Novel Aldimine-Type Schiff Bases of 4-Amino-5-[(3,4,5-trimethoxyphenyl)methyl]-1,2,4-triazole-3-thione/thiol: Docking Study, Synthesis, Biological Evaluation, and Anti-Tubulin Activity

Article abstract of DOI:10.1002/ardp.201600021

The present study was planned to design some novel aldimine-type Schiff bases bearing 3,4,5-trimethoxyphenyl and 1,2,4-triazole-3-thione/thiol as potential tubulin polymerization inhibitors. The obtained results of the molecular docking study using the tubulin complex (PDB code: 1SA0) showed that compounds H-25 and H-26 were well fitted in the colchicine binding site of tubulin with binding energies of ?8.68 and ?8.40 kcal/mol, respectively, in comparison to the main ligand (?8.20 kcal/mol). In parallel, molecular simulations were also performed on five other 3,4,5-trimethoxyphenyl-containing ligand targets including hsp90, VEGFR2, and human and microbial (Staphylococcus aureus and Candida albicans) dihydrofolate reductase, among which H-17, H-45, H-27, H-02, and H-19 were the most suitable compounds, respectively. Evaluation of the cytotoxic effect of the most efficient compounds of the docking steps (H-25) revealed IC₅₀ values of 12.48 ± 1.10, 4.25 ± 0.22, 3.33 ± 0.31, and 9.71 ± 0.75 μM against the HT1080, HT29, MCF-7, and A549 cell lines, respectively, compared to doxorubicin (12.69 ± 1.23, 6.12 ± 0.47, 3.51 ± 0.32, and 6.40 ± 0.31 μM, respectively). The in vitro tubulin polymerization investigation launched compounds H-25 and H-26 as potent antitubulin agents due to their IC₅₀ values of 0.17 ± 0.01 and 10.93 ± 0.43 μM, respectively.

Full text of DOI:10.1002/ardp.201600021

ARC HH PHARM
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Archiv der Pharmazie
Full Paper
Novel Aldimine-Type Schiff Bases of 4-Amino-5-[(3,4,5-
trimethoxyphenyl)methyl]-1,2,4-triazole-3-thione/thiol: Docking
Study, Synthesis, Biological Evaluation, and Anti-Tubulin Activity
1
1
1
2
Alieh Ameri , Ghadamali Khodarahmi , Farshid Hassanzadeh , Hamid Forootanfar ,
1
,3
and Gholam-Hosein Hakimelahi
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Isfahan University of Medical Sciences,
Isfahan, Iran
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical
Sciences, Kerman, Iran
2
3
Institute of Chemistry, Academia Sinica, Nankang, Taipei, Taiwan
The present study was planned to design some novel aldimine-type Schiff bases bearing 3,4,5-
trimethoxyphenyl and 1,2,4-triazole-3-thione/thiol as potential tubulin polymerization inhibitors. The
obtained results of the molecular docking study using the tubulin complex (PDB code: 1SA0) showed
that compounds H-25 and H-26 were well fitted in the colchicine binding site of tubulin with binding
energies of ꢀ8.68 and ꢀ8.40 kcal/mol, respectively, in comparison to the main ligand (ꢀ8.20 kcal/mol).
In parallel, molecular simulations were also performed on five other 3,4,5-trimethoxyphenyl-
containing ligand targets including hsp90, VEGFR2, and human and microbial (Staphylococcus aureus
and Candida albicans) dihydrofolate reductase, among which H-17, H-45, H-27, H-02, and H-19 were
the most suitable compounds, respectively. Evaluation of the cytotoxic effect of the most efficient
compounds of the docking steps (H-25) revealed IC50 values of 12.48 ꢁ 1.10, 4.25 ꢁ 0.22, 3.33 ꢁ 0.31,
and 9.71 ꢁ 0.75 mM against the HT1080, HT29, MCF-7, and A549 cell lines, respectively, compared to
doxorubicin (12.69 ꢁ 1.23, 6.12 ꢁ 0.47, 3.51 ꢁ 0.32, and 6.40 ꢁ 0.31 mM, respectively). The in vitro
tubulin polymerization investigation launched compounds H-25 and H-26 as potent antitubulin
agents due to their IC50 values of 0.17 ꢁ 0.01 and 10.93 ꢁ 0.43 mM, respectively.
Keywords: Anticancer activity / Antifungal activity / Antimicrobial activity / Inhibitors / Triazoles
Received: January 23, 2016; Revised: May 7, 2016; Accepted: May 20, 2016
DOI 10.1002/ardp.201600021
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
of mitotic spindle, maintenance of cell shape, cell migration,
and vesicles transportation launched them as an attractive
target for development of novel anticancer agents [1–3].
The tubulin polymerization inhibitors interfere with micro-
tubule dynamics via binding to three sites of (i) the vinca site;
The pivotal role of microtubules, the hollow, long, and
cylindrical cytoskeletal filaments composed of ab-tubulin
heterodimers, in various cellular processes such as formation
(
(
ii) the taxane domain; and (iii) the colchicine binding site
CBS) of tubulin among which the latest group (CBS inhibitors,
CBSIs) are the only category which do not have a typical drug
in clinical use for cancer therapy [4–6]. The selective antitumor
and antivascular activities of combretastatin A-4 (CA-4)
Correspondence: Dr. Ghadamali Khodarahmi, Department of
Medicinal Chemistry, Faculty of Pharmacy, Isfahan University of
Medical Sciences, 81746-73461 Isfahan, Iran.
E-mail: khodarahmi@pharm.mui.ac.ir
(
Fig. 1), a natural product obtained from the bark of South
African tree Combretum caffrum which are able to bind to
Fax: þ98-31-36680011
the colchicine site of tubulin, encouraged investigators to
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
1

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
Figure 1. The structures of combretastatin A-4, colchicine, podophyllotoxin, noscapine, nocodazole, and rotenone, main ligand of
the docking protein targets, and other structures.
design and synthesize novel analogs of this natural agent
in order to enhance both its potency and efficiency [7–9].
There are many reports on the modification of ring A, ring B,
and/or the ethylene bridge structure connecting the rings
derivatives such as pyrazole, imidazole, isoxazole, indole,
thiophene, thiadiazole, and triazole, has been widely
designed in order to evaluate the antiproliferative activity
of such substituted combretastatins [5, 15]. Due to the
considerable biological activities of the five-membered ring
1,2,4-triazoles like analgesic, antimicrobial, antitubercular,
anti-inflammatory, and antineoplastic activities [16–19],
this heterocycle and their derivatives have been incorpo-
rated into a wide variety of therapeutically interesting
drugs among which vorozole, letrozole, and anastrozole
(as aromatase inhibitor) are currently being used for the
treatment of breast cancer [16, 20]. Literature review
revealed the antitumor and tumor-associated carbonic
anhydrase (isoenzymes I, II, and IX) inhibitory activities of
the thione- and thiol-substituted 1,2,4-triazole ring systems
[16, 21, 22]. In addition, the inhibitory activity of substituted
1,2,4-triazoles-5-thione toward the tyrosine kinase activity
of the epidermal growth factor receptor (EGFR) has been
reported by Abou-Seri et al. [23]. Hasan et al. [24]
synthesized a series of 1,2,4-triazole containing compounds
and reported weak to moderate DHFR (dihydrofolate
[
1, 10]. Literature review revealed that alteration of 3,4,5-
trimethoxy phenyl (TMP) group (which sits in the pocket
of b-tubulin) mainly led to decrease of cytotoxic activity
of combretastatin derivatives [11–13]. Furthermore, this
unique ring (TMP) has been found in the structure of a wide
range of compounds exhibiting cytotoxic, antimicrobial, and
anti-inflammatory (via reduction of TNF-a and IL-6 produc-
tion or inhibition of COX-1 and COX-2) activity [1]. For
example, Salum et al. [14] designed, synthesized, and
evaluated the biological activities of TMP derivatives of
chalcones. They observed that the TMP analog was the most
efficient chalcone causing microtubule destabilization
and mitotic arrest in human cervical cancer cells and inhibit
in the growth of human leukemia cell lines as well as
suppression of cell migration of human breast cancer
(
compound 1) (Fig. 1) [14]. On the other hand, replacing
the double bound of CA-4 with different diaryl-heterocyclic
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
2

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
reductase) inhibitory activity with the IC50 value range of
The main aim of the present study was introducing of novel
antitubulin agent that interacted with colchicine pocket of
ab-tubulin. Some novel aldimine Schiff base derivatives of
4-amino-5-[(3,4,5-trimethoxyphenyl)methyl]-1,2,4-triazole-3-
thione/thiol (Fig. 2) were designed and synthesized according
to the results of molecular docking simulation of tubulin.
Furthermore, the docking study of these hybrid multi-
functional structures on other protein targets containing
heat shock protein (hsp90), vascular endothelial growth
factor receptor 2 (VEGFR2), DHFR (even human (hDHFR) and
microbial derived) was also conducted. Finally, the cytotoxic
effect, tubulin polymerization inhibitory activity, and anti-
microbial properties of the synthesized compounds were
also determined.
5
–40 mM (compound 2) (Fig. 1) [24]. The hybrid structures
of triazole ring and TMP moiety were also introduced as
antitumor (compound 3) [8], antimicrobial (compound 4)
[
25], and antimitotic agents (compound 5) [26] (Fig. 1). On
the other hand, the remarkable biological effects of aldimine-
type Schiff base especially those bearing 1,2,4-triazole
template such as antitumor, tyrosine kinase inhibitory effect,
anticonvulsant, antidepressant, lipid-lowering, and anti-
hypertensive as well as antimalarial, antifungal, and antiviral
activities introduced Schiff base as considerable functional
groups [27, 28]. In addition, some Schiff bases are known
to play an important role in physiological chemistry such as
vision processing and enzymatic activity [29].
Figure 2. The structures of 4-amino-3-[(3,4,5-trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-5-thione (Tn), 4-amino-5-
(3,4,5-trimethoxyphenyl)methyl]-4H-1,2,4-triazole-3-thiol (Tl) and aldimine-type Schiff base structures (code and atom numbers).
[
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
3

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
Results and discussion
The obtained molecular docking results of all Schiff bases,
compound 8, CBSIs (colchicine, noscapine, rotenone, noco-
dazole, and CA-4) (Fig. 1), and DAMA-colchicine (the main
ligand of ab-tubulin) are summarized in Table 1. The TMP ring
of DAMA-colchicine and colchicine interacted with Cysb241
by binding energies of ꢀ8.2 and ꢀ8.12 kcal/mol, respectively
(Fig. 3a and b, correspondingly). Noscapine interacted
with CBS by binding energy of ꢀ8.07 kcal/mol; the O atom
of dioxolane ring and methoxy group of A ring with Asna101
(2.144 A) and Cysb241 (1.829 A), respectively (Fig. 3c and
Table 1). Therefore, the conformer interactions better than
noscapine (ꢀ8.07 kcal/mol, the binding energy ranks of 1–30)
were reported (Table 1). In the case of CA-4, the methoxy
group (TMP ring) and hydroxyl (B ring) displayed H-bond
Molecular docking
In order to introduce novel potential CBSIs, 56 of 4-amino-
5
-[(3,4,5-trimethoxyphenyl)methyl]-1,2,4-triazole-3-thione/
thiol derivatives as aldimine-type Schiff bases including
compounds H-01 to H-28 and compounds H-29 to H-56 which
were introduced as E and Z isomers, respectively (Fig. 2), were
designed and the molecular docking simulation was per-
formed for the X-ray crystal structure of tubulin-colchicine:
Stathmin-like domain complex (PDB code: 1SA0) using
AutoDock 4.2 software [30]. All of the designed compounds
wereclassifiedintofivegroupsincluding(i)compoundsH-01to
H-03, H-15 to H-17, H-29 to H-31, and H-43 to H-45 which
̊
̊
̊
̊
bearing 3-OH, 3-NH
2
, and 3-nitro substitutes on methoxy
interaction with Cysb241 (2.049 A) and Vala181 (2.11 A),
respectively, by binding energy of ꢀ6.86 kcal/mol (Fig. 3d and
Table 1).
phenyl ring; (ii) compounds with hydroxy and/or ethoxy
groups on B ring, compounds H-04 to H-07, H-18 to H-21,
H-32 to H-35, and H-46 to H-49; the two recent groups (i and ii
groups) contained H bond acceptor or donor group similar
to those of CA-4 or colchicine; (iii) and (iv) chloro and
dichloro substituted compounds, respectively, with electron
withdrawing groups [31], compounds H-08 to H-10, H-22 to
H-24, H-36 to H-38, and H-50 to H-52 and H-11 to H-13, H-25
to H-27, H-39 to H-41, and H-53 to H-55, respectively; and
In the first group of Schiff bases, the TMP rings of
compounds with 3-OH-4-methoxy substitutes (compounds
H-01, H-15, H-29, and H-43) interacted with Cysb241 by
binding energies of ꢀ8.01, ꢀ7.78, ꢀ7.35, and ꢀ7.44 kcal/mol,
relatively. Replacing group with amine moiety, compounds
H-02, H-16, H-30, and H-44, was not favorable and Cysb241-
TMP interactions were observed by DG of ꢀ7.85, ꢀ7.86, ꢀ6.67,
and ꢀ7.46 kcal/mol, respectively. In the cases of the nitro
group, such replacement was even more disastrous because
among all the first conformer of this category (H-03, H-17,
H-31, and H-45) only compound H-03 (binding energy
of ꢀ7.43 kcal/mol) was able to interact with Cysb241 via
its TMP ring. In addition, the thiol form of this analog,
compound H-17, displayed different H-bond interactions
(nitro group and 4-methoxy group of the TMP ring as well as
(
v) compounds H-14, H-28, H-42, and H-56 without substitutes
on B ring (Fig. 2). Because of the multi-pharmacophore
moieties in the scaffold of the above designed compounds
containing TMP ring [1], 1,2,4-triazole-5-thione [22], and
azomethine (CH–N) [29] groups, other protein targets (hsp90,
VEGFR2, human, Staphylococcus aureus, and Candida albicans
DHFR, by PDB codes of 3MNR, 3CJF,1PD8, 2W9S, and 3QLS,
respectively), with their main ligands bearing TMP ring (Fig. 1),
were also included in the docking studies. The obtained
docking results of all compounds (the .dlg file results) were
sorted by cluster ranks and binding energy for all targets.
̊
̊
the SH group with Leub251 (2.172 A), Sera178 (1.765A),
̊
and Asnb258 (2.135A), respectively) by binding energy
of ꢀ7.94 kcal/mol. In this group, only compound H-31
(
Z isomer of the thione form) was exhibiting better
Tubulin
binding energy (ꢀ8.38 kcal/mol) compared to colchicine
(ꢀ8.12 kcal/mol) and DAMA-colchicine (ꢀ8.2 kcal/mol) while
displaying H-bond interaction between nitro group and
Glya144 (1.819 A). Therefore, none of the compounds in
this group were selected for synthesis step.
The second group (ii) including hydroxy and/or ethoxy
substituted compounds, did not display efficient interactions
(Cysb241-TMP interaction and/or binding energy) with CBS.
In this group, only compounds H-20 and H-21 favorably
interacted with CBS by DG binding of ꢀ8.35 and ꢀ8.19 kcal/
mol, respectively, comparted to colchicine (binding energy of
ꢀ8.12 kcal/mol). These two compounds exhibited different
interaction patterns with CBS including H-bond interactions
of SH moiety, nitro group, and 4-methoxy group of the TMP
Literature review revealed that there are at least three
pharmacophore models for CBSIs. The first model consists of
three hydrogen bond acceptors (Cysb241, Leub252, Aspb251,
Alab250, and Vala181), one hydrogen bond donor (Thra179),
two hydrophobic centers, and one planar group [32, 33]. The
second model is composed of one hydrogen bond acceptor
̊
(
Asna101), one hydrogen bond donor, one hydrophobic
feature, and one aromatic ring feature [34] while the
third model is made up of three hydrogen bond acceptors
(
(
Vala181, Leub252, and Cysb241), a hydrogen bond donor
Thra179), and a hydrophobic skeleton with four hydrophobic
centers [35]. There are many reports about the critical role
of Cysb241 for formation of H-bond and/or hydrophobic
interactions of the TMP ring containing structures [4, 36–38].
So, in the present study interaction between Cysb241 and
TMP ring (designated as Cysb241-TMP interaction) of
the designed compounds together with docking binding
energy were considered to select the most appropriate
compounds.
̊
̊
ring with Asnb258 (2.079 A), Cysb241 (1.979 A), and Sera178
̊
(1.793 A) for compound H-20 and nitro group, as well as N3
of C ring, and 5-methoxy group of the TMP ring with
̊
̊
̊
Cysb241 (2.181 A), Vala181 (2.085 A), and Asna101 (1.996 A)
for compound H-21. So, none of them were selected for
synthesis step.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
4

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
Table 1. The docking study results of tubulin (PDB code: 1SA0), aldimine-type Schiff base derivatives, DAMA-colchicine,
standard inhibitors and compound 8 (Tn, Tl).
c)
e)
f)
Cluster
rank
DG
Num.
Clus.
K
i
H-Bonding
Residues
near TMP
a)
b)
d)
̊
g)
Rank
Compound
H-26
(kcal/mol)
(mM)
(distance, A)
1
1
1
ꢀ8.68
45
0.44
B: Cys241: HG ꢀ O3 (2.215)
Leu255, Leu248, Met59,
Val315, Ala316, Ala317,
Ala354, Val318
Leu248, Leu255,
Met259, Ile378, Ala316,
Ala317, Thr314
2
H-12
ꢀ8.63
30
0.47
B: Cys241: HG ꢀ O3 (2.193)
3
4
5
H-12
H-26
H-37
2
2
1
ꢀ8.62
ꢀ8.46
ꢀ8.42
49
44
27
0.48
0.63
0.68
B: Cys241: HG ꢀ O1 (2.006)
B: Cys241: HG ꢀ O1 (1.836)
A: Thr179: O ꢀ H1 (2.1)
Leu248, Leu255, Val318
Leu248, Met259, Val318
Leu255, Leu248, Ala354,
Val318, Met259, Thr353
Leu255, Leu248, Asn249,
Lys254
B: Cys241: HG ꢀ O1 (2.049)
A: Ala317: O ꢀ H2 (1.966)
6
7
H-54
H-25
1
1
ꢀ8.41
1
0.69
0.69
B: Ala250: HN ꢀ O1 (2.156)
B: Cys241: HG ꢀ O1 (1.958)
B: Ala250: HN ꢀ N2 (2.187)
B: Cys241: HG ꢀ O1 (1.920)
B: Asn258: O ꢀ H2 (2.04)
A: Asn101: HD ꢀ O2 (2.197)
ꢀ8.4
41
Ala250, Leu242, Leu248
8
9
H-11
H-23
1
1
ꢀ8.39
ꢀ8.39
35
32
0.70
0.71
Leu248, Leu255, Leu242
Leu255, Lys254, Asn258
0
1
0
1
H-31
H-20
1
1
ꢀ8.38
1
0.72
0.76
A: Gly144: HN ꢀ O2 -Nitro (1.819)
Val181, Asn258, Lys254,
Lys352, Leu248, Ala180,
Thr179, Ser178
Val181, Ser178, Lys352,
Thr353
1
ꢀ8.35
13
B: Asn258: O ꢀ H2 (2.079)
B: Cys241: HG ꢀ O (3-OC
2 5
H ) (1.979)
A: Ser178: HG ꢀ O1 (1.793)
1
2
H-26
4
ꢀ8.32
13
0.80
A: Thr179: O ꢀ H2 (2.09)
Leu248, Met259, Val318,
Ala354
B: Cys241: HG ꢀ O1 (2.022)
1
1
1
3
4
5
H-27
H-20
H-20
1
2
3
ꢀ8.28
ꢀ8.25
ꢀ8.22
8
9
0.85
0.9
B: Asn258: OD1 ꢀ H2 (1.825)
B: Cys241: HG ꢀ O3 (2.03)
A: Asn101: HD ꢀ O1 (2.244)
Leu248, Leu255, Ala354,
Ala316, Ala317, Met259
Ala180, VAl181, Asn258,
Lys254, Leu255, Leu248
Ala180, Thr179, VAl181,
Asn258, Lys352
14
0.94
A: Ser178: HG ꢀ O3 (2.213)
A: Val181: HN ꢀ N2 (2.171)
B: Cys241: HG ꢀ O (3-OC
2 5
H ) (1.977)
B: Cys241: HG ꢀ O3 (2.179)
1
1
1
6
7
8
H-24
1
1
2
ꢀ8.2
ꢀ8.2
ꢀ8.19
43
6
0.98
0.98
0.99
Leu248, Ile378, Ala316,
Ala317, Ala354
Leu255, Leu248, Val318,
Ala354, Ala316
Ala250, Lys254, Leu255,
Asn101, Leu248, Asn258
DAMA-
colchicine
A: Thr179: O ꢀ H4 (2.013)
B: Cys241: HG ꢀ O1 (1.889)
A: Asn101: HD ꢀ O1 (2.13)
h)
H-25
12
A: Val181: HN ꢀ N3 (2.122)
B: Asn258: O ꢀ H2 (2.126)
B: Asn258: O ꢀ H1 (1.955)
A: Asn101: HD ꢀ O1 (2.103)
1
9
0
H-22
H-21
1
1
ꢀ8.19
ꢀ8.19
7
0.99
0.10
Lys254, Leu248, Asn258,
Met259
Lys254, Leu255, Asn258
2
19
B: Cys241: HG ꢀ O (4-OCH
2
-) (2.181)
A: Val181: HN ꢀ N3 (2.085)
A: Asn101: HD ꢀ O3 (1.996)
B: Cys241: HG ꢀ O1 (2.033)
B: Cys241: HG ꢀ O1 (1.989)
2
2
1
2
H-12
H-10
4
1
ꢀ8.15
ꢀ8.14
8
137
1.06
1.08
Leu248, Leu255, Val318
Ala250, Leu255, Leu248,
Met259, Lys352
2
3
H-52
1
ꢀ8.14
5
1.08
B: Ala317: O ꢀ H2 (2.042)
B: Ala250: HN ꢀ O1 (2.228)
Lys254, Ala250, Asn249,
Leu248, Leu255
(Continued)
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
5

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
Table 1. (Continued)
c)
e)
f)
Cluster
rank
DG
Num.
Clus.
K
i
H-Bonding
Residues
near TMP
a)
b)
d)
̊
g)
Rank
Compound
H-54
(kcal/mol)
(mM)
(distance, A)
2
4
2
ꢀ8.13
5
1.10
A: Ala317: O ꢀ H2 (2.011)
B: Cys241: HG ꢀ N2 (2.17)
Leu255, Leu248,
Met259, Lys254, Ala250,
Asn258
2
2
2
5
6
7
Colchicine
1
5
2
ꢀ8.12
ꢀ8.11
ꢀ8.1
9
5
9
1.11
1.14
1.15
B: Cys241: HG ꢀ O1 (1.956)
B: Cys241: HG ꢀ O1 (2.014)
Leu255, Ala316, Val318,
Ala354
Leu248, Leu255, Ala250,
Ile378
Leu255, Leu248, Ala250,
Leu242, Val238, Ala354,
Ile378, Thr353
H-12
DAMA-
colchicine
B: Thr353: O ꢀ H4 (2.02)
B: Cys241: HG ꢀ O1 (2.058)
2
2
3
8
9
0
H-25
H-25
H-22
3
4
2
ꢀ8.09
ꢀ8.08
ꢀ8.07
5
6
1.18
1.19
1.22
B: Asn258: O ꢀ H2 (2.04)
Lys254, Leu255, Asn101,
Met259, Asn258
Ala250, Leu248, Ala354,
Ala316, Lys352
Leu255, Lys254, Asn101,
Asn258, Met259
A: Asn101: HD ꢀ O2 (1.911)
B: Asn258: OD1 ꢀ H2 (2.129)
B: Cys241: HG ꢀ O2 (2.125)
A: Asn101: HD ꢀ O1 (2.176)
A: Val181: HN ꢀ N3 (2.232)
B: Asn258: O ꢀ H2 (2.091)
A: Asn101: HD ꢀ O8 (2.144)
B: Cys241: HG ꢀ O2 (1.829)
B: Ala250: HN ꢀ O4 (2.211)
B: Lys254: HZ ꢀ O1 (2.015)
A: Asn101: HD ꢀ O5 (1.906)
A: Thr179: O ꢀ H3 (2.187)
A: Val181: HN ꢀ O5 (2.11)
B: Cys241: HG ꢀ O3 (2.049)
B: Cys241: HG ꢀ O2 (2.019)
13
3
3
7
4
6
6
6
1
Noscapine
Rotenone
Nocodazole
CA-4
1
ꢀ8.07
ꢀ8
36
248
166
60
16
6
1.21
1.37
Leu255, Leu248, Val318,
Ala317, Ala316
Leu 248, Ala250,
Asn249, Lys254,
–
i)
6
1
3
1
1
1
1
4
ꢀ7.81
ꢀ6.86
ꢀ5.89
ꢀ5.74
ꢀ5.48
1.87
54
60
69
85
9.44
Leu255, Val318, Ala354,
Ala317, Ala316, Lys352
Met259, Leu255, Ala250,
Ile378, Ala316
Asn101, Lys254, Leu255,
Ala250
Tl
48.34
62.25
96.48
Tn
B: Asn258: O ꢀ H1 (2.100)
B: Cys241: HG ꢀ O2 (2.036)
Tn
5
Leu255, leu248, Ala250,
Val318
a)
b)
c)
Sorting based on binding energy.
No. of cluster.
Estimated DG binding.
Number of conformers in this cluster.
i
Estimated inhibition constant, K (mM).
d)
e)
f)
̊
Chain: Residue with H-bonding: Group ꢀ atom no. (distance, A).
Residues without H-bonding near TMP ring in hydrophobic pocket.
N-Deacetyl-N-(2-mercaptoacetyl)-colchicine, the main ligand in tubulin (PDB code: 1SA0).
p–cation interaction between A ring and Lysb254.
g)
h)
i)
According to the reported QSAR results of Kandakatla
et al. [31], where the presence of electron withdrawing
and hydrophobic groups introduced as a crucial factor for
tubulin inhibition together with the unfavorable obtained
results of colchicine/CA-4 mimicking B ring compounds (the
groups i and ii of the designed compounds as described
above), the chloro- and dichloro-substituted compounds
colchicine (energy bindings of ꢀ8.12 kcal/mol) and DAMA-
colchicine (energy bindings of ꢀ8.2 kcal/mol) (Table 1). The
Z isomer forms of 2-Cl substituted compounds displayed
interactions between the TMP ring and Cysb241 by binding
energy of ꢀ7.36 (thione form) and ꢀ7.49 kcal/mol (thiol form).
On the other hand, compound H-08 (the thione E isomeric
form) exhibited this interaction by DG of ꢀ8.00 kcal/mol.
Compared to the recent category, all of 3-Cl substituted
compounds represented the same interaction by binding
energy of ꢀ8.06, ꢀ7.95, and ꢀ7.81 kcal/mol for H-37, H-51,
and H-23–H-09, respectively. The obtained results exhibited
(
groups iii and iv) were also included in the docking studies.
The E isomer forms of 4-Cl substituted compounds exhibited
Cysb241-TMP interactions by energy bindings of ꢀ8.14 and
ꢀ
8.2 kcal/mol for H-10 (the thione form) and H-24 (the
thiol tautomer), respectively, which was similar to those of
that the order of Cysb241-TMP interaction of this group was
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
6

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
Figure 3. Interactions between ab-tubulin (PDB code: 1SA0, the colchicine binding site (CBS)) and compounds. The a and b chains
were showed in turquoise and yellow color, respectively. Residues labels of a chain and b chain are indicated by black and red color,
respectively. H-bonding interactions were showed by green dots and wired spheres. (a) Purple structure, the first conformer
of DAMA-colchicine; (b) green structure, the first conformer of colchicine; (c) violet structure, the first conformer of noscapine;
(
d) red structure, the first conformer of CA-4; (e) blue structure, the first conformer of H-25; (f) blue structure, the first conformer of
H-11; (g) blue structure, the first conformer of H-26; (h) blue structure, the first conformer of H-12; (i) blue structure, the first
conformer of H-27.
as follows: 4-Cl > 3-Cl > 2-Cl substituted compounds (Table 1).
Thus, 4-Cl and 3-Cl substituted compounds were selected for
synthesis step.
Among all designed compounds, the dichloro substituted
compounds, group iv, represented to be the best conformers.
The compound H-25, the thiol E isomeric form of 2,3-diCl
compounds, interacted with Cysb241 (4-methoxy of the TMP
(Fig. 3h and Table 1). The replacement of 2,4-diCl by 2,6-diCl
substituted negatively affected the binding energy (ꢀ8.68
to ꢀ7.83 kcal/mol compared to ꢀ8.28 to ꢀ7.75 kcal/mol, respec-
tively). In the last category, the compound H-27 interacted
with Cysb241 by binding energy of ꢀ8.28kcal/mol (Fig. 3i
and Table 1). Consequently, all of the dichloro-substituted
compounds were selected for synthesis step.
Compound 8 (Tl and Tn) interacted with Cysb241 of CBS by
energy binding of ꢀ5.89 (the first cluster) and ꢀ5.48 kcal/mol
(the fourth cluster), respectively, which showed that their
binding energies were not comparable to other compounds
(Table 1). Totally, 705 levels of energy sorting rank were
displayed in the docking analyses among which the energy
ranks of 699 and 705 belonged to the compound H-18 (the
cluster rank 90) and compound Tn (the cluster rank 28) by
binding energy of ꢀ5.15 and ꢀ4.84 kcal/mol, respectively. To
sum up and based on the obtained results, compounds
containing 2,4-, 2,3-, 2,6-dichloro, 3-, and 4-chloro substitutes
on B ring were chosen to synthesize.
̊
̊
ring, 1.958 A) and Alab250 (N atom of C ring, 2.187 A) by
binding energy of ꢀ8.4 kcal/mol (Fig. 3e) while its thione
form, H-11, showed Cysb241-TMP interaction by DG of
ꢀ
8.39 kcal/mol (Fig. 3f). The compound H-26, the thiol E
isomeric form of 2,4-diCl substituted compounds, was the
best compound between all of the investigated compounds
compared to DAMA-colchicine and colchicine (Table 1).
This compound was well overlaid on the colchicine structure
as same as compound H-25 (Fig. 3g) and interacted with
Cysb241 by binding energy of ꢀ8.68 kcal/mol (Table 1).
The thione form of this compound, H-12, also displayed
the same interaction by binding energy of ꢀ8.63 kcal/mol
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
7

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
Other protein targets
respectively) by binding energy of ꢀ9.25 kcal/mol and also
exhibited p–p and p–cationic interaction (the TMP ring with
Phe138 and B ring with Lys58, respectively) (Fig. 4a and
Table 2). The nine of total 25 ranks belonged to the 3-nitro-4-
methoxy substituted compounds (H-17, H-03, H-45, and H-31)
(Table 2). The obtained results indicated that the presence
of methoxy, hydroxy, and ethoxy in the structures was
necessary and led to well burying of structures into the active
site. The compound H-27, 2,6-dichloro substituted, interacted
All 56 compounds in two E and Z forms of thione and thiol
tautomeric structures were also simulated on other protein
targets with main ligand structures bearing the TMP moiety
(
Fig. 1) including hsp90, VEGFR2, hDHFR, S. aureus and C.
albicans DHFR (PDB code: 3MNR, 3CJF, 1PD8, 2W9S, and 3QLS,
respectively). The obtained docking results of all compounds
(
the .dlg file results) were sorted by cluster ranks and binding
energy for all targets (Tables 2 and 3).
̊
with Tyr139 (methoxy group of the TMP ring, 2.162 A) and
̊
Hsp90
H O molecule (SH group, 1.983 A) (Fig. 4b). The TMP ring of
2
The docking results indicated that the main ligand (binding
energy of ꢀ13.14 kcal/mol) interacted with the active site
better than all of the designed compounds (Table 2). The best
compound, H-17, displayed H-bond interaction between
this compound and H-17 were completely flip flapped and
overlaid on the main ligand structure (Fig. 4c).
VEGFR2
̊
methoxy group of B ring and Lys58 (1.872 A) as well as nitro
The obtained results exhibited that all of the Schiff bases
were weaker than the main ligand (binding energy of
̊
and SH groups with two H O molecules (1.723 and 2.239 A,
2
Table 2. Docking study results of interaction between hsp90 and VEFGR2 and aldimine Schiff base derivatives/ligands/
standard inhibitors (the first 25 clusters).
Hsp90
VEGFR2
c)
DG
DG
a)
b)
Rank
Compound
Cluster rank
(kcal/mol)
Compound
Cluster rank
(kcal/mol)
d)
e)
1
2
3
4
5
6
7
8
9
Ligand
1
2
3
4
1
1
1
1
2
3
1
1
1
6
1
1
1
1
4
2
1
1
1
2
1
ꢀ13.14
ꢀ9.59
ꢀ9.53
ꢀ9.39
ꢀ9.25
ꢀ9.15
ꢀ9.09
ꢀ9.06
ꢀ9.03
ꢀ8.94
ꢀ8.92
ꢀ8.88
ꢀ8.87
ꢀ8.85
ꢀ8.84
ꢀ8.83
ꢀ8.79
ꢀ8.76
ꢀ8.76
ꢀ8.72
ꢀ8.71
ꢀ8.7
Ligand
1
2
4
1
1
1
1
1
1
5
1
1
1
2
1
1
1
2
1
2
1
1
1
2
4
ꢀ8.93
ꢀ8.3
Ligand
Ligand
Ligand
H-17
H-03
H-45
H-18
H-17
H-17
H-20
H-31
H-27
Ligand
Ligand
H-54
H-02
H-04
H-17
H-31
H-25
H-16
H-19
H-03
H-48
Ligand
Ligand
H-45
H-17
H-53
H-54
H-31
H-25
Ligand
Ligand
H-49
H-26
H-26
H-21
H-27
H-52
H-25
H-40
H-53
H-51
H-19
H-50
H-54
H-25
H-48
ꢀ8.04
ꢀ7.98
ꢀ7.67
ꢀ7.63
ꢀ7.55
ꢀ7.53
ꢀ7.52
ꢀ7.45
ꢀ7.39
ꢀ7.34
ꢀ7.33
ꢀ7.32
ꢀ7.3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
6
6
0
1
2
3
4
5
6
7
8
ꢀ7.29
ꢀ7.29
ꢀ7.25
ꢀ7.23
ꢀ7.22
ꢀ7.2
9
0
1
2
3
4
5
54
ꢀ7.15
ꢀ7.11
ꢀ7.1
ꢀ8.69
ꢀ8.66
ꢀ8.65
ꢀ7.06
ꢀ3.89
105
98
H-17
67
ꢀ3.91
a)
b)
c)
Sorting based on binding energy.
No. of cluster.
Estimated DG binding.
d)
2
-[(3,4,5-trimethoxyphenyl)amino]-4-(2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)benzamide, main ligand of hsp90
(
PDB code: 3MNR).
e)
4
N -(3-methyl-1H-indazol-6-yl)-N2-(3,4,5-trimethoxyphenyl)pyrimidine-2,4-diamine, main ligand of VEGFR-2 (PDB code: 3CJF).
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
8

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
Table 3. Docking study results of DHFRs (human and microbial) with aldimine-type Schiff base derivatives, main ligand,
and standard inhibitors in the first 25 cluster rank.
hDHFR (1PD8)
DHFR (2W9S) S. aureus
DHFR (3QLS) C. albicans
c)
Cluster
rank
DG
Cluster
rank
DG
Binding
Cluster
rank
DG
Binding
a)
b)
Rank
Compound
(kcal/mol)
Compound
Compound
1
2
3
4
5
6
7
8
9
H-27
H-13
H-03
H-17
H-12
H-11
H-13
H-25
H-26
H-26
H-27
H-11
H-12
H-25
H-27
H-21
H-17
H-03
H-07
H-26
H-08
1
1
1
1
1
1
2
1
1
2
2
2
2
2
3
1
2
2
1
3
1
1
3
1
2
ꢀ10.21
ꢀ10.16
ꢀ10.15
ꢀ10.15
ꢀ10.14
ꢀ10.11
ꢀ10.07
ꢀ10.05
ꢀ9.99
ꢀ9.99
ꢀ9.96
ꢀ9.93
ꢀ9.92
ꢀ9.91
ꢀ9.91
ꢀ9.86
ꢀ9.83
ꢀ9.82
ꢀ9.82
ꢀ9.81
ꢀ9.8
H-02
H-02
H-01
H-16
H-11
H-07
H-10
H-33
H-09
H-12
H-02
H-02
H-02
H-47
H-01
1
2
1
1
1
1
1
1
1
1
3
4
5
1
2
1
4
1
1
1
1
2
2
4
5
ꢀ10.08
ꢀ9.97
ꢀ9.57
ꢀ9.57
ꢀ9.55
ꢀ9.52
ꢀ9.51
ꢀ9.47
ꢀ9.46
ꢀ9.42
ꢀ9.41
ꢀ9.38
ꢀ9.38
ꢀ9.38
ꢀ9.34
ꢀ9.31
ꢀ9.28
ꢀ9.27
ꢀ9.23
ꢀ9.22
ꢀ9.21
ꢀ9.16
ꢀ9.15
ꢀ9.14
ꢀ9.14
H-19
H-18
H-23
H-17
H-03
H-27
H-03
H-17
1
1
1
1
1
1
2
2
1
3
1
3
1
1
1
4
2
2
5
1
1
1
2
3
2
1
ꢀ9.62
ꢀ9.53
ꢀ9.49
ꢀ9.44
ꢀ9.24
ꢀ9.19
ꢀ9.18
ꢀ9.09
ꢀ9.04
ꢀ9.03
ꢀ9.03
ꢀ8.92
ꢀ8.91
ꢀ8.83
ꢀ8.81
ꢀ8.76
ꢀ8.74
ꢀ8.64
ꢀ8.63
ꢀ8.62
ꢀ8.53
ꢀ8.44
ꢀ8.43
ꢀ8.42
ꢀ8.41
ꢀ8.18
H-22
H-03
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
5
6
8
1
2
3
4
6
6
6
6
7
0
1
2
3
4
Trimetrexate
H-17
H-28
Ligand
d)
5
6
7
8
9
H-11
H03
H-28
Ligand
H-17
H-25
H-02
H-26
H-15
H-11
H-25
Methotrexate
H-39
0
1
2
H-24
H-25
H-07
H-01
H-01
e)
Ligand
ꢀ9.75
ꢀ9.74
ꢀ9.74
ꢀ9.72
3
4
5
H-11
H-22
H-08
Trimetrexate
H-02
H-11
Methotrexate
1
0
0
Trimetrexate
Methotrexate
1
1
ꢀ9.4
ꢀ9.31
29
20
55
17
07
08
35
36
18
Trimetrexate
Trimethoprim
1
1
ꢀ8.17
ꢀ7.59
f)
Trimethoprim
1
ꢀ7.12
ꢀ4.72
Trimethoprim
1
ꢀ7.81
H-48
Methotrexate
94
125
ꢀ3.94
ꢀ2.43
H-03
Methotrexate
50
101
ꢀ4.96
ꢀ3.18
H-46
86
a)
b)
c)
Sorting based on binding energy.
No. of cluster.
Estimated DG binding.
d)
6
-Methyl-5-[3-methyl-3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine, main ligand of C. albicans DHFR
(
PDB code: 3QLS).
e)
f)
2
1
,4-Diamino-5-methyl-6-[(3,4,5-trimethoxy-N-methylanilino)methyl]pyrido[2,3-d]pyrimidine, main ligand of hDHFR (PDB code
PD8).
The main ligand of S. aureus DHFR (PDB code: 2W9S).
ꢀ
8.93 kcal/mol) (Table 2). The interactions of Cys917 (N atom
compounds were more suitable for interaction with VEGFR2
compared to E isomers (Table 2).
̊
of C ring and SH group, 2.05 and 1.877 A, respectively) and
̊
Asn921 (nitro groups, 1.955 and 2.118 A) were observed for
compound H-45 (3-niro-4-methoxy substituted) (Fig. 4d). The
compound H-53 (2,3-dichloro substituted), interacted with
Cys917 (N atom of C ring, 2.203 A) and Asn921 (4-methoxy
DHFR
hDHFR: All of the compounds in their first cluster rank were
better than trimethoprim (binding energy of ꢀ7.81 kcal/mol)
while the E isomeric structures almost exhibited well binding
energies compared to Z isomers (Table 3). The E isomer of
̊
̊
group of the TMP ring, 2.081 A) (Fig. 4e) while the main ligand
̊
interacted with Cys917 (N atom of pyrimidine ring, 2.006 A)
Fig. 4f). The Z isomer of mono- or dichloro-substituted
(
compounds bearing 2,6-diCl, 3-nitro-4-OCH
3
, 2,4-diCl, 2,3-
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
9

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
Figure 4. The first conformer interaction of compounds in active sites of hsp90 (PDB code: 3MNR) and VEGFR2 (PDB code: 3CJF). The a
chains and water molecules are showed in olive green and yellow, respectively. H-bonding interactions are showed by green dots
and wired spheres. The p–p and p–cation interactions are displayed by wired cylinder and cone, respectively. (a) Pink structure,
compound H-17 (hsp90); (b) pink structure, H-27 (hsp90); (c) blue structure, main ligand (hsp90); (d) pink structure, H-45 (VEGFR2);
(e) pink structure, H-53 (VEGFR2); (f) blue structure, main ligand (VEGFR2).
th
diCl, and 3-OCH
2
O-4 groups on B ring in the both tautomeric
3,4-diethoxy substituted compound in 94 cluster rank (the
forms displayed favorable binding energies (range of ꢀ10.12
to ꢀ9.81 kcal/mol) compared to the main ligand, trimetrexate,
and methotrexate (ꢀ9.75, ꢀ9.4, and ꢀ9.31 kcal/mol, respec-
tively) and compounds with 2,6-diCl moiety (H-27 and H-13)
were the best of them (Table 3). The SH group of compound
rank of 607) (Table 3). The docking results displayed that the E
isomer form of compounds bearing 3-NH -4-OCH substituted
2 3
(H-02 and H-16) were well buried in the active site of
this protein target (Table 3). They occupied six of 25 levels
by energies range of ꢀ10.08 to ꢀ9.38 kcal/mol (Table 3).
Compound H-02 displayed H-bond interactions between NH
moiety and TMP ring with Ser49 and His23, respectively,
and also p–p interaction with Phe92 by its C ring (Fig. 5d).
The DGs of the E isomer forms of 2,3-diCl, 4-Cl, 3-Cl, and
2,4-diCl substituted compounds (ꢀ9.55, ꢀ9.51, ꢀ9.46, and
ꢀ9.42 kcal/mol, respectively) were highest compared to
methotrexate (ꢀ9.23 kcal/mol) (Table 3). The best structure of
this group was compound H-11, 2,3-diCl substituted, inter-
acted by its C ring with Ser49 and Phe92 as H-bond and p–p
interactions, respectively (Fig. 5e), while the main ligand
displayed only p–pinteraction with Phe31 (Fig. 5f). In conclusion,
the compounds with amine, hydroxy, methoxy, and/or ethoxy
groups exhibited well-binding energies compared to chloro-
substituted compounds against S. aureus DHFR (Table 3).
̊
H-27 interacted with Aspa21 by H-bond interaction (1.978 A)
and its B ring showed p–p interaction with indole ring of
Trpa24 in the active site (Fig. 5a) while its TMP ring did not
display such p–p interaction with Phea31 as compared to the
main ligand (Fig. 5b). Compound H-13, the thione form of
H-27, only displayed p–p interaction with indole ring of
Trpa24 in the active site without H-bond interaction (Fig. 5c).
This conformer was well overlaid on conformer of H-27 while
both the compounds were flip flapped compared to the main
ligand (their TMP rings overlaid on pyrido[2,3-d]pyrimidine
ring of the main ligand) (Fig. 5d). The last rank (635 levels)
th
among of all the Schiff base derivatives belonged to the 50
cluster rank of compound H-03 by binding energy of
ꢀ
4.96 kcal/mol (Table 3). The obtained results confirmed that
the compounds containing chloro group(s) were favorable
as hDHFR inhibitors among which the two substituted
compounds were more appropriate compared to the single
substituted ones (Table 3).
C. albicans DHFR: According to the molecular docking results
of the first cluster, all of the compounds were found to show
higher binding energies (in the range of ꢀ9.62 to ꢀ7.12 kcal/
mol) compared to trimethoprim (the binding energy of
ꢀ7.12 kcal/mol) (Table 3). The best compounds were E isomer
forms and also the compounds with methoxy group displayed
well-binding energies (Table 3). Among the mentioned
S. aureus DHFR: The binding energies of all of the compounds
were better than trimethoprim (binding energy ꢀ7.59 kcal/
mol) and also E isomeric form of them was better than
trimetrexate and trimethoprim (binding energies of ꢀ8.17
and ꢀ7.59 kcal/mol, respectively) (Table 3). The worst binding
energy belonged to the compound H-48, Z thiol isomer of
category compounds bearing 3-nitro-4-OCH
3
substituted,
H-17 (the thiol form), and H-03 (the thione form) involved
eight of 25 levels by energies range of ꢀ9.44 to ꢀ8.63 kcal/mol
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
10

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
Figure 5. The first conformer interaction of compounds with active sites of human (PDB code: 1PD8), S. aureus (PDB code: 2W9S), and
C. albicans DHFR (PDB code: 3QLS). The a chains are showed in olive green. H-bonding interactions are showed by green dots
and wired spheres. The p–p interactions are displayed by wired cylinder. (a) Pink structure, compound H-27 (hDHFR); (b) blue
structure, main ligand (hDHFR); (c) turquoise structure, H-13 (hDHFR); (d) pink, blue, and turquoise structure, H-27, main ligand,
and H-13, respectively (overlaid, in hDHFR); (e) pink structure, H-02 (S. aureus DHFR); (f) pink structure, H-11 (S. aureus DHFR);
(g) pink structure, H-19 (C. albicans DHFR); (h) pink structure, H-23 (C. albicans DHFR).
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
11

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
(
Table 3). Compound H-19 (4-OH-3-OC
2
H
5
substituted) was
34.05 ꢁ 2.57, 43.48 ꢁ 1.58, 9.09 ꢁ 0.81, 19.44 ꢁ 2.07, and
41.89 ꢁ 1.54 mM, respectively (Table 4). In the case of
compound H-25, the obtained IC50 ꢁ S.E. against HT1080,
HT29, MCF-7, and A549 were 12.48 ꢁ 1.10, 4.25 ꢁ 0.22,
3.33 ꢁ 0.31, and 9.71 ꢁ 0.75 mM, respectively which was well
comparedtodoxorubicin(12.69ꢁ 1.23, 6.12ꢁ 0.47, 3.51ꢁ 0.32,
and 6.40 ꢁ 0.31 mM, relatively) (Table 4). This compound
(H-25) was also more potent (IC50 ꢁ S.E., 3.33 ꢁ 0.31 mM) than
colchicine (IC50 ꢁ S.E., 4.08 ꢁ 0.31 mM) against the breast
cancer cell line (MCF-7) (Table 4). The second most active
compound was H-26 (mean IC50 ꢁ S.E. of 19.44 ꢁ 2.07 mM)
which represented IC50 ꢁ S.E. of 13.67 ꢁ 2.03 mM on hepato-
cellular carcinoma cell line (HepG2) compared to H-25
(15.66 ꢁ 0.77 mM) (Table 4). The order of tubulin binding
energies (2,4-dichloro > 3-chloro > 2,3-dichloro > 2,6-dichloro >
4-chloro) of these compounds and their cytotoxicity effects (2,3-
dichloro > 2,4-dichloro > 3-chloro > 2,6-dichloro > 4-chloro) was
almost similar (Tables 1 and 4).
The same results were reported by Hu et al. [10] where
a series of 1,3,4-oxadiazoline as CA-4 analogs was designed
and synthesized among which chloro-substituted compounds
represented good cytotoxic effect by IC50 of 4.32 ꢁ 0.22,
2.36 ꢁ 0.16, and 4.93 ꢁ 0.39 mM against cell lines of HepG2,
MCF-7, and B16-F10, respectively. In another study conducted
by Aly et al. [40], a series of new 1-(3,4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxylic acid
analogs as tubulin polymerization inhibitor agents was
designed and synthesized. They found that the most potent
compound was able to arrest the growth of cell lines of
HepG2, MCF-7, DLD-1, and HL60 by IC50 value of 15.43 ꢁ 0.91,
7.79 ꢁ 0.82, 27.59 ꢁ 1.81, and 12.97 ꢁ 0.88 mM, respectively.
the best compound by binding energy of ꢀ9.62 kcal/mol
̊
(
(
Table 3) which interacted with Met25 (2.163 A), Leu29
1.723 A), Ile112 (2.122 A), and Glu32 (1.911 A) by 4-methoxy
̊
̊
̊
group of the TMP ring, N atom of C ring, OH moiety of B ring,
and SH group, respectively (Fig. 5g). The binding energies of
compounds H-19, H-18, H-23, H-17, H-03, H-27, and H-22
(
ꢀ9.62, ꢀ9.53, ꢀ9.49, ꢀ9.44, ꢀ9.24, ꢀ9.19, and ꢀ9.04 kcal/mol,
respectively) were favorable compared to trimetrexate,
main ligand, methotrexate, and trimethoprim (ꢀ9.03,
ꢀ
8.83, ꢀ8.18, and ꢀ7.12 kcal/mol, respectively) (Table 3).
Compounds H-23, H-27, and H-22 (3-Cl, 2,6-diCl, and 3-Cl,
respectively) of chloro-substituted category, were placed in
the first 25 energy ranks (Table 3) compared to the main
ligand (Fig. 5i). The compound H-23 displayed all of the
mentioned H-19 interactions, except for Ile112 interaction by
th
binding energy of ꢀ9.49 kcal/mol (Fig. 5h). The 86 cluster
ranks of compound H-46 (Z isomer) was the worst rank of
th
energy (718 ranks) (Table 3).
Finally, the obtained result of molecular docking simula-
tions indicated that chloro(s) groups were the most suitable
inhibitors for tubulin (H-26, H-12, H-37, H-25, H-11, and H-24),
hsp90 (H-27, H-54, and H-25), VEFGR2 (H-53, H-54, H-25,
H-39, H-26, H-27, H-52, H-40, H-51, and H-50), hDHFR (H-27,
H-13, H-12, H-11, H-25, H-26, and H-08), and microbial
DHFR (H-11, H-10, H-09, H-12, H-25, H-39, H-24, and H-25)
(
2
Tables 1–3). Therefore, the compounds bearing 2,3-, 2,4-, and
,6-dichloro, 3-, and 4-chloro substituted were selected for
synthesis step.
Chemistry
The synthesis procedure of the selected aldimine-type Schiff
base derivatives is outlined in Scheme 1. Compound 8 was
synthesized by the modified fusion method described by
Ayta cS et al. [39]. Briefly, the mixture of compounds 6 and 7
was melted and stirred at two steps of (i) 5.5 h at 130–140°C
and (ii) 20 min at 160–170°C. Thereafter, boiling water was
added to the reaction mixture while vigorously stirred
followed by recrystallization of compound 8 from methanol
Tubulin polymerization studies
In order to confirm whether the cytotoxic effects of the
selected synthesized compounds were related to tubulin
interaction, the inhibitory activity of compounds H-25 and
H-26 against tubulin assembly was demonstrated. Colchicine
and paclitaxel were applied as reference controls able to
inhibit and induce tubulin polymerization, respectively. The
obtained results (Fig. 6) showed that both the applied
compounds were able to elongate the in vitro tubulin
polymerization procedure among which compound H-25
was the most efficient one and it seemed that it was more
potent compared to the reference compound colchicine in
the both applied concentrations of 5 and 10 mM. The order
of inhibitory action toward tubulin polymerization was
H-25 (10 mM, TPI% of 90.32) > H-25 (5 mM, TPI% of 87.10) >
colchicine (5 mM, TPI% of 73.39) > H-26 (20 mM, TPI% of 66.13)
(Fig. 6). Based on the higher TPI% in the presence of H-25 and
H-26, the IC50 of these two compounds were evaluated and
found to be 0.17 ꢁ 0.01 and 10.93 ꢁ 0.43 mM, respectively,
compared to that of colchicine (0.19 ꢁ 0.01 mM). The obtained
results of simulation docking studies of compound H-25 were
in agreement with the attained results of experimental
tubulin polymerization assay. As presented in Fig. 3e, the A
ring of compound H-25 was able to interact with Cysb241 via
(
Scheme 1). For synthesis of the Schiff bases, a few drops of
HCl (conc.) were added to the mixture of compound 8 and
suitable aldehydes in absolute ethanol and refluxed at 90–
1
00°C for 6–10 h. The Schiff bases were then obtained by
recrystallization from ethanol and fully characterized by FT-IR,
1
13
H NMR, C NMR, and mass spectral data.
Please see the Supporting Information files for the InChI
codes and the diverse spectra of the investigated compounds.
Biological activity
Cytotoxicity assay
The in vitro cytotoxic effects of compounds 8, Schiff base
derivatives, doxorubicin, colchicine, and methotrexate
against HT1080, HepG2, HT29, MCF-7, and A549 cancer cell
lines were evaluated by MTT assay (Table 4). The compounds
8, H-09, H-24, H-25, H-26, and H-27 inhibited the above-
mentioned cell lines by mean IC50 ꢁ S.E. of 167.85 ꢁ 14.04,
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
12

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
Scheme 1. General procedure scheme for the synthesis of compounds: Reagents and conditions: (a) 1) dropwise, stirred at 0–5°C,
) refluxed for 3 h; (b) fusion 140 and 170°C for 6 h in two steps; (c) ethanol (abs.), HCl (conc.), refluxed for 6–10 h.
2
H-bonding by 4-methoxy group of the TMP ring as similar as
colchicine (Fig. 3b) and DAMA-colchicine (Fig. 3a). Further-
more, the C ring of the mentioned compound involved in
formation of an additional H-bond with Alab250 (Fig. 3e).
However, in the case of compound H-26, the best compound
according to binding energy and Cysb241-TMP interaction
was observed with 3-methoxy group (Fig. 3g). It seems that
behind the related position of the methoxy moieties of the
TMP ring for H-bond interaction with Cysb241 in the CBS
pocket, formation of an additional H-bond with Alab250 is
important for tubulin polymerization inhibition. This model
is similar to pharmacophore model described by Nguyen
et al. [32, 33] where the mentioned residue (Alab250) was
launched as hydrogen bond acceptor.
Antimicrobial assay
Table 5 summarized the attained results of antimicrobial
investigations of five aldimine-type Schiff bases, compound 8,
and standard antibacterial (ciprofloxacin) and antifungal
(fluconazole) agents against three Gram-positive and four
Gram-negative bacterial pathogens as well as three yeast
strains. Compound H-25 as the most effective compound
inhibited S. aureus and M. luteus at concentrations below
8 mg/mL (Table 5). The growth of B. subtilis and all four
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
13

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
Table 4. In vitro cytotoxic activities and tubulin polymerization inhibitory (IC50 ꢁ S.E. mM) of the synthesized compounds
and the standard controls.
a)
IC50 TPI
Compound
HT1080
HepG2
HT29
MCF-7
A549
Mean
(mM ꢁ SE)
b)
8
H-09
H-24
H-25
H-26
H-27
Doxorubicin
Colchicine
Methotrexate
75.59 ꢁ 20.53
20.08 ꢁ 3.62
73.60 ꢁ 0.41
12.48 ꢁ 1.10
39.44 ꢁ 4.87
41.92 ꢁ 0.99
12.69 ꢁ 1.23
3.76 ꢁ 0.31
0.74 ꢁ 0.05
125.00 ꢁ 11.99
21.72 ꢁ 2.68
36.32 ꢁ 1.76
15.66 ꢁ 0.77
13.67 ꢁ 2.03
39.32 ꢁ 1.29
6.74 ꢁ 0.48
5.10 ꢁ 0.27
0.29 ꢁ 0.02
214.39 ꢁ 8.48
30.87 ꢁ 3.84
20.02 ꢁ 0.37
4.25 ꢁ 0.22
20.62 ꢁ 1.11
55.07 ꢁ 1.08
6.12 ꢁ 0.47
3.35 ꢁ 0.05
0.39 ꢁ 0.04
144.74 ꢁ 13.45
55.52 ꢁ 1.70
48.56 ꢁ 2.29
3.33 ꢁ 0.31
12.12 ꢁ 1.98
32.59 ꢁ 1.71
3.51 ꢁ 0.32
4.08 ꢁ 0.31
0.19 ꢁ 0.01
279.49 ꢁ 15.74
42.05 ꢁ 1.02
38.88 ꢁ 3.08
9.71 ꢁ 0.75
11.33 ꢁ 0.35
40.54 ꢁ 2.67
6.40 ꢁ 0.31
2.87 ꢁ 0.06
0.43 ꢁ 0.01
167.85 ꢁ 14.04
34.05 ꢁ 2.57
43.48 ꢁ 1.58
9.09 ꢁ 0.81
19.44 ꢁ 2.07
41.89 ꢁ 1.54
7.09 ꢁ 0.56
3.83 ꢁ 0.20
0.40 ꢁ 0.03
nd
nd
nd
0.17 ꢁ 0.01
10.93 ꢁ 0.43
nd
nd
0.19 ꢁ 0.01
nd
a)
b)
Tubulin polymerization inhibition.
Not determined.
studied Gram-negative bacterial strains and two Candida
pathogens (C. albicans and C. utilis) were inhibited by this
compound at concentration of 16 mg/mL (Table 5). However,
the MIC values of all tested compounds were higher than
standard antibacterial (ciprofloxacin) and antifungal (flucon-
azole) (Table 5). C. neoformans was the most resistant
pathogen among all tested microbial species representing
the highest MIC values (Table 5). Compound 8 was the
weakest compound compared to other synthesized com-
pounds against all employed bacterial and fungal strains
the designed compounds to the CBS of tubulin heterodimer.
In addition, the electron deficient ring positively affected on
the fitting of the designed structures into the CBS. It seems
that interaction between 4-methoxy group of TMP ring and
Cysb241 play a considerable role for burying the designed
compounds in the CBS of tubulin. Such finding was observed
for H-25 and H-26 (the most efficient compounds of molecular
simulation studies) which were the most active structures in
the cytotoxic assay and tubulin polymerization investigation.
These two compounds displayed better binding energies
compared to colchicine as standard compound. Compound
H-25 was the most effective one able to strongly inhibit the
in vitro tubulin polymerization by IC50 of 0.17 ꢁ 0.01 mM in
comparison to that of the colchicine (0.19 ꢁ 0.01 mM). This
compound also inhibited the growth of cancer cell lines as
well as microbial pathogens at micromolar concentration.
(
Table 5).
Conclusion
To sum up, the obtained results of the present study showed
that the presence of TMP ring and 1,2,4-triazole-3-thione/
thiol were the most important factors affecting the binding of
Figure 6. Effects of H-25 and H-26 on tubulin
polymerization. Each compound was added to
tubulin solution (4 mg/mL) prepared in P-GEM
(
general tubulin buffer containing 10.2% glyc-
erol supplemented with 1 mM GTP) followed
by monitoring of A340 using a Synergy 2 multi-
mode plate reader during 60 min (intervals of
3
0 s) incubation at 37°C.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
14

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
Table 5. The minimum inhibitory concentration (MIC, mg/mL) of the synthesized compounds and the standard controls
against the tested microorganisms.
Gram-positive bacteria
Gram-negative bacteria
Yeast strains
S.
aureus
B.
subtilis
M.
luteus
P.
K.
E.
coli
S.
typhi
C.
C.
utilis
C.
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
Compound
aeruginosa
pneumoniae
albicans
neoformans
8
H-09
H-24
H-25
H-26
H-27
Ciprofloxacin
Fluconazole
256
64
32
<8
16
32
2
512
128
64
16
32
32
4
256
64
64
<8
16
32
1024
128
128
16
32
64
1024
256
64
16
32
64
2
512
256
64
16
32
64
2
1024
128
64
16
16
32
2
>1024
256
64
16
16
512
256
128
16
16
64
–
>1024
>1024
1024
32
256
512
–
32
–
1.33
–
2.67
–
–
–
–
–
–
8
8
16
a)
b)
c)
d)
e)
f)
Staphylococcus aureus (ATCC 29213).
Bacillus subtilis (ATCC 6051).
Micrococcus luteus (ATCC 4698).
Pseudomonas aeruginosa (ATCC 27853).
Klebsiella pneumoniae (ATCC 13883).
Escherichia coli (ATCC 25922).
Salmonella typhi (ATCC 6539).
Candida albicans (ATCC 10231).
Candida utilis (ATCC 9950).
Cryptococcus neoformans (ATCC 32045).
g)
h)
i)
j)
Experimental
target and ligand for the first conformer in any cluster was
considered up to binding energy of ꢀ8.07kcal/mol (binding
energy of noscapine), while for other protein targets results of .
dlg file for any compounds was sorted based on the lowest
energy compared to the main ligands or standard inhibitors
and only 25 of the primary cluster rank were reported.
Molecular docking
The ligand-flexible docking studies were performed using
AutoDock 4.2 software [30] by Lamarckian Genetic Algorithm
(
LGA). The X-ray crystallographic structures of tubulin-
colchicine: Stathmin-like domain complex, hsp90, hDHFR,
VEGFR2, S. aureus and C. albicans DHFR were gained from
protein data bank (http://www.rcsb.org/) with PDB codes of
Chemistry
3,4,5-Trimethoxyphenyl acetic acid was provided by Alfa
Aesar Co. (Karlsruhe, Germany). All other applied chemicals
were attained from commercial suppliers of Merck chemicals.
The purity of the compounds was assessed by thin-layer
chromatography (TLC) (Merck silica gel-coated aluminum
sheets (Darmstadt, Germany). Melting points were identified
with an electrothermal 9200 capillary melting point appara-
tus. Bruker ALPHA FT-IR spectrometer was used for IR spectra
1
SA0, 3MNR, 1PD8, 3CJF, 2W9S, and 3QLS, respectively, and
then unnecessary parts (protein ligands, non-important chains
and co-enzymes, and crystallographic waters except for hsp90)
were removed. The protein targets were prepared using
prepare PDB file for docking programs (http://swift.cmbi.ru.
nl/servers/html), and other preparations were completed by
Auto-DockTools1.5.6(ADT)[41]. Proteintargetswereprepared
after calculation of Kollman charges atom and deleted non-
polar hydrogens. For ligands, structures were drawn using
MarvinSketch 6.2.2 [42] andoptimized by HyperChem software
with PM3 semi-empirical method. Protein targets and ligands
were prepared based on the tutorial of AutoDockTools [41].
The grid maps of proteins were calculated by AutoGrid. The
coordinates of center in grid box was superposed on root point
on intrinsic ligands with a grid box of 40 ꢂ 40ꢂ 40 points in x, y,
and z directions. All of the default parameters remained
1
13
(KBr discs). Nuclear magnetic resonance ( H and C NMR)
spectra were measured on Bruker (400.13 MHz for H and
100.61 MHz for C at 25°C) spectrometers using CDCl
1
1
3
3
and/or
DMSO-d as solvent and TMS as internal standard. All
6
chemical shift values were measured as d (ppm). Splitting
patterns are as follows: S, singlet; d, doublet; m, multiplet; b,
broad; dd (doublet in doublet). Mass spectra were recorded
on an Agilent technologies 5973 network mass selective
detector (MSD) operating at an ionization potential of 70 eV.
The column chromatography was carried out using standard
silica gel 60 (70–230 mesh) obtained from Merck chemicals.
̊
unchanged. A tolerance of 1 A root-mean-square deviations
RMSD) was used for clustering of docked conformations, and
(
2
1
50 docking runs measured for each ligand. AutoDockTools
.5.6 software[43] was used foranalysis of docking interactions
General method for the preparation of aldimine-type
Schiff base derivatives
General procedure for the synthesis of thiocarbazide (6): Ten
in the complex protein-ligand conformations. The docking
results of tubulin was performed according to clustering
histogram of docking log file (.dlg) and interactions between
milliliters of carbon disulfide was added dropwise for 30 min
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
15

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
to the chill solution of hydrazine hydrate 80% (30 mL) and the
obtained mixture was stirred for 45 min, followed by refluxing
in water bath for 3 h. Afterward, the yellowish solution was
cooled, and the gained solids was filtered and dried. The pure
compound 6 was attained after four times recrystallization
from water (Scheme 1).
washed with cold water, dried, and recrystallized from
ethanol to give a series of novel Schiff bases (Scheme 1).
4-{[(3-Chlorophenyl)methylidene]amino}-3-[(3,4,5-
trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-
5-thione (H-09),
Needle white crystal; yield 69%, m.p. 170–172°C
4-{[(3-chlorophenyl)methylidene]amino}-5-[(3,4,5-
trimethoxyphenyl)methyl]-4H-1,2,4-triazole-3-thiol (H-23)
White solid; yield 59%; m.p. 174–175.6°C (ethanol); FT-IR ymax
General procedure for the synthesis of 4-amino-3-[(3,4,5-
trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-
-thione (Tn),
-amino-5-[(3,4,5-trimethoxyphenyl)methyl]-4H-1,2,4-
triazole-3-thiol (Tl) (8)
ꢀ
1
(cm ): 3102.27, 3056.20 (aromatic C–H stretching), 2943.31,
2837.42 (aliphatic C–H stretching), 2784.50 (SH), 1595.55
(C–N), 1565.73 (C–C), 1332.93 (C–N), 1290.85 (C–S), 1248.79
5
4
1
(C–O), 1128.44 (C–O), 757.31 (C–Cl); H NMR (400.13 MHz,
An equimolar mixture of thiocarbazide (6) and 2-(3,4,5-
trimethoxyphenyl)acetic acid (7) was firstly melted on an oil
bath for 5.5 h at 130–140°C and then at 160–170°C for 20 min.
Consequently, the boiling water was then added and stirred
vigorously up to RT for 30 min. The mixture was then stirred at
CDCl
4-OCH
3
) d (ppm): 3.69 (s, 6 H, 3-, 5-OCH
3
in A ring), 3.73 (s, 3 H,
3
in A ring), 4.05 (s, 2 H, –CH
2
–), 6.46 (s, 2 H, 2-, 6-H in A
0
ring), 7.36 (t, J ¼ 7.83 Hz, 1 H, 5 -H in B ring), 7.42–7.46 (m, 1 H,
0
0
4 -H in B ring), 7.59 (d, J ¼ 7.71 Hz, 1 H, 6 -H in B ring), 7.84 (s,
0
1 H, 2 -H in B ring), 10.39 (s, 1 H, –N–CH–), 11.03 (s, 1 H, SH/NH
1
3
5–10°C for 1 h followed by filtering and drying. Compound (8)
3
tautomer in C ring); C NMR (100.61 MHz, CDCl ) d (ppm):
was then attained by recrystallization from methanol
31.78 (–CH
2
–), 56.02 (3-, 5-OCH
3
), 60.84 (4-OCH
3
), 105.99 (C2,
0
0
(
Scheme 1).
C6 in A ring), 127.42 (C6 in B ring), 127.61 (C2 in B ring),
129.78 (C1 in A ring), 130.33 (C5 in B ring), 132.38 (C4 in B
ring), 134.39 (C3 in B ring), 135.26 (C1 in B ring), 137.19 (C4 in
A ring), 151.69 (C1 in C ring), 153.37 (C3, C5 in A ring), 158.60
(–N–CH–), 162.31 (C4 in C ring), EI-MS (%): m/z calcd. for
0
0
Milky white crystal; yield 43%, m.p. 172.2–173.8°C (metha-
ꢀ1
0
0
nol); FT-IR ymax (cm ): 3297.10, 3242.50 (N–H stretching),
071.23, 3016.14 (aromatic C–H stretching), 2926.71, 2835.05
3
(
(
(
aliphatic C–H stretching), 2749.6 (SH), 1606.09 (C–N), 1590.84
C–C), 1332.16 (C–N), 1242.79, 1124.32 (C–O); H NMR
1
þ
þ
C
19
H
19ClN
4
O
3
S [M] , 418.90; found 418.5 [M] (22.25), 419.6
þ
þ
þ
400.13 MHz, CDCl
3
) d (ppm): 3.77 (s, 3 H, 4-OCH
.79 (s, 6 H, 3-, 5-OCH in A ring), 3.97 (s, 2 H, –CH
H, NH ), 6.47 (s, 2 H, 2-, 6-H in A ring), 11.26 (s, 1 H, SH/NH
3
in A ring),
–), 4.56 (s,
[Mþ1] (6.18), 420.5 [Mþ2] (8.92), 421.5 [Mþ3] (1.88),
þ
þ
3
2
3
2
422.5 [Mþ4] (0.63), 356.5 [C18
H
17ClN
4
O
2
]
(2.03), 296.5
þ
þ
2
[C12
H
N
16 4
O
3
S]
(6.33), 281.4 [C12
H
15 3
N O
3
S]
(100.00),
(12.13),
13
þ
þ
þ
tautomer in C ring); C NMR (100.61 MHz, CDCl
1.43 (–CH –), 56.21 (3-, 5-OCH ), 60.87 (4-OCH
C2,C6 in A ring), 129.64 (C1 in A ring), 137.35 (C4 in A ring),
3
) d (ppm):
), 106.03
266.4 [C12
H
14
14
10
12
N
2
O
3
S] (92.76), 249.4 [C12
H N
15 3
O
3
]
3
(
2
3
3
234.4 [C12
206.4 [C10
180.4 [C10
H
H
H
N
N
O
2
O
3
]
]
(8.75), 223.3 [C11
(7.35), 191.3 [C10
(11.99), 176.4 [C10
H
13NO
2
S]
(6.87),
þ
11 2 2
H N O ] (14.39),
þ
2
O
3
þ
2þ
1
52.02 (C1 in C ring, 153.47 (C3, C5 in A ring), 167.34 (C4 in C
3
]
H
10NO
10NO
] (7.11), 148.4
]
(4.27),
þ
þ
þ
2
ring); EI-MS: m/z calcd. for C12
H
16
N
4
O
3
S [M] , 296.35; found
167.4 [C
9
H
11
O
3
] (1.63), 164.4 [C
9
H
þ
þ
þ
þ
þ
þ
2
96.4 [M]
(100.00), 297.4 [Mþ1] (16.15), 298.4 [Mþ2]
[C
9
H10NO ] (9.23), 139.3 [C
7
H
6
ClN] (15.07), 137.3 [C
H
7 4
ClN]
þ
þ
þ
þ
þ
(
(
(
(
(
6.07), 281.3 [C12
56.79), 264.3 [C12
14.63), 234.3 [C12
3.32), 206.3 [C10
H
15
N
3
O
3
S] (30.09), 280.4 [C12
H
H
H
H
14
N
3
O
3
S]
(46.59), 134.3 [C
8
H
8
NO ] (9.73), 121.3 [C
8
H
9
O ] (6.61),
þ
þ
þ
þ
þ
H
H
16
N
4
O
O
3
]
]
(4.67), 249.3 [C12
(4.33), 221.3 [C12
(11.31), 192.3 [C10
] (8.83), 167.3 [C
15
N
13
N
12
N
3
O
2
O
2
O
3
3
2
]
111.3 [C
6
H
4
Cl ] (17.91), 102.3 [C
7
H
4
N ] (15.07), 89.3
þ
þ
þ
þ
þ
14
N
2
3
]
[C
H
2 5
N
2
S ] (15.25), 77.3 [C
H
6 5
] (9.84), 75.3 [CH
3
N
2
S ] (21.26),
þ
þ
þ
H
10
N
2
O
3
]
]
63.3 [CH
5
NS] (8.99), 59.3 [CHNS] (5.53), 51.3 [C
4
H
3
] (7.54);
þ
þ
14.53), 181.4 [C10
H
13
O
3
9
H
11
O
3
] (1.87),
4 3
Anal calcd. for C19H19ClN O S: C, 54.48; H, 4.57; N, 13.37; S,
7.65. Found: C, 54.35; H, 4.36; N, 13.55; S, 7.45.
þ
þ
1
64.3 [C
9
H
10NO
2
] (5.66), 148.3 [C
9
H
10NO ] (6.43), 134.3
þ
þ
þ
[
C
H
8 8
NO ] (8.52), 121.3 [C
8
H
H
9
O ] (3.67), 105.3 [C
7
H
5
O ] (3.53),
þ
þ
þ
9
1.3 [C
7
H
7
] (4.07), 78.3 [C
6
6
] (9.43), 77.3 [C
6
H
5
] (7.31), 63.3
4-{[(4-Chlorophenyl)methylidene]amino}-3-[(3,4,5-
trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-
5-thione (H-10),
þ
þ
þ
[
CH
5
NS] (4.76), 60.2 [CH
2
NS ] (13.58), 51.3 [C
4
H
3
] (5.07),
þ
4
5.2 [CHS ] (3.63); Anal calcd. for C12
16
H N
4
O
3
S: C, 48.64;
H, 5.44; N, 18.91; S, 10.82. Found: C, 48.51; H, 5.25; N, 18.59;
S, 11.09.
4-{[(4-chlorophenyl)methylidene]amino}-5-[(3,4,5-
trimethoxyphenyl)methyl]-4H-1,2,4-triazole-3-thiol (H-24)
White solid; yield 67%; m.p. 197.2–197.7°C (ethanol); FT-IR
ymax (cm ): 3098.63, 3059.65 (aromatic C–H stretching),
2944.52, 2841.14 (aliphatic C–H stretching), 2775.15 (SH),
ꢀ
1
General method for the preparation of aldimine-type
Schiff bases
For synthesis of Schiff base derivatives, an equimolar mixture
of compound 8 and chloro- or dichloro-substituted aromatic
aldehyde (10 mmol) were dissolved in 60 mL of absolute
ethanol (and three drops of HCl conc.), and refluxed in water
bath for 6–10 h. Then, the reaction mixture was treated with
cold water and ice crush; the precipitated solid was filtered,
1594.60 (C–N), 1334.54 (C–N), 1287.59 (C–S), 1249.55 (C–O),
1
1124.74 (C–O), 749.10 (C–Cl); H NMR (400.13 MHz, CDCl
3
)
d (ppm): 3.67 (s, 6 H, 3-, 5-OCH
4.04 (s, 2 H, –CH –), 6.44 (s, 2 H, 2-, 6-H in A ring), 7.40
(d, J ¼ 8.48 Hz, 2 H, 2 -, 6 -H in B ring), 7.73 (d, J ¼ 8.48 Hz, 2 H,
3 3
in A ring), 3.72 (s, 3 H, 4-OCH ),
2
0
0
0
0
3 -, 5 -H in B ring), 10.29 (s, 1 H, (–N–CH–), 11.03 (s, 1 H, SH/NH
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
16

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
1
3
þ
þ
þ
3
tautomer in C ring); C NMR (100.61 MHz, CDCl
3
) d (ppm):
[C
7
H
3
ClN ] (14.14), 134.3 [C
8
H
8
NO ] (9.60), 123.3 [C
6
H
9
N
]
þ
þ
3
1.75 (–CH –), 56.01 (3-, 5-OCH C), 60.84 (4-OCH ), 106.07 (C2,
C6 in A ring), 129.41 (C3 , C5 in B ring), 129.80 (C2 , C6 in B
ring), 129.84 (C1 in B ring), 131.00 (C1 in A ring), 137.20 (C4 in
A ring), 138.78 (C4 in B ring), 151.56 (C4 in C ring), 153.33 (C3,
2
3
3
(11.91), 119.3 [C
8 9
H
N] (7.01), 111.3 [C
6 4
H
Cl ] (6.22), 109.3
S ] (8.54),
NS] (6.13), 55.3
0
0
0
0
þ
þ
þ
[C
4
H
5
N
4
] (13.58), 103.3 [C
7
H
H
5
5
N] (6.63), 100.3 [C
](10.52), 63.3[CH
2
H
2
N
3
0
þ
þ
þ
91.3[C
7
H
7
] (9.04), 77.3[C
6
5
0
þ
þ
þ
[C
2
H
3
N
2
] (6.37), 43.3 [CH
3
N
2
] (7.10), 41.3 [CHN
2
] (6.10);
C5 in A ring), 159.54 (–N–CH–), 162.15 (C4 in C ring); EI-MS (%):
Anal calcd. for C19
7.07. Found: C, 50.29; H, 3.89; N, 12.48; S, 6.91.
H
18Cl
N
O
S: C, 50.34; H, 4.00; N, 12.36; S,
2
4
3
þ
m/z calcd. for C19
H
19ClN
4
O
3
S [M] , 418.90; found 418.4
þ
þ
þ
[
[
(
(
(
(
(
M] (18.89), 419.4 [Mþ1] (4.97), 420.4 [Mþ2] (7.40), 421.4
þ
þ
þ
þ
þ
þ
þ
Mþ3] (1.798), 422.4 [Mþ4] (0.46), 296.3 [C12
H
16
N
N
4
O
O
3
S]
S]
4-{[(2,4-Dichlorophenyl)methylidene]amino}-3-[(3,4,5-
trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-
5-thione (H-12),
þ
2.23), 281.3 [C12
H
15
N
H
13NO
3
O
3
S] (100.00), 266.3 [C12
H
14
2
3
þ
þ
87.33), 249.3 [C12
15
N
3
O
3
]
(8.65), 234.3 [C12
H
14
N
N
2
O
O
O
3
]
3
]
3
]
7.31), 223.3 [C11
H
2
S] (6.45), 206.3 [C10
H
10
2
4-{[(2,4-dichlorophenyl)methylidene]amino}-5-[(3,4,5-
trimethoxyphenyl)methyl]-4H-1,2,4-triazole-3-thiol (H-26)
Milky white solid; yield 68%; m.p. 231.2–233.5°C (ethanol); FT-
þ
5.17), 191.3 [C10
H
11
N
2
O
2
]
(11.71), 180.3 [C10
H
152
þ
þ
8.78), 164.3 [C
9
H
10NO
2
] (4.83), 148.3 [C
9
H
10NO ] (7.42),
þ
þ
ꢀ1
1
39.2 [C
7
6
H ClN] (12.78), 137.2 [C
H
7 4
ClN] (39.68), 134.3
IR ymax (cm ): 3085.92, 3051.15 (aromatic C–H stretching),
þ
þ
þ
[
(
[
C
8 8
H NO ] (6.79), 111.2 [C
6
H
4
Cl ] (11.67), 102.3 [C
7
H
4
N ]
2929.33, 2897.44, 2827.71 (aliphatic C–H stretching), 2750
(SH), 1580.00 (C–N), 1337.08 (C–N); 1321.38 (C–S), 1239.55,
1130.77 (C–O), 784.67 (C–Cl); H NMR (400.13 MHz, CDCl
3 3
(ppm): 3.70 (s, 6 H, 3-, 5-OCH in A ring), 3.73 (s, 3 H, 4-OCH
þ
þ
13.97), 89.3 [C
2
H
5
N
2
S ] (11.89), 77.3 [C
6
H
5
] (7.96), 75.3
þ
þ
þ
1
CH
3
N
2
S ] (16.02), 63.3 [CH
5
NS] (8.17), 51.3 [C
H
4 3
] (8.75),
3
) d
in
þ
4
4 3
5.3 [CHS ] (3.57); Anal calcd. for C19H19ClN O S: C, 54.48;
H, 4.57; N, 13.37; S, 7.65. Found: C, 54.64; H, 4.38; N, 13.58;
S, 7.44.
A ring), 4.05 (s, 2 H, –CH
2
–), 6.44 (s, 2 H, 2-, 6-H in A ring), 7.24–
0
0
7.29 (m, 1 H, 5 -H in B ring), 7.43 (d, J ¼ 2.06 Hz, 1 H, 3 -H in B
0
ring), 7.97 (d, J ¼ 8.48 Hz, 1 H, 6 -H in B ring), 10.85 (s, 1 H,
13
4
-{[(2,3-Dichlorophenyl)methylidene]amino}-3-[(3,4,5-
–N═CH–), 11.32 (s, 1 H, SH/NH tautomer in C ring); C NMR
trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-
(100.61 MHz, CDCl
3
) d (ppm): 31.75 (–CH
2
–), 56.07 (3-, 5-OCH
3
),
0
5
4
-thione (H-11),
-{[(2,3-dichlorophenyl)methylidene]amino}-5-[(3,4,5-
60.87 (4-OCH
3
), 106.04 (C2,C6 in A ring), 127.66 (C5 in B ring),
0
0
128.22 (C6 in B ring), 129.24 (C1 in B ring), 129.76 (C1 in A
ring), 130.26 (C3 in B ring), 137.21 (C2 in B ring), 137.26 (C4 in
0
0
trimethoxyphenyl)methyl]-4H-1,2,4-triazole-3-thiol (H-25)
Light gray crystal; yield 44%; m.p. 215–216.3°C (ethanol);
0
A ring), 138.91 (C4 in B ring), 151.57 (C1 in C ring), 153.39 (C3,
C5 in A ring), 155.71 (–N═CH–), 162.43 (C4 in C ring); EI-MS
ꢀ
1
FT-IR ymax (cm ): 3242.12 (N–H stretching), 3059.88, 3019.28
þ
(
(
aromatic C–H stretching), 2994.40, 2961.68, 2938.36, 2840.18
aliphatic C–H stretching), 1595.03 (C–N), 1579.75, 1556.23,
(%): m/z calcd. for C19
H
18Cl
N
2 4
O
3
S [M] , 453.34; found 452.4
þ
þ
þ
þ
[MꢀH] (9.72), 453.5 [M] (2.99), 454.4 [Mþ1] (6.98), 455.4
þ
þ
3
1
(
506.80 (C–C), 1334.36 (C–N); 1268.01 (C–S), 1241.90, 1127.34
[Mþ2] (1.71),456.4[Mþ3] (1.48),417.4[C19
4
H18ClN O S ](3.07),
1
þ
þ
C–O), 784.79 (C–Cl); H NMR (400.13 MHz, CDCl
3
) d (ppm):
in A ring),
–), 6.44 (s, 2 H, 2-H, 6-H in A ring), 7.24 (t,
368.7 [C18
H
16
16
14
14
N
N
4
N
2
N
4
O
O
2
O
3
3
S] (0.59), 333.5 [C15
H
17
N
3
O
4
O
3
S ] (2.11),
þ
þ
3
4
.69 (s, 6 H, 3-, 5-OCH
.05 (s, 2 H, –CH
3
in A ring), 3.73 (s, 3 H, 4-OCH
3
304.4 [C14
H
S] (6.39), 281.4 [C12
H
15
N
3
S] (100.00),
þ
þ
2
0
266.4 [C12
H
S] (86.82), 249.4 [C12
H
H
15
N
3
O
4
S]
3
]
(8.15),
(17.89),
þ
þ
þ
J ¼ 7.96 Hz, 1 H, 5 -H in B ring), 7.56 (dd, J ¼ 7.96, 1.54 Hz, 1 H,
234.3 [C12
H
2
O
3
]
(7.87), 230.4 [C11
10
N
0
0
þ
4
4
1
-H in B ring), 7.94 (dd, J ¼ 7.96, 1.54 Hz, 1 H, 6 -H in B ring),
223.3 [C11
H
13NO
]
(8.63), 173.2 [C
2
S] (6.40), 212.3 [C12
þ
13 3
(11.70), 181.3 [C10H O ]
H
12
N
]
(5.91), 191.3
11
12
2
2þ
0.98 (s, 1 H, –N–CH–), 11.09 (s, 1 H, SH/NH tautomer in C ring);
[C10
H
H
N
O
(8.27), 180.3
1
3
þ
þ
þ
C NMR (100.61 MHz, CDCl
3
) d (ppm): 31.77 (–CH
2
–), 56.06 (3-,
[C10
3
O ]
7 4
H
Cl
2
N ] (12.24), 171.2 [C
9
H
7
N
4
]
0
þ
þ
5
-OCH
3
), 60.87 (4-OCH
3
), 106.05 (C2,C6 in A ring), 125.63 (C5
(16.86), 161.3 [C
9
7 2
H NO ]
(7.27), 148.3 [C
9
H
10NO ] (7.60),
0
þ
þ
in B ring), 127.41 (C6 in B ring), 129.74 (C1 in A ring), 132.84
136.3 [C
7
H
3
ClN ] (9.83), 134.3 [C
8
H
8
NO ] (6.86), 119.3
0
0
0
þ
4þ
þ
(
C1 in B ring), 133.65 (C4 in B ring), 134.30 (C3 in B ring),
[C
8
9
H N] (9.89), 109.3 [C
4
H
5
N
]
(7.64), 103.3 [C
7
H
5
N]
0
þ
6
5þ
H ]
1
34.74 (C2 in B ring), 137.26 (C4 in A ring), 151.64 (C1 in C
(20.99), 91.3 [C
7
H
7
]
(8.65), 77.3 [C
NS] (6.37), 55.3 [C
(17.51), 69.3
3
5
H N
2þ
] (5.50), 63.3 [CH
5
þ
2 3
H N
þ
2
] (6.31),
] (9.38); Anal calcd. for
S: C, 50.34; H, 4.00; N, 12.36; S, 7.07. Found: C,
ring), 153.39 (C3, C5 in A ring), 156.39 (–N–CH–), 162.55 (C4 in
[C
þ
þ
þ
2
C ring); EI-MS (%): m/z calcd. for C19
H
18Cl
2
N
4
O
3
S [M] , 453.34;
51.3 [C H
4 3
] (7.23), 43.3 [CH
3
N
þ
þ
þ
found 452.4 [MꢀH] (14.38), 453.5 [M] (4.12), 454.4 [Mþ1]
C H18Cl N O
19 2 4 3
þ
þ
(
[
10.50), 455.4 [Mþ2] (2.48), 456.4 [Mþ3] (2.08), 430.7
50.40; H, 3.85; N, 12.52; S, 6.89.
þ
þ
C
19
H
23Cl
2
N
H
H
H
H
H
2
O
3
S ] (1.33), 417.4 [C19
H18ClN
4
O
3
S ] (16.31),
þ
þ
3
3
2
2
2
1
1
68.7 [C18
04.4 [C14
66.4 [C12
34.3 [C12
06.3 [C10
81.4 [C10
71.2 [C
16
N
4
O
3
S] (1.31), 324.4 [C17
H
16
N
4
OS] (3.88),
4-{[(2,6-Dichlorophenyl)methylidene]amino}-3-[(3,4,5-
trimethoxyphenyl)methyl]-4,5-dihydro-1H-1,2,4-triazole-
5-thione (H-13),
þ
þ
þ
þ
þ
16
14
14
10
13
N
4
O
2
S] (0.81), 281.4 [C12
S] (91.45), 249.4 [C12
H
15
N
3
O
3
S] (100.00),
þ
N
2
O
3
H
H
H
15
N
3
O
3
]
(10.83),
þ
N
N
2
O
3
]
]
(8.16), 223.3 [C11
(7.53), 191.3 [C10
(10.49), 173.2 [C
13NO
2
S] (6.67),
4-{[(2,6-dichlorophenyl)methylidene]amino}-5-[(3,4,5-
trimethoxyphenyl)methyl]-4H-1,2,4-triazole-3-thiol (H-27)
Pale yellow solid; yield 46%; m.p. 207.7–209.5°C (ethanol); FT-
þ
2
O
3
11
N
2
O
2
þ
] (13.09),
N ] (19.05),
(8.16), 148.3
þ
H
O
3
]
7
H
4
Cl
2
þ
þ
ꢀ1
9
H
7
N
4
] (27.24), 161.4 [C
9
7
H NO
2
]
IR ymax (cm ): 3267.35 (N–H stretching), 3011.33 (aromatic
þ
þ
[
C
9
H
10NO ] (8.46), 147.3 [C
9
H
9
NO]
(8.69), 136.3
C–H stretching), 2970.19, 2921.44 (aliphatic C–H stretching),
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
17

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
1
(
(
592.29 (C–N), 1577.51, 1509.98 (C–C), 1336.03 (C–N); 1308.84
Tubulin polymerization assay
1
C–S), 1244.48, 1123.71 (C–O), 774.99 (C–Cl),
H
NMR
in A
The influence of the selected compound(s) on the polymeri-
zation of the tubulin was conducted using the method
described by supplier (Cytoskeleton, Denver, CO, USA).
Briefly, the purified porcine brain tubulin was firstly re-
400.13 MHz, CDCl
3
) d (ppm): 3.69 (s, 6 H, 3-, 5-OCH
3
ring), 3.74 (s, 3 H, 4-OCH
3
in A ring), 4.05 (s, 2 H, –CH
2
–), 6.47 (s,
0
2
7
(
H, 2-, 6-H in A ring), 7.23–7.30 (m, 1 H, 4 -H in B ring), 7.34–
0
0
.41 (m, 2 H, 3 , 5 -H in B ring), 10.61 (br. s., 1 H, –N–CH–), 11.02
2
suspended in ice cold tubulin glycerol buffer (0.5 mM MgCl ,
13
s, 1 H, SH/NH tautomer in C ring), C NMR (100.61 MHz,
CDCl ) d (ppm): 31.49 (–CH –), 56.00 (3-, 5-OCH ), 60.84
4-OCH ), 106.17 (C2, C6 in A ring), 129.18 (C2 , C6 in B ring),
1 mM EGTA, 80 mM PIPES pH 6.9, 1 mM GTP, and 10.2% (v/v)
glycerol) and 100 mL of the prepared mixture was immedi-
ately added to the desired well of a half volume 96-well
microplate containing the designated concentrations of
the selected synthesized analog or vehicle followed by
recording the related A340 nm during 60 min (intervals of 30 s)
incubation at 37°C using a Synergy 2 multi-mode plate
reader (Biotek, Winooski, VT, USA). The percent of tubulin
polymerization inhibition (TPI%) was considered using the
following equation:
3
2
3
0
0
(
1
3
0
0
0
29.34 (C3 , C5 in B ring), 129.80 (C1 in A ring), 131.74 (C4 in B
ring), 135.79 (C1 in A ring), 137.15 (C4 in A ring), 152.09 (C1
in C ring), 153.29 (C3, C5 in A ring), 155.56 (–N–CH–), 159.52
þ
(
C4 in C ring); EI-MS (%): m/z calcd. for C19
H
18Cl
2
N
4
O
3
S [M] ,
þ
þ
4
4
4
3
2
2
2
1
[
(
[
[
53.34; found 452.4 [MꢀH]
(10.36), 453.5 [M] (3.26),
þ
þ
þ
54.4 [Mþ1] (7.22), 455.4 [Mþ2] (1.93), 456.4 [Mþ3] (1.60),
þ
þ
29.5 [C19
44.5 [C18
81.4 [C12
49.3 [C12
23.3 [C11
H
H
H
H
H
H
22Cl
2
N
2
O
3
S] (1.41), 417.4 [C19
H
H
H
18ClN
12ClN
4
O
3
S ] (5.06),
] (1.11),
S] (85.79),
þ
þ
þ
20
15
15
N
2
O
3
S] (0.96), 319.5 [C14
S] (100.00), 266.3 [C12
(8.80), 234.3 [C12
S] (5.60), 206.3 [C10
4
O
3
ꢂ
ꢀ
ꢁꢃ
þ
þ
þ
MaxAT
MaxAc
N
N
3
O
3
14
N
2
O
3
TPI% ¼ 1 ꢀ
ꢂ 100
þ
3
O
3
]
H
H
O
14
N
N
2
O
O
3
]
(7.14),
(4.61),
þ
13NO
2
10
2
3
]
þ
3þ
91.3 [C10
11
N
2
O
2
] (9.44), 181.3 [C10
H
13
] (7.05), 171.2
where MaxAT and MaxAC were regarded as maximum
absorbance of the test and control, respectively. All above-
mentioned procedure was performed in triplicate and
þ
þ
þ
C
H
9 7
N
4
] (7.97), 148.3 [C
9
H
10NO ] (5.99), 136.3 [C7H3ClN ]
þ
þ
6.20), 123.3 [C
6
H
9
N
3
]
(3.75), 109.2 [C
4
H
5
N
4
] (3.47), 105.3
] (4.31), 63.3
þ
þ
þ
þ
C
CH
7
H
7
N] (4.34), 91.3 [C
7
H
7
] (4.23), 77.3 [C
6
H
5
applied for calculation of IC50.
þ
þ
5
NS] (2.48), 55.3 [C
2
H
3
N
2
] (2.68), 51.3 [C
4
H
3
] (2.58),
S: C, 50.34;
þ
4
3.3 [CH
3
N
2
] (3.01); Anal calcd. for C19
H18Cl
2
N
4
O
3
Microplate antimicrobial assay
H, 4.00; N, 12.36; S, 7.07. Found: C, 50.42; H, 3.82; N, 12.25;
S, 6.86.
In order to evaluate the antimicrobial activity of the prepared
compounds, a modified microdilution assay protocol [44, 45]
was performed. At first, all applied microbial strains (Table 5)
were sub-cultured overnight in a suitable medium (Mueller–
Hinton agar for bacterial strains and Sabouraud dextrose
agar for fungal pathogens) to get the fresh culture of
each pathogen followed by cultivation of each bacterial or
fungal strain in the broth medium to reach 0.5 McFarland
Biological activity
Cell culture and cytotoxicity assay
Five human cancer cell lines including MCF-7 (breast cancer),
HT1080 (fibrosarcoma), HT29 (colorectal cancer), HepG2 (liver
hepatocellular carcinoma), and A549 (lung cancer) were
obtained from the Iranian Biological Resource Center (IBRC)
Tehran, Iran. The cells were maintained in DMEM medium
supplemented with FBS (10%, v/v) and antibiotics [penicillin
8
standard turbidity (1.5 ꢂ l0 CFU/mL). Thereafter, serial dilu-
tion (8–1024 mg/mL) of each tested compound was prepared
in a 96-well round-bottom microplate and each bacterial or
fungal pathogen was separately inserted into the desired
(
100 units/mL) and streptomycin (100 mg/mL)] at 37°C in a CO
2
4
incubator (5% CO ). In order to determine the cytotoxic effect
2
well to reach final concentration of 10 CFU/mL. Afterwards,
of the synthesized compounds, the cells were harvested in the
exponential phase of growth, seeded separately into 96-well
tissue culture plates (15000 cells per well) and allowed to
adhere for 24 h. Thereafter, each compound was added to the
desired wells to reach final concentration range of 300 mM to
the prepared microplates were incubated at 37°C for 24 h
in the case of bacterial strains and 48 and 72 h for Candida
species and Cryptococcus neoformans, respectively, followed
by addition of 40 mL INT, (2-(4-iodophenyl)-3-(4-nitrophenyl)-
5-phenyl tetrazolium chloride solution (0.6 mg/mL) to each
well, incubating for further 20 min at 37°C, and recording
the absorbance at 490 nm. MICs were then determined
based on the well concentration before the well in which
microbial growth (represented absorbance at 490 nm) was
observed. Un-inoculated media and inoculated media
containing each microbial strain were applied as negative
and positive control, respectively. In addition, the antibacte-
rial agent ciprofloxacin (concentration range of 0.5–8 mg/mL)
and antifungal agent fluconazole (final concentration of
1–16 mg/mL) were used as standard control. All mentioned
experiments were performed in triplicate and mean of the
obtained results was reported.
1
95 nM followed by 24 h incubation at above-mentioned
conditions. In parallel, doxorubicin (final concentration range
of 0.5–16 mM), colchicine (final concentration range of 0.016–
16 mM), and methotrexate (final concentration range of
0.031–8 mM) were applied as positive controls. Afterward,
20 mL DMEM medium containing MTT (5 mg/mL) was added to
each well and incubated for 4 h. Consequently, the medium
was replaced with 100 mL of DMSO, and the optical densities
were then determined at 570 nm. The IC50 values were then
calculated by nonlinear regression analysis of the obtained
results and expressed as mean ꢁ S.E. MTT assay was performed
in three independent replicates.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
18

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
Anti-Tubulin Activity of Novel Aldimine-Type Schiff Bases
ARC HH PHARM
Archiv der Pharmazie
This work was supported financially by the grant number
[17] M. Koparir, C. Orek, P. Koparir, K. Sarac, Spectrochim.
Acta A Mol. Biomol. Spectrosc. 2013, 105, 522–531.
[18] A. A. Othman, M. Kihel, S. Amara, Arabian J. Chem. 2014,
doi: 10.1016/j.arabjc.2014.09.003
1
90068 from Isfahan University of Medical Sciences, Isfahan,
Iran. The authors would like to appreciate Prof. Mohammad
Ali Faramarzi for his kindly consultations.
[
19] Nithinchandra, B. Kalluraya, S. Aamir, A. R. Shabaraya,
Eur. J. Med. Chem. 2012, 54, 597–604.
The authors have declared no conflict of interest.
[
20] K. Sztanke, T. Tuzimski, J. Rzymowska, K. Pasternak,
M. Kandefer-Szersze nꢀ , Eur. J. Med. Chem. 2008, 43,
References
404–419.
[
21] A. T. Mavrova, D. Wesselinova, Y. A. Tsenov, P. Denkova,
Eur. J. Med. Chem. 2009, 44, 63–69.
[
1] A. S. Negi, Y. Gautam, S. Alam, D. Chanda, S. Luqman,
J. Sarkar, F. Khan, R. Konwar, Bioorg. Med. Chem. 2015,
[22] SS . G. K u€ cS u€ kg u€ zel, P. CS ıkla-S u€ zg u€ n, Eur. J. Med. Chem.
2015, 97, 830–870.
23, 373–389.
[
2] Y.-H. Lo, Y.-T. Lin, Y.-P. Liu, T.-H. Duh, P.-J. Lu, M.-J. Wu,
Eur. J. Med. Chem. 2013, 62, 526–533.
[23] S. M. Abou-Seri, Eur. J. Med. Chem. 2010, 45, 4113–
4121.
[
[
3] L. A. Amos, Semin. Cell Dev. Biol. 2011, 22, 916–926.
4] J.-H. Jiang, C.-H. Zheng, C.-Q. Wang, J. Wang, W. Tian,
C. Yang, Y.-L. Song, Y. Hu, J. Zhu, Y.-Y. Zhou, Chin. Chem.
Lett. 2015, 26, 607–609.
[24] G. S. Hassan, S. M. El-Messery, F. A. Al-Omary, S. T. Al-
Rashood, M. I. Shabayek, Y. S. Abulfadl, E.-S. E. Habib,
S. M. El-Hallouty, W. Fayad, K. M. Mohamed, Eur. J. Med.
Chem. 2013, 66, 135–145.
[
5] A. Kamal, G. B. Kumar, S. Polepalli, A. B. Shaik, V. S.
Reddy, M. K. Reddy, C. Reddy, R. Mahesh, J. S. Kapure,
N. Jain, ChemMedChem 2014, 9, 2565–2579.
[25] A. Saeed, A. Mumtaz, J. Saudi. Chem. Soc. 2015, doi:
10.1016/j.jscs.2015.04.004.
[26] R. Romagnoli, P. G. Baraldi, M. K. Salvador, F. Prencipe,
V. Bertolasi, M. Cancellieri, A. Brancale, E. Hamel,
I. Castagliuolo, F. Consolaro, J. Med. Chem. 2014, 57,
6795–6808.
[27] N. H. N. Moorthy, U. B. Vittal, C. Karthikeyan,
V. Thangapandian, A. Venkadachallam, P. Trivedi, Ara-
bian J. Chem. 2013, doi: 10.1016/j.arabjc.2013.12.021.
[28] J. A. Makawana, C. B. Sangani, L. Lin, H.-L. Zhu, Bioorg.
Med. Chem. 2014, 24, 1734–1736.
[
[
6] A.Massarotti,A.Coluccia,R.Silvestri,G.Sorba,A.Brancale,
ChemMedChem 2012, 7, 33–42.
7] L. M. Greene, S. Wang, N. M. O’boyle, S. A. Bright, J. E.
Reid, P. Kelly, M. J. Meegan, D. M. Zisterer, Oncol. Rep.
2013, 29, 2451–2458.
[
[
8] P.-L. Zhao, A.-N. Duan, M. Zou, H.-K. Yang, W.-W. You,
S.-G. Wu, Bioorg. Med. Chem. Lett. 2012, 22, 4471–
4474.
9] M. Banimustafa, A. Kheirollahi, M. Safavi, S. K. Ardestani,
[29] K. Sztanke, A. Maziarka, A. Osinka, M. Sztanke, Bioorg.
Med. Chem. 2013, 21, 3648–3666.
H. Aryapour, A. Foroumadi, S. Emami, Eur. J. Med. Chem.
2
013, 70, 692–702.
[30] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput. Chem.
2009, 30, 2785–2791.
[
[
10] Y. Hu, X. Lu, K. Chen, R. Yan, Q.-S. Li, H.-L. Zhu, Bioorg.
Med. Chem. 2012, 20, 903–909.
11] Z. Wen, J. Xu, Z. Wang, H. Qi, Q. Xu, Z. Bai, Q. Zhang,
K. Bao, Y. Wu, W. Zhang, Eur. J. Med. Chem. 2015, 90,
[31] N. Kandakatla, G. Ramakrishnan, J. Karthikeyan,
R. Chekkara, Orient. J. Chem. 2014, 30, 1083–1098.
[32] T. L. Nguyen, C. McGrath, A. R. Hermone, J. C. Burnett,
D. W. Zaharevitz, B. W. Day, P. Wipf, E. Hamel, R. Gussio,
J. Med. Chem. 2005, 48, 6107–6116.
[33] O. Zefirova, A. Diikov, N. Zyk, N. Zefirov, Russ. Chem.
Bull. 2007, 56, 680–688.
[34] M.-m. Niu, J.-y. Qin, C.-p. Tian, X.-f. Yan, F.-g. Dong,
Z.-q. Cheng, G. Fida, M. Yang, H. Chen, Y.-q. Gu, Acta
Pharmacol. Sin. 2014, 35, 967–979.
184–194.
[
[
[
12] A. Chaudhary, P. Sharma, G. Bhardwaj, V. Jain, P.
Bharatam, B. Shrivastav, R. Roy, Med. Chem. Res. 2013,
22, 5654–5669.
13] A. Novoa, N. Pellegrini-Mo €ı se, S. Bourg, S. Thoret,
J. Dubois, G. Aubert, T. Cresteil, Y. Chapleur, Eur. J.
Med. Chem. 2011, 46, 3570–3580.
14] L. B. Salum, W. F. Altei, L. D. Chiaradia, M. N. S. Cordeiro,
R. R. Canevarolo, C. P. S. Melo, E. Winter, B. Mattei, H. N.
Daghestani, M. C. Santos-Silva, T. B. Creczynski-Pasa,
R. A. Yunes, J. A. Yunes, A. D. Andricopulo, B. W. Day,
R. J. Nunes, A. Vogt, Eur. J. Med. Chem. 2013, 63,
[35] C. Da, S. L. Mooberry, J. T. Gupton, G. E. Kellogg, J. Med.
Chem. 2013, 56, 7382–7395.
[36] G. La Regina, R. Bai, A. Coluccia, V. Famiglini, S. Pelliccia,
S. Passacantilli, C. Mazzoccoli, V. Ruggieri, L. Sisinni,
A. Bolognesi, J. Med. Chem. 2014, 57, 6531–6552.
[37] N. R. Penthala, H. Zong, A. Ketkar, N. R. Madadi,
V. Janganati, R. L. Eoff, M. L. Guzman, P. A. Crooks, Eur. J.
Med. Chem. 2015, 92, 212–220.
[38] N. M. O’Boyle, J. K. Pollock, M. Carr, A. J. Knox,
S. M. Nathwani, S. Wang, L. Caboni, D. M. Zisterer,
M. J. Meegan, J. Med. Chem. 2014, 57, 9370–9382.
501–510.
[
[
15] R. Romagnoli, P. G. Baraldi, O. Cruz-Lopez, M. Tolomeo,
A. D. Cristina, R. M. Pipitone, S. Grimaudo, J. Balzarini,
A. Brancale, E. Hamel, Bioorg. Med. Chem. Lett. 2011, 21,
746–2751.
16] J. K. Sahu, S. Ganguly, A. Kaushik, Chin. J. Nat. Med.
013, 11, 456–465.
2
2
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
19

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–20
A. Ameri et al.
ARC HH PHARM
Archiv der Pharmazie
[
[
[
39] S. P. Ayta cS , B. Tozkoparan, F. B. Kaynak, G. Aktay,
Institute Molecular Graphics Laboratory, California
2008, p. 56.
€
€
O. G o€ kta sS , S. Un u€ var, Eur. J. Med. Chem. 2009, 44,
528–4538.
4
[42] ChemAxon, MarvinSketch 6.2.2, in, 2014.
[43] M. F. Sanner, J. Mol. Graphics Mod. 1999, 17, 57–61.
[44] A. S. Ahmed, E. E. Elgorashi, N. Moodley, L. J. McGaw, V.
Naidoo, J. N. Eloff, J. Ethnopharmacol. 2012, 143, 826–839.
[45] Z. Aumeeruddy-Elalfi, A. Gurib-Fakim, F. Mahomoodally,
Ind. Crop. Prod. 2015, 71, 197–204.
40] O. M. Aly, E. A. Beshr, R. M. Maklad, M. Mustafa,
A. M. Gamal-Eldeen, Arch. Pharm. Chem. Life Sci. 2014,
347, 658–667.
41] R. Huey, G. M. Morris, Using AutoDock4 with
AutoDockTools: A Tutorial, in, The Scripps Research
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
20