Natural product inspired allicin analogs as novel anti-cancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.01.057

A series of novel analogs of Allicin (S-allyl prop-2-ene-1-sulfinothioate) present in garlic has been synthesized in high yield. Synthesized 23 compounds were evaluated against different breast cancer cells (MDA-MB-468 and MCF-7) and non-cancer cells (WI38). Four compounds (3f, 3h, 3m and 3u) showed significant cytotoxicity against cancer cells whereas nontoxic to the normal cells. Based on the LD₅₀ values and selectivity index (SI), compound 3h (S-p-methoxybenzyl (p-methoxyphenyl)methanesulfinothioate) was considered as most promising anticancer agent amongst the above three compounds. Further bio-chemical studies confirmed that compound 3h promotes ROS generation, changes in mitochondrial permeability transition and induced caspase mediated DNA damage and apoptosis.

Full text of DOI:10.1016/j.bioorg.2019.01.057

Accepted Manuscript
Natural product inspired Allicin analogs as novel anti-cancer agents
Ishani Bhaumik, Kunal Pal, Utsab Debnath, Parimal Karmakar, Kuladip Jana,
Anup Kumar Misra
PII:
S0045-2068(18)31180-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.057
YBIOO 2761
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
20 October 2018
11 January 2019
25 January 2019
Please cite this article as: I. Bhaumik, K. Pal, U. Debnath, P. Karmakar, K. Jana, A. Kumar Misra, Natural product
inspired Allicin analogs as novel anti-cancer agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.01.057
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Natural product inspired Allicin analogs as novel anti-cancer
agents
Ishani Bhaumik Kunal Pal, 1, Utsab Debnath , Parimal Karmakar , Kuladip Jana *, Anup
1
,
1
2
1,
,
1
Kumar Misra *
Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII M, Kolkata
7
00054, India
Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, India
Email: akmisra69@gmail.com; kuladip@jcbose.ac.in
*

Contributed equally.
Abstract:
A series of novel analogs of Allicin (S-allyl prop-2-ene-1-sulfinothioate) present in garlic has
been synthesized in high yield. Synthesized 23 compounds were evaluated against different
breast cancer cells (MDA-MB-468 and MCF-7) and non-cancer cells (WI38). Four
compounds (3f, 3h, 3m and 3u) showed significant cytotoxicity against cancer cells whereas
nontoxic to the normal cells. Based on the LD50 values and selectivity index (SI), compound
3
h (S-p-methoxybenzyl (p-methoxyphenyl)methanesulfinothioate) was considered as most
promising anticancer agent amongst the above three compounds. Further bio-chemical
studies confirmed that compound 3h promotes ROS generation, changes in mitochondrial
permeability transition and induced caspase mediated DNA damage and apoptosis.
Key words: Sulfenyl sulfoxide; allicin, garlic; anti-cancer; ROS; caspase 3; apoptosis.
Introduction
In spite of enormous technological and social development, cancer is still one of the most
common diseases of concern and a leading cause of human suffering and death [1,2]. The
alarming rise in incidence of new types of cancer and the public burden represents a crisis for
public health and health systems worldwide [3]. Detailed analyses of pathways and
mechanisms for the spreading of cancer and identification of several antitumor agents have
led to significant developments in the prevention and treatment of cancer [4,5].
Chemotherapy, irradiation and immunotherapy are the gold standard approaches for the
treatment of cancer worldwide in spite of their toxicity. In the second line, use of natural
products from plants and animals and their derivatives have produced remarkable leads for
the treatment of cancer [6-10]. Due to the toxicity of currently used therapeutics for the
treatment of various types of tumors, several natural products and their structural derivatives

have been evaluated for their potential to act as anti-cancer agents [11,12]. A wide range of
natural products e.g. terpenoids [13,14], flavonoids [15], alkaloids [16], polyesters,
polyphenols [17] and other secondary metabolites have shown promising anticancer
properties. Several attempts were made to functionalize the naturally occurring molecules or
preparation of their analogs using modern synthetic strategies for the development of
potential chemotherapeutic agents from the natural products against cancer with higher
efficacy and lower toxicity [18-21]. Hence the current thrust is to develop alternate strategies
of therapeutics, primarily through the chemical modifications of compounds isolated from
natural sources or by conjugating some compounds with an appropriate pharmacophore to
increase efficacy and minimize toxicity.
Garlic (Allium sativum) contains several organosulfur compounds, which protects it from the
attack of bacteria, fungi and animals [22]. Alliin is one of the natural constituents of garlic,
which is a sulfoxide containing amino acid [23]. During the process of chopping or crushing
of fresh garlic, alliin is converted into allicin (allylsulfenyl allylsulfoxide or S-allyl prop-2-
ene-1-sulfinothioate) by the action of alliinase enzyme, which is responsible for the aroma of
garlic [24]. Recently, garlic (Allium sativum) extracts and garlic-derived compounds have
received increasing attention due to their antiproliferative activities against different types of
cancer cells [25,26]. Allicin is a thioester of sulfenic acid, which is relatively unstable and
changes into disulfide derivatives such as diallyl disulfide [27]. It appears as a colorless oil
with distinctive malodorous smell. Although Allicin is a chiral compound it occurs naturally
as a racemate [28]. In the past, Allicin has been shown to possess several medicinal potential
such as anticancer, antioxidant and antimicrobial properties by inducing apoptosis, regulation
of cell cycle progression and signal transduction [29-31]. Taking clue from the biological
activities of allicin, it is pertinent to synthesize novel analogs of allicin using a generalized
reaction condition to evaluate their pharmaceutical potential as anti-cancer agents. Therefore,
efficient synthesis and biological evaluation of a series of allicin analogs are presented herein.
In addition to the sulfide sulfoxide analogs of allicin, a series of selenium substituted analogs
were also prepared with an expectation of getting better biological response. In the recent
past several selenium containing natural product analogs showed highly promising anti-
cancer potential [32-36]. Furthermore, several biochemical and microscopic experiments
have been carried out to establish the anti-cancer potential of the most promising compound
as well as its mode of action.

Results and discussion
Chemistry
Synthesis of alkyl/arylsulfenyl alkyl/arylsulfoxide derivative
The target compounds were prepared using a two step reaction sequence. A series of alkyl
and aryl disulfide derivatives were prepared in high yield following the reaction conditions
reported earlier [37] (Scheme 1). Treatment of a series of alkyl halides with an equimolar
combination of Na
dialkyl disulfides in high yield. In another experiment, treatment of aryl thiols with 65%
nitric acid in CH Cl at room temperature [38] led to the formation of diaryl disulfides in
satisfactory yield (Scheme 1).
2
S·9H
2
O and CS in DMF at room temperature resulted in the formation of
2
2
2
.
CS , Na S 9 H O
2
2
2
RX
a-1m
R : Alkyl, glycosylalkyl; X : Cl, Br, I
RSSR
DMF, RT, 2-5 min
2
a-2m
1
6
5% HNO3
RSSR
n-2s
RSH
CH Cl , RT, 0.5-2 h
2
2
2
1
n-1s
R : Aryl
Scheme 1: Preparation of alkyl and aryldisulfide derivatives [37,38].
Having the disulfides at hand, efforts have been given for the controlled oxidation of
disulfide bond to achieve a single sulfoxide moiety in the molecules. In a set of initial
experiments, dibenzyl disulfide (1g) was treated with a variety of oxidizing agents [such as
H
2
O
2
, KMnO
4
, m-chloroperbenzoic acid (mCPBA), sodium periodate (NaIO ) etc.] in
4
different stoichiometric quantity in commonly used organic solvents (Table 1). After a series
of experimentation it was observed that use of 1.0 equiv. of mCPBA in dichloromethane
could furnish the desired product efficiently without formation of by products in 1 h at ‒ 20
°C. Dichloromethane was found to be superior among several organic solvents used in this
reaction. Following the optimized reaction condition, a series of alkyl/arylsulfenyl
alkyl/arylsulfoxide derivatives (3a-3s) were prepared and characterized by spectroscopic
analysis (Scheme 2, Table 2).

mCPBA (1.0 equiv)
O
o
CH Cl , -20 C
2
2
RSSR
a-2s
R = alkyl, aryl, glycosylalkyl
RSSR
2
3a-3s
Scheme 2: Preparation of alkyl/arylsulfenyl alkyl/arylsulfoxide derivatives.
Table 1: Optimization for the preparation of benzylsulfenyl benzylsulfoxide.
O
Oxidising agent
BnSSBn
BnSSBn
o
Solvent, - 20 C - RT
(1g)
(2g)
Sl.
Oxidizing Equiv. Solvent Time Yield
No. agent
(h)
(%)
1
2
3
4
5
6
7
8
9
1
1
mCPBA
1.0
0.5
0.5
0.2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
CH
CH
CH
CH
2
2
2
2
Cl
Cl
Cl
Cl
2
1
74
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
2
2
4
3
46
45
20
10
25
8
2
2
Toluene 3
Toluene 7
THF
3
CH
CH
CH
CH
3
2
2
2
OH 4
-
H
2
O
2
Cl
Cl
Cl
2
4
4
4
-
0
1
KMnO
NaIO
4
2
-
4
2
-
After achieving the sulfenylsulfoxide derivatives, attention was given towards the preparation
of their selenium analogs. For this purpose, alkyl diselenides were prepared from the alkyl
halides following a recently developed aqueous medium reaction condition [39]. Treatment
of alkyl halides with potassium selenocyanate (KSeCN) in water followed by alkaline
hydrolysis resulted in the formation of dialkyl diselenides in satisfactory yield. Following
optimized reaction condition in the case of disulfides (Table 1), diselenide derivatives were
treated with equimolar quantity of mCPBA in CH
temperature. Surprisingly no required oxidized product was obtained and the diselenide
remained unaffected. After screening a series of other oxidants [e.g. H , KMnO and
sodium periodate (NaIO )] satisfactory yield of the selenenyl selenoxide derivative was
obtained in satisfactory yield by the treatment with NaIO (1.0 equiv.) in a phase transfer
2
Cl at low temperature to ambient
2
2
O
2
4
4
4

reaction condition using CH
2
Cl and water (5:1) in the presence of tetrabutylammonium
2
bromide (TBAB) at room temperature [40]. Following the optimized reaction condition a
series of selenenyl selenoxide derivatives were prepared in good yield and characterized
using spectroscopic analysis (Scheme 3, Table 3).
(
i) KSeCN, TBAB
H O, 65 C
2
NaIO , TBAB
4
o
CH Cl -H O
O
2
2
2
(
ii) K PO , H O
RT
3
4
2
RX
t-1w
R: Alkyl, aryl; X: Cl, Br
RSeSeR
t-2w
RSeSeR
1
2
3t-3w
Scheme 3: Preparation of alkyl/aryl diselenide derivatives and their selective oxidation.
Table 2: Preparation of alkyl/arylsulfenyl alkyl/arylsulfoxide derivatives.
Sl. RSSR (2)  RS(O)SR (3)
Time (h)
Yield (%)
Ref
No. R:
3
7,38
1
1.0
1.0
72
70
(
3a)
4
1
2
(
3b)
4
4
2
3
3
4
1.0
1.0
74
69
(
3c)
(
3d)
4
4
5
1.0
72
1
3
(3e)
(3f)
6
7
1.0
1.0
68
74
-
AcO
MeO
4
5
(
3g)
8
9
1.0
2.0
73
66
-
(
3h)
-
-
(
3i)
1
0
1.5
68
O
4
OMe
(
3j)

11
12
13
AcO OAc
1.5
1.5
1.5
65
64
68
-
-
-
O
O
AcO
2
OAc
OAc
(3k)
O
AcO
O
AcO
2
OAc
O
(3l)
2
O
AcO
AcO
OAc
(3m)
4
6
7
1
1
1
1
4
5
6
7
1.0
1.0
1.0
1.0
72
70
78
75
(
3n)
4
Me
(
3o)
3p)
-
O N
2
(
-
(
3q)
4
8
1
1
8
9
1.5
1.5
73
71
N
(3r)
N
-
N
N
N
(
3s)
Table 3: Preparation of alkyl/arylselenenyl alkyl/arylselenoxide derivatives.
Sl. RSeSeR (2)  RSe(O)SeR (3)
Time Yield Ref
No. R:
(h)
(%)
1
1.0
74
-
-
-
(
3t)
2
3
1.0
1.0
74
73
MeO
(
3u)
(
3v)
4
9
4
1.0
74
(
3w)

Biology
Selective cytotoxicity of synthesized compounds towards the cancer cells
The synthesized allicin analogs (3a-w) along with etoposide were assessed for their ability to
block proliferation of MDA-MB-468 [human triple negative (ER-, PR- and HER2-) breast
cancer cells], MCF-7 [human (ER+, PR+ and HER2-) breast cancer cells] and WI-38 cells
(
lung fibroblast cells). The above mentioned breast cancer cells are extensively used for the
screening to potential anti-breast cancer agents. For this experiment, cells were treated with
varied concentrations of the compounds (0-50 µM) and cell proliferation was measured by
MTT assay [50, 51]. The anti-proliferative activities of the tested compounds in terms of
LD50 were presented in Table 4. From the MTT assay it was observed that four compounds
(
3f, 3h, 3m and 3u) showed significantly higher efficacy in inhibiting the proliferation of
MDA-MB-468 cells with LD50 values of 18.87 ±1.89 µM, 11.34±2.11 µM, 22.38 ± 4.22 µM
and 14.11±4.13 µM respectively. Similar efficacy of 3f, 3h, 3m and 3u was observed in
MCF-7 cells with LD50 value of 22.36±2.11 µM, 13.01±2.98 µM, 25.92±3.11 M and
2
6.28±5.87 µM and for MCF-7 cells respectively. Subsequently, when the selectivity index
(
SI) compounds were calculated by comparing the cytotoxic LD50 value of the compounds in
normal cell (WI-38) versus cancer cells (MDA-MB-468 and MCF-7) and it was observed that
compound 3h possessed highest SI (SI = 4.59±0.71 and 4.00±0.54) as compared to
compound 3m (SI = 3.56±0.69 and 3.08±0.24), compound 3f (SI = 2.90±0.14 and 2.45±0.08)
and compound 3u (SI = 3.68±1.12 and 1.97±0.23). Etoposide, a prevalent chemotherapeutic
drug for breast cancer therapy has been considered as positive control [52]. Based on the
preliminary data, compound 3h was considered as a potent anti-cancer agent on the basis of
its highest selectivity index (Figure 1).
Table 4: The LD50 (M) values of the compounds 3a-w for the inhibition of proliferation of
cancer cell lines.
Sl. Compound
No.
MDA-MB-
68
MCF-7
WI38
Selectivity
index (SI)
in MDA-
MB-468
Selectivity
index (SI)
in MCF-7
4
1
2
3
4
5
3a
3b
3c
3d
3e
26.43±2.65
52.13±3.98
39.85±3.65 56.77 ±5.77 2.14±0.004 1.42±0.017
33.21±4.49 64.32 ±7.65 1.23±0.053 1.93±0.02
66.78 ±2.98 64.58±3.76 87.66 ±3.19 1.31±0.013 1.35±0.02
29.78 ±3.11 40.68±4.98 23.89 ±5.89 0.80±0.125 0.58±0.08
53.12 ±3.87 38.92±5.46 67.34 ±6.19 1.27±0.028 1.73±0.03

6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3f
18.87 ±1.89 22.36±2.11 54.89± 3.12 2.90 ± 0.14 2.45±0.08
3g
3h
3i
54.32 ±2.59 47.89±1.34 71.34±2.19 1.31±0.021 1.48±0.013
11.34±2.11
39.32±1.87
31.56±2.91
13.01±2.98 52.12±3.17 4.59 ± 0.71 4.00±0.54
54.68±5.66 62.11 ±8.76 1.57±0.15
37.65±4.89 89.77 ±6.77 2.84±0.06
1.13±0.04
2.38±0.09
0.91±0.08
0.79±0.05
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
3j
3k
3l
32 .651.80 44.41±1.90 40 .61±2.10 1.24±0.01
33.38 ±3.22 56.77±2.11 44.87 ±4.65 1.34±0.01
3m
3n
3o
3p
3q
3r
3s
22.38 ± 4.22 25.92±3.11 79 .88±2.55 3.56 ± 0.69 3.08±0.24
36.78 ±2.89 51.98±2.19 64.81±8.54 1.76±0.02
24.87±4.32 32.11±2.32 57.66 ±3.18 2.31±3.18
1.25±0.10
1.79±0.02
1.32±0.01
1.47±0.04
1.24±0.08
0.91±0.08
1.31±0.00
1.97±0.23
1.29±0.01
0.89±0.03
1.14±0.04
28.87 ±2.13 46.43±3.56 61.34±4.97 2.12±0.01
61.23 ±1.95 59.88±6.75 88.11 ±7.28 1.43±0.07
32.11 ±2.18 49.76±3.66 62.11 ±8.21 1.93±0.13
31.20 ±2.11 58.46±3.18 53.21 ±7.65 1.70±0.13
32.18 ± 3.42 49.88±4.56 65.43 ±5.89 2.03±0.04
14.11 ±4.13 26.28±5.87 51.98 ±3.98 3.68± 1.12
20.11 ±1.32 33.68±2.87 43.41±4.13 2.16±0.05
29.43 ±2.08 55.42±5.98 49.77±6.89 1.69±0.12
3t
3u
3v
3w
ETOPOSIDE 27.88 ±4.32 39.63±3.21 45.31 ±2.11 1.63±0.20

Figure 1: Assessment of dose dependent cytotoxicity of compound 3h on triple negative
breast cancer cell lines, MDA-MB-468, MCF-7 and WI38 (non-cancer cells) for 24 h (panel
A); 48 h (panel B) and 72 h (panel C). (D) LD50 values of compound 3h on triple negative
breast cancer cell lines, MDA-MB-468, MCF-7 and WI38 (non-cancer cells). The data is the
average of three experiments ± SD. ∗ = represents P value < 0.05, ∗∗ = represents P value <
0
.01.
In order to study the mechanism of action of the cytotoxicity against the cancer cells of the
most promising compound 3h based on the selectivity index (SI = 4.59±0.71 in MDA-MB-
4
68 and 4.00±0.54 in MCF-7), the induction of apoptosis was measured using Annexin V-
FITC/PI staining [53]. After treatment of MDA-MB-468 and MCF-7 cells with different
concentrations of compound 3h (0, 5, 10 and 15 µM) for 24 h, the flow cytometry analysis
showed significant induction of apoptosis in a concentration dependent manner in the cancer
cells in comparison with the non-cancer cells (WI38). Quite evidently, compound 3h was
able to induce more than 60% apoptosis in MDA-MB-468 and 58% in MCF-7 cells at 15 µM
dose as shown in Figure 2. From the AnnexinV-FITC/PI staining on the cancer cells (MDA-
MB-468 and MCF-7) and non-cancer cell lines (WI38), it was observed that there is no

substantial apoptosis in case of the non-cancer cells treated with compound 3h while in case
of the cancer cells the percentage of apoptotic cell is quite high. Therefore, it was decided to
identify the mechanism of the induction of apoptosis in cancer cells by compound 3h using a
series of biochemical and microscopic analysis.
Figure 2: The dot plot of Annexin V-FITC/PI for the evaluation of apoptosis in MDA-MB
4
68 and MCF-7 and WI38 (non-cancer cells) treated with the compound 3h. 1% H
2
2
O has
been used as positive control. The data is the average of three experiments ± SD. ∗ =
represents P value < 0.05, ∗∗ = represents P value < 0.01.

Treatment of compound 3h promotes ROS generation, mitochondrial permeability
transition (MPT) in cancer cells.
Generation of reactive species (ROS) [54] is a critical phenomenon linked with several
antiproliferative processes. Therefore, ROS production in response to the treatment with
compound 3h was measured by 2,7-dichlorofluorescin diacetate (DCFDA) staining method
[
55]. The cancer cells were treated with varying doses (0, 5, 10 and 15 µM) of compound 3h
for 12 h followed by 30 min DCFDA (100 µM) staining and carried out microscopic analysis.
The photomicrographs demonstrated a concentration dependent increase in the intensity of
the green color in response to compound 3h, which suggested significant generation of ROS
was induced by compound 3h in MDA-MB-468 and MCF-7 cells as shown in Figure 3.
Figure 3: Fluorescence microscopic image of cells treated with compound 3h and
spectrophotometric fluorescence intensity of the dose-dependent generation of cellular ROS
using DCFDA assay. 1% H
2
2
O has been used as positive control. The data is the average of
three experiments ± SD. ∗ = represents P value < 0.05, ∗∗ = represents P value < 0.01.

Since ROS plays critical role in changing the mitochondrial permeability transition (MPT),
the effect of compound 3h on mitochondrial damage has been investigated by JC-1 staining
which showed a drastic alteration of the redox status of the cellular mitochondria in response
to compound 3h in a concentration and time dependent manner (Figure 4). The shift of
fluorescence from red to green or a decrease in the red/green ratio indicated the increase in
the mitochondrial permeability in response to compound 3h. Altogether these results imply
that compound 3h mediated generation of ROS may have decisive role in the mitochondrial
permeability transition (MPT).

Figure 4: Dose dependent mitochondrial membrane potential by JC1 staining of (A) MDA-
MB 468 cells and (B) MCF-7 cells treated with compound 3h. 1% H
2
2
O has been used as
positive control.
DNA fragmentation in cancer cells mediated by compound 3h
Earlier reports demonstrated that ROS generation and change in MPT are directly linked with
DNA damage and apoptosis, which is an imperative process that controls cellular growth
[
56]. The association of DNA fragmentation in apoptosis mediated by compound 3h has also
been studied. Firstly, the DAPI staining was done to study apoptotic nuclear morphology of
MDA-MB 468 and MCF-7 cells after treatment with compound 3h at different doses (0, 5, 10
and 15 µM) for 12 h. The untreated cells did not show any shrinking of nucleus or

polynuclear fragmentation whereas the polynuclear fragmentation and shrinking of nucleus
was clearly observed in the treated cells (Figure 5).
Figure 5: The treatment with compound 3h induces apoptosis in MDA-MB 468 and MCF-7
cells as is depicted in cellular morphology study by DAPI staining. The arrows indicate the
polynuclear fragmentation and shrinking of nucleus. 1% H
control. The data is the average of three experiments ± SD. ∗ = represents P value < 0.05, ∗∗
represents P value < 0.01.
2
O
2
has been used as positive
=
The quantitative analysis also confirmed around 70% and 58% apoptotic cells in MDA-MB-
68 and MCF-7 cells at 15 µM respectively which were highly significant compared to the
4
corresponding control cells (5% and 6% respectively) as mention in Figure 5. Since
compound 3h mediates the generation of ROS and induces DNA damage in the cancer cells,
Comet Assay [57] was performed using MDA-MB-468 and MCF-7 cells to assess DNA
damage in response to compound 3h. It was observed that the fluorescence is confined
mostly to the nucleus because of the undamaged DNA in control cells whereas, negatively
charged DNA fragments were released from the nucleus and migrated towards the anode

leading to a prolonged tail formation in compound 3h treated cells due to DNA damage. It
was further observed that upon treatment with compound 3h at concentration (at 15 µM) for
2
4 h, the tail lengths were 76±1.11 nm and 81±1.54 nm respectively. The increase of tail
length is an indication of DNA damage in cells resulting from the treatment of untreated cells
which had no or negligible tail length (Figure 6).
Figure 6: Comet assay of MDA-MB 468 and MCF-7 cells following treatment with
compound 3h at the doses 5 µM, 10 µM and 15 µM. 1% H
control. The data is the average of three experiments ± SD. ∗ = represents P value < 0.05, ∗∗
represents P value < 0.01.
2
O
2
has been used as positive
=
In addition to the earlier mentioned microscopic analysis, the western blot analyses [58] of
different apoptotic proteins in MDA-MB-468 and MCF-7 cells were carried out after
treatment with compound 3h. The increase in the expression of pro-apoptotic protein Bax
was observed whereas the expression of anti-apoptotic protein Bcl-2 was decreased
significantly both in MDA-MB-468 as well as in MCF-7 cells treated with compound 3h at
1
5 µM dose. Moreover, treatment with compound 3h showed cleavage mediated activation of
caspase 3, which suggested caspase mediated apoptosis. Therefore, altogether these results

suggest that change in Bax/Bcl-2 ratio as well as caspase 3 plays a critical role in compound
h mediated apoptosis of cancer cells (Figure 7).
3
Figure 7: Western blot analysis of apoptotic proteins like cleaved Caspase-3, Bax and Bcl2
in (a) MDA-MB-468 and (b) MCF-7 suggesting the induction of apoptotic pathway upon
treatment of the cells with compound 3h. The data is the average of three experiments ± SD.
∗
= represents P value < 0.05, ∗∗ = represents P value < 0.01.
In silico studies on the binding interaction of selected compounds
with caspase-3 enzyme
In present study, molecular docking was performed to explain the structure activity
relationship (SAR) through the correlation in between the biological activity of selected
active compounds (compound 3f, 3h and 3u) and their binding affinities towards the active
domain of caspase-3 enzyme. It is clear that hydrogen bond interaction with the binding
pocket of caspase-3 enzyme play an essential role for their caspase-3 inhibitory activity
(
Figure 8). However, during docking study Gibbs free energy (ΔG) indicates that π-π
stacking and CH-π hydrophobic interactions with surrounding amino acids (i.e. Tyr 204, Trp
06, Arg 207 and Phe 256) of the binding site are also crucial to increase the biological
2
activity (Table 5, Figure8). The 3D docked conformations of compound 3h and compound 3u
showed the importance of the presence of p-methoxybenzyl group at two side arms of the
core moiety respectively. This group is used to provide common π-π stacking hydrophobic as

well as H-bond interactions with Phe 256 and Ser 251 of caspase-3 enzyme (compound 3h,
ΔG = 8.65 kcal/mol,LD50: 11.34±2.11; compound 3u, ΔG = 8.19 kcal/mol, LD50
4.11±4.13). Furthermore, additional π-π stacking interaction of compound 3h with Arg 207
:
1
induces its better potential activity against the enzyme. Absence of aromatic groups in
compound 3f clearly specifies its lower binding affinity towards the enzyme which makes a
direct correlation with their anti-cancer activity (ΔG = 7.57kcal/mol, LD50: 18.87±1.89). In
addition, Figure 8 also specified the individual superimposed structures of selected
compounds 3f, 3h and 3u along with the co-crystal ligand of PDB ID: 1GFW. This study
may provide information regarding the accurate binding pose required for better inhibitory
activity against caspase-3 enzyme. In this case, compound 3f superimposed only with one
part (indoline-2,3-dione group) of the co-crystal ligand whereas, compound 3u superimposed
with the another part (ethoxybenzene group) of the co-crystal ligand. Therefore, compound 3f
and 3u showed reduced antagonistic activity due to lacking for hydrophobic interactions with
two important amino acids i.e. Thr 62 and Ser 65. However, compound 3h covered up the
whole co-crystal ligand moiety after superimposition and showed its binding affinity towards
important amino acids of the binding pocket (Table 5). These superimposed structures give
significant information to compare their anti-cancer activity.
Table 5: Binding interactions of selected compounds with caspase-3 enzyme.
Binding
Sl.
No. no.
Compound
H-bond
interaction sites
affinity
Hydrophobic interaction sites
(
Kcal/mol)
Ser 120, Gly 122, Cys 163, Gly
165, Thr 166, Trp 206, Tyr 204,
Ser 205
Arg 64, His 121,
Gln 161, Arg 207
1
3f
7.57
Thr 62, Ser 65, His 121, Cys 163,
Tyr 204, Trp 206, Ser 209, Asp
2
3
3h
3u
8.65
8.19
Ser 251
2
53, Phe 256
Tyr 204, Ser 205, Trp 206, Arg
07, Asn 208, Asp 253, Phe 256
Asn 208, Ser 251
2

Figure 8: Molecular docking study of three selected compounds (3f, 3h and 3u) with
caspase-3 enzyme co-crystal (PDB ID: 1GFW). Figure (A), (B) and (C) shows docked
conformations of compound 3f (pink), 3h (white) and 3u (green) into the binding pocket of
caspase-3. Red dotted lines (---) indicate the H-bond interactions. Figure (D), (E) and (F)
pointed out the 2D diagram of compounds 3f, 3h and 3u respectively. Additionally, the
superimposed structures of co-crystal ligand (cyan) with all three selected individual
molecules are represented by (G), (H) and (I).
Conclusion
In summary, a series of alkyl/arylsulfenyl sulfoxide derivatives (allicin analogs) were
prepared using a generalized reaction condition in satisfactory yield. In addition, some
selenium containing analogs have also been prepared in good yield. The synthesized
compounds (3a-w) were evaluated against cancer cells (MDA-MB-468 and MCF-7 cells) and
non-cancer cells (WI38). Four compounds (3f, 3h, 3m and 3u) were found promising in
terms of the cytotoxic activities against cancer cells and less cytotoxicity against non-cancer
cells (WI38). Among four active compounds, compound 3h has been considered as the best

based on the selectivity index. A number of microscopic experiments and fluorescence
studies confirmed that compound 3h exhibits anti-cancer activities against several cancer
cells following apoptotic pathway. A docking study has also been carried out to establish the
structure-activity relationship of the active compounds.
Experimental
General methods
All reactions were monitored by thin layer chromatography over silica gel coated TLC plates.
1
13
Silica gel 230-400 mesh was used for column chromatography. H and C NMR spectra
were recorded on a Bruker Avance 500 MHz spectrometer using CDCl as solvent and TMS
3
as the internal reference unless stated otherwise. Chemical shift values are expressed in 
ppm. ESI-MS were recorded on a Micromass mass spectrometer. Elementary analysis was
carried out on a Carlo Erba analyzer. Biological experiments were carried out in a Shimadzu
UV-2401PC spectrophotometer. Commercially available grades of organic solvents of
adequate purity were used in many reactions.
Breast cancer cell lines- MDA-MB-468, MCF-7 (human breast cancer cells), and WI38
(
human lung fibroblast) cells were obtained from the central cell repository of National
Center for Cell Science (NCCS), Pune, India and cultured as suggested by the supplier. All
the above cell lines were cultured either in RPMI 1640 or DMEM, containing 10% FBS,
1
1
mM sodium pyruvate, 2mM L-glutamine, non–essential amino acids, 100 units/L penicillin,
00mg/L streptomycin and 50mg/L gentamycin sulfate at 37ºC with 5% CO
2
.
The cell culture media along with ingredients were purchased from HiMedia (India). FBS
was procured from Gibco (USA). DMSO was purchased from SRL (India). An annexin V-
FITC apoptosis assay kit was bought from BD Bioscience (India). A Caspase 3 Assay Kit
(
ab39401) and Human Apoptosis Array Kit were purchased from AbCam (UK) and R&D
Systems (USA), respectively.
General experimental condition for the preparation of alkyl/arylsulfenyl sulfoxide
derivatives (allicin analogs): To a solution of disulfide derivatives (2a-s) (1.0 mmol) in
CH
and the reaction mixture was stirred at the same temperature for appropriate time (Table 2).
The reaction mixture was poured into water and extracted with CH Cl (2 × 25 mL) and
organic layer was washed with satd. aq. NaHCO3. The combined organic layers were dried
2
Cl
2
(3 mL) was added m-chloroperbenzoic acid (1.0 mmol) in CH
2
Cl
2
(2 mL) at ‒ 20 °C
2
2

over Na
2
4
SO , filtered and concentrated under reduced pressure. The crude product was
purified over SiO
2
using hexane-EtOAc (1:2) as eluant to give the pure products (Table 2).
General experimental condition for the preparation of selenenyl selenoxide derivatives:
To the solution of diselenide derivative (1.0 mmol) in CH Cl (5 mL) were added NaIO (1.0
2
2
4
mmol) and TBAB (0.2 mmol) in water (1 mL) at room temperature and the reaction mixture
was stirred at the same temperature for appropriate time (Table 3). The reaction mixture was
poured into water and extracted with CH
satd. aq. NaHCO3. The combined organic layers were dried over Na
concentrated under reduced pressure. The crude product was purified over SiO
2
Cl
2
(2 × 25 mL) and organic layer was washed with
SO , filtered and
using
2
4
2
hexane-EtOAc (1:2) as eluant to give the pure selenenyl selenoxide derivative (Table 3).
Spectral data of synthesized compounds
1
S-Propyl propane-1-sulfinothioate (3a): Colorless oil; H NMR (500 MHz, CDCl
3
): δ 3.19-
3
.02 (m, 4 H, 2 SCH
2
), 1.96-1.65 (m, 4 H, 2 CH
2
), 1.10, 1.05 (2 t, J = 7.5 Hz each, 2 x 3 H, 2
1
3
CH
3
); C NMR (125 MHz, CDCl ): δ 57.9 (1 C), 41.1 (1 C), 24.4 (1 C), 17.3 (1 C), 13.2 (2
3
+
C); m/z: 167.0 [M+1] ; Anal. Calcd. for C
3.18; H, 8.60.
6
H14OS
2
(166.30): C, 43.33; H, 8.49; found: C,
4
1
S-Isopropyl propane-2-sulfinothioate (3b): Colorless oil; H NMR (500 MHz, CDCl
3
): δ
1
3
2
.91-2.89 (m, 2 H), 1.05-1.02 (2 t, J = 5.5 Hz each, 12 H, 4 CH
3
); C NMR (125 MHz,
+
CDCl
3
): δ 28.2 (2 CH), 21.8 (4 CH
3
); m/z: 167.0 [M+1] ; Anal. Calcd. for C
6
H14OS
2
(
166.30): C, 43.33; H, 8.49; found: C, 43.17; H, 8.60.
1
S-Butyl butane-1-sulfinothioate (3c): Colorless oil; H NMR (500 MHz, CDCl
3
): δ 3.17-
3
.08 (m, 4 H), 1.81-1.71 (m, 4 H), 1.48-1.37 (m, 4 H), 0.94-0.86 (2 t, J = 5.5 Hz each, 6 H, 2
1
3
CH
3
); C NMR (125 MHz, CDCl ): δ 55.9 (1 C), 32.8, 32.5 (2 C), 25.4 (1 C), 21.7, 21.5 (2
3
+
C), 10.3 (2 C); m/z: 195.0 [M+1] ; Anal. Calcd. for C
8
H18OS
2
(194.35): C, 49.44; H, 9.33;
found: C, 49.26; H, 9.50.
1
S-3-Methylbut-2-enyl 3-methylbut-2-ene-1-sulfinothioate (3d): Colorless oil; H NMR
(
500 MHz, CDCl
3
): δ 5.01-4.88 (m, 4 H), 3.58-3.48 (m, 2 H), 3.31-3.21 (m, 2 H), 1.78 (2 s, 6
1
3
H, 2 CH
3
); C NMR (125 MHz, CDCl ): δ 115.7, 115.3 (2 C=C, 4 C), 46.4 (2 CH ), 21.0,
3
2
+
2
0.9 (2 CH
3
); m/z: 219.0 [M+1] ; Anal. Calcd. for C10
H18OS
2
(218.37): C, 55.00; H, 8.31;
found: C, 54.83; H, 8.50.
1
S-Tetradecyl tetradecane-1-sulfinothioate (3e): White solid; m.p. 54–55 °C (EtOH); H
NMR (500 MHz, CDCl ): δ 3.19-3.06 (m, 2 H), 1.86-1.59 (m, 2 H), 1.48-1.20 (m, 48 H),
3

1
3
0
.88 (t, J = 5.5 Hz each, 6 H, 2 CH
3
); C NMR (125 MHz, CDCl ): δ 56.3 (1 C), 32.9-28.6
3
+
(
(
24 C), 23.5 (1 C), 22.7 (1 C), 14.1 (1 C); m/z: 475.3 [M+1] ; Anal. Calcd. for C28
H58OS
2
474.88): C, 70.82; H, 12.31; found: C, 70.67; H, 12.48.
1
S-2-Acetoxyethyl 2-acetoxyethanesulfinothioate (3f): Colorless oil; H NMR (500 MHz,
1
3
CDCl
3
): δ 4.61-4.32 (m, 4 H), 3.48-3.34 (m, 4 H), 2.09 (s, 3 H); C NMR (125 MHz,
+
CDCl
3
): δ 63.2 (1 C), 57.5 (1 C), 54.9 (1 C), 33.7 (1 C), 20.7 (2 C); m/z: 255.0 [M+1] ; Anal.
Calcd. for C
8
H
14
5
O S
2
(254.32): C, 37.78; H, 5.55; found: C, 37.61; H, 5.70.
1
S-Benzyl phenylmethanesulfinothioate (3g): Colorless oil; H NMR (500 MHz, CDCl
3
): δ
1
3
7
.38-7.25 (m, 10 H, Ar-H), 4.34-4.24 (2 ABq, J = 13.0 Hz each, 4 H, 2 PhCH
2
); C NMR
+
(
125 MHz, CDCl
3
): δ 136.5-127.6 (Ar-C), 62.1 (1 C), 36.0 (1 C); m/z: 263.0 [M+1] ; Anal.
Calcd. for C14
H
14OS (262.39): C, 64.08; H, 5.38; found: C, 63.95; H, 5.50.
2
1
S-p-Methoxybenzyl (p-methoxyphenyl)methanesulfinothioate (3h): Colorless oil; H
NMR (500 MHz, CDCl
3
): δ 7.27-7.19 (m, 4 H, Ar-H), 6.84 (d, J = 10.5 Hz, 2 H, Ar-H), 6.81
d, J = 8.5 Hz, 2 H, Ar-H), 4.29-4.19 (2 dd, J = 7.5, 4.5 Hz each, 4 H, 2 CH ), 3.82, 3.78 (2 s,
): δ 132.0-114.2 (Ar-C), 61.5 [S(O)CH ], 55.2 (2
(
2
1
3
6
H, 2 OCH
3
); C NMR (125 MHz, CDCl
3
2
+
OCH
3
), 35.7 (SCH
2
); m/z: 323.0 [M+1] ; Anal. Calcd. for C16
H
18
O
3
S
2
(322.44): C, 59.60; H,
5
.63; found: C, 59.44; H, 5.78.
1
S-(Naphthalen-2-yl)methyl (naphthalen-2-yl)methanesulfinothioate (3i): Colorless oil; H
NMR (500 MHz, CDCl ): δ 7.85-7.72 (m, 7 H, Ar-H), 7.53-7.41 (m, 7 H, Ar-H), 4.45-4.26
3
1
3
(
m, 4 H); C NMR (125 MHz, CDCl
3
): δ 134.5-126.1 (Ar-C), 62.7 (1 C), 45.5 (1 C); m/z:
(362.50): C, 72.89; H, 5.00; found: C, 72.72; H,
+
3
5
63.0 [M+1] ; Anal. Calcd. for C22
.16.
H18OS
2
S-4-(p-Methoxyphenoxy)butyl
4-(p-methoxyphenoxy)butane-1-sulfinothioate
): δ 6.80-6.78 (m, 8 H, Ar-H), 4.44 (t, J = 6.0 Hz, 4
), 3.78 (s, 6 H, 2 OCH ), 2.01-1.99 (m, 8 H, 4 CH ),
(3j):
1
Colorless oil; H NMR (500 MHz, CDCl
3
H, 2 CH
2
), 3.97 (t, J = 6.0 Hz, 4 H, 2 CH
2
3
2
1
3
C NMR (125 MHz, CDCl
3
): δ 133.7-114.6 (Ar-C), 76.9 (2C), 67.7 (2 C), 55.6 (2 C), 25.8
+
(
2 C), 24.6 (2 C); m/z: 453.1 [M+1] ; Anal. Calcd. for C23
H
32
O
5
2
S (452.62): C, 61.03; H,
7
.13; found: C, 60.87; H, 7.26.
S-2-(2,3,4,6-Tetra-O-acetyl--D-galactopyranosyl)ethyl
2-(2,3,4,6-tetra-O-acetyl--D-
galactopyranosyl)ethane-1-sulfinothioate (3k): This compound is obtained as mixture of
two regio-isomers, spectral value of major isomer is given below.
1
White solid; m.p. 57–58 °C [EtOH]; H NMR (500 MHz, CDCl
3
): δ 5.37 (br s, 2 H, 2 H-4),
5
.18 (t, J = 9.0 Hz, 2 H, 2 H-2), 5.01 (t, J = 9.0 Hz, 2 H, 2 H-3), 4.58-4.50 (2 d, J = 9.0 Hz, 2

H, 2 H-1), 4.30-4.25 (m, 1 H, 1 OCH), 4.21-4.09 (m, 5 H, 2 H-6ab, 1 OCH), 4.07-3.87 (m, 1
H, 1 OCH), 3.87-3.90 (m, 2 H, 2 H-5), 3.89-3.81 (m, 1 H, 1 OCH), 3.41-3.30 (m, 4 H, 2
1
3
SCH
2
), 2.19-1.99 (4 s, 24 H, 4 OCOCH
3
); C NMR (125 MHz, CDCl ): δ 170.2-169.1
3
(
OCOCH ), 101.5-101.1 (4 C-1, 2 regioisomer), 70.9 (4 C), 69.1 (2 C), 68.7 (2 C), 68.5 (2 C),
3
6
7.0 (2 C), 63.0 (2 C, SCH
2
), 62.7 (2 C, 61.1 (2 C, OCH
2
2
), 56.0, 55.8 (2 C, SCH ), 20.8-20.5
+
(
OCOCH
found: C, 46.10; H, 5.40.
S-2-(2,3,4,6-Tetra-O-acetyl--D-glucopyranoside)ethyl
3
); m/z: 853.1 [M+Na] ; Anal. Calcd. for C32
H
46
O
21 2
S
(830.82): C, 46.26; H, 5.58;
2-(2,3,4,6-tetra-O-acetyl--D-
glucopyranosyl)ethane-1-sulfinothioate (3l): This compound is obtained as mixture of two
regio-isomers, spectral value of major isomer is given below.
1
Colorless oil; H NMR (500 MHz, CDCl
3
): δ 5.18 (t, J = 9.0 Hz, 2 H, 2 H-2), 5.06 (t, J = 8.5
Hz, 2 H, 2 H-3), 5.00-4.96 (m, 2 H, 2 H-4), 4.60-4.51 (m, 2 H, 2 H-1), 4.30-4.09 (m, 6 H, 2
H-6ab, 2 H-5), 4.08-3.79 (m, 2 H, OCH ), 3.75-3.78 (m, 2 H, OCH ), 3.39-3.29 (m, 4 H, 2
SCH ), 2.09-2.00 (4 s, 24 H, 4 OCOCH ); C NMR (125 MHz, CDCl ): δ 170.4-169.1
OCOCH ), 101.0-100.6 (4 C-1, 2 regio-isomer), 72.7 (4 C-2, 2 regio-isomer), 72.0 (4 C-3, 2
regio-isomer), 71.1 (4 C-4, 2 regio-isomer), 69.2 (4 C-6, 2 regio-isomer), 68.2 (4 C-5, 2
2
2
1
3
2
3
3
(
3
regio-isomer), 63.0 (SCH
2
), 61.7 (OCH
(830.82): C, 46.26; H, 5.58; found: C, 46.06; H, 5.40.
2-(2,3,4-tri-O-acetyl--L-
2
), 55.8 (SCH
2
), 20.9-20.5 (OCOCH
3
); m/z: 853.1
+
[
M+Na] ; Anal. Calcd. for C32
H
46
O S
21 2
S-2-(2,3,4-Tri-O-acetyl--L-rhamnopyranosyl)ethyl
rhamnopyranosyl)ethane-1-sulfinothioate (3m): This compound is obtained as mixture of
two regio-isomers, spectral value of major isomer is given below.
1
Colorless oil; H NMR (500 MHz, CDCl
3
): δ 5.29-5.21 (m, 4 H, 2 H-1, 2 H-2), 5.07-5.01 (m,
2
H, 2 H-4), 4.80-4.78 (m, 2 H, 2 H-3), 4.25-4.16 (m, 2 H, 2 H-5), 4.05-3.78 (m, 4 H 2
OCH ), 3.44-3.32 (m, 4 H, 2 SCH ), 2.15-1.96 (4 s, 24 H, 4 OCOCH ), 1.28-1.22 (m, 6 H, 2
); C NMR (125 MHz, CDCl ): δ 169.9-169.7 (OCOCH ), 97.8-97.5 (4 C-1, 2 regio-
isomer), 71.0-70.7 (4 C-2, 2 regio-isomer), 69.6-69.5 (4 C-3, 2 regio-isomer), 69.0-68.9 (4 C-
, 2 regio-isomer), 67.7-67.3 (OCH , 2 regio-isomer), 66.9-66.8 (4 C-5, 2 regio-isomer), 60.3
OCH , 2 regio-isomer), 55.4-55.3 (SCH
1.0-20.7 (OCOCH ), 17.4 (CCH
2
2
3
1
3
CH
3
3
3
4
2
(
2
2
, 2 regio-isomer), 32.7 (SCH , 2 regio-isomer),
2
+
2
3
3
); m/z: 737.1 [M+Na] ; Anal. Calcd. for C28
H
42
O S
17 2
(
714.75): C, 47.05; H, 5.92; found: C, 46.86; H, 6.10.
1
S-Phenyl benzenesulfinothioate (3n): Colorless oil; H NMR (500 MHz, CDCl ): δ 7.60-
3
1
3
7
.21 (m, 10 H, Ar-H); C NMR (125 MHz, CDCl
3
): δ 143.0-127.2 (Ar-C); m/z: 235.0
+
[
M+1] ; Anal. Calcd. for C12
H
10OS
2
(234.33): C, 61.50; H, 4.30; found: C, 61.37; H, 4.50.

1
S-p-Tolyl 4-methylbenzenesulfinothioate (3o): Colorless oil; H NMR (500 MHz, CDCl
3
):
1
3
δ 7.80-7.09 (m, 8 H, Ar-H), 2.42, 3.38 (2 s, 6 H, 2 CH
44.6-124.6 (Ar-C), 21.6-21.1 (2 CH ); m/z: 263.0 [M+1] ; Anal. Calcd. for C14
262.39): C, 64.08; H, 5.38; found: C, 63.92; H, 5.55.
S-4-Nitrophenyl 4-nitrobenzenesulfinothioate (3p): Colorless oil; H NMR (500 MHz,
CDCl ): δ 8.33 (d, J = 9.0 Hz, 2 H, Ar-H), 8.25 (d, J = 9.0 Hz, 2 H, Ar-H), 7.81 (d, J = 9.0
3
); C NMR (125 MHz, CDCl
3
): δ
+
1
3
H14OS
2
(
1
3
1
3
Hz, 2 H, Ar-H), 7.63 (d, J = 9.0 Hz, 2 H, Ar-H); C NMR (125 MHz, CDCl ): δ 147.9-124.5
3
+
(
Ar-C); m/z: 325.9 [M+1] ; Anal. Calcd. for C12
H N
8 2
O
5
2
S (324.33): C, 44.44; H, 2.49; found:
C, 44.28; H, 2.65.
S-Naphthalen-2-yl naphthalene-2-sulfinothioate (3q): White solid; m.p. 93-94 °C (EtOH);
1
13
H NMR (500 MHz, CDCl
3
): δ 7.94-7.12 (m, 14 H, Ar-H); C NMR (125 MHz, CDCl ): δ
3
+
1
4
39.7-122.4 (Ar-C); m/z: 335.0 [M+1] ; Anal. Calcd. for C20
H
14OS (334.45): C, 71.82; H,
2
.22; found: C, 71.70; H, 4.35.
1
S-Pyridin-2-yl pyridine-2-sulfinothioate (3r): Colorless oil; H NMR (500 MHz, CDCl
3
): δ
8
3
.72-8.70 (m, 1 H, Ar-H), 8.51-8.49 (m, 1 H, Ar-H), 7.93-7.89 (m, 1 H, Ar-H), 7.86-7.72 (m,
1
3
H, Ar-H), 7.60-7.50 (m, 1 H, Ar-H), 7.39-7.32 (m, 1 H, Ar-H); C NMR (125 MHz,
+
CDCl
3
): δ 159.9-119.8 (Ar-C); m/z: 237.0 [M+1] ; Anal. Calcd. for C10
H
8
N
2
OS
2
(236.31): C,
5
0.83; H, 3.41; found: C, 50.70; H, 3.60.
1
S-1-Phenyl-1H-tetrazol-5-yl 1-phenyl-1H-tetrazole-5-sulfinothioate (3s): Colorless oil; H
1
3
NMR (500 MHz, CDCl
3
): δ 7.75-7.50 (m, 10 H, Ar-H); C NMR (125 MHz, CDCl
3
): δ
+
1
2
40.6-121.2 (Ar-C); m/z: 371.0 [M+1] ; Anal. Calcd. for C14
.72; found: C, 45.25; H, 2.60.
H
10
N
8
OS
2
(370.41): C, 45.40; H,
1
Se-Benzyl phenylmethaneseleninoselenoate (3t): White solid; m.p. 103-104 °C (EtOH); H
1
3
NMR (500 MHz, D
125 MHz, D
(357.93): C, 47.21; H, 3.96; found: C, 47.10; H, 4.10.
Se-p-Methoxybenzyl (p-methoxyphenyl)methaneseleninoselenoate (3u): Colorless oil; H
NMR (500 MHz, CDCl ): δ 7.31-7.21 (m, 4 H, Ar-H), 6.89-6.82 (m, 4 H, Ar-H), 5.02 (d, J =
), 4.78 (d, J = 11.5 Hz, 1 H, SeCH ), 4.63-4.54 (m, 2 H, SeCH ), 3.80 (s,
); C NMR (125 MHz, CDCl ): δ 158.7-112.7 (Ar-C), 71.4 (SeCH ), 63.6
(417.95): C,
2
O): δ 7.50-7.42 (m, 10 H, Ar-H), 4.36-4.17 (m, 4 H, 2 CH
2
); C NMR
+
(
2
O): δ 129.7-127.3 (Ar-C), 59.9 (2 CH
2
); m/z: 358.9 [M+1] ; Anal. Calcd. for
C
14
H14OSe
2
1
3
1
6
0.5 Hz, 1 H, SeCH
2
2
2
1
3
H, 2 OCH
), 55.5 (2 OCH
6.17; H, 4.36; found: C, 46.05; H, 4.50.
3
3
2
+
(
SeCH
2
3
); m/z: 418.9 [M+1] ; Anal. Calcd. for C16
H
18
O
3
Se
2
4
Se-(Naphthalen-2-yl)methyl (naphthalen-2-yl)methaneseleninoselenoate (3v): White
1
solid; m.p. 109-110 °C (EtOH); H NMR (500 MHz, CDCl
3
): δ 8.00-7.89 (m, 7 H, Ar-H),

1
3
7
1
.63-7.50 (m, 7 H, Ar-H), 4.18-4.10 (m, 4 H, 2 SeCH
2
); C NMR (125 MHz, D O): δ 127.7-
2
+
24.7 (Ar-C), 64.0 (SeCH ), 63.4 (SeCH
2
2
); m/z: 458.9 [M+1] ; Anal. Calcd. for C22
H18OSe
2
(
457.96): C, 57.91; H, 3.98; found: C, 57.80; H, 4.15.
1
S-Phenyl benzeneseleninoselenoate (3w): White solid; m.p. 128-129 °C (EtOH); H NMR
(
500 MHz, D
2
O): δ 7.79-7.76 (m, 2 H, Ar-H), 7.76-7.70 (m, 2 H, Ar-H), 7.60-7.49 (m, 6 H,
1
3
+
Ar-H); C NMR (125 MHz, D
2
O): δ 132.9-124.5 (Ar-C); m/z: 330.9 [M+1] ; Anal. Calcd.
for C12
H10OSe
2
(329.90): C, 43.92; H, 3.07; found: C, 43.80; H, 3.20.
MTT assay for checking cytotoxicity of compounds
In order to determine the effect of the synthesized compounds on cell proliferation, MTT
assay was performed [50,51]. Briefly, MDA-MB-468, MCF-7 and WI38 cells were grown in
9
5
6 well plates in presence of the synthesized derivatives (0-50 µM) for 24 h, 48 h and 72 h in
% FBS containing phenol red free DMEM. The cells were washed thrice with PBS to make
them clear from the test agents. MTT (200 µL; 0.5 mg/mL dissolved in phosphate buffer
saline (PBS) having a pH value of 7.4) were added to each well and incubated at 37 °C for 4
h in a humidified incubator containing 5% CO . The purple colored formazan crystals formed
2
in the wells were dissolved in DMSO and absorbance was measured at 570 nm with a
microplate reader.
Apoptotic cell quantification by AnnexinV-FITC staining
Induction of apoptosis was measured by flow cytomety after AnnexinV-FITC/PI staining
6
[
53] using BD Bioscience kit. Briefly, post treatment 0.5×10 cells with compound 3h were
washed in ice-cold 1× PBS and resuspended in 100 µL of binding buffer and incubated with
µL of annexin V-FITC and 5 µL of PI for 15 min at room temperature in a dark place as per
5
manufacturer’s guidelines. Flow cytometric analysis was immediately performed using a
FACS Verse instrument (BD).
ROS estimation by DCFDA method
Intracellular ROS was measured using DCFDA method [55]. Briefly, MDA-MB-468 and
MCF-7, cells were treated with 5 µM, 10 µM and 15 µM doses of compound 3h for 12 h.
After the treatment, the media was discarded and cells were washed thrice with ice cold
Hanks balanced salt solution (HBSS) and incubated with DCFDA (100 µM final) for 30 min
at 37 °C. Then, cells were lysed with alkaline solutions and fluorescence intensity was

measured at excitation of 485 nm and emission at 520 nm (Biotek). The estimation of ROS
was measured by both fluorometer as well as by microscope.
Mitochondrial membrane potential measurement by JC1 staining
Assessment of mitochondrial permeability was measured by JC1 staining as described earlier
5
5]. Mitochondria depolarization is specifically indicated by JC-1 dye, which is a cationic
dye that exhibits potential-dependent accumulation in mitochondria, indicated by a
fluorescence emission shift from red to green as the mitochondrial damages and loses its
membrane potential. MDA-MB-468 and MCF-7 cells were treated with 5 µM, 10 µM and 15
µM of compound 3h. After the treatment, the cells were washed with phosphate buffered
saline (PBS) and incubated with (10 µg/ml) JC-1 for 30 min in dark at 37 C. Finally images
were captured under a fluorescent microscope (Leica, Wetzlar, Germany).
Apoptotic nuclear morphology study by DAPI staining
Nuclear morphology of MDA-MB-468 and MCF-7 cells was studied by DAPI staining. After
exposure of compound 3h (5 µM, 10 µM and 15 µM) for 12 h, cells were washed three times
with 1 × PBS and stained with 4′,6-diamidino- 2-phenylindole (DAPI) in vectashield (0.2 g
−
1
ml , Vector Laboratories Inc.). Nuclear morphology was observed under fluorescence
microscope.
Comet Assay for checking DNA damage
Comet assay (also known as Single Cell Gel Electrophoresis Assay) is a facile method for
measuring DNA strand breaks in eukaryotic cells. The term “comet” refers to the pattern of
DNA migration through the electrophoresis gel, which often resembles a comet [57]. Frosted
slide pre-coated with 200 µL of 0.8% normal melting point of compound 3h rose in PBS and
left briefly to solidify. Cover slips were removed and MDA-MB-468 and MCF 7 cells (90
5
µL) (3×10 ) were washed and re-suspended in 100 µL of low melting point agarose that was
in turn pipetted onto the normal melting point agarose layer spread with cover slips and left
briefly to solidify. Trypsin solution (50 µL) was added onto the slide and left for 30 min at 37
ºC. Slides were then immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA,10
mM Tris-HCl, pH-16, 1%Triton X-100, 10% DMSO) and incubated at 4 ºC for 1 h. Then the
slides were equilibrated in freshly prepared electrophoresis buffer (1 mM EDTA, 300 mM
NaOH, pH-9.1) for 20 mins at 40 ºC before performing electrophoresis. Electrophoresis was

done for 20 mins at 25V, 300 mA at room temperature. The slides were drained and washed
three times with freshly prepared neutralizing buffer (0.4M Tris-HCl, pH-7.5) for 20-30
mins.The slides were stained with 40 µl of 20 µg/mL of ethidium bromide and kept in dark
until use. During the examination, the slides were washed with PBS three times and mounted
with mounting medium and observed directly under fluorescence microscope. A total of
about 50 cells per slide were analyzed for comet parameters using an image analysis system.
Western blot
Briefly, cell lysates were prepared from MDA-MB-468 and MCF7 cells with RIPA buffer
method post treatment with various doses of compound 3h. For immunoblotting, 20-35µg
proteins were resolved on 8%-15% SDS-PAGE, transferred onto PVDF membrane
(
Millipore,USA), blocked with 5% BSA in 1×TBST, and probed with respective primary
antibodies followed by corresponding secondary antibodies. Finally blots were developed
into X-Ray films by ECL method.
Molecular docking study of three selective compounds
Molecular docking study was carried out through Autodock 4.2 software [59]. Before
docking, all the structures (Compound 3f, 3h and 3u) of the inhibitors were made in
Chemdraw 10 and optimize their geometry using the default minimization algorithm. For
docking study, the protein coordinates of Caspase-3 (PDB code: 1GFW) [60] were prepared
in Autodock. The molecules were located in the ligand area by using fit atom method [61].
The docking was made in Autodock by setting the grid size for the search as 60×60×60Å
dispersed around the ligand binding site along with default grid spacing (0.375Å) [58]. The
molecules were allowed for flexible docking in the protein. 100 docking runs were performed
for each molecule and the best docked poses were isolated by identifying their Gibbs free
energies (ΔG) of the population. The interactions between protein and docked molecules
were visualized in Autodock, Pymol and Discovery Studio Visualizer [61-63]. To validate
docking experiments, all extracted ligands of all co-crystals were docked individually with
their proteins to compare protein binding coordinates with the crystal forms and also identify
the binding affinity. Furthermore, standard drug molecule was docked in the same protein
coordinates of the co-crystal structure for comparison study with selected synthesized
molecules.

Statistical analysis
All the experiments were conducted in triplicate. Data were represented as mean±SD.
Data were subjected to One-way ANOVA analysis following students t test using GraphPad
Prism 6.0 (USA). The data presented as the average of three experiments ± SD. ∗ =
represents P value < 0.05, ∗∗ = represents P value < 0.01.
Acknowledgements
I.B. and K.P. thank CSIR, India for providing Junior and Senior Research Fellowships
respectively and U.D. thanks SERB, India for N-PDF fellowship. This work was supported
by SERB, New Delhi, India (AKM) [Project No. EMR/2015/000282].
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