Alkyne-azide cycloaddition analogues of dehydrozingerone as potential anti-prostate cancer inhibitors: Via the PI3K/Akt/NF-kB pathway

Article abstract of DOI:10.1039/c7md00267j

Herein, we report the isolation and synthetic modification of dehydrozingerone (DHZ, 1), a secondary metabolite present in the rhizome of Zingiber officinale. We synthesized O-propargylated dehydrozingerone, which was subsequently coupled by alkyne-azide cycloaddition (3-20) using click chemistry. The compounds (1-20) were evaluated for their in vitro cytotoxic activity in a panel of three cancer cell lines. Among all the DHZ derivatives, 3, 6, 7, 8, 9 and 15 displayed potent cytotoxic potential with an IC₅₀ value ranging from 1.8-3.0 μM in MCF-7, PC-3 and HCT-116 cell lines. Furthermore, compound 7 has proven to be the most potent cytotoxic compound in all the three distinct cancer cell lines and also demonstrated significant anti-invasive potential in prostate cancer. The mechanistic study of compound 7 showed that it not only suppressed the AKT/mTOR signalling which regulates nuclear transcription factor-NF-kB but also augmented the expression of anti-invasive markers E-cadherin and TIMP. Compound 7 significantly decreased the expression of pro-invasive markers vimentin, MMP-2 and MMP-9, respectively. This study underscores an efficient synthetic approach employed to evaluate the structure-activity relationship of dehydrozingerone (1) in search of potential new anticancer agents.

Full text of DOI:10.1039/c7md00267j

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RESEARCH ARTICLE
Alkyne–azide cycloaddition analogues of
dehydrozingerone as potential anti-prostate
Cite this: DOI: 10.1039/c7md00267j
cancer inhibitors via the PI3K/Akt/NF-kB pathway†
a
Chetan Kumar,‡a Reyaz Ur Rasool,‡bc Zainab Iqra,c Yedukondalu Nalli,
Prabhu Dutt,a Naresh K. Satti,a Neha Sharma,a Sumit G. Gandhi,d
c
a
*
*
Anindya Goswami and Asif Ali
Herein, we report the isolation and synthetic modification of dehydrozingerone (DHZ, 1), a secondary me-
tabolite present in the rhizome of Zingiber officinale. We synthesized O-propargylated dehydrozingerone,
which was subsequently coupled by alkyne–azide cycloaddition (3–20) using click chemistry. The com-
pounds (1–20) were evaluated for their in vitro cytotoxic activity in a panel of three cancer cell lines. Among
all the DHZ derivatives, 3, 6, 7, 8, 9 and 15 displayed potent cytotoxic potential with an IC50 value ranging
from 1.8–3.0 μM in MCF-7, PC-3 and HCT-116 cell lines. Furthermore, compound 7 has proven to be the
most potent cytotoxic compound in all the three distinct cancer cell lines and also demonstrated signifi-
cant anti-invasive potential in prostate cancer. The mechanistic study of compound 7 showed that it not
only suppressed the AKT/mTOR signalling which regulates nuclear transcription factor-NF-kB but also aug-
mented the expression of anti-invasive markers E-cadherin and TIMP. Compound 7 significantly decreased
the expression of pro-invasive markers vimentin, MMP-2 and MMP-9, respectively. This study underscores
an efficient synthetic approach employed to evaluate the structure–activity relationship of dehydrozinger-
one (1) in search of potential new anticancer agents.
Received 24th May 2017,
Accepted 2nd October 2017
DOI: 10.1039/c7md00267j
rsc.li/medchemcomm
effect of 1 against cancer cells,10,11 so far, no molecular mech-
anisms of its action on cell growth/invasion have been de-
1. Introduction
Dehydrozingerone (4-hydroxy-3-methoxybenzalacetone, DHZ,
Fig. 1) is isolated from rhizomes of ginger (Zingiber
officinale)1 and can be synthesized by simple aldol condensa-
tion of vanillin and acetone.2 DHZ, which is a recognized bio-
synthetic intermediate and degradant of curcumin,3,4 shows a
structural resemblance to curcumin, as both bear styryl ke-
tone moieties with similar substitutions on the phenyl ring.5
DHZ (1) has shown significant therapeutic potential with pro-
found biological properties, such as anti-inflammatory, anti-
oxidant6,7 antifungal8 and anti-mycobacterial activity.9 Al-
though few preliminary reports unveiled the antiproliferative
scribed. Motohashi et al.12–14 have investigated the anti-
mutagenic activities of DHZ and its synthetic analogs and
reported that 4-O-benzalacetone substituted analogues of DHZ
revealed the strongest antimutagenic activity. These results
disclosed that ring substitution with a group such as 4-hy-
droxyl, methoxyl or methyl increased the antimutagenic activ-
ity. In light of the above studies, we selected the modification
in the aromatic core of DHZ for the development of the SAR.
We have synthesized 4-O-propargylated dehydrozingerone by
alkyne–azide cycloaddition (3–20) demonstrating moderately
high yields (85–93%) by means of click chemistry. This study
aimed to investigate the in vitro cytotoxic potential of
dehydrozingerone derivatives15 in prostate cancer cells.
a Natural Product Chemistry Division, India. E-mail: asifali@iiim.ac.in;
Fax: +91 191 25693331; Tel: +91 191 2569222
b Academy of Scientific & Innovative Research (AcSIR), Anusandhan Bhawan, 2
Rafi Marg, New Delhi-110001, India
Prostate cancer is considered as one of the leading causes
of all cancer deaths in most developed countries.16 The
c Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine,
Canal Road, Jammu-180001, India. E-mail: agoswami@iiim.ac.in
d Plant Biotechnology division, CSIR-Indian Institute of Integrative Medicine,
Canal Road, Jammu, India
† Electronic supplementary information (ESI) available: 1D and 2D NMR and
HRESIMS spectra of all new compounds. NMR spectral data of compounds
(1–20) along with HRMS spectral data. See DOI: 10.1039/c7md00267j
‡ Contributed equally.
Fig. 1 Pharmacophore of (DHZ) 1 for antimutagenic/cytotoxic activity.
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phosphatidylinositol 3-kinase (PI3K)/AKT pathway is constitu-
tively hyperactivated in prostate cancer prognosis and is an
effective target for therapeutic development.17 By modulating
diverse signaling pathways, AKT is implicated in the metasta-
sis progression in an advanced condition of the disease.18
The direct target of hyperactive AKT, nuclear transcription
factor-KB (NF-kB), has been correlated with increased meta-
static potential in prostate cancer and is associated with me-
tastasis and invasion in many tumors.19 The recent literature
demonstrates that NF-kB is constitutively activated in most
types of prostate cancers. NF-kB promotes the migration and
invasion of prostate cancer cells by a number of NF-kB-
regulated gene products.20 The current study demonstrates
the anti-invasive and anti-motility activity of dehydrozinger-
one derivative 7 in the human prostate cancer line (PC-3).
2. Results and discussion
2.1. Chemistry
Dehydrozingerone (DHZ, 1) is known as feruloylmethane
which displays versatile therapeutic activities against various
types of diseases and is used as anti-infective, anticancer,
antiviral, and anti-hypertensive agents.4–6,9 Due to the pro-
found biological potential of 1, we explored its chemistry and
biological activities by modifications of its structure and
synthesised the C4-aryl substituted 1,2,3-triazoles of 1 by al-
kyne–azide cycloaddition. The aromatic ring substituted 1-az-
ido derivatives (a–r) were synthesised via Scheme 1.15
Synthesis of alkyne–azide
cycloaddition derivatives of 1
Scheme 1 Synthesis of substituted 1-azido derivatives.
The compound 1 C4-hydroxyl was O-propargylated, affording
intermediate 2 which was further coupled with different
substituents of azide by employing facile click chemistry15 Biological assay
to
obtain
distinct
derivatives
of
1,2,3-triazol-4-yl-
2.2. Anti-proliferative activity of compound 7
dehydrozingerone (3–20). The appearance of 1H NMR sig-
nals at δH 4.81 (2H) and 2.54 (1H) confirmed the condensa-
tion of propargyl bromide. The signals at δC 56.57 are at-
tributed to the carbon of the active methylene inserted
after O-propargylation of 1. 1H NMR of all the triazole deriv-
atives (3–20) has shown a characteristic singlet for the
triazole proton in the range δH 7.8–8.44. In addition, 13C
NMR spectra exhibited characteristic signals for the triazole
ring carbon at δC 119.5–122.7 and 149.3–144.6, respectively.
The synthetic yields, molecular weight, HPLC data and
NMR spectra of all the compounds have been described in
the experimental section.
For SAR studies, compounds (2–20) were screened for
their in vitro cytotoxicities against three human cancer cell
lines. As is evident from Table 1, 4-O-substituted alkyne–azide
cycloaddition derivatives 3, 6, 7, 8, 9 and 15 showed the
highest cytotoxic potential probably due to the change in
pharmacophore, which facilitated the binding of the ligand
in the receptor pocket.
The cell viability assay (MTT) was performed on a panel of cancer
cell lines. The cells were exposed to the corresponding derivatives
for 48 h with a concentration range of 100 nM to 100 μM. A con-
sistent cytotoxic activity was found in compound 7 against
prostate cancer (PC-3) and HCT-116 (human colon cancer) cell
lines with an IC50 concentration of 3.073 μM and 2.002 μM. As
shown in Table 1, compounds 3, 8 and 15 showed an IC50 value
of 2.028, 1.8 and 2.19 μM respectively in MCF-7 (breast cancer cell)
while the IC50 values of 6 and 9 were found to be 2.013 and 2.045
μM respectively in PC-3 cell lines. In addition, to verify the cyto-
toxic effects of 7 on normal prostate cells (BPH-1), MTT results
showed an IC50 value higher than 100 μM indicating the safety
profile of this compound 7 in normal cells (data not shown).
2.3. Anti-motility and anti-invasive activities of 7
Prostate cancer invasion and metastasis remains the most
critical determinant of resectability and hence survival.21
Here, we aimed to study the effect of 7 on the invasive
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Table 1 The IC50 values (μM) of dehydrozingerone derivatives in a panel
of cancer cell lines. Data were compared with untreated control, and
IC50 values were expressed as the mean
experiments
SD of three independent
S. no
PC-3 (IC50
)
SD
HCT-116 (IC50
)
SD
MCF-7 (IC50
)
SD
1
2
3
4
5
6
7
8
43.21 0.5
40.23 0.5
24.22 0.3
50.34 0.2
42.11 0.2
2.013 0.3
3.073 0.3
31.56 0.4
2.045 0.2
76.81 0.2
46.74 0.2
58.12 0.2
67.23 0.1
59.22 0.5
47.98 0.5
56.81 0.3
80.67 0.4
78.56 0.3
56,89 0.5
59.23 0.2
34.78 0.5
49.74 0.4
37.67 0.1
>100
54.65 0.1
65.38 0.4
2.028 0.5
65.78 0.3
75.85 0.1
33.67 0.2
26.58 0.1
1.8 0.2
29.14 0.5
86.69 0.5
>100 0.3
>100 0.1
87.37 0.2
>100 0.2
2.19 0.2
65.38 0.5
86.37 0.5
>100
>100
21.34 0.1
2.002 0.1
29.55 0.2
25.65 0.3
>100
96.34 0.5
76.57 0.5
57.78 0.5
78.9 0.1
39.00 0.1
65.25 0.4
76.22 0.5
67.33 0.3
45.36 0.5
76.36 0.3
9
10
11
12
13
14
15
16
17
18
19
20
65.48 0.3
>100
Fig. 2 Effects of compound 7 on motility and cell scattering aptitude
of PC3 cells. (A and B) The wound healing assay was performed by
treating PC-3 cells with increasing concentrations of 7 which were
consecutively assessed for the degree of wound healing. Photographs
of scratched areas were taken at 20× magnification (C and D). PC-3
cells were treated with indicated concentrations of 7 along with or
without VEGF to check its effects on cell scattering. Following 24 h of
incubation with 7, individual colonies were observed under an inverted
microscope and were photographed. The data shown are representa-
tive of three independent experiments. SD **P < 0.01.
IC50 values are expressed in μM.
potential of PC-3 cells in vitro. To examine any effect of 7 on
the motility of PC-3 cells, a wound healing assay was
performed. It was observed that 7 significantly inhibited the
cell motility in a concentration dependent manner and there
was a characteristic wound closure due to the migration of
PC-3 cells in vehicle treated wells (Fig. 2A and B). Cell scatter-
ing is an inherent incident associated with metastatic cancer
cells.22 Here, we evaluated the effects of 7 on PC-3 cell scat-
tering and the results demonstrated that 7 efficiently blocked
the cell scattering in the presence of growth factor VEGF
(Vascular endothelial growth factor) (Fig. 2C and D).
Since the PC-3 cell line is considered as an aggressive cell
line due to PTEN mutation and having the ability to invade
through the extracellular matrix to colonize at the secondary
tumor sites,23 we sought to inspect the effect of 7 on cell in-
vasion through the Boyden chamber invasion assay. The re-
sults showed that 7 subdued the cell invasion potential of
PC-3 cells in a dose dependent manner (Fig. 3A and B). Col-
ony formation was totally restrained by 7 in PC-3 cells as well
(Fig. 3C and D). Further, we investigated the apoptosis induc-
ing property of compound 7 in PC-3 cells by studying the mi-
tochondrial membrane potential loss and casapase-3/7 activa-
tion through JC-1 staining and the Caspase-Glo assay,
respectively. The results showed that compound 7 induced
2.4. Compound 7 suppresses Akt-mTOR signalling
In prostate cancer, AKT is considered as a major player
for tumor growth and metastasis. Also in the majority of
prostate cancers, NF-kB through phosphatidylinositol 3-ki-
nase (PI3K) dependent activation of IKK is constitutively
activated, leading to the stimulation of direct downstream
MMPs via transcriptional regulation.24 Therefore, by west-
ern blotting, we analyzed the effect of 7 on the phos-
phorylation of AKT and the corresponding NF-kB levels.
The results showed that the expression of NF-kB was sig-
nificantly attenuated in a dose dependent manner and
the phosphorylation of AKT was strongly impeded by 7
treatments but showed negligible effects on total AKT
(Fig. 4A). This was in contrast to the insignificant effects
of the parent compound 1 on the phosphorylation of
AKT (Fig. S1C†).
Furthermore, it is well known that NF-kB is regulated
by the PI3K/AKT pathway via mTOR activation.19 So
here, we studied the effects of 7 on mTOR and S6K ac-
tivation through western blotting. The results revealed
that the phosphorylation of mTOR was attenuated by 7
along with the downregulation of S6K, and no character-
istic change in the expression of total mTOR was
observed.
apoptosis in
a
concentration-dependent manner, as
suggested by increased JC-1 monomer formation and de-
creased JC-1 aggregate formation (mitochondrial membrane
potential loss). The JC-1 results were further validated by an
increase in caspase 3/7 activity (Fig. S1A and B†). These col-
lective results clearly support the anti-metastatic, anti-
invasive and anti-proliferative potential of 7 in PC-3 cells.
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Fig. 4 Compound 7 inhibits PI3K/Akt/NF-KB signaling. (A and B) PC-3
cells were treated with indicated concentrations of 7. Whole cell ly-
sates were checked for the expressions of indicated proteins by west-
ern blotting. β-Actin was used as a loading control and the data shown
are representative of three independent experiments.
Fig. 3 Effects of compound 7 on the invasive and colony formation
ability of PC-3 cell lines. (A) Boyden chamber assay was performed to
check the effect of 7 on the invasive ability of PC-3 cells. Following incu-
bation with compound 7 for the indicated time point, invaded cells were
counted and images were captured at 20× magnification. (B) Bar graphs
are representative of the number of invasive cells per field. (C and D) To
check the effects on colony formation, PC-3 cells were exposed to dif-
ferent concentrations of 7 and incubated for five days. After the incuba-
tion, the cells were stained with crystal violet; stained colonies were
counted randomly, quantified and images were taken under an inverted
microscope at 20× magnification. Data from three independent experi-
ments were subjected to statistical analysis. SD **P < 0.01, *p < 0.05.
Fig. 5 Compound 7 abrogates MMP-2 and MMP-9 gelatinase activity
and expression. (A) Gelatin zymography was carried out to check the
effects of 7 on MMP-2 and MMP-9 activity. BSA was used as a loading
control. (B) PC-3 cells were seeded in six-well plates and treated with
indicated concentrations of 7. Whole cell lysates were checked for the
expressions of indicated proteins by western blotting. β-Actin was used
as loading control and the data shown are representative of three in-
dependent experiments.
2.5. Compound 7 abrogates MMP 2 and MMP 9 gelatinase
activity as well as their expression
Matrix metalloproteinases (MMPs) play a major role in metas-
tasis and invasion.25 To check the consequences of 7 on MMP
activation, zymography was performed with decreasing concen-
trations of 7. The results showed that matrix gelatin degrada-
tion was inhibited significantly in aggressive PC-3 cells with in-
creased concentrations of 7 (Fig. 5A). Among all MMPs, MMP-2
and MMP-9 play a critical role in cancer progression; so in or-
der to check the effect of 7 on the expression of MMPs, western
blotting experiments were performed and the results showed
that MMP-2 and MMP-9 expressions were significantly attenu-
ated by 7. TIMP-1 is a well known marker involved in the nega-
tive regulation of pro-invasive and pro-metastatic MMPs. So, by
employing western blotting we demonstrated that 7 strongly
amplified the expression of TIMP-1 in a dose dependent man-
ner (Fig. 5B). In higher incidences of metastasis and advanced
tumors, the expression and localization of E-cadherin to the
cell membrane are lost; both loss of E-cadherin and
overexpression of MMPs contribute to sturdy tumors.26 Our re-
sults demonstrated that treatment of PC-3 cells with 7 elevated
the expression of E-cadherin, an epithelial marker, along with a
steady decline in the expression of vimentin, a mesenchymal
marker (Fig. 5B).
3. Conclusions
Distinct dehydrozingerone derivatives (2–20) were synthesized
via the alkyne–azide cycloaddition reaction with good yield.
All these synthetic derivatives were evaluated for their in vitro
cytotoxicities in three distinct human cancer cell lines. The
compounds containing 4-O-substituted alkyne–azide on the
aromatic
ring
attached
to
2,6-difluorophenyl
(3),
4-methoxyphenyl (6), 3-methoxyphenyl (8), 2-methoxyphenyl
(9), and 2-chlorophenyl (15) derivatives were shown to be 16–
18-fold more potent than 1 against human cancer cell lines,
viz. PC-3 and MCF-7, while 2,6-dichlorophenyl (7) showed bet-
ter potency than 1 in all the cancer cell lines tested. Com-
pounds of this class appear amenable to further modifica-
tions and will prove useful for prevention and treatment of
cancer proliferation. Collectively, it can be concluded from
the above studies that compound 7 blocks invasion and me-
tastasis of PC-3 cells by abrogating NF-KB dependent
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Scheme 2 Synthesis of alkyne–azide cycloaddition derivatives (3–20).
expression and activation of MMP-2 and MMP-9 via the sup- 4. Experimental section
pression of the PI3K/AKT pathway. So, it is implied that de-
rivative 7 of dehydrozingerone could be explored as an anti-
4.1. Chemistry
metastatic/anti-invasive agent against malignant prostate
cancer.
High resolution mass spectra were obtained using an Agilent
6540 (Q-TOF) high resolution mass spectrometer in
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electrospray (ESI MS) mode. 1H NMR spectra were recorded
(Bruker Avance) at 400 and 500 MHz while 13C NMR spectra
were recorded at 125 MHz in CDCl3 and CD3OD. The chemi-
cal shift values were reported in δ (ppm) and coupling con-
stants in Hertz. Tetramethylsilane (TMS) was used as an
internal standard. All reactions were monitored by thin layer
chromatography (TLC) on silica gel 60F254 (0.25 mm thick,
Merck) with spots visualized by UV (254 and 366 nm) and
anisaldehyde reagent was used as the development agent.
Dehydrozingerone (DHZ 1) was isolated from the rhizome of
Zingiber officinale while other reagents were purchased com-
mercially and used without further purification, unless other-
wise stated.
standard MTT dye uptake method according to a procedure
described previously.27
4.5. Clonogenic assay
The clonogenic assay was performed according to a previ-
ously described method.21 Briefly, PC-3 cells (1 × 103 cells per
well) were plated in 6-well tissue culture grade plates. The
culture medium was changed after 24 h and then a new me-
dium was added. Cells were then exposed to various concen-
trations of conjugate 7 along with the vehicle DMSO for 5
days.
4.6. Scratch motility (wound healing) assay
The assay was performed as described previously.28 Briefly, in
a 6-well plate PC-3 cells (5 × 105 cells per well) were seeded
and allowed to form a confluent monolayer for 24 h, and
then serum starved for another 24 h. After that, the mono-
layer was scratched with a sterile pipette tip (200 μL), washed
with a serum-free medium and photographed (time 0 h).
Cells were successively treated with medium containing low
serum (1.0%) in the presence of different concentrations of 7
along with the vehicle DMSO for 24 h. Scratched areas were
progressively photographed with a Nikon D3100 inverted
microscope camera (20× magnification). The percentage of
wound closure was estimated using the following equation:
% wound closure = [1 − (wound area at t1/wound area at t0) ×
100%], where t1 is the time after wounding and t0 is the time
immediately after wounding.
4.2. Synthesis of compounds (2–20)
Compound 1 (1 g, 5.2 mmol) was dissolved in dry acetone
(50 mL), and then anhydrous Na2CO3 (2.1 g, 20 mmol) was
added and stirred for 15 minutes at 0 °C under a nitrogen at-
mosphere. Propargyl bromide (1.4 equiv.) was gradually
added (Scheme 1) and stirred for 4–5 h. Subsequently, the re-
action mixture was diluted with brine water and extracted
thrice with ethyl acetate. Finally, the combined extract passed
through Na2SO4 and evaporated under vacuum afforded the
crude product, which was further purified using a silica col-
umn to obtain intermediate compound 2 in excellent yield
(92%). Furthermore, compound 2 was dissolved in a mixture
of t-butyl alcohol and water (3 mL, 1 : 1) followed by the suc-
cessive addition of substituted 1-azido derivatives (1.2 equiv.),
sodium ascorbate (0.5 equiv.) and CuSO4·5H2O (0.1 equiv.).
The reaction mixture was stirred at room temperature for 1–2
h (Scheme 2). Subsequently, the mixture was diluted with wa-
ter (10 mL) and extracted with ethyl acetate (3 × 10 mL) dried
over Na2SO4, and concentrated under vacuum, affording the
crude product, which was subjected to purification by flash
column chromatography leading to the isolation of alkyne–
azide cycloaddition derivatives (2–20).
4.7. Matrigel invasion assay
The effect of 7 on cell invasion was determined using a BD
Biocoat Tumor Invasion Assay System (BD Bioscience, Bed-
ford, MA) according to the instruction of the manufacturer.
Briefly, PC-3 cells (1.2 × 106 cells per well) were treated with
different concentrations of 7 or the vehicle DMSO. The
Matrigel invasion assay was performed following a previously
described method.29
4.3. Cell culture and antibodies
4.8. Cell scattering assay
PC3, MCF-7 and HCT-116 cell lines were purchased from the
European Collection of Cell Culture (ECACC). Cells were cul-
tured in RPMI 1640/DMEM containing 10% FBS, 70 mg L−1
penicillin plus 0.1 g L−1 streptomycin and were incubated at
37 °C with 95% air and 5.0% CO2. Antibodies were obtained
from the following commercial sources: anti-AKT, anti-NF-kB,
anti-MMP-2, anti-MMP-9, anti-E-cadherin, anti-mTOR, anti-S6
K and anti-TIMP-1 from Santa Cruz Biotechnology (Santa
Cruz, CA), anti-p-AKT and anti p-mTOR from Cell Signaling
(Cell Signaling Technologies, MA) and anti-beta actin from
Sigma Chemical (St Louis, MO).
The cell scattering assay was performed as described previ-
ously.28 Briefly, PC-3 cells were seeded at a density of 1000
cells per well in 6-well plates. After 3–4 days of incubation,
small, cohesive, and discrete colonies were formed. Cells
were then stimulated with VEGF (20 mg ml−1) in the presence
of various concentrations of 7 in growth medium. Following
overnight incubation, colonies were observed under a bright
field microscope. Photographs were taken for individual
colonies.
4.9. Immunoblotting
4.4. Cell viability assay
PC-3 cells were exposed to the indicated concentrations of 7
along with DMSO. Cells were then lysed with lysis buffer and
an equal amount of protein from each sample was subjected
to western blot analysis as described previously.30
MCF-7, PC-3 and HCT-116 cells (2.5 × 103 cells per well) were
plated in a 96-well tissue culture plate. The cells were treated
with different concentrations of indicated compounds by the
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4.10. Gelatin zymography
1H), 5.93 (s, 1H), 3.94 (s, 3H), 2.37 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 198.57, 148.45, 147.04, 143.91, 126.88, 124.92,
123.52, 114.95, 109.53, 55.98, 27.26.
The gelatinase activity of MMP-2 was assessed by a method
standardized previously.29 Briefly, PC-3 cells in
a
4.14.2. (E)-4-(3-Methoxy-4-(prop-2-yn-1-yloxy)phenyl)but-3-
en-2-one (2). TLC, Hex : EtOAc (7 : 3), Rf 0.6, colourless amor-
phous solid, yield: 1.1 g (92.19%).1H NMR (400 MHz, CDCl3)
δ 7.49 (t, J = 15.8 Hz, 1H), 7.13 (dd, J = 8.3, 1.9 Hz, 1H), 7.09
(d, J = 1.9 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.68–6.52 (m, 1H),
4.81 (d, J = 2.4 Hz, 2H), 3.92 (s, 3H), 2.54 (t, J = 2.4 Hz, 1H),
2.38 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 198.06, 149.63,
149.36, 143.21, 128.52, 125.89, 122.38, 113.41, 110.25, 77.96,
76.35, 56.57, 55.94, 27.80. HRMS (ESI+) calculated for
C14H15O3 [M + H]+: 231.1016, found: 231.1013. HPLC purity:
99.0%.
subconfluent culture (70–80% cell density of confluent cul-
ture) were refreshed with a new medium and kept for incuba-
tion with increasing concentrations of 7 for 48 h. The condi-
tional media obtained from both treated and untreated
samples were employed for protein estimation and equal
amounts of total proteins (20 mg) were mixed with sample
buffer (2.0% SDS, 25% glycerol, 0.1% bromophenol blue and
60 mM Tris-HCl, pH 6.8). Gelatin zymography of the samples
was carried out using 7.5% SDS-polyacrylamide gels
containing 0.1% gelatin at 100 V for 3 h at 4 °C. The gels
were rinsed accordingly with washing buffer (2.0% Triton
X-100 in dd water) at room temperature to remove SDS,
followed by incubation overnight at 37 °C in TCNB buffer (50
mM Tris-HCl, pH 8.0, 10 mM CaCl2, 0.02% NaN3). The gels
were stained with Coomassie blue R-250 (Sigma) (0.125%
Coomassie blue R-250, 50% methanol, 10% acetic acid) for
1–1.5 h and destained with a destaining solution (20% meth-
anol, 10% acetic acid, 70% dd water). The gelatinase activity
was detected by observing unstained bands on a blue back-
ground on Coomassie stained gels.
4.14.3. (E)-4-(4-((1-(2,6-Difluorophenyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)but-3-en-2-one (3). TLC, CHCl3 :
MeOH (95 : 5), Rf (0.4), white crystalline solid, yield: 81.79 mg
1
(81.57%). H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.57–7.45
(m, 2H), 7.21–7.10 (m, 5H), 6.64 (d, J = 16.2 Hz, 1H), 5.46 (s,
2H), 3.94 (s, 3H), 2.40 (s, 3H). 13C NMR (125 MHz, CDCl3) δ
198.36, 149.86, 143.73, 143.33, 131.46, 128.37, 125.71, 122.81,
113.95, 113.40, 112.67, 112.64, 112.51, 112.48, 110.28, 91.41,
77.28, 77.02, 76.77, 66.07, 62.95, 59.10, 55.96, 53.43, 50.88,
44.31, 27.36. HRMS (ESI+) calculated for C20H32F2N2O3 [M +
H]+: 386.1311, found: 368.2380. Purity: 94.78%.
4.11. JC-1 staining
4.14.4. (E)-4-(4-((1-(2-(Hydroxymethyl)-phenyl)-1H-1,2,3-
For JC-1 staining, treated cells (1 × 106 cells per well) were
washed with chilled PBS and then 1 mM JC-1 (Thermo
Fisher) solution was added to the cells so that the final con-
centration of JC-1 was 1 μM. The cells were accordingly incu-
bated at 37 °C for 30 min and analysed under fluorescence
microscope for JC-1 aggregates and monomers according to
the standard protocol as per manufacturer's instructions.
triazol-4-yl)methoxy)-3-methoxy-phenyl)but-3-en-2-one
(4).
TLC, CHCl3 : MeOH (95 : 5), Rf (0.3) yellowish solid, yield:
1
85.29 mg (86.42%). H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H),
7.63 (d, J = 7.9 Hz, 1H), 7.48 (ddd, J = 16.2, 13.3, 5.2 Hz, 3H),
7.38 (d, J = 7.7 Hz, 1H), 7.12 (s, 2H), 7.09 (s, 1H), 6.61 (d, J =
16.2 Hz, 1H), 5.41 (s, 2H), 4.47 (s, 2H), 3.90 (s, 3H), 2.36 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 198.73, 149.77, 143.25,
135.86, 135.60, 131.61, 130.18, 129.20, 128.40, 125.75, 124.45,
122.78, 113.77, 110.27, 62.91, 61.85, 55.97, 29.70, 27.38.
HRMS (ESI+) calculated for C21H22N3O4 [M + H]+: 380.1605,
found: 380.1598. HPLC purity: 99.0%.
4.12. Caspase3/7-Glo assay
Briefly, cells were seeded in a 96-well plate at a density of 5 ×
103 cells per well for 24 h. The caspase 3/7 reagent and sub-
strate mix was prepared as per the manufacturer's protocol
(Promega). After proper treatment of the cells, approximately
100 μl of the caspase 3/7 reagent and substrate mix was
added and incubated for 25 min. After the incubation, the lu-
minescence was read using a Tecan plate reader.
4.14.5. (3E)-4-(3-Methoxy-4-((1-(4-(phenyldiazenyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)but-3-en-2-one (5). TLC,
CHCl3 : MeOH (95 : 5), Rf 0.4, orange colour solid, yield: 105.2
1
mg (89.19%). H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.09
(d, J = 8.7 Hz, 2H), 7.95 (dd, J = 8.0, 1.5 Hz, 2H), 7.91 (d, J =
8.8 Hz, 2H), 7.57–7.51 (m, 3H), 7.48–7.43 (m, 1H), 7.13 (s,
2H), 7.10 (s, 1H), 6.64–6.59 (m, 1H), 5.43 (s, 2H), 3.93 (s, 3H),
2.37 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 198.45, 152.34,
149.93, 144.68, 143.24, 138.37, 131.75, 129.23, 128.35, 125.74,
124.39, 123.11, 122.81, 121.16, 120.98, 113.56, 110.12, 62.92,
56.00, 27.31. HRMS (ESI+) calculated for C26H24N5O3 [M +
H]+: 454.1874, found: 454.1879. HPLC purity: 94.61%.
4.14.6. (E)-4-(3-Methoxy-4-((1-(4-methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)but-3-en-2-one (6). TLC, CHCl3 :
MeOH (95 : 5), Rf 0.5, creamy white solid, yield: 87.37 mg
(88.52%). 1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 11.4 Hz,
1H), 7.61 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 16.2 Hz, 1H), 7.17–
7.09 (m, 2H), 7.08 (d, J = 7.6 Hz, 1H), 7.03–6.99 (m, 2H), 6.60
(d, J = 16.2 Hz, 1H), 5.40 (s, 2H), 3.90 (d, J = 4.9 Hz, 3H), 3.86
4.13. Statistical analysis
Data were expressed as the mean standard error of three in-
dependent experiments. Statistical analyses were performed
using Graph Pad Prism version 5. Comparisons between two
groups were conducted using unpaired Student's t-test. ‘p’
values <0.05 were considered as statistically significant.
4.14. Characterization of synthesized compounds
4.14.1. (E)-4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one
(1). TLC, Hex : EtOAc (7 : 3), Rf 0.4, yellowish crystalline solid.
1H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 16.2 Hz, 1H), 7.12–
7.03 (m, 2H), 6.93 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 16.2 Hz,
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(d, J = 0.9 Hz, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
198.30, 159.98, 149.98, 149.75, 144.19, 143.32, 130.39, 128.11,
125.64, 122.82, 122.27, 121.47, 114.83, 113.63, 110.22, 62.96,
55.97, 55.65, 27.37. HRMS (ESI+) calculated for C21H22N3O4
[M + H]+: 380.1605, found: 380.1599. HPLC purity: 99.0%.
4.14.7. (E)-4-(4-((1-(2,6-Dichlorophenyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)but-3-en-2-one (7). TLC, CHCl3 :
MeOH (95 : 5), Rf 0.4, yellow crystalline solid, yield: 97.22 mg
8.20 (s, 1H), 8.00–7.94 (m, 2H), 7.44 (d, J = 16.2 Hz, 1H),
7.13–7.06 (m, 3H), 6.60 (d, J = 16.2 Hz, 1H), 5.41 (s, 2H), 3.91
(s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 198.18,
149.83, 143.06, 125.87, 125.59, 122.70, 121.03, 120.60, 113.75,
110.41, 62.84, 56.00, 27.41. HRMS (ESI+) calculated for
C20H19N4O5 [M + H]+: 395.1350, found: 395.1353. Purity:
90.06%.
4.14.12. (E)-4-(3-Methoxy-4-((1-(p-tolyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)but-3-en-2-one (12). TLC, CHCl3 : MeOH
1
(89.52%). H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.51 (d, J
1
= 1.8 Hz, 1H), 7.49 (s, 1H), 7.46 (d, J = 7.2 Hz, 1H), 7.43 (t, J =
2.5 Hz, 1H), 7.10 (s, 2H), 7.09 (s, 1H), 6.61 (d, J = 16.2 Hz,
1H), 5.47 (s, 2H), 3.91 (s, 3H), 2.37 (s, 3H).; 13C NMR (100
MHz, CDCl3) δ 198.28, 150.06, 149.82, 143.94, 143.29, 133.88,
131.84, 128.89, 128.49, 125.75, 125.26, 122.75, 114.59, 110.46,
63.33, 56.00, 27.37. HRMS (ESI+) calculated for C20H18Cl2N3O3
[M + H]+: 418.0714, found: 418.0727. HPLC purity: 99.0%.
4.14.8. (E)-4-(3-Methoxy-4-((1-(3-methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)but-3-en-2-one (8). TLC, CHCl3 :
MeOH (95 : 5), Rf 0.6, creamy white solid, yield: 90.57 mg
(95 : 5), Rf 0.5, white solid, yield: 73.33 mg (77.57%). H NMR
(400 MHz, CDCl3) δ 8.05 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.45
(d, J = 16.2 Hz, 1H), 7.31 (d, J = 8.3 Hz, 2H), 7.13 (dd, J = 6.8,
4.9 Hz, 2H), 7.10–7.07 (m, 1H), 6.60 (d, J = 16.2 Hz, 1H), 5.40
(s, 2H), 3.90 (d, J = 5.1 Hz, 3H), 2.42 (s, 3H), 2.36 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 202.14, 153.71, 153.57, 148.09,
147.14, 142.93, 138.49, 134.06, 132.03, 129.47, 126.64, 125.09,
124.36, 117.47, 114.04, 66.79, 59.85, 31.14, 24.92. HRMS
(ESI+) calculated for C21H22N3O3 [M + H]+: 364.1656, found:
364.1653. HPLC purity: 96.70%.
1
(91.77%). H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.50–7.37
4.14.13. (E)-4-(4-((1-(3,4-bisIJTrifluoromethyl)phenyl)-1H-
(m, 2H), 7.33 (t, J = 2.2 Hz, 1H), 7.26–7.23 (m, 1H), 7.13 (t, J =
4.8 Hz, 2H), 7.09 (s, 1H), 6.98 (dd, J = 8.4, 1.7 Hz, 1H), 6.61
(d, J = 16.2 Hz, 1H), 5.41 (s, 2H), 3.92 (s, 3H), 3.88 (s, 3H),
2.37 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 198.68, 160.53,
149.63, 144.18, 142.94, 137.78, 130.46, 128.17, 125.28, 122.37,
121.42, 115.01, 113.40, 112.78, 110.25, 106.39, 62.77, 55.97,
55.66, 27.53. HRMS (ESI+) calculated for C21H22N3O4 [M +
H]+: 380.1605, found: 380.1602.
1,2,3-triazol-4-yl)methoxy)-3-methoxy
phenyl)but-3-en-2-one
(13). TLC, CHCl3 : MeOH (95 : 5), Rf 0.4, white solid, yield:
1
108.55 mg (85.94%). H NMR (400 MHz, CDCl3) δ 8.24 (d, J =
7.6 Hz, 3H), 7.96 (s, 1H), 7.45 (d, J = 16.2 Hz, 1H), 7.15–7.06
(m, 3H), 6.62 (d, J = 16.2 Hz, 1H), 5.43 (s, 2H), 3.93 (s, 3H),
2.37 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.61 (s), 143.05
(s), 128.65 (s), 125.89 (s), 122.68 (s), 122.35 (s), 120.97 (s),
120.45 (s), 113.82 (s), 110.46 (s), 77.33 (s), 77.02 (s), 76.70 (s),
62.88 (s), 55.99 (s), 27.39 (s). HRMS (ESI+) calculated for
C22H18F6N3O3 [M + H]+: 486.1247, found: 486.1236. HPLC pu-
rity: 99.0%.
4.14.14. EthylIJE)-4-(4-((2-methoxy-4-(3-oxobut-1-en-1-yl)-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoate (14). TLC,
CHCl3 : MeOH (95 : 5), Rf 0.45, white solid, yield: 89.22 mg
(81.38%). 1H NMR (400 MHz, CDCl3) δ 8.24–8.18 (m, 2H),
8.16 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 16.2 Hz, 1H),
7.12 (s, 2H), 7.09 (s, 1H), 6.61 (d, J = 16.2 Hz, 1H), 5.42 (s,
2H), 4.42 (q, J = 7.1 Hz, 2H), 3.91 (d, J = 5.9 Hz, 3H), 2.37 (s,
3H), 2.17 (s, 2H), 1.59 (s, 2H), 1.42 (t, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 198.10, 165.24, 149.94, 145.06,
144.97, 143.25, 143.16, 139.90, 139.79, 131.33, 130.86, 128.45,
125.78, 122.74, 121.06, 119.96, 113.57, 110.41, 62.94, 61.22,
55.65, 27.03, 14.10. HRMS (ESI+) calculated for C23H24N3O5
[M + H]+: 422.1710, found: 422.1716. HPLC purity: 99.0%.
4.14.15. Synthesis of (E)-4-(4-((1-(2-chlorophenyl)-1H-1,2,3-
4.14.9. (E)-4-(3-Methoxy-4-((1-(2-methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)but-3-en-2-one (9). TLC, CHCl3 :
MeOH (95 : 5), Rf 0.6, creamy white solid, yield: 82.90 mg
1
(84.0%). H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.78 (d, J =
7.9 Hz, 1H), 7.44 (dd, J = 15.7, 12.1 Hz, 2H), 7.17 (t, J = 7.5
Hz, 1H), 7.10 (dd, J = 14.5, 7.7 Hz, 4H), 6.61 (d, J = 16.2 Hz,
1H), 5.43 (s, 2H), 3.90 (d, J = 14.2 Hz, 6H), 2.37 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 198.47, 151.14, 150.11, 149.87,
143.35, 143.02, 130.16, 128.11, 125.60, 125.48, 125.41, 122.83,
121.36, 113.99, 112.22, 110.37, 62.64, 55.66, 27.03. HRMS
(ESI+) calculated for C21H22N3O4 [M + H]+ 380.1605, found:
380.1599. HPLC purity: 99.0%.
4.14.10. (E)-N-(4-(4-((2-Methoxy-4-(3-oxobut-1-en-1-yl)-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl) benzamide (10).
TLC, CHCl3 : MeOH (95 : 5), Rf 0.4, white solid, yield: 110.71
1
mg (90.84%). H NMR (400 MHz, MeOD) δ 8.31 (s, 1H), 7.99–
7.92 (m, 4H), 7.78–7.72 (m, 2H), 7.60–7.48 (m, 5H), 7.21–7.14
(m, 3H), 6.65 (d, J = 16.2 Hz, 1H), 5.39 (s, 2H), 3.94 (s, 3H),
2.40 (s, 3H). 13C NMR (125 MHz, MeOD) δ 199.99, 167.59,
149.92, 149.66, 144.45, 144.05, 139.50, 134.43,, 132.59,
131.83, 128.50, 128.12, 127.46, 125.17, 123.05, 121.98, 121.61,
121.10, 113.49, 110.40, 62.41, 55.74, 26.82. HRMS (ESI+) cal-
culated for C27H25N4O4 [M + H]+: 469.1870, found: 469.1874.
HPLC purity: 85.24%.
4.14.11. (E)-4-(3-Methoxy-4-((1-(4-nitrophenyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)but-3-en-2-one (11). TLC, CHCl3 :
MeOH (95 : 5), Rf 0.4, yellow crystalline solid, yield: 90.81 mg
(88.28%). 1H NMR (400 MHz, CDCl3) δ 8.44–8.38 (m, 2H),
triazol-4-yl)methoxy)-3-methoxyphenyl)but-3-en-2-one
(15).
TLC, CHCl3 : MeOH (95 : 5), Rf 0.3, white solid, yield: 89.92 mg
1
(90.16%). H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.74–7.39
(m, 5H), 7.11 (d, J = 15.7 Hz, 3H), 6.61 (d, J = 16.2 Hz, 1H), 5.44
(s, 2H), 3.91 (s, 3H), 2.37 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
198.15, 150.00, 143.60, 143.18, 134.84, 130.80, 130.71, 128.57,
128.30, 127.94, 127.78, 125.71, 125.13, 122.74, 114.06, 110.43,
63.07, 55.82, 27.19. HRMS (ESI+) calculated for C20H19ClN3O3
[M + H]+: 384.1109, found: 384.1114. HPLC purity: 99.0%.
4.14.16. (E)-4-(4-((1-([1,1′-Biphenyl]-4-yl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-but-3-en-2-one
(16).
TLC,
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CHCl3 : MeOH (95 : 5), Rf 0.6, white solid, yield: 91.58 mg
(82.75%). H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.80 (dd,
113.92, 110.49, 62.89, 55.98, 27.35. HRMS (ESI+) calculated
for C20H19N3O4 [M + H]+: 366.1448, found: 366.1450. HPLC
purity: 99.0%.
1
J = 26.0, 8.6 Hz, 6H), 7.65 (d, J = 7.2 Hz, 3H), 7.51 (t, J = 7.5
Hz, 3H), 7.18–7.11 (m, 3H), 6.64 (d, J = 16.2 Hz, 1H), 5.46 (s,
2H), 3.95 (d, J = 4.8 Hz, 4H), 2.40 (s, 3H). 13C NMR (100 MHz, Conflicts of interest
CDCl3) δ 198.23, 149.84, 144.56, 143.25, 142.01, 139.58,
The authors declare no competing interests.
136.03, 129.01, 128.40, 128.03, 127.10, 125.73, 122.80, 121.17,
120.90, 113.79, 110.36, 63.03, 56.08, 56.01, 55.16, 40.62,
29.78, 27.36. HRMS (ESI+) calculated for C15H6F2N2O2 [M +
H]+: 285.0470, found: 285.0482. HPLC purity: 86.04%.
Acknowledgements
We are thankful to the Ministry of AYUSH for the financial as-
sistance in the GAP-2143 project. We are also thankful to
Deepika Singh for NMR spectral data analysis. The manu-
script bears the Institutional publication No. IIIM/2164/2017.
4.14.17. (E)-4-(4-((2-Methoxy-4-(3-oxobut-1-en-1-yl)-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzaldehyde (17). TLC,
CHCl3 : MeOH (95 : 5), Rf 0.35, creamy white solid, yield: 89.71
1
mg (91.38%). H NMR (400 MHz, CDCl3) δ 10.09 (s, 1H), 8.22
(d, J = 5.0 Hz, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.97 (d, J = 8.5 Hz,
2H), 7.47 (d, J = 16.2 Hz, 1H), 7.16–7.10 (m, 3H), 6.63 (d, J =
16.2 Hz, 1H), 5.44 (s, 2H), 3.94 (s, 3H), 2.39 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 198.32, 190.58, 149.73, 149.67, 145.11,
143.21, 140.82, 136.14, 131.37, 128.39, 125.74, 122.75, 121.10,
120.56, 113.59, 110.25, 62.78, 55.95, 29.70, 27.39. HRMS
(ESI+) calculated for C21H20N3O4 [M + H]+ 378.1448, found:
378.1444. HPLC purity: 94.64%.
4.14.18. (E)-4-(3-Methoxy-4-((1-phenyl-1H-1,2,3-triazol-4-yl)-
methoxy)phen-yl)but-3-en-2-one (18). TLC, CHCl3 : MeOH
(95 : 5), Rf 0.5, white crystalline solid, yield: 75.92 mg
(83.53%). 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 10.6 Hz,
1H), 7.71 (d, J = 8.1 Hz, 2H), 7.52 (dd, J = 8.3, 7.0 Hz, 2H),
7.47–7.39 (m, 2H), 7.11 (dd, J = 12.8, 11.2 Hz, 3H), 6.60 (d, J =
16.2 Hz, 1H), 5.41 (s, 2H), 3.91 (s, 3H), 2.36 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 198.13, 149.88, 144.50, 143.26, 136.98,
129.81, 128.96, 128.30, 125.71, 122.80, 121.25, 120.65, 113.75,
110.32, 63.14, 56.08, 27.48. HRMS (ESI+) calculated for
C20H20N3O3 [M + H]+: 350.1494, found: 350.1500. Purity:
95.70%.
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4.14.19. (E)-4-(4-((1-(3-Bromo-5-fluorophenyl)-1H-1,2,3-
triazol-4-yl)meth-oxy)-3-methoxy-phenyl)-but-3-en-2-one (19).
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1
99.27 mg (85.66%). H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H),
7.57–7.45 (m, 2H), 7.21–7.10 (m, 5H), 6.64 (d, J = 16.2 Hz,
1H), 5.46 (s, 2H), 3.94 (s, 3H), 2.40 (s, 3H). 13C NMR (125
MHz, CDCl3) δ 198.36, 149.86, 143.73, 143.33, 131.46, 128.37,
125.71, 122.81, 113.95, 113.40, 112.67, 112.64, 112.51, 112.48,
110.28, 91.41, 77.28, 77.02, 76.77, 66.07, 62.95, 59.10, 55.96,
53.43, 50.88, 44.31, 27.36. HRMS (ESI+) calculated for
C20H18BrFN3O3 [M + H]+: 446.0510, found: 446.0506. Purity:
97.53%.
4.14.20. (E)-4-(4-((1-(2-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)-
methoxy)-3-methoxyphenyl)but-3-en-2-one (20). TLC, CHCl3 :
MeOH (9 : 1), Rf 0.5, yellowish white solid, yield: 82.17 mg
(86.44%). 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 5.1 Hz,
1H), 7.69 (dd, J = 8.0, 1.6 Hz, 1H), 7.55 (t, J = 8.1 Hz, 1H),
7.37–7.27 (m, 1H), 7.19 (dt, J = 13.2, 4.9 Hz, 3H), 7.02 (ddd, J
= 15.4, 8.3, 4.2 Hz, 2H), 6.65 (d, J = 16.2 Hz, 1H), 5.38 (s, 2H),
3.91 (d, J = 5.2 Hz, 3H), 2.40 (d, J = 5.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 198.17, 149.89, 149.66, 149.32, 144.06,
143.10, 129.94, 125.84, 122.67, 120.95, 120.30, 119.57, 119.52,
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