Synthesis of newer 1,2,3-triazole linked chalcone and flavone hybrid compounds and evaluation of their antimicrobial and cytotoxic activities

Article abstract of DOI:10.1016/j.ejmech.2016.02.041

The present study was carried out in an attempt to synthesize a new class of antimicrobial and antiplasmodial agents by copper catalyzed click chemistry to afford 25 compounds 10-14(a-e) of 1,4-disubstituted-1,2,3-triazole derivatives of chalcones and flavones. The structures of the newly synthesized compounds were established by elemental analysis, IR, ¹H NMR, ¹³C NMR and Mass spectral data. The newly synthesized compounds were evaluated for their antibacterial activity against Gram positive bacteria (Staphylococcus aureus, Enterococcus faecalis), Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella boydii, Klebsiella pneumoniae) and antifungal activity against (Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Dermatophyte) as well as molds (Aspergillus niger, Aspergillus fumigatus). The antiplasmodial and cytotoxic activities of these compounds were also evaluated against human malaria parasite Plasmodium falciparum strain 3D7 and human hepato-cellular carcinoma cells (Huh-7), respectively. Compounds 10a, 10c, 10d, 12c and 14e showed promising antibacterial activity while compounds 10e, 11d, 11e, 12c, 13a, 13b, 13e, 14a and 14d showed good antifungal activity as compared to the corresponding standard drugs. Compound 10b was found to be the most active against Plasmodium falciparum while the remaining compounds showed moderate to weak antiplasmodial activity. However, cytotoxic activities of all compounds were found ineffective against Huh-7 cells.

Full text of DOI:10.1016/j.ejmech.2016.02.041

Accepted Manuscript
Synthesis of Newer 1,2,3-Triazole Linked Chalcone and Flavone Hybrid Compounds
and Evaluation of their Antimicrobial and Cytotoxic Activities
Rama Kant, Dharmendra Kumar, Drishti Agarwal, Rinkoo Devi Gupta, Ragini Tilak,
Satish Kumar Awasthi, Alka Agarwal
PII:
S0223-5234(16)30122-2
10.1016/j.ejmech.2016.02.041
EJMECH 8392
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2015
Revised Date: 15 February 2016
Accepted Date: 16 February 2016
Please cite this article as: R. Kant, D. Kumar, D. Agarwal, R.D. Gupta, R. Tilak, S.K. Awasthi, A.
Agarwal, Synthesis of Newer 1,2,3-Triazole Linked Chalcone and Flavone Hybrid Compounds and
Evaluation of their Antimicrobial and Cytotoxic Activities, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.02.041.
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ACCEPTED MANUSCRIPT
Graphical abstract

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Synthesis of Newer 1,2,3-Triazole Linked Chalcone and Flavone Hybrid Compounds and
Evaluation of their Antimicrobial and Cytotoxic Activities
a
b
c
c
b
Rama Kant , Dharmendra Kumar , Drishti Agarwal , Rinkoo Devi Gupta , Ragini Tilak ,
Satish Kumar Awasthi d,*, Alka Agarwal a,*
a
Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University,
Varanasi 221005, UP, India.
b
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University,
Varanasi 221005, UP, India.
c Faculty of Life Sciences and Biotechnology, South Asian University, Delhi 110021, India.
d
Chemical Biology Research Laboratory, Department of Chemistry, University of Delhi, Delhi
110007, India.
* Corresponding authors: E-mail: agarwal.dralka@gmail.com, skawasthi@chemistry.du.ac.in;
Telephone: +915426702173
Abstract
The present study was carried out in an attempt to synthesize a new class of antimicrobial
and antiplasmodial agents by copper catalyzed click chemistry to afford 25 compounds 10-14(a-
e) of 1,4-disubstituted-1,2,3-triazole derivatives of chalcones and flavones. The structures of the
1
newly synthesized compounds were established by elemental analysis, IR, H NMR, 13C NMR
and Mass spectral data. The newly synthesized compounds were evaluated for their antibacterial
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activity against Gram positive bacteria (Staphylococcus aureus, Enterococcus faecalis), Gram
negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella boydii, Klebsiella
pneumoniae) and antifungal activity against (Candida albicans, Candida tropicalis, Candida
parapsilosis, Cryptococcus neoformans, Dermatophyte) as well as molds (Aspergillus niger,
Aspergillus fumigatus). The antiplasmodial and cytotoxic activities of these compounds were
also evaluated against human malaria parasite Plasmodium falciparum strain 3D7 and human
hepato-cellular carcinoma cells (Huh-7), respectively. Compounds 10a, 10c, 10d, 12c and 14e
showed promising antibacterial activity while compounds 10e, 11d, 11e, 12c, 13a, 13b, 13e, 14a
and 14d showed good antifungal activity as compared to the corresponding standard drugs.
Compound 10b was found to be the most active against Plasmodium falciparum while the
remaining compounds showed moderate to weak antiplasmodial activity. However, cytotoxic
activities of all compounds were found ineffective against Huh-7 cells.
Key words; 1,2,3-Triazole, Chalcone, Antibacterial activity, Antifungal activity, Antiplasmodial
activity, Cytotoxicity
1. Introduction
The organic compounds containing chalcone and flavone scaffold as a core unit exhibit
various biological and pharmaceutical activities [1-3]. They are important as structural motifs
among biologically active molecules and also for combinatorial assembly of heterocyclic
scaffolds [4-6]. Chalcones containing several functional groups showed a wide spectrum of
biological activities such as antimicrobial [7,8], antimalarial [9,10], anticancer [11,12], anti-
inflammatory [13], antileishmanial [10,14], antiprotozoal [15], anti-HIV [16], antioxidant [17]
and antiulcer [18] activities. Flavones and their derivatives have also been found to display
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antioxidant [19], antimicrobial [20], anticancer [21], antimalarial [22], anti-inflammatory [23],
antiulcer [24], antileishmanial [25] and anti-HIV [26] properties. Due to its remarkable
bioactivities and structural novelty, much more effort has been devoted to the synthesis of
chalcone and flavone analogues [1,2].
1,2,3-Triazoles have received attention not only in organic chemistry but also in medicinal
chemistry due to their easy synthesis by copper-catalyzed click reaction as well as numerous
biological activities [27-33]. In recent years, 1,2,3-triazoles have gained special attention in the
drug discovery because several drug molecules contain 1,2,3-triazole group such as Tazobactam,
Cephalosporin and Cefatrizine. They are clinically used for the treatment of bacterial infections.
A number of compounds were synthesized with varied biological activities by the combination
of 1,2,3-triazoles with other pharmacophores via click chemistry. For example, a series of 1,2,3-
triazole bearing chalcone showed notable antimalarial activity against the D10, Dd2 and W2
strains of Plasmodium falciparum [34], a family of 1,2,3-triazole tethered β-lactam-chalcone
bifunctional hybrids exhibited moderate to good cytotoxic activity [35], 1,2,3-triazole analogues
of flavone displayed antimicrobial activity [36] and estrogen receptor alpha-positive breast
cancer inhibitors [37]. It is well known that the combination of two or more types of
pharmacophores into one molecule could afford a new entity with increased bioactivities [38,39].
As part of ongoing research work aimed towards the development of small molecules as
therapeutic agents [40-42], here we report the synthesis, antimicrobial, antiplasmodial and
cytotoxic activities of 1,2,3-triazole linked chalcone and flavone hybrids.
2. Result and discussion
2.1. Chemistry
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A synthetic strategy was followed for the synthesis of novel 1,2,3-triazole derivatives of
chalcones and flavones as shown in Scheme 1. The series of triazole derivatives 10-14(a-e) were
synthesized in various steps. The compound 1 was prepared by the simple methylation of 2,4,6-
trihydroxyacetophenone with (CH3)2SO4 and K2CO3 in dry acetone at reflux. This compound
was a common intermediate to all molecules being synthesized. In next step, 3,4-
dihydroxybenzaldehyde was reacted with propargyl bromide in presence of NaH at 0 °C to room
temperature to obtain mono and di-alkyne derivatives (2a-c). The mono-alkyne derivates (2a and
2b) were again methylated with (CH3)2SO4 to yield compound 3a and 3b. Then, the compound 1
was condensed with propargylated-benzaldehyde (3a, 3b and 2c) in the basic medium to give
respective 2-hydroxychalcones (4, 6 and 7). The mono and dipropargylated-2-hdroxychalcone
(4 and 7) was enough to react with I2 in DMSO to give corresponding flavones (5 and 8). In the
final step, the chalcones (4, 6 and 7) and flavones (5 and 8) were further reacted with substituted
aromatic azides (9a-e) via copper catalyzed [3+2] azide-alkyne cycloaddition reaction to afford
25 compounds 10-14(a-e) [Table 1]. The details of general synthetic procedure of all compounds
are mentioned in the experimental section. The structure of all synthesized compounds 10-14(a-
1
e) were confirmed by elemental analysis, IR, H NMR, 13C NMR and mass spectral data. The
structure of compound 10e was also confirmed by X-ray crystallographic analysis (Fig. 1).
2.2. Biological activity
2.2.1. Antimicrobial activity
A small library of 25 compounds 10-14(a-e) of 1,4-disubstituted-1,2,3-triazole containing
either one or two triazole units linked with chalcone and flavone scaffolds by exploiting copper
catalyzed [3+2] azide-alkyne cycloaddition [32] were synthesized. All the synthesized
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compounds 10-14(a-e) were screened for their antimicrobial activity (MIC) against various
bacterial and fungal strains. The potentiality of the compounds as antibacterials was appraised
for their antibacterial studies against various Gram positive such as Staphylococcus aureus
(ATCC 25323), Enterococcus faecalis (ATCC 29212) and Gram negative such as Escherichia
coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27893), Shigella boydii ((clinical isolate)
and Klebsiella pneumoniae (ATCC 27736) strains. The activity result as MIC is summarized in
Table 3. It is an interesting observation that the results of the antibacterial activity of these
compounds appeared to be related to the nature and position of substituents on the phenyl ring
and as well as on the structural variation and isomeric effects in the skeleton moieties. It is
evident from Table 3 that seven compounds viz. 10a, 10c, 10d, 11c, 11d, 12c and 14e were
found more potent with either equal or more MIC as compared to control drug ciprofloxacin.
DMSO was also taken in a control experiment which showed no effect in the experiment.
The compound 10a containing 2-chloro-4-fluoro substituted benzene ring showed potent
inhibitory activity against S. aureus and E. faecalis with MIC 6.25 µg/mL which is similar to the
standard drug ciprofloxacin (MIC 6.25 µg/mL). Further, the same compound also exhibited good
activity with MIC 12.5 µg/mL in E. coli, P. aeruginosa and S. boydii while in K. pneumoniae, it
showed moderate activity with MIC 25 µg/mL. The compound 10b having 3-chloro-4-fluoro
substituted benzene ring was found to have moderate to weak activity against all the tested
strains at a MIC range of 25-100 µg/mL. The compounds 10c and 10d having 2,4-difluoro and 2-
chloro substituted benzene ring, respectively showed most potent activity against E. coli and S.
boydii at MIC 6.25 µg/mL. Further, these compounds were also found active against S. aureus
and P. aeruginosa with MIC 12.5 µg/mL. Again, the compound 10d was found to be the most
potent with MIC 6.25 µg/mL while the compound 10c exhibited good activity with MIC 12.5
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µg/mL against E. faecalis. Further, the compound 10c showed MIC 12.5 µg/mL while
compound 10d showed MIC 25 µg/mL against strain K. pneumoniae. The compounds 11c and
11d were also found to possess good activity against S. boydii with MIC 12.5 µg/mL and showed
moderate to weak activity in the range of 25-100 µg/mL or ˃ 100 µg/mL against remaining
strains. Furthermore, compound 12c containing 2,4-difluoro substituted benzene showed
excellent potency against E. faecalis, E. coli and S. boydii with MIC 6.25 µg/mL. It also
exhibited potent antibacterial activity against S. aureus, P. aeruginosa and K. pneumoniae with
MIC 12.5 µg/mL. Moreover, the compounds 12d to 14d were found weak or inactive with MIC
50-100 µg/mL or >100 µg/mL against all strains. The compound 14e having 4-chloro substituted
benzene ring was found most active against E. coli with MIC 6.25 µg/mL while remaining
strains showed good to moderate susceptibility with MIC in the range of 12.5-25 µg/mL. In
general, control drug ciprofloxacin showed MIC 6.25 µg/mL against all the tested
microorganisms.
It is obvious from the analysis of activity results that electron withdrawing groups such as
chloro, fluoro and also their combination has strong effects in rendering the antibacterial activity.
This observation is supported by the highest activity shown by the five compounds 10a, 10c,
10d, 12c and 14e against both the Gram positive and Gram negative bacterial strains while
compound 10a and 10d showed MIC 25 µg/mL against K. pneumoniae. Further, the positions of
these groups in benzene ring also play a critical role towards activity. For example, compound
10a containing chloro and fluoro groups at position-2 and 4 in benzene ring, showed potency
while the compound 10b with chloro and fluoro groups at position-3 and 4 in benzene ring lost
activity. Again, the compound 10d with chloro group at 2-position in benzene ring showed best
potency against all strains. While the compound 10e containing chloro group at 4-position in
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benzene ring lost the potency. However, it is reversed in triazole containing flavones. For
example, the compound 14e containing chloro group at position-4 in benzene ring has potential
activity with MIC 6.25 µg/mL in E. coli while in other strains compound showed MIC 12.5-25
µg/mL. The compound 14d with chloro group at 2-position lost the activity and exhibited MIC
>100 µg/mL in all tested strains. Thus, it is hypothesized that the chloro group at position-2 is
critical for antibacterial activity in chalcone triazoles while the chloro group at position-4 is
important for antibacterial activity in flavone triazoles. The presence of fluoro group has
significant effect on antibacterial activity in chalcone triazoles but in flavone triazoles, it lost the
activity.
Another interesting observation is based on the isomeric effects and structural variation in
the skeleton moieties. The compounds 10a-e are structural isomers of compounds 11a-e
synthesized from isomeric skeleton moieties 4 and 6, while compounds 10-14(a) and 10-14(d)
are position isomers of compounds 10-14(b) and 10-14(e), respectively. It is evident from the
screening results shown in Table 3 that the triazole compounds 10a-e synthesized from the
skeleton moiety 4 showed most potent inhibitory activity as compare to the compounds 11a-e
synthesized from skeleton moiety 6. Again the isomeric effects have been observed in other
systems. For example, the compounds 10(a and d), 11(a and d) and 13(a and d) showed better
potency against bacterial strains as compare to the compounds 10(b and e), 11(b and e) and 13(b
and e), respectively. Further, the structural variations in the skeleton moieties also play a critical
role toward activity. For example, the triazole compounds 13-14(a-e) synthesized from the
flavones (5 and 8) showed negligible activity except 14e as compare to the compounds
synthesized from chalcones (4 and 7). In order to ascertain this hypothesis, more synthesis of
diversified compounds using multifunctional substituted phenyl ring were needed. More
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systematic structure activity relationship (SAR) study is needed involving large number of
compounds to get molecule for in vivo studies which may be eventually a clinical candidate for
future.
Further, these compounds 10-14(a-e) were also screened for their antifungal activity
against six fungal strains such as Candida albicans (ATCC 90028), Candida albicans (clinical),
Candida tropicalis (ATCC 750), Candida parapsilosis (ATCC 22019), Cryptococcus
neoformans (clinical), Dermatophyte (clinical) and two molds Aspergillus niger (clinical) and
Aspergillus fumigatus (clinical). The results of antifungal activity of tested compounds were
found to be somewhat different from their antibacterial activity as shown in Table 4. These
compounds showed weak to moderate antifungal activity as compared to the standard drug
fluconazole. It is evident from antifungal data that nine compounds viz. 10e, 11d, 11e, 12c, 13a,
13b, 13e, 14a and 14d were found more potent as compared to the MIC of other compounds
with standard drug.
The compound 10a containing 2-chloro-4-fluoro substituted benzene ring showed
moderate to weak activity with MIC range of 25-100 µg/mL and ˃100 µg/mL while fluconazole
has MIC in the range of 0.50-4.00 µg/mL against all tested strains in the present study. The
compound 10c having 2,4-difluoro substituted benzene ring was found moderate potential
against C. parapsilosis with MIC 25 µg/mL while in remaining strains, it showed weak activity
with MIC 50 µg/mL or ˃100 µg/mL. The compound 10e containing 4-chloro substituted benzene
ring was found to be the most potential activity against C. albicans (clinical) and C. tropicalis
with MIC 6.25 µg/mL. Again, the compound 10e also showed good activity against four strains
C. albicans, C. parapsilosis, C. neoformans and Dermatophyte with MIC 12.5 µg/mL, while in
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A. niger and A. fumigatus strains, it showed moderate activity with MIC 25 µg/mL. The
compounds 11d and 11e having 2-chloro and 4-chloro substituted benzene ring, respectively
were found to be the most potent with MIC 6.25 µg/mL in C. tropicalis. Further, the same
compounds also showed MIC 12.5 µg/mL in C. albicans (clinical), C. parapsilosis, C.
neoformans and Dermatophyte. Again, the compound 11d was found to be the most potential
active with MIC 6.25 µg/mL in C. albicans while the compound 11e exhibited good activity with
MIC 12.5 µg/mL. Further, the compound 11d showed MIC 12.5 µg/mL while compound 11e
showed MIC 25 µg/mL against A. niger and A. fumigatus. The compound 12c containing 2,4-
difluoro substituted benzene ring showed potent antifungal activity against both species of C.
albicans and C. neoformans with MIC 6.25 µg/mL while moderate activity was observed with
other strains in the range of 12.5-25 µg/mL.
The flavone compounds 13a and 13b having 2-chloro-4-fluoro and 3-chloro-4-fluoro
substituted benzene ring respectively, showed good activity against four strains viz. C. albicans,
C. albicans (clinical), C. tropicalis and C. parapsilosis with MIC 12.5 µg/mL while fluconazole
showed MIC 0.50 µg/mL. Further, the compound 13a showed good activity again C.
neoformans, A. niger and A. fumigates with MIC 12.5 µg/mL except Dermatophyte with MIC 25
µg/mL. Again, the compound 13b was found to be the most potent against C. neoformans with
MIC 6.25 µg/mL while against Dermatophyte, A. niger and A. fumigates, it showed good to
weak activity with MIC 12.5-50 µg/mL. The compound 13e containing 4-chloro substituted
benzene ring showed good antifungal activity against C. albicans (clinical), C. neoformans and
Dermatophyte with MIC 12.5 µg/mL while against the remaining strains showed MIC 50
µg/mL. Further, the compound 14a having 2-chloro-4-fluoro substituted benzene ring showed
potent inhibitory activity with MIC 6.25 µg/mL against two strains C. tropicalis and
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Dermatophyte while other remaining strains showed MIC 12.5 µg/mL. Again, the compound
14d containing 2-chloro atom in benzene ring also showed reasonable good activity with MIC
6.25-12.5 µg/mL against all strains. The rest compounds were found to be weak or inactive
against all strains with MIC 50-100 µg/mL or ˃100 µg/mL. Fluconazole was taken as a standard
drug which showed MIC value in between 0.50-4.00 µg/mL against various strains.
It is obvious from the above antifungal screening results that presence of chloro group in
phenyl ring of one triazole unit chalcones shows potent antifungal activity, while difluoro-
compounds enhance antifungal activity in two triazole linked chalcones. Further, the
combination of chloro and fluoro atoms in benzene ring has better antifungal activity than
monohalogen compounds in one triazole linked flavones. But, the presence of 2-chloro or its
combination with fluoro group in phenyl ring showed potent activity than other groups and their
combinations in flavones contained two triazole units. Interestingly, the flavone compounds
containing difluoro substituted phenyl rings were found to be inactive against antifungal as well
as antibacterial activity. Further, the screening of these compounds will also help to predict
structural variations, isomeric as well as position effect of functional groups on antimicrobial
activity. Moreover, multifunctional substituted phenyl rings were used to generate large number
of diverse compounds for in vitro antifungal activity. Thus, more systematic design and their
synthesis of compounds with various functional groups are needed to establish a meaningful
structure activity relationship (SAR). This relationship needs to be done on most potent molecule
to find a lead molecule which can be evaluated for in vivo activity.
2.2.2. Antiplasmodial activity
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1,2,3-Triazole derivatives of chalcones and flavones were evaluated in vitro against the
erythrocytic stages of P. falciparum (3D7 strain). Results compiled in Table 5 show that many
compounds exhibited moderate activities. The most potent compound was 10b having 3-chloro-
4-fluoro substituted benzene ring, with an IC50 of 2.74 µg/mL while artemisinin showed IC50
1.117 ± 0.076 ng/mL. The compounds from chalcone series 10c having 2,4-difluoro substituted
phenyl ring and 10e having 4-chloro substituted benzene showed IC50 of 3.58 µg/mL and 5.08
µg/mL, respectively. The compound 10d containing 2-chloro substituted benzene ring in
chalcone series showed IC50 6.78 µg/mL. These compounds also showed promising antibacterial
activity. Similarly, the compound 11a containing 2-chloro-4-fluoro substituted benzene ring in
chalcone series showed IC50 4.92 µg/mL. The compounds of flavones series 13a having 2-
chloro-4-fluoro substituted benzene ring showed IC50 3.85 µg/mL. Moreover, compound of same
series 13b having 3-chloro-4-fluoro substituted benzene ring and compound 13c having 2,4-
difluoro substituted benzene ring exhibited IC50 4.48 µg/mL and 4.79 µg/mL, respectively. The
compound 13e having 4-chloro substituted benzene ring in flavone series showed IC50 5.19
µg/mL. The standard drug artemisinin showed IC50 1.117 ± 0.076 ng/mL. Rest of the compounds
displayed moderate to weak antiplasmodial activities, as compared to the standard drug
artemisinin.
2.2.3. Cytotoxicity
In order to validate the therapeutic value of compounds in the present study, toxicity on
mammalian cells was assessed against Huh-7 cell line; a hepatocyte derived cellular carcinoma
cell line (Table 5). All the compounds are devoid of any considerable cytotoxicity at the highest
test concentration (100 ꢀg/mL). These results further support the significance of this study.
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3. Conclusion
The objective of the present study was to design, synthesis and screen the antimicrobial,
antiplasmodial and cytotoxic activities of new triazole derivative of chalcone and flavone
analogs with hope of discovering new structural entities. An efficient synthetic strategy was
developed for synthesis of 1,2,3-triazole linked chalcone and flavone hybrid compounds. A small
library of 25 compounds were synthesized with different functionalities by exploiting click
chemistry and screened them against various Gram positive and Gram negative bacterial as well
as fungal strains. In addition to this, antiplasmodial and cytotoxic activities of synthesized
compounds were also carried out. The compound 10a, 10c and 10d of chalcone series showed
promising antibacterial activity with MIC 6.25 ꢀg/mL against S. auresus (ATCC 25323), E.
faecalis (ATCC 29212), E. coli (ATCC 35218) and S. boydii (clinical isolate) which is similar to
ciprofloxacin MIC 6.25 ꢀg/mL. The compound 12c contain two triazole moieties in chalcone
series showed promising antibacterial activity with MIC 6.25 ꢀg/mL against E. faecalis (ATCC
29212), E. coli (ATCC 35218) and S. boydii (clinical isolate). Similarly, the compound 10e
exhibited promising antifungal activity with MIC 6.25 ꢀg/mL against C. albicans (Clinical) and
C. tropicalis (ATCC 750). The compounds 11d and 12c also showed potent antifungal activity
with MIC 6.25 ꢀg/mL against C. albicans (ATCC 90028). Other compounds showed moderate
to weak activity against all tested fungal strains.
The compound 10b showed IC50 2.74 ꢀg/mL in vitro against the erythrocytic stages of P.
falciparum (3D7 strain). Rest of the compounds showed weak to moderate antiplamodial
activity. These compounds were also evaluated for cytotoxicity in vitro against the Huh-7 cell
line and exhibited no cytotoxic activity with CC50 values higher than 100 µg/mL.
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Results obtained in this study clearly demonstrate compounds derived from chalcone and
flavones moiety exhibited better antimicrobial activity. This is a preliminary result and to reach
more appropriate conclusion 2nd and 3rd generation compounds should be synthesized in order to
establish meaningful structure activity relationship (SAR).
This study, reporting the high yield of synthesis of novel compounds combining two types
of pharmacophores into one molecule paves the way for further optimization aimed at lowering
of their effective concentrations.
4. Experimental
All the chemicals and solvents were purchased from Sigma-Aldrich and E. Merck (India).
The chemicals were used as received. The reactions were monitored by thin layer
chromatography (TLC) on precoated silica gel 60 F254 (mesh) and spots were visualized with
UV light or iodine chamber. Merck silica gel (230-400 mesh) was used for column
chromatography. Melting points of all synthesized compounds were determined by using open
capillary method and may be uncorrected. NMR spectra were recorded on JEOL GS-400 model
1
FT-NMR (400 MHz for H and 100 MHz for 13C) spectrometer and processed with Delta
software. The chemical shifts were measured in parts per million (ppm) on the delta (δ) scale
relative to the resonance of tetramethylsilane (TMS) as the internal reference. Infrared spectra
were recorded in KBr pellets in the range of 4000-500 cm-1 at room temperature using Perkin-
Elmer 400 FT-IR spectrometer. X-ray analysis was carried out on an Oxford Diffraction
Xcalibur four-circle diffractometer with Eos CCD detector. Mass spectra were obtained on
Agilent Technologies 6530 Accurate Mass Q-TOF LC/MS Mass spectrometer. Elemental
analysis was done on a Perkin-Elmer Model 240B automatic analyzer.
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4.1. Synthesis of 1-(2-hydroxy-4,6-dimethoxyphenyl)ethan-1-one (1)
A mixture of 1-(2,4,6-trihydroxyphenyl)ethan-1-one (2.50 g, 15 mmol), (CH3)2SO4 (2.85
mL, 30 mmol) and anhydrous K2CO3 (4.56 g, 33 mmol) in dry acetone (60 mL) was refluxed for
10 h. The reaction mixture was concentrated and poured on to ice-water (100 mL). The resulting
precipitate was filtered off and washed with cold water. The obtained residue was purified by
silica gel column chromatography with hexane: ethyl acetate (95: 5) as eluent.
1
White solid, Yield: 54%, M.P: 78-80 °C; H NMR (CDCl3, 400 MHz, ppm) δ: 14.03 (s,
1H,-OH), 6.06-6.05 (d, J = 2.20 Hz, 1H, Ar-H), 5.93-5.92 (d, J = 2.20 Hz, 1H, Ar-H), 3.86, 3.82
(s, each 3H, 2 × -OCH3), 2.61 (s, 3H, -COCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 203.17 (-
C=O); 167.56; 166.06; 162.88; 105.97; 93.43; 90.72; 55.53 (-OCH3); 50.86 (-OCH3); 32.91 (-
CH3).
4.2. Propargylation of 3,4-dihydroxybenzaldehyde
In a dry two-neck flask with a nitrogen inlet, NaH (1.39 g, 57.91 mmol) was added into
anhydrous DMSO (20 mL) in small portions under stirring. The solution was cooled to 0 °C. The
anhydrous DMSO (25 mL) containing 3,4-dihydroxybenzaldehyde (4.0 g, 28.96 mmol) was
added drop wise into the flask. The reaction mixture was then stirred at room temperature for 30
min. Propargyl bromide (3.87 mL, 43.44 mmol) was added drop wise and the mixture was stirred
further 45 h at room temperature. The reaction was checked by TLC, showing the formation of
three products due to visualization of spots on TLC plate. The reaction mixture was poured onto
ice-water (300 mL) and neutralized by 1 M HCl solution. The products was extracted with
dichloromethane, washed with brine solution (3 × 200 mL) and dried over Na2SO4. The solvent
was removed under vacuum to afford a brownish crude product. The crude product was purified
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by silica gel column with an eluent of hexane: ethyl acetate (95: 5) to give three products (2a, 2b
and 2c).
4.2.1. 3-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (2a)
1
Colorless crystals, Yield: 29%, M.P: 66-68 °C; H NMR (CDCl3, 400 MHz, ppm) δ: 9.86
(s, 1H, -CHO), 7.48-7.47 (d, J = 1.44 Hz, 1H, Ar-H), 7.46-7.43 (dd, J = 8.04, 1.48 Hz, 1H, Ar-
H), 7.12-7.10 (d, J = 8.04 Hz, 1H, Ar-H), 5.83 (s, 1H, -OH), 4.87-4.86 (d, J = 2.20 Hz, 2H, -
13
OCH2), 2.62-2.61 (t, J = 2.20 Hz, 1H, -C≡CH); C NMR (CDCl3, 100 MHz, ppm) δ: 191.03 (-
CHO); 149.60; 146.33; 131.31; 124.02; 114.80; 111.86; 56.81 (-OCH2).
4.2.2. 4-hydroxy-3-(prop-2-yn-1-yloxy)benzaldehyde (2b)
1
Colorless crystals, Yield: 9%, M.P: 93-95 °C; H NMR (CDCl3, 400 MHz, ppm) δ: 9.84
(s, 1H, -CHO), 7.53-7.52 (d, J = 1.36 Hz, 1H, Ar-H), 7.49-7.47 (dd, J = 8.24, 1.36 Hz, 1H, Ar-
H), 7.09-7.07 (d, J = 8.24 Hz, 1H, Ar-H), 6.29 (s, 1H, -OH), 4.85-4.84 (d, J = 2.28 Hz, 2H, -
13
OCH2), 2.61-2.60 (t, J = 2.28 Hz, 1H, -C≡CH); C NMR (CDCl3, 100 MHz, ppm) δ: 190.70 (-
CHO); 151.87; 145.08; 129.75; 127.98; 115.06; 110.91; 56.97 (-OCH2).
4.2.3. 3,4-bis(prop-2-yn-1-yloxy)benzaldehyde (2c)
Colorless crystals, Yield: 21%, M.P: 99-101 °C; 1H NMR (CDCl3, 400 MHz, ppm) δ: 9.89
(s, 1H, -CHO), 7.58-7.57 (d, J = 2.32 Hz, 1H, Ar-H), 7.55-7.52 (dd, J = 8.24, 1.84 Hz, 1H, Ar-
H), 7.19-7.17 (d, J = 8.24 Hz, 1H, Ar-H), 4.87-4.86 (d, J = 2.76 Hz, 2H, -OCH2), 4.84-4.83 (d, J
= 2.28 Hz, 2H, -OCH2), 2.58-2.57 (t, J = 2.28, Hz, 1H, -C≡CH), 2.56-2.55 (t, J = 2.72 Hz, 1H, -
13
C≡CH); C NMR (CDCl3, 100 MHz, ppm) δ: 190.69 (-CHO); 152.59; 147.83; 130.70; 126.75;
113.07; 112.45; 77.63 (-C≡CH); 76.53 (-C≡CH); 56.71 (-OCH2); 56.64 (-OCH2).
15

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4.3. Methylation of 3-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (2a) and 4-hydroxy-3-(prop-
2-yn-1-yloxy)benzaldehyde (2b).
A mixture of benzaldehyde (2a or 2b, 1.20 g, 6.81 mmol), anhydrous K2CO3 (2.82 g, 20.43
mmol) and (CH3)2SO4 (0.97 mL, 10.33 mmol) in dry acetone (20 mL) was refluxed for 8 h. The
reaction mixture was concentrated and then poured on to ice-water (50 mL). The resulting
precipitate was filtered off and washed with cold water. The obtained product was sufficient pour
for further reactions.
4.3.1. 3-methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (3a)
1
White solid, Yield: 70%, M.P: 88-90 °C; H NMR (CDCl3, 400 MHz, ppm) δ: 9.88 (s, 1H,
-CHO), 7.48-7.46 (dd, J = 8.28, 1.80 Hz, 1H, Ar-H), 7.45-7.44 (d, J = 1.80 Hz, 1H, Ar-H), 7.16-
7.14 (d, J = 8.28 Hz, 1H, Ar-H), 4.87-4.86 (d, J = 2.72 Hz, 2H, -OCH2), 3.95 (s, 3H, -OCH3),
13
2.57-2.56 (t, J = 2.28 Hz, 1H, -C≡CH); C NMR (CDCl3, 100 MHz, ppm) δ: 190.87 (-CHO);
152.11; 150.04; 130.94; 126.23; 112.60; 109.48; 56.61 (-OCH2); 56.03 (-OCH3).
4.3.2. 4-methoxy-3-(prop-2-yn-1-yloxy)benzaldehyde (3b)
White solid, Yield: 75%, M.P: 68-70 °C; 1H NMR (CDCl3, 400 MHz, ppm) δ: 9.87 (s, 1H,
-CHO), 7.56-7.55 (d, J = 1.84 Hz, 1H, Ar-H), 7.54-52 (dd, J = 8.24, 1.84 Hz, 1H, Ar-H), 7.03-
7.01 (d, J = 8.24 Hz, 1H, Ar-H), 4.84-4.83 (d, J = 2.28 Hz, 2H, -OCH2), 3.97 (s, 3H, -OCH3),
13
2.56-2.54 (t, J = 2.28 Hz, 1H, -C≡CH); C NMR (CDCl3, 100 MHz, ppm) δ: 190.06 (-CHO);
154.23; 146.61; 129.22; 126.66; 111.23; 110.23; 77.00 (-C≡CH); 75.79 (-C≡CH); 55.94 (-
OCH2); 55.52 (-OCH3).
16

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4.4. General procedure for the synthesis of chalcones (4, 6 and 7).
A mixture of the 1-(2-hydroxy-4,6-dimethoxyphenyl)ethan-1-one (1, 1 eq) and the
corresponding substituted benzaldehyde (3a, 3b and 2c, 1 eq) in ethanol (4.0 mL/mmol of
acetophenone 1) was stirred at room temperature during 10 min. Then, the aqueous solution of
KOH (3 eq, 20% w/v) was added to reaction mixture. The reaction mixture was stirred at room
temperature until benzaldehyde consumption as monitored by TLC. After completion of the
reaction, HCl (10% v/v aqueous solution) was added until neutral. In all cases a yellow
precipitate of chalcone was formed, which was filtered and dried. The products were purified
using column chromatography with an eluent of chloroform : hexane (1 : 1).
4.4.1. (2E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-[3-methoxy-4-(prop-2-yn-1-
yloxy)phenyl]prop-2-en-1-one (4)
Yellow solid, Yield: 66%, M.P: 145-147 °C; IR (KBr) cm-1: 3238, 2918, 2854, 2122,
1
1618, 1579, 1546, 1505, 1438, 1361, 1256, 1205, 1136, 1112, 1012, 980, 812, 757; H NMR
(DMSO-d6, 400 MHz, ppm) δ: 13.35 (s, 1H, -OH), 7.67-7.63 (d, J = 15.36 Hz, 1H, -COC=CH),
7.61-7.57 (d, J = 15.40 Hz, 1H, -COCH=C), 7.32 (s, 1H, Ar-H), 7.30-7.28 (d, J = 8.08 Hz, 1H,
Ar-H), 7.08-7.06 (d, J = 8.80 Hz, 1H, Ar-H), 6.14 (s, 1H, Ar-H), 6.11 (s, 1H, Ar-H), 4.85-4.84
(d, J = 2.20 Hz, 2H, -OCH2), 3.87, 383, 3.80 (s, each 3H, 3 × -OCH3), 3.61-3.59 (t, J = 2.20 Hz,
1H, -C≡CH); 13C NMR (DMSO-d6, 100 MHz, ppm) δ: 192.24 (-CO); 165.23; 165.13; 161.69;
149.28; 148.63; 142.67; 128.51; 125.69; 122.14; 113.73; 111.30; 106.44; 93.85; 91.00; 78.93 (-
C≡CH); 78.52 (-C≡CH); 56.10 (-OCH2); 55.97 (-OCH3); 55.58 (-OCH3); 55.54 (-OCH3).
17

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4.4.2. (2E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-[4-methoxy-3-(prop-2-yn-1-
yloxy)phenyl]prop-2-en-1-one (6)
Yellow solid, Yield: 74%, M.P: 174-176 °C; IR (KBr) cm-1: 3212, 2920, 2851, 2113,
1
1628, 1585, 1554, 1517, 1441, 1221, 1165, 1114, 1022, 983, 823, 766; H NMR (CDCl3, 400
MHz, ppm) δ: 14.37 (s, 1H, -OH), 7.82-7.78 (d, J = 15.60 Hz, 1H, -COC=CH), 7.76-7.72 (d, J =
15.60 Hz, 1H, -COCH=C), 7.33-7.32 (d, J = 1.84 Hz, 1H, Ar-H), 7.24-7.22 (d, J = 7.32 Hz, 1H,
Ar-H), 6.91-6.89 (dd, J = 8.24, 2.20 Hz, 1H, Ar-H), 6.10-6.09 (d, J = 1.84 Hz 1H, Ar-H), 5.95-
5.94 (d, J = 2.76 Hz, 1H, Ar-H), 4.81-4.80 (d, J = 1.80 Hz, 2H, -OCH2), 3.90 (s, 6H, 2 × -OCH3),
382 (s, 3H, -OCH3), 2.54-2.53 (t, J = 2.76 Hz, 1H, -C≡CH); 13C NMR (DMSO-d6, 100 MHz,
ppm) δ: 192.17 (-CO); 165.42; 165.34; 161.76; 151.43; 146.60; 142.82; 127.33; 125.27; 124.17;
112.28; 112.05; 106.31; 93.87; 91.04; 79.13 (-C≡CH); 78.53 (-C≡CH); 56.13 (-OCH2); 55.85 (-
OCH3); 55.66 (-OCH3).
4.4.3. (2E)-3-[3,4-bis(prop-2-yn-1-yloxy)phenyl]-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-
1-one (7)
Yellow solid, Yield: 50%, M.P: 169-171 °C; IR (KBr) cm-1: 3277, 3219, 2918, 2849,
1
2111, 1626, 1582, 1557, 1508, 1212, 1139, 1015, 817, 761; H NMR (DMSO-d6, 400 MHz,
ppm) δ: 13.45 (s, 1H, -OH), 7.70-7.66 (d, J = 14.68 Hz, 1H, -COC=CH), 7.61-7.57 (d, J = 15.64
Hz, 1H, -COCH=C), 7.44-7.43 (d, J = 1.96 Hz, 1H, Ar-H), 7.34-7.31 (dd, J = 8.80, 1.96 Hz, 1H,
Ar-H), 7.12-7.10 (d, J = 7.84 Hz, 1H, Ar-H), 6.16-6.15 (d, J = 1.48 Hz, 1H, Ar-H), 6.12-6.11 (d,
J = 1.96 Hz, 1H, Ar-H), 4.91-4.90 (d, J = 1.96 Hz, 2H, -OCH2), 4.88-4.87 (d, J = 1.96 Hz, 2H, -
OCH2) 3.89, 381 (s, each 3H, 2 × -OCH3), 3.64-3.63 (t, J = 1.96 Hz, 1H, -C≡CH), 3.62-3.61 (t, J
= 1.96 Hz, 1H, -C≡CH); 13C NMR (DMSO-d6, 100 MHz, ppm) δ: 192.14 (-CO); 165.37; 161.77;
18

ACCEPTED MANUSCRIPT
148.99; 146.92; 142.45; 128.22; 125.76; 123.52; 113.88; 112.68; 106.32; 93.87; 91.02; 79.02 (-
C≡CH); 78.84 (-C≡CH); 78.65 (-C≡CH); 78.57 (-C≡CH); 56.11 (-OCH2); 55.99 (-OCH2); 55.87
(-OCH3); 55.62 (-OCH3).
4.5. General procedure for the synthesis of flavones (5 and 8).
2-Hydroxychalcone (4 or 7, 1eq) was dissolved in DMSO (8.0 mL/mmol of 2-
hydroxychalcone) and I2 (0.05 eq) was added. The reaction mixture was refluxed for 1 h. Then,
the mixture was cooled to room temperature and poured into water. The product was extracted
with ethyl acetate, washed with brine solution until neutrality and dried with Na2SO4. The
solvent was evaporated in vacuo and the product was purified by chromatographic column with
ethyl acetate: hexane (1 : 1) as eluent.
4.5.1. 5,7-dimethoxy-2-[3-methoxy-4-(prop-2-yn-1-yloxy)phenyl]-4H-chromen-4-one (5)
Brown solid, Yield: 65%, M.P: 148-150 °C; IR (KBr) cm-1: 3305, 3015, 2918, 2848, 2148,
1
1638, 1602, 1215, 1159, 749, 665; H NMR (CDCl3, 400 MHz, ppm) δ: 7.49-7.47 (d, J = 8.56
Hz, 1H, Ar-H), 7.32 (s, 1H, Ar-H), 7.11-7.09 (d, J = 8.60 Hz, 1H, Ar-H), 6.59 (s, 1H, -COCH=C
of flavone), 6.53 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 4.82 (s, 2H, -OCH2), 3.94 (s, 6H, 2 × -
OCH3), 389 (s, 3H, -OCH3), 2.53 (s, 1H, -C≡CH); 13C NMR (DMSO-d6, 100 MHz, ppm) δ:
175.63 (-CO); 163.60; 160.20; 159.45; 159.11; 149.30; 149.14; 124.09; 118.92; 113.55; 109.43;
108.25; 107.29; 96.17; 93.39; 78.81 (-C≡CH); 78.63 (-C≡CH); 56.01 (-OCH2); 55.92 (-OCH3);
55.88 (-OCH3).
4.5.2. 2-[3,4-bis(prop-2-yn-1-yloxy)phenyl]-5,7-dimethoxy-4H-chromen-4-one (8)
19

ACCEPTED MANUSCRIPT
White solid, Yield: 74%, M.P: 160-162 °C; IR (KBr) cm-1: 3301, 3210, 3006, 2916, 2851,
2120, 1638, 1602, 1514, 1260, 1159, 1016, 749, 664; 1H NMR (CDCl3, 400 MHz, ppm) δ: 7.56-
7.53 (dd, J = 8.56, 2.48 Hz, 1H, Ar-H), 7.52-7.51 (d, J = 2.44 Hz, 1H, Ar-H), 7.15-7.12 (d, J =
8.56 Hz, 1H, Ar-H), 6.59 (s, 1H, -COCH=C of flavone), 6.54-6.53 (d, J = 2.44 Hz, 1H, Ar-H),
6.37-6.36 (d, J = 2.44 Hz, 1H, Ar-H), 4.82-4.81 (d, J = 2.44 Hz, 4H, 2 × -OCH2), 3.94, 390, (s,
13
each 3H, 2 × -OCH3), 2.56-2.54 (m, 2H, 2 × -C≡CH); C NMR (DMSO-d6, 100 MHz, ppm) δ:
175.77 (-CO); 163.71; 160.27; 159.40; 159.17; 149.55; 146.96; 123.90; 119.89; 113.83; 111.49;
108.27; 107.36; 96.20; 93.40; 79.06 (-C≡CH); 78.88 (-C≡CH); 78.81 (-C≡CH); 78.71 (-C≡CH);
56.34 (-OCH2); 56.09 (-OCH2); 55.99 (-OCH3).
4.6. General procedure for the synthesis of azides (9a-e)
The aniline (1 eq) was dissolved in 6 N HCl solution (10 mL/mmol of aniline) at room
temperature and cooled up to 0 °C, followed by addition of NaNO2 (1.2 eq) in small portions
under stirring. After 10 min of stirring at same temperature, sodium azide (1.2 eq) was added to
the reaction mixture. This mixture was further stirred at room temperature for 3 h. The reaction
was worked up by extraction with chloroform. The organic layer was washed with brine solution
and dried over Na2SO4. After evaporation of the solvent, the crude product (9a-e) was pure
enough for further reactions. All azides were stored at -20 °C.
4.7. General procedure for the synthesis of 1,2,3-triazole building blocks[10-14(a-e)]
The alkyne (4-8, 1 eq) and various aromatic azide (9a-e, 1.2 eq for 4-6, 2.4 eq for 7 and 8)
were dissolved in N,N-dimethylformamide (20 mL/mmol of alkyne). To this reaction mixture,
the solution of sodium ascorbate (0.4 eq in minimum water) was added, followed by copper (II)
20

ACCEPTED MANUSCRIPT
sulfate pentahydrate solution (0.2 eq in minimum water). The heterogeneous mixture was stirred
vigorously at room temperature until alkyne consumption as monitored by TLC. After
completion of the reaction, the mixture was poured onto ice-water and the precipitate was
collected by filtration. The desired product was purified by column chromatography using an
eluent of 2% methanol in chloroform.
4.7.1. (2E)-3-(4-{[1-(2-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-3-
methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (10a)
IR (KBr) cm-1: 3081, 3009, 2938, 2849, 1619, 1580, 1556, 1504, 1254, 1209, 1158, 1034,
1
818, 752; H NMR (CDCl3, 400 MHz, ppm) δ: 14.35 (s, 1H, -OH), 8.02 (s, 1H, -C=CH of
triazole), 7.80-7.76 (d, J = 15.60 Hz, 1H, -COC=CH), 7.73-7.69 (d, J = 15.60 Hz, 1H, -
COCH=C), 7.59-7.55 (dd, J = 9.16, 5.48 Hz, 1H, Ar-H), 7.31-7.29 (dd, J = 7.80, 2.76 Hz, 1H,
Ar-H), 7.21-7.18 (dd, J = 8.24, 1.84 Hz, 1H, Ar-H), 7.17-7.14 (m, 1H, Ar-H), 7.13-7.10 (m, 2H,
Ar-H), 6.08-6.07 (d, J = 2.28 Hz, 1H, Ar-H), 5.94-6.93 (d, J = 2.28 Hz, 1H, Ar-H), 5.41 (s, 2H, -
OCH2), 3.90, 389, 3.81 (s, each 3H, 3 × -OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.36 (-
CO); 168.37; 166.07; 162.37; 149.60; 149.37; 143.78; 142.27; 129.55; 129.19; 129.10; 125.90;
125.23; 121.99; 118.22; 117.96; 115.41; 115.18; 113.98; 111.33; 106.26; 93.78; 91.23; 62.90 (-
OCH2); 55.88 (-OCH3); 55.78 (-OCH3); 55.55 (-OCH3); Elemental analysis for C27H23ClFN3O6:
Calculated: C 60.06, H 4.29, N 7.78; Found: C 59.95, H 4.31, N 7.82; LC-MS (m/z): 540.13
[M+H]+.
4.7.2. (2E)-3-(4-{[1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-3-
methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (10b)
1
IR (KBr) cm-1: 3020, 1619, 1585, 1557, 1506, 1212, 741, 667; H NMR (CDCl3, 400
MHz, ppm) δ: 14.29 (s, 1H, -OH), 7.98 (s, 1H, -C=CH of triazole), 7.78-7.72 (m, 2H, Ar-H + -
21

ACCEPTED MANUSCRIPT
COC=CH ), 7.67-7.65 (d, J = 15.28 Hz, 1H, -COCH=C), 7.56-7.54 (m, 1H, Ar-H), 7.26-7.22 (t,
J = 8.52 Hz, 1H, Ar-H), 7.17-7.14 (dd, J = 8.56, 1.84 Hz, 1H, Ar-H), 7.06-7.03 (m, 2H, Ar-H),
6.05-6.04 (d, J = 2.44 Hz, 1H, Ar-H), 5.90-5.89 (d, J = 2.44 Hz, 1H, Ar-H), 5.35 (s, 2H, -OCH2),
13
3.87, 3.84, 3.77 (s, each 3H, 3 × -OCH3); C NMR (CDCl3, 100 MHz, ppm) δ: 192.22 (-CO);
165.23; 165.18; 161.70; 158.14; 149.55; 149.20; 143.66; 142.76; 133.48; 128.26; 125.53;
123.49; 122.49; 121.04; 120.96; 120.85; 120.66; 118.21; 117.98; 113.52; 111.25; 106.43; 93.90;
91.02; 61.48 (-OCH2); 56.13 (-OCH3); 55.62 (-OCH3); 55.53 (-OCH3); Elemental analysis for
C27H23ClFN3O6: Calculated: C 60.06, H 4.29, N 7.78; Found: C 59.96, H 4.32, N 7.76; LC-MS
(m/z): 540.13 [M+H]+.
4.7.3. (2E)-3-(4-{[1-(2,4-difluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-3-methoxyphenyl)-1-
(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (10c)
IR (KBr) cm-1: 2961, 2917, 2849, 1621, 1583, 1556, 1509, 1458, 1258, 1213, 1030, 815,
1
750; H NMR (CDCl3, 400 MHz, ppm) δ: 14.36 (s, 1H, -OH), 8.14-8.13 (d, J = 2.04 Hz, 1H, -
C=CH of triazole), 7.95-7.89 (m, 1H, Ar-H), 7.82-7.78 (d, J = 15.44 Hz, 1H, -COC=CH), 7.75-
7.71 (d, J = 15.44 Hz, 1H, -COCH=C), 7.23-7.20 (dd, J = 8.72, 2.00 Hz, 1H, Ar-H), 7.14-7.11
(m, 2H, Ar-H), 7.09-7.03 (m, 2H, Ar-H), 6.11-6.10 (d, J = 2.00 Hz, 1H, Ar-H), 5.96-6.95 (d, J =
13
2.00 Hz, 1H, Ar-H), 5.42 (s, 2H, -OCH2), 3.92, 390, 3.83 (s, each 3H, 3 × -OCH3); C NMR
(CDCl3, 100 MHz, ppm) δ: 192.39 (-CO); 168.38; 166.08; 162.39; 149.57; 149.38; 144.32;
142.31; 129.55; 126.24; 126.14; 125.90; 124.34; 124.26; 122.02; 113.83; 112.77; 112.52;
111.32; 106.28; 105.66; 105.39; 105.13; 93.79; 91.25; 62.78 (-OCH2); 55.89 (-OCH3); 55.80 (-
OCH3); 55.58 (-OCH3); Elemental analysis for C27H23F2N3O6: Calculated: C 61.95, H 4.43, N
8.03; Found: C 61.91, H 4.46, N 8.11; LC-MS (m/z): 524.16 [M+H]+.
22

ACCEPTED MANUSCRIPT
4.7.4. (2E)-3-(4-{[1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-3-methoxyphenyl)-1-(2-
hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (10d)
IR (KBr) cm-1: 3009, 2938, 2846, 1617, 1582, 1555, 1505, 1249, 1209, 1153, 1030, 750;
1H NMR (CDCl3, 400 MHz, ppm) δ: 14.38 (s, 1H, -OH), 8.09 (s, 1H, -C=CH of triazole), 7.83-
7.79 (d, J = 15.28 Hz, 1H, -COC=CH), 7.76-7.72 (d, J = 15.28 Hz, 1H, -COCH=C), 7.63-7.60
(m, 1H, Ar-H), 7.58-7.56 (m, 1H, Ar-H), 7.47-7.44 (m, 2H, Ar-H), 7.24-7.21 (dd, J = 8.56, 2.44
Hz, 1H, Ar-H), 7.16-7.14 (d, J = 7.92 Hz, 1H, Ar-H), 7.12-7.11 (d, J = 1.8 Hz, 1H, Ar-H), 6.11-
6.10 (d, J = 2.44 Hz, 1H, Ar-H), 5.96-5.95 (d, J = 2.44 Hz, 1H, Ar-H), 5.45 (s, 2H, -OCH2), 3.92,
3.91, 3.83 (s, each 3H, 3 × -OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.40 (-CO); 168.38;
166.07; 162.40; 149.62; 149.44; 142.34; 134.77; 130.85; 130.77; 130.73; 129.54; 128.60;
127.93; 127.87; 127.76; 125.89; 125.63; 125.19; 122.02; 119.66; 114.04; 111.39; 106.30; 93.80;
91.25; 62.97 (-OCH2); 55.91 (-OCH3); 55.80 (-OCH3); 55.57 (-OCH3); Elemental analysis for
C27H24ClN3O6: Calculated: C 62.13, H 4.63, N 8.05; Found: C 62.08, H 4.66, N 8.13; LC-MS
(m/z): 522.14 [M+H]+.
4.7.5. (2E)-3-(4-{[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-3-methoxyphenyl)-1-(2-
hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (10e)
1
IR (KBr) cm-1: 3020, 2919, 2852, 1617, 1582, 1551, 1505, 1255, 1215, 750; H NMR
(CDCl3, 400 MHz, ppm) δ: 14.29 (s, 1H, -OH), 8.00 (s, 1H, -C=CH of triazole), 7.76-7.72 (d, J =
15.28 Hz, 1H, -COC=CH), 7.69-7.65 (d, J = 15.88 Hz, 1H, -COCH=C), 7.63-7.60 (d, J = 9.16
Hz, 2H, Ar-H), 7.44-7.42 (d, J = 7.92 Hz, 2H, Ar-H), 7.17-7.14 (dd, J = 8.56, 1.84 Hz, 1H, Ar-
H), 7.07-7.05 (m, 2H, Ar-H), 6.05-6.04 (d, J = 2.44 Hz, 1H, Ar-H), 5.90-5.89 (d, J = 2.44 Hz,
13
1H, Ar-H), 5.35 (s, 2H, -OCH2), 3.87, 3.84, 3.77 (s, each 3H, 3 × -OCH3); C NMR (CDCl3,
100 MHz, ppm) δ: 192.37 (-CO); 168.39; 166.09; 162.38; 149.49; 149.32; 142.26; 135.37;
23

ACCEPTED MANUSCRIPT
134.73; 129.96; 129.53; 125.93; 121.98; 121.73; 121.14; 113.65; 111.31; 106.28; 93.80; 91.26;
62.83 (-OCH2); 55.89 (-OCH3); 55.79 (-OCH3); 55.58 (-OCH3); Elemental analysis for
C27H24ClN3O6: Calculated: C 62.13, H 4.63, N 8.05; Found: C 62.10, H 4.65, N 8.09; LC-MS
(m/z): 522.14 [M+H]+.
4.7.6. (2E)-3-(3-{[1-(2-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-4-
methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (11a)
IR (KBr) cm-1: 3077, 3012, 2938, 2838, 1617, 1580, 1554, 1509, 1258, 1210, 1154, 1027,
1
821, 752; H NMR (CDCl3, 400 MHz, ppm) δ: 14.40 (s, 1H, -OH), 8.00 (s, 1H, -C=CH of
triazole), 7.82-7.79 (d, J = 15.24 Hz, 1H, -COC=CH), 7.74-7.70 (d, J = 15.88 Hz, 1H, -
COCH=C), 7.59-7.55 (dd, J = 9.16, 5.48 Hz, 1H, Ar-H), 7.40-7.39 (d, J = 1.84 Hz, 1H, Ar-H ),
7.31-7.28 (dd, J = 7.92, 2.44 Hz, 1H, Ar-H), 7.20-7.18 (dd, J = 8.56, 1.84 Hz, 1H, Ar-H), 7.17-
7.12 (m, 1H, Ar-H), 6.90-6.88 (d, J = 7.92 Hz, 1H, Ar-H), 6.08-6.07 (d, J = 2.44 Hz, 1H, Ar-H),
5.95-6.94 (d, J = 1.84 Hz, 1H, Ar-H), 5.44 (s, 2H, -OCH2), 3.95, 390, 3.82 (s, each 3H, 3 × -
OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.44 (-CO); 168.38; 166.09; 162.57; 151.68;
147.61; 142.34; 129.20; 129.11; 128.64; 125.56; 125.28; 124.49; 118.23; 117.96; 115.39;
115.17; 113.10; 111.58; 106.30; 93.74; 91.20; 63.11 (-OCH2); 55.99 (-OCH3); 5.96 (-OCH3);
55.54 (-OCH3); Elemental analysis for C27H23ClFN3O6: Calculated: C 60.06, H 4.29, N 7.78;
Found: C 60.01, H 4.26, N 7.82; LC-MS (m/z): 540.13 [M+H]+.
4.7.7. (2E)-3-(3-{[1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-4-
methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (11b)
IR (KBr) cm-1: 2922, 2846, 1624, 1582, 1552, 1509, 1440, 1265, 1210, 1157, 1033, 818,
1
763; H NMR (CDCl3, 400 MHz, ppm) δ: 14.33 (s, 1H, -OH), 7.99 (s, 1H, -C=CH of triazole),
7.78-7.73 (m, 2H, Ar-H + -COC=CH ), 7.69-7.65 (d, J = 15.88 Hz, 1H, -COCH=C), 7.56-7.52
24

ACCEPTED MANUSCRIPT
(m, 1H, Ar-H), 7.33-7.32 (d, J = 1.84 Hz, 1H, Ar-H), 7.26-7.21 (t, J = 8.56 Hz, 1H, Ar-H), 7.16-
7.13 (dd, J = 8.52, 1.84 Hz, 1H, Ar-H), 6.86-6.84 (d, J = 8.56 Hz, 1H, Ar-H), 6.04-6.03 (d, J =
1.84 Hz, 1H, Ar-H), 5.91-5.90 (d, J = 2.44 Hz, 1H, Ar-H), 5.36 (s, 2H, -OCH2), 3.91, 3.87, 3.77
13
(s, each 3H, 3 × -OCH3); C NMR (CDCl3, 100 MHz, ppm) δ: 192.44 (-CO); 168.40; 166.12;
162.57; 156.79; 151.55; 147.41; 142.28; 128.72; 125.67; 124.31; 123.15; 120.36; 120.29;
117.78; 117.55; 112.75; 111.63; 106.34; 93.79; 91.24; 62.97 (-OCH2); 56.01 (-OCH3); 55.95 (-
OCH3); 55.55 (-OCH3); Elemental analysis for C27H23ClFN3O6: Calculated: C 60.06, H 4.29, N
7.78; Found: C 60.00, H 4.27, N 7.74; LC-MS (m/z): 540.13 [M+H]+.
4.7.8. (2E)-3-(3-{[1-(2,4-difluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-4-methoxyphenyl)-1-
(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one ne (11c)
1
IR (KBr) cm-1: 2958, 2917, 2846, 1607, 1575, 1504, 1209, 1142, 1009, 822; H NMR
(CDCl3, 400 MHz, ppm) δ: 14.32 (s, 1H, -OH), 8.08-8.07 (d, J = 2.72 Hz, 1H, -C=CH of
triazole), 7.88-7.82 (m, 1H, Ar-H), 7.78-7.74 (d, J = 14.68 Hz, 1H, -COC=CH), 7.70-7.66 (d, J =
14.68 Hz, 1H, -COCH=C), 7.37 (s, 1H, Ar-H), 7.16-7.13 (dd, J = 8.24, 1.84 Hz, 1H, Ar-H),
7.02-6.97 (m, 2H, Ar-H), 6.86-6.84 (d, J = 8.28 Hz, 1H, Ar-H), 6.04-6.03 (d, J = 2.76 Hz, 1H,
Ar-H), 5.91-6.90 (d, J = 1.80 Hz, 1H, Ar-H), 5.37 (s, 2H, -OCH2), 3.91, 386, 3.77 (s, each 3H, 3
× -OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.17 (-CO); 168.50; 165.93; 162.20; 149.62;
149.29; 144.15; 142.10; 129.37; 126.21; 126.05; 125.73; 124.26; 121.81; 113.67; 112.62;
111.15; 106.15; 105.30; 93.91; 91.04; 62.62 (-OCH2); 55.95 (-OCH3); 55.73 (-OCH3); 55.52 (-
OCH3); Elemental analysis for C27H23F2N3O6: Calculated: C 61.95, H 4.43, N 8.03; Found: C
61.91, H 4.45, N 8.07; LC-MS (m/z): 524.16 [M+H]+.
4.7.9. (2E)-3-(3-{[1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-4-methoxyphenyl)-1-(2-
hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (11d)
25

ACCEPTED MANUSCRIPT
IR (KBr) cm-1: 3015, 2917, 2852, 1617, 1582, 1557, 1510, 1260, 1215, 1153, 1031, 817,
1
750; H NMR (CDCl3, 400 MHz, ppm) δ: 14.39 (s, 1H, -OH), 8.06 (s, 1H, -C=CH of triazole),
7.83-7.79 (d, J = 15.28 Hz, 1H, -COC=CH), 7.75-7.71 (d, J = 15.88 Hz, 1H, -COCH=C), 7.61-
7.58 (m, 1H, Ar-H), 7.56-7.53 (m, 1H, Ar-H), 7.44-7.42 (m, 3H, Ar-H), 7.20-7.18 (dd, J = 8.56,
1.84 Hz, 1H, Ar-H), 6.91-6.89 (d, J = 7.96 Hz, 1H, Ar-H), 6.09-6.08 (d, J = 1.84 Hz, 1H, Ar-H),
5.96-5.95 (d, J = 2.44 Hz, 1H, Ar-H), 5.45 (s, 2H, -OCH2), 3.96, 3.91, 3.82 (s, each 3H, 3 × -
OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.45 (-CO); 168.36; 166.06; 162.58; 151.70;
147.65; 142.37; 134.77; 130.84; 130.75; 128.67; 128.63; 127.90; 127.74; 125.53; 125.22;
124.53; 113.05; 111.55; 106.30; 93.72; 91.17; 63.16 (-OCH2); 55.97 (-OCH3); 55.52 (-OCH3);
Elemental analysis for C27H24ClN3O6: Calculated: C 62.13, H 4.63, N 8.05; Found: C 62.09, H
4.60, N 8.08; LC-MS (m/z): 522.14 [M+H]+.
4.7.10. (2E)-3-(3-{[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]methoxy}-4-methoxyphenyl)-1-(2-
hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (11e)
IR (KBr) cm-1: 2958, 2917, 2846, 1611, 1575, 1544, 1504, 1264, 1198, 1142, 978, 815,
1
755; H NMR (CDCl3, 400 MHz, ppm) δ: 14.33 (s, 1H, -OH), 8.00 (s, 1H, -C=CH of triazole),
7.78-7.74 (d, J = 15.24 Hz, 1H, -COC=CH), 7.69-7.65 (d, J = 15.28 Hz, 1H, -COCH=C), 7.62-
7.60 (d, J = 8.52 Hz, 2H, Ar-H), 7.44-7.42 (d, J = 9.16 Hz, 2H, Ar-H), 7.35-7.34 (d, J = 1.80 Hz,
1H, Ar-H), 7.16-7.13 (dd, J = 8.52, 2.44 Hz, 1H, Ar-H), 6.86-6.84 (d, J = 8.52 Hz, 1H, Ar-H),
6.04-6.03 (d, J = 2.44 Hz, 1H, Ar-H), 5.91-5.90 (d, J = 2.44 Hz, 1H, Ar-H), 5.37 (s, 2H, -OCH2),
3.91, 3.86, 3.77 (s, each 3H, 3 × -OCH3); 13C NMR (DMSO-d6, 100 MHz, ppm) δ: 192.23 (-
CO); 165.22; 161.69; 149.61; 149.22; 142.79; 142.47; 131.20; 130.21; 128.24; 127.26; 125.51;
122.40; 118.01; 117.74; 115.75; 115.52; 113.62; 111.17; 106.44; 93.91; 91.05; 61.41 (-OCH2);
56.05 (-OCH3); 55.62 (-OCH3); 55.53 (-OCH3); Elemental analysis for C27H24ClN3O6:
26

ACCEPTED MANUSCRIPT
Calculated: C 62.13, H 4.63, N 8.05; Found: C 62.15, H 4.60, N 8.10; LC-MS (m/z): 522.14
[M+H]+.
4.7.11. (2E)-3-[3,4-bis({[1-(2-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy})phenyl]-1-
(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (12a)
IR (KBr) cm-1: 3077, 3011, 2924, 2851, 1622, 1582, 1557, 1508, 1264, 1213, 1030, 816,
749; 1H NMR (CDCl3, 400 MHz, ppm) δ: 14.28 (s, 1H, -OH), 7.99, 7.95 (s, each 1H, 2 × -C=CH
of triazole), 7.77-7.73 (d, J = 15.28 Hz, 1H, -COC=CH), 7.67-7.63 (d, J = 15.24 Hz, 1H, -
COCH=C), 7.54-7.49 (m, 2H, Ar-H), 7.32-7.31 (d, J = 1.84 Hz, 1H, Ar-H ), 7.25-7.21 (m, 2H,
Ar-H), 7.18-7.15 (dd, J = 8.56, 1.84 Hz, 1H, Ar-H), 7.11-7.05 (m, 3H, Ar-H), 6.04-6.03 (d, J =
2.44 Hz, 1H, Ar-H), 5.90-5.89 (d, J = 2.44 Hz, 1H, Ar-H), 5.36, 5.35 (s, each 2H, 2 × -OCH2),
3.89, 3.77 (s, each 3H, 2 × -OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.38 (-CO); 168.40;
166.17; 163.91; 162.54; 161.37; 150.28; 148.27; 141.92; 131.26; 131.22; 130.18; 130.15;
129.83; 129.19; 129.10; 126.17; 125.33; 125.30; 124.02; 118.23; 117.97; 115.41; 115.40;
115.18; 114.81; 114.69; 106.30; 93.78; 91.23; 63.39 (-OCH2); 63.08 (-OCH2); 55.96 (-OCH3);
55.56 (-OCH3); Elemental analysis for C35H26Cl2F2N6O6: Calculated: C 57.15, H 3.56, N 11.43;
Found: C 57.11, H 3.58, N 11.46; LC-MS (m/z): 735.13 [M+H]+.
4.7.12. ((2E)-3-[3,4-bis({[1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy})phenyl]-
1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-o (12b)
IR (KBr) cm-1: 2922, 2846, 1627, 1582, 1555, 1505, 1255, 1215, 1030, 817, 754; 1H NMR
(DMSO-d6, 400 MHz, ppm) δ: 13.44 (s, 1H, -OH), 8.67, 8.65 (s, each 1H, 2 × -C=CH of
triazole), 7.82-7.77 (m, 3H, Ar-H + -COCH=CH ), 7.71-7.60 (m, 3H, Ar-H), 7.52-7.45 (m, 3H,
Ar-H ), 7.36-7.30 (m, 2H, Ar-H), 6.14 (s, 1H, Ar-H), 6.12 (s, 1H, Ar-H), 5.35, 5.32 (s, each 2H,
2 × -OCH2), 3.89, 3.81 (s, each 3H, 2 × -OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.44 (-
27

ACCEPTED MANUSCRIPT
CO); 168.28; 166.10; 163.91; 162.46; 161.37; 150.33; 148.20; 142.02; 131.22; 130.26; 130.07;
129.83; 129.19; 129.09; 126.29; 125.20; 123.93; 118.29; 118.06; 115.72; 115.29; 114.74;
114.55; 106.21; 93.90; 91.14; 63.51 (-OCH2); 63.93 (-OCH2); 55.96 (-OCH3); 55.56 (-OCH3);
Elemental analysis for C35H26Cl2F2N6O6: Calculated: C 57.15, H 3.56, N 11.43; Found: C 57.11,
H 3.58, N 11.40; LC-MS (m/z): 735.13 [M+H]+.
4.7.13. (2E)-3-[3,4-bis({[1-(2,4-difluorophenyl)-1H-1,2,3-triazol-4-yl]methoxy})phenyl]-1-(2-
hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (12c)
1
IR (KBr) cm-1: 3086, 3014, 2924, 2851, 1617, 1582, 1556, 1518, 1260, 1215, 753; H
NMR (CDCl3, 400 MHz, ppm) δ: 14.33 (s, 1H, -OH), 8.14-8.13 (d, J = 2.44 Hz, 1H, -C=CH of
triazole), 8.12 (s, 1H, -C=CH of triazole), 7.92-7.85 (m, 2H, Ar-H), 7.83-7.79 (d, J = 15.88 Hz,
1H, -COC=CH), 7.73-7.69 (d, J = 15.24 Hz, 1H, -COCH=C), 7.39 (s, 1H, Ar-H), 7.23-7.21 (dd,
J = 8.56, 1.84 Hz, 1H, Ar-H), 7.13-7.11 (d, J = 8.56 Hz, 1H, Ar-H), 7.08-6.98 (m, 4H, Ar-H),
6.09-6.08 (d, J = 1.80 Hz, 1H, Ar-H), 5.96-6.95 (d, J = 2.44 Hz, 1H, Ar-H), 5.40 (s, 4H, 2 × -
OCH2), 3.95, 3.82 (s, each 3H, 2 × -OCH3); 13C NMR (CDCl3, 100 MHz, ppm) δ: 192.43 (-CO);
168.40; 166.18; 163.83; 163.73; 162.57; 150.25; 148.37; 141.95; 129.89; 126.27; 126.24;
126.17; 124.32; 124.26; 123.95; 114.73; 114.52; 112.73; 112.50; 106.37; 105.62; 105.37;
105.13; 93.82; 91.27; 63.37 (-OCH2); 63.05 (-OCH2); 55.94 (-OCH3); 55.57 (-OCH3); Elemental
analysis for C35H26F4N6O6: Calculated: C 59.83, H 3.73, N 11.96; Found: C 59.79, H 3.77, N
11.92; LC-MS (m/z): 703.19 [M+H]+.
4.7.14. (2E)-3-[3,4-bis({[1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl]methoxy})phenyl]-1-(2-
hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one (12d)
IR (KBr) cm-1: 3015, 2928, 2847, 1622, 1577, 1557, 1495, 1250, 1215, 1031, 745, 664; 1H
NMR (CDCl3, 400 MHz, ppm) δ: 14.39 (s, 1H, -OH), 8.09, 8.06 (s, each 1H, 2 × -C=CH of
28