Synthesis and antiproliferative activity of conformationally restricted 1,2,3-triazole analogues of combretastatins in the sea urchin embryo model and against human cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2013.12.015

A series of 1,5-diaryl- and 4,5-diaryl-1,2,3-triazole derivatives of combretastatin A4 were synthesized and evaluated as antimitotic microtubule destabilizing agents using the sea urchin embryo model. Structure-activity relationship studies identified compounds substituted with 3,4,5- trimethoxyphenyl and 3,4-methylenedioxy-5-methoxyphenyl ring A and 4-methoxyphenyl ring B as potent antiproliferative agents with high cytotoxicity against a panel of human cancer cell lines including multi-drug resistant cells. 4,5-Diaryl-1,2,3-triazoles (C-C geometry) were found to be considerably more active than the respective 1,5-diaryl-1,2,3-triazoles (N-C geometry). Compound 10ad′ induced G₂/M cell cycle arrest and apoptosis in human T-leukemia Jurkat cells via caspase 2/3/9 activation and downregulation of the antiapoptotic protein XIAP. A mitotic catastrophe has been evaluated as another possible cell death mode.

Full text of DOI:10.1016/j.bmc.2013.12.015

Accepted Manuscript
Synthesis and antiproliferative activity of conformationally restricted 1,2,3-tri-
azole analogues of combretastatins in the sea urchin embryo model and against
human cancer cell lines
Dmitry V. Demchuk, Alexander V. Samet, Natalia B. Chernysheva, Vladimir
I. Ushkarov, Galina A. Stashina, Leonid D. Konyushkin, Mikhail M. Raihstat,
Sergei I. Firgang, Alex A. Philchenkov, Michael P. Zavelevich, Ludmila M.
Kuiava, Vasyl F. Chekhun, Dmitry Yu. Blokhin, Alex S. Kiselyov, Marina N.
Semenova, Victor V. Semenov
PII:
S0968-0896(13)01009-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.12.015
BMC 11277
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
13 October 2013
1 December 2013
6 December 2013
Please cite this article as: Demchuk, D.V., Samet, A.V., Chernysheva, N.B., Ushkarov, V.I., Stashina, G.A.,
Konyushkin, L.D., Raihstat, M.M., Firgang, S.I., Philchenkov, A.A., Zavelevich, M.P., Kuiava, L.M., Chekhun,
V.F., Blokhin, D.Y., Kiselyov, A.S., Semenova, M.N., Semenov, V.V., Synthesis and antiproliferative activity of
conformationally restricted 1,2,3-triazole analogues of combretastatins in the sea urchin embryo model and against
human cancer cell lines, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2
013.12.015
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Synthesis and antiproliferative activity of conformationally restricted 1,2,3-triazole analogues of
combretastatins in the sea urchin embryo model and against human cancer cell lines
a
a
a
a
Dmitry V. Demchuk , Alexander V. Samet , Natalia B. Chernysheva , Vladimir I. Ushkarov , Galina
a
a
a,b
a
A. Stashina , Leonid D. Konyushkin , Mikhail M. Raihstat , Sergei I. Firgang , Alex A. Philchenkov
c
c
c
c
d
,
Michael P. Zavelevich , Ludmila M. Kuiava , Vasyl F. Chekhun , Dmitry Yu. Blokhin , Alex S.
e
b,f
a,b,
Kiselyov , Marina N. Semenova , Victor V. Semenov
*
a
N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russian
Federation
b
Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus
c
R. E. Kavetsky Institute of Experimental Oncology, Pathology and Radiobiology, National
Academy of Sciences of Ukraine, 45 Vasylkivska Street, 03022 Kyiv, Ukraine
d
N. N. Blokhin Cancer Research Center, 24 Kashirskoe Shosse, 115478 Moscow, Russian Federation
e
ChemDiv, 6605 Nancy Ridge Drive, San Diego, CA 92121, USA
f
N. K. Kol’tsov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow, Russian
Federation
Dmitry V. Demchuk
Alexander V. Samet
Natalia B. Chernysheva
Vladimir I. Ushkarov
Galina A. Stashina
Leonid D. Konyushkin
Mikhail M. Raihstat
Sergei I. Firgang
Alex A. Philchenkov
Michael P. Zavelevich
Ludmila M. Kuiava
Vasiliy F. Chekhun
Dmitry Yu. Blokhin
Alex S. Kiselyov
demchuk@ioc.ac.ru
sametav@ioc.ac.ru
info@chemblock.com
ushkarov@inbox.ru
galina_stashina@chemical-block.com
LeonidK@chemical-block.com
mr@chemical-block.com
cbi@chemical-block.com
a_philch@onconet.kiev.ua
butenko@onconet.kiev.ua
kuyavalm@ukr.net
chekhun@onconet.kiev.ua
blokhin@yandex.ru
akiselyov@chemdiv.com
ms@chemical-block.com
vs@zelinsky.ru
Marina N. Semenova
Victor V. Semenov
*
To whom correspondence should be addressed. Tel: +7 (916) 620-9584. Fax: +7 (499) 137-2966. E-mail: vs@zelinsky.ru
1

Abbreviations:
Bcl-2, B-cell lymphoma 2; CA2, combretastatin A-2; CA4, combretastatin A-4; CA4P, CA4 disodium
phosphate; MoAb, monoclonal antibody; PARP-1, poly(ADP-ribose) polymerase-1; PI, propidium
iodide; SAR, structure-activity relationship; XIAP, X-linked inhibitor of apoptosis
Key words:
1
,2,3-Triazole
Apoptosis
Caspases
Plant polyalkoxybenzenes
Microtubule destabilizing agents
Sea urchin embryo
2

Abstract
A series of 1,5-diaryl- and 4,5-diaryl-1,2,3-triazole derivatives of combretastatin A4 were synthesized
and evaluated as antimitotic microtubule destabilizing agents using the sea urchin embryo model.
Structure-activity relationship studies identified compounds substituted with 3,4,5-trimethoxyphenyl
and 3,4-methylenedioxy-5-methoxyphenyl ring A and 4-methoxyphenyl ring B as potent
antiproliferative agents with high cytotoxicity against a panel of human cancer cell lines including
multi-drug resistant cells. 4,5-Diaryl-1,2,3-triazoles (C–C geometry) were found to be considerably
more active than the respective 1,5-diaryl-1,2,3-triazoles (N–C geometry). Compound 10ad' induced
2
G /M cell cycle arrest and apoptosis in human T-leukemia Jurkat cells via caspase 2/3/9 activation and
downregulation of the antiapoptotic protein XIAP. A mitotic catastrophe has been evaluated as another
possible cell death mode.
3

1
. Introduction
Natural antimitotic stilbenes represented by combretastatins A-1, A-2 and A-4 (Fig. 1; CA1,
CA2, and CA4) are constituents of the bark of the African willow tree Combretum caffrum Kuntze
1–3
Combretaceae). These molecules are strong inhibitors of tubulin polymerization interacting with the
(
4
,5
colchicine binding site of tubulin. CA4 and its derivatives are potently cytotoxic against a wide
variety of human solid tumor and hematological malignancies both in vitro and in vivo. Water-soluble
phosphorylated prodrugs CA4 disodium phosphate (CA4P, Zybrestat) and combretastatin A-1 disodium
6
–9
phosphate (Oxi4503) (Fig. 1) are currently in clinical trials as antitumor vascular disrupting agents.
However, these drugs affect cardiovascular system, cause acute pain, and display other serious side
1
0–12
effects limiting their therapeutic utility.
A significant efforts were dedicated to the design and
development of novel conformationally restricted combretastatin analogues with reduced general
1
3,14
toxicity and improved on-target efficacy profiles.
Insert Figure 1
Numerous reports suggest that only cis-conformation of combretastatins and its analogues is
1
3–15
relevant to their cytotoxic, antimitotic, and antitubulin activity.
Unfortunately, isomerization to the
inactive trans-configuration happens spontaneously upon exposure to heat, light, and protic media.
Importantly, cis-trans biotransformation has also been reported as one of metabolic pathways of CA4 in
1
liver microsomes. A relatively simple chemical structure of combretastatins provides opportunity for
6
the development of a large number of biologically active stilbenes by ‘locking’ the olefinic bond into
1
3,14
the respective non-isomerizable scaffold.
For example, various 5-membered N-containing
1
heterocyclic templates including 4,5-diaryloxazoles, isoxasoles,
7
18,19
20
diarylthiazoles, thiazol-2(3H)-
2
ones, imidazol-2-ones, pyrazolones, pyrazoles,
1
22
23
24,25
26
tetrazoles, 1,2,4- , and 1,2,3-triazoles
27,28
29–34
were successful isosteres of the cis-olefin. The resulting compounds retained potency of the parent CA4
and exhibited favorable stability ex vivo and in vivo.
4

In addition to the cis-olefine linker, both the 3,4,5-trimethoxy substituted ring A and p-methoxy
1
3,14
group in the ring B are essential for the antimitotic antitubulin effects of combretastatin series.
The
position of 3,4,5-trimethoxyphenyl (A) and p-methoxyphenyl (B) rings relative to the heterocyclic
1
isostere was shown to be important for biological activity. ortho-Substituted diaryl azoles consistently
3
3
1,33,35
displayed markedly higher antiproliferative activity than their meta-analogues.
Moreover, nature
of the bond (N vs C linking) between these aryl substituents and N-heterocycle dramatically affected the
3
0,31,33
28 36
1,2,4-triazole, and tetrazole series.
activity of regioisomers, as shown for 1,2,3-triazole,
Specifically, the attachment of the trimethoxy substituted ring A to nitrogen yielded considerably more
potent compounds.
When analyzing the literature on heterocyclic analogues of combretastatins, we noticed that the
systematic structure-activity relationship (SAR) linking the effect of a substituent in the heterocyclic
bridge (amino-, alkyl-, and other groups) to the cytotoxic properties of the resultant molecule are
somewhat scarce and controversial. For instance, it was found that the introduction of CH
3
or NH
2
3
groups into triazole bridge of 3,4-diaryl-1,2,4-triazoles resulted in the loss of activity. However, 4,5-
7
2
4,25
21
diaryl-3-aminopyrazoles
cytotoxicity, cell cycle arrest in G2/M phase, and inhibition of tubulin polymerization. Similarly, both
,4-diaryl-5-amino- and 4,5-diaryl-3-aminoisoxazoles exhibited high antimitotic microtubule
destabilizing activity, whereas N-acetylation of the NH group in these series yielded ambiguous
and 3,4-diaryl-5-aminoisoxazoles were reported to display significant
3
2
2
1,38
results.
Considering the above literature evidence, we aimed to study the impact of substitution in the i)
heterocycle isostere; ii) aromatic rings A and B, and iii) relative position of the A and B rings on the
biological activity of cis-restricted combretastatin analogues. Ortho-diaryl-substituted 1,2,3-triazoles
(
Fig. 1) featuring aromatic rings connected to either N-C (1,5-geometry) or C-C (4,5-geometry) atoms
of the heterocycles were selected for several reasons. First, 1,5-diaryl-1,2,3-triazoles were described to
3
0–34,39
be potent cytotoxic agents affecting tubulin polymerization.
5
Second, a single position in 1,5-

diaryl-1,2,3-triazoles is available for substitution minimizing the number of possible regioisomers.
2
9,39
Third, biological activity of 4,5-diaryl-1,2,3-triazoles was not reported in great detail.
Particularly,
CA4 and its derivatives were reported to kill malignant cells by inducing different modes of cell death
3
5,36,40
41
necrosis, autophagy, and mitotic catastrophe. However, the question
42
43
including apoptosis,
remains, is there any relationship between cell type, cell death mechanism, and chemical structure of
antimitotic stilbenes.
In the present study we decided to: (i) conduct structure-activity relationship (SAR) studies for
1
,5-diaryl-1,2,3-triazole series in order to identify the optimized substitution pattern within this
structural class; (ii) estimate the antiproliferative and microtubule destabilizing activity using
4
4
phenotypic sea urchin embryo assay ; (iii) compare antimitotic activity of 1,5- and 4,5-diaryl-1,2,3-
triazoles, and (iv) assess cytotoxicity and apoptosis inducing properties of these series in cultured
human cancer cells. Triggering of apoptosis was studied using Jurkat human leukemia T-cells and its
multi-drug resistant subclone Jurkat A4.
The resistance of tumors to chemotherapeutics, including microtubule targeting drugs, is a
substantial problem in oncology. In 2001, Jurkat/A4 subclone was obtained by exposure of Jurkat
human leukemia T-cells to agonistic monoclonal antibodies (MoAb) against the death receptor Fas
4
5
(
CD95/APO-1) and selection of survived cells lacking functional Fas on their surface. Jurkat/A4 cells
displayed resistance toward several anticancer drugs such as etoposide, doxorubicin, and cycloplatam,
as well as to various apoptotic stimuli, including UV-irradiation, lethal doses of X-rays,
oxidative stress (H O and menadione), protein kinase inhibitor staurosporin, and tumor
2
2
necrosis factor-related apoptosis-inducing ligand TRAIL (despite the presence of the TRAIL
receptor DR5). Therefore, Jurkat/A4 cells represent a promising model to study molecular mechanisms
of multi-drug resistance,
in particular, inability to execute apoptotic cell death, that may be useful in screening for compounds
capable of killing resistant cells.
6

Previously it was shown that the sea urchin embryo model provides reliable and reproducible
evaluation of solubility in salt-containing medium (seawater), cell permeability, antimitotic potential,
and general toxicity/embryotoxicity of tested molecules together with their mode of action. The assay
includes (i) fertilized egg test for antimitotic activity assessed by cleavage alteration/arrest and (ii)
behavioral monitoring of a free-swimming hatched blastulae exposed to compounds. Specific changes
of swimming pattern, namely, cessation of forward movement, settlement to the bottom of the culture
vessel, and rapid spinning of an embryo around the animal–vegetal axis suggests a microtubule
destabilizing activity caused by a molecule (video illustrations are available at
http://www.chemblock.com). The reliability of data obtained by the sea urchin embryo assay has been
4
6–48
verified by multiple conventional cell-based and tubulin polymerization assays.
2
2
. Results and discussion
.1. Chemical synthesis
Polymethoxy substituted 1,5-diaryltriazoles 5–10 have been prepared via reported procedures
from the respective aldehydes (Scheme 1). These were prepared as described earlier from the natural
4
9,50
substrates isolated from the dill and parsley seed extracts.
Polymethoxy-4,5-diaryl-1,2,3-triazole
analogues of combretastatin 14ad', 14bd', and 14cd' have been prepared as described on Scheme 2.
Insert Schemes 1 and 2
1
H-4,5-Diaryl-1,2,3-triazoles were prepared from corresponding benzaldehydes following a
series of synthetic steps including 1) treatment of X with Ohira-Bestmann reagent; 2) Sonogashira
coupling of the resulting acetylenes with 4-iodoanizole, and 3) [3+2]-cycloaddition of the acetylenes
with benzylazide under high pressure and hydrogenation of the benzyl group. Our attempts to reduce
reaction pressure or to use catalytic conditions to promote [3+2] cycloaddition step were unsuccessful.
Specifically, the highest yields of the targeted 1H-4,5-diaryl-1,2,3-triazoles were secured when a
solution of 1,2-diarylalkyne (2 mmol) and azide (2.60 mmol) in DMF (1 mL) was heated at 100 ºC and
1
5 kbar for 7 h (see Experimental section). Notably, the reaction did result in equal ratio (ca. 1:1)
7

regioisomers as evidenced by the NMR-analysis of crude reaction mixtures. In a representative
experiment, the reaction mixture (isomers of 1-benzyl-4(5)-(4-methoxyphenyl)-5(4)-(3,4,5-
trimethoxyphenyl)-1H-1,2,3-triazoles) was separated using column chromatography to furnish pure
compound 13a'd. Its structure was determined unequivocally using NOESY experiment.
2
We observed an inter-space interaction of the NCH with protons H-2''',6''' allowing to assign the
1
structure of 13a'd. The H NMR signals of isomers in other mixtures were assigned in a similar way.
2
Removal of the PhCH fragment was accomplished by hydrogenation of the crude reaction mixtures at
2
0 bar. Since harsh reaction conditions required to access a series of 1H-4,5-diaryl-1,2,3-triazoles
precluded us from completing the expanded structure-activity studies, we focused on synthesizing
several representative compounds that were expected to parallel activities of the respective 1,5-
diaryltriazoles. The main goal was to conduct a direct biological comparison of the most active
analogues within these two classes of regioisomers.
2
2
.2. Biological evaluation
.2.1. Antimitotic activity in the phenotypic sea urchin embryo assay
The antiproliferative microtubule destabilizing effects of targeted o-diaryl-1,2,3-triazoles were
4
4
studied in the phenotypic sea urchin embryo assay. CA2 and CA4 served as reference compounds.
The results are shown in Table 1.
Insert Table 1.
As evidenced from Table 1, 1,5-diaryl-1,2,3-triazoles 10ad', 10bd', and 5de', and 4,5-diaryl-
1
,2,3-triazoles 14ad', 14bd', and 14cd' strongly affected sea urchin embryo cleavage and swimming
behavior, causing embryo spinning. These phenotypic changes are directly related to antimitotic
microtubule destabilizing activity of these compounds as shown by us earlier. The most potent
molecules were substituted with trimethoxyphenyl- (10ad') or 3,4-methylenedioxy-5-methoxyphenyl-
(
10bd') (ring A) and p-methoxyphenyl groups (ring B). Tetramethoxy substitution in the ring A (10cd')
markedly reduced activity of the resultant molecules. Compounds 10da' and 5fa' could be considered
8

as antitubulin agents as well. Although they did not cause embryo spinning, the formation of tuberculate
4
4,46,47
eggs typical of microtubule destabilizers was observed.
Our sea urchin embryo data revealed that the substitution in the 1,2,3-triazole ring (5ad'–9ad')
was not tolerated as compared to the respective unsubstituted molecule 10ad'. The introduction of 4-
NH
functionalities at C4 position afforded inactive compounds 5ad'–8ad' (Tабл. 1). It is worth noting that
,5-diaryl-1,2,3-triazoles with trimethoxyphenyl ring A tethered to the N atom (9ad' and 10ad') were
consistently more active than the respective regioisomers (trimethoxyphenyl substituent for the ring B,
2 2 3
group (9ad') decreased activity, whereas addition of CN, CONH , COOCH , and COOH
1
9
da' and 10da'). This fact is in a good agreement with the published evidence for the importance of
3
0,31,33
trimethoxyphenyl ring A connection to a nitrogen atom in 1,5-diaryl-1,2,3-triazoles.
4
,5-Diaryl-1,2,3-triazoles 14ad' and 14bd' (C–C geometry) were found to be considerably more
active than the respective 1,5-diaryl-1,2,3-triazoles 10ad' and 10bd' (N–C geometry). Similarly,
compound 14cd' displaying the tetra-substituted ring A exhibited only weak antiproliferative effect.
2
.2.2. In vitro cancer cell growth inhibition
,5-Diaryl-1,2,3-triazoles 5aa', 5dd', 5fa', 5fe', 6aa', 6ad', 7da', 8ad', 8da', 9da', and 10bd',
1
and 4,5-diaryl-1,2,3-triazoles 14ad', 14bd', and 14cd' have been further selected for cytotoxicity screen
against 60 human tumor cell lines (Developmental Therapeutics Program at NCI). As anticipated, the
data obtained by this cell-based assay correlated well with the sea urchin embryo observations (Tables 1
and 2). Namely, triazoles 5aa', 5dd', 5fe', 6aa', 6ad', 7da', 8ad', and 8da', inactive in the sea urchin
embryos, did not inhibit cancer cell growth at concentrations up to 10 µM. Compounds 5fa' and 9da'
showed only weak antiproliferative effects in both experimental approaches. Similarly, highly potent
antimitotic 4,5-diaryl-1,2,3-triazoles (14ad', 14bd', and 14cd') together with 1,5-diaryl-1,2,3-triazole
1
0bd' showed significant cytotoxicity against human cancer cells. Notably, GI50 values for the most
potent triazoles 14ad' and 14bd' were less than 10 nM for 50 and 10 tumor cell lines, respectively, of
the 60 tested (Table 2). It should be pointed out that newly synthesized compounds 14ad' and 14bd'
9

significantly affected growth of several cancer cells otherwise insensitive to CA4. These included colon
cancer HT-29, ovarian cancer OVCAR-5, renal cancer 786-0 and TK-10 cell lines (Table 2). Similar to
the sea urchin embryo assay results, diaryltriazole 14bd' with the C–C geometry was more potent than
its N–C regioisomer 10bd'. As anticipated from the sea urchin embryo tests, tetramethoxy-substituted
derivative (14cd') was less potent than the respective trimethoxy analogue in the cell based assay as
well.
To the best of our knowledge, there is a single report dealing with the biological evaluation of
4
,5-diaryl-1,2,3-triazoles (C–C geometry). According to the published data, cytotoxicity of the
combretastatin analogue substituted with 3,4,5-trimethoxyphenyl- (ring A) and 4-aminophenyl- (ring B)
2
9
groups was lower than expected with the IC50 value of 56 µM for murine B6 melanoma cells. This
observation further emphasizes the importance of p-OCH substitution in the ring B. According to
previous SAR studies of combretastatin derivatives, the removal of p-OCH or its replacement with
OH, Cl, Br, NO , and OAc functionalities reduced the cytotoxicity. Moreover, the introduction of p-
3
1
5
3
2
NO
2
group resulted in inactive molecule.
Insert Table 2.
It should be noted that compounds 10bd', 14ad', 14bd', and 14cd' showed higher cytotoxicity
against multi-drug resistant human ovarian cells MDR NCI/ADR-RES overexpressing P-glycoprotein
than against the parent cell line OVCAR-8 (Table 2). This observation suggests that the selected
molecules were not substrates of this transmembrane transporter and are not likely to be effluxed.
1
,5-Diaryl-1,2,3-triazole 10ad' that altered sea urchin embryo development at 0.02 µM by
microtubule destabilizing mode of action, was reported to inhibit polymerization of purified tubulin.
This compound displayed potent antiproliferative activity against human cancer cells with IC50 of 0.027
3
1
3
µM for K562 human leukemia cells and IC50 of 0.020–0.053 µM for 4 human cancer cell lines
1
3
including multi-drug resistant cells. Similar to the SAR results from the sea urchin embryo assay, the
9
1
0

3
respective regioisomer 10da' was less active with IC50 of 4 µM for K562 human leukemia cells and
1
3
0
IC50 > 1 µM for SH-SY5Y human neuroblastoma cells.
2
.2.3. Induction of apoptosis in Jurkat and multi-drug resistant Jurkat A4 cells
A summary of data on cytotoxic activity of 10ad' against Jurkat human leukemia T-cells and its
multi-drug resistant subclone Jurkat A4 is presented in Figure 2. CA4P was used as a reference
compound. In Jurkat cells, 10ad' showed cytotoxicity similar to that of CA4P with EC50 of 30 nM and
1
5 nM, respectively. Notably, 10ad' had similar activity in the sea urchin embryo assay (cleavage
alteration EC = 20 nM, Table 1). In contrast, both 10ad' and CA4P were much less toxic in Jurkat/A4
cells with multi-drug resistant phenotype exhibiting the death fraction less than 20 % even at
concentration of 1 µM. Nevertheless, the delayed death of Jurkat/A4 cells was observed after two-hour
exposure to 10ad' followed by seven days cultivation in drug-free medium (data not shown). Based on
these cytotoxicity results, agents 10ad' and CA4P were subsequently used at their EC50 concentrations
in Jurkat cells, whereas Jurkat/A4 cells were exposed to 10-times higher concentrations for both
compounds.
Insert Figures 2 and 3.
We next assessed the effect of compound 10ad' on the cell cycle distribution. It was found that
1
2 0 1
0ad' induced cell cycle arrest at G /M phase with concomitant decrease of G /G phase in both Jurkat
and Jurkat/A4 cells, similarly to CA4P (Fig. 3). However, the effective concentrations of both 10ad'
and CA4P in Jurkat/A4 cells were 10-times higher. Our results suggested that 10ad' exhibited
antimitotic activity comparable with that of CA4P in both parental and multi-drug resistant leukemia
cells.
Research over the last two decades has provided convincing evidence that the cell cycle arrest at
2
G /M phase induced by CA4 and its derivatives eventually leads to cell death via apoptosis and mitotic
2
0,55
catastrophe.
To examine possible mechanism(s) of cell death caused by 10ad'¸ we analyzed
morphological changes in Jurkat and Jurkat/A4 cells after exposure to this compound. It was discovered
1
1

that both 10ad' and CA4P caused marked alterations in chromosome segregation. Namely, we observed
formation of micronuclei and small cell fragments containing unequally segregated chromosomes
enclosed by cell membrane (Fig. 4). Such nuclear alterations could be considered as indicators of
5
6
mitotic catastrophe. In addition, detection of condensed and fragmented nuclei typical for compounds
that affect mitotic spindle organization was suggestive of the early stages of apoptosis. In Jurkat cells,
the morphological features of apoptosis became more evident with the increase of concentration for
both molecules (data not shown).
To gain insight into the mechanisms underlying the cytotoxic effect of 10ad', apoptogenic
activity of this compound was examined. CA4P was used as a reference molecule. As shown in Figure
5
, sub-G1 population became evident in Jurkat cells treated with 10ad' (30 nM, 48 h) but not in multi-
drug resistant Jurkat/A4 cells even at 10-times higher concentration of the compound (300 nM). Similar
results were obtained with CA4P in both cell lines.
Insert Figures 4 and 5.
Caspases are known to play a pivotal role in both initiation and execution of apoptotic cell
5
7,58
death.
Caspase-9 is a major initiator caspase for intrinsic (receptor-independent) apoptotic signaling
pathway. Caspase-2 is unique in this class as it combines the properties of both initiator and effector
caspases. On the one hand, its structure and activation mode resemble those of caspase-9. Caspase-9
organization features a long prodomain containing a CARD sequence and procaspase autoprocessing.
On the other hand, its substrate specificity is similar to that of effector caspases-3 and -7. Both intrinsic
and extrinsic (receptor-mediated) apoptotic signaling pathways lead to activation of the effector
caspase-3, which is the main protease that degrades the cell.
To assess the role of caspases as apoptosis markers in cell death caused by 10ad', Jurkat and
Jurkat/A4 cells were pretreated with the pancaspase peptide inhibitor z-VAD-fmk for 3 h followed by
the addition of 10ad'. Figure 6 demonstrates that pretreatment with z-VAD-fmk decreased the
percentage of hypodiploid Jurkat cells, but not Jurkat/A4 cells exposed to 10ad' or CA4P. The
1
2

involvement of initiator caspases in 10ad'- or CA4P-induced apoptosis was further examined by
Western blot analysis (Fig. 7A). In Jurkat cells, the band corresponding to the active form of caspase-9
was evident after 48 h exposure to either 10ad' or CA4P. The involvement of caspase-9 in cell death
3
5
induced by 10ad' has been also shown recently in PC12 rat pheochromocytoma cells. The active form
of caspase-2 was expressed both in 24 h and 48 h samples. The band intensity was slightly increased for
samples with longer exposure time. Activation of neither caspase-9 nor caspase-2 was observed in
Jurkat/A4 cells treated with either 10ad' or CA4P (Fig. 7A). Improtantly, caspase-2 activation is a
5
6,59
prerequisite of mitotic catastrophe,
induced death of Jurkat cells.
Insert Figures 6 and 7.
suggesting possible contribution of mitotic catastrophe in 10ad'-
Proteins of IAP and Bcl-2 families are known to be the critical regulators of apoptosis.
Particularly, X-linked inhibitor of apoptosis expression (XIAP) and Bcl-2 antiapoptotic proteins that
block cell death machinery are involved in the development of drug resistance of tumor cells. As
6
0
reported previously, XIAP undergoes cleavage by active caspase-3. Therefore we analyzed the effect
of compound 10ad' on XIAP and Bcl-2 expression in Jurkat leukemia cells. It was found that 10ad' as
well as CA4P after 24 h treatment caused the reduction of XIAP band density in Jurkat cell line but not
in the multi-drug resistant counterpart. Similarly, we reported previously the decreased XIAP
6
1
expression upon etoposide-triggered apoptosis in Jurkat but not in Jurkat/A4 cells. In contrast to
XIAP, no detectable changes in the levels of antiapoptotic Bcl-2 expression in either Jurkat or Jurkat/A4
cells have been found (Fig. 7B).
Next, to explore the role of effector caspase-3 in 10ad'- and CA4P-induced apoptosis, flow
cytometry and FITC-conjugated active caspase-3 MoAb were used. As evidenced from Figure 8, the
percentage of cells with active form of caspase-3 increased in a time-dependent manner in Jurkat but
not Jurkat/A4 cells exposed to both CA4P and 10ad'. We examined also whether 10ad'-induced
apoptosis was associated with cleavage of caspase substrate PARP-1. Figure 8 demonstrates that
1
3

apoptosis induction in Jurkat cells at 30 nM of 10ad' was accompanied by PARP-1 processing, as
evident by flow cytometric analysis with MoAb that specifically recognizes the cleaved form of PARP-
1
. In contrast, no PARP-1 cleavage was observed in Jurkat/A4 cells exposed to 10ad' at 10-times higher
concentration.
Taken together, our results provide evidence that both 10ad' and CA4P are potent apoptosis
inducers in Jurkat cells. Accumulation of Jurkat cells in G /M phase in the presence of 10ad' was
associated with the increased number of hypodiploid apoptotic sub-G1 cells. Caspase-9 and -2
2
6
2,63
activation caused by 10ad' or CA4P is in agreement with the recent data
that point to the role of
these caspases in the cell death induced by different CA4 derivatives. Moreover, the involvement of
caspase-2 in apoptosis induced by both microtubule-destabilizing and microtubule-stabilizing agents of
6
4
other chemical classes has been reported. The fragmentation of PARP-1 in 10ad'- treated Jurkat cells
served as another proof of caspase-dependent cytotoxicity caused by this molecule. The observed
decrease of XIAP expression in Jurkat cells after treatment with 10ad' or CA4P correlates well with the
literature data suggesting that apoptotic cell death induced by CA4 and/or its analogues coincided with
6
3,65
the downregulation of XIAP protein.
The expression of antiapoptotic Bcl-2 protein was unaffected
by 10ad' or CA4P treatment, similar to the reported lack of effect of CA4P on Bcl-2 expression levels
6
6
in WSU-CLL cells. Thus, the regulation of XIAP but not Bcl-2 expression may be involved in 10ad'-
and CA4P-induced apoptosis in Jurkat cells. It should be noted that in addition to caspase-dependent
apoptosis caused by 10ad' and CA4P an alternative form(s) of cell death could be operative, since the
suppression of apoptosis by pancaspase inhibitor z-VAD-fmk was incomplete. Specific morphological
alterations as well as caspase-2 involvement suggested that mitotic catastrophe could represent one of
nonapoptotic routes of cell death in Jurkat cells exposed to 10ad'.
Jurkat/A4 cells with multi-drug resistant phenotype were characterized by altered components of
6
cell death machinery. Although both 10ad' and CA4P induced mitotic arrest in Jurkat/A4 cells
1
followed by delayed death, we could not observe any increase in the hypodiploid cell fraction,
1
4

activation of caspase-2, -3, and -9, downregulation of XIAP expression, and induction of PARP-1
cleavage, all characteristic features of apoptosis. The identification of cell death mode in Jurkat/A4 cells
treated by microtubule destabilizing compound 10ad' requires further investigations using a diverse
6
7
panel of biochemical and functional assays.
. Conclusions
Based on results of the in vivo sea urchin embryo assay, cytotoxicity panel of 60 human tumor
cell lines, and Jurkat cells activity data, we identified 1,5-diaryl-1,2,3-triazole derivatives 10ad' and
3
1
0bd' and 4,5-diaryl-1,2,3-triazole derivatives 14ad' and 14bd' featuring 3,4,5-trimethoxyphenyl and
3
,4-methylenedioxy-5-methoxyphenyl ring A, respectively, as potent antimitotic microtubule
destabilizing agents. At low nanomolar concentrations these compounds blocked proliferation of
multiple human tumor cell lines including multi-drug resistant P-glycoprotein overexpressing cells.
Triazoles 14ad' and 14bd' (C–C geometry) were found to be considerably more active than the
respective 1,5-diaryl-1,2,3-triazoles 10ad' and 10bd' (N–C geometry). The mechanism of cell death
under exposure of triazoles has been investigated with compound 10ad' in human T-leukemia Jurkat
2
cells. This molecule induced G /M cell cycle arrest and apoptosis via caspase 2/3/9 activation and
downregulation of the antiapoptotic protein XIAP. In addition to apoptosis, micronucleated cell
formation together with caspase-2 activation indicated possible involvement of mitotic catastrophe. In
multi-drug resistant Jurkat/A4 cells, 10ad' also exhibited lethal effect by unknown mechanism.
Considering these encouraging data from phenotypic and mechanistic studies, 10ad', 10bd', 14ad', and
1
4bd' may prove to be lead candidates for further in vivo studies to assess its potential as an anti-tumor
agents.
4
4
. Experimental section
.1. Chemistry
NMR spectra were collected on a Bruker DR-500 instrument [working frequencies of 500.13
1 13
MHz ( H) and 75.47 ( C)]. Mass spectra were obtained on a Finnigan MAT/INCOS 50 instrument (70
1
5

eV) using direct probe injection. Elemental analysis was accomplished with the automated Perkin-
Elmer 2400 CHN microanalyzer. The compound purity has been determined by NMR, HPLC, and
elemental analyses. Purity of targeted compounds was determined to be ≥ 95%.
Alkyl 1,2,3-triazole-4-carboxylates 7 and 1,2,3-triazole-4-carbonitriles 5 were prepared from
5
2
arylazides and β-ketoesters (β-ketonitriles) as previously described.
.1.1. General procedure for the synthesis of arylamines (3, 9) by Curtius rearrangement
A suspension of the acid (20 mmol) in SOCl (5 mL) was refluxed for 2.5 h, excess of SOCl
was thoroughly removed with abs. benzene (2 × 4 mL) affording acid chloride. After dilution with
acetone (88 mL), a solution of NaN (3.25 g, 50 mmol) in water (11 mL) was added dropwise at 0– -2
C for 20 min with stirring. The reaction mixture was further stirred at 0 °C for 1 h, warmed to rt, then
the solvent was thoroughly removed at 30–35 °C in vacuo. The residue was diluted with water (30 mL),
and targeted acyl azide was extracted with CH Cl (3 × 50 mL). The combined extracts were washed
with water (3 × 50 mL), dried by filtration through cotton plug, the solvent was removed, and the
residue was thoroughly dried over P at 30 °C. The resulting acyl azide (yield 95–96%) was
suspended in abs. EtOH (15 mL), warmed slowly to 85–90 °C with stirring (N evolution was
4
2
2
3
°
2
2
2
O
5
2
observed), and refluxed for 5 h affording urethane (yield ca. 94%) solution. The solvent was removed in
vacuo, then aqueous NaOH (1N, 60 mL) and EtOH (8 mL) were added. The mixture was refluxed for 8
2 2
h, EtOH was removed, and the residue was extracted with CH Cl (3 × 50 mL). The combined extract
was washed with water (3 × 50 mL), dried, and the solvent was removed. The targeted arylamines 3 or
1
4
7
7
,2,3-triazolamines 9 were washed with MeOH.
.1.1.1. Intermediate ethyl 7-methoxy-1,3-benzodioxol-5-yl-carbamate. White solid, yield 100%, mp
1
1–73 °C. H NMR (DMSO-d
6
): δ 1.22 (t, J = 7.1 Hz, 3H, CH
3 2 3
CH O), 3.77 (s, 3H, OCH ), 4.10 (k, J =
.1 Hz, 2H, OCH
2
CH
3
), 5.92 (s, 2H, OCH O), 6.76 (d, J = 1.9 Hz, 1H, H-4), 6.78 (d, J = 1.9 Hz, 1H, H-
2
1
6

+
), 9.47 (s, 1H, NH). EIMS m/z 239 [M] (84), 211 (32), 193 (69), 180 (15), 167 (41), 166 (100), 148
6
(
7), 136 (8), 124 (17), 124 (11), 122 (10), 108 (9), 94 (8), 79 (23), 68 (11), 53 (19).
4
.1.1.2. 7-Methoxy-1,3-benzodioxol-5-amine (3b). White solid, yield 57% (starting from benzoic
6
8 1
acid), mp 85–86 °C (H
2
O) (lit. 85–86 °C). H NMR (DMSO-d
O), 5.83 (d, J = 1.9 Hz, 1H, H-6), 5.86 (d, J = 1.9 Hz, 1H, H-4). EIMS m/z 167
M] (100), 152 (1), 138 (3), 124 (17), 122 (12), 110 (8), 95 (14), 80 (51), 68 (15), 65 (6), 52 (21). Anal.
Calcd for C NO : C, 57.48; H, 5.43; N, 8.38. Found: C, 57.65; H, 5.49; N, 8.23.
.1.1.3. Intermediate ethyl 4,7-dimethoxy-1,3-benzodioxol-5-yl-carbamate. White solid, yield
6 3
): δ 3.72 (s, 3H, OCH ), 4.80 (s, 2H,
2 2
NH ), 5.75 (s, 2H, OCH
+
[
8
H
9
3
4
1
7
8
1
1
00%, mp 85–90 °C. H NMR (DMSO-d
6
): δ 1.22 (t, J = 7.1 Hz, 3H, CH
3
CH
2
O), 3.75 (s, 3H, OCH
3
-
), 3.78 (s, 3H, OCH
3
-4), 4.08 (k, J = 7.1 Hz, 2H, OCH
2
CH
3
), 5.98 (s, 2H, OCH
2
O), 6.83 (s, 1H, H-6),
+
.49 (s, 1H, NH). EIMS m/z 269 [M] (100), 254 (15), 224 (4), 223 (18), 208 (29), 196 (17), 182 (53),
66 (18), 152 (11), 136 (12), 124 (13), 110 (17), 95 (10), 93 (10), 82 (11), 80 (16), 68 (32), 65 (25), 53
(
34).
.1.1.4. 5-Amino-4,7-dimethoxy-1,3-benzodioxole (3c). White solid, yield 86% (starting from benzoic
4
6
9 1
acid), mp 70–71 °C (lit. 70–72 °C). H NMR (DMSO-d
6
): δ 3.70 (s, 3H, OCH
3
3
-7), 3.72 (s, 3H, OCH -
+
O), 5.94 (s, 1H, H-6). EIMS m/z 197 [M] (94), 182 (100), 166
4
), 4.56 (s, 2H, NH ), 5.81 (s, 2H, OCH
2
2
(
(
4), 154 (11), 152 (19), 137 (10), 124 (34), 109 (10), 96 (16), 94 (9), 92 (7), 81 (19), 68 (32), 65 (16), 53
35). Anal. Calcd for C : C, 54.82; H, 5.62; N, 7.10. Found: C, 54.88; H, 5.69; N, 7.17.
.1.2. General procedure for the synthesis of arylazides (4)
9 4
H11NO
4
51
Prepared according to published procedure and used without further purification.
1
.1.2.1. 6-Azido-4-methoxy-1,3-benzodioxole (4b). White solid, yield 85%, mp 70–72 °C. H NMR
4
(
DMSO-d
6
): δ 3.83 (s, 3H, OCH
3
2
-4), 6.01 (s, 2H, OCH O), 6.37 (d, J = 1.9 Hz, 1H, H-5), 6.47 (d, J =
+
.9 Hz, 1H, H-7). EIMS m/z 193 [M] (12), 165 (44), 150 (14), 107 (6), 94 (5), 92 (7), 77 (15), 68 (12),
1
6
3
8 7 3 3
4 (19), 55 (57), 53 (100). Anal. Calcd for C H N O : C, 49.74; H, 3.65; N, 21.75. Found: C, 49.64; H,
.61; N, 21.84.
1
7

1
.1.2.2. 5-Azido-4,7-dimethoxy-1,3-benzodioxole (4c). White solid, yield 62%, mp 68–70 °C. H
4
NMR (DMSO-d
6
3 3 2
): δ 3.77 (s, 3H, OCH -4), 3.83 (s, 3H, OCH -7), 6.03 (s, 2H, OCH O), 6.32 (s, 1H, H-
+
). EIMS m/z 223 [M] (26), 195 (63), 194 (27), 180 (41), 152 (12), 150 (27), 138 (10), 136 (10), 124
6
(
47), 122 (31), 96 (56), 94 (43), 83 (69), 82 (44), 79 (42), 78 (18), 68 (38), 67 (38), 64 (34), 53 (100).
Anal. Calcd for C : C, 48.43; H, 4.06; N, 18.83. Found: C, 48.37; H, 4.03; N, 18.89.
.1.3. General procedure for the synthesis of carbonitriles (5)
9
9 3 4
H N O
4
5
2
Prepared according to published procedure.
4
.1.3.1. 1,5-Bis(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carbonitrile (5aa'). White solid, yield 79%,
1
mp 184–185 °C (EtOH). H NMR (DMSO-d
6
): δ 3.66 (s, 6H, OCH
3
3
-3',5'), 3.70 (s, 6H, OCH -4', 4''),
+
-3'',5''), 6.81 (s, 2H, H-2',6'), 6.93 (s, 2H, H-2'',6''). EIMS m/z 426 [M] (44), 398
3
.71 (s, 6H, OCH
3
(
(
27), 384 (23), 383 (100), 367 (10), 325 (9), 231 (7), 152 (7), 137 (11), 122 (6), 120 (6), 109 (14), 107
5), 104 (6), 92 (6), 88 (6), 81 (17), 77 (10). Anal. Calcd for C21 : C, 59.15; H, 5.20; N, 13.14.
22 4 6
H N O
Found: C, 59.29; H, 5.28; N, 13.01.
4
.1.3.2. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carbonitrile (5ad').
1
White solid, yield 81%, mp 145–146 °C (EtOH). H NMR (DMSO-d
6
): δ 3.67 (s, 6H, OCH
3
-3',5'), 3.71
(
s, 3H, OCH
3
-4'), 3.79 (s, 3H, OCH
3
-4''), 6.87 (s, 2H, H-2',6'), 7.08 (d, J = 8.5 Hz, 2H, H-3'',5''), 7.40 (d,
+
J = 8.5 Hz, 2H, H-2'',6''). EIMS m/z 366 [M] (30), 340 (1), 338 (45), 323 (100), 307 (31), 295 (10), 292
(
(
5), 280 (25), 265 (32), 249 (10), 237 (23), 209 (10), 194 (8), 178 (8), 171 (94), 152 (17), 145 (92), 139
10), 137 (31), 133 (10), 128 (15), 122 (15), 116 (34), 114 (33), 109 (46), 107 (17), 102 (20), 92 (16),
8
9 (21), 81 (56), 77 (33), 76 (34), 66 (97). Anal. Calcd for C19
18 4 4
H N O : C, 62.29; H, 4.95; N, 15.29.
Found: C, 62.21; H, 4.88; N, 15.35.
4
.1.3.3. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carbonitrile (5da').
1
White solid, yield 84%, mp 126–128 °C (EtOH). H NMR (DMSO-d
6
): δ 3.64 (s, 6H, OCH
3
-3'',5''),
3
7
.70 (s, 3H, OCH
3
-4''), 3.81 (s, 3H, OCH
3
-4'), 6.75 (s, 2H, H-2'',6''), 7.11 (d, J = 8.9 Hz, 2H, H-3',5'),
+
.46 (d, J = 8.9 Hz, 2H, H-2',6'). EIMS m/z 366 [M] (34), 338 (10), 324 (20), 323 (100), 295 (10), 280
1
8

(
6), 265 (11), 263 (5), 251 (6), 237 (8), 209 (15), 194 (8), 171 (11), 166 (6), 144 (7), 139 (5), 132 (5),
1
1
4
1
8
04 (15), 102 (11), 92 (44), 88 (17), 77 (47), 64 (34). Anal. Calcd for C19
5.29. Found: C, 62.32; H, 4.99; N, 15.34.
18 4 4
H N O : C, 62.29; H, 4.95; N,
.1.3.4. 1,5-Bis(4-methoxyphenyl)-1,2,3-triazole-4-carbonitrile (5dd'). White solid, yield 75%, mp
1
25–126 °C (EtOH). H NMR (DMSO-d
6
): δ 3.79 (s, 3H, OCH
3
-4''), 3.81 (s, 3H, OCH
3
-4'), 7.06 (d, J =
.8 Hz, 2H, H-3'',5''), 7.08 (d, J = 8.9 Hz, 2H, H-3',5'), 7.34 (d, J = 8.8 Hz, 2H, H-2'',6''), 7.42 (d, J = 8.9
+
Hz, 2H, H-2',6'). EIMS m/z 306 [M] (23), 291 (1), 278 (62), 264 (17), 263 (100), 235 (34), 220 (14),
1
92 (23), 171 (35), 164 (7), 145 (82), 130 (13), 128 (10), 116 (33), 114 (30), 107 (12), 102 (27), 92
(
14 4 2
92), 90 (29), 88 (19), 77 (97). Anal. Calcd for C17H N O : C, 66.66; H, 4.61; N, 18.29. Found: C,
6
4
6.73; H, 4.66; N, 18.21.
.1.3.5. 1-(4-Methoxyphenyl)-5-(3-methoxyphenyl)-1,2,3-triazole-4-carbonitrile (5de'). White solid,
1
yield 73%, mp 90–92 °C (EtOH). H NMR (DMSO-d
6
): δ 3.69 (s, 3H, OCH
3
-4'), 3.81 (s, 3H, OCH
3
-
3
''), 6.97 (d, J = 7.7 Hz, 1H, H-4''), 7.00 (d, J = 2.4 Hz, 1H, H-2''), 7.08 (d, J = 8.9 Hz, 2H, H-3',5'), 7.10
(
dd, J = 7.7, 2.4 Hz, 1H, H-6''), 7.42 (t, J = 7.7 Hz, 1H, H-5''), 7.42 (d, J = 8.9 Hz, 2H, H-2',6'). EIMS
+
m/z 306 [M] (45), 278 (100), 263 (100), 248 (9), 247 (7), 235 (98), 232 (12), 220 (22), 192 (44), 171
(
(
10), 164 (10), 139 (11), 130 (11), 128 (11), 116 (25), 114 (32), 102 (27), 92 (81), 88 (16), 77 (68), 64
78). Anal. Calcd for C17 : C, 66.66; H, 4.61; N, 18.29. Found: C, 66.76; H, 4.65; N, 18.20.
.1.3.6. 1-(3-Chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carbonitrile (5fa'). White
14 4 2
H N O
4
1
solid, yield 78%, mp 130–131 °C (EtOH). H NMR (DMSO-d
6 3
): δ 3.64 (s, 6H, OCH -3'',5''), 3.71 (s,
3
H, OCH -4''), 6.77 (s, 2H, H-2'',6''), 7.48 (d, J = 7.8 Hz, 1H, H-4'), 7.60 (t, J = 7.8 Hz, 1H, H-5'), 7.70
3
+
d, J = 7.8 Hz, 1H, H-6'), 7.76 (s, 1H, H-2'). EIMS m/z 372 [M+2] (14), 370 [M] (41), 329 (30), 327
+
(
(
88), 299 (15), 283 (5), 269 (11), 255 (7), 231 (9), 215 (8), 213 (21), 203 (6), 201 (7), 178 (9), 171 (10),
1
62 (6), 151 (6), 145 (8), 144 (9), 113 (30), 111 (82), 104 (22), 102 (18), 100 (10), 88 (33), 77 (11), 76
(
4 3
38), 75 (100). Anal. Calcd for C18H15ClN O : C, 58.31; H, 4.08; Cl, 9.56; N, 15.11. Found: C, 58.24;
H, 4.03; Cl, 9.62; N, 15.15.
1
9

4
.1.3.7. 1-(3-Chlorophenyl)-5-(3-methoxyphenyl)-1,2,3-triazole-4-carbonitrile (5fe'). White solid,
1
yield 72%, mp 100–102 °C (EtOH). H NMR (DMSO-d
6
): δ 3.70 (s, 3H, OCH -3''), 6.99 (d, J = 8.9 Hz,
3
1
8
H, H-4''), 7.02 (s, 1H, H-2''), 7.14 (d, J = 8.9 Hz, 1H, H-6''), 7.45 (t, J = 8.9 Hz, 1H, H-5''), 7.47 (d, J =
.0 Hz, 1H, H-4'), 7.59 (t, J = 8.0 Hz, 1H, H-5'), 7.69 (d, J = 8.0 Hz, 1H, H-6'), 7.72 (s, 1H, H-2'). EIMS
+
m/z 312 [M+2] (10), 310 [M] (36), 284 (23), 282 (74), 267 (6), 247 (14), 241 (13), 239 (39), 232 (10),
+
2
8
03 (9), 171 (12), 128 (10), 116 (23), 114 (41), 113 (25), 111 (69), 102 (18), 101 (12), 90 (15), 88 (15),
7 (11), 76 (31), 75 (100). Anal. Calcd for C16 O: C, 61.84; H, 3.57; Cl, 11.41; N, 18.03. Found:
H11ClN
4
C, 61.76; H, 3.52; Cl, 11.44; N, 18.07.
.1.4. General procedure for the synthesis of 1,5-diaryl-1,2,3-triazole-4-carboxamides (6)
4
The corresponding 1,2,3-triazole-4-carbonitrile (2 mmol) was dissolved in conc. H SO (1 mL)
4
2
and kept for 10 h. The reaction mixture was poured onto ice (10 g). The resulting precipitate was
filtered off and recrystallized from 50% aqueous MeOH.
4
6
4
4
.1.4.1. 1,5-Bis(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carboxamide (6aa'). White solid, yield
1
6%, mp 199–200 °C. H NMR (DMSO-d
6
): δ 3.62 (s, 6H, OCH
3
-3'',5''), 3.67 (s, 12H, OCH
3
-3',4',5',
+
). EIMS m/z 444 [M] (68),
''), 6.73 (s, 2H, H-2',6'), 6.77 (s, 2H, H-2'',6''), 7.50 and 7.93 (s, 2H, CONH
2
16 (15), 402 (9), 401 (46), 373 (33), 358 (35), 342 (8), 327 (5), 315 (8), 300 (10), 284 (6), 356 (5), 218
5), 209 (40), 207 (13), 206 (14), 193 (70), 179 (11), 168 (82), 165 (24), 152 (21), 150 (18), 148 (15),
45 (12), 140 (19), 137 (134), 135 (16), 134 (15), 133 (12), 122 (22), 120 (18), 119 (12), 109 (42), 107
22), 104 (15), 93 (21), 92 (22), 81 (49), 77 (36), 66 (63), 53 (40), 44 (100). Anal. Calcd for
: C, 56.75; H, 5.44; N, 12.61. Found: C, 59.86; H, 5.49; N, 12.54.
(
1
(
21 24 4 7
C H N O
4
.1.4.2. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carboxamide (6ad').
1
White solid, yield 69%, mp 150–151 °C. H NMR (DMSO-d
6
): δ 3.64 (s, 6H, OCH
3
-3',5'), 3.67 (s, 3H,
OCH
3
-4'), 3.76 (s, 3H, OCH
3
-4''), 6.70 (s, 2H, H-2',6'), 6.93 (d, J = 8.6 Hz, 2H, H-3'',5''), 7.29 (d, J =
+
). EIMS m/z 384 [M] (30), 356 (10), 341 (39), 313
8
.6 Hz, 2H, H-2'',6''), 7.45 and 7.88 (s, 2H, CONH
2
(
30), 298 (32), 283 (6), 270 (6), 255 (16), 240 (9), 212 (6), 209 (8), 194 (29), 168 (21), 166 (8), 152 (80,
2
0

1
4
4
46 (26), 140 (11), 137 (15), 134 (26), 119 (15), 109 (25), 100 (11), 91 (21), 81 (27), 77 (25), 66 (44),
4 (100). Anal. Calcd for C19 : C, 59.37; H, 5.24; N, 14.58. Found: C, 59.46; H, 5.28; N, 14.52.
.1.5. General procedure for the synthesis of 1,5-diaryl-1,2,3-triazole-4-carboxylates (7)
20 4 5
H N O
5
2
Prepared according to published procedure.
4
.1.5.1. Methyl 5-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carboxylate
1
7ad'). White solid, yield 93%, mp 145–147 °C. H NMR (DMSO-d
(
6
): δ 3.64 (s, 6H, OCH
3
-3',5'), 3.68
(
s, 3H, OCH
3
-4'), 3.76 (s, 3H, OCH
3
-4''), 3.77 (s, 3H, CO
2
CH
3
), 6.73 (s, 2H, H-2',6'), 6.97 (d, J = 8.8
+
Hz, 2H, H-3'',5''), 7.32 (d, J = 8.8 Hz, 2H, H-2'',6''). EIMS m/z 399 [M] (42), 371 (60), 356 (100), 340
(
11), 328 (24), 312 (28), 298 (13), 297 (14), 285 (11), 282 (20), 270 (18), 254 (29), 209 (10), 204 (10),
95 (35), 194 (21), 170 (10), 166 (11), 156 (11), 152 (14), 146 (14), 140 (10), 137 (19), 135 (45), 121
20), 119 (23), 109 (29), 107 (17), 102 (11), 92 (18), 81 (42), 77 (39). Anal. Calcd for C20 : C,
1
(
21 3 6
H N O
6
4
0.14; H, 5.30; N, 10.52. Found: C, 60.22; H, 5.34; N, 10.48.
.1.5.2. Ethyl 1-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carboxylate (7a'd).
1
White solid, yield 78%, mp 145–147 °C (EtOH). H NMR (DMSO-d
6
): δ 1.17 (t, J = 7.1 Hz, 3H,
CH
3
CH
2
), 3.63 (s, 6H, OCH
3
-3'',5''), 3.67 (s, 3H, OCH
3
-4''), 3.78 (s, 3H, OCH -4'), 4.24 (k, J = 7.1 Hz,
3
2
H, OCH
2
CH
3
), 6.71 (s, 2H, H-2'',6''), 7.03 (d, J = 8.9 Hz, 2H, H-3',5'), 7.37 (d, J = 8.9 Hz, 2H, H-2',6').
+
EIMS m/z 413 [M] (25), 385 (16), 370 (68), 312 (15), 282 (11), 254 (12), 239 (8), 224 (13), 195 (20),
1
93 (10), 183 (15), 179 (14), 170 (11), 165 (16), 163 (12), 150 (53), 135 (28), 127 (9), 121 (14), 107
(
23 3 6
24), 104 (17), 92 (72), 90 (16), 77 (100). Anal. Calcd for C21H N O : C, 61.01; H, 5.61; N, 10.16.
Found: C, 61.14; H, 5.69; N, 10.01.
4
8
2
.1.5.3. Ethyl 1-(3-chlorophenyl)-5-phenyl-1,2,3-triazole-4-carboxylate (7fg'). White solid, yield
1
5%, mp 135–137 °C. H NMR (DMSO-d
6 3 2
): δ 1.14 (t, J = 7.1 Hz, 3H, CH CH O), 4.22 (k, J = 7.1 Hz,
H, OCH CH ), 7.35 (dd, J = 8.1, 2.0 Hz, 1H, H-4'), 7.39 (d, J = 6.5 Hz, 2H, H-2'',6''), 7.41 (t, J = 6.5
2
3
Hz, 2H, H-3'', 5''), 7.44 (t, J = 6.5 Hz, 1H, H-4''), 7.49 (t, J = 8.1 Hz, 1H, H-5'), 7.57 (t, J = 2.0 Hz, 1H,
+
H-2'), 7.58 (dd, J = 8.1, 2.0 Hz, 1H, H-6'). EIMS m/z 329 [M+2] (12), 327 [M] (34), 302 (4), 300 (2),
+
2
1

2
99 (12), 271 (5), 255 (8), 229 (18), 227 (27), 226 (36), 214 (33), 208 (11), 165 (12), 154 (16), 145
47), 129 (14), 118 (61), 113 (12), 111 (30), 105 (33), 89 (10), 77 (13), 75 (28). Anal. Calcd for
: C, 62.30; H, 4.31; Cl, 10.82; N, 12.82. Found: C, 62.42; H, 4.35 Cl, 10.78; N, 12.76.
(
17 3 2
C H14ClN O
4
.1.5.4. Methyl 1-(7-methoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-1,2,3-triazole-4-
1
carboxylate (7bd'). White solid, yield 97%, mp 165–167 °C. H NMR (DMSO-d
6
): δ 3.72 (s, 3H,
OCH
3
-7'), 3.76 (s, 3H, OCH
3
-4''), 3.78 (s, 3H, COOCH
3
), 6.07 (s, 2H, OCH
2
O), 6.68 (d, J = 1.9 Hz, 1H,
H-6'), 6.77 (d, J = 1.9 Hz, 1H, H-4'), 6.96 (d, J = 8.8 Hz, 2H, H-3'',5''), 7.31 (d, J = 8.8 Hz, 2H, H-2'',6'').
+
EIMS m/z 383 [M] (28), 355 (64), 340 (29), 312 (18), 296 (90), 281 (19), 266 (21), 254 (12), 251 (8),
2
38 (7), 236 (6), 225 (12), 208 (10), 204 (14), 195 (10), 193 (98), 179 (100), 167 (10), 164 (12), 152
(
31), 151 (31), 148 (31), 145 (15), 135 (69), 133 (20), 123 (32), 121 (51), 119 (85), 116 (25), 102 (23),
9
4
4
5 (78), 93 (39), 78 (70). Anal. Calcd for C19
.49; N, 11.15
17 3 6
H N O : C, 59.53; H, 4.47; N, 10.96. Found: C, 59.78; H,
.1.5.5. Methyl 1-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-1,2,3-triazole-4-
carboxylate (7cd'). This was used without further purification.
4
.1.6. General procedure for the synthesis of 1,5-diaryl-1,2,3-triazole-4-carboxylic acids (8)
A mixture of the corresponding ester (2.5 mmol) and KOH in EtOH (15%, 2.5 mL) was stirred
overnight at rt, and EtOH was removed. The residue was dissolved in water (20 mL) and acidified with
HCl (~18%) to pH 5–6. The resulting precipitate was filtered off and washed with water to neutral pH.
4
.1.6.1. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carboxylic acid (8ad').
1
White solid, yield 94%, mp 175–177 °C (decomp.). H NMR (DMSO-d
6
): δ 3.64 (s, 6H, OCH
3
-3',5'),
3
7
.67 (s, 3H, OCH
3
-4'), 3.79 (s, 3H, OCH
3
-4''), 6.72 (s, 2H, H-2',6'), 6.96 (d, J = 8.8 Hz, 2H, H-3'',5''),
+
H). EIMS m/z 385 [M] (1), 357 (1), 341 (10), 313
.31 (d, J = 8.8 Hz, 2H, H-2'',6''), 12.97 (s, 1H, CO
2
(
36), 298 (100), 283 (16), 270 (10), 255 (25), 240 (22), 212 (12), 210 (35), 179 (17), 175 (22), 152 (14),
1
7
47 (78), 137 (22), 135 (21), 119 (41), 109 (34), 107 (16), 103 (11), 92 (20), 89 (15), 81 (48), 79 (20),
7 (42). Anal. Calcd for C19 : C, 59.22; H, 4.97; N, 10.90. Found: C, 59.28; H, 5.01; N, 10.85.
19 3 6
H N O
2
2

4
.1.6.2. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-carboxylic acid (8da').
1
White solid, yield 96%, mp 192–195 °C (decomp.). H NMR (DMSO-d
6
): δ 3.62 (s, 6H, OCH
3
-3'',5''),
3
7
.67 (s, 3H, OCH
3
-4''), 3.78 (s, 3H, OCH
3
-4'), 6.70 (s, 2H, H-2'',6''), 7.03 (d, J = 8.9 Hz, 2H, H-3',5'),
+
.36 (d, J = 8.9 Hz, 2H, H-2',6'), 13.02 (s, 1H, COOH). EIMS m/z 385 [M] (3), 342 (12), 313 (15), 298
(
100), 270 (10), 255 (11), 240 (13), 226 (11), 224 (10), 212 (11), 193 (14), 184 (18), 169 (10), 165 (22),
63 (14), 150 (25), 146 (12), 141 (16), 135 (30), 134 (18), 122 (14), 120 (13), 119 (12), 107 (27), 104
11), 92 (76), 79 (22), 77 (75). Anal. Calcd for C19 : C, 59.22; H, 4.97; N, 10.90. Found: C,
1
(
19 3 6
H N O
5
4
9.28; H, 5.02; N, 10.85.
.1.6.3. 1-(3-Chlorophenyl)-5-phenyl-1,2,3-triazole-4-carboxylic acid (8fg'). White solid, yield 95%,
1
mp 165–167 °C (decomp.). H NMR (DMSO-d
6
): δ 7.33 (d, J = 8.0 Hz, 1H, H-4'), 7.38 (d, J = 7.4 Hz,
2
H, H-2'',6''), 7.38 (t, J = 7.4 Hz, 2H, H-3'', 5''), 7.43 (t, J = 7.4 Hz, 1H, H-4''), 7.48 (t, J = 8.0 Hz, 1H,
+
H-5'), 7.55 (s, 1H, H-2'), 7.56 (d, J = 8.0 Hz, 1H, H-6'),13.12 (s, 1H, COOH). EIMS m/z 301 [M+2]
+
4), 299 [M] (10), 273 (5), 271 (14), 257 (8), 255 (23), 229 (37), 227 (100), 214 (22), 208 (13), 192
(
(
45), 190 (27), 165 (61), 163 (16), 154 (17), 150 (14), 145 (40), 124 (11), 118 (26), 117 (20), 116 (32),
13 (11), 111 (53), 105 (22), 102 (11), 90 (33), 89 (81), 76 (31), 75 (69). Anal. Calcd for
: C, 60.11; H, 3.36; Cl, 11.83; N, 14.02. Found: C, 60.16; H, 3.39; Cl, 11.79; N, 13.97.
1
15 3 2
C H10ClN O
4
.1.6.4. 1-(7-Methoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carboxylic
1
acid (8bd'). White solid, yield 80%, mp 196–198 °C (decomp.). H NMR (DMSO-d
6
): δ 3.71 (s, 3H,
OCH
Hz, 1H, H-4'), 6.95 (d, J = 8.7 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.7 Hz, 2H, H-2'',6''), 12.95 (s, 1H,
COOH). EIMS m/z 325 [M-CO ] (15), 297 (84), 282 (91), 226 (10), 194 (31), 164 (39), 151 (16), 148
17), 147 (17), 146 (19), 136 (26), 119 (24), 95 (44), 93 (21), 91 (22), 78 (37), 63 (32), 50 (45), 44
100). Anal. Calcd for C18 : C, 58.54; H, 4.09; N, 11.38. Found: C, 58.56; H, 3.90; N, 11.22.
.1.6.5. 1-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-1,2,3-triazole-4-
carboxylic acid (8cd'). White solid, yield 76% (on 2 steps starting from azide 4c), mp 189–191 °C
3 3 2
-7'), 3.77 (s, 3H, OCH -4''), 6.07 (s, 2H, OCH O), 6.67 (d, J = 1.6 Hz, 1H, H-6'), 6.75 (d, J = 1.6
2
(
(
15 3 6
H N O
4
2
3

1
(
decomp.). H NMR (DMSO-d
'), 6.09 (s, 2H, OCH O), 6.94 (d, J = 8.7 Hz, 2H, H-3'',5''), 6.99 (s, 1H, H-6'), 7.28 (d, J = 8.7 Hz, 2H,
H-2'',6''), 12.96 (s, 1H, COOH). EIMS m/z 355 [M-CO ] (26), 327 (60), 312 (90), 297 (15), 281 (16),
69 (9), 254 (8), 281 (16), 269 (9), 254 (8), 252 (8), 241 (6), 224 (11), 194 (13), 166 (90), 156 (13), 147
23), 146 (28), 135 (23), 121 (14), 119 (20), 106 (17), 95 (13), 93 (26), 91 (20), 89 (18), 77 (16), 65
33), 63 (20), 53 (77), 44 (100). Anal. Calcd for C19 : C, 57.14; H, 4.29; N, 10.52. Found: C,
6 3 3 3
): δ 3.46 (s, 3H, OCH -4'), 3.76 (s, 3H, OCH -4''), 3.78 (s, 3H, OCH -
7
2
2
2
(
(
17 3 7
H N O
5
6.88; H, 4.06; N, 10.84.
.1.7. General procedure for the synthesis of 1,5-diaryl-1,2,3-triazole-4-amines (9)
,5-Diaryl-1,2,3-triazole-4-amines 9 were prepared according to General procedure for the
synthesis of arylamines 3, 9 (see above).
4
1
4
.1.7.1. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-amine (9ad'). White
1
solid, yield 68%, mp 172–174 °C. H NMR (DMSO-d
6
): δ 3.63 (s, 6H, OCH
3
-3',5'), 3.68 (s, 3H, OCH
3
-
4
7
'), 3.76 (s, 3H, OCH
3
-4''), 4.84 (s, 2H, NH
2
), 6.60 (s, 2H, H-2',6'), 6.96 (d, J = 8.8 Hz, 2H, H-3'',5''),
+
.18 (d, J = 8.8 Hz, 2H, H-2'',6''). EIMS m/z 356 [M] (15), 328 (77), 313 (49), 300 (15), 298 (26), 297
(
83), 282 (11), 270 (12), 255 (15), 227 (11), 221 (13), 194 (31), 180 (26), 179 (23), 178 (18), 164 (12),
63 (21), 161 (20), 152 (14), 148 (11), 137 (18), 134 (100), 109 (30), 107 (13), 105 (14), 94 (19), 81
26), 77 (39). Anal. Calcd for C18 : C, 60.66; H, 5.66; N, 15.72. Found: C, 60.54; H, 5.61; N,
1
(
20 4 4
H N O
1
4
5.83.
.1.7.2. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole-4-amine (9da'). White
1
solid, yield 65%, mp 170–172 °C. H NMR (DMSO-d
6
): δ 3.61 (s, 6H, OCH
3
-3'',5''), 3.65 (s, 3H,
OCH
3
-4''), 3.79 (s, 3H, OCH
3
-4'), 4.98 (s, 2H, NH ), 6.43 (s, 2H, H-2'',6''), 7.03 (d, J = 8.9 Hz, 2H, H-
2
+
',5'), 7.28 (d, J = 8.9 Hz, 2H, H-2',6'). EIMS m/z 356 [M] (5), 328 (25), 313 (25), 300 (9), 270 (5), 239
3
(
5), 194 (25), 180 (18), 161 (23), 135 (77), 134 (100), 120 (19), 105 (31), 92 (53), 77 (64). Anal. Calcd
: C, 60.66; H, 5.66; N, 15.72. Found: C, 60.88; H, 5.62; N, 15.55.
20 4 4
for C18H N O
2
4

4
1
2
1
2
.1.7.3. 1-(3-Chlorophenyl)-5-phenyl-1,2,3-triazole-4-amine (9fg'). White solid, yield 41%, mp 107–
1
10 °C. H NMR (DMSO-d
6
): δ 5.05 (s, 2H, NH
2
), 7.22 (d, J = 7.0 Hz, 2H, H-2'',6''), 7.24 (dd, J = 7.9,
.0 Hz, 1H, H-4'), 7.34 (t, J = 7.0 Hz, 1H, H-4''), 7.39 (t, J = 7.0 Hz, 2H, H-3'', 5''), 7.45 (t, J = 2.0 Hz,
+
H, H-2'), 7.48 (t, J = 7.9 Hz, 1H, H-5'), 7.53 (dd, J = 7.9, 2.0 Hz, 1H, H-6'). EIMS m/z 272 [M+2] (2),
+
70 [M] (8), 244 (4), 242 (14), 227 (1), 216 (7), 214 (21), 207 (100), 180 (9), 165 (17), 141 (20), 140
(
29), 139 (64), 138 (74), 113 (19), 111 (59), 104 (86), 89 (19), 77 (63), 75 (81). Anal. Calcd for
: C, 62.11; H, 4.10; Cl, 13.10; N, 20.70. Found: C, 62.02; H, 4.06; Cl, 13.03; N, 20.83.
14 4
C H11ClN
4
.1.8. General procedure for the synthesis of 1,5-diaryl-1,2,3-triazoles (10)
The corresponding acid 8 (1 mmol) was heated slightly above its melting point until the gas
evolution ceased (1–2 min), then cooled to yield pure 1,2,3-triazoles.
4
.1.8.1. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1,2,3-triazole (10ad'). White solid, yield
3
1
1
9
6 3
8%, mp 125–127 °C (lit. 126 °C). H NMR (DMSO-d ): δ 3.68 (s, 6H, OCH -3',5'), 3.72 (s, 3H,
OCH
3
-4'), 3.76 (s, 3H, OCH
3
-4''), 6.74 (s, 2H, H-2',6'), 6.98 (d, J = 8.8 Hz, 2H, H-3'',5''), 7.27 (d, J =
+
.8 Hz, 2H, H-2'',6''), 8.04 (s, 1H, H-4). EIMS m/z 341 [M] (68), 313 (81), 298 (100), 282 (12), 255
8
(
30), 240 (24), 212 (23), 152 (14), 146 (63), 137 (15), 122 (11), 120 (11), 109 (23), 103 (11), 91 (26),
8
5
4
1
9 (17), 81 (31), 77 (27). Anal. Calcd for C18
.58; N, 12.39.
19 3 4
H N O : C, 63.33; H, 5.61; N, 12.31. Found: C, 63.24; H,
.1.8.2. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole (10da'). White solid, yield
1
00%, mp 145–147 °C. H NMR (DMSO-d
6
): δ 3.61 (s, 6H, OCH
3
-3'',5''), 3.66 (s, 3H, OCH
3
-4''), 3.82
(
s, 3H, OCH
3
-4'), 6.57 (s, 2H, H-2'',6''), 7.10 (d, J = 8.8 Hz, 2H, H-3',5'), 7.38 (d, J = 8.8 Hz, 2H, H-
+
',6'), 8.12 (s, 1H, H-4). EIMS m/z 341 [M] (33), 313 (9), 298 (100), 270 (7), 240 (9), 226 (8), 206 (5),
2
1
1
4
84 (10), 146 (7), 107 (7), 92 (25), 79 (16), 77 (31). Anal. Calcd for C18
2.31. Found: C, 63.27; H, 5.56; N, 12.37.
19 3 4
H N O : C, 63.33; H, 5.61; N,
.1.8.3. 1-(7-Methoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-1,2,3-triazole (10bd'). White
1
solid, yield 89%, mp 112–114 °C. H NMR (DMSO-d
6
): δ 3.76 (s, 6H, OCH
3
-7',4''), 6.11 (s, 2H,
2
5

OCH
2
O), 6.67 (d, J = 1.9 Hz, 1H, H-6'), 6.78 (d, J = 1.9 Hz, 1H, H-4'), 6.98 (d, J = 8.8 Hz, 2H, H-
+
'',5''), 7.26 (d, J = 8.8 Hz, 2H, H-2'',6''), 8.01 (s, 1H, H-4). EIMS m/z 325 [M] (52), 297 (65), 282
3
(
(
100), 254 (11), 224 (11), 151 (30), 146 (47), 120 (13), 103 (15), 95 (56), 93 (20), 91 (37), 89 (26), 78
34), 77 (27). Anal. Calcd for C17 : C, 62.76; H, 4.65; N, 12.92. Found: C, 62.83; H, 4.68; N,
15 3 4
H N O
1
2.85.
4
.1.8.4. 1-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-1,2,3-triazole (10cd').
1
White solid, yield 76%, mp 125–127 °C. H NMR (DMSO-d
6
): δ 3.39 (s, 3H, OCH
3
-4'), 3.75 (s, 3H,
OCH
3
-4''), 3.81 (s, 3H, OCH
3
-7'), 6.13 (s, 2H, OCH
2
O), 6.93 (s, 1H, H-6'), 6.98 (d, J = 8.8 Hz, 2H, H-
+
'',5''), 7.26 (d, J = 8.8 Hz, 2H, H-2'',6''), 8.03 (s, 1H, H-4). EIMS m/z 355 [M] (70), 327 (59), 312
3
(
100), 297 (11), 281 (24), 269 (14), 268 (11), 252 (10), 243 (10), 182 (6), 156 (21), 146 (54), 135 (20),
21 (19), 117 (11), 106 (20), 103 (15), 95 (19), 93 (32), 91 (36), 89 (35), 77 (41). Anal. Calcd for
: C, 60.84; H, 4.82; N, 11.83. Found: C, 60.97; H, 4.87; N, 11.73.
.1.8.5. 1-(3-Chlorophenyl)-5-phenyl-1,2,3-triazole (10fg'). White solid, yield 99%, mp 75–77 °C. H
NMR (DMSO-d ): δ 7.32 (m, 2H, H-2'',6''), 7.37 (d, J = 7.9 Hz, 1H, H-4'), 7.43 (m, 3H, H-3'',4'',5''),
.55 (t, J = 7.9 Hz, 1H, H-5'), 7.61 (s, 1H, H-2'), 7.63 (d, J = 7.9 Hz, 1H, H-6'), 8.15 (s, 1H, H-4).
1
18 17 3 5
C H N O
1
4
6
7
+
+
EIMS m/z 257 [M+2] (19), 255 [M] (58), 229 (38), 227 (100), 192 (36), 165 (43), 116 (14), 113 (12),
11 (43), 102 (12), 90 (25), 89 (61), 76 (25), 75 (59). Anal. Calcd for C14 : C, 65.76; H, 3.94;
Cl, 13.86; N, 16.43. Found: C, 65.68; H, 3.92; Cl, 13.79; N, 16.57.
.1.9. General procedure for the synthesis of arylalkynes (11)
CO (4.15 g, 30 mmol) was added to the stirred solution of corresponding benzaldehyde (15
mmol) and 1-diazo-2-oxopropylphosphonate (3.69 g, 18 mmol) in dry methanol (55 mL) at 0°С (ice-
1
H10ClN
3
4
K
2
3
7
0
water bath). The reaction mixture was stirred overnight in ice-water bath, then methanol was
evaporated, and the residue was dissolved in 15 mL of cold water. The precipitate was filtered off,
washed with water, and dried in vacuo to yield arylalkyne as colorless crystals. The targeted arylalkynes
1
1 were used without further purification.
2
6

3
.1.9.1. 5-Ethynyl-1,2,3-trimethoxybenzene (11a). White solid, yield 99%, mp 68–70 °C (lit. 68–70
1
4
1
C). H NMR (CDCl
°
3
3
): δ 3.03 (s, 1H, CH), 3.86 (s, 9H, 3 x OCH ), 6.73 (s, 2H, H-4,6). EIMS m/z 192
+
[
M] (100), 177 (58), 162 (3), 149 (26), 134 (25), 119 (16), 117 (15), 106 (8), 89 (9), 75 (9), 63 (29).
Anal. Calcd for C11 : C, 68.74; H, 6.29. Found: C, 68.60; H, 6.23.
.1.9.2. 6-Ethynyl-4-methoxy-1,3-benzodioxole (11b). White solid, yield 91%, mp 63–64 °C. H
12 3
H O
71
1
4
NMR (CDCl
3
): δ 2.96 (s, 1H, CH), 3.88 (s, 3H, OCH
3
2
), 5.98 (s, 2H, OCH O), 6.66 (d, J = 1.4 Hz, 1H,
+
H-5), 6.70 (d, J = 1.4 Hz, 1H, H-7). EIMS m/z 176 [M] (100), 161 (7), 131 (30), 103 (13), 75 (44), 63
(
13). Anal. Calcd for C10
8 3
H O : C, 68.18; H, 4.58. Found: C, 68.32; H, 4.65.
1
.1.9.3. 5-Ethynyl-4,7-dimethoxy-1,3-benzodioxole (11c). White solid, yield 90%, mp 54–56 °C. H
4
NMR (CDCl
3
3 3 2
): δ 3.20 (s, 1H, CH), 3.85 (s, 3H, OCH -4), 4.00 (s, 3H, OCH -7), 6.00 (s, 2H, OCH O),
+
.64 (s, 1H, H-5). EIMS m/z 206 [M] (100), 191 (60), 161 (13), 135 (13), 133 (22), 118 (10), 107 (23),
6
1
4
4
05 (27), 103 (11), 101 (15), 92 (38), 90 (26), 83 (23), 77 (67). Anal. Calcd for C11
.89. Found: C, 63.94; H, 4.81.
10 4
H O : C, 64.07; H,
.1.10. General procedure for the synthesis of 1,2-diarylalkynes (12)
3 4
Diethylamine (14.5 mL) was added with syringe to the mixture of (PPh ) Pd (0.168 g, 0.146
mmol), CuI (0.055 g, 0.291 mmol), 4-iodoanizole (3.41 g, 14.57 mmol), and arylalkyne (14.57 mmol)
under Ar. The solution was stirred at 50 °C for 2 h and overnight at rt, then diethylamine was
evaporated, and the residue was diluted with water. Diarylalkynes 12ad' and 12bd' as crude solid
deposits were filtered off, washed with water, and dried in vacuo, yielding pale yellow products. Oily
1
2cd' was extracted from the mixture with ethyl acetate, dried over MgSO
under reduced pressure, and the residue was dried in vacuo, yielding pale yellow diarylalkyne.
2ad', 12bd', and 12cd' were purified by flash chromatography on silica gel (toluene–heptane–
ethylacetate = 1:1:0.4) and crystallized from methanol to afford colorless crystals.
.1.10.1. 1,2,3-Trimethoxy-5-(4-methoxyphenylethynyl)benzene (12ad'). Сolorless solid, yield 97%,
4
, the solvent was removed
1
4
7
mp 114–115 °C (lit. 119–121 °C). H NMR (CDCl
2
1
3
3 3
): δ 3.83 (s, 3H, OCH -4), 3.87 (s, 3H, OCH -4'),
2
7

3
2
.88 (s, 6H, 2 x OCH
3
-3,5), 6.76 (s, 2H, H-2,6), 6.88 (d, J = 8.8 Hz, 2H, H-3',5'), 7.47 (d, J = 8.8 Hz,
+
H, H-2',6'). EIMS m/z 298 [M] (100), 283 (77), 268 (2), 255 (16), 240 (8), 225 (16), 223 (11), 212
(
(
12), 195 (9), 169 (21), 149 (27), 134 (18), 126 (19), 119 (15), 113 (14), 91 (8), 85 (19), 69 (29), 63
10). Anal. Calcd for C18 : C, 72.47; H, 6.08. Found: C, 72.34; H, 6.00.
.1.10.2. 4-Methoxy-6-[(4-methoxyphenyl)ethynyl]-1,3-benzodioxole (12bd'). Сolorless solid, yield
18 4
H O
4
1
8%, mp 88–90 °C. H NMR (CDCl
8
3
3 3
): δ 3.83 (s, 3H, OCH -4'), 3.92 (s, 3H, OCH -4), 5.99 (s, 2H,
OCH
.44 (d, J = 8.9 Hz, 2H, H-2',6'). EIMS m/z 282 [M] (100), 267 (38), 239 (14), 237 (10), 181 (9), 166
18), 152 (15), 150 (16), 141 (36), 138 (33), 113 (18), 91 (19), 87 (17), 86 (17), 75 (15), 63 (16). Anal.
Calcd for C17 : C, 72.33; H, 5.00. Found: C, 72.49; H, 5.07.
.1.10.3. 4,7-Dimethoxy-5-[(4-methoxyphenyl)ethynyl]-1,3-benzodioxole (12cd'). Colorless solid,
2
O), 6.69 (d, J = 1.4 Hz, 1H, H-5), 6.73 (d, J = 1.4 Hz, 1H, H-7), 6.87 (d, J = 8.9 Hz, 2H, H-3',5'),
+
7
(
14 4
H O
4
1
yield 92%, mp 58–60 °C. H NMR (CDCl
3
3 3
): δ 3.82 (s, 3H, OCH -4'), 3.86 (s, 3H, OCH -7), 4.03 (s,
3
H, OCH
3
-4), 6.00 (s, 2H, OCH
2
O), 6.66 (s, 1H, H-6), 6.86 (d, J = 8.8 Hz, 2H, H-3',5'), 7.46 (d, J = 8.8
+
Hz, 2H, H-2',6'). EIMS m/z 312 [M] (100), 297 (23), 282 (3), 267 (4), 239 (7), 198 (16), 183 (9), 171
(
10), 156 (31), 151 (11), 147 (7), 113 (6), 82 (13), 63 (13). Anal. Calcd for C18
Found: C, 69.09; H, 5.10.
.1.11. General procedure for the synthesis of isomer mixture of 1-benzyl-4(5),5(4)-diaryl-1,2,3-
triazoles (13)
For the procedure a barostat with the capacity of 40 tons and operating temperatures up to 200
16 5
H O : C, 69.22; H, 5.16.
4
7
C was used. A solution of 1,2-diarylalkyne (2 mmol) and benzylazide (0.346 g, 2.60 mmol) in DMF
3
°
(
1 mL) was poured into a Teflon vial and inserted into barostat thick-walled steel cylinder. The reaction
mixture was heated at 100 °C for 7 h under 15 kbar, and then was cooled to rt overnight. Solvent was
evaporated in vacuo (20 °C, 0.1 mbar). The residue was purified by column chromatography on silica
gel (eluent toluene–heptane–ethylacetate = 2:1:1) to yield a mixture of isomers in ratio ~1:1 to 1:1.5 as
a colorless viscous oil, which was used for the next hydrogenation step.
2
8

4
.1.11.1. Mixture of 1-benzyl-4-(4-methoxyphenyl)-5-(-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole
(
13a'd) and 1-benzyl-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (13ad').
Colorless viscous oil, yield 88%. Ratio of isomers: 13ad' : 13a'd = 1 : 1.3. In a representative
experiment, the reaction mixture (1-benzyl-4(5)-(4-methoxyphenyl)-5(4)-(3,4,5-trimethoxyphenyl)-1H-
1
,2,3-triazole) was separated using column chromatography to yield a single isomer. Its structure was
determined unequivocally using NOESY experiment. Specifically, we observed an inter-space
1
with protons H-2''',6''' allowing to assign the structure 13a'd. The H NMR
interaction of the NCH
2
signals of isomers in other mixtures were assigned in a similar way.
4
.1.11.2. 1-Benzyl-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (13ad').
1
Colorless viscous oil, yield 38%. H NMR (CDCl
3
3 3
): δ 3.65 (s, 6H, 2 × OCH -3'',5''), 3.81 (s, 3H, OCH -
4
3
4
'''), 3.85 (s, 3H, OCH
3
-4''), 5.40 (s, 2H, NCH ), 6.84 (s, 2H, H-2'',6''), 6.95 (d, J = 8.7 Hz, 2H, H-
2
''',5'''), 7.06 (d, J = 7.5 Hz, 2H, H-2',6'), 7.08 (d, J = 8.7 Hz, 2H, H-2''',6'''), 7.28 (m, 3H, H-3',4',5').
.1.11.3. 1-Benzyl-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (13a'd).
1
White solid, yield 50%, mp 136–138 °C. H NMR (CDCl
3
): δ 3.61 (s, 6H, 2 × OCH
), 6.26 (s, 2H, H-2''',6'''), 6.83 (d, J = 8.7 Hz,
H, H-3'',5''), 7.07 (d, J = 7.5 Hz, 2H, H-2',6'), 7.28 (m, 3H, H-3',4',5'), 7.56 (d, J = 8.7 Hz, 2H, H-
3
-3''',5'''), 3.79 (s,
3
2
2
3
2
1
2
3 3 2
H, OCH -4''), 3.92 (s, 3H, OCH -4'''), 5.41 (s, 2H, NCH
1
3
'',6''). NMR С (CDCl
3
, 125.76 MHz): δ 52.12 (ОСН
3
-4'''), 55.22 (ОСН
3
-4''), 56.02 (2 × ОСН
3
-
''',5'''), 61.01 (СН N), 107.09 (С-2''',6'''), 113,89 (С-3'',5''), 123.01 (С-1''), 123.50 (С-1'''), 127.28 (C-
2
',6'), 127.80 (C-3',5'), 128.07 (C-4'), 128.73 (С-2'',6''), 132.93 (C-4), 136.02 (C-1'), 138.76 (C-5),
+
44.16 (C-4'''), 153.61 (C-3''',5'''), 159.23 (C-4''). EIMS m/z 431 [M] (17), 340 (1), 313 (20), 312 (100),
96 (2), 282 (4), 266 (5), 251 (4), 178 (4), 140 (7), 119 (6), 91 (70), 65 (21). Anal. Calcd for
C
25
H
25
N
3
O
4
: C, 69.59; H, 5.84; N, 9.74. Found: C, 69.76; H, 5.91; N, 9.58.
.1.11.4. Mixture of 1-benzyl-5-(4-methoxyphenyl)-4-(7-methoxy-1,3-benzodioxol-5-yl)-1H-1,2,3-
triazole (13bd') and 1-benzyl-4-(4-methoxyphenyl)-5-(7-methoxy-1,3-benzodioxol-5-yl)-1H-1,2,3-
4
2
9