Triazolopyridinyl-acrylonitrile derivatives as antimicrotubule agents: Synthesis, in vitro and in silico characterization of antiproliferative activity, inhibition of tubulin polymerization and binding thermodynamics

Article abstract of DOI:10.1016/j.ejmech.2017.09.065

In this paper we report the synthesis, in vitro anticancer activity, and the experimental/computational characterization of mechanism of action of a new series of E isomers of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives (6c-g, 7d-e, 8d-e, 9c-f, 10d-e, 11d-e). All new compounds are endowed with moderate to interesting antiproliferative activity against 9 different cancer cell lines derived from solid and hematological human tumors. Fluorescence-based assays prove that these molecules interfere with tubulin polymerization. Furthermore, isothermal titration calorimetry (ITC) provides full tubulin/compound binding thermodynamics, thereby ultimately qualifying and quantifying the interactions of these molecular series with the target protein. Lastly, the analysis based on the tight coupling of in vitro and in silico modeling of the interactions between tubulin and the title compounds allows to propose a molecular rationale for their biological activity.

Full text of DOI:10.1016/j.ejmech.2017.09.065

Accepted Manuscript
Triazolopyridinyl-acrylonitrile derivatives as antimicrotubule agents: Synthesis, in
vitro and in silico characterization of antiproliferative activity, inhibition of tubulin
polymerization and binding thermodynamics
Irene Briguglio, Erik Laurini, Maria Antonietta Pirisi, Sandra Piras, Paola Corona,
Maurizio Fermeglia, Sabrina Pricl, Antonio Carta
PII:
S0223-5234(17)30780-8
10.1016/j.ejmech.2017.09.065
EJMECH 9780
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 July 2017
Revised Date: 27 September 2017
Accepted Date: 28 September 2017
Please cite this article as: I. Briguglio, E. Laurini, M.A. Pirisi, S. Piras, P. Corona, M. Fermeglia, S.
Pricl, A. Carta, Triazolopyridinyl-acrylonitrile derivatives as antimicrotubule agents: Synthesis, in vitro
and in silico characterization of antiproliferative activity, inhibition of tubulin polymerization and binding
thermodynamics, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.065.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Triazolopyridinyl-acrylonitrile derivatives as antimicrotubule agents: synthesis,
in vitro and in silico characterization of antiproliferative activity, inhibition of
tubulin polymerization and binding thermodynamics
a,1
b,c,1
a,
a
a
Irene Briguglio , Erik Laurini , Maria Antonietta Pirisi , Sandra Piras , Paola Corona , Maurizio
b,c
b,c,*
a,*
Fermeglia , Sabrina Pricl , Antonio Carta
a
Dipartimento di Chimica e Farmacia, Università degli Studi di Sassari, via Muroni 23A, 07100 Sassari, SS,
Italy
b
c
Molecular Simulation Engineering (MOSE) Laboratory, DEA, University of Trieste, Piazzale Europa 1,
3
4127 Trieste, Italy
National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-
DEA, University of Trieste, 34127 Trieste, Italy
*
1
Corresponding authors. acarta@uniss.it (A. Carta), sabrina.pricl@dia.units.it (S. Pricl).
These authors equally contributed to this work.
Abstract
In this paper we report the synthesis, in vitro anticancer activity, and the
experimental/computational characterization of mechanism of action of a new series of E isomers of
triazolo[4,5-b/c]pyridin-acrylonitrile derivatives (6c-g, 7d-e, 8d-e, 9c-f, 10d-e, 11d-e). All new
compounds are endowed with moderate to interesting antiproliferative activity against 9 different
cancer cell lines derived from solid and hematological human tumors. Fluorescence-based assays
prove that these molecules interfere with tubulin polymerization. Furthermore, isothermal titration
calorimetry (ITC) provides full tubulin/compound binding thermodynamics, thereby ultimately
qualifying and quantifying the interactions of these molecular series with the target protein. Lastly,
the analysis based on the tight coupling of in vitro and in silico modelling of the interactions
between tubulin and the title compounds allows to propose a molecular rationale for their biological
activity.
Keywords:
Triazolo[4,5-b/c]pyridin-acrylonitriles,
Antiproliferative
activity,
Tubulin
polymerization, Molecular modeling, Isothermal titration calorimetry, Fluorescence-based assays
1. Introduction
Incontrollable cell proliferation constitute the basis for malignant tumor emergence. Various
chemotherapeutic agents can reach different molecular targets affecting the proliferation process.

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Among these, structurally complex natural products such as paclitaxel, vinblastine, and vincristine
are potent microtubules-targeting drugs [1] widely used in the treatment of cancer. Microtubules are
cytoskeletal polymers that play a key role in the regulation of processes such as segregation of
chromosomes during mitosis, intracellular transport and cellular shape. Their basic structural unit is
constituted by assembled/disassembled α,β-tubulin heterodimers in a dynamic system. Specific
binding sites for inhibitors can be identified on the heterodimers [2]. Consequent to this interaction,
inhibitors suppress tubulin dynamic instability and interfere with microtubule functions, including
mitotic spindle formation. This event blocks mitosis and arrests the cell cycle in the G2/M phase,
ultimately leading to apoptosis [3].
Microtubule-targeting agents are classified into two categories: 1) molecules binding to the
vinblastine and colchicine binding site and inhibiting tubulin assembly are defined as microtubule
destabilizing agents; 2) molecules binding to the tubulin paclitaxel-binding site are defined as
microtubule-stabilizing agents [4]. Actually, colchicine and its structurally analogues are not used in
clinical settings as they are too toxic at therapeutic doses. However, synthetic compounds able to
bind to the colchicine-binding site are currently undergoing clinical development and evaluation in
patients with a wide array of cancers [5].
Recently we reported the synthesis and evaluation as antiproliferative agents of some benzotriazol-
(1)-yl-3-phenylacrylonitrile derivatives [6, 7], and demonstrated that this series of compounds act
on the G2/M phase of the cell cycle. This behavior was consistent with a possible tubulin binding
activity; accordingly, we confirmed this hypothesis using competition experiments in which the
reference
compound
(E-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(benzo[d][1,3]dioxol-5-
yl)acrylonitrile strongly displaced radio labeled colchicine from its tubulin binding site, with an IC50
value lower than colchicine itself (0.85 µM and 1.02 µM, respectively) [8].
Molecular modeling studies of the putative binding mode of this series of compounds on tubulin
further supported this finding [8]. As previously observed, substitutions with electron donor (i.e
methyl group) and electro withdrawing (i.e. chlorine) groups on the phenyl moiety of benzotriazole
did not lead to enhanced activity, unsubstituted benzotriazole remaining the most promising
scaffold. Actually, with the twofold goal of improving the antiproliferative potency and the
physicochemical properties of these derivatives, we modified the main scaffold and substituted the
benzotriazole group with a 3H-[1,2,3]triazolo[4,5-b]pyridine or a 3H-[1,2,3]triazolo[4,5-c]pyridine
moiety.
Indeed, we speculated that the steric hindrance exerted by the presence of a bulky group (e.g.,
methyl or chlorine) on the benzotriazole moiety could be more deleterious to the biological activity
of the resulting compounds than their electronic contribution. So we decided to preserve an

ACCEPTED MANUSCRIPT
electron-withdrawing group on this moiety, maintaining at the same time the steric hindrance only
on the other phenyl, to speed up the Knoevenagel condensation and to obtain E-stereoselective
synthesis, as previously observed [6-9]. For these reasons, we chose to introduce a nitrogen on the
phenyl side of benzotriazole moiety, simultaneously evaluating the relevance of its relative position.
The electron-withdrawing nitrogen in pyridine makes the deprotonated intermediate of the
Knoevenagel condensation more stable, affording exclusively the thermodynamically more stable
E-derivatives. Spectral and analytical data obtained for all new compounds are in accordance with
those of the previously described counterparts [6-9], and support the assigned chemical structure.
We finally synthesized a series of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives, maintaining the
same feature in the heterocyclic moiety, while the phenyl-moiety of the side chain was
functionalized using a variety of groups chosen from those endowed with the most antiproliferative
activity in the previous series (Br, CH , OCH , 3,4,5(OCH ) ), or totally substituted with a naphtyl
3
3
3 3
moiety, as depicted in Figure 1.
Figure 1. General chemical structures for the triazolo[4,5-b/c]pyridin-acrylonitrile derivatives synthesized in this work.
All new derivatives were synthesized following a consolidated chemistry [9]. From each
triazolopyridine intermediate (1a,b of Scheme 1), we obtained 3 series of E isomers from
substitution on each of the N atoms (N1-3) of the triazole moiety (2-4a,b of Scheme 1). All
compounds were evaluated for antiproliferative activity against a panel of cell lines derived from
hematological and solid human tumors. To investigate whether the antiproliferative activities of the
synthesized compounds were related to an interaction with tubulin, in vitro inhibition of tubulin
polymerization was determined using a fluorescence-based assay. The preliminary determination of
the dissociation constant K of the new compounds from tubulin was carried out in vitro by
d
fluorescence spectroscopy. The values of K and the tubulin binding site were then confirmed for all
d
compounds by isothermal titration calorimetry (ITC). Finally, a molecular modelling study was
carried out to draw a molecular rationale for the binding of these new series of molecules to tubulin.

ACCEPTED MANUSCRIPT
2. Chemistry
The intermediate compound 3H-[1,2,3]triazolo[4,5-b]pyridine (1a) was commercially available
while the 3H-[1,2,3]triazolo[4,5-c]pyridine (1b) was prepared in good yield via diazotation reaction
with NaNO starting from the pyridine-3,4-diamine. The 3H-[1,2,3]triazolo[4,5-b/c]pyridine-1,2,3-
2
carbonitrile derivatives (2-4a,b) were in turn prepared by nucleophilic substitution reaction of
triazolo derivatives (1a,b) with chloroacetonitrile in dimethylformamide (DMF) in the presence of
triethylamine (Et N) (Scheme 1), following a procedure reported in details in our previous work [9].
3
In particular the yields of the condensation of 1a and 1b with chloroacetonitrile afforded the
mixture of compounds 2-4a, in 78 % in the ratio 2a (24%), 3a (16%), 4a (38%); and compounds 2-
4b, in 63 % in the ratio 2b (19%), 3b (16%), 4b (28%).
Scheme 1: Synthesis of intermediate 3H-[1,2,3]triazolo[4,5-b/c]pyridine-1,2,3-carbonitrile (2-4a,b).
Finally, the synthesis of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives (6c-g, 7d-e, 8d-e, 9c-f,
10d-e, 11d-e) was accomplished by Knoevenagel condensation of intermediates (2-4a,b) with the
appropriate commercially available aldehydes (5c-g), in toluene at reflux in the presence of Et N
3
(Scheme 2) [6]. In this new series, between the two possible geometric isomers (E/Z), E-isomers
were obtained as the sole product. Spectral and analytical data obtained for all new compounds
were in accordance with those of previously described derivatives [6, 11, 12] and supported the
assigned chemical structure.

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Scheme 2: Synthesis of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives 6c-g, 7d-e, 8d-e, 9c-f, 10d-e and 11d-e.
3. In vitro cell growth inhibition assay
To characterize the tumor cell growth inhibitory profile of all new synthesized compounds 6c-g, 7d-
e, 8d-e, 9c-f, 10d-e and 11d-e, their antiproliferative activity against a panel of 9 cell lines derived
from different human tumors was evaluated (Table 1). From these data, a structure-activity
relationship could be established based on the variation of the position of the pyridine N atom, and
of the nature and position of the substituent R on the molecular scaffold (Scheme 1). As can be seen
by comparing corresponding data for (6-8) and (9-11) molecular series, the position of the pyridine
N atom only marginally affects the activity of the relevant compounds, those belonging to the 6-8
series being slightly more active than the corresponding 9-11 derivatives (e.g., compare EC (6e) =
5
0
1
0.3 - 36.8 µM and EC (9e) = 13.3 – 41.4 µM, Table 1). On the contrary, for both compound
50
series (6-8) and (9-11) the presence of the R substituent either at position 1 or 3 of the triazole ring
provides better antiproliferative activity with respect to those carrying R at position 2 in all cell
lines. Thus, taking the HeLa cell line as a proof-of-concept, the range of EC values for compound
50
6
d (0.97 µM, Table 1), featuring the substituent R = 4-CH -C H at position 1, are significantly
3 6 4
lower than those of the corresponding isomer 7d (25.1 µM, Table 1), in which the same R locates at
position 2 of the triazole ring. Also, the derivatives featuring bulkier Rs at positions 1 of
triazolo[4,5-b]pyridine and ]triazolo[4,5-c]pyridin derivatives, i.e., 2-naphtyl (6f, EC = 17.2 µM
5
0
and 9f, EC = 22.2 µM) and 3,4,5-(CH O)-C H (6g, EC = 13.8 µM) show significantly lower
50
3
6
2
50
potency with respect to all other compounds bearing less sterically hindered groups at the same sites
Table 1).
(

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Table 1. Half-maximal inhibitory concentration (EC , µM) of compounds (6-8) and (9-11) in different human cancer
50
cell lines. EC values are the means of three independent determinations.
5
0
c
f
Pos.
a
b
CCRF-
CEM
d
e
LCLC
103H
g
h
i
Cpd
R
HeLa
DU145
MCF-7
A-427
DAN-G
5637
1.9
HL-60
"
N"
0
.65
0.98
± 0.31
0.76
± 0.15
7.1
± 0.7
1.2
± 0.6
2.1
± 0.4
15.4
± 2.2
12.2
± 1.9
6
c
1
4-Br-C
H
6
4
±
0.14
± 0.6
0
±
.97
0.23
1.4
± 0.6
0.99
± 0.18
10.9
± 2.1
1.6
± 0.6
1.9
± 0.2
18.9
± 2.1
2.2
± 0.5
10.9
± 1.3
6
d
1
1
1
4-CH -C
3
6
H
4
4
-CH O-
10.3
± 1.2
12.7
± 2.2
7.4
± 0.6
25.6
± 1.3
8.9
± 0.4
10.2
± 1.0
36.8
± 2.3
12.6
± 1.8
27.9
± 2.0
3
6
e
C H
6
4
1
±
7.2
1.9
39.2
± 1.7
15.6
± 2.2
59.8
± 2.6
17.4
± 1.1
17.1
± 1.3
78.3
± 3.4
19.4
± 1.2
68.3
± 2.8
6
f
2-Naphtyl
,4,5-
3
1
±
3.8
1.4
15.8
± 1.5
11.3
± 1.6
29.1
± 1.8
11.6
± 1.0
13.8
± 1.1
40.1
± 2.8
12.5
± 1.1
37.1
± 1.6
6
g
1
(CH O)
C H
3
3
-
6
2
2
±
5.1
2.3
22.4
± 1.9
32.6
± 2.3
48.5
± 2.0
39.6
± 1.6
37.5
± 1.9
88.1
± 4.2
33.1
± 1.7
78.3
± 3.1
7
d
2
2
3
3
4-CH
3
-C
6
H
4
4
4
-CH O-
38.6
± 2.5
31.6
± 1.8
33.7
± 1.7
67.8
± 2.4
45.8
± 1.3
51.4
± 2.2
90.6
± 3.9
49.2
± 2.3
69.5
± 2.9
3
7
e
C H
6
4
3
±
.3
0.8
2.7
± 1.1
1.8
± 0.5
15.4
± 1.9
3.9
± 0.2
2.2
± 0.3
26.7
± 1.5
4.8
± 0.3
31.1
± 2.2
8
d
4-CH
3
-C
6
H
4
-CH O-
11.1
± 1.3
12.6
± 1.4
8.5
± 0.9
31.0
± 2.3
10.7
± 1.8
12.6
± 1.5
49.8
± 2.0
15.3
± 1.2
49.9
± 2.4
3
8
e
C H
6
4
c
f
Pos.
N"
a
b
CCRF-
CEM
d
e
LCLC
103H
g
h
i
Cpd
R
HeLa
DU145
MCF-7
A-427
DAN-G
5637
HL-60
"
1
±
.6
0.8
1.2
± 0.3
1.2
± 0.3
8.5
± 0.4
2.8
± 0.8
3.2
± 0.7
20.0
± 2.6
2.9
± 0.2
17.5
± 2.2
9
c
1
4-Br-C
6
H
4
2
±
.3
0.3
2.8
± 0.4
2.5
± 0.2
14.4
± 1.9
3.5
± 0.6
4.1
± 0.4
19.2
± 2.2
3.3
± 0.4
18.6
± 2.0
9
d
1
1
1
2
2
3
4-CH -C
3
6
H
4
4
-CH O-
13.1
± 1.6
13.8
± 1.9
9.9
± 0.7
29.5
± 1.8
10.1
± 1.0
12.9
± 1.1
41.4
± 2.5
17.1
± 1.5
32.9
± 2.1
3
9
e
C H
6
4
2
±
2.2
1.4
36.7
± 1.5
22.1
± 1.8
68.6
± 2.2
24.4
± 1.3
27.1
± 1.7
81.5
± 2.9
23.6
± 1.7
70.5
± 2.2
9
f
2-Naphtyl
4-CH -C
3
±
2.0
2.6
33.7
± 1.8
30.2
± 2.1
44.8
± 2.9
41.2
± 2.1
41.5
± 1.8
90.9
± 3.9
35.2
± 1.1
85.1
± 3.6
1
0d
0e
1d
1e
3
6
H
4
4
4
-CH O-
44.1
± 2.7
41.1
± 2.2
43.6
± 2.0
77.3
± 2.7
52.1
± 2.8
57.7
± 3.0
98.8
± 3.1
50.6
± 2.1
72.8
± 3.1
3
1
C H
6
4
9
±
.5
1.1
8.2
± 1.6
7.8
± 0.3
28.2
± 2.1
9.9
± 1.1
7.5
± 0.9
41.4
± 1.8
10.6
± 1.2
39.3
± 2.5
1
4-CH
3
-C
6
H
4
-CH O-
17.6
± 1.7
15.8
± 1.2
16.3
± 1.1
38.6
± 3.0
17.7
± 2.2
16.9
± 1.8
57.2
± 2.6
20.1
± 2.1
59.3
± 2.8
3
1
3
C H
6
4
j
0.027
0.018
0.021
0.061
0.045
0.070
0.059
0.053
0.0086
Colchicine
-
±
0.003 ± 0.002 ± 0.003 ± 0.005 ± 0.001 ± 0.005 ± 0.006 ± 0.009 ± 0.008
a
b
c
d
HeLa = ACC-57 (cervix carcinoma); DU145 = ACC-261 (prostate carcinoma); CCRF-CEM = ACC-240 (T-cell leukemia); MCF-
e
f
7
= ACC-115 (breast adenocarcinoma); A-427 = ACC-234 (small cell lung carcinoma); LCLC 103H = ACC-384 (large cell lung
carcinoma); DAN-G = ACC-249 (pancreas carcinoma); 5637 = ACC-35 (urinary bladder carcinoma); HL-60 = ACC-3 (acute
myeloid leukemia); Colchicine = reference compound.
g
h
i
j
Within the entire pool of synthesized molecules, compound 6c in which R = 4-Br-C H was found
6
4
to be the most potent antiproliferative agent, with EC values in the range 0.65-15.4 µM towards
5
0

ACCEPTED MANUSCRIPT
all cell lines. In detail, compound 6c inhibited the growth of HeLa cells with a half-maximal
inhibitory concentration EC = 0.65 ± 0.14 µM (Figure 2A). Similarly, inhibition of DU145 and
50
CCRF-CEM cell growth was achieved by the same compound with EC of 0.98 ± 0.31 and 0.76 ±
5
0
0.15 µM, respectively. Good antiproliferative activity for 6c was also observed against breast
(MCF-7), small (A-427)/non small (LCLC 103H) cell lung, and urinary bladder (5637) carcinoma
cell lines (Table 1), whilst moderate activity (10 -20 µM) was recorded in the case of acute myeloid
leukemia (HL-60) and pancreatic carcinoma cell lines (DAN-G, Figure 2B).
Figure 2. Compound 6c inhibition of proliferation of HeLa (A) and DAN-G (B) cells.
Nonspecific cytotoxicity of anticancer drugs may constitute a major obstacle in cancer
chemotherapy. Accordingly, we also preliminarily determined the effect of the two most promising
compounds, 6c and 6d, on the proliferation activity of three normal cell lines (i.e., human peripheral
mononuclear blood cells (HPMBC), human pulmonary fibroblasts (HPF), and human prostate
epithelial cells (HPEC), Table S1). Comparison of the relevant EC50 values with those of the
pertinent malignant cell lines (Table 1) reveals that both compounds are endowed - on average –
with substantial less normal cell cytotoxicity (28.5-35 x for HPEC, 14-17 x for HPF, and 40-55 x
for HPEC, respectively).
4. In vitro tubulin polymerization assay
To investigate whether the antiproliferative activities of compounds (6-8) and (9-11) were related to
an interaction with tubulin, they were assayed for their inhibitory effects on tubulin polymerization
(
ITP) in vitro using a fluorescence-based tubulin polymerization assay. As shown in Figure 3, the
hit compound 6c was able to inhibit the rate and extent of tubulin assembly in a concentration-
dependent manner, the half-maximal inhibition (ITP IC ) occurring at 17.2 ± 0.9 µM.
5
0
Figure 3. (A) In vitro concentration-dependent inhibition of tubulin polymerization by compound 6c (ITP IC = 17.2 ±
50
0
.9 µM). Symbol legend: ò, 0 µM; <, 5 µM; ▲, 10 µM; ø, 15 µM; ô, 20 µM; £, 30 µM; r, 40 µM. (B)

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Determination of 6c ITP IC value by sigmoidal fitting of steady state levels of tubulin assembly from panel A vs. drug
5
0
concentration. Three independent sets of experiments were performed; however, only one of the sets is shown for
clarity.
Compounds 6d was also found to inhibit tubulin assembly with an ITP IC of 23.9 ± 1.2 µM. In the
5
0
(9-11) series, only compound 9c was endowed with appreciable tubulin depolymerization activity,
with ITP IC = 29.1 ± 1.5 µM. Under similar conditions, four reference compounds - colchicine,
5
0
nocodazole, podophyllotoxin, and vinblastine sulfate - exhibited significantly more potent
microtubule depolymerization activity, with ITP IC of 1.32 ± 0.23, 0.81 ± 0.07, 0.26 ± 0.09, and
50
0.15 ± 0.07 µM, respectively. Accordingly, compounds 6c, 6d, and 9c can be classified as moderate
tubulin polymerization inhibitors. Of note, we observe that the determined concentrations required
to inhibit tubulin polymerization by these three compounds are much higher than those required for
cytotoxicity, a phenomenon however well documented in the literature [see, for instance, 13-16].
All other compounds had ITP IC values > 30 µM; as such, they were regarded to have only weak
5
0
activity as inhibitors of tubulin polymerization.
Tubulin polymerization inhibitors usually interfere with the G2/M phase of the cell cycle by altering
the dynamics of tubulin polymerization, ultimately triggering cell death via apoptotic pathways.
Therefore, we considered interesting to study the effect exerted by the two lead compounds, 6c and
6d, on cell cycle progression by flow cytometry experiments (Figure S2). The results show that
untreated (control) prostate carcinoma cells DU145, taken as a proof-of-concept, distributed in the
Sub-G1 (8.7%), G0/G1 (78%), S (3.6%), and G2/M (9.5%) phase. On the other hand, both
compounds arrested most of the cells in the G2/M phase (71.1% and 69.3% for 6c and 6d,
respectively, Figure S2). Collectively, these results are consistent with the indication that these
molecules can act as efficient mitotic blockers.

ACCEPTED MANUSCRIPT
. Preliminary determination of the dissociation constant of compounds 6c to tubulin in vitro
by fluorescence spectroscopy
5
According to the results presented above, 6c was able to prevent the assembly of tubulin with ITP
EC < 20 µM. Therefore, we performed a preliminary investigation to determine whether 6c
50
directly interacted with tubulin. Compound 6c quenched the tryptophan fluorescence of tubulin in a
concentration-dependent fashion, thereby confirming the interaction between the small molecule
and the protein (Figure 4A).
Figure 4. (A) Quenching of intrinsic tryptophan fluorescence of tubulin by different concnetrations of compound 6c.
Symbol legend:
µM. (B) Differential change in fluorescence intensity of tubulin upon binding of compound 6c. Three independent
sets of experiments were performed; however, only one of the sets is shown for clarity.
ò, 0 µM; <, 5 µM; ▲, 10 µM; ø, 15 µM; ô, 20 µM; £, 30 µM; r, 40 µM. Tubulin concentration =
2
The dissociation constant (K ) of compound 6c from tubulin - obtained by fitting the maximum
d
tubulin fluorescence intensity change as a function of compound concentration (Figure 4B) - was
found to be equal to 19.6 ± 1.0 µM. Based on this preliminary information and with the twofold
purpose of obtaining 1) accurate K values for all compounds and 2) a full thermodynamic
d
characterization of compound/tubulin interactions (indispensible for validation of the modeling
results, vide infra), further binding studies were conducted by ITC.
6. Determination of the dissociation constant and tubulin binding site of compounds (6-8)
and (9-11) by Isothermal Titration Calorimetry (ITC)
In order to investigate the tubulin binding affinities of all (6-8) and (9-11) compounds in detail,
thermodynamic studies were conducted using isothermal titration calorimetry (ITC). The averaged
dissociation constant (K ), binding free energy (∆G ), enthalpy (∆H ) and entropy terms (-T∆S )
d
b
b
b
resulting from three independent ITC measurements on each compound are listed in Table 2, while
Figure 5 displays some representative ITC results and fitting curves for compounds belonging to
both (6-8) and (9-11) series.

ACCEPTED MANUSCRIPT
Table 2. ITC determined thermodynamic data of compounds (6-8) and (9-11) binding to tubulin. ∆G = ∆H –T∆S .
b
b
b
∆
G = RT lnK . n = number of binding site. All experiments were run in triplicate. Errors on ∆H are within 5%.
b
d
b
Pos.
T∆Sb
(kcal/mol)
Cpd
R
K (µM) ∆G (kcal/mol) ∆H (kcal/mol)
n (-)
d
b
b
"
N"
6
c
1
4-Br-C
6
H
4
16.4
-6.75
-6.57
-5.24
-4.82
-5.10
-4.56
-4.40
-5.97
-5.17
-9.21
-8.96
-7.35
-6.87
-7.16
-6.81
-6.75
-7.84
-7.23
2.46
2.39
2.11
2.05
2.06
2.25
2.35
1.87
2.06
0.98
1.00
0.95
0.97
1.01
0.96
1.02
0.98
0.99
6
d
1
1
1
1
2
2
3
3
4-CH
4-CH
3
-C
6
H
4
22.0
192
385
241
581
763
58.4
217
6
e
3
O-C
6
H
4
6
f
2-Naphtyl
3,4,5-(CH O) -C
4-CH -C
4-CH O-C
4-CH -C
4-CH O-C
6
7
g
3
3
H
6 2
d
3
6
H
4
7
e
3
6
H
4
4
8
d
3
6
H
4
8
e
3
6
H
Pos.
T∆Sb
(kcal/mol)
Cpd
R
K (µM) ∆G (kcal/mol) ∆H (kcal/mol)
n (-)
d
b
b
"
N"
9
c
1
4-Br-C
4-CH
4-CH O-C
6
H
4
33.9
-6.30
-5.91
-5.09
-4.83
-4.59
-4.41
-5.29
-8.63
-7.79
-7.13
-6.91
-6.75
-6.59
-7.41
2.33
1.88
2.03
2.08
2.16
2.18
2.12
1.00
0.97
0.97
0.96
1.03
0.99
0.95
9
d
1
1
1
2
2
3
3
3
-C
6
H
4
64.5
246
376
554
749
175
9
9
e
3
6
H
4
g
2-Naphtyl
4-CH -C
4-CH O-C
4-CH -C
4-CH O-C
1
0d
0e
1d
1e
colchicine
3
6
H
4
1
3
6
H
4
4
1
3
6
H
4
1
3
6
H
283
-5.00
-6.98
1.98
1.59
0.98
1.01
0.058
-10.21
-11.80
Figure 5. Representative ITC binding isotherms for 6c (A), 6f (B), 7e (C), 9c (D), 9f (E), and 10e (F) titrations into
tubulin solutions. Panel A, insert: example of ITC raw data.

ACCEPTED MANUSCRIPT
For the two series of compounds, the interaction with tubulin resulted in an exothermic ligand-
binding event, with K values in the range 16.4 - 763 µM and 33.6 - 749 µM for the (6-8) and (9-
d
1
1) compounds, respectively. Importantly, the ITC-derived K value for compound 6c (16.4 µM) is
d
nicely in agreement with the value obtained from the corresponding tubulin fluorescence quenching
experiment (19.6 ± 1.0 µM). Generally speaking, the tubulin-compound binding is driven by the
simultaneous participation of stabilizing van der Waals and hydrophobic interactions (∆H < 0),
b
while entropy slightly disfavor complex formation (T∆S > 0). The conformational entropy change
b
is commonly unfavorable in protein-ligand binding event, as the binding process involves the loss
of configurational degrees of freedom for both the drug molecule and the protein, resulting in an
ultimate negative change in conformational entropy. However, the presence of the planar bicyclic
scaffold characterizing all present compounds confers a degree of overall molecular rigidity, which
reflects only in a confined, unfavorable entropic contribution to ligand binding.
The last row in Table 2 shows the binding thermodynamic parameters for colchicine, used as a
reference compound. The corresponding K value of 58 nM, in line with previous findings, confirm
d
that all title compounds were consistently weaker than colchicine in their interaction with tubulin.
To understand whether the cytotoxic effect of the present (6-8) and (9-11) derivatives is dependent
on their antimicrotubule activity, we next examined the relationship between the K values
d
determined by the ITC tubulin binding assay and EC50 values in the cell-based assays. Since
cytotoxicity tests were performed on 9 different human tumor cell lines, for the purpose of this
comparison a single EC50,av value, corresponding to half-maximal inhibitory concentration value
averaged over all cell lines, was employed. As shown in Figure 6, the resulting average EC vs. K
d
5
0
2
plot show a highly positive correlation (R = 0.980), suggesting a strong correlation between (6-8)
and (9-11) compounds tubulin binding and cytotoxic potency. Therefore, we could conclude that
the cytotoxic activity of both (6-8) and (9-11) derivatives is largely attributed to their
antimicrotubule action.

ACCEPTED MANUSCRIPT
Figure 6. Correlation between tubilin binding activity (K ) and average cytotoxic activity (EC50,av) of compounds (6-8)
d
(ò) and (9-11) (▲).
Microtubule-destabilizing compounds often bind tubulin by nesting into specific binding sites. In a
previous work [8] we assessed the capability of a series of related compounds to compete with
colchicine or paclitaxel for binding to tubulin by resorting to a competitive scintillation proximity
assay. According to that study [8], we found that those compounds competitively inhibited
3
3
[
H]colchicine binding to biotinylated tubulin while they did not compete with [ H]paclitaxel. In
this work, however, we resorted again to ITC to examine whether the present compounds do
compete either with colchicine or paclitaxel for the respective binding sites. With respect to the
competitive scintillation proximity assay, the ITC-based experiments offers several advantages,
including a clear determination of eventual binding site competition and, above all, to avoid
working with radioligands.
Accordingly, we investigated the binding of compound 6c to purified tubulin in the presence of
colchicine or paclitaxel using ITC. Panel A of Figure 7 shows the typical exothermic profiles
obtained for tubulin titration by 6c at 37°C either in the presence of colchicine (thick black data) or
in the presence of paclitaxel (light gray data). Figure 7 B shows the corresponding binding
isotherms, i.e. the integrated heats of reaction as a function of the 6c/tubulin molar ratio, in these
two conditions.
Figure 7. A: ITC raw data obtained by titrating 6c into a solution containing tubulin in the presence of colchicine (thick
black curve) or paclitaxel (light gray curve). B: corresponding integrated heats of reaction in the presence of colchicine
(
<) or in the presence of paclitaxel (ò).

ACCEPTED MANUSCRIPT
As can be easily seen from Figure 7B, in the presence of colchicine the tubulin binding of 6c is
completely abrogated. Colchicine is a very tight tubulin binder, with K = 58 nM (Table 2).
d
Accordingly, 6c (K = 16.4 µM) it is not able to displace colchicine from its tubulin binding site, as
d
expected. Moreover, the fact that the titration of the tubulin-colchicine complex with 6c returned a
completely flat curve is a clear indication that, upon saturation of all tubulin binding sites by
colchicine, 6c is no longer able to interact with the protein, i.e., 6c binds tubulin at the colchicine
binding site. On the contrary, the binding isotherm resulting from the titration of tubulin by 6c in
the presence of paclitaxel (Figure 6B, circles) is practically coincident with that obtained in its
absence (Figure 4A). Accordingly, 6c does not interact with tubulin by binding at the paclitaxel
binding pocket.
7. Molecular modeling rationale for the binding of compounds (6-8) and (9-11) to tubulin
In order to determine the structural basis of the biological results discussed above, the binding
ability of the synthesized compounds to the colchicine binding site of α,β-tubulin was investigated
using a consolidated molecular modeling procedure based on the combination of docking and
Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA)-based scoring techniques.
Specifically, for this analysis we considered a subset of 3 compounds for each series, namely 6c, 6f,
7e, 9c, 9f, and 10e, as representative of strong, moderate, and weak tubulin binders, respectively
(Table 2 and Figure 5). Table 3 reports the results of this in silico analysis. As seen by comparing
the entries in Tables 2 and 3, all ligands are correctly ranked for affinity, the magnitudes of the
calculated ∆Gb,calc (and of the corresponding Kd,calc) values being in excellent agreement with those
determined experimentally.
Table 3. In silico binding thermodynamics of a subset of compounds (6-8) and (9-11) on human tubulin. The calculated
value for colchicine is also reported for comparison.
Cpd Pos. "N"
R
Kd,calc (µM)
17.8
∆Gb,calc (kcal/mol)
-6.70 ± 0.27
∆Hb,calc (kcal/mol) T∆Sb,calc (kcal/mol)
6
c
1
1
2
4-Br-C
6
H
4
-23.07± 0.15
-18.88 ± 0.11
-18.09 ± 0.13
16.37 ± 0.23
14.04 ± 0.21
13.58 ± 0.19
6
f
2-Naphtyl
4-CH O-C
371
-4.84 ± 0.24
7
e
3
6
H
4
635
-4.51 ± 0.23

ACCEPTED MANUSCRIPT
Cpd Pos. "N"
R
Kd,calc (µM)
37.3
∆Gb,calc (kcal/mol)
-6.24 ± 0.19
∆Hb,calc (kcal/mol) T∆Sb,calc (kcal/mol)
9
c
1
1
2
4-Br-C
6
H
4
-22.16 ± 0.12
-18.57 ± 0.13
-17.23 ± 0.11
-24.11± 0.12
15.92± 0.15
13.83 ± 0.23
12.86 ± 0.22
13.99 ± 0.18
9
f
2-Naphtyl
4-CH O-C
436
-4.74 ± 0.26
1
0e
3
6
H
4
798
-4.37 ± 0.25
colchicine
0.067
-10.12± 0.22
It is interesting to observe that, with respect to the experimental values (Table 2), both ∆Hb,calc and –
T∆Sb,calc are substantially overestimated by the MM/PBSA calculations (Table 3); yet, since a
parallel shift is observed between experimental and computed ∆H and –T∆S, (see Figure S1) their
difference (i.e., ∆Gb,calc) is ultimately close to the relevant experimental value (∆Gb).
Concerning the binding mode of these new acrylonitrile derivatives, it has to be noticed that all
compounds are provided with the chemical functions required for mapping the 3D pharmacophore
model previously developed for tubulin inhibitors with a similar scaffold [8]. Indeed, taking the new
lead compound 6c as a meaningful example, all pharmacophore elements could be easily fitted by
the proper molecular features, as shown in Figure 8A. Specifically, the triazolopyridinyl ring
matches both the hydrophobic aromatic (HYAr) and the first hydrogen bond acceptor (HBA)
features, while the cyano nitrogen atom overlays the second HBA. Finally, the 4-Br-C H enyl
6
4
substituent maps the ring aromatic (RA) feature, presenting the aromatic ring in the proper
conformation to optimize this molecular requirement (Figure 8A).
Figure 8. A: Mapping of compound 6c onto the 3D pharmacophore model developed for structurally-related tubulin
inhibitors [8]. The 3D pharmacophoric hypothesis features are portrayed as meshed spheres, color-coded as follows:
light green, HBA; light blue, HYAr; light orange, RA. Compound 6c is shown as atom-colored sticks-and-balls (gray,
C; blue, N; dark red, Br; white, H). B: Equilibrated MD snapshots of tubulin/6c complex. The image is a zoomed view
of the binding site. The ligand is portrayed as in panel A, while the protein residues mainly involved in the interaction
with 6c are highlighted as colored sticks and labelled. H-bonds interactions are shown as dotted black lines. Hydrogen
atoms, water molecules, and ions are omitted for clarity.

ACCEPTED MANUSCRIPT
Our Molecular Dynamics (MD) simulations that allowed us to give a precise description of the
binding mode of these new compounds have confirmed this interactions pattern. In fact, its
combination with the relevant per-residue decomposition analysis of the binding enthalpy (∆Hres)
provided both the identification of the tubulin residues mainly involved in ligand binding and the
nature underlying each specific interaction (Figure 8B and Figure 9A).
Figure 9. A: Comparison of the per-residue binding enthalpy decomposition (∆H ) for compounds 6c and 7e in
res
complex with tubulin. Only those amino acids critical for the binding are shown. B: Superposition of equilibrated
binding pose of 6c (purple) and 7e (red) in the tubulin binding cavity.
As can be seen in Figure 8B, the p-bromophenyl ring of 6c is perfectly encased in the protein
binding pocket lined by residues βL242, βL248, βA250, βl252, βK254 and βL255, and the deriving
hydrophobic stabilizing interactions provided an overall contribution of -5.01 kcal/mol to the
binding (Figure 9A). Furthermore, the donor guanidinium group of βK352 on the receptor finds its
acceptor counterpart in the triazole N3 of the inhibitor, forming a persistent hydrogen bond (average
dynamic distances (ADL) = 2.1 ± 0.1 Å) which yields a corresponding stabilizing contribution of
∆Hres = -2.26 kcal/mol. The triazolepyridinyl moiety is embedded in a hydrophobic pocket
consisting of the side chains of αA180, αV181, βV257, βM259, and βV351 which all concur to
foster a network of hydrophobic interactions with the ligands (∑∆H = -2.77 kcal/mol, Figure 9A).
res
Finally, the cyano N atom is engaged in a permanent hydrogen bond with the hydroxyl group of the
side chain of βT314 (ADL = 2.3 ± 0.2 Å; ∆H = -1.98 kcal/mol).
res
The same analysis applied to the other tested compounds, allowed us to confirm the structure-
activity relationship proposed above (based on the in vitro results), and the related experimental
data. First, all compounds demonstrated similar binding modes within the same tubulin binding
pocket, the position of the substituted acrylonitrile moiety with respect to the triazolpyridinyl ring
(N1, N2, or N3) modulating their protein binding affinity. Indeed, compounds featuring

ACCEPTED MANUSCRIPT
substitutions at the N1 position are endowed with the highest tubulin affinity; yet, substitutions at
position N3 is also well tolerated, the pyridine nitrogen atom not being involved in any specific
interaction. On the other hand, compound 7e (taken as a representative example of the N2
substituted derivatives) exhibits a substantially lower affinity compared to e.g., 6c, as the
conformation it assumes in the tubulin binding site (Figure 9B) does not satisfy all molecular
determinants requested by the pharmacophore model. Specifically, the orientation of the chemical
groups of 7e involved in hydrogen bonds with tubulin residues βT314 and βK352 leads to a weaker
interactions both in terms of dynamics stability (ADL = 2.8 ± 0.1 Å and ADL = 2.7 ± 0.3 Å for
βT314 and βK352, respectively) and enthalpic contribution (∆HβT315 = -1.55 kcal/mol and
∆HβK252 = -1.77 kcal/mol, Figure 9A). This, in turn, locally reflects into a sub-optimal
encasement of 7e in both protein hydrophobic pockets (thereby resulting into a lower stabilization
energy of the protein residues directly involved in such specific interactions (Figure 9A), and
globally into a less favorable ∆Gb,calc value for this molecule (Table 3).
8. Conclusions
In this work we presented the synthesis and in vitro anticancer activity of a new series of E isomers
of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives (6c-g, 7d-e, 8d-e, 9c-f, 10d-e, 11d-e). All
compounds were found to exert moderate to interesting antiproliferative activities against 9
different cancel cell lines derived from solid and hematological human tumors. Fluorescence-based
assays proved that these molecules interfere with tubulin polymerization, and isothermal titration
calorimetry led to full tubulin/compound binding thermodynamics determination (i.e., K , ∆G ,
d
b
∆
H , and Τ∆S values). Coupling all experimental information with in silico modelling of the
b β
interactions between tubulin and the title compounds allowed to conclude that the position of the
pyridine nitrogen atom does not exert a significant influence on their binding affinity toward their
target protein. On the contrary, the position of the different R substituents on the three N atoms of
the triazole ring is the major molecular determinant for the biological activity of these molecules,
the N1 and N3 position leading to more potent compounds with respect to those presenting the
same Rs at the N2 position.
9
9
. Experimental section
.1. Synthetic methods
Melting points were determined by a Kofler hot stage or Digital Electrothermal apparatus, and are
1
13
uncorrected. H and C-NMR spectra were recorded on a Varian XL-200 and on a Bruker Avance
III 400 NanoBay instruments, using TGC-MS as internal standard. The chemical shift values are

ACCEPTED MANUSCRIPT
reported in ppm (δ) and coupling constants (J) in Hertz (Hz). Signal multiplicities are represented
by: s (singlet), d (doublet) and m (multiplet). GC/MS spectra were performed on combined HP
5790 GC/MS and on a triple quadrupole (API 2000, Applied Biosystem MDS SCIEX) apparatuses.
Column chromatography was performed using 230-400 mesh silica gel (Merck silica gel 60). Light
petroleum refers to the fraction with b.p. 40-60 °C. The analytical results for C, H, N and chlorine
were within 0.4% of the theoretical values.
9.1.1. Starting materials, intermediates and known compounds
3
H-[1,2,3]triazolo[4,5-b]pyridine (1a), 3,4-diaminopyridine, aldehydes, 2-chloroacetonitrile,
triethylamine, acetic acid, organic solvents and inorganic reagents were commercially available.
3
H-[1,2,3]triazolo[4,5-c]pyridine (1b) was prepared following a consolidated procedure [10].
1
9
.1.1.1. 3H-[1,2,3]triazolo[4,5-c]pyridine (1b). Yield: 83%. M.p.: 81 °C. H-NMR (DMSO-d ): δ
6
1
3
9
.47(s, 1H, CH-4); 8.47(d, 1H, CH-7, J = 8 Hz); 7.88 (d,1H, CH-6, J = 4.2 Hz). C-NMR (CDCl ):
3
δ 143.21 (C), 141.72 (CH), 138.56 (CH), 128.34 (C), 111.45 (CH). GC/MS: 121 (M+H). Anal.
Calcd for (C H N ): C, 50.00; H, 3.36; N, 46.65. Found: C, 49.92; H, 3.27; N, 46.72.
5
4
4
9.1.2. General procedure for the preparation of the intermediates 2a-b, 3a-b, 4a-b.
2
g (16.6 mmol) of the appropriate 3H-[1,2,3]triazolo[4,5-b/c]pyridine (1a,b) were dissolved in 20
ml of dimethylformamide (DMF). 1.95 g (19.4 mmol) 2.68 ml of triethylamine and 2 g (26.5 mmol)
.69 ml of chloroacetonitrile were added to the resultant solution. The whole mixture was heated
1
under reflux for 12 h. After cooling, the triethylamine hydrochloride formed was filtered, and the
solution was evaporated to dryness. The residue, dissolved in ether, was washes with water.
Through evaporation of the extract, a residue containing the three isomers 2-4a or 2-4b was
obtained. Isomers were separated by flash chromatography, elution with light petroleum-ethyl
acetate 80:20 afforded desired compounds.
1
9
.1.2.1. 3H-[1,2,3]triazolo[4,5-b]pyridine-3-carbonitrile (2a). Yield: 24%. M.p.: 56.8-57 °C. H-
NMR (DMSO-d ): δ 8.86 (d, 1H, CH-5. J = 4.0 Hz); 8.66 (d, 1H, CH-7. J = 8.8 Hz); 7.61 (m, 1H,
6
1
3
CH-6); 6.14 (s, 2H, CH ). C-NMR (CDCl ): δ 151.53 (CH); 144.76 (C); 136.18 (C); 129.18 (CH);
2
3
1
20.96 (CH); 114.73 (C); 34. 27 (CH ). GC/MS: 146 (M+H). Anal. Calcd for (C H N ): C, 49.66;
2 6 3 5
H, 2.08; N, 48.26. Found: C, 49.56; H, 2.19; N, 48.23.
.1.2.2. 2H-[1,2,3]triazolo[4,5-b]pyridine-2-carbonitrile (3a). Yield: 16%. M.p.: 98.6-100 °C. H-
NMR (DMSO-d ): δ 8.89 (d, 1H, CH-5, J = 4.0 Hz); 8.53 (d, 1H, CH-7, J = 8.8 Hz); 7.61-7.55 (m,
1
9
6

ACCEPTED MANUSCRIPT
1
3
1
H, CH-6); 6.34 (s, 2H, CH ). C-NMR (CDCl ): δ 155.15 (C); 153.21 (CH); 128.01 (C); 136.52
2 3
(
C); 127.57 (CH); 123.41(CH); 114.06 (C); 44.57 (CH ). GC/MS: 146 (M+H). Anal. Calcd for
2
(
C H N ): C, 49.66; H, 2.08; N, 48.26. Found: C, 49.61; H, 2.10; N, 48.18.
6
3
5
9.1,2,3. 1H-[1,2,3]triazolo[4,5-b]pyridine-1-carbonitrile (4a). Yield: 38%. M.p.: 160-160.5 °C.
1
H-NMR (DMSO-d ): δ 8.80 (d, 1H, CH-5, J = 4 Hz); 8.50 (d, 1H, CH-7, J = 8.4); 7.74-7.70 (m,
6
1
3
1
H, CH-6); 6.22 (s, 2H, CH ). C-NMR (CDCl ): δ 156.54 (C); 148.89 (CH); 125.16 (C); 123.30
2 3
(
CH); 120.12 (CH); 114.79 (C); 36.38 (CH ). GC/MS: 146 (M+H). Anal. Calcd for (C H N ): C,
2
6
3
5
4
9
9.66; H, 2.08; N, 48.26. Found: C, 49.59; H, 2.15; N, 48.24.
1
.1.2.4. 3H-[1,2,3]triazolo[4,5-c]pyridine-3-carbonitrile (2b). Yield: 19%. M.p.: 70-71 °C. H-
NMR (DMSO-d ): δ 9.50 (s, 1H, CH-4); 8.59 (d, 1H, CH-7, J = 2.8 Hz); 8.17 (d, 1H, CH-6 J =
6
1
3
6
1
Hz); 6.30 (s, 2H, CH ). C-NMR (DMSO): δ 148.26 (C); 142.51 (CH); 136.06 (CH); 129.57 (C);
2
14.70 (C); 112.90 (CH); 36.49 (CH ). GC/MS: 146 (M + H). Anal. Calcd for (C H N ): C, 49.66;
2
6
3
5
H, 2.08; N, 48.26. Found: C, 49.61; H, 2.10; N, 48.29.
.1.2.5. 2H-[1,2,3]triazolo[4,5-c]pyridine-2-carbonitrile (3b). Yield: 16%. M.p.: 99-100 °C. H-
NMR (DMSO-d ): δ 9.58 (s, 1H, CH-4); 8.51 (d, 1H, CH-7, J = 6 Hz); 7.98 (d, 1H, CH-6, J = 6Hz);
1
9
6
1
3
6
.41 (s, 2H, CH ). C-NMR (DMSO): δ 146.14 (C); 145.61 (CH); 143.21 (CH); 141.22 (C); 113.91
2
(
C); 111.59 (CH); 44.55 (CH ). GC/MS: 146 (M + H). Anal. Calcd for (C H N ): C, 49.66; H, 2.08;
2 6 3 5
N, 48.26. Found: C, 49.65; H, 2.03; N, 48.26.
.1.2.6. 1H-[1,2,3]triazolo[4,5-c]pyridine-1-carbonitrile (4b). Yield: 28%. M.p.: 80-81 °C. H-
NMR (DMSO-d ): δ 9.56 (s, 1H, CH-4); 8.68 (d, 1H, CH-7, J = 5.8 Hz); 8.01 (d, 1H, CH-6 J =
1
9
6
1
3
4
.6Hz); 6.21(s, 2H, CH ). C-NMR (DMSO): δ 145.39 (CH); 144.16 (CH); 142.30 (C); 136.10 (C);
2
1
14.73 (C); 105.30 (CH); 33.51 (CH ). GC/MS: 146 (M + H). Anal. Calcd for (C H N ): C, 49.66;
2
6
3
5
H, 2.08; N, 48.26. Found: C, 49.62; H, 2.04; N, 48.30.
9.1.3. General procedure for the preparation of [1,2,3]triazolo[4,5-b/c]pyridin-acrylonitrile
derivatives.
To a solution of appropriate triazolo[4,5-b/c]pyridine-carbonitrile (2a-b, 3a-b, 4a-b) (1.4 mmol)
and Et N (4.0 mmol) in toluene (5 mL) stirred at room temperature, a solution of a selected
3
aldehyde 5c-g (14 mmol) in the same solvent (3 mL) was added drop wise. The whole mixture was
finally heated under reflux for 24 h. the solution was evaporated to obtain a crude residue
containing a mixture of three E-isomers. The desired compounds were obtained by chromatography
on silica gel column using light petroleum-ethyl acetate as eluent.
9.1.3.1.
(E)-2-(1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)-3-(4-bromophenyl)acrylonitrile
(6c).
1
Yield: 53%. M.p.: 174-175 °C. H-NMR (CDCl ): δ 8.87 (d, 1H, CH-5, J = 4.4 Hz); 8.38 (d, 1H,
3

ACCEPTED MANUSCRIPT
CH-7, J = 8.0 Hz), 8.04 (s, 1H, CH); 7.80 (d, 2H, CH 2′-6′, J = 3.8 Hz); 7.70 (d, 2H, CH-3′-5′, J =
1
3
3
.8 Hz); 7.61 (m, 1H, CH-6). C-NMR (CDCl ): δ 157.85 (C); 149.31 (CH); 138.98 (CH); 132.83
3
(2 CH); 130.99 (2 CH); 129.09 (C); 127.02 (C); 124.21 (C); 123.65 (CH); 119.45 (CH); 113.16 (C),
1
06.68 (C). GC/MS: 326 (M+H). Anal. Calcd for (C H BrN ): C, 51.56; H, 2.47; Br, 24.50.
14 8 5
Found: C, 51.60; H, 2.51; Br, 24.47.
5.1.3.2. (E)-2-(1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)-3-(p-tolyl)acrylonitrile (6d). Yield: 53%.
1
M.p. = 111-112 °C. H-NMR (DMSO-d ): δ 8.86 (d, 1H, CH-5. J = 4.4 Hz); 8.60 (d, 1H, CH-7. J =
6
8.0 Hz), 8.27 (s, 1H, CH=); 7.91 (d, 2H, CH-2',6', J = 7.8 Hz); 7.78 (m, 1H, CH-6); 7.45 (d, 2H,
13
CH-3′,5′, J = 7.8 Hz); 2.49 (s, 3H, CH ). C-NMR (CDCl ): δ 157.76 (C); 149.16 (CH); 144.82
3
3
(C); 143.48 (CH); 130.21 (2 CH); 129.86 (2 CH); 127.44 (C); 124.33 (C); 123.46 (CH); 119.45
(
CH); 113.69 (C); 105.02 (C); 21.65 (CH ). GC/MS: 262 (M+H). Anal. Calcd for (C H N ): C,
3
15 11
5
6
9
8.95; H, 4.24; N, 26.80. Found: C, 68.99; H, 4.26; N, 26.78.
.1.3.3. (E)-2-(1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)-3-(4-methoxyphenyl)acrylonitrile (6e).
1
Yield: 35%. M.p. = 65.5-66 °C. H-NMR (DMSO-d ): δ 8.86 (d, 1H, CH-5. J = 4.0 Hz); 8.58 (d,
6
1H, CH-7, J = 8.0 Hz), 8.23 (s, 1H, CH=); 7.80 (d, 2H, CH-2',6', J = 4.2 Hz); 7.76 (m, 1H, CH-6);
1
3
7
.20 (d, 2H, CH-3′,5′, J = 4.2 Hz); 7.18 (m, 1H, CH, J =4.2 Hz); 3.88 (s, 3H, CH ); C-NMR
3
(
CDCl ): δ 162.99 (C); 149.36 (CH); 141.29 (CH); 132.29 (2 CH); 131.71 (C); 124.40 (C); 123.25
3
(
CH); 122.73 (C); 119.65 (CH); 115.27 (2 CH); 114.08 (C); 103.22 (C); 55.64 (CH ). GC/MS: 278
3
(
M+H). Anal. Calcd for (C H N O): C, 64.97; H, 4.00; N, 25.26. Found: C, 65.00; H, 4.03; N,
1
5
11
5
2
9
5.24.
.1.3.4. (E)-2-(1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)-3-(naphthalen-2-yl)acrylonitrile (6f).
1
Yield: 20%. M.p.: 186-187 °C. H-NMR (CDCl ): δ 8.87 (d, 1H, CH-5, J = 2.0 Hz); 8.41 (d, 1H,
3
CH-7, J = 4.2 Hz); 8.34 (s, 1H, CH); 8.22 (s, 1H, CH-2′); 8.11 (d, 1H, CH-8′); 8.01-7.91 (m, 3H,
1
3
napht); 7.66-7.60 (m, 3H, H-6 + 2H napht.). C-NMR (CDCl ): δ 157.85 (C); 149.24 (CH); 140.89
3
(
CH); 134.88 (C); 132.90 (C); 132.17 (CH); 129.44 (CH); 128.99 (CH); 128.64 (CH); 128.23 (CH);
27.96 (CH); 127.67 (C); 127.39 (CH); 125.30 (C); 124.49 (CH); 123.54 (CH); 119.53 (CH);
13.69 (C); 105.93 (C). GC/MS: 298 (M+H). Anal. Calcd for (C H N ): C, 72.72; H, 3.73; N,
1
1
2
9
1
8
11
5
3.56. Found: C, 72.69; H, 3.88; N, 23.61.
.1.3.5.
(E)-2-(1H-[1,2,3]triazolo[4,5-b]pyridin-1-yl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile
1
(
6g). Yield: 35%. M.p.: 158-159 °C. H-NMR (CDCl ): δ 8.78 (d, 1H, CH-5. J = 8.4 Hz); 8.36 (d,
3
1H, CH-7, J = 8.4 Hz), 7.97 (s, 1H, CH); 7.61 (m, 1H, CH-6); 7.27 (s, 2H, CH-2′,5′); 3.97 (s, 3H,
13
CH ); 3.96 (s, 6H, 2 CH ). C-NMR (CDCl ): δ 157.72 (C); 153.56 (2 C); 149.19 (CH); 141.88
3
3
3
(C); 141.22 (CH); 125.24 (C); 124.35 (C); 123.53 (CH); 119.44 (CH); 113.88 (C); 107.55 (2 CH);

ACCEPTED MANUSCRIPT
1
6
9
04.83 (C); 61.15 (CH ); 56.36 (2 CH ). GC/MS: 338 (M+H). Anal. Calcd for (C H N O ): C,
3
3
17 15
5
3
0.53; H, 4.48; N, 20.76. Found: C, 60.57; H, 4.51; N, 20.74.
.1.3.6. (E)-2-(2H-[1,2,3]triazolo[4,5-b]pyridin-2-yl)-3-(p-tolyl)acrylonitrile (7d). Yield: 26 %.
1
M.p.: 200-201 °C. H-NMR (CDCl ): δ 8.89 (d, 1H, CH-5, J = 2.6 Hz); 8.61 (s, 1H, CH); 8.31 (d,
3
1H, CH-7, J = 8.6 Hz); 7.91 (d, 2H, CH-2′,6′, J = 8.8 Hz); 7.44-7.43 (m, 1H, CH-6); 7.34 (d, 2H,
13
CH-3′,5′, J = 8.8 Hz); 2.45 (s, 3H, CH ). C-NMR (CDCl ): δ 155.61 (C); 153.84 (CH); 143.62 (2
3
3
C); 138.89 (CH); 137.53 (C); 130.36 (2 CH); 130.19 (2 CH); 127.32 (CH); 123.47 (CH); 112.82
C); 111.15 (C); 21.80 (CH ). GC/MS: 262 (M+H). Anal. Calcd for (C H N ): C, 68.95; H, 4.24;
(
3
15 11
5
N, 26.80. Found: C, 68.99; H, 4.20; N, 26.83.
9.1.3.7. (E)-2-(2H-[1,2,3]triazolo[4,5-b]pyridin-2-yl)-3-(4-methoxyphenyl)acrylonitrile (7e).
1
Yield: 27 %. M.P.: 160-162 °C. H-NMR (DMSO-d ): δ 8.93 (d, 1H, CH-5, J = 2.6 Hz); 8.76 (s, 1H,
6
CH=); 8.56 (d, 1H, CH-7, J = 8.6 Hz); 8.11 (d, 2H, CH-2′,6′, J = 8.8 Hz); 7.62 (m, 1H, CH-6); 7.22
1
3
(
d, 2H, CH-3′,5′, J = 8.8 Hz); 3.88 (s, 3H, CH ). C-NMR (CDCl ): δ 163.09 (C); 154.25 (CH);
3 3
1
1
42.95 (C); 138.57 (CH); 137.60 (C); 132.88 (2 CH); 127.23 (CH); 123.80 (CH); 123.31 (C);
15.31 (2 CH); 113.21 (C); 109.61 (C); 55.63 (CH ). GC/MS: 278 (M+H). Anal. Calcd for
3
(
C H N O): C, 64.97; H, 4.00; N, 25.26. Found: C, 64.01; H, 4.03; N, 25.22.
15 11 5
9.1.3.8. (E)-2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-3-(p-tolyl)acrylonitrile (8d). Yield: 53%.
1
M.p. = 95-96 °C. H-NMR (DMSO-d ): δ 8.96 (d, 1H, CH-5. J = 4.4 Hz); 8.81 (d, 1H, CH-7, J =
6
8.6 Hz), 8.59 (s, 1H, CH); 7.94 (d, 2H, CH-2',6', J = 8.2 Hz); 7.7 (m, 1H, CH-6, J = 4.4 Hz, J = 8.6
1
3
Hz); 7.4 (d, 2H, CH-3',5', J = 8.2 Hz); 2.46 (s, 3H, CH ). C-NMR (CDCl ): δ 151.03 (CH); 144.16
3
3
(C); 142.71 (C); 139.38 (CH); 137.66 (C); 130.13 (2 CH); 130.01 (2 CH); 129.84 (CH); 127.85 (C);
1
20.94 (CH); 113.45 (C); 105.76 (C); 21.72 (CH ). GC-MS: 262 (M+H). Anal. Calcd for
3
(
C H N ): C, 68.95; H, 4.24; N, 26.80. Found: C, 68.98; H, 4.21; N, 26.83.
15 11 5
9.1.3.9. (E)-2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-3-(4-methoxyphenyl)acrylonitrile (8e).
1
Yield: 28%. M.p.: 144-145 °C. H-NMR (DMSO-d ): δ 8.91 (d, 1H, CH-5, J = 4.9 Hz); 8.76 (d, 1H,
6
CH-7, J = 8.2 Hz), 8.45 (s, 1H, CH=); 7.99 (d, 2H, CH-2',6', J = 8.0 Hz); 7.68 (m, 1H, CH-6); 7.19
1
3
(
d, 2H, CH-3',5', J = 8.0 Hz); 3.88 (s, 3H, CH ). C-NMR (CHCl ): δ 162.70 (C); 151.93 (CH);
3 3
145.18 (C); 139.67 (CH); 137.57 (C); 131.92 (2 CH); 129.37 (CH); 123.12 (C); 120.84 (CH);
1
14.64 (2 CH); 113.87 (C); 103.95 (C); 55.56 (CH ). GC/MS: 278 (M+H). Anal. Calcd for
3
(
C H N O): C, 64.97; H, 4.00; N, 25.26. Found: C, 64.99; H, 3.97; N, 25.19.
1
5
11
5
9.1.3.10. (E)-2-(1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-3-(4-bromophenyl)acrylonitrile (9c).
1
Yield: 36%. M.p. = 169-170 °C. H-NMR (CDCl ): δ 9.56 (s,1H, CH-4); 8.74 (d, 1H, CH-6, J = 5.6
3
Hz); 7.99 (s, 1H, CH); 7.88 (d, 1H, CH-7, J = 5.6 Hz); 7.80 (d, 2H, CH-3′,5′, J = 8.4 Hz); 7.70 (d,
1
3
2
H, CH-2′,6′, J = 8.4 Hz). C-NMR (CDCl ): δ 146.77 (CH); 145.34 (CH); 143.44 (C); 139.34
3

ACCEPTED MANUSCRIPT
(CH); 135.16 (C); 132.85 (2xCH); 131 (2xCH); 128.97 (C); 127.14 (C); 114.17 (C); 106.36 (C);
1
2
9
05.03 (CH). GC/MS: 326 (M+H). Anal. Calcd for (C H BrN ): C, 51.56; H, 2.47; Br, 24.50; N,
14 8 5
1.47. Found: C, 51.60; H, 2.51; Br, 24.52; N, 21.43.
.1.3.11. (E)-2-(1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-3-(p-tolyl)acrylonitrile (9d). Yield: 51%.
1
M.p. = 180-181 °C. H-NMR (DMSO-d ): δ 9.64 (s, 1H, CH-4); 8.74 (d, 1H, CH-6, J = 5.4 Hz),
6
8.27 (s, 1H, CH); 8.1 (d, 1H, CH-7, J = 5.8 Hz); 7.92 (d, 2H, CH=, J = 7.8 Hz); 7.45 (d, 2H, CH=, J
1
3
=
7.8 Hz); 2.42 (s, 3H, CH ). C-NMR (CDCl ): δ 146.86 (CH); 145.32 (CH); 143.52 (C); 143.36
3 3
(C); 141.58 (CH); 135.19 (C); 130.23 (2 CH); 129.95 (2 CH); 127.33 (C); 114.08 (C); 105.05 (CH);
1
2
9
04.63 (C); 21.80 (CH ). GC/MS: 262 (M+H). Anal. Calcd for (C H N ): C, 68.95; H, 4.24; N,
3
15 11
5
6.80. Found: C, 69.98; H, 4.21; N, 26.76.
.1.3.12. (E)-2-(1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-3-(4-methoxyphenyl)acrylonitrile (9e).
1
Yield: 94%. M.p.: 195-196.7°C. H-NMR (DMSO-d ): δ 9.63 (s, 1H, CH-4); 8.73 (d, 1H, CH-6, J =
6
6
2
.2 Hz), 8.22 (s, 1H, CH); 8.07 (d, 1H, CH-7, J = 6.2 Hz); 8.02 (d, 2H, CH=, J = 8.8 Hz); 7.21 (d,
1
3
H, CH=, J = 8.8 Hz); 3.88 (s, 3H, CH ). C-NMR (CDCl ): δ 163.07 (C); 146.39 (CH); 145.26
3
3
(CH); 145.20 (CH); 144.01 (C); 141.62 (CH); 135.37 (C); 132.04 (2 CH); 122.61 (C); 114.99 (2
CH); 113.89 (C); 105.01(CH); 102.81 (C); 55.29 (CH ). GC/MS: 278 (M+H). Anal. Calcd for
3
(
C H N O): C, 64.97; H, 4.00; N, 25.26. Found: C, 64.99; H, 3.97; N, 25.21.
15 11 5
9.1.3.13. (E)-2-(1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-3-(naphthalen-2-yl)acrylonitrile (9f).
1
Yield: 43 %. M.p. = 188-189 °C. H-NMR (CDCl ): δ 9.57 (s, 1H, CH-4); 8.74 (d, 1H, CH-6, J =
3
6.0 Hz); 8.33 (s, 1H, CH); 8.15 (s, 1H, CH); 8.10 (d, 1H, CH-7, J = 6.0 Hz); 8.01-7.57 (m, 6H, CH).
1
3
C- NMR (CDCl ): δ 146.62 (CH); 145.29 (CH); 143.42 (C); 141.22 (CH); 135.28 (C); 134.90 (C);
3
1
1
32.96 (C); 132.21 (CH); 129.46 (CH); 128.07 (CH); 128.79 (CH); 127.96 (CH); 127.48 (CH);
24.45 (CH); 114.14 (C); 113.49 (C); 105.81 (C); 105.13 (CH). GC/MS: 298 (M+H). Anal. Calcd
for (C H N ): C, 72.72; H, 3.73; N, 23.56. Found: C, 72.68; H, 3.80; N, 23.54.
1
8
11
5
9
4
1
2
.1.3.14. (E)-2-(2H-[1,2,3]triazolo[4,5-c]pyridin-2-yl)-3-(p-tolyl)acrylonitrile (10d). Yield:
1
6.9%. M.p. = 194-195°C. H-NMR (DMSO-d ): δ 9.65 (s, 1H, CH-4); 8.84 (s, 1H, CH,), 8.56 (d,
6
H, CH-6. J = 5.4 Hz); 8.03 (d, 1H, CH-7, J = 5.4 Hz); 8.00(d, 2H, CH-2′,6′, J = 8.8 Hz); 7.45 (d,
1
3
H, CH-3′,5′, J = 8.8 Hz); 2.42 (s, 3H, CH ). C-NMR (CDCl ): δ 146.74 (C); 145.57 (CH); 144.00
3
3
(C); 143.84 (CH); 141.76 (C); 139.70 (CH); 130.46 (2 CH); 129.95 (2 CH); 127.02 (C); 112.81 (C);
1
1.84 (CH); 110.94 (C); 21.84 (CH ). GC/MS: 262 (M+H). Anal. Calcd for (C H N ): C, 68.95;
3
15 11
5
H, 4.24; N, 26.80. Found: C, 68.93; H, 4.27; N, 26.79.
9.1.3.15. (E)-2-(2H-[1,2,3]triazolo[4,5-c]pyridin-2-yl)-3-(4-methoxyphenyl)acrylonitrile (10e).
1
Yield: 56%. M.p.: 171-172 °C. H-NMR (DMSO-d ): δ 9.65 (s, 1H, CH-4); 8.83 (s, 1H, CH,), 8.55
6
(d, 1H, CH-6. J = 6 Hz); 8.12 (d, 2H, CH-2′,6′, J = 8.6 Hz); 8.04 (d, 1H, CH-7, J = 6.2 Hz); 7.22 (d,

ACCEPTED MANUSCRIPT
1
3
2
1
1
H, CH-3′,5′, J=8.6 Hz); 3.89 (s, 3H, CH3). C-NMR (DMSO-d ): δ 163.34 (C); 146.71 (C);
6
45.42 (CH); 143.75 (CH); 141.73 (C); 139.32 (CH); 132.68 (2 CH); 122.34 (C); 115.05 (2 CH);
13.21 (C); 111.47 (CH); 109.38 (C); 55.66 (CH ). GC-MS: 278 (M+H). Anal. Calcd for
3
(
C H N O): C, 64.97; H, 4.00; N, 25.26. Found: C, 65.00; H, 4.02; N, 25.29.
15 11 5
9.1.3.16. (E)-2-(3H-[1,2,3]triazolo[4,5-c]pyridin-3-yl)-3-(p-tolyl)acrylonitrile (11d). Yield: 16%.
1
M.p. = 152–153 °C. H.NMR (CDCl ): δ 9.50 (s, 1H, CH-4); 8.68 (d, 1H, CH-6, J = 5.6 Hz); 8.06
3
(d, 1H, CH-7, J = 5.6 Hz); 7.96 (s, 1H, CH); 7.86 (s, 2H, CH-2′,6′, J = 8 Hz); 7.37 (d, 2H, CH-3′,5′,
1
3
J = 8 Hz); 2.47 (s, 3H, CH ). C-NMR (CDCl ): δ 149.98 (C); 143.87 (C); 142.73 (CH); 141.91
3
3
(CH); 134.82 (CH); 130.2 (2 CH); 130.0 (2 CH); 129.15 (C); 128.80 (C); 114.51 (CH); 113.65 (C);
1
2
9
04.79 (C); 21.82 (CH ). GC/MS: 262 (M+H). Anal. Calcd for (C H N ): C, 68.95; H, 4.24; N,
3
15 11
5
6.80. Found: C, 69.00; H, 4.26; N, 26.78.
.1.3.17. (E)-2-(1H-[1,2,3]triazolo[4,5-c]pyridin-1-yl)-3-(4-methoxyphenyl)acrylonitrile (11e).
1
Yield: 30%. M.p. = 161-162 °C. H-NMR (DMSO-d ): δ 9.56 (s, 1H, CH-4); 8.66 (d, 1H, CH-6, J =
6
5.8 Hz), 8.33 (s, 1H, CH); 8.25 (d, 1H, CH-7, J = 5.8 Hz); 8.04 (d, 2H, CH-2′,6′, J = 9 Hz); 7.21 (d,
13
2
H, CH-3′,5′, J = 9 Hz); 3.88 (s, 3H, CH ). C-NMR (DMSO-d ): δ 162.42 (C); 148.66 (C); 143.06
3
6
(CH); 141.74 (CH); 136.35 (CH); 135.43 (C); 132.41 (2 CH); 128.74 (C); 122.71 (C); 114.87 (2
CH); 114.54 (C); 113.53 (CH); 105.98 (C); 55.62 (CH ). GC/MS = 278 (M+H). Anal. Calcd for
3
(
C H N O): C, 64.97; H, 4.00; N, 25.26. Found: C, 65.01; H, 4.03; N, 25.30.
15 11 5
9.2. Cytotoxicity assay
All cancer cell lines were obtained from the German Collection of Microbiology and Cell Culture
DSMZ, Braunschweig, FRG).
(
Human peripheral mononuclerar blood cells (P10588), human pulmonary fibroblasts (P10551), and
human prostate epithelial cells (P10953) were purchased from Innoprot (Derio, Spain).
Exponentially growing cells derived from human hematological tumors - CCRF-CEM (ACC-240,
5
T-cell leukemia) and HL-60 (acute myeloid leukemia) - were seeded at an initial density of 1 x 10
cells/mL in 96 well plates in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS),
100 units/mL penicillin G and 100  g/mL streptomycin. Human cell lines derived from solid
tumors - HeLa (ACC-57, cervix carcinoma), DU145 (ACC-261, prostate carcinoma), MCF-7
(ACC-115, breast adenocarcinoma), A-427 (ACC-234, small cell lung carcinoma), LCLC 103H
(ACC-384, large cell lung carcinoma), DAN-G (ACC-249, pancreas carcinoma), and 5637 (ACC-
5
3
5, urinary bladder carcinoma) - were also seeded at 1x10 cells/mL in 96 well plates in specific
media supplemented with 10% FCS and antibiotics as above. Cell cultures were then incubated at
7°C in a humidified, 5% CO atmosphere in the absence or presence of serial dilutions of test
3
2

ACCEPTED MANUSCRIPT
compounds. Cell viability was determined after 96 h at 37°C by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT) method.
Each test was prepared in triplicate, and the experiments were carried out three times. EC values
5
0
were obtained by nonlinear regression (GraphPad Prism v. 6.0) and represent the concentration at
which cell growth was inhibited by 50%.
9.3. In vitro tubulin polymerization assay
The purity of tubulin was estimated to be >97% using Coomassie Brilliant Blue stained SDS-
PAGE. Tubulin was re-suspended in a buffer containing 25 mM PIPES, 1 mM EGTA, and 3 mM
MgCl with 1 M glutamate and was incubated without or with different concentrations of title
2
compounds for 10 min on ice. After 10 min, 1 mM GTP was added to the reaction mixtures, and
subsequently, the assembly kinetics of tubulin was monitored at 350 nm (37 °C) for 20 min using
Spectramax M2e multi-mode microplate reader (Molecular Devices, CA, USA). Each test was run
in triplicate. The steady-state tubulin assembly level in the absence of inhibitor was set to 100%.
ITP IC values were obtained by sigmoidal fitting of steady state levels of tubulin assembly vs.
5
0
drug concentration and represent the concentration for 50% inhibition of the maximum tubulin
polymerization level. Three independent sets of experiments were performed.
9.4. Cell cycle analysis
To determine the effect of compounds 6c and 6d on the cell cycle, cells were seeded in 12-well
5
plates at a density of 10 cells/mL and allowed to attach for 24 h. Cells were then incubated in the
presence or absence (DMSO) of compounds 6c and 6d at a concentration of 2  M for 24 h. DMSO
was used for control. Next, cells were collected, washed and fixed in 70% ethanol in PBS at -20 °C.
After leaving overnight, the fixed cells were pelleted and stained with Propidium Iodide (25
mg/ml)) in the presence of RNase A (40 mg/mL) containing 0.1% Triton X-100 for 30 min at 37 °C
in dark. About 10,000 events were analyzed using a FACSCalibur flow cytometry system (BD
BioSciences, San Jose, CA, USA). Distribution of cells in cell cycle was determined using the BD
FACStation Software (v. 6.1).
9.5. Preliminary determination of the dissociation constant of compounds 6c to tubulin in
vitro by fluorescence spectroscopy
Tubulin was incubated in the absence and presence of different concentrations of compound 6c for
10 min at 37 °C. Tryptophan fluorescence of tubulin in the presence and absence of compound 6c
was monitored using an excitation wavelength of 295 nm in a 0.3 cm path length cuvette (FP-6500

ACCEPTED MANUSCRIPT
spectrofluorometer JASCO, Tokyo, Japan). The dissociation constant K for the binding interaction
d
of 6c and tubulin was calculated by fitting the change in fluorescence data using GraphPad Prism
software (v. 6.0). Three independent sets of experiments were performed.
9.6. Determination of the dissociation constant and tubulin binding site of compounds (6-8)
and (9-11) by Isothermal Titration Calorimetry (ITC).
Isothermal titration calorimetry (ITC) experiments were performed with a MicroCal PEAQ-ITC
calorimeter (Malvern, UK) at 37°C (cell volume = 280 µL). Thermodynamics of Tubulin/ligand
complex formation was investigated in PEM buffered solutions with 0.1 mM GDP, as stabilizer
agents. Specifically, a solution of tubulin (50µM, sample cell) was titrated with 19 step-by-step
injections of 2µL volume of each compound (600 µM, syringe). Solutions and buffer were degassed
for 30 min at room temperature under stirring at 750 rpm prior to each experiment. All experiments
were run in triplicate. Data acquisition and analysis were performed using inbuilt Origin 7.0 to
determine the binding stoichiometry (n) and thermodynamic parameters of the binding reaction.
9.7. Molecular modeling
All simulations were carried out using the Pmemd modules of Amber 16 [17], running on own
CPU/GPU calculation cluster. Molecular graphics images were produced using the UCSF Chimera
package (v.1.10) [18]. Chimera is developed by the Resource for Biocomputing, Visualization, and
Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311).
All other graphs were obtained using GraphPad Prism (v. 6.0).
9.7.1 Docking procedure
The optimized structures of title compounds were docked into the tubulin binding pockets using the
optimized 3D structure of the tubulin taken from our previous work [8]. All docking experiments
were performed with Autodock 4.2.6/Autodock Tools 1.4.6 [19] on a win64 platform. The resulting
docked conformations were clustered and visualized; then, the structure of each resulting complex
characterized by the lowest Autodock interaction energy in the prevailing cluster was selected for
further modeling.
9
.7.2. Molecular dynamics simulations
Each ligand/tubulin complexes obtained from the docking procedure was further refined in Amber
6 using the quenched molecular dynamics (QMD) method as previously described [see, for
example, 8, 20-22 and reference therein]. Next, the best energy configuration of each complex
1

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resulting from QMD was subsequently solvated by a cubic box of TIP3P water molecules [23]
extending at least 10 Å in each direction from the solute. The system was neutralized and the
solution ionic strength was adjusted to the physiological value of 0.15M by adding the proper
+
-
amounts of Na and Cl ions. Each solvated system was relaxed (500 steps of steepest descent
followed by 500 other conjugate-gradient minimization steps) and then gradually heated to the
target temperature of 37°C in intervals of 50 ps of constant volume-constant temperature (NVT)
molecular dynamics (MD) simulations (Verlet integration method, time step = 1.0 fs). The
Langevin thermostat was used to control temperature. During this phase of MD, the protein was
restrained with a force constant of 2.0 kcal/(mol Å), and all simulations were carried out with
periodic boundary conditions. Subsequently, the density of the system was equilibrated via MD
runs in the isothermal-isobaric (NPT) ensemble, with a time step of 1 fs. All restraints on the
protein atoms were then removed, and each system was further equilibrated using NPT MD runs at
37°C. Three equilibration steps were performed (4 ns each, time step = 2.0 fs). System stability was
monitored by the fluctuations of the root-mean-square-deviation (rmsd) of the simulated position of
the backbone atoms of tubulin with respect to those of the initial protein model. The equilibration
phase was followed by a data production run consisting of 50 ns of MD simulations in the NVT
ensemble. Data collection was performed on over the last 20 ns of each equilibrated MD trajectory
were considered for statistical data collections. 1000 trajectory snapshots were analyzed for each
compound/tubulin complex.
9.7.3. Free energy of binding analysis
The free energy of binding ∆G and its major components (∆H and T∆S ) between the selected
b
b
b
compounds and tubulin was estimated by resorting to the well-validated Molecular
Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) approach [24] implemented in Amber
16. The per residue binding free energy decomposition (interaction spectra) was carried out using
the Molecular Mechanics/Generalized Boltzmann Surface Area (MM/GBSA) approach [25,26], and
was based on the same snapshots used in the binding free energy calculation.
Acknowledgments
S.P. and E.L. gratefully acknowledge the Associazione Italiana per la Ricerca sul Cancro (AIRC)
for the generous financial support (grant IG 2015 Id.17413 to S.P.).
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Highlights
We present a series of E isomers of triazolo[4,5-b/c]pyridin-acrylonitrile derivatives
All the derivates have been investigated for their antiproliferative activity
Fluorescence-based assays prove that derivatives interfere with tubulin polymerization.
Isothermal titration calorimetry provides full tubulin/compound binding thermodynamics.
In silico modelling of interactions between tubulin and the title compounds is reported