Synthesis and biological evaluation of some novel triazole hybrids of curcumin mimics and their selective anticancer activity against breast and prostate cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2016.07.053

The anti-cancer property of curcumin, an active component of turmeric, is limited due to its poor solubility, stability and bioavailability. To enhance its efficacy, we designed a novel series of twenty-four monocarbonyl curcumin analogue–1,2,3-triazole c

Full text of DOI:10.1016/j.bmcl.2016.07.053

Accepted Manuscript
Synthesis and biological evaluation of some novel triazole hybrids of curcumin
mimics and their selective anticancer activity against breast and prostate cancer
cell lines
Dhanaraju Mandalapu, Karan S. Saini, Sonal Gupta, Vikas Sharma, Mohd.
Yaseen Malik, Swati Chaturvedi, Veenu Bala, Hamidullah, Subhadra Thakur,
Jagdamba P. Maikhuri, Muhammad Wahajuddin, Rituraj Konwar, Gopal Gupta,
Vishnu Lal Sharma
PII:
S0960-894X(16)30781-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.053
BMCL 24097
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
31 March 2016
27 June 2016
22 July 2016
Please cite this article as: Mandalapu, D., Saini, K.S., Gupta, S., Sharma, V., Yaseen Malik, Mohd., Chaturvedi, S.,
Bala, V., Hamidullah, Thakur, S., Maikhuri, J.P., Wahajuddin, M., Konwar, R., Gupta, G., Sharma, V.L., Synthesis
and biological evaluation of some novel triazole hybrids of curcumin mimics and their selective anticancer activity
against breast and prostate cancer cell lines, Bioorganic & Medicinal Chemistry Letters (2016), doi:http://dx.doi.org/
1
0.1016/j.bmcl.2016.07.053
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Synthesis and biological evaluation of some novel triazole hybrids of curcumin
mimics and their selective anticancer activity against breast and prostate
#
cancer cell lines
a
b
a, e
b
c
Dhanaraju Mandalapu, Karan S. Saini, Sonal Gupta, Vikas Sharma, Mohd. Yaseen Malik, Swati
c
a,
e
b
d
b
Chaturvedi, Veenu Bala,
Wahajuddin, Rituraj Konwar, Gopal Gupta, Vishnu Lal Sharma *
Hamidullah, Subhadra Thakur, Jagdamba P. Maikhuri, Muhammad
c
b
b
a,e
a
b
c
Medicinal & Process Chemistry Division, Endocrinology Division, Pharmacokinetics and Metabolism Division,
d
CSIR-Central Drug Research Institute, Sitapur road, Lucknow-226031 (India), Medicinal Chemistry, National
Institute of Pharmaceutical Education and Research (NIPER), Raebareli-229010 (India), Academy of Scientific and
e
Innovative Research (AcSIR), New Delhi-110001 (India).
Abstract:
The anti-cancer property of curcumin, an active component of turmeric, is limited due to its poor
solubility, stability and bioavailability. To enhance its efficacy, we designed a novel series of
twenty-four monocarbonyl curcumin analogue–1,2,3-triazole conjugates and evaluated their anti-
cancer activity towards endocrine related cancers. The new compounds (17-40) were synthesized
through CuAAC click reaction and SAR analysis carried out. Out of these all, compound 17
showed most significant anti-cancer activity against prostate cancer cells with IC50 values of
8
.8μM and 9.5μM in PC-3 and DU-145 cells respectively. Another compound 26 showed
significant anti-cancer activity against breast cancer cells with IC50 of 6 μM, 10 μM and 6.4 μM
in MCF-7, MDA-MB-231 and 4T1 cells, respectively while maintaining low toxicity towards
non-cancer originated cell line, HEK-293. Compound 17 and 26 arrested cell cycle and induced
mitochondria-mediated apoptosis in cancer cells. Further, both of these compounds significantly
down-regulated cell proliferation marker (PCNA), inhibited activation of cell survival protein
(
Akt phosphorylation), upregulated pro-apoptotic protein (Bax) and down-regulated anti-
apoptotic protein (Bcl-2) in their respective cell lines. In addition, in vitro stability, solubility and
plasma binding studies of the compound 17 and 26 showed them to be metabolically stable.
Thus, this study identified two new curcumin monocarbonyl-1,2,3-triazole conjugate compounds
with more potent activity than curcumin against breast and prostate cancers.
Keywords: Curcumin mimics, 1,2,3-Triazole, Endocrine cancers, AKT-pathway, Hybridization.
1

*Corresponding author: Medicinal and Process Chemistry Division, CSIR-Central Drug Research
Institute, Sector 10, Jankipuram ext., Lucknow, Uttar Pradesh 226031, India. Tel.: 91-522-2772450; Ext.
4671; Fax: 91-522-2771941 E-mail address: vl_sharma@cdri.res.in; vlscdri@gmail.com
#
CDRI Manuscript No. 284/2015/VLS
Cancer is a group of heterogeneous disease, commonly characterized by uncontrolled cell
division that can further progress towards mortality through cancer cells acquiring ability to
invade other vital organs. After cardiovascular disease, cancer is the most life threatening disease
that causes one out of eight deaths worldwide. An estimated 13.2 million cancer related deaths
and ~21.4 million new cancer cases are expected by 2030 [1]. National Institutes of Health (NIH)
has projected medical expenditures to reach $158 billion in the year 2020 with total cost
estimates of $895 billion worldwide. The highest medical costs associated with the two
endocrine related cancers–breast cancer ($16.5 billion) in women and prostate cancer ($12
billion) in men [2]. Among women, breast cancer is the leading cause of cancer deaths
worldwide, with nearly 1.67 million new cases diagnosed accounting more than 25% of all the
cancers in 2012. On the other hand, prostate cancer is the second leading cause of cancer deaths
in men, contributing to ~15% of the total number of new cases diagnosed in 2012 [3]. In cancer
related deaths breast cancer and prostate cancer accounted for 6.4% and 3.7% respectively in
2
012 [4]. The incidence of these two cancers has significantly increased in developed countries
like United States and is constantly rising in developing countries [5]. However, significant
progress has been made in diagnosis and therapeutic management of cancers in the last several
years due to increased knowledge and innovations. Nevertheless, all currently available
chemotherapeutic agents suffer from undesirable off-target effects and frequently develop
resistance. The majority of successful anti-cancer drugs have been discovered from natural
resources and development of natural product derived semi-synthetic anti-cancer agents is a
promising area for any cancer drug discovery programs [6]. Curcumin, (Figure 1) an active
component of Cucuma longa (turmeric), has not only been shown to prevent carcinogenesis, but
also known to have direct therapeutic benefits in various cancers [7]. Turmeric is a culinary spice
and medicine regularly consumed in Eastern Asian countries, and thought to contribute to the
massive difference in the rates of prostate cancer incidences between Eastern (less than 10 per
1
00,000 in Asia) and Western (120 per 100,000 in Northern America) countries [8]. The anti-
cancer effect of curcumin is due its moderate effect on multiple growth factor pathways in the
2

cell, and its multiple weak effects result into a remarkable aggregate effect, which is valuable in
complex diseases like cancer [9]. However, low bioavailability and rapid plasma clearance limits
clinical application of curcumin. The metabolic instability of curcumin is found to be due to the
β–diketone moiety. This was successfully circumvented by replacing the β–diketone moiety with
single carbonyl group to synthesize monocarbonyl curcumin analogue with improved efficacy
and stability [10-13]. The anti-cancer activity of monocarbonyl curcumin analogue against
endocrine related breast cancer [14] and prostate cancer is due to inhibition of AKT, HER2/neu,
and signal transducer and activator of transcription 3 (STAT3) signaling pathways [15].
Figure 1: Structures of Curcumin (1), monocarbonyl curcumin analogues (2), 1,2,3-triazole containing
anti cancer molecules (3-6) and envisaged monocarbonyl curcumin analogue–1,2,3-triazole conjugates
(prototype 1 and 2).
Molecular hybridization is a common strategy used for developing new chemotherapeutic agents
uniting two or more active pharmacophores into a conjugated single molecule and thereby
creating distinct novel chemical entities [16-22]. These novel conjugates may modulate multiple
cellular pathways simultaneously through synergistic action with minimum redundant side
effects compared to single-target drugs [23]. 1,2,3–triazoles conjugation increases metabolic
stability, water solubility and infuse capability to readily interact with biological targets through
3

hydrogen bonding and dipole interactions [24]. Their synthetic accessibility through click
chemistry and widely reported pharmacological effects [25-28] makes it appealing to anti-cancer
drug design. In fact, 1,2,3–triazoles conjugates have already been reported to exhibit potent anti-
cancer activity [29-33] and carboxyamidotriazole [34], a 1,2,3-triazole bearing anti-cancer drug,
is now available in the market (Fig. 1). On the other hand, dithiocarbamates are reported as
effective anti-cancer agents [35-37].
As part of our ongoing interest in developing novel hybrid therapeutic agents against endocrine
related cancers [38] and generating better synthetic analog of curcumin, we designed and
synthesized a novel series of hybrid scaffolds by combining monocarbonyl curcumin analogue
with 1,2,3-triazoles (fig. 1, prototype1). In prototype 2, the dithiocarbamate moiety was
incorporated to achieve better biological activity. Compounds were synthesized and biologically
evaluated against breast cancer and prostate cancer cell lines using MTT assay along with
curcumin as positive control. This letter reports the effect of these new analogs on cancer cell
viability, cell cycle and apoptosis along with their SAR.
Synthesis: The general procedure for the synthesis of the intermediates (3E,5E)–3,5–
bis(substitutedbenzylidene)–1–(prop–2–ynyl)piperidin–4–one (8–10) and (3E,5E)–prop–2–ynyl
3
,5–bis(substitutedbenzylidene)–4–oxopiperidine–1–carbodithioate (14–16) have been illustrated
in Scheme 1. (3E,5E)–3,5–bis(substitutedbenzylidene)piperidin–4–one (5-7) were synthesized by
reacting substituted benzaldehydes (1-3) with 4–piperidinone hydrochloride hydrate (4) in
presence of small amount of conc.HCl in glacial acetic acid at room temperature followed by
basifying with potassium carbonate in acetone:water (5:1) in good yields [39]. Furthermore,
(
3E,5E)–3,5–bis(substitutedbenzylidene)–1–(prop–2–ynyl)piperidin–4–one
(8–10)
were
obtained by treating 5–7 with propargyl bromide in acetone at room temperature. The synthesis
of hitherto unknown dithiocarbamate potassium salts (11–13) were obtained by reacting 5–7 with
o
carbon disulfide, aq. KOH in DCM at 0 C. 11–13 further gave rise intermediates (3E,5E)–prop–
2
–ynyl 3,5–bis(substitutedbenzylidene)–4–oxopiperidine–1–carbodithioate (14–16) by reacting
o
with propargyl bromide in water:acetone (5:1) at 10–15 C.
4

Scheme 1: Reagents and conditions: (a) glacial acetic acid, Conc. HCl, rt, 48 h; (b) K
2
CO
3
,
o
acetone:water (5:1), 20 C, 2 h; (c) propargyl bromide (1 eq.), acetone, K
2
CO
3
, rt, 5–6 h; (d)
o
carbon disulfide (1.3 eq.), aq. KOH, DCM, 0 C, 2 h; (e) propargyl bromide (1 eq.),
o
water:acetone (5:1), 10–15 C, 1 h.
Scheme 2: Reagents and conditions: (a) CuSO
THF:water (1:1), rt, 1–1.5 h.
4
.5H
2
O (0.05 eq.), sodium ascorbate (0.1 eq.),
5

The final compounds, (3E,5E)–1–((1–(substitutedbenzyl)–1H–1,2,3–triazol–4–yl)methyl)–3,5–
bis(substitutedbenzylidene) piperidin–4–ones (prototype 1; 17–28) and (3E,5E)–(1–
(
substitutedbenzyl)–1H–1,2,3–triazol–4–yl)methyl
oxopiperidine–1–carbodithioates (prototype 2; 29–40) were obtained by reacting a mixture of 8–
0 or 14–16 (scheme 1) with substituted benzyl azides in presence of catalytic amount of
CuSO .5H O and sodium ascorbate in a mole ratio of 2:2:0.2:0.1 in 1:1 volumes of THF/H O for
–1.5 h in good yields.
3,5–bis(substitutedbenzylidene)–4–
1
4
2
2
1
Biological Evaluation: The anti-cancer activity of the two prototypes synthesized (17–40) were
evaluated against human breast cancer cell lines (MCF–7 and MDA–MB–231), mouse mammary
tumor cell line (4T1), human prostate cancer cell lines (PC–3 and DU–145) and non–cancer
originated human embryonic kidney cell line (HEK–293) using MTT assay. Cells were treated
with compounds for 24 h and curcumin was used as a standard drug. Interestingly as illustrated
in table 1, all the compounds except 34, 35 and 40 induced loss of cell viability of breast cancer
cells. Out of the twenty four compounds evaluated, sixteen compounds (17-21, 24-28, 30, 33, 36-
3
9) exhibited anti-cancer activity with IC50 6.00 µM–100 µM against MCF–7 cells. Of these
fourteen compounds (17-20, 25-28, 30, 33 and 36-39) demonstrated 1 to 13.8 fold higher activity
than standard curcumin (IC50 83.1 µM) at IC50 values ranges from 6 µM–81.6 µM. Whereas
against MDA–MB–231 cells, nineteen compounds (17-22, 24-32, 36-39) were active (IC50
9
8
.00µM–100.7µM). Among these fourteen compounds 17-22, 25-28, 30 and 36-38 showed up to
.3 fold higher activity than curcumin (IC 50 75.3 µM). In case of 4T1 mouse mammary tumor
cells, all the test compounds except 34, 35 and 40 were active at IC50 5.3µM–69.7µM of which
sixteen compounds (17, 19-23, 25-31 and 37-39) were more active (up to 9.3 fold) than standard
curcumin. Compound 26 showed most significant activity against all the breast cancer cell lines.
The activity data against prostate cancer cell line PC-3 revealed that seventeen compounds (17-
2
8, 30, 33-35, 39 and 40) were active with IC50 7.1–80.0 µM while against DU–145 cells
eighteen compounds (17-30, 33, 34, 38 and 39) were active at IC50 values 4.3–74.6 µM. Ten
compounds 17, 19, 20, 23-28 and 33 against PC–3 cells and another ten compounds (17, 19-21,
2
3-28, 30) against DU-145 cells showed up to 5.7 and 9.5 fold better activity than curcumin
(
PC–3, 39.0 µM and DU-145, 41 µM) respectively. On the other hand, twenty compounds (17–
3
0, 33–35, 38 and 40) exhibited cell growth inhibitory activity against prostate cancer cells either
6

PC-3 or DU–145 while fifteen (17–28, 30, 33 and 34) of these were active against both the cell
lines.
a
Table 1. In vitro anti-cancer activity (IC50) of all the synthesized compounds (17-40) in µM.
Prostate cancer cell
lines
Normal
cell line
Breast cancer cell lines
MDA-
1
2
Comp.
R
R
MCF-7
4T1
PC-3
DU-145
HEK
MB-231
2-F 28.7 ± 1.6 10 ± 1.1 18.0 ± 0.9 8.8 ± 0.2
4-F
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-CH
4-CH
4-CH
4-CH
4-OCH
4-OCH
4-OCH
4-OCH
2-F
3
3
3
3
9.5 ± 0.2
>100
ND
29 ± 1.8 26.8 ± 1.1 57.2 ± 1.8 65.6 ± 2.1 49.7 ± 1.3
2-Cl 66.7 ± 2.0 29 ± 0.6 23.2 ± 1.7 8.9 ± 0.5 10.5 ± 0.1 91.5 ± 0.4
3-Cl
2-F
4-F
2-Cl
3-Cl
2-F
10 ± 1.1
≥100
9 ± 0.5
29.3 ± 1.1 29.3 ± 1.8 33.1 ± 1.2 73.5 ± 1.5
67.2 ± 0.5 39.4 ± 1.6 39.9 ± 1.5 32.6 ± 0.9
70.7 ± 0.6 49.3 ± 1.5 67.5 ± 2.9 59.4 ± 1.2
3
3
3
3
>100
ND
ND
>100
>100
99 ± 1.7
6 ± 1.2
>100
98 ± 0.9 56.6 ± 1.9 32.5 ± 1.1 15.4 ± 0.7
16 ± 1.3
11.4 ± 0.9 36.3 ± 0.9 17.8 ± 1.7
>100
6.5 ± 1.2 19.7 ± 1.0 10.6 ± 0.2 41.2 ± 1.3
2-F
2-F
2-F
4-F
6.0 ± 1.0 10.0 ± 1.6 6.4 ± 1.9 19.4 ± 0.5 8.7 ± 0.3
46.0 ± 0.7
33.2 ± 2.0
33.1 ± 1.0
ND
2-Cl
3-Cl
2-F
10 ± 1.6 16.5 ± 0.4 5.3 ± 0.9
7.1 ± 0.3
6.8 ± 0.3
>80
7.5 ± 0.6
4.3 ± 0.6
80 ± 2.2
12 ± 1.0
>100
12 ± 0.3
91.5 ± 1.3 28.4 ± 1.6
6.1 ± 0.5
4-CH
4-CH
4-CH
4-CH
4-OCH
4-OCH
4-OCH
4-OCH
2-F
3
3
3
3
4-F 81.6 ± 1.3 45.6 ± 0.9 29.3 ± 1.1 45.5 ± 4.7 38.8 ± 4.9
ND
ND
ND
ND
ND
ND
>100
ND
2-Cl
3-Cl
>100
>100
93.2 ± 1.7 49.3 ± 1.5
83 ± 1.9 53.4 ± 1.3
>80
>80
>80
>80
3
3
3
3
2-F 79.4 ± 1.3
>100
>100
>100
49.9 ± 1.2 31.5 ± 1.2 54.5 ± 1.8
4-F
2-Cl
3-Cl
>100
>100
>100
>100
45.6 ± 0.8 74.6 ± 1.9
60.1 ± 1.4
>80
>80
>80
>80
32 ± 1.1
32 ± 0.5 69.7 ± 2.0
2-F 51.6 ± 1.6 57.6 ± 1.5 30.7 ± 1.2
4-F 56.9 ± 1.3 58.8 ± 1.1 9.9 ± 1.5
2-Cl 78.7 ± 2.1 100 ± 2.0 43.6 ± 0.8
>80
2-F
2-F
2-F
>80
35.7 ± 3.5 51.3 ± 1.3
>80
80 ± 1.1
39 ± 3.2
46.3 ± 1.0
>80
41 ± 2.9
1.6±0.21
ND
ND
94.1 ± 1.4
7.2±1.12
40±4.51
5.1±1.15
3-Cl
>100
>100
>100
Curcumin
Doxorubicin
5 Fluorouracil
Nocodazole
83.1 ± 4.4 75.3 ± 2.8 49.4 ± 1.4
Standa
rds
0.13±0.22 1.6±0.23 0.99±0.13 1.4±0.34
15±.23
0.42±0.32
19±2.51
1.1±1.2
23±3.52
1.3±0.23
13.3±1.54 24.2±2.67
2.5±1.11 2.2±0.54
Data are expressed in terms of Mean ± SE of three independent experiments; ND- Not tested
a
Out of all the compounds evaluated compound 26 showed most significant anti-cancer activity
against all the breast cancer cell lines. Similarly, compound 17 turned out to be the most active
against all prostate cancer cell lines (Table 1). Interestingly, all the compounds did not induced
significant cytotoxicity against non-cancer originated cell lines (HEK-293) suggesting specificity
of the compounds towards cancer cells.
7

Structure–activity relationship (SAR): Against MCF-7 cells the 2-fluoro substitution (25-28) at
1
R position of prototype 1 was more desirable and the order of preference was 4-methyl (17-20)
2
1
followed by 4-methoxy (21-24) groups irrespective of the R substitution. When R is 2-fluoro
2
(
25-28) then a fluoro substituent (25, 26) at R position was found to be more favorable than
1
chloro group (27, 28). Against PC-3 cells the preference of substituent at R was again 2-fluoro
2
(
(
25-28) > 4-methyl (17-20) > 4-methoxy (21-24) groups. While at R position chloro substituent
19, 20; 23, 24; 27, 28) was more desirable over fluoro substituent (17, 18; 21, 22; 25, 26). 2-
1
choloro substitution was better with 4-methyl at R (19) while 3-chloro was better over 2-chloro
1
substituent with 4-methoxy or 2-fluoro at R (24, 28). In the prototype 2 which contained an
additional dithiocarbamate group between Curcumin and 1,2,3-triazole scaffolds the order of
1
2
activity with respect to substituent at R was 2-fluoro > 4-methoxy > 4-methyl groups. At R
position fluoro substitution seemed to be more favorable over chloro substitution as out of twelve
compounds (29-40), four compounds (30, 33, 37, 38) with fluoro substituent were active while
with chloro substitution (36, 39) only two were found to be active. Whereas against PC-3 cell
1
line, at R position 4-methoxy substituent (33-36) was more desirable over 4-methyl (29-32) and
2
2
-fluoro groups (37-40). A fluoro substituent at R position (33, 34) gave much better activity
1
1
with 4-methoxy at R position. The SAR study revealed that both the ring substitutions R
2
(
phenyl ring in monocarbonyl curcumin analogue scaffold) and R (1,2,3-triazole scaffold) were
detrimental for activity. The SAR study (table 1) illustrated that monocarbonyl curcumin
analogue-1,2,3-triazole conjugate (prototype 1; 17-28) was more desirable scaffold as compared
to dithiocarbamate incorporated monocarbonyl curcumin analogue-1,2,3-triazole conjugates
(
prototype 2; 29-40). This biological activity of two prototypes further supported the importance
of the active scaffold in this study. It is interesting to observe that twelve compounds (17-21, 24-
8, 30 and 33) were active against both PC-3 and MCF-7 cancer cells.
2
Out of all the compounds evaluated, compounds 17 and 26 were selected as lead molecules for
further investigation due to their potent activity against prostate cancer and breast cancer cells
respectively and better cytotoxicity profile towards non-malignant cell line (HEK-293). In order
to investigate the effect of compounds 17 and 26 on clonogenic potential on PC-3 and MCF-7
cells respectively, cells were treated for 24 h and colony formation was assessed. Tumor is
composed of heterogeneous cell types and some subpopulation may contain long term clonal
renewal capacity which is often considered as a source of tumor refractoriness towards
8

chemotherapy. Clonogenic potential is described as the cell renewal capacity or tumorigenic
potential of cancer cells after chemotherapy or radiation therapy is withdrawn. Compounds 26
th
efficiently inhibited MCF-7 colony formation and no colonies were seen in MCF-7 cells till 7
day of evaluation. Compound 17 significantly and dose-dependently retarded colony formations
in PC-3 cells though complete inhibition was not observed (Fig. 2).
Figure 2: Effect of compounds on colony formation (A) MCF-7 cells were treated with different
concentrations of compounds 26 for 24 h (B) PC-3 cells were treated with different concentrations of
compounds 17 for 24 h. At the end of treatment, medium were replaced with complete medium and cells
allowed to grow for 7 days. Colony formation was checked by staining with 0.1% crystal violet.
Further the effect of compounds 17 and 26 on cell cycle progression was assessed. Dysregulation
of the cell cycle check points and over expression of proteins associated with cell cycle
regulation are the hallmark of malignancy. Therefore, compounds that are able to arrest cell
cycle progression have beneficial effect in cancer treatment [40]. To check the effect of most
active compounds 17 and 26 on cell cycle phase distribution following treatment of 24 h,
distribution of cell cycle phases was evaluated using PI staining method. Compound 26 induced
significant decrease in G0/G1 population and subsequent increase in S phase dose dependently in
9

MCF-7 cells (Fig. 3A). Whereas, compound 17 increased the cell population at G2/M of cell
cycle in PC-3 cells (Fig. 3B).
Figure 3: Effect of compound 26 and 17 on cell cycle progression in MCF-7 and PC-3 cells
respectively. (A) MCF-7 cells were treated with compound 26 (B) PC-3 cells were treated with compound
17 for 24 h, cells were harvested, stained with PI and acquired by flow-cytometer.
In addition, phase contrast microscopy was used to evaluate the effect of compounds 26 and 17
on morphological changes on MCF-7 and PC-3 cells respectively. Results showed that both the
compounds caused significant decrease in numbers as well as alteration of cellular morphology
in MCF-7 as well as in PC-3 cells (Fig. 4) suggestive of cytotoxicity against cancer cells. The
intrinsic morphology of cells was altered and eventually detached from surface upon exposure at
higher concentrations.
Apoptosis maintain natural balance between healthy cell survival and death of excess, damaged
or abnormal cells [38]. However, the balance often inclines towards abnormal cell survival in
cancer due to inhibition or evasion of apoptosis. Therefore, in order to evaluate induction of
apoptosis following treatment with compound 17 and 26, AnnexinV-FITC/PI dual staining assay
was carried out [44]. MCF-7 cells were treated with various concentrations of compound 26
(
3µM, 6µM and 9µM) and PC-3 cells with compound 17 (4.8 µM and 8.8 µM) for 24h. There
+
-
was a dose dependent increase of early apoptosis (AnnexinV / PI ) and subsequent decrease of
10

-
-
live cells (AnnexinV / PI ) following compound treatment in MCF-7 as well as in PC-3 cells
-
+
(
Fig. 5). In contrast, no significant change in necrotic population (AnnexinV / PI ) was seen with
both compounds (Fig. 5). These data clearly indicate that compound 26 and 17 induced apoptosis
mediated cell death in MCF-7 and PC-3 cells respectively.
Figure 4: Morphological changes induce by compound 26 and 17 in MCF-7 and PC-3 cells
respectively. (A) MCF-7 and (B) PC-3 cells treated with different concentration of 26 and 17 for 24 h and
images were captured by using Nikon microscope at 10X magnification.
Figure 5: Compound 26 and 17 induces apoptosis in MCF-7 and PC-3 cells. (A) MCF-7 cells treated
with compound 26 and (B) PC-3 cells were treated with 17 for 24 h. Cells were harvested, stained with
Annexin V-FITC/PI and samples were acquired with flow-cytometer.
11

Curcumin conjugates were known to induce apoptosis in breast cancer cells via loss of
mitochondrial membrane potential (MMP) [41]. Loss of MMP or mitochondrial depolarization is
a common indication of involvement of intrinsic pathway of apoptosis. Therefore, cells were
treated with different concentrations of most active compounds 17 and 26 for 24 h and alteration
in MMP was measured by flowcytometry-based JC-1 staining method [43]. Compound 26 and
1
7 induced dose dependent increase of green population (suggestive of depolarized
mitochondrial membrane) in MCF-7 and PC-3 cells respectively (Fig. 6). The increase of green
population and decrease of red population (suggestive of polarized mitochondrial membrane)
clearly indicated the involvement of mitochondria in compound 26 and 17 induced cell death in
both cell lines.
Figure 6: Effect of Compound 26 and 17 on mitochondrial membrane potential in MCF-7 and PC-3
cells. (A) MCF-7 cells treated with compound 26 (B) PC-3 cells were treated with Compound 17 for 24 h.
Cells were harvested, stained with JC-1 and samples were acquired by flow cytometry.
In order to investigate the effect of compound 26 and 17 on cellular signaling pathways that
regulate cell proliferation, survival and apoptosis, western blotting was carried. There was a
decrease of proliferating cell nuclear antigen (PCNA), an important cell cycle regulatory protein
in both the MCF-7 and PC-3 cells following compound treatment (Fig. 7). However, effect was
more promising in case of PC-3 cells. Furthermore, increased expression of pro-apoptotic
markers, Bax and decrease expression of anti-apoptotic markers, Bcl-2 was observed following
compound treatment for 24 h in both cell lines (Fig. 7).
12

Figure 7: Effect of 26 and 17 on cellular signaling in MCF-7 and PC-3 cells. (A) MCF-7 cells were
treated with compound 26 (B) PC-3 cells were treated with 17 for 24 h. Cells were harvested and protein
expression was checked by Western blotting.
Among the signaling pathways implicated for endocrine cancers, activated Akt plays an
important role in cell proliferation and it has been shown to be constitutively over expressed in
cancers. It was widely reported that phytochemicals like, curcumin and other curcumin mimics
down regulate the Akt phosphorylation [12, 42]. Phosphorylation of Akt, an indicator of cell
survival was drastically inhibited in both the cell lines with treatment of compounds (Fig. 6).
Compound 26 and 17 at concentration of 9 µM and 8.8 µM respectively, resulted in complete
inhibition of Akt phosphorylation in respective MCF-7 and PC-3 cells (fig. 6). Collectively,
these data suggest that compound 26 and 17 modulate cellular signaling of both breast cancer
cells and prostate cancer cells resulting in inhibition of cell proliferation and induction of
apoptosis.
In addition, in vitro stability and plasma binding of compounds 17 and 26 was assessed. In
simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), compound 17 was 92% and
3
0% intact in SGF after 1 hr and SIF after 2 hr, respectively. The other compound 26 was 85%
and 65% stable under the established experimental conditions after 1 hr and 2 hr respectively.
13

Figure 8:
1
1
20
00
SGFStability of 17
8
6
4
2
0
0
0
0
0
0
20
40
60
80
time(min)
Figure 8: In vitro SGF and SIF stability of compounds 17 and 26
In plasma stability study, the percentage of compound remaining intact at different time intervals
after incubation was determined by the following formula.
Concentration of drug after incubation
%
Drug remaining =
× 100
Concentration of drug before incubation
Both the compounds were found to be stable in plasma as 90% of 17 and 96% compound 26 was
intact after 2 hr of incubation.
14

Figure 9: In vitro plasma stability of compounds 17 and 26
In the in vitro metabolic study, the compound 17 was metabolised instantaneously as 66%
compound was intact after 5 min in presence of microsomes where the other compound 26 found
to be 46% intact after after 5 min. Whereas the parent compound (curcumin) was found to be
metabolised instantaneously as only 45% was found (Table 2). Metabolic stability of curcumin-
1
,2,3-triazole conjugates were found to be ≥ to that of the curcumin in case of 17 and less in case
of 26 based on the results obtained with curcumin as a control.
Concentration of drug after incubation
Concentration of drug before incubation
%
Drug remaining =
× 100
Elimination rate constant was obtained from natural log of % parent compound remained versus
time plot, and the parameters estimated were as in vitro half-life, intrinsic clearance (Clint
)
intrinsic hepatic clearance (Clint.H), respectively. Following formulae were used:
15

Table 2. In vitro metabolic stability data of compounds 17, 26 and curcumin.
Curcumin
Compound 17
0.044
Compound 26
0.088
-1
0.051
K (min )
1
0
3.68
.101
Half-Life (min)
15.89
7.52
Intrinsic Clearance (mL/min×mg)
Hepatic Clearance (L/h/kg)
0.087
0.176
182.39
157.52
316.8
In plasma protein binding study the percent protein binding (%) was estimated from the percent
compound remaining in the supernatant (plasma) after adding the charcoal. Analysis was done
using Ultra Performance Liquid Chromatography (LC-30AD) with photodiode array (SPD-
M20A) detection system. The extent of protein binding of drug at time (t=0) was calculated and
found to be 96.99% for 17 and 81.24% for 26.
Further solubility of the compounds 17, 26 and curcumin was found to be 1.12, 2.08 and 0.06
mg/L, respectively. Solubility of the compounds (17 and 26) was found to be 18 and 34 times
higher than curcumin.
In conclusion, this study reports synthesis and biological evaluation of new monocarbonyl
curcumin analogue-1,2,3-triazole conjugates. This series of hybrid compounds exhibited potent
anti-cancer activity against endocrine related breast and prostate cancer. All the compounds
inhibited proliferation either one or both type of the cancer cells. SAR revealed that
incorporation of dithiocarbamate group (prototype-2) in the active monocarbonyl curcumin
analogue-1,2,3-triazole scaffold (prototype 1) resulted in reduced activity. It was also found that
1
among the more active compounds 17-28, 2-fluoro and 4-methyl groups at R position
maintained good activity against breast and prostate cancer cells, respectively. Among the
compounds synthesized, twelve compounds (17-21, 24-28, 30 and 33) were found to be active
against both breast and prostate cancer cells. Compound 17 showed remarkable activity against
prostate cancer cells with high safety index (>11) whereas compound 26 showed remarkable
activity against breast cancer with good safety index (>7.6). The most active compounds (17 and
2
6) of the series induced cell cycle arrest, caused mitochondrial mediated apoptosis by
modulating the expression levels of various cellular signaling proteins like Akt, PCNA Bax and
16

Bcl-2 in both breast and prostate cancer cells. In addition, in vitro stability, plasma binding and
solubility studies showed that compound 17 and 26 were relatively stable.
In summary, we identified two novel monocarbonyl curcumin analogue-1,2,3-triazole
conjugates, 17 and 26 with more potent activity than curcumin against endocrine-related cancers.
Compounds 17 and 26 induced loss of cell viability, arrest of cell cycle progression and induced
apoptosis by inhibiting Akt phosphorylation in human breast and prostate cancer cells,
respectively. Our findings demonstrated that these two compounds could serve as potential lead
molecules for the future development of novel cancer chemotherapeutic agents against breast and
prostate cancers.
Acknowledgement
We acknowledge Mrs. Tara Rawat (Senior Technical Officer) for technical assistance and SAIF
Division for spectral data. We acknowledge CSIR (D.M., K.S. and Y.M.), ICMR (V.B. and
S.G.), UGC (V.S.), DBT (H) and DST (S.C.) for research fellowships. This study was partly
supported by the ICMR, New Delhi, and CSIR-Network project, CSIR, New Delhi, INDIA.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://......
1
13
(
Synthetic procedures for compounds (5-40), scan copies of HNMR and C NMR spectral data
of compounds 8-40, HRMS spectra of 17-40 associated with this article and biological methods).
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Research Highlights:
1
2
3
4
. Curcumin mimic–1,2,3-triazole conjugates with two prototypes were synthesized.
. Compounds 17 and 26 displayed significant anticancer activity than curcumin.
. Compounds 17 and 26 induced mitochondrial-mediated apoptosis and cell cycle arrest.
. Also significantly down regulated phospho-Akt, PCNA, Bcl-2 and upregulated Bax.
21

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