Design, synthesis and comparative analysis of triphenyl-1,2,3-triazoles as anti-proliferative agents

Article abstract of DOI:10.1016/j.ejmech.2020.112813

Herein, a series of triaryl-1,2,3-triazoles, in order to check cytotoxicity on breast cancer cell lines have been synthesized with pendent benzyl ring to mimic the phenolic A ring of Tamoxifene. The biological results indicated that most of the compounds

Full text of DOI:10.1016/j.ejmech.2020.112813

European Journal of Medicinal Chemistry 207 (2020) 112813
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and comparative analysis of triphenyl-1,2,3-triazoles
as anti-proliferative agents
,
e
,
,
,
, e
Divya Dheer a 1, Chittaranjan Behera b 1, Davinder Singh a e, Mohd Abdullaha c
,
Gousia Chashoo d, Sandip B. Bharate c e, Prem N. Gupta b **, Ravi Shankar a
,
,
e
,
, e, *
a Bio-organic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India
b PK-PD Toxicology & Formulation Division, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India
c Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India
d Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India
e Academy of Scientific and Innovative Research (AcSIR), New Delhi, 110025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, a series of triaryl-1,2,3-triazoles, in order to check cytotoxicity on breast cancer cell lines have
been synthesized with pendent benzyl ring to mimic the phenolic A ring of Tamoxifene. The biological
results indicated that most of the compounds possessed comparative anti-proliferative activities in both
ER þ ve (MCF-7) and ERꢀve (MDA-MB-231) breast cancer cell lines. Among synthesized derivatives, five
Received 22 July 2020
Received in revised form
18 August 2020
Accepted 2 September 2020
Available online 9 September 2020
compounds 8f, 8i, 8j, 8n and 8p showed anti-proliferative activities at <5
m
M against MCF-7 cell line and
three compounds 8e, 8f and 8j show IC50 value greater than 30
m
M in FR-2 cells (normal cell). Moreover,
to understand the mechanistic behavior of the selective compound 8f, various studies performed viz.
surface morphological changes by bright field microscopic examination, nuclear morphological alter-
ation by DAPI staining, measurement of intracellular ROS level and determination of change in mito-
chondrial membrane potential. It was observed that, the selective compound 8f associated with higher
ROS generation along with decrease in mitochondrial membrane potential in addition to surface and
nuclear morphological alterations such as reduction in number and shrinkage of cells coupled with
nuclear blabbing indicating sign of apoptosis. Further, molecular docking study in comparison to
Keywords:
Breast cancer
1,2,3-Triazole
Estrogen receptor
Molecular docking studies
Apoptosis
Morphological alteration
tamoxifen was also carried out to investigate the interaction of 8f with ER-a which favors its possible
mode of anticancer action.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
compounds containing 1,2,3-1H-triazole tethered with various
natural products have shown promising in vitro anticancer results
[6e9]. Some of the triazoles, namely, anastrozole (Arimidex) (1)
and letrozole (Femara) (2) are clinically available third-generation
aromatase inhibitors that exhibits opposed effects on the ER (es-
trogen receptor) expression of breast cancer cells, showing superior
therapeutic effects over antiestrogens. These triazoles inhibit the
aromatase enzyme by binding of the N-4 nitrogen of the triazole
ring with the heme iron atom of the CYP19 enzyme complex also
proving contraindication in premenopausal women including
pregnant and lactating women [10]. Therefore, there comes a need
for novel compounds which can overcome such limitations.
Nitrogen-based heterocycles are structural motifs of biological
interest in various disciplines, namely, anticancer, antinociceptive,
antipyretic, antimicrobial, antiepileptic, antituberculotic, antiviral,
anticoagulant as well antiplatelet agents and several synthesized
compounds have been approved by the FDA [1,2]. Towards this
direction, triazoles have turned out to be the most effective moi-
eties against different tumor cells [3e5]. Some of the hybrid
* Corresponding author. Bio-organic Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Jammu, 180001, India.
ERb has also shown clinical advancement in the prevention of
** Corresponding author. PK-PD Toxicology & Formulation Division, CSIR-Indian
Institute of Integrative Medicine, Jammu, 180001, India.
E-mail addresses: pngupta@iiim.res.in (P.N. Gupta), rshankar@iiim.res.in
(R. Shankar).
colorectal carcinoma [11,12]. Non-steroidal drugs which interact
with ER have already proven useful in contraceptives, uterine
dysfunction, osteoporosis as well as for the treatment of breast
cancer [13]. Some of the other examples of triphenylethylene based
1
Both authors contributed equally.
https://doi.org/10.1016/j.ejmech.2020.112813
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
SERMs include raloxifene (3), nafoxidine (4) and lasofoxifene (5).
On the other hand, Tamoxifen (6) has been the drug of choice for
last 30 years for estrogen related positive breast tumors. Tamoxifen
being cheaper compared to other anti-estrogen drugs can very well
increase the cure rate by 10% especially in case of low- and middle-
income countries but the selectively of combined actions of ago-
nists and antagonists has been minimal. Novel molecules having
increased selectively signify the most active biological potential
accompanied by least adverse outcomes. In fact, some of the es-
estradiol that are suitable to bind with its receptor. The 1,2,3-
triazole ring represents the central core of the whole compound
and three phenyl rings existed perfectly arranged to prove the ideal
geometry of molecules further imitating PPT framework. Compared
to PPT, the main differences in our present work are (i) the presence
of extra nitrogen in the core that can enhance an interaction of
molecule with receptors; (ii) the existence of bendable benzyl
group (ring 1) that may help in adaptation of a favorable confor-
mation change within the receptor cavity (Fig. 2) [22]. Furthermore,
basic amino chains at para position of ring 2 ease the association of
ER proteins to give desired effect.
trogen receptor dependent a- (7) or b- (8) agonists are already in
clinical trials and Katzenellenbogen group have also innovated the
concept of antiestrogens with a wide variety of pyrazole based
molecules as depicted in Fig. 1.
2. Results and discussion
Numerous literature reports have shown triphenylethylene as
well as benzothiopene derivatives to be successful ER molecules
and the triaryl substituted pyrazole-related ER antagonists exhib-
iting remarkable therapeutic potential is also widely recognized
[14]. For instance, Katzenellenbogen and group in 1999 explored a
novel series of triaryl-substituted pyrazole based ligand molecules
showing high affinity for ER [15]. According to the study, pro-
pylpyrazoletriol (PPT) (7) was observed to be the best compound of
2.1. Chemistry
Initially, 5-iodo-1,4-disubstituted-1,2,3-triazoles (3a-3d) were
synthesized through copper (I)- catalyzed version of azide-alkyne
cycloaddition reaction, using phenylacetylene, I2 in DMF and
benzyl bromide, sodium azide in triethylamine, along with the
emergence of in situ 1-iodophenylacetylene and aryl azide (Scheme
1) [22]. In our previous work, we were able to synthesize 3a-3d as
well as 4a-4d in-situ from benzyl bromide 1 and phenylacetylene 2
in the ratio of 80:20 approximately. Infact, I2 as well as phenyl-
the series which binded well with 410-fold affinity for ER-
a re-
ceptor [16]. Continuing their research, they discovered that the
above pyrazole ER agonist molecule, if converted into its antagonist
by embracing them with a basic side chain, might act in the similar
manner, as in case of raloxifene. Based on the above study, C (5)
piperidinyl-ethoxy substituted pyrazole exhibited 20-fold higher
acetylene 2 were shaken in aqueous medium in addition to CuI/
b-
CD -cyclodextrin) for half an hour. The emergence of 1-
(b
iodophenyacetylene was confirmed through its isolation and
characterization by proton NMR. Thenceforth, benzyl bromide 1
plus NaN3 were stirred in the same flask in the presence of elevated
heat conditions close to 90 OC. Surprisingly, 1-Benzyl-5-iodo-4-
phenyl-1H-1,2,3-triazole 3a was yielded as major compound
(79%). On the other hand, 1-Benzyl-4-phenyl-1H-1,2,3-triazole 4a
was obtained as minor compound when all the reactants were
added synchronously. Based on NMR characterization, presence of
iodine in triazole derivatives 3a-3d was confirmed through a sharp
affinity towards ER-a better than ER-b subtype [17]. Later, Hajela
group in 2008 investigated new tetrazolyl indole derivatives con-
taining N-ethyl amino moiety and observed 100% contraceptive
efficacy at animal dose 10 mg/kg [18]. The same group synthesized
3,4,6-triaryl-2-pyranones [19] as well as hydrazone [20] derivatives
as a new class of anti-breast tumor compounds. Recently, Katze-
nellenbogen group has again derivatized a large library of triaryl
pyrazoles which hold a great promise as anti-inflammatory drugs
[21]. Based on these observations, we have designed 1,2,3-triazoles
as the cyclic prototype of triaryl substituted pyrazoles corre-
sponding to triphenylethylene derivatives in adherence to specified
positioning having three phenyl substitutions for therapeutic
benefits.
singlet at
pounds 4a-4d, proton NMR showed the presence of a resonance
peak at 7.6 as singlet but absence of 76 in carbon spectrum.
d76 in carbon spectrum. Whereas, in 5H-triazole com-
d
d
In the next step, Suzuki reaction was carried out in the presence
of 4-hydroxyphenylboronic acids to form (6a-6d) (Scheme 2) [23].
Simultaneously, without iodo-triazole compound 4 obtained as
byproduct in the same reaction mixture, has been further utilized
to get 6a via tosylation after having 4-(1-benzyl-4-phenyl-1H-1,2,3-
triazol-5-yl)phenyl 4-methylbenzenesulfonate 5 as an intermedi-
ate (Scheme 2) [24,25].
The rationale behind the designing of 1,2,3-triazole based
compounds was to construct a scaffold having specified arrange-
ment bearing three phenyl substitutions in accordance to the re-
ported leads such as propylpyrazoletriol (PPT) (7), tamoxifene and
Further in the method, alkyl chains were propagated with the
help of various alkylating reagents to obtain 1-benzyl-5-(4-(2-
Fig. 1. Chemical structures of non-steroidal antiestrogens and triazole based aroma-
Fig. 2. Structural resemblance of 1,2,3-triazole based molecule to tamoxifen, estradiol
tase inhibitors.
and propylpyrazoletriol (PPT).

D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
3
(6e), which was confirmed by proton NMR with the absence of
another set of 4 protons. No phenyl-H compound was found
which was further confirmed by mass [MþH]þ 268.1078 (Scheme
4).
2.2. Biological activity
2.2.1. Antitumoral activity against breast & colon tumor cells
The designed derivatives (6a-6e) and (8a-8p) were investigated
for antitumoral activity and results indicated that most of the
compounds possessed comparative anti-proliferative activities in
MCF-7, MDA-MB-231 and HCT-116 cancer cells using SRB assay.
Most synthesized compounds inhibited the growth of cancer cells
Scheme 1. Synthesis of iodo and without iodo-triazoles. Reaction and conditions: 1
(1.0 mmol), 2 (1.0 mmol), NaN3 (2.0 mmol), I2 (1.5 mmol),
b-CD (1.0 mol %) CuI (10 mol
%), Water (5 mL), open flask, 90 ꢁC, 2e4 h.
at IC50 of less than 50
compounds 8f (3.5 M), 8i (4. 7
(4.5 M) and 8q (3.3 M) showed anti-proliferative activities at less
than 5 M against MCF-7 cell line and three compounds 8e
(30.8 M), 8f (34.8 M) and 8j (29.0 M) showed IC50 value > 30 mM
mM. Among synthesized derivatives, six
m
m
M), 8j (3.5 M), 8n (3.9 M), 8p
m
m
m
m
m
m
m
m
in FR-2 cells. Five of these, 8e, 8f, 8g, 8i and 8j inhibited the growth
of both MCF-7 as well as MDA-MB-231 cell lines at IC50 below
20 mM (Table 1). Three of these, 8b, 8m and 8n were active against
MCF-7 cell line selectively. Dimethoxy derivative of compound 8q
was also synthesized and evaluated against MCF-7 cell line but no
improvement was observed therefore, its activity against another
cell line was not evaluated. N-debenzylated compound 8q also
showed decrease in activity which confirmed the importance of N-
benzyl group in biological action. Activity of compound 8f was
evaluated towards MCF-7, MDA-MB-231 as well as HCT-116 tumor
Scheme 2. Synthesis of triaryl-substituted 1,2,3-triazoles. Reaction and Conditions:
(a) 3 (1.38 mmol), PhB(OH)2 (2.07 mmol), K2CO3 (3.45 mmol), PdCl2(PPh3)2 (10 mol%),
DMF:H2O (2.5:0.5) 6 mL; (b) 3b or 3c (1 mmol), 1 M BCl3 in DCM (5 mmol), ꢀ60 ꢁC,
2e4 h; (c) 4 (1.27 mmol), 4-bromophenyl 4-methylbenzenesulfonate (0.85 mmol),
Pd(OAc)2 (4.0 mol%), PCy3 (8.0 mol%), K2CO3 (2 equiv., 1.69 mmol), toluene (2 mL), N2
atm., 22 h, 120 ꢁC; (d) 5 (1 mmol), aq. NaOH solution (1 M), acetone (10 mL), reflux,
4e5 h.
cells at different concentrations of 0.1, 1, 10, 50 and 100 mM. Se-
lective destruction of tumor cells while guarding development of
healthy cells plays a pivotal characteristic among cytoprotectives.
Nine active derivatives were investigated against FR-2 (normal
breast epithelial) cell line at density of 10,000 cell/well for possible
cytotoxicity. As shown in Table 1, the IC50 values of compounds 8e,
chloroethoxy)phenyl)-4-phenyl-1H-1,2,3-triazole 7a (7a-7f). Next
step included the amination of above compounds, with different
amines like pyrrolidine, piperidine, diethylamine or morpholine to
form final active compounds (8a-8p). Desired methodology has
been depicted below (Scheme 3).
Hydroxy functional group plays a very important role to
enhance biological activity therefore; we have tried demethyla-
tion of active synthesized dimethoxy compounds 6d to check the
effect of methoxy group in biological activity. Compound 6d was
treated with BBr3 in DCM at ꢀ78 ꢁC to get demethylated com-
pound but unfortunately, we obtained N-debenzylated compound
8f and 8j were found to be greater than 30
which further showed remarkably higher activity as compared to
MCF-7 (5.7, 3.5 and 3.5 M), and MDA-MB-231 (15.28, 15.54 and
9.61 M respectively), demonstrating that these molecules 8e, 8f
mM in FR-2 cell line
m
m
and 8j having reduced toxicity towards healthy human cells
compared to tumor cells.
The selected test compounds were screened against normal
breast epithelial cell line FR-2 and cytotoxic selectivity of these
compounds was evaluated by calculating the selectivity index
based on the below mentioned formula:
non-cancerous cells
cancer cells
SI ¼ IC
/IC
50
50
Infact, SI greater than and equals to 10 has been marked as se-
lective molecule [26]. In Table 1, selectivity index of nine com-
pounds has been mentioned, out of which only compound 8f
having SI ꢂ 10 was considered to be a selective one and therefore
used for further investigation. The selectivity index of active com-
pounds has also been discussed in Table 1. In fact, the increase in
the percent growth inhibition has been observed with the increase
in dose of compound 8f (Fig. 3).
Scheme 3. Synthesis of aminated triaryl-substituted 1,2,3-triazoles. Reaction and
Conditions: (a) 6 (0.15 mmol), halo alkanes (0.30 mmol), K2CO3 (0.30 mmol), acetone
(10 mL), reflux, 4e5 h; (b) 7 (1.12 mM), amine (2 equiv), TBAI (10 mg), dry DMF
(15 mL), 70e75 ꢁC, 7 h.
Scheme 4. Reaction and Conditions: (a) BCl3, DCM, ꢀ70 ꢁC, rt, 6 h

4
D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
Table 1
Cytotoxic activities of target molecules against breast and colon cancer, normal cell lines along with their selectivity index.
Compd No
IC50 (Mean SD,
MCF-7
mM)
MDA-MB-231
HCT-116
FR-2
Selectivity Index (SI)
6a
6b
6c
6d
6e
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
53.2 0.87
77.5 2.76
68.6 1.74
22.6 0.66
>100
23.36 1.71
>100
42.51 2.06
>100
94.42 2.72
34.88 2.06
95.87 3.25
83.05 2.8
40.3 0.79
52.67 0.39
52.86 1.02
15.28 0.35
15.54 0.6
19.43 0.24
29.1 0.36
10.32 0.49
9.61 0.67
40.86 1.48
99.76 1.08
46.51 1.56
28.64 2.05
30.2 1.17
16.92 1.18
ND
74.18 4.1
38.31 1.07
71.52 0.83
51.68 1.81
25.53 1.77
33.23 2.61
20.73 2.11
71.19 2.08
30.43 3.1
21.17 2.8
23.37 2.2
36.5 2.36
13.74 2.06
39.66 2.7
27.03 2.54
29.60 2.83
83.11 3.3
28.37 3.6
19.39 2.4
ND
45.97
2
7.1 0.25
27.21 0.85
33.3 1.2
5.7 0.3
3.5 2.26
8.5 0.4
12.8 3.89
4.7 0.25
3.5 0.2
46.17 2.03
57 1.34
8.8 1.36
3.95 0.15
34.4 1.2
4.56 0.12
3.33 0.11
9.53 1.3
14.5 1.49
2.04
30.8 0.88
34.8 1.17
20.66 1.18
5.4
9.94
2.43
17.63 1.4
29.07 2.33
3.75
8.3
8l
8m
8n
8o
8p
8q
12.08 1.27
10.13 1.3
1.37
2.5
17.53 2.1
22.3 2.8
3.9
Tamoxifen citrate
10.71 1.55
ND
2.33
Note: ND ¼ not determined.
Fig. 3. Graphical representation of growth inhibition on different concentrations of
compound 8f againts MCF-7,MDZ-MB-231 as well as HCT-116 tornor cells assessed
through SRB assay.
Fig. 4. Comparison of cell inhibition of compound 8f and Tarnoxifen citrate against FR-
2 cell line.
Compound 8f was found selective against normal breast
epithelial FR-2 cells with a selectivity index of 9.94 for 8f and 2.33
for tamoxifen hence, exhibiting better cytotoxicity against MCF-
7 cells (Fig. 4).
comparison to the compound 8f dose against MCF-7 cell line.
2.2.2. Assessment of cellular death caused due to compound 8f
using bright field microscopy
2.2.3. Compound 8f altered nuclear morphology assessed by DAPI
staining
Using bright field microscopy, the surface morphological
changes were assessed by treating with compound 8f in MCF-
7 cells (Fig. 5). Random fields under bright light were examined
following to the treatment with 8f compound at various concen-
trations against MCF-7 cells for 48 h using inverted microscope. On
viewing the wells of the six well tissue culture plate, visible
rounding, shrinking of cell size and finally reduced cell number
with disturbed morphology in comparison to the untreated control
cells were visible among treated batches justifying inhibition of cell
with compound 8f. In addition to these, the positive control groups
In the present study, DAPI staining helped to distinguish be-
tween the normal and apoptotic cells by the nuclear morpho-
logical changes caused after the treatment with 8f compound
against MCF-7 cell line in a concentration dependent manner.
The morphological changes occurred can be visualized using
fluorescence microscopy (Fig. 6). The nuclei of untreated cells
appeared like more or less rounded structures while the treat-
ment groups (8f) including tamoxifen showed chromatin
condensation, nuclear blabbing and formation of apoptotic
bodies. The nuclear morphological changes except the untreated
control clearly suggested that MCF-7 cells had undergone
also show cellular death at 9.5 mM dose which was quiet high in

D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
5
Fig. 5. Bright field microscopic examination of untreated and treated cells revealed surface morphology. (A) The untreated cells possess microvilli on cell surface and typical patchy
formed structures. (B) Treated with standard drug tamoxifen at 9.5 mM showed apoptotic behavior in MCF-7 cells (C) After treatment with compound 8f with 3.5 mM concentration
for 48 h causes shrinkage, loss of microvilli, and apoptosis.
Fig. 6. DAPI staining assay of MCF-7 cells. The cells upon treatment with compound 8f for 48 h stained with DAPI and examined for morphological changes. (A) Untreated cells
represent normal rounded nuclear morphology whereas (B) tamoxifen 9.5 M showed typical apoptotic bodies formed (C) MCF-7 cells treated with 3.5 M of compound 8f showed
nuclear morphological changes with visibly reduction in cell number and condensed chromatin structure. Arrows depicts the formation of apoptotic bodies.
m
m
apoptosis. This is the first report for the induction of apoptosis
caused due to the anticancer compound 8f.
a key feature of apoptosis.
2.2.5. Compound 8f induces depletion of mitochondrial membrane
potential (MMP) in MCF-7 cell lines
2.2.4. Treatment of 8f molecule to enhanced intracellular ROS
production in MCF-7 cells
Reduction of MMP
(DJm) is the main characteristic of
Higher level of reactive oxygen species generation is a prime
indication of apoptosis in cancer cells. In the current study, MCF-
7 cells were incubated with DCFDA dye and intracellular ROS
observed using a fluorescence microscope. Higher amount of ROS
was produced in the positive control group. In a similar way, the
amount of ROS was increased after the treatment with com-
apoptosis. To assess the effect of 8f molecule, loss of MMP was
observed through fluorescence microscopy using rhodamine-123
staining (Fig. 8). After 48 h treatment with compound 8f against
MCF-7 cells, it was observed that the significant reduction in MMP
in 3.5 mM concentration treatment group was due to the decrease in
their fluorescence intensities which clearly justified the mito-
chondrial membrane destabilization in comparison to untreated
cells. The result was quiet similar in case of tamoxifen treatment
group. The MMP loss for compound 8f has also not been reported in
earlier studies and the data indicated that apoptosis was induced by
treatment with 8f molecule due to the loss of MMP.
pound 8f at 3.5 mM concentration. Observation through fluores-
cence microscope is a qualitative means of ROS generation in
MCF-7 breast cancer cells where a sharp increase in fluores-
cence intensity was observed (Fig. 7). This study indicated that
compound 8f triggered ROS generation in MCF-7 cells which are
Fig. 7. (a) Detection of intracellular ROS production in MCF-7 cells. (A) Least amount of ROS generated in untreated cells which is a basic feature of cancer cells (B) H2O2 (0.05%) used
as a positive control and acts as a ROS inducing agent (C) The cells were treated with compound 8f of desired concentration for 48 h. The fluorescence intensity of DCFHDA was also
found increased in 3.5 mM concentration of compound 8f after treatment.

6
D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
Fig. 8. Loss of MMP of 8f derivative treated MCF-7 cell lines was determined through rhodamine-123 staining and observed under fluorescence microscope. (A) The untreated
groups demonstrated intact mitochondria in comparison to the treated groups which prompted a significant decrease in MMP (B) tamoxifen treated group (9.5 M) was taken as
standard positive control drug (C) represents compound 8f treated group at 3.5 M concentration for 48 h.
m
m
Fig. 9. Interaction of 4-hydroxytamoxifen (grey) and 8f (purple) with ER-
a (PDB: 3ERT). (A) Surface view of ER-a protein showing the active site; (B) Overlay of 4-hydroxytamoxifen
and 8f in the active site of ER- ; (C) Interactions of 4-hydroxytamoxifen with ER-
a
a
active site; and (D) Interactions of 8f with ER-a active site
In order to rationalize the activity profile, next we carried out
the molecular docking study of computationally energy mini-
mized conformation of active compound 8f with structurally
3. Conclusion
In this research work, we have synthesized triaryl-substituted
1,2,3-triazoles in order to check cytotoxicity on breast and colon
cancer cell lines. The biological results indicated that among all
the compounds screened against different cell lines, compound
related drug moiety, 4-hydroxy tamoxifen (OHT) in ER a. Infact,
compound 8f displayed strong binding at the active site with the
dock score of ꢀ9.97 while the co-crystallized ligand 4-
hydroxytamoxifen has a dock score of ꢀ13.61. The overlay im-
age of 8f with 4-hydroxytamoxifen has indicated the perfect
alignment of these two ligands with each other as shown in
Fig. 9. The N-benzyl and phenyl ring of 8f were precisely
superimposed over phenolic and phenyl ring of 4-
hydroxytamoxifen, respectively and the triazole moiety of 8f
occupied the junction of three aryl rings containing C]C bond.
The key H-bonding interaction with Thr-347 of 4-
hydroxytamoxifen was also observed in 8f. The favorable inter-
8f induced apoptosis at IC50 dose chosen at 3.5 mM concentration
in MCF-7 cells. Moreover, this diethyl amine substituted 8f
molecule inhibited cell proliferation effectively in both breast
and colon cancer cell line variants. As per the screening of 8f
against normal breast epithelial cell line FR-2, the study observed
the lack of cytotoxic effect at 3.5 mM concentration nearly indi-
cating it to be 10-fold safer from their IC50 value against MCF-
7 cells and far better than the tamoxifen. The mechanistic studies
revealed that the compound 8f triggered apoptosis in breast
cancer cells which is a mechanism of autolysis of cells. Therefore,
this work displays an important prospective towards futuristic
action of 8f with ER-
a may be indicating that modulation of ER-a
activity could be its possible mode of anticancer action.

D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
7
approaches and possibilities for target molecule 8f as a suitable
chemotherapeutic agent.
75.63 (C), 55.33 (OCH3), 54.38 (CH2) ppm; HRMS (ESI): calcd. for
C
16H14IN3O [MþH]þ, 392.0215; found: 392.0254.
4. Experimental section
4.1.1.4. 5-Iodo-1-(4-methoxybenzyl)-4-(4-methoxyphenyl)-1H-1,2,3-
triazole (3d). Yield: 85% (1.35 g), yellow solid; M. p.: 126e127 ꢁC; 1H
4.1. Chemistry
NMR (400 MHz, CDCl3)
6.97 (d, J ¼ 8.9, 2H), 6.87 (d, J ¼ 8.7, 2H), 5.57 (s, 2H), 3.84 (s, 3H),
3.78 (s, 3H);13C NMR (101 MHz, CDCl3)
159.92 (C), 159.76 (C),
d
7.85 (d, J ¼ 8.9, 2H), 7.28 (d, J ¼ 8.7, 2H),
General Information 1H and 13C NMR spectra in CDCl3 and
d
CD3OD were recorded on Bruker Avance III-400 MHz, using
(CH3)4Si as an internal standard. Chemical shifts (d) are expressed
150.10 (C), 129.42 (2CH), 128.82 (2CH), 126.53 (C), 122.87 (C), 114.28
(2CH), 113.98 (2CH), 75.38 (C), 55.32 (2OCH3), 53.95 (CH2) ppm;
HRMS (ESI): calcd. for C17H16IN3O2 [MþH]þ, 422.0321; found:
422.0370.
in parts per million referenced to the residual solvent (i.e., 1H
7.24 ppm, 13C 77.1 ppm for CDCl3; 1H 3.35, 4.78 ppm, 13C 49.3 ppm
for CD3OD). Signal multiplicity is expressed as follows: s (singlet),
br s (broad singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet). J values are given in hertz (Hz). For the HRMS measurement,
Q-TOF was used. All reactions and purity of the synthesized com-
pounds were monitored by TLC using silica gel 60 F254 aluminium
plates. Visualization was accomplished by UV light, exposure to
iodine vapours and by treating the plates with dragendorff reagent
followed by heating. Unless otherwise indicated, materials and
solvents were purchased and used without further purification.
4.1.1.5. 1-Benzyl-4-phenyl-1H-1,2,3-triazole
(230 mg), white solid; M. p.: 135e137 ꢁC;1H NMR (400 MHz, CDCl3)
7.81e7.78 (m, 2H), 7.66 (s, 1H), 7.41e7.36 (m, 5H), 7.33e7.30(m,
3H), 5.57 (s, 2H); 13C NMR (101 MHz, CDCl3)
148.26 (C), 134.72 (C),
(4). Yield:
20%
d
d
130.58 (C), 129.16 (2CH), 128.79 (3CH), 128.15 (CH), 128.06 (2CH),
125.72 (2CH), 119.45 (CH), 54.24 (CH2); HRMS [ESI]: m/z calculated
for C15H13N3 [MþH]þ 236.1143, found: 236.1189.
4.1.1.6. 4-Bromophenyl 4-methylbenzenesulfonate. To a solution of
4.1.1. General procedure for the synthesis of iodo-triazoles (3a-3d,
4)
4-bromophenol (400 mg, 2.30 mmol, 1.0 equiv.) in DCM (2 mL) at
0
ꢁC were added tosyl chloride (485 mg, 2.54 mmol, 1.1 equiv.)
A suspension of phenylacetylene 1 (500 mg, 4.89 mmol, 1.0
equiv.) and iodine (1.86 g, 7.33 mmol, 1.5 equiv.) was added to a
portion-wise and pyridine (205 mL, 2.54 mmol, 1.1 equiv.) dropwise.
The reaction mixture was warmed to room temperature and stirred
for 4 h. Distilled water (20 mL) and saturated aqueous NH4Cl
(10 mL) were added, and the mixture was extracted with CH2Cl2
(3 ꢃ 60 mL). The combined organic fractions were washed with
brine, dried (Na2SO4), and concentrated in vacuo. The purification
of the crude material was thus obtained by flash chromatography
(silica gel, 10% EtOAc in n-hexane) afforded bromobenzene tosylate
as a white solid. Yield: 85% (644 mg); M. p.: 77e78 ꢁC; 1H NMR
mixture of CuI (91 mg, 10 mol %) and
becyclodextrin (46 mg,
1.0 mol %) dissolved in water (20 mL) and stirred for 30 min at room
temperature. A red homogeneous solution was formed, which
confirmed the formation of 1-iodophenylacetylene as revealed by
TLC analysis. Thereafter, benzyl bromide 2 (581
equiv.), sodium azide (636 mg, 9.78 mmol, 2.0 equiv.) and trie-
thylamine (205 L, 1.46 mmol, 0.3 equiv.) were added to the reac-
mL, 4.89 mmol, 1.0
m
tion mixture and reaction temperature was raised up to 90 ꢁC for
4 h. After completion of reaction as monitored by TLC, the reaction
mixture was diluted with water (20 mL). The aqueous solution was
extracted with EtOAc (3 ꢃ 10 mL) and combined organic layer was
washed with saturated Na2S2O3 solution, dried over anhydrous
Na2SO4, and evaporated in vacuum. The residue was purified by
column chromatography using (10% EtOAc: petroleum ether) to
afford the desired product.
(400 MHz, CDCl3)
d
7.72(d, J ¼ 8.4 Hz, 2H), 7.43 (d, J ¼ 8.9 Hz, 2H),
7.35(d, J ¼ 8.1 Hz, 2H), 6.89 (d, J ¼ 6.8 Hz, 2H), 2.48 (s, 3H); 13C NMR
(101 MHz, CDCl3)
d 148.66 (C), 145.60 (C), 132.71 (2CH), 132.18 (C),
129.84 (2CH), 128.53 (2CH), 124.14 (2CH), 120.54 (C), 21.69 (CH3)
ppm; HRMS (ESI): calcd. for C13H11BrO3S [MþH]þ, 328.9625; found:
328.1437.
4.1.1.7. 4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenyl 4-
methylbenzenesulfonate (5). A suspension of Pd(OAc)2 (8.0 mg,
4.0 mol%), PCy3 (19 mg, 8.0 mol%), K2CO3 (235 mg, 1.69 mmol, 2
equiv.), 4 (200 mg, 0.85 mmol, 1 equiv.) and 4-bromophenyl 4-
methylbenzenesulfonate (415 mg, 1.27 mmol, 1.50 equiv.) in
toluene (2 mL) was stirred under N2 for 24 h at 120 ꢁC. Reaction was
quenched with cold water and extracted with Et2O (2 ꢃ 50 mL). The
combined organic layers were washed with aqueous NH4Cl
(50 mL), H2O (50 mL) and brine (50 mL), dried over Na2SO4 and
concentrated in vacuum. The remaining residue was purified by
column chromatography (silica gel, 50% EtOAc in n-hexane) to
afford (5) as an off-white solid. Yield: 75% (307 mg); M. p.:
4.1.1.1. 1-Benzyl-5-iodo-4-phenyl-1H-1,2,3-triazole (3a). Yield: 79%
(1.23 g), white solid; M. p.: 138e140 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.87 (d, J ¼ 8.4 Hz, 2H), 7.40e7.36 (m, 2H), 7.28e7.26 (m, 6H), 5.60
(s, 2H); 13C NMR (125 MHz, CDCl3)
d
150.23 (C), 134.30(C), 130.21
(C), 128.94 (2CH), 128.63 (CH), 128.56 (2CH), 128.52 (CH), 127.83
(2CH), 127.46 (2CH), 76.45 (C), 54.42 (CH2); HRMS [ESI]: m/z
calculated for C15H12IN3 [MþH]þ 362.0109, found: 362.0129.
4.1.1.2. 5-Iodo-1-(4-methoxybenzyl)-4-phenyl-1H-1,2,3-triazole
(3 b). Yield: 72% (1.38 g), off-white solid; M. p.: 115e117 ꢁC; 1H
NMR (400 MHz, CDCl3)
(d, J ¼ 8.6 Hz, 2H), 6.81 (d, J ¼ 8.7 Hz, 2H), 5.53 (s, 2H), 3.72 (s, 3H);
13C NMR (101 MHz, CDCl3)
159.79 (C), 150.19 (C), 130.32 (C),
d
7.90e7.82 (m, 2H), 7.43e7.28 (m, 3H), 7.22
103e105 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.73(d, J ¼ 8.4 Hz, 2H),
7.49e7.44 (m, 2H), 7.33(d, J ¼ 8.0 Hz, 2H), 7.26e7.23 (m, 6H), 7.05 (s,
d
4H), 6.96 (dd, J ¼ 7.5, 1.9 Hz, 2H), 5.38 (s, 2H), 2.46 (s, 3H); 13C NMR
129.43 (2CH), 128.56 (CH), 128.51 (2CH), 127.49 (2CH), 126.45 (C),
114.30 (2CH), 76.03 (C), 55.31 (OCH3), 54.00 (CH2) ppm; HRMS
(ESI): calcd. for C16H14IN3O [MþH]þ, 392.0215; found: 392.0254.
(101 MHz, CDCl3) d 150.48 (C), 145.76 (C), 144.91 (C), 135.07 (C),
132.48 (C), 132.25 (C), 131.56 (2CH), 130.58 (C), 129.84 (2CH), 128.79
(2CH), 128.51 (2CH), 128.47 (2CH), 128.31 (CH), 127.92 (CH), 127.42
(2CH), 126.85 (C), 126.75 (2CH), 123.27 (2CH), 52.28 (CH2), 21.74
(CH3) ppm; HRMS (ESI): calcd. for C28H23N3O3S [MþH]þ, 482.1494;
found: 482.1534.
4.1.1.3. 1-Benzyl-5-iodo-4-(4-methoxyphenyl)-1H-1,2,3-triazole (3c).
Yield: 80% (1.18 g), off-white solid; M. p.: 107e109 ꢁC; 1H NMR
(400 MHz, CDCl3)
5H), 7.03e6.95 (m, 2H), 5.66 (s, 2H), 3.85 (s, 3H); 13C NMR
(101 MHz, CDCl3) 159.95 (C), 150.18 (C), 134.46 (C), 128.90 (2CH),
128.82 (2CH), 128.46 (CH), 127.82 (2CH), 122.80 (C), 113.99 (2CH),
d
7.91e7.84 (m, 2H), 7.33 (ddd, J ¼ 11.2, 8.5, 6.7 Hz,
4.1.2. General procedure for Suzuki reactions (6a-6e)
A mixture of 3a (500 mg, 1.38 mmol, 1 equiv.), phenylboronic
acid (287 mg, 2.07 mmol, 1.5 equiv.), K2CO3 (476 mg, 3.45 mmol, 2.5
d

8
D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
equiv.), and Pd(PPh3)2Cl2 (97 mg, 0.13 mmol, 0.1 equiv.) in DMF:H2O
(2.5:0.5, 6 mL) was stirred for 4 h at 80 ꢁC. Then the resultant
mixture was poured into H2O (15 mL) and was extracted with
CH2Cl2 (3 ꢃ 15 mL). The combined organic layers were washed with
brine (15 mL) and dried over Na2SO4. The solvent was removed
under vacuum, and the residue was purified by column chroma-
tography [silica gel, 30% EtOAc in petroleum ether] to give (6a-6e);
4.1.3.1. 1-Benzyl-5-(4-(2-chloroethoxy)phenyl)-4-phenyl-1H-1,2,3-
triazole (7a). Yield: 85% (183 mg); 1H NMR (400 MHz, CDCl3)
7.57
d
(dd, J ¼ 8.0, 1.5 Hz, 2H), 7.27e7.21 (m, 6H), 7.05 (dd, J ¼ 7.4, 5.3 Hz,
4H), 6.94 (d, J ¼ 8.7 Hz, 2H), 5.38 (s, 2H), 4.26 (t, 2H), 3.84 (t, 2H). 13
C
NMR (101 MHz, CDCl3)
d 159.19 (C), 144.54 (C), 135.50 (C), 133.55
(C), 131.57 (2CH), 131.02 (C), 128.72 (2CH), 128.44 (2CH), 128.14
(CH), 127.67 (CH), 127.44 (2CH), 126.68 (2CH), 120.45 (C), 115.31
(2CH), 68.08 (CH2), 51.95 (CH2), 41.76 (CH2) ppm; HRMS (ESI): calcd.
for C23H20ClN3O [MþH]þ, 390.1328; found: 390.1371.
4.1.2.1. 4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenol
(6a).
Yield: 85% (385 mg), white solid; M. p.: 180e182 ꢁC; 1H NMR
4.1.3.2. 1-Benzyl-5-(4-(3-chloropropoxy)phenyl)-4-phenyl-1H-1,2,3-
(400 MHz, CDCl3)
d
7.58 (dd, J ¼ 7.8, 1.5 Hz, 2H), 7.28e7.24 (m, 6H),
triazole (7b). Yield: 88% (217 mg); 1H NMR (400 MHz, CDCl3)
d 7.58
7.07 (d, J ¼ 3.6 Hz, 2H), 6.98 (q, J ¼ 8.7 Hz, 4H), 5.42 (s, 2H); 13C NMR
(dd, J ¼ 8.1, 1.4 Hz, 2H), 7.24 (dd, J ¼ 5.0, 2.6 Hz, 6H), 7.04 (d,
(101 MHz, CDCl3)
d 157.50 (C), 144.45 (C), 135.36 (C), 134.08 (C),
J ¼ 8.8 Hz, 4H), 6.94 (d, J ¼ 8.7 Hz, 2H), 5.38 (s, 2H), 4.14 (t, 2H), 3.75
131.57 (2CH), 130.81 (C), 128.73 (2CH), 128.47 (2CH), 128.17 (CH),
127.75 (CH), 127.53 (2CH), 126.75 (2CH), 119.04 (C), 116.42 (2CH),
52.01 (CH2) ppm; HRMS (ESI): calcd. for C16H14IN3O [MþH]þ,
328.1405; found: 328.1443.
(t, 2H), 2.28e2.22 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 159.77 (C),
144.44 (C), 135.60 (C), 133.77 (C), 131.49 (2CH), 131.17 (C), 128.72
(2CH), 128.45 (2CH), 128.12 (CH), 127.64 (CH), 127.47 (2CH), 126.69
(2CH), 119.88 (C), 115.23 (2CH), 64.50 (CH2), 51.90 (CH2), 41.41
(CH2), 32.21 (CH2) ppm; HRMS (ESI): calcd. for C24H22ClN3O
[MþH]þ, 404.1485; found: 404.1524.
4.1.2.2. 4-((4,5-Diphenyl-1H-1,2,3-triazol-1-yl)methyl)phenol (6 b).
Yield: 80% (335 mg), pale-yellow solid; M. p.: 172e173 ꢁC; 1H NMR
(400 MHz, CDCl3)
d 7.58e7.41 (m, 6H), 7.25e7.23 (m, 2H), 7.17 (d,
4.1.3.3. 1-Benzyl-5-(4-(4-chlorobutoxy)phenyl)-4-phenyl-1H-1,2,3-
J ¼ 6.8, 2H), 6.90 (d, J ¼ 8.5, 2H), 6.73 (d, J ¼ 8.5, 2H), 5.32 (s, 2H); 13
C
triazole (7c). Yield: 90% (229 mg); 1H NMR (400 MHz, CDCl3)
d 7.60
NMR (101 MHz, CDCl3)
d 155.95 (C), 144.53 (C), 133.84 (C), 130.75
(dd, J ¼ 8.1, 1.5 Hz, 2H), 7.25 (dt, J ¼ 8.9, 5.1 Hz, 6H), 7.10e7.01 (m,
(C), 130.20 (2CH), 129.75 (C), 129.26 (2CH), 129.21 (2CH), 128.49
(2CH), 127.86 (C), 127.78 (C), 127.16 (C), 126.76 (2CH), 115.69 (2CH),
51.69 (CH2) ppm; HRMS (ESI): calcd. for C21H17N3O [MþH]þ,
328.1405; found: 328.1441.
4H), 6.92 (d, J ¼ 8.8 Hz, 2H), 5.39 (s, 2H), 4.04 (t, 2H), 3.64 (t, 2H),
2.07e1.95 (m, 4H); 13C NMR (126 MHz, CDCl3)
d 159.85 (C), 144.45
(C), 135.56 (C), 133.77 (C), 131.46 (2CH), 131.08 (C), 128.73 (2CH),
128.45 (2CH), 128.13 (CH), 127.64 (CH), 127.47 (2CH), 126.66 (2CH),
119.62 (C), 115.09 (2CH), 67.13 (CH2), 51.89 (CH2), 44.74 (CH2), 29.30
(CH2), 26.61 (CH2) ppm; HRMS (ESI): calcd. for C25H24ClN3O
[MþH]þ, 418.1641; found: 418.1683.
4.1.2.3. 4-(1-Benzyl-5-phenyl-1H-1,2,3-triazol-4-yl)phenol
Yield: 84% (351 mg); pale-yellow solid; M. p.: 166e168 ꢁC; 1H NMR
(400 MHz, CDCl3) 7.47e7.38 (m, 5H), 7.26e7.24 (m, 3H), 7.13 (d,
J ¼ 6.9, 2H), 7.02 (dd, J ¼ 6.6, 2.8, 2H), 6.73 (d, J ¼ 8.7, 2H), 5.40 (s,
2H); 13C NMR (101 MHz, CDCl3)
155.70 (C), 144.53 (C), 135.37 (C),
(6c).
d
4.1.3.4. 1-Benzyl-5-(4-((5-chloropentyl)oxy)phenyl)-4-phenyl-1H-
d
1,2,3-triazole (7d). Yield: 92% (242 mg); 1H NMR (400 MHz, CDCl3)
133.17 (C), 130.18 (2CH), 129.63 (CH), 129.16 (2CH), 128.71 (2CH),
128.32 (2CH), 128.16 (CH), 127.92 (C), 127.52 (2CH), 123.27 (C),
115.53 (2CH), 52.11 (CH2) ppm; HRMS (ESI): calcd. for C21H17N3O
[MþH]þ, 328.1405; found: 328.1414.
d
7.58 (dd, J ¼ 8.1, 1.6 Hz, 2H), 7.26 (t, 6H), 7.04 (d, J ¼ 8.7 Hz, 4H),
6.91 (d, J ¼ 8.8 Hz, 2H), 5.39 (s, 2H), 4.02 (t, 2H), 3.59 (t, 2H),
1.93e1.82 (m, 4H), 1.67 (dd, J ¼ 13.0, 5.8 Hz, 2H). 13C NMR (126 MHz,
CDCl3)
d 159.99 (C), 144.45 (C), 135.59 (C), 133.79 (C), 131.43 (2CH),
131.13 (C), 128.70 (2CH), 128.41 (2CH), 128.10 (CH), 127.59 (CH),
127.47 (2CH), 126.66 (2CH), 119.56 (C), 115.11 (2CH), 67.76 (CH2),
51.87 (CH2), 44.83 (CH2), 32.30 (CH2), 28.53 (CH2), 23.56 (CH2) ppm;
HRMS (ESI): calcd. for C26H26ClN3O [MþH]þ, 432.1798; found:
432.1822.
4.1.2.4. 4-(1-(4-Methoxybenzyl)-4-(4-methoxyphenyl)-1H-1,2,3-
triazol-5-yl)phenol (6d). Yield: 88% (404 mg), yellow solid; M. p.:
127e129 ꢁC; 1H NMR (400 MHz, CDCl3)
d
7.49 (d, J ¼ 8.9, 2H), 6.99
(d, J ¼ 9.1, 6H), 6.78 (dd, J ¼ 12.2, 8.8, 4H), 5.33 (s, 2H), 3.76 (d,
J ¼ 4.2, 6H);13C NMR (101 MHz, CDCl3)
d 159.47 (C), 159.22 (C),
4.1.3.5. 1-(4-(3-Chloropropoxy)benzyl)-4,5-diphenyl-1H-1,2,3-
157.75 (C), 144.30 (C), 133.22 (C), 131.62 (2CH), 129.11 (2CH), 128.05
(2CH), 127.52 (C), 123.54 (C), 119.02 (C), 116.45 (2CH), 114.07 (2CH),
113.94 (2CH), 55.28 (OCH3), 55.21 (OCH3), 51.55 (CH2). ppm; HRMS
(ESI): calcd. for C23H21N3O3 [MþH]þ, 388.1616; found: 388.1657.
triazole (7e). Yield: 87% (214 mg); 1H NMR (400 MHz, CDCl3)
d 7.54
(dd, J ¼ 7.7, 1.8, 2H), 7.47 (dd, J ¼ 14.3, 7.3, 2H), 7.26e7.23 (m, 4H),
7.16 (d, J ¼ 6.8, 2H), 6.96 (d, J ¼ 8.6, 2H), 6.77 (d, J ¼ 8.7, 2H), 5.34 (s,
2H), 4.07 (t, 2H), 3.73 (t, 2H), 2.24e2.18 (m, 2H); 13C NMR (101 MHz,
CDCl3)
d 158.61 (C), 144.55 (C), 133.70 (C), 130.99 (C), 130.19 (2CH),
4.1.2.5. Synthesis of 4-(5-(4-methoxyphenyl)-4H-1,2,3-triazol-4-yl)
129.67 (CH), 129.18 (2CH), 129.10 (2CH), 128.43 (2CH), 128.04 (C),
127.72 (C), 127.67 (CH), 126.73 (2CH), 114.65 (2CH), 64.35 (CH2),
51.59 (CH2), 41.43 (CH2), 32.22 (CH2) ppm; HRMS (ESI): calcd. for
phenol (6e). Yield: 30% (21 mg), semi-solid; 1H NMR (400 MHz,
CDCl3)
d
7.46 (d, J ¼ 15.1, 4H), 6.88 (d, J ¼ 15.9, 4H), 3.84 (s, 3H); ESI-
MS (m/z): 268.25 [MþH]þ, 374.35 [MþH]þ; HRMS (ESI): calcd. for
C
24H22ClN3O [MþH]þ, 404.1485; found: 404.1519.
C
15H13N3O2 [MþH]þ, 268.1041; found: 268.1078.
4.1.3.6. 5-(4-(3-Chloropropoxy)phenyl)-1-(4-methoxybenzyl)-4-(4-
4.1.3. General procedure for the synthesis of desired halide with
different aliphatic chains (7a-7f)
Compound 6 (200 mg, 6.11 mmol, 1.0 equiv.) was alkylated with
ώ-dihaloalkanes (152 mL, 1.83 mmol, 3.0 equiv.) in the presence of
anhydrous K2CO3 (337 mg, 2.44 mmol, 4.0 equiv.), in dry acetone
(10 mL), was refluxed for 4e5 h. On completion, the reaction
mixture was cooled and filtered. The filterate was concenterated
and was purified by column chromatography [silica gel, 30% EtOAc
in petroleum ether] to give (7) as yellow oil.
methoxyphenyl)-1H-1,2,3-triazole (7f). Yield: 91% (217 mg); 1H
NMR (400 MHz, CDCl3)
d
7.48 (d, J ¼ 8.8, 2H), 7.06 (d, J ¼ 8.7, 2H),
6.97 (dd, J ¼ 18.1, 8.6, 4H), 6.79 (dd, J ¼ 8.7, 7.1, 4H), 5.31 (s, 2H), 4.17
(t, 2H), 3.80 (d, J ¼ 6.3, 2H), 3.77 (s, 6H), 2.33e2.24 (m, 2H);13C NMR
(126 MHz, CDCl3) d 159.61 (C), 159.38 (C), 159.11 (C), 144.36 (C),
132.74 (C), 131.57 (2CH), 129.00 (2CH), 127.96 (2CH), 127.64 (C),
123.71 (C), 120.08 (C), 115.10 (2CH), 114.01 (2CH), 113.87 (2CH),
64.32 (CH2), 55.29 (OCH3), 55.21 (OCH3), 51.45 (CH2), 41.47 (CH2),
32.17 (CH2) ppm; HRMS (ESI): calcd. for C26H26ClN3O3 [MþH]þ,

D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
9
464.1696; found: 464.1743.
HRMS (ESI): calcd. for C28H30N4O [MþH]þ, 439.2453; found:
439.2496.
4.1.4. General procedure for amination (8a-8p)
A solution of 7a (100 mg, 0.25 mmol, 1.0 equiv.), piperidine
4.1.4.6. 3-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)-
(101
mL, 1.02 mmol, 4.0 equiv.), and tetrabutylammonium iodide
N,N-diethylpropan-1-amine (8f). Yield: 70% (76 mg); 1H NMR
(92 mg, 0.25 mmol, 1.0 equiv.) in dry DMF (1.5 mL) was heated at
70e75 ꢁC with stirring for 7 h. The reaction mixture was diluted
with ethyl acetate, washed with water, dried over sodium sulphate
and concentrated. The concentrate was chromatographed on basic
alumina using 10% MeOH/DCM obtained as light-brown oil.
(400 MHz, CDCl3)
d
7.56 (dd, J ¼ 8.0,1.6, 2H), 7.28e7.21 (m, 6H), 7.05
(d, J ¼ 8.7, 4H), 6.91 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.11 (t, 2H), 3.05 (t,
2H), 2.98 (q, J ¼ 7.2, 4H), 2.28e2.21 (m, 2H), 1.31 (t, 6H); 13C NMR
(101 MHz, CDCl3)
d 159.53 (C), 144.50 (C), 135.55 (C), 133.67 (C),
131.53 (2CH), 131.11 (C), 128.73 (2CH), 128.43 (2CH), 128.14 (CH),
127.64 (CH), 127.45 (2CH), 126.69 (2CH), 120.06 (C), 115.16 (2CH),
65.59 (CH2), 51.93 (CH2), 49.30 (CH2), 47.02 (2CH2), 25.02 (CH2),
9.81 (2CH3) ppm; HRMS (ESI): calcd. for C28H32N4O [MþH]þ,
441.2610; found: 441.2647.
4.1.4.1. 1-(2-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
ethyl)piperidine (8a). Yield: 70% (79 mg); 1H NMR (400 MHz,
CDCl3)
d
7.57 (dd, J ¼ 8.1, 1.6, 2H), 7.28e7.24 (m, 6H), 7.07e7.02 (m,
4H), 6.95e6.91 (m, 2H), 5.39 (s, 2H), 4.17 (t, 2H), 2.85 (t, 2H), 2.58 (s,
4H), 1.68e1.63 (m, 4H), 1.51e1.47 (m, 2H); 13C NMR (101 MHz,
4.1.4.7. 1-(3-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
CDCl3)
d
159.80 (C), 144.49 (C), 135.60 (C), 133.77 (C), 131.44 (2CH),
propyl)piperidine (8g). Yield: 71% (79 mg); 1H NMR (400 MHz,
131.15 (C), 128.73 (2CH), 128.44 (2CH), 128.13 (CH), 127.62 (CH),
127.50 (2CH) 126.69 (2CH), 119.77 (C), 115.30 (2CH), 66.09 (CH2),
57.83 (CH2), 55.14 (2CH2), 51.93 (CH2), 25.78 (2CH2), 24.07 (CH2)
ppm. HRMS (ESI): calcd. for C28H30N4O [MþH]þ, 439.2498; found:
439.2489.
CDCl3)
d
7.57 (dd, J ¼ 8.0, 1.6, 2H), 7.28e7.23 (m, 6H), 7.07e7.02 (m,
4H), 6.91 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.07 (t, 2H), 2.65 (t, 2H), 2.57
(s, 4H), 2.14e2.07 (m, 2H), 1.73e1.68 (m, 4H), 1.50 (t, 2H); 13C NMR
(101 MHz, CDCl3)
d 160.04 (C), 144.44 (C), 135.62 (C), 133.82 (C),
131.41 (2CH), 131.19 (C), 128.70 (2CH), 128.41 (2CH), 128.09 (CH),
127.58 (CH), 127.48 (2CH), 126.67 (2CH), 119.56 (C), 115.21 (2CH),
66.57 (CH2), 55.82 (CH2), 54.58 (2CH2), 51.88 (CH2), 26.56 (CH2),
25.70 (2CH2), 24.24 (CH2) ppm; HRMS (ESI): calcd. for C29H32N4O
[MþH]þ, 453.2610; found: 453.2661.
4.1.4.2. 1-Benzyl-4-phenyl-5-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-
1H-1,2,3-triazole (8b). Yield: 72% (78 mg); 1H NMR (400 MHz,
CDCl3)
4H), 6.94 (d, J ¼ 8.8, 2H), 5.39 (s, 2H), 4.24 (t, 2H), 3.08 (t, 2H), 2.84
(s, 4H), 1.93e1.90 (m, 3H); 13C NMR (101 MHz, CDCl3)
159.55 (C),
d
7.57 (dd, J ¼ 8.0, 1.5, 2H), 7.29e7.23 (m, 6H), 7.06e7.03 (m,
d
4.1.4.8. 4-(3-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
144.51 (C), 135.57 (C), 133.68 (C), 131.49 (2CH), 131.13 (CH), 128.72
(2CH), 128.42 (2CH), 128.12 (CH), 127.61 (CH), 127.48 (2CH), 126.68
(2CH), 120.05 (C), 115.28 (2CH), 66.52 (CH2), 54.84 (CH2), 54.78
(2CH2), 51.92 (CH2), 23.50 (2CH2) ppm; HRMS (ESI): calcd. for
propyl)morpholine (8h). Yield: 69% (78 mg); 1H NMR (400 MHz,
CDCl3)
d
7.57 (dd, J ¼ 7.9, 1.5, 2H), 7.27e7.24 (m, 6H), 7.07e7.03 (m,
4H), 6.92 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.07 (t, 2H), 3.73 (t, 4H), 2.56 (t,
2H), 2.50 (s, 4H), 2.05e1.98 (m, 2H); 13C NMR (101 MHz, CDCl3)
C
27H28N4O [MþH]þ, 425.2297; found: 425.2342.
d 160.01 (C), 144.46 (C), 135.61 (C), 133.79 (C), 131.44 (2CH), 131.16
(C), 128.71 (2CH), 128.41 (2CH), 128.10 (CH), 127.60 (CH), 127.46
(2CH), 126.69 (2CH), 119.62 (C), 115.17 (2CH), 66.94 (2CH2), 66.30
(CH2), 55.52 (CH2), 53.76 (2CH2), 51.87 (2CH2), 26.38 (CH2) ppm;
HRMS (ESI): calcd. for C28H30N4O2 [MþH]þ, 455.2402; found:
455.2446.
4.1.4.3. 4-(2-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
ethyl)morpholine (8c). Yield: 67% (75 mg); 1H NMR (400 MHz,
CDCl3)
4H), 6.93 (d, J ¼ 8.8, 2H), 5.39 (s, 2H), 4.16 (t, 2H), 3.76 (t, 4H), 2.85 (t,
2H), 2.62 (t, 4H); 13C NMR (101 MHz, CDCl3)
159.75 (C), 144.51 (C),
d
7.57 (dd, J ¼ 8.0, 1.6, 2H), 7.28e7.24 (m, 6H), 7.05 (d, J ¼ 8.7,
d
135.61 (C), 133.69 (C), 131.47 (2CH), 131.17 (C), 128.69 (2CH), 128.39
(2CH), 128.10 (CH), 127.60 (CH), 127.46 (2CH), 126.70 (2CH), 119.97
(C), 115.28 (2CH), 66.91 (2CH2), 66.07 (CH2), 57.62 (CH2), 54.18
(2CH2), 51.90 (CH2) ppm; HRMS (ESI): calcd. for C27H28N4O2
[MþH]þ, 441.2246; found: 441.2294.
4.1.4.9. 1-Benzyl-4-phenyl-5-(4-(4-(pyrrolidin-1-yl)butoxy)phenyl)-
1H-1,2,3-triazole (8i). Yield: 70% (75 mg); 1H NMR (400 MHz,
CDCl3)
d
7.57 (dd, J ¼ 8.0, 1.5, 2H), 7.28e7.24 (m, 6H), 7.07e7.03 (m,
4H), 6.90 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.05 (t, 2H), 3.31 (s, 4H),
3.18e3.14 (m, 2H), 2.18 (t, 6H), 1.927e1.90 (m, 4H); 13C NMR
(101 MHz, CDCl3)
d 159.60 (C), 144.50 (C), 135.58 (C), 133.68 (C),
4.1.4.4. 2-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)-
131.54 (2CH), 131.14 (C), 128.74 (2CH), 128.43 (2CH), 128.14 (CH),
127.63 (CH), 127.46 (2CH), 126.70 (2CH), 120.02 (C), 115.13 (2CH),
67.01 (CH2), 55.43 (CH2), 53.77 (2CH2), 51.91 (CH2), 26.78 (CH2),
23.41 (2CH2), 22.98 (CH2) ppm; HRMS (ESI): calcd. for C29H32N4O
[MþH]þ, 453.2610; found: 453.2649.
N,N-diethylethanamine (8d). Yield: 71% (77 mg); 1H NMR
(400 MHz, CDCl3)
d
7.57 (dd, J ¼ 8.0,1.6, 2H), 7.28e7.23 (m, 6H), 7.05
(d, J ¼ 8.7, 4H), 6.93 (d, J ¼ 8.8, 2H), 5.39 (s, 2H), 4.20 (t, 2H), 3.06 (t,
2H), 2.83 (q, J ¼ 7.1, 4H), 1.18 (t, 6H); 13C NMR (101 MHz, CDCl3)
d
159.49 (C), 144.52 (C), 135.57 (C), 133.67 (C), 131.50 (2CH), 131.13
(C), 128.72 (2CH), 128.42 (2CH), 128.12 (CH), 127.61 (CH), 127.47
(2CH), 126.68 (2CH), 120.07 (C), 115.25 (2CH), 65.96 (CH2), 51.93
(CH2), 51.60 (CH2), 47.89 (2CH2), 11.06 (2CH3) ppm; HRMS (ESI):
calcd. for C27H30N4O [MþH]þ, 427.2453; found: 427.2489.
4.1.4.10. 4-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)-
N,N-diethylbutan-1-amine (8j). Yield: 71% (77 mg); 1H NMR
(400 MHz, CDCl3)
d
7.57 (dd, J ¼ 8.0, 1.5, 2H), 7.28e7.24 (m, 6H),
7.07e7.04 (m, 4H), 6.90 (d, J ¼ 8.7, 2H), 5.40 (s, 2H), 4.05 (t, 2H), 3.16
(q, J ¼ 7.3, 4H), 3.09 (t, 2H), 2.12e2.04 (m, 2H), 1.95e1.88 (m, 2H),
4.1.4.5. 1-Benzyl-4-phenyl-5-(4-(3-(pyrrolidin-1-yl)propoxy)
1.41 (t, 6H); 13C NMR (101 MHz, CDCl3)
d 159.60 (C), 144.50 (C),
phenyl)-1H-1,2,3-triazole (8e). Yield: 73% (79 mg); 1H NMR
135.57 (C), 133.67 (C), 131.55 (2CH), 131.11 (C), 128.73 (2CH), 128.43
(2CH), 128.13 (CH), 127.63 (CH), 127.44 (2CH), 126.69 (2CH), 120.02
(C), 115.08 (2CH), 67.00 (CH2), 51.91 (CH2), 51.39 (CH2), 46.59
(2CH2), 26.64 (CH2), 20.87 (CH2), 8.64 (2CH3) ppm; HRMS (ESI):
calcd. for C29H34N4O [MþH]þ, 455.2766; found: 455.2804.
(400 MHz, CDCl3)
d
7.56 (dd, J ¼ 8.0, 1.6, 2H), 7.27e7.22 (m, 6H), 7.05
(d, J ¼ 8.8, 4H), 6.91 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.13 (t, 2H), 3.19 (t,
4H), 2.42e2.35 (m, 2H), 2.12 (t, 2H); 13C NMR (101 MHz, CDCl3)
d
159.35 (C), 144.52 (C), 135.54 (C), 133.61 (C), 131.57 (2CH), 131.12
(C), 128.73 (2CH), 128.42 (2CH), 128.14 (CH), 127.63 (CH), 127.45
(2CH), 126.70 (2CH), 120.29 (C), 115.17 (2CH), 65.24 (CH2), 53.98
(2CH2), 53.08 (CH2), 51.93 (CH2), 26.18 (CH2), 23.45 (2CH2) ppm;
4.1.4.11. 1-(4-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
butyl)piperidine (8k). Yield: 70% (78 mg); 1H NMR (400 MHz,

10
D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
CDCl3)
d
7.57 (dd, J ¼ 8.1, 1.5, 2H), 7.28e7.24 (m, 6H), 7.05 (dt, J ¼ 4.3,
(C), 144.33 (C), 132.81 (C), 131.50 (2CH), 129.00 (2CH), 127.93 (2CH),
127.69 (C), 123.79 (C), 119.79 (C), 115.10 (2CH), 114.00 (2CH), 113.84
(2CH), 66.34 (CH2), 55.29 (OCH3), 55.20 (OCH3), 54.19 (2CH), 53.10
(CH2), 51.41 (CH2), 28.44 (CH2), 23.45 (2CH) ppm; HRMS (ESI):
calcd. for C30H34N4O3 [MþH]þ, 499.2664; found: 499.2692.
3.0, 4H), 6.90 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.05 (t, 2H), 3.01 (dd,
J ¼ 25.2, 17.0, 6H), 2.20e2.03 (m, 6H), 1.93e1.86 (m, 2H), 1.70 (s,
2H); 13C NMR (101 MHz, CDCl3)
d 159.66 (C), 144.52 (C), 135.60 (C),
133.71 (C), 131.56 (2CH), 131.15 (C), 128.76 (2CH), 128.45 (2CH),
128.15 (CH), 127.64 (CH), 127.48 (2CH), 126.72 (2CH), 120.01 (C),
115.14 (2CH), 67.11 (CH2), 57.44 (CH2), 53.43 (2CH2), 51.93 (CH2),
26.80 (CH2), 22.80 (2CH2), 22.31 (CH2) ppm; HRMS (ESI): calcd. for
4.1.4.17. N,N-diethyl-3-(4-(1-(4-methoxybenzyl)-4-(4-
methoxyphenyl)-1H-1,2,3-triazol-5-yl)phenoxy)-propan-1-amine
C
30H34N4O [MþH]þ, 467.2766; found: 467.2810.
(8q). Yield: 73% (79 mg); 1H NMR (400 MHz, CDCl3)
d 7.48 (d,
J ¼ 8.8 Hz, 2H), 7.04 (d, J ¼ 8.6 Hz, 2H), 6.96 (dd, J ¼ 18.3, 8.6 Hz, 4H),
6.78 (dd, J ¼ 8.5, 6.2 Hz, 4H), 5.31 (s, 2H), 4.07 (t, J ¼ 6.3 Hz, 2H), 3.77
(s, 6H), 2.67e2.62 (m, 2H), 2.57 (dd, J ¼ 14.3, 7.1 Hz, 4H), 1.46 (dt,
J ¼ 15.4, 7.8 Hz, 2H), 1.05 (t, J ¼ 7.1 Hz, 6H); 13C NMR (101 MHz,
4.1.4.12. 4-(4-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
butyl)morpholine (8l). Yield: 68% (76 mg); 1H NMR (400 MHz,
CDCl3)
d
1H NMR (400 MHz, CDCl3)
d
7.57 (dd, J ¼ 8.0, 1.5, 2H),
7.28e7.24 (m, 6H), 7.07e7.03 (m, 4H), 6.91 (d, J ¼ 8.7, 2H), 5.39 (s,
CDCl3) d 161.40 (C), 160.85 (C), 160.55 (C), 145.73 (C), 134.25 (C),
2H), 4.03 (t, 2H), 3.72 (t, 4H), 2.49e2.42 (m, 6H), 1.89e1.83 (m, 2H),
132.90 (2CH), 130.40 (2CH), 129.34 (2CH), 129.14 (C), 125.30 (C),
121.19 (C), 116.57 (2CH), 115.45 (2CH), 115.28 (2CH), 67.88 (OCH2),
56.67(OCH3), 56.59 (OCH3), 52.82 (CH2), 50.79 (CH2), 48.47 (2CH2),
28.37 (CH2), 13.07 (2CH3) ppm; ESI-MS (m/z): 501.0 [MþH]þ.
1.75e1.69 (m, 2H); 13C NMR (101 MHz, CDCl3)
d 160.06 (C), 144.46
(C), 135.62 (C), 133.80 (C), 131.44 (2CH), 131.17 (C), 128.70 (2CH),
128.40 (2CH), 128.09 (CH), 127.59 (CH), 127.47 (2CH), 126.69 (2CH),
119.57 (C), 115.13 (2CH), 67.86 (CH2), 66.92 (2CH2), 58.57 (CH2),
53.70 (2CH2), 51.88 (CH2), 27.19 (CH2), 23.03 (CH2) ppm; HRMS
(ESI): calcd. for C29H32N4O2 [MþH]þ, 469.2559; found: 469.2591.
4.2. Experimental protocols
4.2.1. Cell lines, cell culture, growth conditions and reagents
4.1.4.13. 1-Benzyl-4-phenyl-5-(4-((5-(pyrrolidin-1-yl)pentyl)oxy)
A panel of human cancer cell lines namely MCF-7(Breast), HCT-
116(Colon) [27e29], MDA-MB-231(Breast), FR-2 (normal breast
epithelial) were purchased from ATCC for screening of (6a-6d) and
(8a-8s) anticancer test molecules. The cell lines were grown in T75
tissue culture flasks in complete growth medium (RPMI-1640 and
phenyl)-1H-1,2,3-triazole (8 m). Yield: 73% (79 mg); 1H NMR
(400 MHz, CDCl3)
d
7.58 (dd, J ¼ 8.0, 1.5, 2H), 7.28e7.24 (m, 6H),
7.07e7.04 (m, 4H), 6.90 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.01 (t, 2H), 3.31
(s, 4H), 3.10 (t, 2H), 2.18 (s, 4H), 2.05e2.01 (m, 2H), 1.90e1.86 (m,
2H), 1.64e1.60 (m, 2H); 13C NMR (101 MHz, CDCl3)
d
159.88 (C),
DMEM) added with 10% FBS, 100 mg/mL streptomycin as well as 100
144.45 (C), 135.59 (C), 133.79 (C), 131.48 (2CH), 131.16 (C), 128.72
(2CH), 128.41 (2CH), 128.11 (CH), 127.60 (CH), 127.47 (2CH), 126.71
(2CH), 119.72 (C), 115.16 (2CH), 67.41 (CH2), 55.49 (CH2), 53.72
(2CH2), 51.89 (CH2), 28.59 (CH2), 25.40 (CH2), 23.67 (CH2), 23.39
(2CH2) ppm; HRMS (ESI): calcd. for C30H34N4O [MþH]þ, 467.2766;
found: 467.2802.
units/mL penicillin in humidified carbon dioxide incubator (New
Brunswick, Galaxy 170R, Eppendorf) at 37 ꢁC, 5% CO2 with 95%
relative humidity. DAPI (40, 6-diamidino-2-phenylindole),
Rhodamine-123, DCFDA (20, 70-dichlorofluorescein diacetate) were
procured from Sigma-Aldrich (St. Louis, MO, USA). Sulforhodamine
B dye purchased from Hi Media. Monolayer cultures of the above
cell lines were trypsinised using 0.25% trypsin/EDTA (1 mM) solu-
tion. After the cells got detached, the activity of trypsin/EDTA so-
lution was stoppedusing complete growth medium and centrifuged
at 1000 rpm for 5 min. Cells were again dispersed in complete
growth medium in tissue culture flasks and incubated in CO2
incubator. When cells attained approx. 50e60% confluency, they
were treated with target compounds dissolved in DMSO and the
untreated control cultures with (DMSO, < 0.2%).
4.1.4.14. 1-(5-(4-(1-Benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phenoxy)
pentyl)piperidine (8n). Yield: 72% (80 mg); 1H NMR (400 MHz,
CDCl3)
d
7.57 (dd, J ¼ 8.0, 1.4, 2H), 7.28e7.24 (m, 6H), 7.07e7.03 (m,
4H), 6.90 (d, J ¼ 8.7, 2H), 5.39 (s, 2H), 4.00 (t, 2H), 2.78 (s, 4H), 2.69
(t, 2H), 2.28 (t, 2H), 1.85e1.79 (m, 6H), 1.58e1.52 (m, 4H); 13C NMR
(126 MHz, CDCl3)
d
¼ 160.01 (C), 144.42 (C), 135.58 (C), 133.82 (C),
131.42 (2CH), 131.13 (C), 128.71 (2CH), 128.41 (2CH), 128.10 (CH),
127.59 (2CH), 126.66 (2CH), 119.49 (C), 115.11 (2CH), 67.71 (CH2),
58.57 (CH2), 54.11 (2CH2), 51.87 (CH2), 28.91 (CH2), 25.39 (CH2),
24.53 (2CH2), 23.96 (CH2), 23.49 (CH2) ppm; HRMS (ESI): calcd. for
4.2.2. Cytotoxicity activity against different cancer cell lines
The in vitro cytotoxicity activity of target compounds (6a-6d)
and (8a-8p) were carried out using SRB (Sulforhodamine B) assay
method reported by Skehan et al. [30] For preliminary screening,
optimum inoculum densities per well of MCF-7 (7000), MDA-MB-
231 (10,000) and HCT-116 (7000) cell lines were seeded in 96-
well flat bottom plates (NUNC). Tamoxifen citrate used as stan-
C
31H36N4O [MþH]þ, 481.2923; found: 481.2963.
4.1.4.15. 4,5-Diphenyl-1-(4-(3-(pyrrolidin-1-yl)propoxy)benzyl)-1H-
1,2,3-triazole (8o). Yield: 70% (76 mg); 1H NMR (400 MHz, CDCl3)
d
7.54 (dd, J ¼ 7.7,1.9, 2H), 7.50e7.45 (m, 2H), 7.25e7.24 (m, 4H), 7.16
(d, J ¼ 6.8, 2H), 6.97 (d, J ¼ 8.5, 2H), 6.74 (d, J ¼ 8.6, 2H), 5.33 (s, 2H),
dard drug for reference. Briefly, 100 mL/well of cell suspensions
4.04 (t, 2H), 3.84 (s, 2H), 3.27 (s, 2H), 2.83 (s, 2H), 2.42 (s, 2H), 2.25
were seeded in 96-well tissue culture plates and incubated for 24 h.
When cells attained 50e60% confluency, then different concen-
trations of anticancer test compounds were incubated and kept for
another 48 h. Next, after the completion of 48 h incubation, the
cells were settled using 50% ice cold trichloroacetic acid (TCA) and
kept at 4 ꢁC for 1 h and the plates were washed thrice in aqueous
(s, 2H), 2.09 (s, 2H); 13C NMR (101 MHz, CDCl3)
d 158.10 (C), 144.54
(C), 133.75 (C), 130.93 (C), 130.16 (2CH), 129.75 (CH), 129.23 (2CH),
129.21 (2CH), 128.45 (2CH), 128.21 (C), 127.96 (C), 127.72 (CH),
126.71 (2CH), 114.54 (2CH), 64.78 (CH2), 53.92 (2CH2), 53.27 (CH2),
51.48 (CH2), 25.76 (CH2), 23.45 (2CH2) ppm; HRMS (ESI): calcd. for
C
28H30N4O [MþH]þ, 439.2453; found: 439.2491.
medium and air dried. Once the plates dried, 100 mL/well SRB dye
added and kept for half an hour at room temperature. Soon after,
the plates were again washed thrice using 1% glacial acetic acid to
eliminate excess unbound SRB and the plates were further air dried.
When the plates were completely dried, the bound SRB was solu-
bilized by adding 100mL/well 10 mM TRIS (tris(hydroxymethyl)
aminomethane) buffer of pH 10.5 and plates were kept on orbital
shaker for 5 min. Lastly, absorbance was recorded at 540 nm in
4.1.4.16. 1-(4-Methoxybenzyl)-4-(4-methoxyphenyl)-5-(4-(3-(pyrro-
lidin-1-yl)propoxy)phenyl)-1H-1,2,3-triazole (8p). Yield: 75%
(80 mg); 1H NMR (400 MHz, CDCl3)
d
7.48 (d, J ¼ 8.9, 1H), 7.08e6.89
(m, 3H), 6.83e6.74 (m, 2H), 5.31 (s, 1H), 4.09 (t, 1H), 3.77 (s, 3H),
2.78e2.70 (m, 1H), 2.65 (s, 2H), 2.13e2.06 (m, 1H), 1.87e1.81 (m,
2H); 13C NMR (126 MHz, CDCl3)
d
¼ 159.83 (C), 159.37 (C), 159.08

D. Dheer et al. / European Journal of Medicinal Chemistry 207 (2020) 112813
11
microplate reader (Tecan Infinite M Nano). IC50 was determined by
4.2.7. Measurement of loss of mitochondrial membrane potential
DJm)
Mitochondrial perturbation occurs due to loss of membrane
potential was studied using dye rhodamine-123 by fluorescence
microscope qualitatively. Human breast cancer (MCF-7) cells
(1ˣ105/mL/well) were seeded in a 6-well tissue culture plates and
Graph Pad Prism 6.0 (Graph Pad Software Inc, San Diego, CA, USA)
software. Percent cell viability and percent inhibition was calcu-
lated using the following formula and were tested at least in trip-
licates [31].
(
% Growth inhibition ¼ 100 - [Absorbance of treated cells/Absor-
treated with 3.5 mM concentration of molecule 8f and tamoxifen
used as standard positive control drug and incubated for 48 h. After
bance of control cells x
100]
treatment, cells were washed with ice cold PBS, later final con-
centration of 0.2
mM rhodamine incubated to all the wells and
finally kept for 20e30 min in dark inside the incubator. Further,
washed with cold PBS and final volume of incomplete media was
added to all the wells and analyzed under fluorescence microscope
(Olympus, 1 ꢃ 70) [35].
4.2.3. Treatment profile and dose selection
On the basis of the data obtained from the screening profile of
different molecules, 8f molecule possesses good anticancer activity
against MCF-7 cell line bearing IC50 value of 3.5
current study we have chosen 3.5 M as an impressive dose of the
anticancer test molecule 8f for 48 h in addition to negative control
(untreated) as well as 9.5 M of standard moiety, tamoxifen citrate
mM. Therefore, in
m
4.2.8. Statistical analysis
The results are interpreted as mean of SD belonging to three
independent tests. Standard deviations were calculated using
Graphpad prism 6.0 (Graph Pad Software Inc., San Diego, CA, USA)
and Microsoft excel.
m
in 6-well tissue culture plates. By taking the above mentioned
doses, further mechanistic studies were performed against MCF-
7 cell line for 48 h time period.
4.2.9. Molecular docking studies
4.2.4. Morphological examination of compound 8f under bright
field microscopy
Morphological examination through bright field microscopy is
the simplest technique to identify the surface morphological
changes of cancer cells after the treatment with anticancer test
molecules by comparing with untreated cells. In this experiment,
The crystal structure of human ER-a (PDB ID: 3ERT) [31e36] [31]
was retrieved from the protein data bank and was subjected to
protein preparation steps under default settings in Schrodinger
software. The ligands were drawn in ChemDraw. The docking
protocol was validated by re-docking the co-crystallized ligand and
comparing its interaction pattern. All steps of docking were as per
default settings of Glide module of Schrodinger software.
MCF-7 cells were treated with IC50 3.5
mM of 8f for 48 h in addition
to untreated control as well as 9.5 M of standard moiety, tamox-
m
ifen in 6-well tissue culture plates. After the treatment, compound
containing exhausted media was discarded and cells were washed
with ice cold PBS. Further, all the wells were incubated with
incomplete growth medium and cells were imaged under bright
field inverted microscope (Olympus 10X) (Fig. 3) [32].
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.2.5. Apoptosis assay through DAPI staining
Acknowledgement
In order to examine apoptotic cell death qualitatively,
morphological variations in chromatin structure were detected
using DAPI staining. Precisely, MCF-7 cells at a density of 1ˣ105 were
seeded in a 6-well tissue culture plates and kept for 24 h to attain
confluency about 50e60% and treatment was given to the plate
with above mentioned concentration of compound 8f and tamox-
ifen used as standard positive control drug kept for another 48 h
incubation. Later, cells were washed with ice cold PBS to remove
dead cell moieties. Again cells were fixed using 70% ethanol for
1 h at room temperature and washed with cold PBS. Finally, the
D.D., D.S. thank to CSIR-New Delhi, ICMR, and C.B., M.D. thank
UGC, India for supporting research fellowship. This research work
was financially supported by DST-EMR/2016/002304 and DST-EEQ/
2016/000102.
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