A first-in-class anticancer dual HDAC2/FAK inhibitors bearing hydroxamates/benzamides capped by pyridinyl-1,2,4-triazoles

Article abstract of DOI:10.1016/j.ejmech.2021.113569

Novel 5-pyridinyl-1,2,4-triazoles were designed as dual inhibitors of histone deacetylase 2 (HDAC2) and focal adhesion kinase (FAK). Compounds 5d, 6a, 7c, and 11c were determined as potential inhibitors of both HDAC2 (IC₅₀ = 0.09–1.40 μM) and F

Full text of DOI:10.1016/j.ejmech.2021.113569

European Journal of Medicinal Chemistry 222 (2021) 113569
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A first-in-class anticancer dual HDAC2/FAK inhibitors bearing
hydroxamates/benzamides capped by pyridinyl-1,2,4-triazoles
,
b
,
, d, **,
Muhamad Mustafa a 1, Amer Ali Abd El-Hafeez c
1, Dalia Abdelhamid a,
Gajanan D. Katkar d, Yaser A. Mostafa e, Pradipta Ghosh d
h, Alaa M. Hayallah b
,
,
f
,
g
,
, e, i
Gamal El-Din A. Abuo-Rahma a
, b, *
a Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, 61519, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Deraya University, Minia, Egypt
c Pharmacology and Experimental Oncology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt
d Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA
e Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, 71526, Egypt
f Department of Medicine, University of California San Diego, La Jolla, CA, USA
g Moores Comprehensive Cancer Center, University of California San Diego, La Jolla, CA, USA
h Veterans Affairs Medical Center, La Jolla, CA, USA
i Pharmaceutical Chemistry Department, Faculty of Pharmacy, Sphinx University, New Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 5-pyridinyl-1,2,4-triazoles were designed as dual inhibitors of histone deacetylase 2 (HDAC2) and
focal adhesion kinase (FAK). Compounds 5d, 6a, 7c, and 11c were determined as potential inhibitors of
Received 6 April 2021
Received in revised form
26 April 2021
Accepted 11 May 2021
Available online 29 May 2021
both HDAC2 (IC50 ¼ 0.09e1.40
m
M) and FAK (IC50 ¼ 12.59e36.11 nM); 6a revealed the highest activity
with IC50 values of 0.09 M and 12.59 nM for HDAC2 and FAK, respectively. Compound 6a was superior to
m
reference drugs vorinostat and valproic acid in its ability to inhibit growth/proliferation of A-498 and
Caki-1 renal cancer cells. Further investigation proved that 6a strongly arrests the cell cycle at the G2/M
phase and triggers apoptosis in both A-498 and Caki-1 cells. Moreover, the enhanced Akt activity that is
observed upon chronic application of HDAC inhibitors was effectively suppressed by the dual HDAC2/FAK
inhibitor. Finally, the high potency and selectivity of 6a towards HDAC2 and FAK proteins were ratio-
nalized by molecular docking. Taken together, these findings highlight the potential of 6a as a promising
dual-acting HDAC2/FAK inhibitor that could benefit from further optimization.
Keywords:
HDAC2
FAK
1,2,4-Triazoles
Anticancer
Molecular docking
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
therapy suffer from unpredictable pharmacokinetic profile, drug-
drug interactions, and physical incompatibilities, leading to un-
The approach “one agent multiple targets” or “multi-target
agents” gained significant support in the field of drug discovery,
and has come to be known as polypharmacology [1]. It is generally
agreed upon that combination therapy has a considerable thera-
peutic relevance; however, such an approach is not without sig-
nificant disadvantages. For example, patients taking combination
predictable and additive side effects and adverse reactions, un-
foreseen drug inactivation or precipitation of the formulation [2].
On the contrary, a single compound acting on dual or multiple
targets can concurrently modulate various tumor pathways to get
desirable therapeutic efficiency without such additive side effects
[3]. Consequently, designing and developing multi-target drugs
implemented an adequate strategy for cancer therapeutics.
Histone deacetylases (HDACs) are important therapeutic targets
because their inhibition can reverse the aberrant epigenetic states
linked to cancer. The several FDA approved inhibitors of HDAC for
cancer treatment are vorinostat (SAHA), romidepsin, belinostat,
panobinostat, and chidamide (Fig. 1) [4]. The HDAC binding pocket
consists of a hydrophobic cap, linker, and zinc binding group (ZBG).
Fortunately, each part of HDAC pharmacophore can be modified to
* Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
macy, Minia University, Minia, 61519, Egypt.,
** Corresponding author. Department of Cellular and Molecular Medicine, Uni-
versity of California San Diego, La Jolla, CA, USA.
E-mail addresses: aam002@health.ucsd.edu (A.A. Abd El-Hafeez), gamal.
aborahma@mu.edu.eg (G.E.-D.A. Abuo-Rahma).
1
Authors have equally contributed to this work.
https://doi.org/10.1016/j.ejmech.2021.113569
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
allow for the hybridization approach. The cap group can be modi-
fied with a variety of hydrophobic moieties, the linker could be
aliphatic or aromatic, and the ZBG could be a carboxylic acid,
hydroxamic acid, 2-aminobenzamide, or thiol [5]. Among HDAC
inhibitions, those that are selective for the HDAC2 isoform are
particularly important because HDAC2 is overexpressed in several
cancers such as prostate, gastric, non-small lung, colon, hepato-
cellular, and renal [5e11]. In fact, selective inhibition of HDAC2
isoform inhibits tumor growth, promotes apoptosis, and results in
G2/M cell cycle arrest and G1 delay [12,13].
Despite their demonstrated anti-tumor efficacy, most of the
studied HDAC inhibitors are associated with the emergence of
resistance upon prolonged administration, such as Akt kinase
activation and STAT3 phosphorylation [14e19]. Several researchers
embarked upon developing dual or multi-target HDAC inhibitors as
a promising research area. Currently, there are several dual/multi
acting HDAC inhibitors in clinical trials such as CUDC-907 (PI3K and
HDAC), 4SC-202 (LSD1 and HDAC), tinostamustine (alkylating agent
and HDAC), and CUDC-101 (EGFR, HER2, HDAC), each combining a
diverse array of targets with HDACs [4,20,21].
TAE226 are potent FAK kinase inhibitors; however, they did not
enter clinical trials because the former did not show remarkable
antiproliferative activity, and the latter affected the glucose blood
levels and glucose metabolism (Fig. 2) [24]. Four FAK inhibitors
completed phase І clinical trials CEP-37440, PF-562271, PND-1186,
and GSK-2256098, while defactinib (VS-6063) completed phase ІІ
clinical trials [25]. Meanwhile, the five-membered FAK kinase in-
hibitors revealed a substantial ability to inhibit FAK and tumor cells
(Fig. 3) [26]. Several discovered five/six-membered FAK kinase in-
hibitors revealed dual/multi-kinase inhibitory activity due to the
various kinases' structural similarities. However, till now, there are
no reported dual acting FAK inhibitors that target another non-
kinase protein.
1,2,4-triazole and pyridine rings constitute useful heterocyclic
moieties in developing several anticancer agents [27,28]. Besides
their known antiproliferative activity, 1,2,4-triazole and pyridine
bearing compounds showed potent FAK and HDAC inhibitory ac-
tivities. Moreover, pyridine moiety was widely used as a surface
recognition group in developing potent HDAC and FAK inhibitors as
well [5,26,29e33].
Focal adhesion kinase (FAK) is an essential cell signaling regu-
lator inside the microenvironment of the tumor. It plays an indis-
pensable role in regulating tumor cell survival, migration,
metastasis, proliferation, and angiogenesis. FAK is recognized as a
promising therapeutic target as it is overexpressed in several tu-
mors [22,23]. The human FAK gene is comprised of three domains:
an amino-terminal FERM domain, central protein kinase, and a
carboxy-terminal focal adhesion targeting (FAT) domain. Of these
domains, the maximum effort has gone into targeting the kinase
domain. The main pharmacophore of the FAK inhibitors is
comprised of diphenyl five/six-membered nitrogenous heterocy-
cles. The six-membered are the early discovered inhibitors and got
the most attention, whereas the five-membered are growing as
quite promising FAK kinase inhibitors. Compounds PF-573228 and
The administration of HDAC inhibitors is associated with Akt
kinase activation, leading to pro-survival cell elicitation effects,
whereas FAK kinase inhibition leads to Akt suppression [14,15,34].
All the reported dual/multi-acting FAK inhibitors targeted FAK and
another different kinase; these multi-kinase targeting compounds
are prone to have side effects. Because of this, the best is to merge
non-kinase targets such as histone deacetylase with FAK kinase;
unlike the non-selective multi-kinase inhibitors, derivatives that
inhibit non-kinase proteins are expected to show high levels of
specificity.
Given this underwhelming performance of HDAC and FAK in-
hibitors created to date, we set out to design and synthesis of 20
novel dual-acting HDAC2/FAK inhibitors that leverage the prom-
ising properties of 1,2,4-triazole and pyridine heterocyclic rings.
Fig. 1. Chemical structures of the FDA approved HDAC inhibitors.
2

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
The design of the dual inhibitors involved using 5-pyridinyl-1,2,4-
triazole moiety as the surface recognition group and five carbon
atom linker (aliphatic or aromatic) was implemented to the
designed structures, while the ZBG was carboxylic acid, hydroxamic
acid, or 2-aminobenzamide (Fig. 4). Moreover, the variation of
substituents (methyl, ethyl, allyl, or phenyl) was made on N4 of the
1,2,4-triazole ring Table 1.
pyridinyl-1,2,4-triazole derivatives were confirmed by 1HNMR,
13CNMR, and mass spectroscopy and their purity were checked
elemental analysis.
2.2. Biological investigation
2.2.1. In vitro antiproliferative activity
The synthesized compounds were evaluated for their anticancer
2. Results and discussion
activity as a single dose (10 mM) against six different cell lines
derived from three tumor subpanels, including colon (HCT116 and
HT-29), leukemia (K562, and KG-1), and renal (A-498 and Caki-1)
cell lines, in which both HDAC2 and FAK are overexpressed, using
the MTT assay (Table 1) [37e39]. Most compounds showed prom-
ising anticancer activity with growth inhibition percentages.
Among them, compounds 5d, 6a, 7c, and 11c were the most potent
against all the tested cells (GI% ¼ 40.12e99.81%). The renal cancer
cell lines A-498 and Caki-1 were the most sensitive toward 5d, 6a,
7c, and 11c (GI% ¼ 89.34e99.81%). Therefore, we tested the effect of
different concentrations (1, 5, 10, 25, 50, 100, 1000, 2000, and
2.1. Chemistry
The synthesis of the 5-pyridinyl-1,2,4-triazole derivatives 5a-d,
6a-d, 7a-d, 10a-d, and 11a-d (Table 1) is outlined in Scheme 1.
Isonicotinic acid hydrazide 1 was treated with the appropriate
isothiocyanate derivative to give thiosemicarbazide 2. Cyclization
of 2 was accomplished through the addition of 2 N NaOH followed
by acidification with conc. HCl to afford the corresponding 1,2,4-
triazole-3-thiol derivatives 3a-d [35]. Alkylation of 3a-d with
ethyl-6-bromohexanoate or ethyl-4-bromomethyl benzoate in the
presence of triethylamine afforded the ester intermediates 4a-
d and 8a-d, respectively. Hydrolysis of the ester derivatives using
LiOH afforded the corresponding carboxylic acid derivatives 5a-
d and 9a-d, respectively. The acid intermediates 5a-d and 9a-
d were treated with N,N0-carbonyldiimidazole (CDI) in acetonitrile
at room temperature. The resultant imidazolide intermediate was
either treated with hydroxylamine hydroxide to yield the target
hydroxamic acid derivatives 6a-d and 10a-d or with 2-
phenylenediamine in the presence of trifluoroacetic acid to afford
the corresponding 2-aminobenzamide derivatives 7a-d and 11a-d,
respectively [30,36]. Chemical structures of the synthesized 5-
4000 mM) of the synthesized compounds on renal cancer cells (A-
498 and Caki-1), using DMSO as a negative control, whereas VPA,
SAHA, and TAE226 were used as positive controls. The half maximal
inhibitory concentration (IC50) was calculated for each compound
(Table 2). Compounds 5d, 6a, 7c, and 11c displayed significant po-
tency (IC50 ¼ 0.95e2.35
mM) against the tested renal cancer cell
lines. Interestingly, 6a showed a substantial ability to inhibit renal
cancer growth; its IC50 values were comparable to TAE226 and
superior to VPA and SAHA. On the other hand, compounds 7a, 5d,
10a, 10c, 10d, 11a, and 11d showed moderate potency
(IC50 ¼ 6.19e20.85
m
M), whereas the derivatives 5b, 5c, 10b, 6b, 6c,
6d, 7d, and 11b showed low potency (IC50 ¼ 76.36->100
mM). These
Fig. 2. Chemical structure of the six-membered FAK kinase inhibitors.
3

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
Fig. 3. Chemical structure of the five-membered FAK kinase inhibitors.
Fig. 4. Design of the dual acting 5-pyridinyl-1,2,4-triazole derivatives.
results showed that the synthesized compounds possess promising
anticancer activity, especially against renal cancer, among them 5d,
6a, 7c, and 11c were the most potent.
presence of 2-aminobenzamide ZBG is essential for the N4 allyl
substituted derivatives to display potent antiproliferative activity.
The structure-activity relationship revealed that the N4 methyl
derivatives showed promising anticancer activity with the car-
boxylic acid, the hydroxamic acid, and the 2-aminobenzamide ZBGs
regardless of the type of the linker (aliphatic or aromatic). Except
for the aliphatic derivatives bearing hydroxamic acid or the 2-
aminobenzamide motifs, the N4 phenyl substituted derivatives
showed considerable antiproliferative activity, while compounds
containing N4 ethyl did not reveal notable activity. Moreover, the
2.2.2. In vitro HDAC inhibition assay
The HDAC (1, 2, 3, 6, and 8) inhibitory activities of the synthe-
sized 5-pyridinyl-1,2,4-triazole derivatives were evaluated by flu-
orogenic enzymatic assays compared to valproic acid (VPA), TSA,
and SAHA as positive controls. The results are summarized in
Table 3. The dual-acting compounds showed strong to weak
inhibitory activities against HDAC (1, 2, 3, 6, and 8). The IC50 values
revealed that the synthesized compounds were more potent
4

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
Table 1
inhibitory activity (IC50 ¼ 0.090
HDAC inhibitors TSA (IC50 ¼ 0.035
m
m
M), compared to the reference
M) and SAHA (IC50 ¼ 0.096 M),
M). Compound 6a was
Growth inhibition (%) of 5-pyridinyl-1,2,4-triazole derivatives 5a-d, 6a-d, 7a-d, 10a-
d, and 11a-d.
m
and more potent than VPA (IC50 ¼ 102.74
m
Cpd
R
HCT116
HT-29
K562
KG-1
A-498
Caki-1
found to be more selective toward HDAC2 than HDAC1 (~137 fold),
HDAC3 (~38 fold), HDAC6 (~154 fold), and HDAC8 (~79 fold). These
data revealed that using the 5-pyridinyl-1,2,4-triazole scaffold, the
five aliphatic/aromatic carbon linker, and various ZBGs improved
HDAC2 selectivity and retained the antiproliferative activity. The
hydroxamic acid derivative 6a, bearing N4 methyl moiety with the
aliphatic chain, showed the most potent HDAC2 selectivity.
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
10a
10b
10c
10d
11a
11b
11c
11d
Methyl
Ethyl
Allyl
Phenyl
Methyl
Ethyl
20.12
2.15
34.19
9.34
21.25
80.48
68.34
9.15
41.28
1.09
71.36
18.64
95.48
9.64
18.25
1.58
2.95
72.73
40.12
4.09
16.12
8.34
44.94
10.36
86.37
15.14
16
14.29
36.08
39.13
54.09
8.67
55.94
7.34
45.28
4.13
11.36
89.34
99.81
5.26
11.06
4.36
45.34
22.34
97.67
4.67
31.84
1.67
44.85
56.91
55.61
1.18
34.69
1.06
3.25
94.8
98.14
2.06
8.62
7.68
50.82
8.91
95.38
1.65
16.25
78.51
60.14
21.35
2.07
3.56
54.36
6.35
90.49
24.51
32.36
1.03
44.13
29.34
18.03
1.85
15.36
67.19
84.17
17.28
5.91
11.25
63.15
3.64
94.18
34.91
72.08
7.12
61.95
50.94
24.11
5.91
Allyl
Phenyl
Methyl
Ethyl
Allyl
2.2.3. In vitro activity against FAK kinase
Phenyl
Methyl
Ethyl
As our rationale is to target HDAC2/FAK proteins, we evaluated
the synthesized 5-pyridinyl-1,2,4-triazole derivatives for their ac-
tivity against the FAK enzyme using Z0-LYTE® technology, which is
based on FRET (Invitrogen/Life Technologies). TAE226 was used as a
positive control. The results in Table 4 showed that all the syn-
thesized compounds have FAK inhibitory activity in the nanomolar
range (IC50 range 12.59e425.31 nM). Interestingly, with agree with
the HDAC2 inhibition results, 5d, 6a, 7c, and 11c were found to be
the most potent anti-FAK derivatives with IC50 values ranging from
12.6 to 36.11 nM. Compound 6a was also the most potent derivative
in this study against FAK kinase (IC50 ¼ 12.59 nM), and its activity
was found to be comparable to the positive anti-FAK reference,
TAE226 (IC50 ¼ 5.20 nM). Besides, the prepared compounds
showed a superior selectivity on FAK when evaluated against five
different tyrosine kinases (Table S1). These results revealed that the
synthesized compounds have potent anti-FAK activity; among
them, 6a is the most potent.
54
8.34
48.19
9.34
Allyl
25.34
42.67
47.91
1.22
70.67
37.92
50.21
61.85
75.16
12.09
94.81
66.94
Phenyl
Methyl
Ethyl
Allyl
Phenyl
80.46
45.36
90.14
19.6
87.46
47.34
97.08
72.54
toward HDAC2 (IC50 range: 0.09e40.07
range: 8.93->300 M), HDAC3 (IC50 range: 3.41->300
(IC50 range: 6.93->300 M), and HDAC8 (IC50 range: 5.32-
>300 M). Compounds 5d, 6a, 7c, and 11c were the most potent
derivatives in this study toward HDAC2 with IC50 values ranging
from 0.09 to 1.4 M. The other compounds showed moderate to low
mM) than HDAC1 (IC50
m
mM), HDAC6
m
m
m
activities. Moreover, compound 6a exhibited comparable HDAC2
Scheme 1. Synthetic approach of the target HDAC2/FAK inhibitors
(a) RNCS (R ¼ methyl, ethyl, allyl, or phenyl), EtOH, 70 ꢀC, 4h; (b) (i) 2 N NaOH, (ii) HCl, 3h; (c) TEA, CH3CN, 70 ꢀC, 8h; (d) (i) LiOH, EtOH, (ii) HCl; (e) CDI, DMF, NH2OH.HCl, rt, 10h; (f)
CDI, DMF, 2-phenylenediamine, TFA, rt, 10h.
5

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
Table 2
Concentration producing 50% growth inhibition (IC50, mM SEM) of the 5-pyridinyl-1,2,4-triazole derivatives on A-498 and Caki-1 renal cancer cells.
Cpd
R
A-498
Caki-1
Cpd
R
A-498
Caki-1
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
Methyl
Ethyl
Allyl
Phenyl
Methyl
Ethyl
12.3 1.2
16.54 2.49
183.34 5.28
164.3 3.16
1.89 0.54
1.23 0.35
112.03 2.05
89.36 2.56
92.36 3.15
9.87 2.49
98.3 5.30
1.34 0.34
214.97 6.01
10a
10b
10c
10d
11a
11b
11c
11d
Methyl
Ethyl
Allyl
Phenyl
Methyl
Ethyl
Allyl
Phenyl
e
20.85 1.19
203.12 8.37
13.25 2.90
8.76 1.25
9.34 1.04
124.36 3.46
1.42 0.34
11.04 2.15
322.07 7.61
9.56 2.03
7.13 1.53
6.97 1.58
80.67 4.93
1.60 0.51
8.35 1.73
2825.37 22.46
32.46 2.07
1.05 0.34
125.02 5.23
85.34 3.97
2.35 0.36
0.95 0.17
149.35 6.49
76.36 2.37
174.19 5.18
13.40 2.31
77.63 3.08
1.19 0.28
Allyl
Phenyl
Methyl
Ethyl
Allyl
Phenyl
6.19 1.05
VPA
SAHA
TAE226
2141.25 40.36
25.08 3.70
0.81 0.14
e
e
341.85 2.79
Table 3
HDAC inhibitory activities of 5-pyridinyl-1,2,4-triazole derivatives (IC50, mM).
Cpd
R
HDAC1
HDAC2
HDAC3
HDAC6
HDAC8
HDAC1/HDAC2 HDAC3/HDAC2 HDAC6/HDAC2 HDAC8/HDAC2
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
10a
10b
10c
10d
11a
11b
11c
11d
VPA
TSA
SAHA
Methyl 8.93 1.25
7.36 0.36
26.39 2.78
10.12 2.10
0.64 0.15
0.09 0.02
28.93 6.35
11.72 2.30
32.36 2.64
14.36 0.82
27.13 1.72
1.40 0.33
84.60 4.32
156.40 5.94 >300
245.79 4.36 >300
21.10 3.17
3.41 0.36
>300
222.41 9.37 287.96 2.81
>300
84.13 1.39
>300
10.98 1.03
>300
56.39 4.39
174.13 8.69 >300
20.70 0.79
17.96 1.72
71.08 2.79
>300
78.39 2.15
11.95 2.34
>300
>300
11.05 4.37
7.10 0.86
1.21
6.99
9.70
13.98
136.78
2.64
>25.60
>9.27
2.80
11.49
5.93
10.65
>11.37
>29.64
17.02
154.11
>9.32
24.57
7.61
0.99
>11.06
6.99
4.12
3.30
>9.87
0.88
0.71
0.85
>7.49
7.78
1.15
2.09
7.14
0.16
1.62
Ethyl
Allyl
184.36 8.36
98.19 4.37
>11.37
>29.64
17.27
78.89
10.37
0.94
>9.27
0.51
7.30
4.19
>7.75
6.84
>9.87
2.54
1.31
24.29
32.97
37.89
10.37
18.98
>9.27
5.86
>11.06
7.84
>7.75
12.96
5.73
1.85
1.84
5.48
7.49
32.88
13.44
1.31
13.14
0.31
Phenyl 8.95 2.04
Methyl 12.31 1.64
10.89 3.09
13.87 1.54
269.72 11.23 >300
Ethyl
Allyl
76.34 2.15
>300
11.06 0.37
>300
7.28 0.49
198.07 5.32 >11.06
5.86 0.81
>300
29.76 1.87
>300
28.37 6.87
12.76 2.51
7.85 3.19
>300
22.49 3.10
5.32 0.79
184.30 5.16 0.81
Phenyl >300
Methyl 40.18 5.02
246.13 5.19
14.20 2.70
>300
9.79 2.16
159.46 2.38
14.36 2.32
Ethyl
Allyl
>300
31.05 3.40
22.18
6.62
3.26
>9.87
1.75
1.54
0.59
>7.49
9.25
2.64
Phenyl 256.36 11.34 38.73 3.71
Methyl 14.2 1.22
4.35 0.76
30.39 2.49
11.19 2.18
9.76 3.90
12.97 0.63
40.07 5.37
1.28 0.46
8.39 1.08
Ethyl
Allyl
>300
19.60 2.72
9.86 1.28
Phenyl 15.03 2.58
Methyl 7.64 2.79
6.93 0.57
11.03 3.76
>300
0.61
Ethyl
Allyl
>300
11.84 1.63
>7.49
17.57
0.63
1.79
28.00
4.38
42.09 4.72
112.75 8.91 9.63 1.48
9.96 1.29
Phenyl 22.13 3.47
e
e
e
84.21 4.18
0.041 0.012
0.062 0.019
102.74 3.94 134.67 2.87 215.01 8.56
0.035 0.010 0.46 0.18
0.096 0.015 0.03 0.012
0.25 0.094
0.015 0.008
0.98
0.42 0.10
1.17
0.65
2.2.4. Inhibition of HDAC2 expression and FAK phosphorylation
using western blot analysis
To further investigate the biochemical effects of the most active
compounds 5d, 6a, 7c, and 11c on HDAC2 and FAK, Western blot
analysis was performed on A-498 and Caki-1 cells. The obtained
significant percentage of cells in G2/M phase of the cell cycle
compared with the untreated cells. To explore whether the anti-
proliferative activity of the synthesized compounds toward renal
cancer cells (A-498 and Caki-1) was accompanied by an increase in
cancer cell apoptosis, the apoptosis of A-498 and Caki-1 cells
treated with different concentrations of the most potent derivative
data were attuned to b-actin expression to exclude the variations.
Among the tested compounds, 6a maintained a promising HDAC2/
FAK inhibitory activity; Treating cells with 6a decreased the
expression level of HDAC2 and FAK phosphorylation (Fig. 5).
Moreover, 6a effectively reduced their expression in A498 than
Caki-1 cells. Interestingly, FAK autophosphorylation was entirely
blocked by treating A-498 and Caki-1 cells with 7c. Taken together,
the mentioned results demonstrate that 6a represses the expres-
sion of HDAC2 and the phosphorylation FAK.
6a (0.75 and 1.5 mM) was analyzed using Annexin V-FITC/PI assay.
As illustrated in Fig. 6C and D, it shows that 50% of A-498 cells
experienced apoptosis with 6a treatment. Moreover, 6a induced
apoptosis in Caki-1 cells to reach 37% at 1.5 mM concentration.
Table 4
The FAK enzymatic activities of the 5-pyridinyl-1,2,4-triazole derivatives (IC50, nM).
Cpd
R
IC50 (nM)
Cpd
R
IC50 (nM)
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
Methyl
Ethyl
Allyl
Phenyl
Methyl
Ethyl
50.30 2.14
76.21 5.36
124.30 8.71
36.11 1.09
12.59 1.41
124.03 5.79
189.36 6.42
425.31 9.54
57.08 3.04
140.64 6.18
14.30 2.73
7d
Phenyl
Methyl
Ethyl
Allyl
Phenyl
Methyl
Ethyl
Allyl
312.11 3.05
64.35 3.75
254.36 5.28
46.12 2.01
22.40 1.39
53.12 2.91
112.15 5.30
15.60 3.10
96.12 5.84
5.20 0.94
2.2.5. Cell cycle and apoptosis analysis
10a
10b
10c
10d
11a
11b
11c
The effect of compound 6a on cell cycle progression in A-498
and Caki-1 cells was analyzed using flow cytometry (Fig. 6A and B).
Compared to the negative control group, the percentages of cells in
G2/M phase increased substantially from 18% to 65%, in A-498 cells
and from 5.5% to 35%, in Caki-1 cells. Also, the percentage of cells in
G1 phase decreased after treatment with 1.5 mM of compound 6a
for 24 h, whereas the percentage of cells in S phase showed minor
changes. Evidently, the derivative 6a induced the arrest of a
Allyl
Phenyl
Methyl
Ethyl
11d
TAE226
Phenyl
e
Allyl
6

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
2.2.6. Relevance of Akt activation in HDAC inhibitor, VPA, resistance
and relevance of Akt suppression in dual HDAC and FAK inhibitors
sensitivity
induce caspase-8, caspase-9, and caspase-3 activation in A-498 cell
line (Table 5). The exposure of A-498 cells to 6a revealed a sub-
stantial activation of caspase-8 and caspase-9 by 3 and 6-folds,
respectively, compared to the negative control. Interestingly, 6a
activated caspase-3 dramatically by 11-folds in A-498 cells
compared to the negative control. The caspases activation could
explain the ability of the synthesized compounds to induce
apoptosis via caspases pathways.
It was reported that the chronic the HDAC inhibitor, VPA,
application causes resistance via activation of Akt, and it is well
known that FAK plays a vital role in Akt activation. Therefore, FAK
inhibition will overcome HDAC inhibitors' resistance via Akt acti-
vation suppression. We hypothesized that the synthesized dual
HDAC2/FAK inhibitors might not allow resistance development
upon prolonged use due to their ability to inhibit FAK and even-
tually suppress Akt activation. To test our hypothesis, A-498 cells
were exposed to a fixed dose of either the synthesized dual HDAC2/
FAK inhibitor 6a or the HDAC inhibitor VPA for 12 weeks, and then
the antiproliferative activities of 6a and VPA were reassessed. There
was no marked change in the IC50 on 6a-pretreated A-498 cells
2.3. Docking study
To further investigate the broad antiproliferative activity of the
hydroxamic acid derivative 6a, molecular docking was performed
on the crystallographic structures of HDAC2 (PDB code: 4LXZ) and
FAK (PDB code: 2JKK), obtained from the Protein Data Bank (PDB)
[42,43]. The 4LXZ entry was selected for HDAC2 because the protein
is bound with the potent HDAC inhibitor SAHA in this structure.
Likewise, the entry 2JKK was chosen for FAK because, in this case,
the protein is complexed with the potent FAK inhibitor TAE226.
Compound 6a showed several significant interactions within
HDAC2 active site, which may account for its potent inhibitory ac-
tivity (Table 6). The hydroxamic acid moiety (ZBG) exhibited a co-
ordinate bond with Zn atom and formed three hydrogen bonds
with the key amino acids His145, Gly154, and Tyr308 (Fig. 9A).
Besides, Leu276 showed a hydrogen bonding interaction with the
N2 of the 1,2,4-triazole ring. Moreover, the hydrophobic amino
(IC50 ¼ 1
m
M) in comparison with its IC50 in cells that were not
pretreated with 6a (0.95
mM, Fig. 7A). In contrast, a significant
change in the IC50 (from 2.1 mM to 3.09 mM) was observed in cells
that were not pretreated with VPA compared to pretreated cells
(Fig. 7B). These data suggest that there is no observed resistance to
6a in A-498 cells, but the cells got resistant to VPA. Previously, re-
searchers showed that the reason for the VPA resistance is due to
Akt activation after chronic use. To elucidate the molecular mech-
anism by which VPA acquires resistance and 6a maintains its
sensitivity, we examined HDAC2, p-FAK, FAK, p-Akt, and Akt pro-
teins expression in A-498 cells pretreated or not pretreated with
either 6a or VPA for 12 weeks for 24 h. The obtained results
revealed that 6a showed a steady ability to inhibit HDAC2 expres-
sion and FAK phosphorylation for 12 weeks in both pretreated and
not pretreated A-498 cells (Fig. 7C). Also, it showed a substantial
ability to inhibit Akt phosphorylation in both pretreated and not
pretreated experiments. On the contrary, the VPA-pretreated cells
showed a drastic overexpression of Akt phosphorylation, accom-
panied by a lower ability to inhibit HDAC2 expression (Fig. 7D).
acids Phe155, Phe210, and His183 showed
the aliphatic linker (methylene groups). The 1,2,4-triazole and
pyridine rings formed two … H contacts with Phe155 and His33
p … H interactions with
p
amino acids, respectively. Additionally, 6a occupied the ATP bind-
ing site of FAK kinase perfectly and formed approximately the same
interactions as TAE226 (Table 7). Favorably, the nitrogen in the
pyridinyl moiety formed a hydrogen bond with Cys502 of the ki-
nase hinge (Fig. 9B). The pyridine ring formed four
p … H contacts
with Ile428, Cys502, Leu501, and Leu553 amino acids. The sulfur
atom made a hydrogen bond with Gly429 amino acid, and the 5-
pyridinyl-1,2,4-triazole moiety pointed toward the gatekeeper
residue Met499. Moreover, the carbonyl oxygen formed two
hydrogen bonds with Gly563 and Asn551 amino acids. The
hydroxamic acid moiety was also in higher proximity to Asp564 of
the DFG motif of the activation loop.
2.2.7. Compound 6a inhibits STAT3 activation
The transcription factor, STAT3, plays a fundamental role in
carcinogenesis, and is promising target for anticancer drug devel-
opment. Moreover, the activation of STAT3 is well correlated with
the overexpression of HDAC and FAK [17,18]. To elucidate whether
the anticancer effects of 6a are associated with STAT3 inhibition,
this study examined the expression of STAT3. Western blotting
results in Fig. 8 showed the ability of 6a to inhibit the STAT3
activation.
3. Conclusion
HDAC inhibitors comprise a diverse library of scaffolds that is
amenable to several modifications, allowing the hybridization
approach. FAK inhibition is emerging as a promising approach to
treat cancer and prevent metastasis. The major discovery we report
here is the rationale design and validation of a series of novel first-
in-class dual HDAC2/FAK inhibitors containing 5-pyridinyl-1,2,4-
triazole. The structural features of the HDAC scaffold included a
surface recognition group (pyridine moiety), a linker (aliphatic or
aromatic five-carbon linker), and a ZBG (carboxylic, hydroxamic
acids, and 2-aminobenzamide). The prepared compounds showed
remarkable HDAC2 selectivity among five tested HDAC isoforms.
Compounds 5d, 6a, 7c, and 11c showed significant dual inhibition
of HDAC2 and FAK enzymes. The same compounds revealed a
promising ability to inhibit six different cancer cells (HCT116, HT-
29, K562, KG-1, A-498, and Caki-1). The hydroxamic acid deriva-
tive 6a displayed the most potent HDAC2/FAK inhibitory activity
and, subsequently, the highest antiproliferative activity. Unlike VPA,
Western blot analysis revealed that 6a pretreated A-498 cells did
not result in Akt activation, thus decreased the HDAC inhibition
related side effects. Besides, 6a inhibited STAT3 phosphorylation,
induced cellular apoptosis, and arrested the cell cycle. It activated
2.2.8. Activation of different caspases as apoptotic executioners
Activation of caspases (2, 3, 6, 7, 8, 9, and 10) is a prominent
biochemical feature representing a central executioner of apoptosis
mediated by various inducers. HDAC/FAK inhibition is associated
with caspase-8, caspase-9, and caspase-3 activation [40,41].
Consequently, Compound 6a was evaluated for its potential to
Fig. 5. Western blot analysis of HDAC2, FAK, and total FAK in A-498 and Caki-1 cells
treated with 5d, 6a, 7c, and 11c (b-actin was used as loading control).
7

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
Fig. 6. Cell cycle analysis and Flow cytometry of A-498 and Caki-1 cells at 0.75 and 1.5
mM concentrations. A) 6a induces G2/M arrest and decreases G1 phase concomitantly; B)
Increasing the percentage of cells in G2/M phase parallel to increasing 6a concentration; C) 6a induces apoptosis in A-498 and Caki-1 cells; D) Percentage of apoptotic cells triggered
by 6a administration.
caspase-8, caspase-9, and caspase-3. Compound 6a occupied
HDAC2 and FAK active sites and formed all the essential in-
teractions. These results suggest that developing a compound that
Elemental analyses were recorded on Shimadzu GC/MS-QP5050A,
Regional center for Mycology and Biotechnology, Al-Azhar Uni-
versity, Cairo, Egypt. Mass spectra were recorded on Advion
compact mass spectrometer (CMS) and reported as mass/charge
(m/z), [ESI-H] samples were dissolved in methanol þ water þ TEA
could target HDAC/FAK proteins is considered
a promising
approach that warrants further investigation in animal models.
and
[ESI
þ
Na]
samples
were
dissolved
in
methanol þ water þ sodium acetate, Nawah scientific center for
4. Experimental methods
research, Almokattam, Cairo, Egypt.
4.1. Chemistry
4.1.1. General procedure for the synthesis of the compounds 3a-d
The 1,2,4-triazole derivatives were prepared according to the
general procedure in literatures [35]. Equimolar quantities of the
isonicotinic acid hydrazide 1 (0.01 mol) and the appropriate iso-
thiocyanate 2 (0.01 mol) were heated under reflux in absolute
ethanol for 4 h. The solvent was evaporated under vacuum and
100 mL of 2 N NaOH solution was added. The solution was refluxed
for 3 h, then, the reaction mixture was cooled and acidified with
conc. HCl. The formed precipitate was filtered off and recrystallized
from ethanol to afford white solid with 92% yield.
All chemicals were purchased from Aldrich, Merck, Alpha Aesar,
and El-Nasr pharmaceutical chemicals companies, and were used
without further purification. Reactions were monitored by thin
layer chromatography (TLC), using Merck9385 pre-coated
aluminum plate silica gel (Kieselgel 60) 5 ꢁ 20 cm plates with a
layer thickness of 0.2 mm. The spots were detected by exposure to
UV-lamp at 254 nm. Melting points were determined on Stuart
electro thermal melting point apparatus and were uncorrected.
NMR spectra were carried out using a Bruker Avance 400 MHz
1HNMR spectrometer and 100 MHz 13CNMR spectrometer (Beni
Sweif, Egypt), using TMS as internal reference. Chemical shifts (
d
4.1.2. General procedure for the synthesis of the compounds 5a-
d and 9a-d
The prepared 1,2,4-triazoles 3a-d were reacted in equimolar
quantities (1.4 mmol) with the substituted esters (1.4 mmol), in
presence of triethylamine (1.7 mmol) as a base and CH3CN as a
values are given in parts per million (ppm) relative to TMS DMSO‑d6
(2.5 and 3.5 ppm for 1HNMR and 76.9 ppm for 13CNMR) and
coupling constants (J) in Hertz. Splitting patterns are designated as
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
8

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
Fig. 7. Effect of the hydroxamic acid derivative 6a on the pretreated A-498 cells. A) GI% of pretreated and not pretreated A-498 cells with 6a; B) GI% of pretreated and not pretreated
A-498 cells with VPA; C) pretreatment of A-498 cells with 6a causes Akt activation suppression; D) pretreatment of A-498 cells with VPA causes significant Akt activation.
The produced precipitate was recrystallized with aqueous ethanol
with 90e95% yield.
4.1.2.1. 6-((4-methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)hex-
anoic acid 5a. White powder (0.38 g, 92% yield); mp 222e224 ꢀC;
1HNMR (400 MHz, DMSO‑d6)
d (ppm): 1.35e1.39 (m, 2H, CH2
aliphatic), 1.45e1.51 (m, 2H, CH2 aliphatic), 1.63e1.69 (m, 2H, CH2
aliphatic), 2.17 (t, 2H, J¼7.21 Hz, CH2 aliphatic), 3.14 (t, 2H,
J¼7.21 Hz, SeCH2), 3.63 (s, 3H, NeCH3), 7.71 (d, 2H, J¼4.81 Hz,
AreH), 8.72 (d, 2H, J¼5.61 Hz, AreH), 11.98 (s, 1H, COOH); 13CNMR
(100 MHz, DMSO‑d6)
d (ppm): 24.49, 27.98, 29.40, 32.44, 32.99,
34.09, 122.76, 134.99, 150.86, 152.74, 153.71, 174.98 (C]O); Anal.
Calcd for C14H18N4O2S: C, 54.88; H, 5.92; N,18.29; S,10.46. Found: C,
55.12; H, 6.16; N, 18.40; S, 10.53; ESI-MS: [M ꢂ H]-, 305.3, found:
305.2.
Fig. 8. Effect of the hydroxamic acid derivative 6a on STAT3 activation. Treatment of A-
498 cells with 6a causes STAT3 activation suppression.
Table 5
Effect of compound 6a on caspase-8, caspase-9, and caspase-3 activation.
4.1.2.2. 6-((4-ethyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)hex-
anoic acid 5b. White powder (0.39 g, 93% yield); mp 205e207 ꢀC;
Compound
Caspase-8
Caspase-9
Caspase-3 concentration, pg/ml
1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 1.19 (t, 3H, J¼7.21 Hz,
Concentration, ng/ml
NeCH2eCH3), 1.34e1.39 (m, 2H, CH2 aliphatic), 1.45e1.51 (m, 2H,
CH2 aliphatic), 1.65e1.71 (m, 2H, CH2 aliphatic), 2.17 (t, 2H,
J¼7.21 Hz, CH2 aliphatic), 3.19 (t, 2H, J¼7.21 Hz, SeCH2), 4.02 (q, 2H,
J¼7.21 Hz, NeCH2eCH3), 7.66 (d, 2H, J¼5.61 Hz, AreH), 8.73 (d, 2H,
J¼5.61 Hz, AreH), 12.03 (s, 1H, COOH); 13CNMR (100 MHz,
6a
Control
0.888
0.274
28.47
4.53
515.40
45.57
solvent to yield the ester derivatives 4a-d and 8a-d. The corre-
sponding carboxylic acid derivatives 5a-d and 9a-d (1.3 mmol),
were obtained through the hydrolysis of 4a-d and 8a-d with 20
equivalents of LiOH solution, followed by acidification with dil. HCl.
DMSO‑d6)
d (ppm): 15.50, 24.49, 28.01, 29.40, 32.94, 34.02, 122.83,
135.14, 151.01, 152.18, 153.12, 174.97 (C]O); Anal. Calcd for
C
15H20N4O2S: C, 56.23; H, 6.29; N,17.49; S,10.01. Found: C, 56.45; H,
6.38; N, 17.71; S, 9.87; ESI-MS: [M ꢂ H]-, 319.4, found: 319.0.
9

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
Table 6
Energy scores (kcal/mol) and binding features for the HDAC2/FAK inhibitor 6a compared to SAHA, within HDAC2 binding site.
Energy score (S) (kcal/mol)
Ligand-receptor interactions
Residue
Compound
SAHA
Type
Length (Ǻ)
Zn
Coordinate bond
Hydrogen bond
2.56
2.65
3.82
4.06
4.44
His145
Phe155
His183
Phe210
p
p
p
… H
… H
… H
ꢂ9.43
ꢂ8.82
Zn
Coordinate bond
Hydrogen bond
Hydrogen bond
Hydrogen bond
Hydrogen bond
2.69
2.70
1.97
1.78
3.86
4.91
4.20
4.0
His145
Gly154
Tyr308
Leu276
His33
6a
p
p
p
… H
… H
… H
His183
Phe155
Fig. 9. Presumptive binding modes of the compound 6a (magenta). A) Docking pose of 6a within HDAC2 active site (PDB code: 4LXZ); B) Docking pose of 6a within the ATP binding
site of FAK (PDB code: 2JKK).
Table 7
Energy scores (kcal/mol) and binding features for the HDAC2/FAK inhibitor 6a compared to TAE226, within the ATP binding site of FAK.
Energy score (S) (kcal/mol)
Ligand-receptor interactions
Compound
TAE226
Residue
Type
Length (Ǻ)
Cys502
Glu500
Ile428
Asp564
Leu501
Leu553
Hydrogen bond Hydrogen bond
2.99
3.24
3.83
3.00
4.45
3.33
ꢂ9.51
Hydrogen bond
Hydrogen bond
p
p
… H
… H
Cys502
Gly429
Ile428
Asn551
Gly563
Cys502
Ile428
Hydrogen bond
Hydrogen bond
Hydrogen bond
Hydrogen bond
Hydrogen bond
3.27
3.69
4.08
3.24
3.74
4.80
4.10
4.48
3.46
6a
ꢂ8.72
p
p
p
p
… H
… H
… H
… H
Leu501
Leu553
4.1.2.3. 6-((4-allyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)hex-
aliphatic), 2.14 (t, 2H, J¼6.41 Hz, CH2 aliphatic), 3.15 (t, 2H,
J¼7.21 Hz, SeCH2), 4.81 (d, 2H, Jcis¼4.81 Hz, NeCH2eCH]CH2),
4.73 (d, 1H, Jcis¼16.83 Hz, eCH]CH2), 5.17 (d, 1H, Jtrans¼10.42 Hz,
eCH]CH2), 5.88e5.97 (m, 1H, CH2eCH]CH2), 7.61 (d, 2H,
anoic acid 5c. Off white powder (0.41 g, 95% yield); mp 171e173 ꢀC;
1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 1.30e1.35 (m, 2H, CH2
aliphatic), 1.43e1.48 (m, 2H, CH2 aliphatic), 1.61e1.66 (m, 2H, CH2
10

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
J¼5.61 Hz, AreH), 8.69 (d, 2H, J¼5.61 Hz, AreH); 13CNMR (100 MHz,
2H, CH2 aliphatic), 1.48e1.55 (m, 2H, CH2 aliphatic), 1.65e1.73 (m,
2H, CH2 aliphatic), 1.96 (t, 2H, J¼7.23 Hz, CH2 aliphatic), 3.20 (t, 2H,
J¼7.28 Hz, SeCH2), 4.72 (d, 2H, Jcis¼4.40 Hz, NeCH2eCH]CH2),
4.79 (d, 1H, Jcis¼17.45 Hz, eCH]CH2), 5.23 (d, 1H, Jtrans¼10.46 Hz,
eCH]CH2), 5.97e6.02 (m, 1H, CH2eCH]CH2), 7.68 (d, 2H,
J¼5.99 Hz, AreH), 8.75 (d, 2H, J¼5.99 Hz, AreH), 8.72 (s, 1H, NH),
DMSO‑d6)
d (ppm): 24.49, 27.98, 29.36, 33.10, 34.03, 47.11, 117.61,
122.52, 132.80, 134.87, 150.96, 152.97, 153.49, 174.94 (C]O); Anal.
Calcd for C16H20N4O2S: C, 57.81; H, 6.06; N, 16.85; S, 9.64. Found: C,
58.04; H, 6.21; N, 16.97; S, 9.75; ESI-MS: [M ꢂ H]-, 331.4, found:
331.0.
10.38 (s, 1H, OH); 13CNMR (100 MHz, DMSO‑d6)
d (ppm): 25.08,
4.1.2.4. 6-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)
27.97, 29.28, 32.57, 33.05, 47.05, 117.55, 121.67, 122.46, 132.72,
hexanoic acid 5d. White crystals (0.43 g, 90% yield); mp
134.79, 150.77, 150.89, 152.91, 153.43, 169.45 (C]O); Anal. Calcd for
180e182 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 1.26e1.32 (m,
C16H21N5O2S: C, 55.31; H, 6.09; N, 20.16; S9.23. Found: C, 55.60; H,
2H, CH2 aliphatic), 1.42e1.48 (m, 2H, CH2 aliphatic), 1.61e1.67 (m,
2H, CH2 aliphatic), 2.15 (t, 2H, J¼7.21 Hz, CH2 aliphatic), 3.12 (t, 2H,
J¼7.21 Hz, SeCH2), 7.28 (d, 2H, J¼4.81 Hz, AreH), 7.42e7.55 (m, 2H,
AreH), 7.52e7.56 (m, 3H, AreH), 8.5 (d, 2H, J¼5.61 Hz, AreH), 12.01
6.23; N, 20.45; S, 9.26; ESI-MS: [MþNa]þ, 370.1, found: 370.1.
4.1.3.4. N-hydroxy-6-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-
yl)thio)hexanamide 6d. White crystals (0.38 g, 85% yield); mp
(s, 1H, COOH); 13CNMR (100 MHz, DMSO‑d6)
d
(ppm): 24.47, 28.01,
163e165 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm): 1.29e1.35 (m,
29.23, 32.38, 34.00, 122.05, 128.20, 130.68, 130.97, 134.05, 134.47,
150.61, 152.70, 153.91, 174.97 (C]O); Anal. Calcd for C19H20N4O2S:
C, 61.94; H, 5.47; N, 15.21; S, 8.70. Found: C, 61.72; H, 5.70; N, 15.44;
S, 8.92; ESI-MS: [M ꢂ H]-, 367.4, found: 367.2.
2H, CH2 aliphatic), 1.46e1.53 (m, 2H, CH2 aliphatic), 1.64e1.72 (m,
2H, CH2 aliphatic), 1.94 (t, 2H, J¼7.29 Hz, CH2 aliphatic), 3.17 (t, 2H,
J¼7.23 Hz, SeCH2), 7.30 (d, 2H, J¼6.24 Hz, AreH), 7.47e7.50 (m, 2H,
AreH), 7.57e7.61 (m, 3H, AreH), 8.56 (d, 2H, J¼6.24 Hz, AreH), 8.73
(s, 1H, NH); 10.38 (s, 1H, OH); 13CNMR (100 MHz, DMSO‑d6)
4.1.3. General procedure for synthesis of 5-pyridinyl-4H-1,2,4-
triazol-3-thiol derivatives 6a-d, 7a-d, 10a-d, and 11a-d
d (ppm): 25.06, 28.00, 29.14, 32.35, 32.56, 121.99, 128.11, 130.62,
130.91, 133.97, 134.40, 150.53, 152.63, 153.85, 169.46 (C]O); Anal.
Calcd for C19H21N5O2S: C, 59.51; H, 5.52; N, 18.26; S, 8.36. Found: C,
59.74; H, 5.59; N, 18.42; S, 8.45; ESI-MS: [MþNa]þ, 406.4, found:
405.9.
The corresponding carboxylic acid derivatives 5a-d and 9a-
d (1.24 mmol) were treated with CDI (1.6 mmol) in dime-
thylformamide or acetonitrile at 40 ꢀC. The resulting imidazolide
derivative was then reacted with hydroxylamine hydrochloride
(1.6 mmol) to yield the corresponding hydroxamic acid derivatives
6a-d and 10a-d, while treating the imidazolide derivatives with 2-
phenylenediamine (1.6 mmol) in presence of TFA (0.13 g,
1.09 mmol) yielded the corresponding 2-aminobenzamide de-
rivatives. After reaction completion (4e10 h), the resulting mixture
was poured on dist. H2O. The formed precipitate was dried and
recrystallized with aq. Methanol (Compounds 7a-c were purified
with flash column using CH2Cl2:MeOH (9:1) as an eluent.
4.1.3.5. N-(2-aminophenyl)-6-((4-methyl-5-(pyridin-4-yl)-4H-1,2,4-
triazol-3-yl)thio)hexanamide 7a. Brown oil (0.40 g, 81% yield);
1HNMR (400 MHz, DMSO‑d6)
d (ppm): 1.49e1.55 (m, 2H, CH2
aliphatic), 1.64e1.72 (m, 2H, CH2 aliphatic), 1.76e1.83 (m, 2H, CH2
aliphatic), 2.38 (t, 2H, J¼7.34 Hz, CH2 aliphatic), 3.26 (t, 2H,
J¼7.21 Hz, SeCH2), 3.72 (s, 3H, NeCH3), 4.93 (s, 2H, NH2), 6.58 (t,1H,
J¼7.52 Hz, AreH), 6.77 (d, 1H, J¼9.41 Hz, AreH), 6.94 (t, 1H,
J¼7.27 Hz, AreH), 7.20 (d, 1H, J¼9.41 Hz, AreH), 7.81 (d, 2H,
J¼6.11 Hz, AreH), 8.81 (d, 2H, J¼6.11 Hz, AreH), 9.20 (s, 1H, C]
4.1.3.1. N-hydroxy-6-((4-methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-
OeNH); 13CNMR (100 MHz, DMSO‑d6)
d (ppm): 25.24, 28.03, 29.36,
yl)thio)hexanamide 6a. White powder (0.35 g, 88% yield); mp
32.37, 33.02, 36.05, 116.35, 116.64, 122.17, 122.68, 123.98, 125.79,
135.64, 142.38, 150.78, 152.69, 153.65, 171.55 (C]O); Anal. Calcd for
141e143 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 1.34e1.41 (m,
2H, CH2 aliphatic), 1.48e1.56 (m, 2H, CH2 aliphatic), 1.66e1.73 (m,
2H, CH2 aliphatic), 1.96 (t, 2H, J¼7.27 Hz, CH2 aliphatic), 3.19 (t, 2H,
J¼7.21 Hz, SeCH2), 3.68 (s, 3H, NeCH3), 7.76 (d, 2H, J¼6.11 Hz,
AreH), 8.77 (d, 2H, J¼6.11 Hz, AreH), 10.38 (s, 1H, OH); 13CNMR
C20H24N6OS: C, 60.58; H, 6.10; N, 21.20; S, 8.09. Found: C, 60.41; H,
6.28; N, 20.98; S, 8.27; ESI-MS: [MþNa]þ, 419.5, found: 419.2.
4.1.3.6. N-(2-aminophenyl)-6-((4-ethyl-5-(pyridin-4-yl)-4H-1,2,4-
(100 MHz, DMSO‑d6)
d
(ppm): 25.08, 27.96, 29.31, 32.38, 32.58,
triazol-3-yl)thio)hexanamide 7b. Brown oil (0.43 g, 86% yield);
32.97, 122.68, 134.92, 150.79, 152.67, 153.65, 169.45 (C]O); Anal.
Calcd for C14H19N5O2S: C, 52.32; H, 5.96; N, 21.79; S, 9.98. Found: C,
52.49; H, 6.13; N, 22.06; S, 10.04; ESI-MS: [MþNa]þ, 344.4, found:
344.2.
1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 1.24 (t, 3H, J¼7.27 Hz,
CH2eCH3), 1.46e1.50 (m, 2H, CH2 aliphatic), 1.60e1.68 (m, 2H, CH2
aliphatic), 1.74e1.81 (m, 2H, CH2 aliphatic), 2.34 (t, 2H, J¼7.34 Hz,
CH2 aliphatic), 3.27 (t, 2H, J¼7.21 Hz, SeCH2), 4.07 (q, 2H, J¼7.27 Hz,
NeCH2eCH3), 4.84 (s, 2H, NH2), 6.53 (t, 1H, J¼7.52 Hz, AreH), 6.72
(d, 1H, J¼9.54 Hz, AreH), 6.87e6.92 (m, 1H, AreH), 7.16 (d, 1H,
J¼9.54 Hz, AreH), 7.71 (d, 2H, J¼6.11 Hz, AreH), 8.77 (d, 2H,
J¼6.11 Hz, AreH), 9.13 (s, 1H, C]OeNH); 13CNMR (100 MHz,
4.1.3.2. 6-((4-ethyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)-N-
hydroxyhexanamide 6b. White powder (0.36 g, 86% yield); mp
233e235 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm): 1.24 (t, 3H,
J¼7.21 Hz, NeCH2eCH3), 1.36e1.42 (m, 2H, CH2 aliphatic),
1.49e1.57 (m, 2H, CH2 aliphatic), 1.68e1.76 (m, 2H, CH2 aliphatic),
1.96 (t, 2H, J¼7.27 Hz, CH2 aliphatic), 3.23 (t, 2H, J¼7.27 Hz, SeCH2),
4.07 (q, 2H, J¼7.21 Hz, NeCH2eCH3), 7.71 (d, 2H, J¼6.24 Hz, AreH),
8.78 (d, 2H, J¼6.24 Hz, AreH), 8.72 (s, 1H, NH), 10.38 (s, 1H, OH);
DMSO‑d6) d (ppm): 15.45, 25.24, 28.06, 29.37, 32.98, 30.05, 116.33,
116.61, 117.72, 122.75, 123.99, 125.76, 126.18, 135.08, 142.36, 150.94,
152.12, 153.06, 171.51 (C]O); Anal. Calcd for C21H26N6OS: C, 61.44;
H, 6.38; N, 20.47; S, 7.81. Found: C, 61.68; H, 6.62; N, 20.71; S, 7.94;
ESI-MS: [MþNa]þ, 433.5, found: 433.3.
13CNMR (100 MHz, DMSO‑d6)
d (ppm): 15.44, 25.09, 28.00, 29.32,
32.58, 32.91, 122.75, 135.08, 150.94, 152.12, 153.06, 169.45 (C]O);
Anal. Calcd for C15H21N5O2S: C, 53.71; H, 6.31; N, 20.88; S, 9.56.
Found: C, 53.98; H, 6.52; N, 20.74; S, 9.67; ESI-MS: [MþNa]þ, 358.4,
found: 357.9.
4.1.3.7. 6-((4-allyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)-N-(2-
aminophenyl)hexanamide 7c. Brown oil (0.42 g, 92% yield); 1HNMR
(400 MHz, DMSO‑d6)
d (ppm): 1.44e1.50 (m, 2H, CH2 aliphatic),
1.60e1.67 (m, 2H, CH2 aliphatic), 1.72e1.79 (m, 2H, CH2 aliphatic),
2.34 (t, 2H, J¼7.34 Hz, CH2 aliphatic), 3.24 (t, 2H, J¼7.21 Hz, SeCH2),
4.72 (d, 2H, Jcis¼4.52 Hz, NeCH2eCH]CH2), 4.78e4.88 (m, 3H,
Jcis¼17.24 Hz, eCH]CH2 and NH2), 5.24 (d, 1H, Jtrans¼10.39 Hz,
eCH]CH2), 5.94e6.03 (m, 1H, CH2eCH]CH2), 6.54 (t, 1H,
4.1.3.3. 6-((4-allyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)-N-
hydroxyhexanamide 6c. Off white powder (0.35 g, 82% yield); mp
143e145 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm): 1.33e1.40 (m,
11

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
J¼7.46 Hz, AreH), 6.72 (d, 1H, J¼9.54 Hz, AreH), 6.90 (t, 1H
J¼7.40 Hz, AreH), 7.16 (d, 1H, J¼7.95 Hz, AreH), 7.69 (d, 2H,
J¼6.24 Hz, AreH), 8.76 (d, 2H, J¼6.24 Hz, AreH), 9.12 (s, 1H, C]
4.1.3.12. N-hydroxy-4-(((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-
3-yl)thio)methyl)benzamide 10d. White powder (0.48 g, 92% yield);
mp 145e147 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm): 4.54 (s, 2H,
OeNH); 13CNMR (100 MHz, DMSO‑d6)
d
(ppm): 24.78, 27.58, 28.88,
SeCH2), 7.32e7.35 (m, 2H, AreH), 7.43e7.51 (m, 4H, AreH),
7.61e7.65 (m, 3H, AreH), 7.74 (d, 2H, J¼5.14 Hz, AreH), 8.61 (d, 2H,
J¼6.11 Hz, AreH), 9.11 (s, 1H, NH), 11.27 (s, 1H, OH); 13CNMR
32.67, 35.60, 46.59, 115.89, 116.18, 117.11, 121.76, 122.02, 123.44,
125.73, 132.28, 133.91, 134.12, 135.20, 141.88, 150.45, 170.79 (C]O);
Anal. Calcd for C22H26N6OS: C, 62.53; H, 6.20; N, 19.89; S, 7.59.
Found: C, 62.77; H, 6.39; N, 20.06; S, 7.73; ESI-MS: [MþNa]þ, 445.5,
found: 445.3.
(100 MHz, DMSO‑d6)
d (ppm): 36.01, 121.98, 127.50, 128.04, 129.49,
130.61, 130.95, 132.37, 133.80, 134.31, 140.86, 150.59, 152.81, 153.22,
164.37 (C]O); Anal. Calcd for C21H17N5O2S: C, 62.52; H, 4.25; N,
17.36; S, 7.95. Found: C, 62.70; H, 4.42; N, 17.59; S, 8.06; ESI-MS:
[M ꢂ H]-, 402.4, found: 402.2.
4.1.3.8. N-(2-aminophenyl)-6-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-
triazol-3-yl)thio)hexanamide 7d. yellowish brown powder (0.48 g,
92% yield); mp 179e181 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm):
4.1.3.13. N-(2-aminophenyl)-4-(((4-methyl-5-(pyridin-4-yl)-4H-
1,2,4-triazol-3- yl)thio)methyl)benzamide 11a. Light yellow crystals
(0.47 g, 91% yield); mp 244e246 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
1.37e1.44 (m, 2H, CH2 aliphatic), 1.57e1.65 (m, 2H, CH2 aliphatic),
1.70e1.78 (m, 2H, CH2 aliphatic), 2.32 (t, 2H, J ¼ 7.40 Hz, CH2
aliphatic), 3.20 (t, 2H, J ¼ 7.21 Hz, SeCH2), 4.83 (s, 2H, NH2), 6.53 (t,
1H, J¼7.52 Hz, AreH), 6.72 (d, 1H, J¼9.54 Hz, AreH), 6.89 (t, 1H
J¼7.58 Hz, AreH), 7.15 (d, 1H, J¼9.54 Hz, AreH), 7.30 (d, 2H,
J¼6.24 Hz, AreH), 7.47e7.50 (m, 2H, AreH), 7.56e7.61 (m, 3H,
AreH), 8.57 (d, 2H, J¼6.24 Hz, AreH), 9.11 (s, 1H, C]OeNH);
d
(ppm): 3.65 (s, 3H, NeCH3), 4.55 (s, 2H, SeCH2), 4.94 (s, 2H, NH2),
6.64 (t, 1H, J¼7.52 Hz, AreH), 6.83 (d, 1H, J¼8.07 Hz, AreH), 7.02 (t,
1H, J¼7.85 Hz, AreH), 7.20 (d, 1H, J¼7.83 Hz, AreH), 7.56 (d, 2H,
J¼6.24 Hz, AreH), 7.78 (d, 2H, J¼4.28 Hz, AreH), 7.97 (d, 2H,
J¼8.31 Hz, AreH), 8.81 (d, 2H, J¼4.28 Hz, AreH), 9.70 (s, 1H, C]
OeNH); 13CNMR (100 MHz, DMSO‑d6)
d (ppm): 32.43, 36.90, 116.57,
13CNMR (100 MHz, DMSO‑d6)
d (ppm): 25.23, 28.07, 29.21, 32.38,
116.71, 122.66, 123.68, 127.02, 127.22, 128.44, 129.38, 134.20, 134.82,
141.24, 143.66, 150.83, 151.87, 153.85, 165.42 (C]O); Anal. Calcd for
36.05, 116.34, 116.63, 121.98, 123.99, 125.75, 126.17, 128.12, 130.61,
130.89, 133.99, 134.41, 142.35, 150.54, 152.63, 153.85, 171.50 (C]O);
Anal. Calcd for C25H26N6OS: C, 65.48; H, 5.71; N, 18.33; S, 6.99.
Found: C, 65.74; H, 5.98; N, 18.50; S, 7.02; ESI-MS: [MþNa]þ, 481.5,
found: 481.3.
C
22H20N6OS: C, 63.44; H, 4.84; N, 20.18; S, 7.70. Found: C, 63.71; H,
4.95; N, 20.37; S, 7.65; ESI-MS: [MþNa]þ, 439.5, found: 439.2.
4.1.3.14. N-(2-aminophenyl)-4-(((4-ethyl-5-(pyridin-4-yl)-4H-1,2,4-
triazol-3-yl)thio)methyl)benzamide 11b. Brown oil (0.39 g, 73%
4.1.3.9. N-hydroxy-4-(((4-methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-
yield); 1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 1.16 (t, 3H, J¼7.34 Hz,
3-yl)thio)methyl)benzamide 10a. White powder (0.36 g, 81% yield);
CH2eCH3), 4.01 (q, 2H, J¼7.34 Hz, CH2eCH3), 4.58 (s, 2H, SeCH2),
4.93 (s, 2H, NH2), 6.61 (t, 1H, J¼7.70 Hz, AreH), 6.80 (d, 1H,
J¼8.19 Hz, AreH), 6.98 (t, 1H, J¼7.70 Hz, AreH), 7.18 (d, 1H,
J¼8.19 Hz, AreH), 7.55 (d, 2H, J¼8.07 Hz, AreH), 7.70 (d, 2H,
J¼5.50 Hz, AreH), 7.96 (d, 2H, J¼8.31 Hz, AreH), 8.78 (d, 2H,
J¼5.50 Hz, AreH), 9.71 (s, 1H, C]OeNH); 13CNMR (100 MHz,
mp 190e192 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm): 3.64 (s, 3H,
NeCH3), 4.53 (s, 2H, SeCH2), 7.51 (d, 2H, J¼6.24 Hz, AreH), 7.75 (d,
2H, J¼6.24 Hz, AreH), 7.79 (d, 2H, J¼4.28 Hz, AreH), 8.83 (d, 2H,
J¼4.28 Hz, AreH), 9.1 (s, 1H, NH), 11.27 (s, 1H, OH); 13CNMR
(100 MHz, DMSO‑d6)
d (ppm): 32.38, 36.84, 122.67, 127.52, 129.94,
132.39, 135.47, 140.49, 150.83, 152.34, 155.07, 164.35 (C]O); Anal.
Calcd for C16H15N5O2S: C, 56.29; H, 4.43; N, 20.51; S, 9.39. Found: C,
56.61; H, 4.56; N, 20.74; S, 9.51; ESI-MS: [M ꢂ H]-, 340.3, found:
340.0.
DMSO‑d6)
d (ppm): 15.44, 36.86, 39.34, 116.59, 116.72, 122.73,
123.70, 127.01, 127.21, 128.46, 129.37, 134.21, 134.99, 141.25, 143.65,
150.98,151.40,153.23,165.42 (C]O); Anal. Calcd for C23H22N6OS: C,
64.17; H, 5.15; N, 19.52; S, 7.45. Found: C, 64.40; H, 5.39; N, 19.78; S,
7.62; ESI-MS: [MþNa]þ, 453.5, found: 453.2.
4.1.3.10. 4-(((4-ethyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)
methyl)-N-hydroxybenzamide 10b. White powder (0.35 g, 76%
4.1.3.15. 4-(((4-allyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)
methyl)-N-(2-aminophenyl)benzamide 11c. Yellowish brown pow-
der (0.51 g, 93% yield); mp 182e184 ꢀC; 1HNMR (400 MHz,
yield); mp 198e200 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d (ppm): 1.17
(t, 3H, J¼7.27 Hz, NeCH2eCH3), 4.03 (q, 2H, J¼7.27 Hz,
NeCH2eCH3), 4.58 (s, 2H, SeCH2), 7.52 (d, 2H, J¼8.31Hz, AreH),
7.73e7.76 (m, 4H, AreH), 8.82 (d, 2H, J¼6.11 Hz, AreH), 9.10 (s, 1H,
DMSO‑d6)
d
(ppm): 4.61 (s, 2H, SeCH2), 4.71 (d, 2H, J¼4.52 Hz,
NeCH2eCH]CH2), 4.79 (d, 1H, Jcis¼17.24 Hz, eCH]CH2), 4.96 (s,
2H, NH2), 5.24 (d, 1H, Jtrans¼10.64 Hz, eCH]CH2), 5.92e6.01 (m,
1H, CH2eCH]CH2), 6.65 (t, 1H, J¼7.58 Hz, AreH), 6.83 (d, 1H,
J¼8.07 Hz, AreH), 7.03 (t, 1H, J¼7.58 Hz, AreH), 7.21 (d, 1H,
J¼7.95 Hz, AreH), 7.59 (d, 2H, J¼8.19 Hz, AreH), 7.71 (d, 2H,
J¼4.65 Hz, AreH), 7.99 (d, 2H, J¼8.19 Hz, AreH), 8.80 (d, 2H,
J¼4.65 Hz, AreH), 9.71 (s, 1H, C]OeNH); 13CNMR (100 MHz,
NHeNH), 11.26 (s, 1H, OH); 13CNMR (100 MHz, DMSO‑d6)
d (ppm):
15.38, 36.89, 39.35, 122.73, 127.54, 129.47, 132.39, 135.00, 141.01,
150.97, 151.39, 153.22, 164.33 (C]O); Anal. Calcd for C17H17N5O2S:
C, 57.45; H, 4.82; N, 19.71; S, 9.02. Found: C, 57.72; H, 5.06; N, 19.97;
S, 9.14; ESI-MS: [M ꢂ H]-, 354.4, found: 353.8.
4.1.3.11. 4-(((4-allyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)
DMSO‑d6) d (ppm): 36.84, 47.07, 116.57, 116.71, 117.71, 122.49,
methyl)-N-hydroxybenzamide 10c. White powder (0.41 g, 86%
123.69, 127.01, 127.20, 128.43, 129.38, 132.62, 134.22, 134.69, 141.15,
143.65, 150.93, 152.20, 153.60, 165.42 (C]O); Anal. Calcd for
yield); mp 194e196 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
d
(ppm): 4.52
(s, 2H, SeCH2), 4.64 (d, 2H, J¼4.65 Hz, NeCH2eCH]CH2), 4.73 (d,
1H, Jcis¼17.24 Hz, eCH]CH2), 5.17 (d, 1H, Jtrans¼10.39 Hz, eCH]
CH2), 5.86e5.94 (m, 1H, CH2eCH]CH2), 7.48 (d, 2H, J¼8.29 Hz,
AreH), 7.66 (d, 2H, J¼6.11 Hz, AreH), 7.70 (d, 2H, J¼8.31 Hz, AreH),
8.76 (d, 2H, J¼6.11 Hz, AreH), 9.05 (s, 1H, NH), 11.21 (s, 1H, OH);
C24H22N6OS: C, 65.14; H, 5.01; N, 18.99; S, 7.24. Found: C, 65.38; H,
5.17; N, 19.23; S, 7.45; ESI-MS: [MþNa]þ, 465.5, found: 465.2.
4.1.3.16. N-(2-aminophenyl)-4-(((4-phenyl-5-(pyridin-4-yl)-4H-
1,2,4-triazol-3-yl)thio)methyl)benzamide 11d. Off white powder
(0.54 g, 91% yield); mp 230e232 ꢀC; 1HNMR (400 MHz, DMSO‑d6)
13CNMR (100 MHz, DMSO‑d6)
d (ppm): 36.83, 47.04, 117.67, 122.49,
127.52,129.48,132.40,132.59,134.69,140.89,150.92,152.20,153.59,
164.34 (C]O); Anal. Calcd for C18H17N5O2S: C, 58.84; H, 4.66; N,
19.06; S, 8.73. Found: C, 59.11; H, 4.83; N, 19.28; S, 8.59; ESI-MS:
[MþNa]þ, 390.4, found: 390.2.
d (ppm): 4.58 (s, 2H, SeCH2), 4.96 (s, 2H, NH2), 6.65 (t, 1H,
J¼7.58 Hz, AreH), 6.83 (d, 1H, J¼7.95Hz, AreH), 7.02 (t, 1H,
J¼7.58 Hz, AreH), 7.21 (d, 1H, J¼7.82 Hz, AreH), 7.35 (d, 2H,
J¼6.24 Hz, AreH), 7.47 (d, 2H, J¼8.19 Hz, AreH), 7.56e7.64 (m, 5H,
12

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
AreH), 7.97 (d, 2H, J¼8.19 Hz, AreH), 8.62 (d, 2H, J¼4.52 Hz, AreH),
45 min. Fluorescence measurements were obtained with Synergy 4
Hybrid microplate reader (Bio-Tek) at excitation wavelength
360 nm and emission wavelength 460 nm. On the other hand, to
determine the ability of the synthesized compounds to inhibit the
activity of HDAC6, we used fluorogenic Assay kit (BPS Biosciences)
per the manufacturer's instructions. Valproic acid, trichostatin A
and SAHA were utilized as inhibitor positive controls. The experi-
ments were repeated at least three times with each experiment
containing three replicates. The IC50 values were determined using
Prism v.8 software.
9.70 (s, 1H, C]OeNH); 13CNMR (100 MHz, DMSO‑d6)
d (ppm):
34.07, 114.63, 114.77, 120.03, 121.77, 125.06, 125.26, 126.10, 126.46,
127.44, 128.70, 129.02, 131.88, 132.35, 139.18, 141.70, 148.64, 148.73,
150.87, 151.26, 163.50 (C]O); Anal. Calcd for C27H22N6OS: C, 67.76;
H, 4.63; N, 17.56; S, 6.70. Found: C, 68.02; H, 4.80; N, 17.82; S, 6.83;
ESI-MS: [MþNa]þ, 501.5, found: 501.2.
4.2. Biological investigations
4.2.1. Cell culture and reagents
Six different cell lines derived from three tumor subpanels,
including colon (HCT116 and HT-29), leukemia (K562, and KG-1),
and renal (A-498 and Caki-1) cell lines were obtained from the
American Type Culture Collection (ATCC; Manassas, VA, USA) and
were cultured in their suitable media containing 10% fetal bovine
4.2.4. Kinase selectivity assays
IC50 values of the synthesized compounds and TAE226 on five
different kinases (FAK, FLT, FRK, IGF-1R, and BTK) were determined
using Z0-LYTE® technology, which is based on FRET (Invitrogen/Life
Technologies).
serum (FBS; Sigma-Aldrich), 100 U/ml penicillin, and 100
mg/ml
streptomycin (Life technologies) in a humidified atmosphere with
5% CO2 at 37 ꢀC. 5-pyridinyl-1,2,4-triazole derivatives were chem-
ically synthesized (5a-d, 6a-d, 7a-d, 10a-d, and 11a-d). All chem-
icals were purchased from Sigma-Aldrich Co., LLC except TAE226
was purchased from Selleck Chemicals (Houston, TX, USA). All
chemicals used in this study were of the analytical or cell-culture
grade.
4.2.5. Western blotting
A-498 and Caki-1 cells were harvested, washed twice with
chilled PBS, and lysed with ice-cold lysis buffer containing 0.1% SDS,
150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mg/ml aprotinin, 5 mg/
ml pefabloc SC (4-(2-Aminoethyl) benzenesulfonyl fluoride hidro-
chloride), a protease inhibitor cocktail, 1% phosphatase inhibitor
cocktail and 50 mM TriseHCl (pH 7.4) after 24 h incubation with the
IC50 value of the potent compounds (5d, 6a, 11c, and 7c) or DMSO
(control). The cell lysates were kept on ice for 30 min after gently
vortex, and then centrifuged at 14,000g for 15 min at 4 ꢀC. The
supernatants were loaded onto SDS-PAGE gel immediately after
protein extraction, or otherwise the supernatant was stored
at ꢂ80 ꢀC until use. The protein concentration was determined by
Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) according
to the manufacturer's instructions. For western blotting analysis,
4.2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
The proliferation assay for six different cell lines derived from
three tumor subpanels, including colon (HCT116 and HT-29), leu-
kemia (K562, and KG-1), and renal (A-498 and Caki-1) cell lines was
quantified using MTT assay. Cells were seeded as 1 ꢁ 104 cells/well
and cultured overnight in a 96-well plate. The cells were treated
with either 10
mМ of 5-pyridinyl-1,2,4-triazole derivatives (5a-d,
40 mg protein-weight of cell lysate were loaded onto a SDS-PAGE
6a-d, 7a-d, 10a-d, and 11a-d), or DMSO as a negative control. After
24 h, the cells washed with phosphate buffered saline (PBS, Invi-
gel. Proteins separated on a SDS-PAGE gel were transferred onto a
PVDF membrane. The PVDF membrane was incubated in blocking
buffer containing either 5% non-fat milk powder or 5% bovine
serum albumin (Sigma-Aldrich) for 1 h. Subsequently, the PVDF
membrane was incubated with primary antibodies for overnight
and followed by HRP-conjugated anti-rabbit IgG (Cell Signaling
Technologies) or HRP- conjugated anti-mouse IgG (Becton Dick-
inson Co, Durham, NC, USA) for 1 h with agitation at room tem-
perature. Finally, the specific bindings to each primary antibody
were detected on an X-ray film (Konica Minolta Medical Imaging,
Wayne, NJ, USA) with ECL prime immunodetection reagent (GE
Healthcare, Little Chalfont, UK).
trogen Gibco) and incubated with 20
ml of MTT solution (2 mg/ml)
for 4 h at 37 ꢀC. Then, 150
ml DMSO was used to solubilize MTT
formazan crystals. Finally, the plates were shaken, and the optical
density was determined at 570 nm using ELISA plate reader (Model
550, Bio-Rad, USA). At least, three independent experiments were
performed. Percentage of growth inhibition was determined as (1ꢂ
[OD of treated cells/OD of control cells]). On the other hand, using
the MTT assay, we tested the effect of different concentrations (1, 5,
10, 25, 50, 100, 1000, 2000, and 4000
mM) of the synthesized
compounds on renal cancer cells (A-498 and Caki-1), using DMSO
as a negative control, whereas VPA, SAHA, and TAE226 were used as
positive controls. The IC50 values were calculated using Prism v.8
software (GraphPad Software Inc., La Jolla, CA).
4.2.6. Cell cycle assay
The flow cytometer was used to support the anti-proliferative
activity of the synthesized compounds towards A-498 and Caki-
1 cells. Briefly, cells at a density of 1 ꢁ 105 cells/mL were cultured
4.2.3. Fluorogenic enzymatic assays for HDAC isoforms
To determine the ability of the synthesized compounds to
inhibit the activity of HDACs, we did invitro optimize fluorogenic
enzymatic assays for HDAC isoforms in 96-well opaque half-area
microplate (Corning). Recombinant, full-length human HDAC1, 2,
3, and 8 (BPS Bioscience, CA, USA) were diluted in assay buffer A
(25 mM TriseHCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and
1 mg/mL BSA, pH 8.0) to give 4, 5, 1, 8.5 ng/mL stock solutions,
respectively. Serial dilutions of inhibitors were made in assay buffer
B (25 mM TriseHCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, pH
with different concentrations of 6a (0.75 and 1.5 mM) or DMSO as a
negative control and incubated for 24 h. The cells were harvested
and washed with PBS. Subsequently, 80% ice cold ethanol was
added to the cell pellets drop by drop with continuous vortexing to
prevent clumping and aggregation of cells and then kept at 20+C
for overnight. Then, the cell pellets washed twice, stained with PI,
and incubated in the dark for 20 min. Flow cytometric analysis was
conducted using FACS Calibur flow cytometer (BD Biosciences, San
Jose, CA, USA).
8.0). Then, we mixed 10
hibitor and incubated for 5 min at room temperature. After incu-
bation, 10 L of HDAC substrate was added and the mixture
incubated for 45 min at room temperature. For HDAC1, 2, 3 and 8,
the reaction was quenched with trypsin and trichostatin A dis-
solved in assay buffer B and incubated at room temperature for
mL of each enzyme stock to 30 mL of in-
4.2.7. Apoptosis assay
m
Induction of apoptosis of A-498 and Caki-1 cells was analyzed
using the annexin V/propidium iodide (PI) staining kit (BioLegend,
San Diego, CA, USA) according to the manufacturer's instructions.
A-498 and Caki-1 cells were treated with different concentrations
13

M. Mustafa, A.A. Abd El-Hafeez, D. Abdelhamid et al.
European Journal of Medicinal Chemistry 222 (2021) 113569
of 6a (0.75 and 1.5
m
M) or DMSO as a negative control and incu-
thus validating the docking using MOE. Compound 6a was then
docked into the binding sites using the Alpha triangle placement
method. Refinement was carried out using Forcefield and scored
using the Affinity dG scoring system. The resulting docking poses
were visually inspected, and the pose of the lowest binding free
energy value was considered.
bated for 24 h. Flow cytometric analysis was conducted using FACS
Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Quan-
titative analysis of the FACS data was performed with FlowJo soft-
ware (FlowJo, Ashland, OR, USA).
4.2.8. Resistance development assays
To examine whether A-498 cells can develop resistance to 6a or
valproic acid, A-498 cells were exposed to 30% of IC50 of 6a or
valproic acid for 12 weeks, followed by treatment with different
5. Statistical analysis
Data are represented as means SEM. Statistical significance
was assessed using one-way analysis of variance (ANOVA)
including a Tukey's test for multiple comparisons. *p < 0.05,
**p < 0.01, and ***p < 0.001. The data shown in the figures are
representative data for three independent experimental results.
concentrations of 6a (0.25, 0.5, 1, 5, and 10 mM) or valproic acid
(VPA; 0.25, 0.5, 1, 2, and 4 mM) or DMSO as a negative control for
24 h. Then, the cells were subjected to the MTT assay to detect cell
growth inhibition or to the Western blot assay to detect HDAC2, p-
FAK, FAK, p-Akt, and Akt proteins expression. Control cell cultures
were not pretreated with 6a or VPA.
Declaration of competing interest
4.2.9. Determination of caspase-8 & caspase-9
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
The determination of caspase-8 and caspase-9 was performed
using human caspase-8 ELISA kit (EIA-4863) and human caspase-9
ELISA kit DRG® (EIA-4860) (DRG International Inc., USA). Cells were
lysed with cell extraction buffer. The lysate was diluted in standard
diluent buffer over the range of the assay and measured for human
caspase-8 and caspase-9 content, separately. For each assay, the
cells were plated in a density of 1.2e1.8 ꢁ 10,000 cells/well in a
Acknowledgements
A.A.A was supported by an NIH-funded Cancer Therapeutics
Training Program (CT2, T32 CA121938) and P.G by the NIH
(CA238042, CA100768 and CA160911).
volume of 100
m
l complete growth medium þ100
mL of the tested
compound per well in a 96-well plate for 24 h before the enzyme
assay for caspase-8 and caspase-9 according to the manufacturer's
protocol. Finally, the absorbance of each microwell was read using a
microplate reader set at 450 nm.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113569.
4.2.10. Determination of caspase-3
The level of human active caspase-3 protein was evaluated using
Invitrogen (Catalog KHO1091) ELISA kit. The manufacturer's in-
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