Arif Khan1, Fatima Naaz1, Rafia Basit2, Deepak Das1, Piyush Bisht3, Majeed Shaikh4, Bilal Ahmad Lone3, Yuba Raj Pokharel3, Qazi Naveed Ahmed4, Shazia Parveen5, Intzar Ali6, Shashank Kumar Singh2, Gousia Chashoo2, Syed Shafi1. 1. Department of Chemistry, School of Chemical and Life Sciences, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India. 2. Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, India. 3. Faculty of Life Sciences and Biology, South Asian University, New Delhi 110021, India. 4. Natural product and Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, India. 5. Faculty of Science, Chemistry Department, Taibah University, Yanbu Branch, Yanbu 46423, Saudi Arabia. 6. Department of Microbiology, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard, New Delhi 110062, India.
Abstract
A series of novel 1,2,3-triazole derivatives of capsaicin and its structural isomer (new natural product hybrid capsaicinoid) were synthesized by exploiting one-/two-point modification of capsaicin without altering the amide linkage (neck). The newly synthesized compounds were screened for their antiproliferative activity against an NCI panel of 60 cancer cell lines at a single dose of 10 μM. Most of the compounds have demonstrated reduced growth between 55 and 95%, whereas capsaicin (10) has shown reduced growth between 0 and 24%. Compounds showing more than 50% growth inhibition were further evaluated for the IC50 value. Among the cell lines tested, lung cancer cell lines (A549, NCI-H460) were found to be more susceptible toward most of the synthesized compounds. Compounds 14g and 14j demonstrated good antiproliferative activity in NCI-H460 with IC50 values of 6.65 and 5.55 μM, respectively, while compounds 18b, 18c, 18f, and 18m demonstrated potential antiproliferative activity in A549 cell lines with IC50 values ranging between 2.9 and 10.5 μM. Among the compounds, compound 18f was found to demonstrate the best activity with an IC50 value of 2.91 μM against A549. Furthermore, 18f induces cell cycle arrest at the S-phase and disrupts the mitochondrial membrane potential, reducing cell migration potential by inducing cellular apoptosis and higher ROS generation along with a decrease in mitochondrial membrane potential in addition to surface and nuclear morphological alterations such as a reduction in the number and shrinkage of cells coupled with nuclear blabbing indicating the sign of apoptosis of A549 non-small cell lung cancer cell lines. Compound 18f has emerged as a lead molecule and may serve as a template for further discovery of capsaicinoid scaffolds.
A series of novel 1,2,3-triazole derivatives of capsaicin and its structural isomer (new natural product hybrid capsaicinoid) were synthesized by exploiting one-/two-point modification of capsaicin without altering the amide linkage (neck). The newly synthesized compounds were screened for their antiproliferative activity against an NCI panel of 60 cancer cell lines at a single dose of 10 μM. Most of the compounds have demonstrated reduced growth between 55 and 95%, whereas capsaicin (10) has shown reduced growth between 0 and 24%. Compounds showing more than 50% growth inhibition were further evaluated for the IC50 value. Among the cell lines tested, lung cancer cell lines (A549, NCI-H460) were found to be more susceptible toward most of the synthesized compounds. Compounds 14g and 14j demonstrated good antiproliferative activity in NCI-H460 with IC50 values of 6.65 and 5.55 μM, respectively, while compounds 18b, 18c, 18f, and 18m demonstrated potential antiproliferative activity in A549 cell lines with IC50 values ranging between 2.9 and 10.5 μM. Among the compounds, compound 18f was found to demonstrate the best activity with an IC50 value of 2.91 μM against A549. Furthermore, 18f induces cell cycle arrest at the S-phase and disrupts the mitochondrial membrane potential, reducing cell migration potential by inducing cellular apoptosis and higher ROS generation along with a decrease in mitochondrial membrane potential in addition to surface and nuclear morphological alterations such as a reduction in the number and shrinkage of cells coupled with nuclear blabbing indicating the sign of apoptosis of A549 non-small cell lung cancer cell lines. Compound 18f has emerged as a lead molecule and may serve as a template for further discovery of capsaicinoid scaffolds.
Despite the recent advances in therapies,
cancer is still the second
leading cause of death and a major cause of morbidity and mortality
worldwide. Lung cancer is responsible for around 20% of all cancer
deaths with an estimated 1.8 million new cases and 1.6 million deaths
annually.[1] Lung cancer represents one of
the most malignant tumors, and non-small cell lung cancer (NSCLC,
accounts for 80–85% of lung cancer cases) is the most aggressive
type of lung cancer.[2−4] Currently, chemotherapy is the standard treatment
for patients with advanced non-small cell lung cancer. The absence
of effective anti-lung cancer drugs makes the mortality of lung cancer
still high. The high toxicity, low tumor specificity, and increasing
resistance to available chemotherapeutic agents[5,6] demand
new drug candidates with high activity and efficacy against lung cancer.[7−9]Due to the invasiveness, toxicity, and ineffectiveness of
current
therapeutic approaches, there has been renewed interest in using natural-product-based
compounds for the treatment of cancer. Over the past few decades,
natural products have gained a lot of interest in cancer drug discovery.
More than 70% of anticancer drugs are directly derived from natural
sources, developed through structural modifications, or inspired by
natural products.[10−12] Inspired by natural product scaffolds, structural
modifications of natural products have evolved as a major area in
drug development. A wide variety of secondary leads had emerged via structural modifications/semisynthetic modifications
of natural products, and several of them are in the market.[10,13]Capsaicin (10) is a major spicy component of
chili
peppers that are consumed as spices in many cultures worldwide. It
has been used medicinally for centuries and is known for its analgesic,[14,15] antioxidant,[16] chemopreventive,[17] chemotherapeutic,[18] antidiabetic,[19] anti-inflammatory,[20] and antiobesity[21] properties. Capsaicin and its derivatives were also found to be
potent inhibitors of bacterial (Staphylococcus aureus SA-1199B) NorA efflux pumps.[22] Capsaicin
has demonstrated in vitro and in vivo anticancer activity against a variety of cancer types.[23−25] Studies also revealed that capsaicin may act as a carcinogen or
co-carcinogen.[26,27] One of the broadly believed mechanisms
is the interaction of capsaicin with transient receptor potential
vanilloids (TRPVs). TRPVs lead to Ca2+-mediated mitochondrial
damage and release cytochrome c that ultimately causes
the cell apoptosis. It was found to be a robust apoptotic agent, but
the low activity profile, toxicity at higher doses, and pungent nature
limit its use as an anticancer agent.[16,23] Capsaicin
is approved as a topical treatment of neuropathic pain. Capsaicin
selectively activates TRPV1, a Ca2+-permeable cationic
ion channel that is enriched in the terminals of selected nociceptors.
The limited analgesic potential for the use of systemically administered
capsaicin studies in animals using local or topical application has
yielded conflicting results. However, the side effects caused by capsaicin
including pungency, rise in blood pressure, itching, musculoskeletal
disorder, hyperalgesia, fatigue, vomiting, transient hypertension,
stinging, and erythema at the application site limit its application
as an oral therapeutic agent.[28−30] Capsaicin, which inhibits VEGF,
should be a key component in the development of novel anticancer therapies
for NSCLC remission. Endothelial cells express VEGFR-2 (tyrosine kinase
receptor), which is an effective target for suppressing tumor cell
proliferation and metastasis, and it plays a crucial role in antiangiogenesis.[23,31−33]On the other hand, the 1,2,3-triazole moiety
is a key pharmacophore
exhibiting a wide range of pharmacological activities.[22,34−39] The 1,2,3-triazole moiety plays a significant role in medicinal
chemistry owing to its capability of forming a hydrogen bond, which
improves its solubility and ability to favorably interact with bimolecular
targets.[40−42] 1,2,3-Triazoles are highly stable to metabolic degradation
as compared to other heterocyclic compounds.[43−45] Several 1,2,3-triazole
tethered natural product scaffolds like oleanolic acid,[46] quinolone,[47] isatin,[47] myrrhanone C,[48] podophyllotoxin,[49] artemisinin,[3] coumarin,[50] and curcumin[51] with
a hydrophobic character have demonstrated potential antiproliferative
activities against lung cancer cell lines (Figure ). Conjugation of the 1,2,3-triazole moiety
evidenced to be one of the important strategies to improve the anticancer
properties of natural scaffolds, and many secondary leads have been
developed by this approach.
Figure 1
Rational for designing novel analogues embedded
with 1,2,3-triazoles.
Rational for designing novel analogues embedded
with 1,2,3-triazoles.The anticancer properties
of capsaicin as a robust
apoptotic inducing
agent and the long hydrophobic side chain present in capsaicin make
it an ideal scaffold for the development of secondary leads against
lung cancer.[5,6,52,53] In view of the low anticancer activity profiles
of capsaicin and the biological importance of the 1,2,3-triazole moiety
toward the development of anticancer secondary leads against lung
cancer, we aim to develop some new 1,2,3-triazole conjugates of capsaicin
through one-/two-point modification of capsaicin as shown below (Figure ).
Figure 2
1,2,3-Triazole tethered
capsaicinoids through one-/two-point modifications.
1,2,3-Triazole tethered
capsaicinoids through one-/two-point modifications.
Results and Discussion
Chemistry
The designer molecules
were synthesized through
one-/two-point modification of capsaicin by employing a multistep
synthetic strategy starting from vanillin as shown in Schemes and 2. The one-point modification was carried at the vanillyl group (head)
of capsaicin, while the two-point modification was carried out by
varying the lipid group (tail) and vanillyl group (head).
Scheme 1
Synthesis
of 1,2,3-Triazole Tethered Capsaicin Derivatives
Scheme 2
Synthesis of 1,2,3-Triazole Tethered Natural Product
Hybrid Capsaicinoids
One-Point Modification
Natural capsaicin (10) was treated with propargyl
bromide to give the propargylated intermediate
(13). Finally, the propargylated intermediate was reacted
with different substituted aromatic azides under Cu-I catalyzed click
chemistry conditions to afford the desired molecules (14a–p) bearing the 1,2,3-triazole moiety (Scheme ). All the synthesized
compounds (14a–p) with their melting
point are illustrated in Table .
Table 1
Chemical Structure of the Synthesized
Compounds (18a–o)
Formation of propargylated capsaicin (13) was confirmed
by the disappearance of a singlet corresponding to the phenolic −OH
peak of capsaicin (10) at δ 8.83 ppm and the appearance
of a doublet corresponding to −CH2– at δ 4.73
ppm and a singlet corresponding to alkynyl −CH (terminal alkyne)
at δ 3.53 ppm in 1H NMR. Formation of 1,4-disubstituted
1,2,3-triazole derivatives (14a–p) through 1,3-dipolar cycloaddition between propargylated capsaicin
(13) and aryl azides was confirmed by the presence of
a singlet corresponding to the triazolyl proton in the range of δ
8.06–8.63 ppm, disappearance of the signal corresponding to
alkynyl −CH at δ 3.53 ppm, and presence of an additional
aromatic signal in 1H NMR. The appearance of signals corresponding
to triazolyl and aromatic carbons in addition to the capsaicin signals[54] in 13C NMR spectra further confirms
the formation of target molecules. Finally, the formation of all the
target molecules was confirmed by mass spectra.
Two-Point
Modification
Vanillin (15) was
reacted with hydroxylamine hydrochloride in the presence of sodium
acetate trihydrate to afford the corresponding oxime (16a). The oxime (16a) was reduced to the corresponding
benzyl amine (16b) by using Zn/CH3COOH. 3-Methoxy-4-hydroxy
benzylamine was coupled with R-(+)-citronellic acid
to afford the hybrid natural product conjugate under EDC·HCl
coupling conditions. The natural product hybrid (16c)
was further reacted with propargyl bromide to yield the propargylated
intermediate (17). This propargylated intermediate was
finally reacted with different substituted aromatic azides under Cu-I
catalyzed click chemistry conditions to afford the new target molecules
(18a–o) bearing the 1,2,3-triazole
moiety (Scheme ).
All the synthesized compounds with their melting point are illustrated
in Table .
Table 2
Chemical Structure of the Synthesized
Natural Product Hybrid Compounds (18a–o)
Formation of vanillin oxime
(16a) from
vanillin was
defined by the presence of a broad doublet corresponding to one proton
at 7.75–7.71 ppm (−N–OH) and 5.90 ppm (−OH).
Reduction of oxime to benzylamine (16b) was confirmed
by the absence of peaks corresponding to aldoxime at 7.75 and 5.90
ppm and the presence of a triplet at δ 3.64–3.67 ppm
corresponding to two protons (−CH2). Formation of
amide (16c) from vanillyl amine and R-(+)-citronellic acid was confirmed by the appearance of two triplets
at 5.47 and 5.07 ppm corresponding to an −NH of amide and an
olefinic proton (HC=C) and other signals at the aliphatic region
corresponding to R-(+)-citronellic acid. Propargylation
of 16c to 17 was recognized by the presence
of a triplet at δ 2.50 ppm corresponding to terminal alkyne
and the presence of additional −CH2 protons at δ
4.73 ppm. Finally, formation of 1,2,3-triazoles (18a–o) from the propargylated intermediate (17) was
affirmed by the presence of a singlet at δ 8.06–8.18
ppm (CDCl3, 1H NMR) corresponding to the −CH–
proton of the 1,2,3-triazole moiety and the absence of signal at δ
2.50 ppm. Finally, formation of the compounds was confirmed by 1H NMR, 13C NMR, LC–MS, and HR-MS.
Biology
Antiproliferative Investigation against 60 Cell Lines
Newly synthesized 1,2,3-triazole conjugates of capsaicin (14a–p) and 1,2,3-triazole conjugates of the structural
isomer of capsaicin (18a–n) were
submitted to the Developmental Therapeutic Program-National Cancer
Institute, Bethesda, USA (www.dtp.nci.nih.gov). These synthesized compounds were selected for the screening at
a single dose of 10 μM and tested against 60 cancer cell lines
under nine different cancer cell types (leukemia, lung, colon, CNS,
melanoma, ovarian, renal, prostate, and breast cancers) with their
subpanels. Screening results of in vitro antiproliferative
activity of the tested compounds were reported as growth percent as
shown in Tables and 4. Compounds 18a, 18c, 18h, 18k, and 18o were screened
against a panel of five human cancer cell lines, viz., breast (MCF-7), colon (HCT-116), lung (A549), pancreas (MiaPaCa),
and prostate (PC-3) at 10 μM concentration, and the results
are reported in percentage growth inhibition (GI) as depicted in Table .
Table 3
Antiproliferative Activity of Synthesized
1,2,3-Triazole Tethered Capsaicin (14a–p) against
an NCI Panel of 60 Human Cancer Cell Lines
cancer panel
subpanel/comp no.
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
leukemia
CCRF-CEM
72.44
87.33
87.87
84.65
84.94
90.24
68.85
79.73
88.58
86.21
94.23
83.65
96.33
87.34
86.83
75.54
K-562
66.66
84.87
76.84
77.13
74.53
81.49
76.27
68.74
82.35
75.37
59.18
76.13
79.71
79.98
78.64
74.64
MOLT-4
81.99
92.80
81.04
86.41
79.33
86.56
80.34
82.62
90.94
88.39
77.45
79.70
92.05
83.54
79.62
78.54
RPMI-8226
60.83
86.29
75.55
78.11
61.80
65.86
53.37
68.40
81.34
65.44
57.35
71.25
79.37
74.10
73.95
67.31
SR
73.55
85.61
68.45
78.92
62.18
78.38
70.36
76.67
81.54
76.32
67.19
77.40
86.62
75.55
87.52
58.25
non-small cell
lung cancer
A549/ATCC
85.32
82.93
84.33
85.90
84.06
91.99
48.99
84.35
91.27
84.82
88.49
85.96
91.04
84.04
88.34
82.16
EKVX
72.02
80.81
82.11
76.21
75.28
95.11
88.25
72.71
79.11
90.82
72.66
89.40
95.93
84.83
82.11
79.28
HOP-62
83.97
71.45
81.95
90.38
80.87
97.38
64.56
84.07
84.95
102.9
88.21
88.73
90.68
86.39
94.51
97.43
HOP-92
77.10
62.44
80.14
78.72
90.32
70.38
13.84
66.16
78.66
80.48
68.79
72.06
82.53
83.54
86.50
80.41
NCI-H226
70.36
86.45
81.77
76.64
89.59
90.88
53.63
74.15
81.34
100.5
86.31
80.50
91.44
75.82
76.71
86.88
NCI-H23
78.67
87.17
93.56
93.19
83.21
87.96
94.56
81.41
95.32
72.14
70.17
96.02
90.08
87.91
89.51
74.91
NCI-H322M
92.15
86.20
89.36
93.25
97.91
94.09
96.15
91.76
90.16
95.29
90.64
92.18
89.07
89.39
90.50
101.0
NCI-H460
79.92
84.09
101.3
101
99.76
91.79
16.74
75.71
103.5
92.96
72.09
104.4
101.5
93.21
102.7
86.45
NCI-H522
65.69
69.17
64.46
69.27
78.21
82.60
75.57
70.15
77.48
88.19
82.50
63.91
65.21
62.09
65.93
80.70
colon cancer
COLO205
108.5
102.4
106.4
110.6
117.9
106.8
72.59
119.9
115.7
117.3
103.1
116.8
106.2
115.2
120.9
106.5
HCC-2998
112.7
104
110.5
98.1
107.5
101.2
94.75
110.2
97.17
94.51
88.79
117.3
91.82
108.4
99.66
100.6
HCT-116
63.68
76.84
100.3
106.8
77.19
41.96
2.97
65.61
97.81
15.41
49.09
82.68
87.14
92.44
90.69
48.16
HCT-15
91.77
91.31
94.11
93.61
96.24
77.90
59.70
89.61
95.97
83.47
94.02
106.1
103.2
103.9
94.42
71.18
HT29
88.41
99.25
98.23
99.43
98.13
88.86
78.99
106.0
106.9
95.56
98.09
98.69
87.19
100.1
109.4
97.85
KM12
95.56
98.56
97.72
99.98
90.03
91.20
81.18
94.49
98.88
91.46
94.30
98.21
102.6
96.05
100.3
97.54
SW-620
88.74
92.82
100.1
96.12
99.76
97.40
41.33
90.98
98.10
98.02
94.70
96.98
102.4
102.4
102.7
95.17
CNS cancer
SF-268
84.04
82.49
87.78
93.32
86.10
92.25
87.80
87.23
95.04
90.86
78.89
92.64
90.52
81.74
93.05
94.60
SF-295
88.30
90.53
99.41
94.64
89.02
103.9
90.90
90.52
98.96
102.1
85.39
107.6
105.4
98.90
100.05
88.82
SF-539
87.11
81.97
104.2
96.14
104.1
94.44
81.86
92.18
104.8
94.55
92.40
106.3
92.01
100.3
103.68
105.0
SNB-19
87.72
59.97
89.74
87.31
97.39
68.23
76.10
81.35
84.84
81.91
80.91
91.56
85.13
77.94
85.68
95.85
SNB-75
76.44
75.84
87.34
79.08
83.94
91.95
49.67
83.46
88.08
94.17
90.92
103.3
95.25
99.45
92.63
90.98
U251
86.89
69.08
85.65
91.90
86.36
85.29
46.69
83.87
93.12
91.16
75.48
90.23
92.80
81.65
89.37
88.35
melanoma
LOX IMVI
85.61
89.42
89.93
87.04
89.50
92.87
60.32
84.23
87.12
78.15
83.68
86.08
94.42
85.10
89.32
84.46
MALME-3M
97.85
98.65
107.4
105.3
98.44
103.1
99.71
103.2
107.7
102.3
102.7
106.2
98.49
109.8
107.95
107.2
M14
90.64
94.14
92.57
92.67
89.72
93.4
97.51
94.05
96.85
95.27
90.17
96.66
94.53
93.33
99.82
93.54
MDA-MB-435
99.29
98.61
102.6
94.22
99.74
101.2
107.0
98.80
100.6
107.7
106.9
109.6
106.5
109.0
107.0
104.3
SK-MEL-2
101.6
86.66
90.98
108.4
91.96
88.40
95.06
111.3
105.5
98.43
95.21
89.80
97.57
91.94
95.17
90.32
SK-MEL-28
106.5
105.7
105.8
104.0
102.4
105.9
107.0
102.5
107.3
104.6
100.4
115.6
108.1
113.7
114.9
112.1
SK-MEL-5
77.63
91.17
89.83
81.64
76.10
93.24
97.06
83.73
91.50
86.97
72.04
83.94
77.50
86.78
83.95
81.53
UACC-257
91.64
90.11
87.93
89.23
82.58
94.42
98.24
90.21
94.96
94.35
90.46
85.25
93.58
88.19
86.37
81.89
UACC-62
67.84
74.98
73.31
75.61
69.29
70.05
84.33
71.93
76.23
81.83
62.99
75.07
87.79
70.18
71.39
75.07
ovarian cancer
IGROV1
93.44
79.91
98.76
104.1
90.41
89.40
58.71
96.98
100.8
100.4
72.65
104.5
103.3
98.46
102.5
98.81
OVCAR-3
104.9
84.40
101.1
92.4
92.69
104.9
106.8
96.37
97.52
108.9
100.1
103
103.1
91.85
101.0
100.4
OVCAR-4
82.64
67.25
87.87
69.52
87.18
94.19
98.59
82.15
84.19
94.94
89.23
99.97
103.4
88.79
88.97
84.94
OVCAR-5
100
99.78
103.5
102.4
99.15
107.3
96.56
97.57
102.6
95.30
116.2
112.8
109.1
108.4
112.13
107.4
OVCAR-8
78.97
57.20
89.57
91.05
82.87
76.93
41.06
71.55
94.5
63.28
79.60
82.16
88.18
76.53
91.14
83.35
NCI/ADR-RES
78.12
76.59
92.04
91.11
28.98
81.79
48.24
70.62
89.77
79.67
71.97
94.61
91.69
87.43
87.71
80.64
SK-OV-3
91.64
78.80
91.28
97.24
76.69
93.55
91.30
86.36
97.84
88.22
74.85
99.76
89.94
94.38
101.6
98.61
renal cancer
786-0
92.52
76.84
93.81
96.31
93.73
73.38
2.94
83.74
97.19
91.65
92.05
102.4
107.6
99.62
100.2
99.82
ACHN
59.66
72.99
74.08
81.98
78.92
81.18
82.95
87.69
72.01
68.45
CAKI-1
88.99
79.62
93.42
58.30
90.66
81.67
85.84
88.42
90.63
74.59
74.31
79.93
79.48
88.54
77.39
94.35
RXF 393
97.98
91
104.9
100.6
99.73
92.69
90.23
95.34
103.1
90.77
105.4
101.0
104.4
102.5
101.0
98.20
SN12C
80.18
84.24
94.47
112.6
91.67
89.51
65.57
82.38
92.65
98.27
83.0
98.02
101.6
89.3
98.54
88.40
TK-10
99.28
105.5
94.57
88.62
92.72
104.9
20.45
103.0
111.3
91.84
117.5
90.25
91.59
100.6
87.44
108.97
UO-31
76.58
72.21
75.19
102.7
60.63
74.73
92.75
86.04
77.11
111.8
67.93
105.0
116.9
75.81
93.93
75.67
prostate cancer
PC-3
79.23
83.59
80.60
77.08
77.97
82.16
70.50
84.14
82.05
79.46
73.69
73.46
80.91
84.17
70.87
78.43
DU-145
98.92
95.88
99.19
86.53
104.7
108.7
62.20
96.08
105.8
44.39
98.83
83.12
85.40
96.95
93.77
107.95
breast cancer
MCF-7
84.88
86.88
97.24
95.33
83.45
74.06
80.73
89.31
97.12
103.0
76.17
98.16
102.3
96.29
102.2
86.88
MDA-MB-231 ATCC
79.50
64.80
80.85
87.29
75.32
88.39
75.16
69.06
78.71
90.83
71.32
95.76
92.66
72.21
88.05
89.27
HS 578T
90.35
74.80
97.20
84.04
95.78
90.76
70.69
77.70
87.70
95.03
90.48
83
69.00
89.68
78.03
90.74
BT-549
86.83
99.35
90.81
91.78
80.40
96.27
80.41
91.05
89.25
91.24
88.08
93.12
79.12
105.0
99.09
93.65
T-47D
71.04
81.08
74.30
87.05
63.99
72.34
84.51
73.75
83.08
76.82
68.37
92.18
95.75
77.41
111.0
73.17
MDA-MB-468
64.91
74.73
88.19
71.60
77.44
72.34
70.22
68.19
92.81
74.86
68.15
75.45
80.43
92.44
79.80
71.77
Table 4
Antiproliferative Activity of Synthesized
1,2,3-Triazole (18a–n) against an
NCI Panel of 60 Human Cancer Cell Lines
PANELNME
comp no.
16c
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18n
leukemia
CCRF-CEM
70.56
59.43
92.94
76.55
40.10
116.05
84.49
81.06
85.57
36.52
97.09
83.96
97.30
58.35
61.14
HL-60(TB)
76.37
93.01
87.80
83.86
41.06
95.54
83.95
110.2
100.27
47.68
96.30
89.26
91.54
70.85
88.43
K-562
89.43
79.40
53.52
60.82
44.62
81.37
51.89
44.88
83.08
51.53
59.00
72.74
81.62
47.60
66.32
MOLT-4
83.43
54.59
71.73
47.68
16.29
84.05
68.65
64.23
80.63
20.66
84.80
67.45
74.84
14.78
69.97
RPMI-8226
84.37
37.90
75.30
56.33
47.56
90.71
77.63
73.14
73.29
39.91
89.81
75.33
81.80
58.26
69.27
SR
90.57
62.33
65.77
61.39
48.53
79.78
27.12
39.18
81.95
45.13
41.51
68.94
73.39
24.73
63.80
non-small cell lung cancer
A549/ATCC
96.36
94.07
57.07
81.77
64.07
59.42
46.49
85.48
56.83
65.65
42.61
76.78
80.64
48.20
78.59
EKVX
86.35
74.29
75.34
51.21
54.00
81.06
77.24
75.25
69.05
58.81
92.14
65.19
78.32
81.03
79.03
HOP-62
85.81
120.28
61.55
89.31
79.25
11.33
95.67
103.84
82.74
78.10
88.76
67.99
46.72
88.63
65.10
HOP-92
83.48
103.88
53.02
71.92
79.43
–3.27
51.60
86.49
76.24
57.55
86.10
55.58
4.63
65.16
45.94
NCI-H226
100.60
79.51
63.78
54.16
59.73
66.35
50.55
80.08
73.48
56.87
72.34
51.40
73.35
54.94
70.24
NCI-H23
94.13
91.88
64.87
72.88
64.51
56.64
68.51
45.49
66.55
62.45
79.35
74.89
68.25
64.54
60.06
NCI-H322M
91.13
106.92
83.48
104.66
91.28
95.25
82.51
92.29
103.37
94.44
90.10
105.20
92.68
94.96
99.60
NCI-H460
103.61
97.03
65.04
88.73
78.43
75.06
45.51
78.17
62.92
84.19
14.61
79.49
89.03
45.78
74.17
NCI-H522
86.86
85.53
62.93
67.33
51.40
55.44
61.51
58.92
80.13
61.88
83.09
66.40
57.14
63.51
41.63
colon
cancer
COLO 205
105.96
99.03
91.10
102.61
71.71
104.99
53.63
121.73
94.43
78.01
74.25
97.16
111.74
95.57
112.90
HCC-2998
108.39
93.30
75.87
94.66
91.88
92.47
93.52
107.31
91.41
106.48
111.05
94.86
100.56
89.49
95.80
HCT-116
94.11
96.71
38.23
72.22
49.66
53.13
29.82
32.09
18.65
49.09
12.49
47.86
60.15
29.43
41.92
HCT-15
100.88
88.04
65.29
81.43
53.84
79.57
29.35
71.79
80.93
62.42
56.79
91.65
82.81
66.16
82.35
HT29
103.79
99.48
79.57
97.34
76.66
82.28
78.27
108.14
100.11
22.55
96.61
102.98
97.40
77.55
94.43
KM12
100.37
96.04
87.72
94.27
62.97
93.06
82.66
86.78
91.32
63.85
84.99
96.37
99.29
71.66
94.59
SW-620
98.70
76.75
97.41
80.24
89.26
29.90
86.83
80.92
92.93
38.71
101.33
92.72
71.03
91.70
CNS cancer
SF-268
77.04
92.33
95.38
85.96
82.41
52.34
58.39
88.77
73.15
78.08
83.86
69.92
58.88
71.57
71.71
SF-295
103.18
88.16
55.55
66.84
56.38
41.76
80.45
70.15
96.45
60.45
72.87
64.41
45.20
54.05
63.24
SF-539
93.56
96.00
67.72
73.07
75.23
66.88
5.30
43.12
69.79
66.99
16.35
68.43
44.60
59.74
51.16
SNB-19
95.32
74.44
78.63
67.87
61.27
49.98
81.31
84.82
73.39
66.43
97.17
53.11
55.68
83.59
57.66
SNB-75
56.78
41.60
60.00
7.99
47.20
78.02
56.57
65.15
10.58
54.57
47.32
U251
98.73
93.80
57.71
102.61
80.61
54.40
48.23
82.82
40.06
62.83
50.97
65.37
61.60
83.86
63.36
melanoma
LOX IMVI
92.96
92.33
59.71
75.06
56.37
80.73
50.61
62.72
71.40
62.00
80.73
81.38
76.70
72.59
74.03
MALME-3M
90.29
88.78
68.35
87.23
68.22
79.26
77.44
76.19
71.40
70.30
80.81
96.02
78.28
75.36
85.41
M14
95.93
85.67
90.80
93.39
55.87
102.02
89.03
88.83
82.48
65.91
92.12
102.20
94.79
88.23
97.23
MDA-MB-435
102.18
97.17
84.94
93.75
66.50
98.16
91.28
88.25
99.53
74.94
74.88
103.84
100.50
81.26
98.30
SK-MEL-2
101.97
91.22
83.62
95.46
53.30
71.68
93.67
64.50
95.86
66.32
110.78
84.61
78.55
79.97
78.04
SK-MEL-28
106.36
99.70
86.95
76.60
69.78
96.28
89.28
87.77
98.47
85.31
91.55
89.56
94.93
85.39
96.54
SK-MEL-5
97.72
77.37
62.77
89.51
5.29
60.43
71.91
35.89
86.20
16.33
78.75
65.24
70.80
31.05
55.65
UACC-257
112.93
101.28
85.53
72.07
60.33
91.96
91.04
69.39
92.03
71.22
103.84
69.92
94.00
68.74
86.34
UACC-62
81.15
64.23
65.67
108.08
39.56
65.59
69.35
53.53
65.91
48.01
72.06
64.41
69.54
56.65
65.15
ovarian cancer
IGROV1
89.12
88.78
80.15
95.28
87.05
72.31
83.14
78.76
106.74
78.93
102.20
97.38
81.86
81.49
89.93
OVCAR-3
94.85
116.94
82.26
70.95
83.57
101.33
60.12
107.92
98.44
80.34
102.85
107.56
105.46
94.05
104.69
OVCAR-4
78.72
95.26
52.67
106.44
33.26
49.69
53.41
63.75
63.65
45.26
45.13
73.07
71.43
55.29
49.55
OVCAR-5
93.28
69.23
80.72
72.31
93.08
91.71
86.36
101.94
98.00
95.61
97.48
101.77
94.92
97.97
105.21
OVCAR-8
99.92
107.77
49.14
57.30
49.81
29.30
46.30
63.55
23.70
48.57
28.11
52.29
66.47
59.99
47.52
NCI/ADR-RES
94.46
81.94
47.96
99.18
24.02
32.86
36.55
17.58
3.67
36.03
44.05
45.40
54.77
14.02
52.06
SK-OV-3
97.77
47.99
72.13
75.06
82.97
26.10
99.00
80.08
95.87
68.44
88.99
85.05
43.56
82.43
64.02
renal cancer
786-0
113.25
84.28
92.28
87.70
38.94
80.43
97.04
71.40
83.46
92.98
68.16
43.58
96.24
68.65
A498
103.70
92.16
98.87
141.89
106.80
58.98
79.95
92.59
91.34
107.83
90.96
136.18
103.28
92.30
104.93
ACHN
96.18
110.84
44.83
80.22
67.59
61.09
71.52
72.71
111.72
80.79
77.61
69.02
65.32
72.75
56.39
CAKI-1
81.89
93.22
45.87
87.58
46.28
31.84
64.04
72.08
69.51
48.65
73.63
81.82
55.08
62.58
28.75
RXF 393
104.03
90.76
56.13
79.87
72.64
14.13
74.78
90.59
83.02
55.93
88.27
52.33
38.41
49.41
61.80
SN12C
82.18
98.10
67.70
79.39
69.58
64.79
38.19
70.68
96.63
73.66
27.14
80.79
76.12
77.75
71.63
TK-10
120.42
88.46
95.63
99.80
93.11
68.70
90.74
104.96
72.43
113.60
101.93
91.77
95.83
105.47
108.92
UO-31
60.69
62.50
30.26
60.54
60.75
42.13
36.50
75.65
71.89
50.93
62.92
prostate cancer
PC-3
83.21
83.14
80.17
76.26
49.41
85.05
65.91
85.51
75.72
48.11
82.47
80.20
87.92
68.16
81.39
DU-145
102.58
92.07
82.81
86.56
90.12
90.48
76.78
86.40
84.49
77.90
100.88
93.09
101.70
73.70
92.71
breast cancer
MCF7
97.19
80.61
51.57
63.32
40.61
67.54
49.58
63.96
74.54
50.45
56.77
77.07
66.90
42.30
60.21
MDA-MB-231/ATCC
79.57
59.59
72.60
59.66
75.74
76.76
71.94
87.20
66.80
81.38
80.58
HS 578T
100.24
71.37
52.51
80.95
48.38
57.02
81.99
68.23
73.67
94.33
33.70
39.51
76.00
73.79
BT-549
95.55
83.27
87.40
66.42
28.16
83.67
71.72
72.57
77.08
24.05
92.35
79.69
70.98
58.51
93.63
T-47D
89.01
66.00
52.46
58.60
9.14
60.21
33.60
46.14
58.91
14.63
32.42
67.90
60.60
35.97
49.34
MDA-MB-468
99.02
66.15
41.27
43.43
28.48
50.44
54.99
41.44
56.62
31.66
64.90
64.96
57.69
30.23
46.73
Table 5
Percent GI on Breast, Colon, Lung,
Pancreas, and Prostate Cell Lines
tissue cell line
type
breast MCF-7
colon HCT-116
lung A549
pancreas MiaPaCa
prostate PC-3
compounds
conc. (μM)
% GI
18a
10
10
37
31
23
03
18b
10
34
41
55
66
28
18c
10
43
78
91
45
15
18d
10
39
26
41
12
09
18e
10
12
38
25
18
06
18f
10
32
45
71
27
28
18g
10
43
32
33
53
05
18h
10
11
15
15
23
14
18i
10
28
25
16
36
01
18j
10
04
50
42
17
21
18k
10
05
22
36
42
11
18l
10
01
23
28
27
23
18m
10
51
47
62
31
05
18n
10
42
26
36
29
18
18o
10
45
26
32
21
03
capsaicin
10
24
0.0
0.0
15
19
Among the capsaicin derivatives
(14a–p), compounds 14f, 14p, 14j, 14g, and 14k illustrated susceptibility
against the HCT-116 colon cancer cell line with 2.97–49.09%
growth of cancer cell. Compound 14g demonstrated better
antiproliferative activity against HOP-92, NCI-H460, HCT-116, 786-0,
and SN12C cancer cell lined with growth of 2.97–20.45%. It
also exhibited moderate antiproliferative activity with growth of
41.33–49.67% against A549/ATCC non-small cell lung cancer,
SW-620 colon cancer, SNB-75 CNS cancer, U251 CNS cancer, OVCAR-5 ovarian
cancer, and OVCAR-8 ovarian cancer cell lines. Herein, compounds 14e and 14j displayed potent activity against
NCI/ADR-RES ovarian cancer and HCT-116 colon cancer with 28.98 and
15.41% growth, respectively. Compound 14j also demonstrated
moderate antiproliferative activity against prostate PC-3 cancer cell
line with a growth of 44.39%. Potent compounds (14g, 14j) that showed excellent activity from the preliminary screening
(NCI antiproliferative data) were further evaluated for their IC50 values.Inspired by the potent antiproliferative activity
of 1,2,3-triazole
tethered capsaicinoids, a series of (18a–o) 1,2,3-triazole derivatives of the structural isomer of
capsaicin, which was derived by the hybrid conjugate of vanillyl amine
and citronellic acid, were prepared and evaluated for their antiproliferative
activity. Antiproliferative data revealed that compounds 18a–n exhibited cytotoxicity against various cancer
cell lines. Compound 18b exhibited excellent activity
against HCT-116, SNB-75, OVCAR-8, NCI/ADR-RES, ACHN, CAKI-1, and MDA-MB-468
with a range of growth of 38.23–49.14%. Compound 18d showed excellent activity against leukemia (CCRF-CEM, HL-60(TB),
K-562, MOLT-4, RPMI-8226, SR), HCT-116, SK-MEL-5, UACC-62, OVCAR-4,
OVCAR-8, NCI/ADR-RES, CAKI-1, UO-31, PC-3, MCF-7, BT-549, T-47D, and
MDA-MB-468 with a growth range of 5.29–49.81%. Moreover, compound 18e also displayed moderate cytotoxicity against the non-small
cell lung cancer HOP-62 cell line; CNS cancer SF-295, SNB-19, and
SNB-75 cell lines; ovarian cancer OVCAR-4, OVCAR-8, NCI/ADR-RES, and
SK-OV-3 cell lines; renal cancer CAKI-1 and RXF 393; and breast cancer
HS 578T cell line with a growth range of 7.99–49.98%. Compound 18f exhibited moderate activity against SR, A549, NCI-H460,
HCT-116, HCT-15, SW-620, SF-539, SNB-75, U251, OVCAR-8, NCI/ADR-RES,
SN12C, MCF7, and T-47D cancer cell lines with a growth range of 5.30–49.58%.
Compound 18g also exhibited good activity against SR,
NCI-H23, HCT-116, SF-539, SK-MEL-28, NCI/ADR-RES, UO-31, T-47D, and
MDA-MB-468 cancer cell lines with a growth range of 17.58–46.14%.
Compound 18h has demonstrated promising activity against
NCI/ADR-RES cell lines with a growth of 3.67%. Among all other synthesized
compounds, only compound 18i demonstrated moderate activity
against the leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226,
SR), colon cancer (HCT-116, HT29), melanoma (SK-MEL-5, UACC-62), ovarian
cell (OVCAR-4, OVCAR-8, NCI/ADR-RES), renal cell (CAKI-1, UO-31),
prostate cancer (PC-3), and breast cancer (BT-549, T-47D, MDA-MB-468)
cell lines with a growth range of 14.05–48.65%. Compound 18f also showed good activity against SR, A549, NCI-H460,
HCT-116, SW-620, SF-539, OVCAR-4, OVCAR-8, NCI/ADR-RES, SN12C, and
T-47D cancer cell lines with a growth range of 10.58–45.13%.
Compounds 18l, 18m, and 18n also showed good activity against K-562, MOLT-4, SR, A549, HOP-92,
NCI-H460, NCI-H522, HCT-116, SNB-75, SF-295, SF-539, SNB-75, SK-OK-3,
SK-MEL-5, OVCAR-4, OVCAR-8, NCI/ADR-RES, CAKI-1, 786–0, RXF
393, MCF7, HS 578T, T-47D, and MDA-MB-468 cancer cell lines with a
growth range of 4.63–49.34%. Antiproliferative data revealed
that compounds 18c and 18f exhibited excellent
cytotoxicity against the lung (A549) cell line with a range of growth
inhibition of 55–91%. Compound 18m showed moderate
activity against breast (MCF-7) and lung (A549) cell lines with a
growth inhibition range of 51–62% (Table ).
Determination of IC50 Values
Compounds (14g, 14j, 18b, 18c, 18f, 18m) showing excellent % GI against non-small
cell lung cancer (A549, NCI-H460), breast cancer (MCF-7), colon cancer
(HCT-116), and ovarian cancer (SKOV-3) cell lines were further evaluated
for their IC50 values using an SRB assay, and the results
are depicted in Table .Among the cell lines tested, lung cancer cell lines NCI-H460
and A549 were found to be more susceptible against these compounds.
Compounds 18b, 18c, 18f, and 18m exhibited good to moderate cytotoxicity against the A549
cancer cell line with IC50 values ranging between 2.91
and 10.55 μM. Among the compounds tested, compounds 18c and 18f demonstrated the best antiproliferative activity
against A549 with IC50 values of 3.68 and 2.91 μM,
respectively, while compound 14g demonstrated moderate
antiproliferative activity against NCI-H460. Compound 18m was found to be moderately active against both MCF-7 and A549 cell
lines with IC50 values of 9.34 and 8.44 μM, respectively.
Dose–response curve of compounds 18b, 18c, 18f, 18m doxorubicin and paclitaxel against A549 and MCF-7 cell lines
is depicted in Supporting Information.
The standard compounds doxorubicin and paclitaxel were screened as
positive control, as shown in Table . Compound 18f was further evaluated for
its toxicity against normal HEK-293 cells (normal kidney cells). The
IC50 of compound 18f was 116-fold higher in
HEK-293 (IC50 = 696 μM) as compared to A549 cells.
Table 6
IC (μM)
Profile of Active Compounds
test compound
non-small
cell Lung cancer
breast cancer
colon cancer
ovarian cancer
HEK-293
A549
NCI-H460
MCF-7
HCT-116
SKOV-3
14g
nt
6.65
nt
8.90
10.63
nt
14j
nt
5.55
nt
10.45
32.50
nt
18b
10.554
nt
nt
nt
nt
nt
18c
3.69
nt
nt
nt
nt
nt
18f
2.91
nt
nt
nt
nt
696
18m
8.44
nt
9.35
nt
nt
nt
capsaicin
nt
30.66
nt
40.16
22.03
nt
paclitaxel
0.03 nM
nt
nt
nt
nt
nt
doxorubicin
nt
4.29
0.18 nM
57.77
25.83
nt
Structure–Activity Relationship (SAR)
On the
basis of the in vitro antiproliferative results (IC50) obtained, SAR of the synthesized compounds was developed
based on the nature and position of the substituent present on the
aromatic ring attached to the 1,2,3-triazole ring. Triazole conjugates
in both series (14a–p and 18a–o) were found to be highly active when compared
to the nonconjugated natural capsaicin and its structural isomer (16c). The lipophilic side chain of 1,2,3-triazole tethered
capsaicinoids greatly influenced the antiproliferative activity. Replacing
the lipophilic side chain of (R)-N-(4-hydroxy-3-methoxybenzyl)-3,7-dimethyloct-6-enamide
with citronellic acid significantly enhanced the activity. Further,
it has been observed that the nature of the substituents on the aromatic
ring connected to the 1,2,3-triazole moiety also influenced the activity.
Compounds with choro/bromo/cyano substituted aromatic moiety demonstrated
better activity. Chloro-substitution led to better activity when compared
to other substitutions. On the basis of the position of the substitution,
the activity profile has been observed as 3,4-dichloro > 3-chloro
> 4-chloro > 2-chloro. On the basis of SAR, the most active
compound
(18f) has been selected for further detailed studies
(Figure ).
Figure 3
SAR for synthesized
1,2,3-triazoles against antiproliferative activity.
SAR for synthesized
1,2,3-triazoles against antiproliferative activity.
Reactive Oxygen Species (ROS) Generation Assay
A higher
level of ROS generation is a prime indication of apoptosis in cancerous
cells. In the current study, A549 cells were incubated with DCFDA
dye, and intracellular ROS was observed using a fluorescence microscope.
A 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 compound 18f at 5, 3, and 1.5 μM concentration.
Observation through fluorescence microscope is a qualitative means
of ROS generation in A549 lung cancer cells where a sharp increase
in fluorescence intensity was observed (Figure ). This study indicated that compound 18f triggered ROS generation in A549 cells, which is a key
feature of apoptosis.
Figure 4
Determination of reactive oxygen species in A549 cells
by paclitaxel
and 18f. H2O2 is used as positive
control. Control group had no green fluorescence, and the ROS level
was low in the control group but higher in the treated groups.
Determination of reactive oxygen species in A549 cells
by paclitaxel
and 18f. H2O2 is used as positive
control. Control group had no green fluorescence, and the ROS level
was low in the control group but higher in the treated groups.
Apoptosis Assay through DAPI Staining
DAPI staining
helped differentiate between normal and apoptotic cells by the nuclear
morphological changes caused in a concentration-dependent manner after
treatment with compound 18f at 5, 3, and 1.5 μM
concentration against the A549 cell line. The morphological changes
that have occurred can be visualized using fluorescence microscopy.
The nuclei of untreated cells appeared as more or less rounded structures,
while the treatment groups (18f) including paclitaxel
showed chromatin condensation, nuclear blebbing, and formation of
apoptotic bodies. The nuclear morphological changes, with the exception
of the untreated control, clearly suggested that A549 cells had undergone
apoptosis (Figure ).
Figure 5
Fluorescence microscopy analysis of DAPI-stained cells was undertaken
to study nuclear alterations and apoptotic body formation, both of
which are also features of apoptosis. Effect of 18f on
nuclear morphology of A549 lung cancer cells with varying concentrations
and paclitaxel as positive control was assessed after DAPI staining.
Arrows represent the changes in the nuclear structure such as chromatin
condensation and nuclear damage.
Fluorescence microscopy analysis of DAPI-stained cells was undertaken
to study nuclear alterations and apoptotic body formation, both of
which are also features of apoptosis. Effect of 18f on
nuclear morphology of A549 lung cancer cells with varying concentrations
and paclitaxel as positive control was assessed after DAPI staining.
Arrows represent the changes in the nuclear structure such as chromatin
condensation and nuclear damage.
Measurement of Loss of Mitochondrial Membrane Potential (ΔΨm)
Reduction of MMP Ψm) is the main characteristic of apoptosis. To assess the effect
of compound 18f, loss of MMP was observed through fluorescence
microscopy using rhodamine-123 staining. After 48 h treatment with
compound 18f against A549 cells, it was observed that
the significant reduction in MMP at 3.0 μM was due to the decrease
in fluorescence intensities that clearly justified the mitochondrial
membrane destabilization in comparison to untreated cells. The result
was quite similar in the case of the paclitaxel treatment group. The
results obtained indicate the probable role of loss of MMP in the
induction of apoptosis by 18f in the A549 cell line (Figure ).
Figure 6
Effect of paclitaxel
and 18f on mitochondrial membrane
potential using rhodamine-123. Exponentially growing A549 cells were
treated with paclitaxel at its IC50 doses and 18f at varying concentrations for 48 h. Paclitaxel was used as positive
control. Cells treated with paclitaxel and 18f at 5 μM
show the maximum loss in the mitochondrial membrane potential.
Effect of paclitaxel
and 18f on mitochondrial membrane
potential using rhodamine-123. Exponentially growing A549 cells were
treated with paclitaxel at its IC50 doses and 18f at varying concentrations for 48 h. Paclitaxel was used as positive
control. Cells treated with paclitaxel and 18f at 5 μM
show the maximum loss in the mitochondrial membrane potential.
Cell Cycle Analysis
To determine
the effect of compound 18f on the cell cycle of A549
cells, cell cycle analysis was
performed using flow cytometry after 48 h of treatment. As shown in Figure , the treatment of
compound 18f induced the accumulation of cells in the
S-phase from 31.78% in control to 50.57%, with a concomitant decrease
in G0–G1 phase reduction from 62.8% in control to 34.32% in
compound 18f treated cells at 10 μM concentration
(Figure ).
Figure 7
Compound 18f induces cell cycle arrest in A549 cells.
Cell cycle analysis of A549 cancer cells treated with compound 18f for 48 h indicated that compound 18f induced
cell cycle arrest at the S-phase. Data presented as mean ± SD
of three independent experiments. **P <
0.01.
Compound 18f induces cell cycle arrest in A549 cells.
Cell cycle analysis of A549 cancer cells treated with compound 18f for 48 h indicated that compound 18f induced
cell cycle arrest at the S-phase. Data presented as mean ± SD
of three independent experiments. **P <
0.01.
Migration Potential of
NCI-H460 and A549 Lung Cancer Cells
To determine the effect
of compounds 14j and 18f on the migration
capacities of NCI-H460 and A549 cells,
a wound healing assay was performed. Compound 14j showed
moderate wound healing and inhibited the colony-forming ability of
NCI-H460 cells (Figures S3 and S4). Our
results demonstrated that doses of compound 18f treatment
at different concentrations significantly inhibited the migration
potential of A549 cells when compared to their respective control
group after 24 h of scratching (Figure ).
Figure 8
Compound 18f inhibits the migration potential
of A549
lung cancer cells. The migration potential of compound 18f treated cells at different concentrations was examined using a wound
healing assay. Images were captured at different time points, and
the wound area was quantified using the ImageJ software. Scale bar,
100 μm; magnification, 10×. *P < 0.05,
**P < 0.01.
Compound 18f inhibits the migration potential
of A549
lung cancer cells. The migration potential of compound 18f treated cells at different concentrations was examined using a wound
healing assay. Images were captured at different time points, and
the wound area was quantified using the ImageJ software. Scale bar,
100 μm; magnification, 10×. *P < 0.05,
**P < 0.01.
Conclusions
A series of thirty-one 1,2,3-triazole tethered
capsaicinoids were
synthesized by employing one-/two-point modifications around the capsaicin
scaffold. All the newly synthesized compounds were evaluated for their
antiproliferative activity against 60 cancer cell lines. Antiproliferative
screening suggested that compounds 14g, 14j, 18b, 18c, 18f, and 18m showed good to moderate activity against cancer cell lines.
The most potent compound (18f) showed good IC50 (2.92 μM) value against A549 non-small cell lung cancer. Compound 18f revealed triggered apoptosis, elevated intracellular ROS
levels, disrupted mitochondrial membrane potential and reduced cell
migration potential of A549 cells in a dose-dependent manner. Further,
compound 18f was found to arrest the cell cycle at the
S-phase and produced significant activity. As a result of the findings
of this investigation, this compound may serve as a template for the
further development of novel capsaicinoid-based anticancer agents
against lung cancer.
Methods
General Procedure
for Synthesis of Propargylation
A
solution of 0.5 g of compound (10) was dissolved in 10
mL of dry acetone followed by the addition of activated K2CO3 (5 equiv). Then propargyl bromide (1.2 equiv) was
added to it and was refluxed for 4–5 h. After the completion
of the reaction (monitored by TLC), the reaction mixture was allowed
to attain room temperature and was poured into ice-cold water to form
a colorless solid that was afterward filtered off under a vacuum to
afford the desired product.
General Procedure for Synthesis
of the 1,2,3-Triazole Ring (14a–p)
The solution of compound
(13) bearing terminal alkyne was dissolved in 15 mL of t-butanol/water (1:1) at ambient temperature. Then 1.5 equiv
of CuSO4·5H2O was added, and the reaction
mixture was stirred for 10 min. Initially, the color of the reaction
mixture was observed to be light blue. Then sodium ascorbate (4 equiv)
was added to the reaction mixture and allowed to stir for15 min. The
color of the reaction mixture changed from blue to dark brown. After
15 min, substituted aromatic azide (1.1 equiv) was added to the reaction
mixture and was allowed to stir for a further 8 h at ambient temperature.
After the completion of the reaction monitored by TLC, the reaction
mixture was poured into the water and extracted with ethyl acetate
(2 × 20 mL). The combined organic layer was dried over anhydrous
sodium sulfate, filtered, and evaporated under reduced pressure to
obtain the final triazole derivative (14a–p), which was recrystallized with ethyl acetate/hexane (70–90%
quantitative yields).
A solution of 1 g of hydroxylamine
hydrochloride and sodium acetate
trihydrate (2 equiv) was dissolved in 25 mL of water. Further on,
vanillin (15, 0.9 equiv) was added to the reaction mixture and refluxed
for 10 min. After the completion of the reaction monitored by TLC,
the reaction mixture was brought to room temperature. The solid precipitate
formed was filtered off under a vacuum and washed with 10 mL of cold
water that led to the colorless solid in pure form in 76.4% quantitative
yield. Melting point (m.p.) = 79.3 °C; 1H NMR (400
MHz, CDCl3, ppm) δ 9.10 (s, 1H), 8.03 (s, 1H), 7.69
(s, 1H), 7.10–7.04 (m, 1H), 6.87 (d, J = 8.4
Hz, 1H), 3.86 (s, 3H); exact mass: 167.06, elemental analysis calculated
for (C8H9NO3): C, 57.48; H, 5.43;
N, 8.38; found: C, 57.47; H, 5.45; N, 8.37.
4-Hydroxy-3-methoxy-phenylamine
(16b)
A solution of 1 g of 4-hydroxy-3-methoxy-benzaldehyde
oxime (16a) in 5 mL of acetic acid was cooled to 10–15
°C.
Then 4.71 g (4 equiv) of activated zinc dust was added to the reaction
mixture and stirred at ambient temperature for a further 3 h. Completion
of the reaction was monitored by TLC, and the reaction mixture was
filtered off to remove excess zinc. Further on, the filtrate was collected
and neutralized with ammonia to yield a white solid precipitate. The
precipitate formed was filtered under a vacuum and washed with 20
mL of cold water to afford the required compound (16b) in pure form (74.0% yield) that was directly used for the next
step without any further purification. 1H NMR (400 MHz,
CDCl3, ppm) δ 6.83 (ddt, J = 8.8,
1.7, 0.9 Hz, 1H), 6.82–6.76 (m, 2H), 6.61 (s, 1H), 3.93 (tt, J = 6.2, 0.9 Hz, 2H), 3.85 (s, 2H), 1.39 (d, J = 12.5 Hz, 1H). Chemical formula: C8H11NO2, elemental analysis: C, 62.73; H, 7.24; N, 9.14. found C,
62.76; H, 7.29; N, 9.19.
General Procedure for N-(4-Hydroxy-3-methoxybenzyl)-3,7-dimethyloct-6-enamide
(16c)
To the DMF (10 mL) solution of R-(+)-citronellic acid (1.2 equiv), 2 equiv of (3-dimethylamino-propyl)-ethyl-carbodiimide
hydrochloride (EDC·HCl) and 0.2 equiv of hydroxybenzotriazole
(HOBt) were added, and the reaction mixture was stirred for 5 min
under an inert atmosphere in a two-neck round-bottom flask. Finally,
the compound (16b) was added in the reaction mixture
to continue at room temperature overnight. The completion of the reaction
was monitored by TLC; the reaction mixture was poured into ice-cold
water and extracted with ethyl acetate (4 × 20 mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered,
and evaporated under a vacuum to afford the impure reaction mixture
that was purified by column chromatography (silica gel) to obtain
the pure natural product hybrid amide. 1H NMR (400 MHz,
CDCl3, ppm) δ 6.86–6.81 (m, 2H), 6.78 (m,
1H), 6.57 (s, 1H), 5.12–5.04 (m, 1H), 4.38 (m, 2H), 3.85 (s,
2H), 2.49 (d, J = 16.2 Hz, 1H), 2.25 (d, J = 16.2 Hz, 1H), 2.10–2.00 (m, 1H), 2.03–1.80
(m, 2H), 1.66 (m, 3H), 1.53 (m, 1H), 1.27 (m, 1H), 0.97 (d, J = 8.1 Hz, 3H); exact mass: 305.20; elemental analysis
calculated for (C18H27NO3): C, 70.79;
H, 8.91; N, 4.59; O, 15.72; found: C, 70.77; H, 9.93; N, 4.57.
A solution of 0.5 g compound (16c) was dissolved in 10 mL of dry acetone followed by the addition of
activated K2CO3 (5 equiv). Then propargyl bromide
(1.2 equiv) was added to it and refluxed for 4–5 h. After the
completion of the reaction (monitored by TLC), the reaction mixture
was allowed to attain room temperature and poured into ice-cold water
to form a colorless solid that afterward was filtered off under a
vacuum to afford the desired product. 1H NMR (400 MHz,
CDCl3, ppm) δ 6.97–6.95 (d, J = 8.1 Hz, 1H), 6.95 (s, 1H), 6.83–6.79 (d, J = 18.2 Hz, 1H), 5.92–5.89 (d, J = 16.2 Hz,
1H), 5.07 (brs, 1H), 4.73 (s, 2H), 4.67 (s, 1H), 4.40–4.39
(d, J = 5.5 Hz , 1H), 4.37–4.36 (d, J = 5.4 Hz, 2H), 3.84 (s, 3H), 2.50 (s, 1H), 2.33 (s, 1H),
2.24–2.15 (m, 2H), 2.03–1.94 (m, 2H), 1.66 (s, 3H),
1.58 (s, 3H), 1.40–1.33 (m, 1H), 1.24–1.18 (m, 1H),
0.95–0.94 (d, J = 8.1 Hz, 3H); IR cm–1: 3280, 3075, 2964, 2924, 2851, 2116, 1638, 1596, 1553, 1513, 1453,
1420, 1378, 1265, 1219, 1140, 1022; exact mass: 343.21, elemental
analysis calculated for (C21H29NO3): C, 73.44; H, 8.51; N, 4.08; O, 13.97; found: C, 73.43; H, 8.54;
N, 4.06.
General Procedure of Formation of the 1,2,3-Triazole
Ring (18a–o)
Same as the
General Procedure
for Synthesis of the 1,2,3-Triazole Ring (14a–p).
In
Vitro Antiproliferative Activity at a Single
Dose
All the synthesized compounds were screened for their
antiproliferative activity against a panel of 60 cancer cell lines
at the National Cancer Institute, Bethesda, MD, USA, as per the standard
procedure given at http://www.dtp.nci.nih.gov. The RPMI 1640 medium (5% fetal bovine serum and 2 mM l-glutamine) was used to grow the human tumor cell lines. All the
tumor cells were incubated into a 96-well microtiter plate. Then this
plate was placed for incubation at 37 °C for 24 h. After those
two plates of each cell line were fixed with TCA in situ, and optical density was measured at this point, which represented
the cell population of each cell line at the time of compound addition
(ODtzero). On the other hand, all the tested compounds were dissolved
in DMSO to yield 400-fold desired final concentration and stored at
−80 °C. These frozen compounds were thawed, and their
aliquot part was diluted to 10–4 M concentration
with the medium containing 50 μg/mL of gentamicin at the time
of compound addition. The control sample was made with DMSO only.
The tested compounds (100 μL) from the aliquot parts were added
to the appropriate 96-well microtiter plate containing 100 μL
of the medium resulting in the required final drug concentrations
of 10–5 and 0 M (control). After the addition of
tested compounds, the 96-well microtiter plate was incubated for 48
h at 100%, 5% CO2, 95% air, and 100% relative humidity.
Cold TCA was used to stop the assay for adherent cells. Further on,
50 mL of 50% (w/v) TCA was used to fix the cell and incubated for
1 h at 4 °C. The supernatant was removed, and the 96-well microtiter
plates were rinsed five times with water and air dried. A 100 mL solution
of the protein binding dye Sulforhodamine B (SRB) was made at 0.4%
(w/v) in 1% acetic acid and added to each well of the plates. These
plates were placed at room temperature for incubation for 10 min and
then washed with 1% acetic acid five times to remove unbound dye.
Then the plates were treated with 10 mM Trizma base so that unbound
dye was solubilized with the Trizma base. The absorbance was measured
at a wavelength of 515 nm on an automated plate reader, and results
for each tested compound were calculated as the percent of tumor growth
of the treated cells in comparison with the untreated control cells.
Optical density (OD) was recorded for the SRB-derived color just before
exposing the cells to the test compound (ODtzero) and after 48 h exposure
to the test compound (ODtest) or the control vehicle (ODctrl).
Cell
Lines, Cell Culture, Growth Conditions, and Reagents
A panel
of human cancer cell lines, namely, MCF-7 (breast), NCI-H460,
A549 (lung), MiaPaCa (pancreas), HCT-116 (colon), and PC-3 (prostate),
was purchased from ATCC. The cell lines were grown in T75 tissue culture
flasks in a complete growth medium (RPMI-1640 and DMEM) added with
10% FBS,100 mg/mL streptomycin, as well as 100 U/mL penicillin in
a humidified carbon dioxide incubator (New Brunswick, Galaxy 170R,
Eppendorf) at 37 °C and 5% CO2 with 95% relative humidity.
DAPI (4′ 6-diamidino-2-phenylindole) and Rhodamine-123, DCFDA
(2′, 7′-dichlorofluorescein diacetate) were procured
from Sigma-Aldrich (St. Louis, MO, USA). Sulforhodamine B dye was
purchased from Hi Media. Monolayer cultures of the above cell lines
were trypsinized using 0.25% trypsin/EDTA (1 mM) solution. After the
cells got detached, the activity of trypsin/EDTA solution was stopped
using the complete growth medium and centrifuged at 900 rpm for 5
min. Cells were again dispersed in the complete growth medium in tissue
culture flasks and incubated in a CO2 incubator. When cells
attained approx. 50–60% confluency, they were treated with
target compounds dissolved in DMSO and the untreated control cultures
with DMSO (<0.2%).
Cytotoxicity Activity against Different Cancer
Cell Lines
The in vitro cytotoxicity activity
of target compounds
(18a–o) was carried out using the
SRB (Sulforhodamine B) assay method reported. For preliminary screening,
optimum inoculum densities per well of A549, NCI-H460, MCF-7, and
HCT-116 cell lines were seeded in 96-well flat-bottom plates (NUNC).
Briefly, 100 mL/well of cell suspensions was seeded in 96-well tissue
culture plates and incubated for 24 h. When cells attained 50–60%
confluency, then different concentrations 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 an aqueous medium and air dried. Once the plates dried,
100 mL/well SRB dye was 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
solubilized by adding 100 mL/well 10 mM TRIS (tris(hydroxymethyl)
aminomethane) buffer at pH 10.5, and plates were kept on an orbital
shaker for 5 min. Lastly, absorbance was recorded at 540 nm in a microplate
reader (Tecan Infinite M Nano). IC50 was determined by
GraphPad Prism 6.[55]
Reactive
Oxygen Species (ROS) Generation Assay
Dye
2′,7″-dichlorofluorescein diacetate (DCFH-DA) was used
to measure intracellular ROS production. DCFH gets transformed into
highly fluorescent 2′,7″-dichlorofluorescein (DCF) in
the presence of an oxidant. In this study, 1 × 105 A549 cells were incubated for 24 h in six-well plates and 1.5, 3,
and 5 μM concentration of molecule 18f and kept
for a further 48 h incubation. H2O2 (0.05%)
was used as the standard positive control and added 1 h before conclusion
of the experiment. The cells were washed with ice-cold PBS, and 10
mM DCFH-DA was added to all the wells for 30 min in the dark. The
plate was further washed with cold PBS, and the final volume of incomplete
media was added to each well and finally observed under an inverted
fluorescence microscope (Olympus IX70).[56,57]
Apoptosis
Assay through DAPI Staining
To examine apoptotic
cell death qualitatively, morphological variations in chromatin structure
were detected using DAPI staining. Precisely, A549 cells at a density
of 1 × 105 were seeded in six-well tissue culture
plates and kept for 24 h to attain confluency of about 50–60%,
treatment was given to the plate with the above-mentioned concentration
of compound 18f and paclitaxel used as standard positive
control drug, and cells were kept for 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 cells were stained with 1 mg/mL
DAPI in the dark for 5 min and washed with ice-cold PBS, the final
volume of PBS was added to each well, and the plate was observed under
an inverted fluorescence microscope (Olympus 1X70).[58]
Measurement of the Loss of Mitochondrial
Membrane Potential
(ΔΨm)
Mitochondrial perturbation due to the loss
of membrane potential was studied using dye rhodamine-123 by a fluorescence
microscope qualitatively. Non-small lung cancer A549 cells (1 ×
105/mL/well) were seeded in six-well tissue culture plates;
treated with 1.5, 3.0, and 5.0 μM concentration of compound 18f and paclitaxel used as the standard positive control drug;
and incubated for 48 h. After treatment, cells were washed with ice-cold
PBS; later, the final concentration of 0.2 mM rhodamine was incubated
to all the wells and finally kept for 20–30 min in the dark
inside the incubator. Further, cells were washed with cold PBS, and
the final volume of incomplete media was added to all the wells and
analyzed under a fluorescence microscope (Olympus 1X70).[59]A549 cancer
cells were treated
with compound 18f and analyzed for the distribution of
cell population in different phases of the cell cycle. After 48 h
of treatment, the cells were harvested and washed with phosphate buffer
saline (PBS). The cells were then fixed with ice-cold 70% ethanol.
After overnight incubation at 4 °C, the cells were washed with
PBS and incubated with propidium iodide solution (20 μg/mL)
containing RNase (10 μg/mL) at 37 °C for half an hour.
The cells were analyzed using a BD FACSverse cytometer. A total of
10, 000 events were analyzed for each sample, and the data were analyzed
using the Modfit LT software.[23]
Wound
Healing Assay
A549 cells (4 × 105) were seeded
and grown within six-well plates until a monolayer
was formed. Then, by using a 200 μL pipette tip, a scratch was
given in the confluent monolayer. Cells were washed with 1× PBS
to remove detached cells before incubating with fresh media with different
compound 18f concentrations (3, 10, and 30 μM)
or DMSO vehicle control. Cell migration across the wound area was
captured at two time intervals (0 and 24 h) with an inverted microscope.
The wound area was measured using the ImageJ software, and the percentage
wound healed area during the course of the assay was calculated after
normalizing the wound area.[23]
Authors: Jamie R Friedman; Stephen D Richbart; Justin C Merritt; Kathleen C Brown; Krista L Denning; Maria T Tirona; Monica A Valentovic; Sarah L Miles; Piyali Dasgupta Journal: Biomed Pharmacother Date: 2019-08-09 Impact factor: 6.529
Figure 1
Figure 2
Scheme 1
Scheme 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8