Synthesis and biological evaluation of novel bavachinin analogs as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2018.01.006

A library of 28 analogs of bavachinin including aliphatic and aromatic ethers, epoxide, chalcone, oxime, semicarbazide, oxime ether and triazole derivatives have been synthesized and evaluated for cytotoxicity against four different human cancer cell lines. Bio-evaluation studies exhibited better cytotoxic profile for many analogs compare to bavachinin. Best results were observed for a 1,2,3-triazole analog (17i) with IC₅₀ values 7.72, 16.08, 7.13 and 11.67 μM against lung (A549), prostate (PC-3), colon (HCT-116) and breast (MCF-7) cancer cell lines respectively. This analog showed three and four fold improvement in cytotoxicity against HCT-116 and A549 cell lines than parent molecule (1). Structure activity relationship (SAR) study for all synthesized analogs was carried out. Further, mechanistic study of the lead molecule (17i) revealed that it inhibits colony formation and in vitro migration of human colon cancer cells (HCT-116). Also, it induced the morphological changes and mediated the apoptotic cell death of HCT-116 cells with perturbance in mitochondrial membrane potential (MMP) and PARP cleavage.

Full text of DOI:10.1016/j.ejmech.2018.01.006

Accepted Manuscript
Synthesis and biological evaluation of novel bavachinin analogs as anticancer agents
Nidhi Gupta, Arem Qayum, Arun Raina, Ravi Shankar, Sumeet Gairola, Shashank
Singh, Payare L. Sangwan
PII:
S0223-5234(18)30006-0
10.1016/j.ejmech.2018.01.006
EJMECH 10083
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 August 2017
Revised Date: 10 December 2017
Accepted Date: 3 January 2018
Please cite this article as: N. Gupta, A. Qayum, A. Raina, R. Shankar, S. Gairola, S. Singh, P.L.
Sangwan, Synthesis and biological evaluation of novel bavachinin analogs as anticancer agents,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.006.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel bavachinin analogs as anticancer agents
Nidhi Gupta, Arem Qayum, Arun Raina, Ravi Shankar, Sumeet Gairola, Shashank Singh, and
Payare L. Sangwan*
O
OH
O
O
N
O
O
O
N
O
N
1
N
Cell lines
IC50
A549 (Lung)
7.72 µΜ
PC-3 (Prostate) 6.08 µΜ
ΗCΤ−116 (Colon) 7.13 µΜ
MCF-7 (Breast) 11.67 µΜ
O
17i
NH2
Design and synthesis of bavachinin analogues as potent cytotoxic agents

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Synthesis and biological evaluation of novel bavachinin analogs as anticancer agents
Nidhi Guptaa, Arem Qayumb,c, Arun Rainaa,c, Ravi Shankar a,c, Sumeet Gairolac,d, Shashank
Singhb,c, and Payare L. Sangwana,c,*
aBioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu-180001,
India
bCancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine Jammu-180001,
India
cAcademy of Scientific and Innovative Research (AcSIR), CSIR-IIIM Campus, Jammu, India
dPlant Science Division, CSIR-Indian Institute of Integrative Medicine Jammu-180001, India
#IIIM manuscript no. IIIM/2168/2017
*Corresponding authors. Tel.: +91 191 2585006-13 Extn. 371; fax +91 191 2586333.
E-mails; plsangwan@iiim.ac.in (P. L. Sangwan)
Keywords: Bavachinin; cytotoxicity; colony formation; in-vitro migration; MMP; PARP

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Abstract
A library of 28 analogs of bavachinin including aliphatic and aromatic ethers, epoxide, chalcone,
oxime, semicarbazide, oxime ether and triazole derivatives have been synthesized and evaluated
for cytotoxicity against four different human cancer cell lines. Bio-evaluation studies exhibited
better cytotoxic profile for many analogs compare to bavachinin. Best results were observed for
a 1,2,3-triazole analog (17i) with IC50 values 7.72, 16.08, 7.13 and 11.67 µM against lung
(A549), prostate (PC-3), colon (HCT-116) and breast (MCF-7) cancer cell lines respectively.
This analog showed three and four fold improvement in cytotoxicity against HCT-116 and A549
cell lines than parent molecule (1). Structure activity relationship (SAR) study for all synthesized
analogs was carried out. Further, mechanistic study of the lead molecule (17i) revealed that it
inhibits colony formation and in vitro migration of human colon cancer cells (HCT-116). Also, it
induced the morphological changes and mediated the apoptotic cell death of HCT-116 cells with
perturbance in mitochondrial membrane potential (MMP) and PARP cleavage.
1. Introduction
Flavanoids are one of largest classes of polyphenolic compounds that occur naturally in plants
which possess broad spectrum of biological activities and are considered as suitable therapeutic
agents against cancer. They generally possess a phenylbenzopyrone structure (C6-C3-C6)
consisting of two aromatic rings, A and B connected by a central pyran ring C. The saturation
level and opening of central pyran ring categorize various classes of flavonoids namely flavones,
flavonols, isoflavones, flavanols, flavanones and flavanonols [1,2]. Unlike other flavonoids,
flavanones like bavachinin 1, hesperetin 2 and naringenin 3 (Fig.1) have been potential sources
to search for new leads in the area of cancer therapy [3,4].
Insert Figure 1.

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The seeds of Psoralea corylifolia are major source of bavachinin [5] and exhibit diverse
pharmacological activities including anticancer [3], PPAR agonist [6], anti-inflammatory [7],
anti-alzheimer [8] and immunomodulatory [9]. Cytotoxic effects of bavachinin have also been
studied on various cancer cell lines [3, 10] and possess 20S proteasome inhibitory activity that
inhibits the signaling action of NFκB leading to cell death via apoptosis [11]. Beside this
bavachinin also inhibited in vitro migration of human KB cells and during in vivo studies in nude
mice with KB xenografts, it significantly reduced tumor volume and CD31 expression [12].
Bavachinin 1 nucleus is capable of undergoing suitable chemical transformation studies at
various key positions available on molecule (Fig. 1). Several bavachinin derivatives have
synthesized and reported for their different biological activities. Chen et al. prepared bavachinin
derivatives 4 and 5 via biotransformation to study their T cell differentiation effects [13]. Dai et
al. reported anticancer activity of bavachinin and its enzymatically synthesized glucoside 6 [14]
while Du et al. reported PPAR-γ agonist activity of several bavachinin analogs among which
compounds 7 and 8 were found as most potent derivatives [15] (Fig. 2). However, further
investigation is desired to develop the potent anticancer agents based on bavachinin.
Insert Figure 2.
Therefore, the natural product bavachinin was isolated in bulk quantity from the seeds of
Psoralea corylifolia for synthesis of its analogs being our interest in isolation and structural
modification of natural products for drug discovery of potent analogs in the area of anticancer
[16-19]. Further the synthesized analogs were screened for cytotoxicity against four different
human cancer cell lines (A549, PC3, HCT-116 and MCF-7). The most active compound was
taken further to study its cytotoxic mode of action.

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2. Results and discussion
2.1. Chemistry
Bavachinin 1 was isolated in gram quantities from the seeds of P. corylifolia and was used for
structural modification studies. The configuration at C-2 an asymmetric center in the molecule
was observed (S) based on NOESY (Fig. S1) which was also confirmed by comparing the
observed specific rotation value [α]D = -20 (c = 1.0, CHCl3) with literature [5, 20]. Different
analogs of bavachinin 1 were synthesized with modifications at ring A, B, C and prenyl chain of
the molecule (Fig. 3) as shown in scheme 1 and 2. Aliphatic ether analogs of bavachinin (9a-e)
were synthesized by reacting 1 with appropriate alkyl halides in acetone in the presence of
potassium carbonate as base [21]. Analogs 9f-i, aromatic ethers of bavachinin were synthesized
through Chan-Lam coupling by reacting 1 with various aryl boronic acids in DCM in the
presence of copper acetate and pyridine [22]. Epoxide analog of bavachinin (10) was synthesized
by reacting 1 with m-chloroperbenzoic acid (m-CPBA) in DCM as per earlier report [23].
NOESY spectra (Fig S2) is provided in supporting information. Compound 11, chalcone analog
of 1 was obtained by treating it with sodium hydride in ethanol. Compound 12, carbonyl
reduction analog was synthesized by reacting 1 with sodium borohydride (NaBH4) in methanol
as per our earlier report [17] and its configuration was found 2S,4R at C-2 and C-4 position on
the basis of 2D NMR (COSY, HSQC and HMBC) experiments including NOESY (Fig S3-8).
Further the configuration was also confirmed through observed specific rotation [α]D = +19 (c =
1.0, CHCl3) which was found in comparison with literature [24]. Compound 12a, double bond
reduced product was prepared by hydrogenation of bavachinin in presence of 10% Pd/C [23].
Compounds 13 and 14, oxime and semicarbazide analogs of bavachinin were synthesized by

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treating 1 with hydroxylamine hydrochloride and semicarbazide hydrochloride respectively in
ethanol as solvent [25].
Insert Figure 3
Insert Scheme 1.
Bavachinin triazoles were synthesized at oxime moiety to prepare anticancer analogs in the light
of literature [19, 26]. For this, methyl ether of bavachinin 9a was reacted with hydroxylamine
hydrochloride to prepare its oxime analog 15. Compound 15 was reacted with propargyl bromide
in acetone in presence of potassium carbonate to furnish compound 16 which was further
subjected to Cu (I) catalyzed 1,3-dipolar cycloaddition reaction (click chemistry) using various
substituted aromatic azides to provide 1,2,3-triazole derivatives 17a-k in excellent yields [27]
(scheme 2). To confirm the configuration of C=N double bond in triazole analogs 17a-k,
NOESY experiment of one compound i.e. 17i was recorded. On interpretation of data, the signal
at δ 5.37 (-OCH2) did not show any correlation with either of protons at C-3 (-CH2) that rule out
the E configuration while the same signal at δ 5.37 (-OCH2) showing weak correlation with
aromatic proton singlet at δ 7.63 (H-5) which indicate Z configuration of C=N (Fig. S9).
Insert Scheme 2.
2.2. Biology
2.2.1. Cell growth inhibition studies
Compound 1 and its synthesized analogs were screened against a panel of four different human
cancer cell lines namely lung (A549), prostate (PC-3), colon (HCT-116) and breast (MCF-7) to
evaluate their cytotoxic potential using SRB assay for 48 hours. Results are shown in table 1 and
values given are the average of triplicate analysis. In preliminary screening at 50 µM, bavachinin
and some of its derivatives including compounds 9e, 9i, 10, 12, 12a, 13, 17d, 17f and 17i
exhibited significant inhibition effects (>50% inhibition) against all the experimental cancer cell

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lines. Compounds 9a-f, 9i, 13, 16, 17f and 17j showed maximum inhibition effects against colon
cancer cells (HCT-116). Compounds 10, 11 12a, and 14 displayed maximum inhibition effects
against breast cancer cells (MCF-7) where as compounds 15 and 17c sensitized lung cancer cells
the most. Overall, compounds 17d and 17i exhibited most potent cytotoxic effects (100%
inhibition) against all the cell lines examined while compounds 9g, 12, 17a, 17b, 17e, 17g and
17h did not possess significant cytotoxicity against any of the cell lines tested.
Insert Table 1.
2.2.2. IC50 and structure activity relationship (SAR) studies
Compounds showing significant cytotoxic effects (>75% growth inhibition) at 50 µM (Table 1)
were further screened at four different concentrations 1, 5, 10 and 30 µM to calculate their IC50
values. The calculated IC50 values are shown in Table 2. Parent molecule showed IC50 values
30.5, 25.4, 20.7 and 19.5 µM against lung, prostate, colon and breast cancer cell lines
respectively. Compound 13 displayed three fold more potent cytotoxicity than parent (1) against
lung cancer cell line (A549). Compound 17i was found to be three and four fold more active than
the parent against lung (A549) and colon (HCT-116) cancer cell lines respectively.
Insert Table 2.
Structure activity relationship (SAR) of compound 1 was established on the basis of growth
inhibition and IC50 data and can be summarized as follows: Alkyl and aryl ether derivatives (9a-
i) affected colon cancer cells (HCT-116) the most than other cell lines tested but did not exhibit
significant anticancer effects compared to the parent (1). Epoxide analog (10) displayed
comparable cytotoxic effects where as oxime (13) and semicarbazide (14) analogs were found to
be more active as compared to the parent against A549 cell line. Chalcone analog (11) inhibited
breast cancer cells the most. Reduction analog (12) did not display any significant cytotoxicity
against any of the examined cell line while double bond reduced prenyl analog (12a) showed

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comparable cytotoxic effects to parent against lung cancer cell line (A549). Oxime ether analog
(16) displayed cytotoxic effects against lung (A549) and colon cancer (HCT-116) cell lines.
Among triazole derivatives (17a-k), analogs with electron releasing groups (17a, 17b and 17h)
did not reveal any significant cytotoxicity except for the analog having –OH substitution at p-
position (17f) showing cytotoxicity to some extent against HCT-116 cell line. Loss of activity in
17e having –CH2OH group at 3-position than 17d having –CH2OH group at 2-position showed
position of substituent is essential for the activity. Analogs with electron withdrawing groups
such as -COC6H5 and -CN (17j and 17k) showed cytotoxicity to some extent but analog having
benzamide moiety (17i) was found to be most potent having IC50 values 7.72, 16.08, 7.13 and
11.67 µM against lung, prostate, colon and breast cancer cell lines respectively, hence chosen for
further cell death mechanistic study.
2.2.3. Compound 17i inhibited cell proliferation during colony formation assay in colon cancer
cells (HCT-116)
In vitro clonogenic assay was performed to measure the cell proliferation capability i.e. the
ability of a single cell to grow into a colony. This assay tests each and every cell in the
population for its ability to undergo unlimited divisions [28]. HCT-116 cells were treated with
different concentrations of 17i at 3.6, 7.2 and 14.4 µM in which colonies were formed after 14
days treatment. It was found that compound 17i significantly decreased colony formation in
colon cancer cells (HCT-116) in a concentration dependent manner as compared to the untreated
control (A) (Fig. 4).
Insert Figure 4
2.2.4. Compound 17i inhibited in vitro cell migration during wound healing assay in HCT-116
cells

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In vitro scratch assay or wound healing assay is a well-developed method for cell migration
studies. In this method, a wound is created in a cell monolayer in order to monitor the cell
migration and proliferation [29]. In this experiment, HCT-116 cells monolayer was scratched and
treated with 17i at different concentrations (3.6, 7.2 and 14.4 µM) for 24h. The wound area was
measured at two different time points 0 and 24h in which the percentage reduction in cell
migration was quantitatively assessed with 0h post scratch. Rate of migration was assessed as
compared to the corresponding control (A). It was observed that cell migration was inhibited in a
concentration dependent manner (Fig. 5).
Insert Figure 5.
2.2.5. Compound 17i induced morphological changes in HCT-116 cells
The colon cancer cells (HCT-116) were treated with 17i at 3.6, 7.2 and 14.4 µM for 24 h. Phase
contrast microscopy was used to assess the morphological changes in the cells. Characteristic
morphological changes were observed in the 17i treated cells in a concentration-dependent
manner (Fig. 6).
Insert Figure 6.
2.2.6. Compound 17i triggered mitochondrial membrane potential (MMP) loss
MMP loss occurs due to disruption in the mitochondrial membrane permeabilisation with the
release of apoptogenic stimuli leading to cell death [30]. The changes in potential of
mitochondrial membrane of HCT-116 cells were analyzed by staining with a fluorescent cationic
dye, rhodamine-123 (RH-123). The rate of fluorescence decay is directly proportional to MMP
loss. When HCT-116 cells treated with different concentrations of 17i (3.6, 7.2 and 14.4 µM
)
dramatic loss in MMP was observed in a concentration dependent manner whereas nuclei of
untreated cells (control) were having intact mitochondria (Fig. 7).

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Insert Figure 7
2.2.7. Compound 17i mediated PARP cleavage
PARP [poly (ADP ribose) polymerase] is a nuclear protein having the main function in repairing
DNA damage in response to cellular stress [31]. Cleaved PARP activity in HCT-116 cells was
analyzed using Western blot analysis where it loses its nick sensor function and becomes inactive
towards DNA damage. PARP cleavage started at a concentration of 3.6 µM and produced 89
KDa c-terminal fragment and continued the cleaved pattern as concentration increased with β-
actin serving as a loading control. This shows that PARP played an important role in apoptotic
cell pathway (Fig. 8).
Insert Figure 8.
3. Conclusion
In conclusion, a novel library of 28 analogs of bavachinin was synthesized and evaluated for
cytotoxicity against a panel of four different human cancer cell lines namely lung (A549),
prostate (PC-3), colon (HCT-116) and breast (MCF-7) along with the parent molecule.
Cytotoxicity results revealed that many compounds exhibit significant cytotoxic effects against
different cancer cell lines. More significantly, compounds 13, 14 and 17i were found to be
promising derivatives with IC50 <20 µM against various cancer cell lines examined. Compound
17i was found as lead compound among the synthesized analogs with three and four fold
improvement in cytotoxicity against colon and lung cancer cell lines as compared to the parent
molecule. Moreover, 17i induced apoptotic cell death through loss of MMP and PARP cleavage.
It also inhibited the colony formation, cell migration and induced the morphological changes in a
concentration dependent manner. All these experiments indicated the potential of compound 17i
to develop as potent anticancer agent.

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4. Experimental section
4.1. Chemistry
All the reagents and solvents used for isolation, purification and synthesis were purchased from
Sigma-Aldrich/E. Merck. All chemical reactions were monitored by TLC on silica gel 60 F254
plates (E. Merck) using 2% ceric ammonium sulphate solution as spraying reagent for detection
of spots. All synthesized derivatives were purified by column chromatography using silica gel
60-120 mesh as stationary phase. NMR spectra were recorded on Bruker DPX 400 and DPX 500
instruments using CD3OD, acetone-d6, CDCl3 as the solvent taking TMS as the internal
standard. The chemical shifts are expressed in δ and coupling constant in Hertz. High Resolution
Mass Spectra (HRMS) were recorded on Agilent Technologies 6540 instrument. Melting points
were determined (uncorrected) with a Buchi melting point apparatus B-545. The optical rotation
[α]D values were measured with a Perkin Elmer 241 polarimeter.
4.1.1. Isolation of Bavachinin (1)
Bavachinin was isolated in gram quantity by repeated column chromatography in 5-8% EtoAc/
Hexane of DCM: MeOH (1: 1) extract prepared from seeds of Psoralea corylifolia. Initially,
Bakuchiol, the major constituents of the extract was isolated [17], after that bavachinin was
obtained as crystalline compound which was characterized by spectroscopic techniques as
reported in literature [5, 20].
4.1.2. General procedure for the synthesis of aliphatic ether analogs (9a-e). Bavachinin (100
mg, 0.30 mmol) was dissolved in acetone and to this K2CO3 (0.30 mmol) and respective alkyl
halides (0.30 mmol) were added. The reaction mixture was stirred at room temperature for 2 h
till the completion (monitored by TLC analysis). After completion, mixture was extracted with

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ethyl acetate and water (3 times). The combined organic layers were dried over sodium sulphate
and concentrated on rotavapour. The crude product obtained was purified by column
chromatography on silica gel 60-120 mesh with 3-5% ethyl acetate/hexane as the eluent to afford
the desired pure products in 90-94% yield. The spectral data of all the derivatives (9a-e) are
given below.
4.1.2.1. 7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-one (9a). Yield:
1
91%, gummy mass, [α]D = -12 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.69 and 1.74
(3H each, s, H-4’’ and H-5’’), 2.75 and 3.02 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 7.3 Hz,
H-1’’), 3.83 and 3.84 (3H each, s, 2 × -OCH3), 5.27 (1H, t, J = 8.0 Hz. H-2’’), 5.37 (1H, dd, J =
13.0, 4.0 Hz, H-2), 6.44 (1H, s, H-8), 6.95 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.39 (2H, d, J =
8.0 Hz, H-2’ and H-6’), 7.68 (1H, s, H-5). 13C NMR (125 MHz, CDCl3): δ 190.86, 164.09,
162.27, 159.97, 132.89, 131.08, 127.71 × 2, 127.1, 124.87, 121.84, 114.2 × 2, 114.04, 98.83,
79.8, 55.72, 55.34, 44.17, 27.8, 25.79 and 17.72. HRMS m/z calcd for C22H25O4 [M + H]+
353.1747, found 353.1735.
4.1.2.2. 2-(4-ethoxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-one (9b). Yield:
1
90%, gummy mass, [α]D = -6 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.42 (3H, t, J =
7.0 Hz, -CH2CH3), 1.69 and 1.74 (3H each, s, H-4’’ and H-5’’), 2.75 and 3.02 (1H each, m, H-
3a and 3b), 3.24 (2H, d, J = 7.3 Hz, H-1’’), 3.83 (3H each, s, -OCH3), 4.04 (2H, q, J = 8.0 Hz, -
CH2CH3), 5.28 (1H, t, J = 8.0 Hz. H-2’’), 5.35 (1H, dd, J = 12.0, 4.0 Hz, H-2), 6.44 (1H, s, H-8),
6.95 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.37 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.67 (1H, s, H-
13
5). C NMR (125 MHz, CDCl3): δ 191.07, 164.09, 162.32, 159.32, 133.0, 130.81, 127.75 × 2,
127.07, 124.84, 121.78, 114.71 × 2, 113.97, 98.82, 79.88, 63.56, 55.76, 44.17, 27.83, 25.89,
17.78 and 14.82. HRMS m/z calcd for C23H27O4 [M + H]+ 367.1904, found 367.1900.

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4.1.2.3. 7-methoxy-6-(3-methylbut-2-en-1-yl)-2-(4-propoxyphenyl)chroman-4-one (9c). Yield:
91%, gummy mass, [α]D = -12 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.04 (3H, t, J =
7.0 Hz, -CH3), 1.69 and 1.74 (3H each, s, H-4’’ and H-5’’), 1.81 (2H, q, J = 8.0 Hz, -CH2CH3),
2.76 and 3.02 (1H each, m, H-3a and 3b), 3.25 (2H, d, J = 8.0 Hz, H-1’’), 3.83 (3H, s, -OCH3),
3.94 (2H, q, J = 8.0 Hz, - CH2CH2CH3), 5.27 (1H, t, J = 8.0 Hz. H-2’’), 5.37 (1H, dd, J = 13.0,
4.0 Hz, H-2), 6.44 (1H, s, H-8), 6.94 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.38 (2H, d, J = 8.0 Hz,
H-2’ and H-6’), 7.67 (1H, s, H-5). 13C NMR (125 MHz, CDCl3): δ 191.07, 164.09, 162.32,
159.52, 132.99, 130.75, 127.73 × 2, 127.06, 124.82, 121.78, 114.74 × 2, 113.97, 98.83, 79.87,
69.61, 55.76, 44.16, 27.82, 25.88, 22.56, 17.78 and 10.54. HRMS m/z calcd for C24H29O4 [M +
H]+ 381.2060, found 381.2064.
4.1.2.4. 2-(4-butoxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-one (9d). Yield:
92%, gummy mass, [α]D = -12 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 0.98 (3H, t, J =
7.0 Hz, -CH3), 1.51 (2H, q, J = 8.0 Hz, -CH2CH3), 1.7 and 1.74 (3H each, s, H-4’’ and H-5’’),
1.78 (2H, m, -CH2CH2CH3), 2.77 and 3.05 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0 Hz,
H-1’’), 3.84 (3H, s, -OCH3), 3.98 (2H, t, J = 8.0 Hz, -CH2CH2CH2CH3), 5.28 (1H, t, J = 8.0 Hz.
H-2’’), 5.39 (1H, dd, J = 12.0, 4.0 Hz, H-2), 6.44 (1H, s, H-8), 6.94 (2H, d, J = 8.0 Hz, H-3’ and
H-5’), 7.37 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.67 (1H, s, H-5). 13C NMR (125 MHz, CDCl3):
δ 191.11, 164.1, 162.32, 159.54, 133.01, 130.72, 127.73 × 2, 127.06, 124.83, 121.77, 114.74 × 2,
113.97, 98.82, 79.88, 67.81, 55.77, 44.16, 27.82, 25.88, 19.25, 17.78, 13.87 and 10.54. HRMS
m/z calcd for C25H31O4 [M + H]+ 395.2217, found 395.2234.
4.1.2.5.
7-methoxy-6-(3-methylbut-2-en-1-yl)-2-(4-(prop-2-yn-1-yloxy)phenyl)chroman-4-one
(9e). Yield: 94%, gummy mass, [α]D = -14 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.7
and 1.75 (3H each, s, H-4’’ and H-5’’), 2.54 (1H, t, J = 4.0 Hz, ≡CH), 2.77 and 3.03 (1H each,

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m, H-3a and 3b), 3.25 (2H, d, J = 8.0 Hz, H-1’’), 3.85 (3H, s, -OCH3), 4.72 (2H, s, -OCH2), 5.28
(1H, t, J = 8.0 Hz. H-2’’), 5.39 (1H, dd, J = 12.0, 4.0 Hz, H-2), 6.45 (1H, s, H-8), 7.05 (2H, d, J
= 8.0 Hz, H-3’ and H-5’), 7.42 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.68 (1H, s, H-5). 13C NMR
(125 MHz, CDCl3): δ 190.97, 164.13, 162.26, 157.82, 133.05, 131.94, 127.76 × 2, 127.08,
124.92, 121.73, 115.16 × 2, 113.95, 98.81, 79.68, 78.33, 75.8, 55.85, 55.79, 44.16, 27.83, 25.9,
17.79. HRMS m/z calcd for C24H25O4 [M + H]+ 377.1747, found 377.1762.
4.1.3. General procedure for the synthesis of aromatic ether analogs (9f-i). To a solution of
bavachinin (0.30 mmol) in dichloromethane, Cu(OAc)2 (0.45 mmol), pyridine (0.60 mmol) and
respective boronic acids (0.90 mmol) were added. The reaction mixture was kept on stirring at
r.t. for 2-3 h till the completion (monitored by TLC analysis). After completion of the reaction,
usual workup and purification was done which afforded compounds 9f-i in 85-90% yield.
4.1.3.1. 7-methoxy-6-(3-methylbut-2-en-1-yl)-2-(4-phenoxyphenyl)chroman-4-one (9f). Yield:
1
85%, gummy mass, [α]D = -18 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.7 and 1.74
(3H each, s, H-4’’ and H-5’’), 2.77 and 3.05 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0 Hz,
H-1’’), 3.85 (3H, s, -OCH3), 5.27 (1H, t, J = 8.0 Hz, H-2’’), 5.44 (1H, dd, J = 12.0, 4.0 Hz, H-2),
6.46 (1H, s, H-8), 7.05 (4H, m, H-3’, H-5’, H-2’’’ and H-6’’’), 7.13 (1H, t, J = 8.0 Hz, H-4’’’),
13
7.35 (2H, m, H-3’’’ and H-5’’’), 7.44 (2H, t, J = 8.0 Hz, H-2’ and H-6’), 7.68 (1H, s, H-5). C
NMR (125 MHz, CDCl3): δ 190.73, 164.15, 162.19, 157.81, 156.79, 133.56, 133.04, 129.86 × 2,
127.87 × 2, 127.12, 125.01, 123.68, 121.75, 119.24 × 2, 118.85 × 2, 113.99, 98.81, 79.69, 55.78,
44.27, 27.82, 25.87, 17.78. HRMS m/z calcd for C27H27O4 [M + H]+, 415.1009 found 415.1029.
4.1.3.2. 2-4-(4-fluorophenoxy)phenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-one (9g).
Yield: 87%, gummy mass, [α]D = -16 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.69 and
1.74 (3H each, s, H-4’’ and H-5’’), 2.78 and 3.01 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0

ACCEPTED MANUSCRIPT
Hz, H-1’’), 3.84 (3H, s, -OCH3), 5.27 (1H, t, J = 8.0 Hz, H-2’’), 5.44 (1H, dd, J = 12.0, 4.0 Hz,
H-2), 6.45 (1H, s, H-8), 7.0 (6H, m, H-3’, 5’, 2’’’, 3’’’, 5’’’ and H-6’’’), 7.42 (2H, t, J = 8.0 Hz,
H-2’ and H-6’), 7.68 (1H, s, H-5). 13C NMR (125 MHz, CDCl3): δ 190.58, 164.14, 162.14,
160.3, 158.17, 152.47, 133.51, 132.98, 127.89 × 2, 127.13, 125.03, 121.77, 120.89, 120.81,
118.26 × 2, 116.53, 116.3, 114.02, 98.8, 79.6, 55.74, 44.24, 27.81, 25.81, 17.74. HRMS m/z
calcd for C27H26FO4 [M + H]+ 433.1810, found 433.1808.
4.1.3.3. 7-methoxy-6-(3-methylbut-2-en-1-yl)-2-(4-(3-nitrophenoxy)phenyl)chroman-4-one (9h).
1
Yield: 88%, gummy mass, [α]D = -22 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.7 and
1.74 (3H each, s, H-4’’ and H-5’’), 2.84 and 3.03 (1H each, m, H-3a and 3b), 3.25 (2H, d, J = 8.0
Hz, H-1’’), 3.87 (3H, s, -OCH3), 5.27 (1H, t, J = 8.0 Hz, H-2’’), 5.48 (1H, dd, J = 12.0, 4.0 Hz,
H-2), 6.48 (1H, s, H-8), ), 7.11 (2H, d, J = 8.0 Hz, H-3’ and 5’), 7.35 (1H, dd, J = 8.0 Hz, 4.0
Hz, H-6’’’), 7.52 (3H, m, H-2’, 6’ and 5’’’), 7.69 (1H, s, H-5), 7.82 (1H, t, J = 4.0 Hz, H-2’’’),
7.96 (1H, m, H-4’’’). 13C NMR (125 MHz, CDCl3): δ 190.54, 164.23, 162.1, 158.13, 155.9,
149.35, 135.4, 133.11, 130.46, 128.28 × 2, 127.13, 125.16, 124.47, 121.68, 119.91 × 2, 118.03,
113.95, 113.11, 98.8, 79.46, 55.82, 44.3, 27.81, 25.86, 17.77. HRMS m/z calcd for C27H26NO6
[M + H]+ 460.1755, found 460.1750.
4.1.3.4. 2-(4-(4-bromophenoxy)phenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-one (9i).
Yield: 89%, gummy mass, [α]D = -18 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.69 and
1.74 (3H each, s, H-4’’ and H-5’’), 2.82 and 3.01 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0
Hz, H-1’’), 3.84 (3H, s, -OCH3), 5.27 (1H, t, J = 8.0 Hz, H-2’’), 5.42 (1H, dd, J = 12.0, 4.0 Hz,
H-2), 6.45 (1H, s, H-8), 6.9 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.03 (2H, d, J = 8.0 Hz, H-2’ and
H-6’), 7.44 (4H, m, H-2’’’, 3’’’, 5’’’ and H-6’’’), 7.68 (1H, s, H-5). 13C NMR (125 MHz,
CDCl3): δ 190.78, 164.24, 162.2, 157.23, 156.1, 134.12, 133.08, 132.82 × 2, 127.98 × 2, 127.15,

ACCEPTED MANUSCRIPT
125.11, 121.71, 120.76 × 2, 119.03 × 2, 116.14, 113.95, 98.8, 79.58, 55.79, 44.24, 27.82, 25.86,
17.78. HRMS m/z calcd for C27H26BrO4 [M + H]+ 493.1009, found 493.1029.
4.1.4. Synthesis of 6-((3,3-dimethyloxiran-2-yl)methyl)-2-(4-hydroxyphenyl)-7-methoxychroman-
4-one) (10). Bavachinin (0.30 mmol) was dissolved in 3 mL DCM at 0oC and to this m-CPBA
(0.45 mmol) in 2 mL DCM was added slowly. The reaction mixture was stirred for 2 h at r.t. as
per earlier report [23]. After completion, the mixture was extracted with ethyl acetate and water.
The organic layer was dried and purified with 5% ethyl acetate/hexane as eluent to afford desired
1
pure product 10 in 78% yield, gummy mass, [α]D = -16 (c = 1.0, CHCl3), H NMR (400 MHz,
CDCl3): δ 1.36 and 1.42 (3H each, s, H-4’’ and H-5’’), 2.81 (3H, m, 2 × H-1’’ and H-3a), 3.09
(2H, m, H-2’’ and H-3b), 3.83 (3H, s, -OCH3), 5.38 (1H, t, J = 8.0 Hz, H-2), 6.46 (1H, s, H-8),
6.78 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.25 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.76 (1H, s, H-
5). 13C NMR (125 MHz, CDCl3): δ 191.76, 164.45, 163.16, 156.96, 134.61, 133.62, 127.92 × 2,
121.1, 115.7 × 2, 113.99, 99.23, 80.05, 63.86, 60.21, 55.93, 43.83, 29.1, 24.76 and 18.88. HRMS
m/z calcd for C21H23O5 [M + H]+ 355.1540, found 355.1571.
4.1.5.
Synthesis
of
(E)-1-(2-hydroxy-4-methoxy-5-(3-methylbut-2-en-1-yl)phenyl)-3-(4-
hydroxyphenyl)prop-2-en-1-one (11). Bavachinin (0.30 mmol) was dissolved in 3.5 mL ethanol
and to this NaH (0.3 mmol) was added and the mixture was kept on stirring at r.t. for half an
hour. After completion of the reaction, usual workup was done and further purified with 7%
1
ethyl acetate/hexane to furnish 11 in 90% yield. H NMR (400 MHz, CDCl3): δ 1.69 and 1.73
(3H each, s, H-4’’ and H-5’’), 3.2 (2H, m, H-3), 3.83 (3H, s, -OCH3), 5.23 (1H, t, J = 8.0 Hz, H-
2), 6.37 (1H, s, H-8), 6.84 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.38 (1H, d, J = 16.0 Hz, H-2’ and
H-6’), 7.34 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.76 (1H, s, H-5). 13C NMR (125 MHz, CDCl3):
δ 191.76, 164.7, 164.09, 159.89, 144.57, 132.85, 130.57 × 2, 129.77, 126.41, 122.19, 121.76,

ACCEPTED MANUSCRIPT
117.05, 115.93 × 2, 113.22, 99.05, 55.54, 27.93, 25.56 and 17.63. HRMS m/z calcd for C21H23O4
[M + H]+ 339.1591, found 339.1594.
4.1.6. Synthesis of 2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-ol (12).
Compound 1 (0.30 mmol) was dissolved in 4 mL methanol and to this NaBH4 (0.45 mmol) was
added slowly at 0oC. The reaction mixture was stirred at r.t. for 1h and after completion of the
reaction, the mixture was concentrated, diluted with water and further extracted with ethyl
acetate. The organic layer was dried and purified with 8% EtOAc/hexane as eluent to provide
desired product 12 in 70% yield, m. p. 141oC, [α]D = +19 (c = 1.0, CHCl3). 1H NMR (400 MHz,
CDCl3): δ 1.71 and 1.73 (3H each, s, H-4’’ and H-5’’), 2.14 and 2.39 (1H each, m, H-3a and 3b),
3.25 (2H, d, J = 8.0 Hz, H-1’’), 3.76 (3H, s, -OCH3), 4.73 (1H, t, J = 4.0 Hz, H-4), 5.15 (1H, t, J
= 8.0 Hz. H-2’’), 5.29 (1H, dd, J = 12.0, 4.0 Hz, H-2), 6.43 (1H, s, H-8), 6.85 (2H, d, J = 8.0 Hz,
13
H-3’ and H-5’), 7.3 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.07 (1H, s, H-5). C NMR (125 MHz,
CDCl3): δ 157.48, 156.95, 153.83, 131.97, 131.16, 127.42 × 2, 127.26, 123.11, 122.36, 117.44,
114.92 × 2, 98.45, 77.11, 64.93, 54.64, 39.78, 27.64, 24.84 and 16.89. HRMS m/z calcd for
C21H24NaO4 [M + H]+ 363.1567, found 363.1569.
4.1.7. Synthesis of 2-(4-hydroxyphenyl)-6-isopentyl-7-methoxy chroman-4-one (12a). To a
solution of bavachinin (0.30 mmol) in methanol, 10% Pd/C was added slowly while stirring at
r.t. The mixture was kept on stirring for 3 h till completion [23, 26]. Then the reaction mixture
was filtered, filtrate was concentrated and purified through silica gel column chromatography
using 20% EtOAc/hexane as eluent which provided 12a in 80 % yield, solid, m. p. 185oC, [α]D =
-21 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 0.93 (6H, d, J = 6.6 Hz, H-4’’ and H-5’’),
1.44 (2H, m, H-2’’), 1.58 (1H, m, H-3’’), 2.54 (2H, m, H-1’’), 2.78 and 3.04 (1H each, m, H-
3a and 3b), 3.83 (3H, s, -OCH3), 5.39 (1H, dd, J = 13.4, 2.8 Hz, H-2), 6.44 (1H, s, H-8), 6.90

ACCEPTED MANUSCRIPT
(2H, d, J = 8.6 Hz, H-3’ and H-5’), 7.26 (1H, s, H-5), 7.35 (2H, d, J = 8.6 Hz, H-2’ and H-6’).
13C NMR (125 MHz, CDCl3): δ 191.5, 164.47, 162.3, 156.25, 131.0, 127.98 × 2, 127.25, 126.36,
115.7 × 2, 113.85, 99.81, 79.83, 55.74, 44.14, 38.85, 27.97, 27.19, 22.58 × 2. HRMS m/z calcd
for C21H25O4 [M + H]+ 341.1751, found 341.1754.
4.1.8. Synthesis of 2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-one
oxime (13). To a solution of bavachinin (0.30 mmol) in ethanol was added pyridine (0.6 mmol)
and NH2OH.HCl (0.45 mmol) dissolved in ethanol slowly at r.t. while stirring. The mixture was
stirred for 1.5h till completion. After the usual work-up, mixture was dried and purified with 7%
EtOAc/hexane as eluent to provide 13 in 85% yield, gummy mass, [α]D = -9.0 (c = 1.0, CHCl3),
1H NMR (400 MHz, CDCl3): δ 1.7 and 1.72 (3H each, s, H-4’’ and H-5’’), 2.68 and 3.45 (1H
each, m, H-3a and 3b), 3.23 (2H, d, J = 8.0 Hz, H-1’’), 3.79 (3H, s, -OCH3), 4.98 (1H, t, J = 8.0
Hz. H-2’’), 5.27 (1H, dd, J = 12.0, 4.0 Hz, H-2), 6.44 (1H, s, H-8), 6.85 (2H, d, J = 8.0 Hz, H-3’
and H-5’), 7.31 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.6 (1H, s, H-5). 13C NMR (125 MHz,
CDCl3): δ 159.73, 156.97, 156.33, 149.85, 131.83, 131.06, 127.6 × 2, 123.99, 123.88, 122.53,
115.17 × 2, 110.38, 99.29, 77.4, 54.98, 30.0, 27.8, 25.14 and 17.05. HRMS m/z calcd for
C21H24NO4 [M + H]+ 354.1700, found 354.1718.
4.1.9. Synthesis of 2-(2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chroman-4-
ylidene)hydrazinecarboxamide (14). To a solution of bavachinin (0.3 mmol) in ethanol was
added pyridine (0.6 mmol) and NH2CONHNH2.HCl (0.45 mmol) dissolved in ethanol slowly at
r.t. while stirring. The mixture was stirred for 1.5 h till completion. After the usual work-up,
mixture was dried and purified with 10% EtOAc/hexane as eluent to provide 14 in 85% yield,
1
white solid, m. p. 151oC, [α]D = +38.0 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.73
(6H, s, H-4’’ and H-5’’), 2.67 and 3.03 (1H each, m, H-3a and 3b), 3.26 (2H, d, J = 8.0 Hz, H-

ACCEPTED MANUSCRIPT
1’’), 3.8 (3H, s, -OCH3), 5.02 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.27 (1H, t, J = 8.0 Hz, H-2’’),
6.45 (1H, s, H-8), 6.87 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.32 (2H, d, J = 8.0 Hz, H-2’ and H-
6’), 7.69 (1H, s, H-5). 13C NMR (125 MHz, CDCl3): δ 161.5, 160.25, 158.65, 158.42, 144.25,
133.5, 131.92, 129.08 × 3, 125.85, 124.1, 116.81 × 2, 113.24, 100.79, 78.7, 56.69, 33.26, 29.53,
26.81 and 18.87. HRMS m/z calcd for C22H26N3O4 [M + H]+ 396.1918, found 396.1901.
4.1.10. Synthesis of (Z)-7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
one oxime (15). To synthesize compound 15, firstly 9a was prepared in gram quantities by
reacting 1 (2.0 g, 5.9 mmol) with CH3I (5.9 mmol) following the same procedure as mentioned
in section 4.1.1. Then, to a solution of compound 9a (1.9 g, 5.39 mmol) in ethanol was added
pyridine (10.79 mmol) and NH2OH. HCl (8.09 mmol) dissolved in ethanol slowly at r.t. while
stirring. The mixture was stirred for 1.5 h till completion. After the usual work-up, mixture was
dried and purified with 8% EtOAc/hexane as eluent to provide 15 in 90% yield, gummy mass,
1
[α]D = 0 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.68 and 1.72 (3H each, s, H-4’’ and
H-5’’), 2.7 and 3.52 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0 Hz, H-1’’), 3.79 and 3.82
(3H each, s, 2 × -OCH3), 5.01 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.28 (1H, t, J = 8.0 Hz. H-2’’),
6.44 (1H, s, H-8), 6.93 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.4 (2H, d, J = 8.0 Hz, H-2’ and H-
6’), 7.56 (1H, s, H-5). 13C NMR (125 MHz, CDCl3): δ 160.08, 159.73, 156.54, 150.2, 132.76,
132.01, 127.73 × 2, 124.32, 123.97, 122.25, 114.1 × 2, 109.81, 90.56, 77.26, 55.47, 55.36, 30.14,
27.91, 25.81 and 17.75. HRMS m/z calcd for C22H26NO4 [M + H]+ 368.1856, found 368.1868.
4.1.11. Synthesis of (Z)-7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-prop-2-yn-1-yl oxime (16). Compound 15 (5.17 mmol) was dissolved in acetone and to this
K2CO3 (5.17 mmol) and propargyl bromide (5.17 mmol) were added. After the usual work-up,
mixture was dried and purified with 8% EtOAc/hexane as eluent to provide 16 in 94% yield,

ACCEPTED MANUSCRIPT
1
colorless solid, m.p. 154oC, [α]D = +5.0 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.73
(6H each, s, H-4’’ and H-5’’), 2.49 (1H, t, J = 4.0 Hz, , ≡CH), 2.7 and 3.43 (1H each, m, H-3a
and 3b), 3.26 (2H, d, J = 8.0 Hz, H-1’’), 3.79 and 3.83 (3H each, s, 2 × -OCH3), 4.77 (2H, d, J =
4.0 Hz, N-O-CH2), 4.9 (1H, dd, J = 8.0 Hz, 4.0 Hz, H-2), 5.28 (1H, t, J = 8.0 Hz, H-2’’), 6.43
(1H, s, H-8), 6.93 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.38 (2H, d, J = 8.0 Hz, H-2’ and H-6’),
7.66 (1H, s, H-5). 13C NMR (125 MHz, CDCl3): δ 160.16, 159.72, 156.7, 150.46, 132.18,
131.91, 127.75 × 2, 124.56, 124.42, 122.71, 114.06 × 2, 109.61, 99.52, 79.95, 77.24, 74.33, 61.6,
55.47, 55.37, 30.94, 28.28, 25.84 and 17.85. HRMS m/z calcd for C25H28NO4 [M + H]+
406.2013, found 406.2037.
4.1.12. General procedure for the synthesis of triazolyl derivatives (17a-k). Compounds (17a-k)
were synthesized by reacting 16 (1 eq) with respective freshly prepared organic azides (1.1 eq) as
per literature [27], in 6 mL water: t-butanol (1:1) mixture followed by the addition of sodium
ascorbate and copper (II) sulphate pentahydrate (10 mol% each). The heterogeneous reaction
mixture was stirred vigorously at room temperature till the completion of reaction. After
completion, the reaction mixture was extracted with ethyl acetate and water. The combined
organic layer was dried, concentrated and purified through column chromatography using 10-
30% EtOAc:hexane mixture as eluent to furnish pure products in excellent yields. The spectral
data of all triazolyl derivatives (17a-k) is given below.
4.1.12.1. Synthesis of (Z)-N-(4-(4-((((7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-
yl)chroman-4-ylidene)amino)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetamide (17a). Yield:
95%, gummy mass, [α]D = +2 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.67 (6H, s, H-
4’’ and H-5’’), 2.13 (3H, s, -NHCOCH3), 2.66 and 3.35 (1H each, m, H-3a and 3b), 3.2 (2H, d, J
= 8.0 Hz, H-1’’), 3.74 and 3.75 (3H each, s, 2 × -OCH3), 4.95 (1H, dd, J = 12.0, 4.0 Hz, H-2),

ACCEPTED MANUSCRIPT
5.23 (1H, t, J = 8.0 Hz. H-2’’), 5.3 (2H, s, -OCH2C-), 6.38 (1H, s, H-8), 6.86 (2H, d, J = 8.0 Hz,
H-3’ and H-5’), 7.31 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.6 and 7.71 (2H each, d, J = 8.0 Hz, H-
2’’’, 3’’’, 5’’’ and 6’’’ ), 7.62 (1H, s, H-5), 8.07 (1H, s, =CH-N-). 13C NMR (125 MHz, CDCl3):
δ 170.31, 160.19, 159.61, 156.62, 150.34, 145.23, 139.39, 132.39, 132.12, 131.85, 127.62 × 2,
124.34, 124.14, 122.53, 121.81, 121.17 × 2, 120.54 × 2, 113.92 × 2, 109.81, 99.47, 77.12, 66.85,
55.27, 55.12, 28.07, 25.54, 23.53, 22.59, 17.52. HRMS m/z calcd for C33H36N5O5 [M + H]+
582.2711, found 582.2699.
4.1.12.2. Synthesis of (Z)-N-(4-(4-((((7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-
yl)chroman-4-ylidene)amino)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)benzamide (17b). Yield:
91%, white solid, m. p. 205oC, [α]D = +5 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.72
(6H, s, H-4’’ and H-5’’), 2.69 and 3.44 (1H each, m, H-3a and 3b), 3.25 (2H, d, J = 8.0 Hz, H-
1’’), 3.78 and 3.8 (3H each, s, 2 × -OCH3), 4.95 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.3 (1H, t, J =
8.0 Hz, H-2’’), 5.38 (2H, s, -OCH2C-), 6.43 (1H, s, H-8), 6.91 (2H, d, J = 8.0 Hz, H-3’ and H-
5’), 7.36 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.5 (2H, t, J = 8.0 Hz, H-3’’’’ and 5’’’’), 7.57 (1H,
t, J = 8.0 Hz, H-4’’’’), 7.66 (1H, s, H-5), 7.72 and 7.83 (2H each, d, J = 8.0 Hz, H-2’’’, 3’’’,
13
5’’’and 6’’’ ), 7.89 (2H, d, J = 8.0 Hz, H-2’’’’ and 6’’’’), 8.0 (1H, s, =CH-N-). C NMR (125
MHz, CDCl3): δ 165.79, 160.14, 159.7, 156.61, 150.06, 145.62, 138.5, 134.46, 133.19, 132.29,
132.24, 131.88, 128.92 × 2, 127.73 × 2, 127.11 × 2, 124.39, 124.26, 122.65, 121.43 × 2, 121.28,
120.99 × 2, 114.04 × 2, 109.78, 99.59, 77.3, 67.37, 55.5, 55.32, 28.23, 25.87, 22.72, 17.86.
HRMS m/z calcd for C38H38N5O5 [M + H]+ 644.2867, found 644.2853.
4.1.12.3. Synthesis of (Z)-(7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-((1-(2-chloropyridin-3-yl)-1H-1,2,3-triazol-4-yl)methyl)oxime (17c). Yield: 90%, gummy
mass, [α]D = +6 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.69 (6H, s, H-4’’ and H-5’’),

ACCEPTED MANUSCRIPT
2.67 and 3.42 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0 Hz, H-1’’), 3.78 and 3.81 (3H
each, s, 2 × -OCH3), 4.96 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.27 (1H, t, J = 8.0 Hz. H-2’’), 5.41
(2H, s, -OCH2C-), 6.42 (1H, s, H-8), 6.91 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.36 (2H, d, J = 8.0
Hz, H-2’ and H-6’), 7.46 (1H, dd, J = 8.0, 4.0 Hz, H-3’’’), 7.64 (1H, s, H-5), 8.04 and 8.54 (1H
each, dd, J = 8.0, 4.0 Hz, H-2’’’and 4’’’), 8.17 (1H, s, =CH-N-). 13C NMR (125 MHz, CDCl3): δ
160.14, 159.7, 156.65, 150.2, 145.29, 144.83, 136.02, 132.24, 132.13, 131.88, 127.7 × 2, 125.07,
124.42 × 2, 124.23, 123.38, 122.64, 114.06 × 2, 109.72, 99.59, 77.24, 67.09, 55.47, 55.34, 30.94,
28.21, 25.8, 17.8. HRMS m/z calcd for C30H31ClN5O4 [M + H]+ 560.2059, found 560.2034.
4.1.12.4. Synthesis of (Z)-(7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-((1-(2-hydroxymethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (17d). Yield: 92%,
1
gummy mass, [α]D = +3 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.7 (6H, s, H-4’’ and
H-5’’), 2.69 and 3.42 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0 Hz, H-1’’), 3.78 and 3.8
(3H each, s, 2 × -OCH3), 4.47 (2H, s, -CH2OH), 4.98 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.27 (1H, t,
J = 8.0 Hz. H-2’’), 5.4 (2H, s, -OCH2C-), 6.43 (1H, s, H-8), 6.91 (2H, d, J = 8.0 Hz, H-3’ and H-
5’), 7.35 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.39 (1H, t, J = 8.0 Hz, H-4’’’), 7.48 (2H, m, H-3’’’
13
and 6’’’), 7.62 (1H, m, H-5’’’), 7.66 (1H, s, H-5), 7.99 (1H, s, =CH-N-). C NMR (125 MHz,
CDCl3): δ 160.17, 159.71, 156.65, 150.15, 145.17, 135.95, 135.6, 132.28, 131.86, 131.55,
130.01, 129.09, 127.73 × 2, 124.61, 124.44, 124.22, 122.65, 114.05 × 2, 109.74, 99.61, 77.23,
67.26, 61.78, 55.48, 55.34, 30.92, 28.22, 25.81, 22.72, 17.82. HRMS m/z calcd for C32H35N4O5
[M + H]+ 555.2602, found 555.2588.
4.1.12.5. Synthesis of (Z)-(7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-((1-(2-hydroxymethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (17e). Yield: 94%,
gummy mass, [α]D = +7 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.7 (6H, s, H-4’’ and

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H-5’’), 2.7 and 3.42 (1H each, m, H-3a and 3b), 3.24 (2H, d, J = 8.0 Hz, H-1’’), 3.77 and 3.8 (3H
each, s, 2 × -OCH3), 4.76 (2H, s, -CH2OH), 4.97 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.28 (1H, t, J =
8.0 Hz. H-2’’), 5.36 (2H, s, -OCH2C-), 6.42 (1H, s, H-8), 6.9 (2H, d, J = 8.0 Hz, H-3’ and H-5’),
7.34 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.38 (1H, t, J = 8.0 Hz, H-5’’’), 7.45 (1H, t, J = 8.0 Hz,
H-4’’’), 7.57 (1H, d, J = 8.0 Hz, H-6’’’), 7.65 (1H, s, H-5), 7.74 (1H, s, H-2’’’), 8.01 (1H, s,
=CH-N-). 13C NMR (125 MHz, CDCl3): δ 160.15, 159.66, 156.61, 150.11, 143.36, 137.1,
132.24, 131.88, 129.78, 127.73 × 2, 126.99, 124.37, 124.3, 122.68, 121.46, 119.49, 118.89,
114.03 × 2, 109.76, 99.58, 77.19, 67.3, 64.2, 55.47, 55.33, 30.88, 28.25, 25.84, 22.72, 17.84.
HRMS m/z calcd for C32H35N4O5 [M + H]+ 555.2602, found 555.2596.
4.1.12.6. Synthesis of (Z)-(7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (17f). Yield: 93%, gummy
mass, [α]D = +4 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.62 (6H, s, H-4’’ and H-5’’),
2.64 and 3.37 (1H each, m, H-3a and 3b), 3.13 (2H, d, J = 8.0 Hz, H-1’’), 3.69 and 3.7 (3H each,
s, 2 × -OCH3), 4.87 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.21 (1H, t, J = 8.0 Hz. H-2’’), 5.3 (2H, s, -
OCH2C-), 6.33 (1H, s, H-8), 6.89 (2H, dd, J = 8.0, 16.0 Hz, H-3’’’ and 5’’’), 6.9 (2H, d, J = 8.0
Hz, H-3’ and H-5’), 7.21 (1H, t, J = 8.0 Hz, H-2’’’), 7.26 (2H, d, J = 8.0 Hz, H-2’ and H-6’),
13
7.57 (1H, s, H-5), 7.98 (1H, s, H-6’’’), 8.04 (1H, s, =CH-N-). C NMR (125 MHz, CDCl3): δ
160.2, 159.66, 158.72, 156.65, 150.43, 145.29, 137.5, 132.26, 131.88, 130.44, 130.42, 127.74 ×
2, 124.4, 124.33, 122.67, 121.17, 116.85, 114.03 × 2, 109.67, 108.98, 99.57, 77.16, 66.82, 55.47,
55.33, 28.25, 25.84, 22.72, 17.84. HRMS m/z calcd for C31H33N4O5 [M + H]+ 541.2384, found
541.2358.
4.1.12.7. Synthesis of (Z)-(7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-((1-(4-chloro-2-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (17g). Yield: 91%,

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gummy mass, [α]D = +6 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.74 (6H, s, H-4’’ and
H-5’’), 2.22 (3H, s, -CH3), 2.72 and 3.47 (1H each, m, H-3a and 3b), 3.26 (2H, d, J = 8.0 Hz, H-
1’’), 3.81 and 3.83 (3H each, s, 2 × -OCH3), 5.03 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.32 (1H, t, J =
8.0 Hz. H-2’’), 5.42 (2H, s, -OCH2C-), 6.45 (1H, s, H-8), 6.94 (2H, dd, J = 8.0, 16.0 Hz, H-3’’’
and 5’’’), 7.31 (2H, d, J = 8.0 Hz, H-3’ and H-5’), 7.39 (3H, m, H-3’’’, 5’’’ and 6’’’), 7.66 (1H,
13
s, H-5), 7.78 (1H, s, =CH-N-). C NMR (125 MHz, CDCl3): δ 160.18, 159.78, 156.61, 150.01,
144.98, 135.59, 135.03, 132.18, 131.93, 131.36, 127.68 × 2, 127.24, 126.99, 124.65, 124.38 × 2,
124.2, 122.67, 114.07 × 2, 109.82, 99.61, 77.22, 67.35, 55.46, 55.33, 30.9, 28.25, 25.84, 17.86,
17.76. HRMS m/z calcd for C32H34ClN4O4 [M + H]+ 573.2263, found 573.2278.
4.1.12.8. Synthesis of (Z)-(7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)chroman-4-
oneO-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (17h). Yield: 92%,
1
gummy mass, [α]D = +12 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.73 (6H, s, H-4’’
and H-5’’), 2.72 and 3.41 (1H each, m, H-3a and 3b), 3.27 (2H, d, J = 8.0 Hz, H-1’’), 3.8, 3.81,
3.9 and 3.92 (3H each, s, 5 × -OCH3), 4.98 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.29 (1H, t, J = 8.0
Hz, H-2’’), 5.4 (2H, s, -OCH2C-), 6.44 (1H, s, H-8), 6.92 (2H, dd, J = 8.0 Hz, H-3’ and 5’), 6.95
(2H, s, H-2’’’ and 6’’’), 7.37 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.66 (1H, s, H-5), 7.68 (1H, s,
=CH-N-). 13C NMR (125 MHz, CDCl3): δ 160.09, 159.72, 156.64, 153.9 × 3, 150.02, 145.45,
132.93, 132.14, 131.9, 127.68 × 2, 124.36, 124.3, 122.71, 121.66, 114.04 × 2, 109.82, 99.61,
98.85, 77.18, 67.4, 61.01, 56.45 × 3, 55.45, 55.33, 30.9, 28.25, 25.77, 17.86. HRMS m/z calcd
for C34H39N4O7 [M + H]+ 615.2813, found 615.2831.
4.1.12.9. Synthesis of (Z)-4-(4-((((7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-
yl)chroman-4-ylidene)amino)oxy)methyl)-1H-1,2,3-triazol-1-yl)benzamide (17i). Yield: 90%,
white solid, m.p. 192oC , [α]D = +8 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ 1.44 (6H, s,

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H-4’’ and H-5’’), 2.42 and 3.13 (1H each, m, H-3a and 3b), 2.98 (2H, d, J = 8.0 Hz, H-1’’), 3.52
and 3.53 (3H each, s, 2 × -OCH3), 4.74 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.0 (1H, t, J = 8.0 Hz. H-
2’’), 5.1 (2H, s, -OCH2C-), 6.16 (1H, s, H-8), 6.64 (2H, dd, J = 8.0 Hz, H-3’ and 5’), 7.09 (2H, s,
H-2’ and 6’), 7.37 (1H, s, H-5), 7.58 (2H, d, J = 8.0 Hz, H-3’’’ and H-5’’’), 7.77 (2H, d, J = 8.0
13
Hz, H-2’’’ and H-6’’’), 7.94 (1H, s, =CH-N-). C NMR (125 MHz, CDCl3): δ 170.64, 161.66,
161.05, 158.05, 151.86, 147.21, 140.58, 135.09, 133.56, 133.28, 130.72 × 2, 129.03 × 2, 125.82,
125.59, 123.98, 122.96, 121.59 × 2, 115.4 × 2, 111.03, 100.96, 78.52, 68.32, 56.75, 56.61, 32.21,
30.9, 26.97, 18.98. HRMS m/z calcd for C32H34N5O5 [M + H]+ 568.2554, found 568.2548.
4.1.12.10. Synthesis of (Z)-4-(4-((((7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-
yl)chroman-4-ylidene)amino)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)(phenyl)methanone (17j).
Yield: 87%, white solid, m. p. 152oC, [α]D = +6 (c = 1.0, CHCl3), 1H NMR (400 MHz, CDCl3): δ
1.72 (6H, s, H-4’’ and H-5’’), 2.71 and 3.42 (1H each, m, H-3a and 3b), 3.26 (2H, d, J = 8.0 Hz,
H-1’’), 3.79 and 3.8 (3H each, s, 2 × -OCH3), 5.0 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.29 (1H, t, J =
8.0 Hz. H-2’’), 5.41 (2H, s, -OCH2C-), 6.43 (1H, s, H-8), 6.91 (2H, dd, J = 8.0 Hz, H-3’ and 5’),
7.36 (2H, d, J = 8.0 Hz, H-2’ and H-6’), 7.52 (2H, t, J = 8.0 Hz, H-3’’’’ and H-5’’’’), 7.62 (1H,
m H-4’’’’), 7.66 (1H, s, H-5), 7.81 (2H, d, J = 8.0 Hz, H-2’’’’ and H-6’’’’), 7.89 (2H, d, J = 8.0
13
Hz, H-3’’’ and H-5’’’), 7.97 (2H, d, J = 8.0 Hz, H-2’’’ and H-6’’’), 8.11 (1H, s, =CH-N-). C
NMR (125 MHz, CDCl3): δ 195.26, 160.19, 159.71, 156.65, 150.2, 146.12, 139.63, 137.5,
137.05, 132.92, 132.28, 131.84, 131.77 × 2, 130.03 × 2, 128.54 × 2, 127.73 × 2, 124.41, 124.24,
122.66, 121.15, 120.02 × 2, 114.05 × 2, 109.71, 99.6, 77.22, 67.3, 55.5, 55.36, 30.92, 28.25,
25.88, 17.87. HRMS m/z calcd for C38H37N4O5 [M + H]+ 629.2758, found 629.2752.
4.1.12.11. Synthesis of (Z)-4-(4-((((7-methoxy-2-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-
yl)chroman-4-ylidene)amino)oxy)methyl)-1H-1,2,3-triazol-1-yl)benzonitrile (17k). Yield: 86%,

ACCEPTED MANUSCRIPT
1
gummy mass, [α]D = +10 (c = 1.0, CHCl3), H NMR (400 MHz, CDCl3): δ 1.72 (6H, s, H-4’’
and H-5’’), 2.71 and 3.42 (1H each, m, H-3a and 3b), 3.25 (2H, d, J = 8.0 Hz, H-1’’), 3.79 and
3.81 (3H each, s, 2 × -OCH3), 4.99 (1H, dd, J = 12.0, 4.0 Hz, H-2), 5.28 (1H, t, J = 8.0 Hz, H-
2’’), 5.41 (2H, s, -OCH2C), 6.43 (1H, s, H-8), 6.92 (2H, dd, J = 8.0 Hz, H-3’ and 5’), 7.36 (2H,
d, J = 8.0 Hz, H-2’ and H-6’), 7.64 (1H, s, H-5), 7.83 and 7.91 (2H each, d, J = 8.0 Hz, H-2’’’,
3’’’, 5’’’ and H-6’’’), 8.06 (1H, s, =CH-N-). 13C NMR (125 MHz, CDCl3): δ 164.09, 163.58,
160.49, 154.1, 150.4, 143.57, 137.72 × 2, 136.04, 135.68, 131.5 × 2, 128.27, 128.06, 126.51,
124.64, 124.49 × 2, 121.51, 117.89 × 2, 116.28, 113.5, 103.45, 81.0, 71.05, 59.31, 59.17, 34.72,
32.06, 29.65, 21.65. HRMS m/z calcd for C32H32N5O4 [M + H]+ 550.2449, found 550.2444.
4.2. Biology
4.2.1. Cell culture and growth conditions
Human cancer cell lines A549 (Lung), PC-3 (Prostate), MCF-7 (Breast) and HCT-116 (Colon)
were obtained from National Cancer Institute (NCI), USA. 5-fluorouracil was used as positive
control and purchased from Sigma-Aldrich (Bangaluru, India). All four cancer cell lines were
grown in tissue culture flasks in complete growth medium (RPMI-1640) supplemented with 10%
fetal bovine serum, 100 µg/mL streptomycin and 100 units/mL penicillin (New Brunswick,
Galaxy 170R, Eppendorf) at 37°C, 5% CO2 and 98% RH.
4.2.2. Cytotoxicity Assay
The SRB assay [32] was executed to assess the cytotoxic potential of the potent inhibitors in
which optimum cell density per well was seeded in 96 well flat bottom plates. 100 µL of cell
suspension of a panel of human cancer cell lines PC-3 (7000), MCF-7 (8000), A549 (7500), and
HCT-116 (7000) was plated. Following 24 h of incubation under culture conditions, the cells

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were exposed to different concentration (1, 5, 10, 30 and 50 µM) of test materials containing
complete growth medium along with 5-fluorouracil as positive control. The plates were kept
under incubation under the same conditions for 48 h at 37°C. Further, cells were fixed with ice
cold TCA for 1 h at 4°C. After 1 h, the plates were washed three times with water and allowed to
air dry. Afterwards, 100 µL of 0.4% SRB dye was added for half an hour at room temperature.
Plates were then washed three times with water followed by 1% v/v acetic acid to remove the
unbound SRB. After drying at room temperature, the bound dye was solubilised by adding 100
µL of 10 mM Tris buffer (pH-10.4) to each well. The plates were retained on the shaker for 5
min in order to dissolve the protein bound dye. OD was taken at 540 nm in a microplate reader
(Thermo Scientific) and IC50 was determined by using GraphPAD Prism Software Version 5.0
(1).
Absorbance of treated cells - Absorbance of Blank
The % of cell viability =
X 100
Absorbance of control cells - Absorbance of Blank
% Growth inhibition = 100 - % of cell viability
4.2.3. Colony formation assay
HCT-116 cells (8x104/mL/well) were seeded and treated with compound 17i at different
concentrations of 3.6, 7.2 and 14.4 µM and incubated for 24 h. Then, the treated cells were
trypsinized, counted and re-seeded at 1000 cells/well in a six well plate. The cells were left in
order to give colonies of >50 cells to assess the clonogenic ability. Thereafter, Cells were fixed
with 1ml of 4% formaldehyde and stained with 0.5% crystal violet. Finally, crystal violet was
aspirated carefully and rinsed with water. Clonogenic survival was expressed as the number of
colony-forming units in treated cultures in comparison to untreated controls [17].
4.2.4. In vitro cell migration assay

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HCT-116 cells were plated at a concentration of 8x105cells/well and allowed to reach a
conflueny upto 70-80%. Then, the cells were serum starved for 24h and monolayer was scraped
in a straight horizontal line with a sterile 200 µL tip. Eventually, cells were treated with different
concentrations (3.6, 7.2 and 14.4 µM) of 17i for 24 h. Wounded areas were gradually
photographed (20x magnification) at 0 and 24 h and the percentage of wound closure was
estimated by the following equation:
% Wound closure = [1-(wound area at 0 h /wound area at 24 h) × 100%]
4.2.5. Phase contrast microscopy
HCT-116 cells were treated with 3.6, 7.2 and 14.4 µM concentration of compound 17i for 24 h.
Cells were simply photographed using phase contrast microscope after treatment.
4.2.6. Mitochondrial membrane potential (MMP)
Cells (1x106/1.5 mL/well) in 12 well plates were treated with 17i at 3.6, 7.2 and 14.4 µM and 5-
FU at 5 µM for 24 h. Rhodamine-123 (10 nM) was added 1 h before experiment termination.
Cells were washed in PBS and immediately analyzed under fluorescence microscopy [18].
4.2.7. Western blot analysis
Cells (HCT-116) were treated with varying concentration of 17i at 3.6, 7.2 and 14.4 µM for 24 h.
After completion of incubation, the cells were trypsinized and washed with cold PBS. The whole
cell lysates were prepared by using RIPA buffer, protease and phosphatase inhibitor cocktail.
The lysates prepared were centrifuged at 12,000 g for 5 min [33]. The total protein concentration
was determined with the Quantipro BCA assay kit (Sigma) in which BSA (bovine serum
albumin) was taken as a standard. An equal amount of protein per lane was resolved by 10%

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SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) and transferred to
PVDF (polyvinylidene-di-fluoride membrane) (Millipore). Non-specific binding was blocked by
using 5% BSA in TBST solution for 1 h at r.t. After blocking, the membrane was probed with
primary PARP (1:1000) and β-actin (1:500) and incubated with the corresponding HRP
conjugated secondary antibodies (CST). Immunoreactive proteins were detected with the ECL
western blotting detection system and exposed to X-ray film.
Acknowledgement
Council of Scientific and Industrial Research (CSIR), New Delhi is well acknowledged for
funding support 12th FYP project BSC-0108). Authors (NG, AQ and AR) are greatly thankful to
CSIR-New Delhi for providing the senior research fellowships. The authors are also thankful to
the staff of instrumentation division of our institute for recording the spectroscopic (NMR and
HRMS) data.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version at
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