ±
1.82
59.51 ±
1.11
75.09 ±
1.25
1
20.96 ± 1.14 39.03
±1.94
46.17 ±
1.09
57.51 ±
2.83
1
28.06 ± 3.49 31.92
±2.32
46.36 ±
3.02
57.51 ±
2.04
±
23.01 ± 2.19 43.59
±3.23
78.70 ±
2.11
66.03 ±
2.56
2
18.12 ± 3.16 33.64 ±
2.42
55.87 ±
1.92
38.62 ±
3.10
1
14.31 ± 2.67 30.25 ±
3.38
40.19 ±
3.11
42.34 ±
2.59
3
13.49 ± 2.72 11.09 ±
1.32
41.32 ±
2.69
43.68 ±
2.45
2
19.51 ± 1.49 40.44
±1.09
82.50 ±
1.89
43.23 ±
1.69
1
9j
33.47 ± 2.46 47.97 ±
2.97
69.38 ±
1.56
44.16 ±
2.93
1
2
5

1
1
1
1
1
0
1
2
3
4
9m
18.42 ±
.53
21.29 ±
.25
30.80 ±
.25
CYC-PHO 29.12 ±
.14
CYC-HEXI 35.29 ±
.07
21.43 ± 2.49 30.32 ±
65.01 ±
1.12
68.81 ±
1.38
2
2.11
18.06 ± 0.61 24.45 ±
1.77
9n
65.30 ±
2.40
55.87 ±
2.17
3
5-FU
25.80 ± 1.48 40.01 ±
1.75
16.65 ±
2.09
42.87 ±
2.13
1
29.00 ± 2.27 36.32 ±
1.12
22.77 ±
1.27
52.30 ±
1.95
2
25.13 ± 2.31 43.30 ±
2.02
18.46 ±
1.45
40.12 ±
1.78
2
5
-FU 5-fluorouracil, CYC-PHO cyclophosphamide, CYC-HEXI cycloheximide.
a
b
5
0% growth inhibition as determined by MTT assay (24h drug exposure). Compounds
tested in triplicate, data expressed as mean value ± SD of three independent experiments.
2
6

Table 3. Minimum inhibitory concentration (MIC) in µg/ml of synthesized compounds
against bacterial strains evaluated by microdilution method.
Sr. No. Compounds Gram positive
Gram negative
S. aureus (µg/ml) E. Coli (µg/ml) C. albicans (µg/ml)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
4a
4b
4c
4d
4f
-
-
125
250
62.5
31.25
125
250
-
-
-
-
-
-
-
-
-
4g
4i
-
-
250
-
125
-
4j
250
125
31.25
62.5
-
4l
-
-
0
1
2
3
4
5
6
7
8
9
4o
4p
4r
5a
5c
6a
6c
9a
9b
9d
125
250
62.5
250
31.25
125
250
-
125
250
250
62.5
250
125
250
-
-
125
625
-
500
62.5
125
125
-
-
-
2
7

2
2
2
2
2
2
2
2
2
2
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
9e
62.5
125
250
250
-
-
-
9g
-
-
9h
-
62.5
9i
62.5
-
9l
-
125
9m
-
-
125
9n
250
62.5
-
250
62.5
-
-
9o
31.25
10b
500
10c
125
62.5
-
62.5
-
-
Cefadroxil
Ampicillin
-
62.5
-
-
Fluconazole -
31.25
2
8

Table 4. Comparative data of Binding energy and Ki for Progesterone (PDB Code:
ZUC) and Estrogen receptor (PDB Code: 3EQM) of most potent compounds.
1
Sr.
no.
Compound Progesterone receptor
(PDB Code: 1ZUC)
Estrogen receptor
(PDB Code: 3EQM)
Binding energy
(kcal/mol)
-11.07
Binding energy
Ki
Ki
(kcal/mol)
(nM)
34.52
32.66
22.10
12.86
(nM)
7.70
3.89
41.31
30.48
2.43
2.29
1.67
1.58
1.37
1.55
1.96
3.54
1.88
-
1
2
3
4
5
6
7
8
9
4d
4e
5a
5b
9a
9c
9e
9g
9h
9i
-10.18
-10.21
-10.44
-10.77
-9.32
-11.47
-10.07
-10.25
146.53 -11.75
-9.63
87.99
49.85
-11.79
-9.96
-11.97
-9.34
143.20 -12.01
176.78 -12.09
232.18 12.02
128.42 -11.88
-9.21
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
-9.05
9j
-9.40
9m
9n
-10.01
-10.35
45.80
25.88
24.20
-
-11.53
-11.90
-
Tanaproget -10.39
Flavone
5-FU
-
-9.31
150.34
115.16
29.26
-4.67
378.20 -5.37
61.31 -6.19
CYC-PHO -5.75
2
9