Design and discovery of new antiproliferative 1,2,4-triazin-3(2H)-ones as tubulin polymerization inhibitors targeting colchicine binding site

Article abstract of DOI:10.1016/j.bioorg.2021.104965

Thirty-five new colchicine binding site inhibitors have been designed and synthesized based on the 1,2,4-triazin-3(2H)-one nucleus. Such molecules were synthesized through a cascade reaction between readily accessible α-amino ketones and phenyl carbazate

Full text of DOI:10.1016/j.bioorg.2021.104965

Bioorganic Chemistry 112 (2021) 104965
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and discovery of new antiproliferative 1,2,4-triazin-3(2H)-ones as
tubulin polymerization inhibitors targeting colchicine binding site
,
,b,
Ibrahim H. Eissaa *, Mohammed A. Dahaba *, Mohamed K. Ibrahima, Nawaf A. Alsaifc, A.
Z. Alanazid, Sally I. Eissae , Ahmed B.M. Mehany , Andre M. Beauchemin
,
f
g
b
´
a Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, 11884, Egypt
b Centre for Catalysis Research and Innovation, Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ontario K1N6N5, Canada
c Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
d Department of pharmacology and toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
e Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
f Department of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Dariyah, Riyadh, 13713, Saudi Arabia
g Department of Zoology, Faculty of Science (Boys), Al-Azhar University, Cairo, 11884, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thirty-five new colchicine binding site inhibitors have been designed and synthesized based on the 1,2,4-triazin-
Anti-cancer
3(2H)-one nucleus. Such molecules were synthesized through a cascade reaction between readily accessible
Colchicine analogues
Tubulin polymerization
Docking
α
-amino ketones and phenyl carbazate as a masked N-isocyanate precursor. The synthesized derivatives are
cisoid restricted combretastatin A4 analogues containing 1,2,4-triazin-3(2H)-one in place of the olefinic bond,
and they have the same essential pharmacophoric features of colchicine binding site inhibitors. The synthesized
compounds were evaluated in vitro for their antiproliferative activities against a panel of three human cancer cell
lines (MCF-7, HepG-2, and HCT-116), using colchicine as a positive control. Among them, two compounds 5i and
6i demonstrated a significant antiproliferative effect against all cell lines with IC50 ranging from 8.2 ꢀ 18.2 µM.
Further investigation was carried out for the most active cytotoxic agents as tubulin polymerization inhibitors.
Compounds 5i and 6i effectively inhibited microtubule assembly with IC50 values ranging from 3.9 to 7.8 µM.
Tubulin polymerization assay results were found to be comparable with the cytotoxicity results. The cell cycle
analysis revealed significant G2/M cell cycle arrest of the analogue 5i in HepG-2 cells. The most active com-
pounds 4i, 4j, 5 g, 5i and 6i did not induce significant cell death in normal human lung cells Wl-38, suggesting
their selectivity against cancer cells. Also, These compounds upregulated the level of active caspase-3 and
boosted the levels of the pro-apoptotic protein Bax by five to seven folds in comparison to the control. Moreover,
apoptosis analyses were conducted for compound 5i to evaluate its apoptotic potential. Finally, in silico studies
were conducted to reveal the probable interaction with the colchicine binding site. ADME prediction study of the
designed compounds showed that they are not only with promising tubulin polymerization inhibitory activity
but also with favorable pharmacokinetic and drug-likeness properties.
1,2,4-Triazin-3(2H)-ones
1. Introduction
(FMF) [3-5]. Additionally, due to the anti-fibrotic and anti-
inflammatory activities, the therapeutic use of colchicine has extended
Microtubule dynamics is an important target for developing anti-
cancer agents [1]. The tubulin heterodimer contains three distinct drug
binding sites: the paclitaxel (taxanes alkaloid), vinblastine (vinca alka-
loid), and colchicine binding sites (CBS) [2]. Colchicine I (Fig. 1) is the
oldest antimitotic agent known and it has been the first line therapy for
the treatment of acute gouty arthritis and familial Mediterranean fever
beyond gouty arthritis, to atherosclerosis, osteoarthritis, and pericarditis
[6-10]. However, due to the efficiency and high inhibition observed for
tubulin polymerization, hundreds of analogues have been prepared
recently to maintain colchicine’s antimitotic abilities but reduce toxicity
to the biological organism [11-16]. Colchicine binding site inhibitors
(CBSIs) exert their biological effects by inhibiting tubulin assembly and
* Corresponding authors at: Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, 11884,
Egypt (I.H. Eissa and M.A. Dahab).
E-mail addresses: Ibrahimeissa@azhar.edu.eg (I.H. Eissa), mohammeddahab@azhar.edu.eg (M.A. Dahab).
https://doi.org/10.1016/j.bioorg.2021.104965
Received 4 September 2020; Received in revised form 7 April 2021; Accepted 3 May 2021
Available online 5 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
suppressing microtubule formation [17]. Colchicine itself binds to
tubulin very tightly, but neither colchicine nor compounds that bind to
the colchicine binding site on tubulin have yet found significant use in
cancer treatment [18]. Combretastatin A-4 (CA-4) II (Fig. 1) has been
identified as a promising anticancer agent that selectively targets tumor
vasculature [19-22]. The activity of CA-4 is hampered by the poor
metabolic stability and the weak water solubility due to the isomeriza-
tion of the active z-olefinic conformation into the corresponding inactive
trans-analogs [23,24]. To overcome this problem, several analogues
have been extensively investigated to retain the potency and efficacy of
CA-4, but with different pharmacokinetic profile [25-32]. Many cyclic
structures have been investigated to replace the olefinic bridge,
including three, five and six-membered rings, e.g., compounds III and IV
[33-41] (Fig. 1). Even fused carbocyclic compounds have also been
advanced as molecules that modify the ethylene bridge of CA-4, e.g.,
compounds V [42-44] (Fig. 1). In the last few decades, 1,2,4-triazine
derivatives have expanded significantly, especially as a vital heterocy-
clic class of antitumor agents. Some of the 1,2,4-triazine derivatives that
displayed potential anticancer activity have undergone clinical trials
[45-49]. Recently, 5,6-diaryl-1,2,4-triazines were prepared and evalu-
ated for their cytotoxic activities. Some of them showed potent cytotoxic
activity e.g., compounds VI (IC50 = 16.10 µM), [50] (Fig. 1). Interest-
ingly, in this approach the prepared compounds lack some of the
essential pharmacophoric features which are important for the activity.
The two phenyl rings have to be substituted with one or more methoxy
groups. These methoxy groups play a dual role for the binding with the
colchicine binding site, since they act as hydrogen bond acceptors and
hydrophobic centers as well (Fig. 3). For this purpose and in continua-
tion to our previous efforts of design and synthesis of new anticancer
agents [51-59], we developed a mild cascade reaction to access some
new 1,2,4-triazin-3(2H)-one derivatives. The synthesized compounds
have the essential pharmacophoric features of CBSIs with different
electronic properties. In silico studies were performed to investigate
their binding pattern with the prospective target (colchicine binding site
of tubulin protein).
Fig. 2. The colchicine binding site having a funnel shape (isolated pocket of
tubulin heterodimers obtained from Protein Data Bank (PDB ID: 1sa0) using
MOE 2014 software).
Thr179α, Asn349β, Asn350β, Lys352β, and Thr314β residues. The 2nd
side of the cavity is bordered by Val238β, Tyr202β, Thr239β, Leu242β,
Leu252β, Cys241β, Ile378β, Leu248β, Val318β, and Leu255β residues.
The 3rd side is surrounded by Asp251β, Ala250β, Met259β, Lys254β,
Asn258β, Ala316β, Thr353β, Ala354β, Ala317β and residues.
According to many reports [60-63], CBSIs have seven main phar-
macophoric features. These include three hydrogen bond acceptors (A1,
A2, and A3), one hydrogen bond donor (D1), two hydrophobic centers
(H1 and H2), and one planar group (R1) (Fig. 3A). The seven pharma-
cophoric points are allocated in two planes separated by an acute angle
of 45◦. The 1st plane comprises the features of A1, D1, H1, and R1. The
2nd plane includes the features of A2, A3, and H2 (Fig. 3B). Colchicine
and combretastatin A-4 as representative examples of CBSIs are con-
sisted of three parts: “A” ring, “B” ring (linker), and “C” ring. Many re-
ports [64-66] revealed that modification of the “B” ring could affect the
cytotoxic activity of the CBSIs (Fig. 3C & 3D).
1.1. Rational drug design
The colchicine binding site is positioned at the interface between the
α
and β subunits of the tubulin protein, having a funnel shape (Fig. 2).
The 1st side of the cavity is surrounded by Val181
α
, Asn101
α
, Ala180
α
,
H3C
O
HN
HO
O
HO
OCH3
OCH3
H3CO
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
H3CO
OCH3
(III)
Colchicine (I)
Combretastatin A-4 (II)
O
RS
N
N
R
N
N
N
N
OCH3
O
N
N
R2
R3
HO
OCH3
OCH3
N
OCH3
OCH3
H3CO
H3CO
(IV)
R1
R4
H3CO
(V)
(VI)
Newly designed compounds
Fig. 1. Chemical structures of some reported CBSIs in which the olefinic bridge of CA-4 is replaced by cyclic structures [40-42,50], and the general structure of our
target compounds.
2

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
B
A
2nd Plane
st Plane
1
H1
H2
A1
A2
R1
A3
D1
D
C
Linker
R1
D1
HO
O
B
HN
R1
H2
Linker
C
A1
O
H1
B
O
A
C
O
H2
A1
O
O
A
H1
O
O
O
A2
O
A2
A-4
Combretastatin
Colchicine
Fig. 3. A) The essential pharmacophoric features of CBSIs: hydrogen bond acceptors (A1, A2 & A3), hydrogen bond donor (D1), hydrophobic centers (H1 & H2), and
planar group (R1) (based on Ref. [60,61]). B) The pharmacophoric model with two planes: the 1st plane (red) (A1, D1, H1 and R1); the 2nd Plane (pink) (A2, A3, and
H2) (based on Ref. [52,60]). C&D) Pharmacophoric points of colchicine and combretastatin A-4 as CBSIs. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
In this study, we replaced the olefinic bridge with 1,2,4-triazin-3
(2H)-one ring to mimic the relative orientation induced by the Z-alkene
configuration of CA-4. Such strategy produced many CA-4 analogs
having the same essential pharmacophoric features of the reported
tubulin inhibitors. The second aim was focused on modification of the
“A” ring, “C” ring, and linker region to study the structure–activity
relationship of the synthesized compounds as new cytotoxic agents
targeting CBS. Accordingly, the “A” ring could be phenyl, para-
methoxyphenyl, 3,5-di methoxyphenyl, or 3,4,5-trimethoxyphenyl
group. The “C” ring could be phenyl or para-methoxyphenyl group.
The linker region was designed to be 1,2,4-triazin-3(2H)-one ring. The
choice of this moiety was based on some considerations. i) The ring itself
comprises HBA and HBD points which act as essential pharmacophoric
features. ii) The non-planar configuration of the ring is consistent with
the non-planar configuration of the middle ring of colchicine.
cascade synthesis of 1,2,4-triazin-3(2H)-ones was reported from readily
accessible α-amino ketones [78,79] and phenyl carbazate as a masked N-
isocyanate precursor, under acidic condition [67]. Phenyl carbazate
were synthesized by the improved method developed by Beauchemin
group [80] through dropwise addition of hydrazine (1.0 M) in THF over
diphenyl carbonate in petroleum ether as solvent (around 90% yield).
Also, α-amino ketones (2a-e) were prepared by simple mixing of two
equivalent of the amine with one equivalent of the corresponding
-haloketone in MeCN and stir overnight at room temperature. Then,
α
acid catalyzed condensation of
α-amino ketones (2a-e) with phenyl
carbazate, followed by intramolecular cyclization on the N-isocyanate
intermediate to give the target compounds 3a-e (Scheme 1).
In this work, we were interested in a complementary approach
involving, i) alkylation of –NH group of the triazine moiety to form a
panel of esters with one or two carbon bridge (4a-j). ii) Hydrolysis of the
synthesized esters to form the corresponding acids (5a-j). iii) Alkylation
of –NH group of the triazine moiety to form a panel of amides with one
carbon or two carbon bridge (6a-j) (Scheme 2).
In a separate approach, we focused on the modification of the 1,2,4-
triazin-3(2H)-one “B” ring of the designed compounds. Such modifica-
tion was achieved by chemical substitutions of esters, acids, or amides
side chains at the NH group. These substituents could also have the
essential HBA and HBD features and possess the same spatial orientation
of acetamide side chain of colchicine. All modification pathways and
molecular design rationale are illustrated and summarized in Fig. 4.
Ester derivatives of 1,2,4-triazin-3(2H)-one (4a-j) were synthesized
via the reaction of 1,2,4-triazin-3(2H)-ones (3a-e) with ethyl bromoa-
cetate or ethyl 3-bromopropionate in DMF in presence of NaH. The role
of NaH was to ease the replacement of relatively stable acidic 1,2,4-
triazin-3(2H)-ones proton and to prevent the accumulation of acid
byproduct which can cause the decomposition of the formed ester. In
case of ethyl bromoacetate the reaction was complete in about 30 min,
while in case of ethyl 3-brompropionate it needed to be heated at reflux
in DMF for around 4 h. The ester derivatives (4a-j) were confirmed by 1H
NMR spectra, which showed the disappearance of the singlet signal of
–NH proton of triazine ring and the appearance of a new two signals (for
acetate) or three signals (for propionate) for the ester methylene proton
2. Results and discussion
2.1. Chemistry
In continuation for our previous work of synthesis of heterocyclic
structures using N-isocyanates as reactive intermediates [67-77], new
1,2,4-triazin-3(2H)-one derivatives have been synthesized. A mild
3

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
* R = H
A)
* R = esters, acids or amides
with one or two carbon bridge
Z-geometry is crucial
R
O
N
N
B
N
R2
R3
O
HO
Design of new CBSIs
A
Replacement of olifinic bridge with
1,2,4-triazin-3-one with different
substituents in 2 position
O
O
C
O
R1
R4
for
Three methoxy groups are
antitumor
Important
antitumor
activity
for
important
effects
antitubulin
and
activity
* Phenyl
* Phenyl
* p-Methoxyphenyl
* 3,5-Dimethoxyphenyl
* 3,4,5-Trimethoxyphenyl
* p-Methoxyphenyl
CA-4
1,2,4-Triazin-3-one derivatives
B)
D1
D1
O
N
O
O
N
NH2
O
N
OH
A3
O
A3
O
A3
O
H2
H2
H2
N
N
N
R1
R1
R1
N
O
N
O
N
O
O
O
O
A2
O
O
A2
O
H1
H1
O
O
O
A2
A1
H1
A1
A1
4i
5i
6i
Fig. 4. A) Rational of molecular design of the new proposed CBSIs based on CA-4. B) Some new 1,2,4-triazin-3(2H)-ones having the essential pharmacophoric
features of the reported CBSIs.
H
O
O
N
OPh
H
R4
3 CH3CN, rt,
H2N
Br
+
N
R4
R3
H2N
O
R1
R1
Citric acid, THF,
argon, rt-reflux
2a-e
R
R2
overnight
R2
N
H
1
2
3
4
2a, 3a
2b, 3b
2c, 3c
2d, 3d
2e, 3e
: R ,R ,R ,R = H
N
O
N
1
2
3
4
: R = OCH3, R ,R ,R = H
R4
R3
1
3
2
4
: R ,R = OCH3,R ,R = H
1
2
4
3
: R ,R ,R = OCH3, R = H
R1
3a-e
1
2
3
4
: R ,R ,R ,R = OCH3
R2
Scheme 1. Syntheses of 1,2,4-triazin-3(2H)-ones using N-isocyanate cascade reaction.
in the range of δ 2.80 – 4.20 ppm. This methylene proton was also
detected in both of 13C NMR and DEPT 135 NMR spectra resonating in
the range of δ 30.0 – 50.0 ppm. Also, 1H NMR spectra showed the
presence of triplet signal at a range of δ 1.20–1.30 ppm corresponding to
the methyl group of the ester.
The acid derivatives (5a-j) were confirmed by 1H NMR spectra, which
showed the disappearance of the methyl and one of the methylene
protons of the ester, with appearance of a new broad singlet signal in a
range of δ 10.50 – 11.00 ppm corresponding to the carboxylic group of
the acid. The disappearance of the ester methylene protons was also
confirmed by 13C NMR and DEPT 135 NMR spectra.
Acid derivatives (5a-j) were prepared by hydrolysis of the corre-
sponding esters (4a-j) via refluxing with NaOH, in CH3OH as solvent.
Amide derivatives of 1,2,4-triazin-3(2H)-one (6a-j) were synthesized
4

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
1
2
3
4
4a:
4b:
4c:
4d:
4e:
4f:
(95%)
R ,R ,R ,R = H, n = 1
O
1
2
3
4
(95%)
(95%)
R = OCH3, R ,R ,R = H, n = 1
1
3
2
4
OCH2CH3
O
R ,R = OCH3,R ,R = H, n = 1
n
1
2
4
3
N
(95%)
R ,R ,R = OCH3, R = H, n = 1
N
1
2
3
4
(95%)
R ,R ,R ,R = OCH3, n = 1
N
R4
1
2
3
4
(75%)
R ,R ,R ,R = H, n = 2
4a-j
R1
R3
4g:
4h:
4i:
(65%)
(82%)
(55%)
1
2
3
4
R = OCH3, R ,R ,R = H, n = 2
R2
1
3
2
4
R ,R = OCH3,R ,R = H, n = 2
1
2
4
3
R ,R ,R = OCH3, R = H, n = 2
1
2
3
4
4j:
(55%)
R ,R ,R ,R = OCH3, n = 2
O
NaOH, CH3OH,
reflux
Br
n
NaH, DMF,
H3CH2CO
O
OH
n
rt - reflux
N
O
N
N
R4
H
5a-j
N
N
O
R1
R3
R2
2
N
R4
1
3
4
5a:
5b:
5c:
5d:
5e:
5f:
(95%)
R ,R ,R ,R = H, n = 1
R1
R3
3a-e
1
2
3
4
(95%)
(95%)
R = OCH3, R ,R ,R = H, n = 1
R2
1
3
2
4
R ,R = OCH3,R ,R = H, n = 1
1
2
4
3
1
2
3
4
(95%)
R ,R ,R = OCH3, R = H, n = 1
3a
(74%)
: R ,R ,R ,R = H
O
1
2
3
4
1
2
3
4
(95%)
R ,R ,R ,R = OCH3, n = 1
3b
3c
3d
3e
(53%)
(74%)
(74%)
: R = OCH3, R ,R ,R = H
X
n
1
2
3
4
H2N
1
3
2
4
(95%)
R ,R ,R ,R = H, n = 2
: R ,R = OCH3,R ,R = H
1
2
3
4
X = I, Cl
NaH, DMF,
rt - 100oC
1
2
4
3
5g:
5h:
5i:
(95%)
(95%)
(95%)
R = OCH3, R ,R ,R = H, n = 2
: R ,R ,R = OCH3, R = H
1
3
2
4
1
2
3
4
R ,R = OCH3,R ,R = H, n = 2
(74%)
: R ,R ,R ,R = OCH3
1
2
4
3
R ,R ,R = OCH3, R = H, n = 2
1
2
3
4
5j:
(95%)
R ,R ,R ,R = OCH3, n = 2
O
NH2
O
n
N
N
N
R4
R3
6a-j
R1
R2
1
2
3
4
1
2
3
4
6f:
6g:
6h:
6i:
(71%)
6a:
6b:
6c:
6d:
6e:
(75%)
R ,R ,R ,R = H, n = 2
R ,R ,R ,R = H, n = 1
1
2
3
4
1
2
3
4
(72%)
R = OCH3, R ,R ,R = H, n = 2
(68%)
(80%)
R = OCH3, R ,R ,R = H, n = 1
1
3
2
4
1
3
2
4
(70%)
(72%)
R ,R ,R = OCH3, R = H, n = 2
R ,R = OCH3,R ,R = H, n = 2
R ,R = OCH3,R ,R = H, n = 1
1
2
4
3
1
2
4
3
(69%)
R ,R ,R = OCH3, R = H, n = 1
1
2
3
4
1
2
3
4
6j:
(70%)
R ,R ,R ,R = OCH3, n = 2
(72%)
R ,R ,R ,R = OCH3, n = 1
Scheme 2. Synthesis of the target compounds 4a-j, 5a-j and 6a-j (Isolated yields in parentheses).
via the reaction of 1,2,4-triazin-3(2H)-ones (3a-e) with iodoacetamide
or 3-chloropropanamide in DMF in presence of NaH. In case of iodoa-
cetamide the reaction was completed in about one hour, while in case of
3-chloropropanamide it needed heating at 100 OC for around 4 h. The
amide derivatives (6a-j) were confirmed by 1H NMR spectra, which
showed the disappearance of the singlet signal of –NH proton of triazine
ring and the appearance of a new singlet signal (for acetamide) or two
triplet signals (for propionamide) for the methylene protons in a range of
δ 2.80 – 4.20 ppm. This methylene proton was also confirmed by both
13C NMR and DEPT 135 NMR spectra resonating in a range of δ 35.0 –
50.0 ppm. Also, 1H NMR spectra showed the presence of two separated
singlet signals at a range of δ 5.70–6.60 ppm corresponding to the amino
group protons.
2.2. Biological evaluation
2.2.1. In vitro anti-proliferative activity
The synthesized compounds were tested for their in vitro cytotoxic
activities using standard MTT method [81,82] against a group of human
cancer cell lines namely, breast cancer (MCF-7), hepatocellular carci-
noma (HepG-2) and colorectal carcinoma (HCT-116). Colchicine was
used as a positive control. The results of cytotoxicity test were reported
as growth inhibitory concentration (IC50) values and summarized in
Table 1.
The tested compounds exhibited different degrees of anti-
proliferative activities against the three tested cell lines. In general,
compounds 3b, 4i, 4j, 5 g, 5i, 6f and 6i showed superior anti-
proliferative activities against the three cell lines with IC50 values
5

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
In bold: the most potent compounds
Table 1
a
IC50 values are the mean ± S.D. (standard deviations) of three separate
In vitro anti-proliferative activities of the tested compounds and tubulin poly-
experiments.
merization inhibition.
Comp.
b
c
NT: stands for (Not Tested)
In vitro cytotoxicity IC50 (µM)a
Tubulin polymerization
These IC50 values were retrieved from reference[85]
This IC50 value were retrieved from reference[86]
MCF-7
HepG-2
HCT116
Inhibition IC50 (µM)a
d
3a
133.9 ±
2.1
167.7 ±
2.3
186.9 ±
2.5
NTb
ranging from 8.2 to 42.2 µM. The cytotoxic activities of compounds 4i,
4j, 5i, and 6i were higher than that of the reference drug, colchicine
(IC50 = 34.3, 27.3 and 31.1 µM against MCF-7, HepG-2 and HCT-116,
respectively).
3b
3c
35.9 ±
1.3
19.9
30.6
285.1 ± 1.73
NTb
0.9
1.2
62.1 ±
1.8
70.1 ±
1.7
83.0 ±
1.9
3d
3e
133.7 ±
2.4
151.0 ±
2.7
211.7 ±
2.9
NTb
2.2.2. Tubulin polymerization assay
77.1 ±
1.7
67.7 ±
1.5
86.0 ±
2.1
55.8 ± 1.22
NTb
To investigate whether the cytotoxic activity of the synthesized
compounds was related to an interaction with the tubulin system,
tubulin polymerization assay was performed for the most cytotoxic
compounds 3b, 3e, 4f, 4i, 4j, 5 g, 5i, 6d, 6f and 6i. The inhibition of
microtubule polymerization was evaluated turbidimetrically using a
fluorescent plate reader [83,84]. Colchicine was used as a positive
control (Table 1).
4a
108.3 ±
2.3
97.0 ±
2.4
160.8 ±
2.8
4b
4c
307.1 ±
5.7
298.4 ±
4.4
344.4 ±
6.1
NTb
213.4 ±
3.5
176.6 ±
3.8
218.9 ±
3.9
NTb
4d
4e
169.1 ±
2.3
154.8 ±
3.6
204.7 ±
2.7
NTb
The results revealed that compounds 4i, 4j, 5i and 6i were the most
potent tubulin polymerization inhibitors with an IC50 values ranging
from 3.9 to 22.3 µM. These compounds had activities higher than that of
colchicine (IC50 = 28.6 µM). We can conclude that the cytotoxic activity
of the synthesized compounds may be due to an interaction with tubulin
and an interference with microtubule assembly.
103.5 ±
3.4
120.1 ±
3.5
145.5 ±
4.2
NTb
4f
101.4 ±
2.2
85.8 ±
2.4
121.4 ±
3.4
33.9 ± 0.95
NTb
4 g
4 h
4i
156.7 ±
3.9
201.2 ±
4.2
196.0 ±
4.6
119.6 ±
3.4
111.8 ±
3.2
156.9 ±
2.8
NTb
20.5
14.4
22.4
17.7 0.75
22.3 0.92
NTb
2.2.3. Cell cycle analysis
1.7
1.1
1.5
Antimitotic drugs induced cell cycle arrest at G2/M phase in various
cancer cell lines [87]. In order to investigate the impact of compound 3e
and 5i on growth inhibition of cancer cells, their activities on the cell
cycle distribution and apoptosis induction were assessed using HepG-2
cell lines according to the method described by Wand et al [88,89]. In
the present work, HepG-2 cell line was treated with compounds 3e and
5i at concentrations of 67.7 and 8.2 µM, respectively for 24 h.
The results revealed that incubation compounds 3e, 5i and colchi-
cine with HepG-2 cells produced marked increase of HepG-2 cells at G2/
M phase from 20.6% to 38.4%, 24.7%, and 47.3%, respectively. On the
other hand, such percentages decreased at S and G0-G1 phases. Such
results indicate that the synthesized compounds caused cell arrest at G2/
M phase. Moreover, the tested compounds as well as colchicine pro-
duced an increase of the cell population at Pre-G1, indicating that
compound 3e and 5i caused apoptotic effect at pre-G1 phase (Table 2
and Fig. 5).
4j
23.1
18.5
25.1
1.5
1.2
1.4
5a
77.3 ±
2.3
85.8 ±
1.9
112.0 ±
2.5
5b
5c
341.6 ±
6.3
328.3 ±
5.7
369.3 ±
7.4
NTb
56.1 ±
2.9
95.7 ±
2.5
107.3 ±
3.5
NTb
5d
5e
63.7 ±
3.8
49.6 ±
2.8
70.4 ±
3.6
NTb
139.2 ±
2.7
158.7 ±
2.9
180.2 ±
2.3
NTb
5f
162.8 ±
1.4
180.8 ±
1.4
226.6 ±
1.3
NTb
5 g
5 h
5i
42.2
23.2
33.1
28.0 0.87
NTb
3.2
3.1
3.8
77.6 ±
3.6
91.9 ±
3.6
123.4 ±
4.5
10.9
8.2
15.7
3.9 0.06
NTb
0.84
0.63
1.2
5j
86.2 ±
2.8
100.7 ±
2.8
139.7 ±
3.3
2.2.4. Annexin V-FITC apoptosis assay
Apoptosis is a programmed cell death which plays a key role in the
maintenance of tissue homeostasis and cell survival [90]. Thus, inducing
apoptosis in cancer cells has emerged as an attractive strategy in cancer
therapy. To further confirm apoptotic effect of compounds 3e, and 5i in
HepG-2 cells, Annexin V and PI double staining assay was performed
[91]. In this test, HepG-2 cells were incubated with compound 3e and 5i
at concentrations of 67.7 and 8.2 µM respectively for 24 h. The results in
Table 3, Fig. 6. revealed that compound 3e induced total apoptotic effect
equal 13.7% which was nine times more than the control cells (1.5%).
Also, it induced early apoptosis by 6.2% and enhanced late apoptosis by
5.3% when compared with the untreated control HepG-2 cells (0.5% and
6a
89.6 ±
2.1
76.6 ±
2.3
107.8 ±
2.6
NTb
6b
6c
346.7 ±
6.2
268.5 ±
5.9
358.6 ±
6.5
NTb
255.2 ±
3.9
216.3 ±
2.5
311.1 ±
4.1
NTb
6d
6e
192.0 ±
2.2
184.7 ±
1.5
229.1 ±
2.3
105.0 ± 1.33
NTb
185.0 ±
3.8
177.8 ±
3.8
203.5 ±
4.9
6f
29.2
35.7 ±
4.1
29.2
37.9 ± 0.98
NTb
3.4
4.6
6 g
6 h
6i
122.7 ±
1.2
131.8 ±
0.71
184.9 ±
1.3
144.2 ±
2.8
164.1 ±
2.7
196.9 ±
4.6
NTb
Table 2
Effect of compound 3e and 5i on cell cycle progression.
16.3
11.2
18.2
7.8 0.15
NTb
0.71
0.42
0.73
Sample
Cell cycle distribution (%)
6j
82.6 ±
2.7
89.1 ±
3.1
119.2 ±
4.1
%G0-G1
%S
%G2-M
%Pre-G1
3e / HepG-2
36.3
45.1
51.6
29.7
25.3
30.3
27.8
22.9
38.4
24.7
20.6
47.3
13.7
13.5
1.5
Colchicine
CA-4
34.3 ±
1.16
27.3 ±
0.97
31.1 ±
1.1
28.6 ± 0.95
2.03 ± 0.41d
5i / HepG-2
Control / HepG-2
Colchicine / HepG-2
0.67 ±
16.04 ±
9.00 ±
22.6
0.13c
0.50c
0.85c
6

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
Control / HepG-2
Colchicine / HepG-2
3e / HepG-2
5i / HepG-2
Fig. 5. HepG-2 cells distribution upon treatment with compound 3e, 5i and colchicine.
fibroblasts) using MTT assay and their IC50 were determined.
Table 3
The results revealed that IC50 doses of all the tested compounds
against WI-38 cells were very high in comparison to their IC50 doses
against the cancer cell lines by six to eleven-fold (Table 4). Such results
indicate that compounds 4i, 4j, 5 g, 5i and 6i have a significant effect in
rapidly proliferating cells but not in normal cells.
Apoptosis and necrosis percent induced by compound 3e, 5i, and colchicine.
Sample
Apoptosis
Total
Necrosis
Early
Late
3e / HepG-2
13.7
13.5
1.5
6.2
4.1
0.5
7.4
5.3
2.2
2.3
0.8
2.3
5i / HepG-2
7.1
Control / HepG-2
Colchicine / HepG-2
0.2
2.2.6. Caspase-3 determination
22.6
12.9
Caspases are the primary drivers of apoptotic cell death [92].
Caspase-3 is the most well-characterized effector caspase which is the
executioner caspase activated by upstream initiator caspases [93]. Thus,
the investigation of this protein expression level is indicative of
apoptosis induction. To find out whether the most promising compounds
4i, 4j, 5 g, 5i and 6i induced caspase-3 dependent apoptosis, HepG-2
cells were treated with such compounds at concentrations of 14.4,
18.5, 23.2, 8.2, and 11.2 µM, respectively for 48 h and the levels of
caspase-3 were assessed. The results revealed that the tested compounds
upregulated the caspase-3 levels from three to six folds as compared to
the control. These findings prove apoptosis induction potential of the
tested compounds (Table 5).
0.2%, respectively). For compound 5i, it induced total apoptotic effect
equal 13.5% which was nine times more than the control cells (1.5%).
Furthermore, it induced early apoptosis by 4.1% and enhanced late
apoptosis by 7.1% when compared with the untreated control HepG-2
(0.5% and 0.2%, respectively).
2.2.5. In vitro cytotoxicity against human normal cell
One of the main problems of cancer chemotherapy is the unwanted
damage to normal cells caused by the high toxicities of anticancer drugs.
The most promising compounds 4i, 4j, 5 g, 5i and 6i were further
investigated in vitro against WI-38 cell line (normal human lung
7

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
Control / HepG-2
Colchicine / HepG-2
3e / HepG-2
5i / HepG-2
Fig. 6. Apoptosis and necrosis percent induced by compound 3e, 5i, and colchicine.
Table 4
Table 5
in vitro cytotoxicity of compounds 4i, 4j, 5 g, 5i and 6i on normal WI-38 cells.
Effect of the compounds 4i, 4j, 5 g, 5i, and 6i on caspase-3 and Bax expression
levels.
Comp.
Comp.
WI-38 IC50 (µM)a
Caspase-3 (Pg/mL)a
Bax (Pg/mL)a
4i
139.2 ± 5.3
179.6 ± 4.1
187.3 ± 5.4
127.1 ± 3.6
170.6 ± 3.5
4j
4i
282.4 ± 5.7
215.6 ± 4.6
226.1 ± 4.2
321.3 ± 6.8
247.8 ± 4.3
60.3 ± 2.5
311.6 ± 1.3
285.8 ± 2.4
308.6 ± 2.3
387.2 ± 2.9
350.4 ± 1.7
56.1 ± 1.1
5 g
5i
4j
5 g
6i
5i
6i
a
IC50 values are the mean ± S.D. (standard deviations) of three separate
Control / HepG-2
experiments.
a
The values (in picogram per mL) given are means ± SD of three experiments.
2.2.7. Bax determination
concentrations of 14.4, 18.5, 23.2, 8.2, and 11.2 µM, respectively for 48
h and their effects on the levels of Bax were determined as illustrated in
Table 5. The results revealed that these compounds boosted the levels of
the pro-apoptotic protein Bax by five to seven folds as compared to the
The apoptotic regulation depends mainly on a family of proteins
responsible for synchronizing the mitochondrial apoptotic pathway
[94], e.g. Bax which induces apoptosis (pro-apoptotic). In this context,
HepG-2 cells were treated with compounds 4i, 4j, 5 g, 5i and 6i at
8

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
control cells, emphasizing that these compounds shifted the cells to-
wards undergoing apoptosis. This data revealed that these compounds
induced cell death by apoptosis in human lung cancer cells.
Table 6
The docking scores of the synthesized compounds, colchicine, and co-
crystallized ligand (DAMA-colchicine) against tubulin.
Comp.
Binding free energy
(kcal/mol)
Comp.
Binding free energy
(kcal/mol)
2.3. In silico studies
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
–33.77
ꢀ 39.46
ꢀ 43.71
ꢀ 47.95
ꢀ 53.51
ꢀ 48.21
ꢀ 49.56
ꢀ 52.38
ꢀ 59.41
ꢀ 65.85
ꢀ 50.41
ꢀ 53.46
ꢀ 58.84
ꢀ 64.54
ꢀ 60.95
ꢀ 45.66
ꢀ 50.77
5e
ꢀ 62.63
ꢀ 46.13
ꢀ 54.47
ꢀ 59.18
ꢀ 60.05
ꢀ 63.04
ꢀ 43.09
ꢀ 46.17
ꢀ 51.58
ꢀ 58.96
ꢀ 59.69
ꢀ 44.61
ꢀ 47.96
ꢀ 49.81
ꢀ 60.02
ꢀ 60.48
ꢀ 42.84
5f
2.3.1. Flexible alignment
5 g
3D- flexible alignment of representative compound 4i with colchi-
cine is presented in Fig. 7. In the figure is possible to observe that, in
general, the structure of compound 4i has a good overlap with the
reference molecule (colchicine).
5 h
5i
5j
6a
6b
6c
2.3.2. Docking studies
6d
Discovery Studio 4.0 software was used for the docking simulations
against 3D structure of tubulin heterodimers obtained from Protein Data
Bank (PDB ID: 1sa0). DAMA-colchicine, the native ligand, and colchi-
cine were used as reference ligands (See Table 6).
6e
4 g
4 h
4i
6f
6 g
6 h
4j
6i
DAMA-colchicine gave a binding energy value of ꢀ 42.84 kcal/mol.
The “A” ring (trimethoxy phenyl) occupied the first pocket of the
colchicine binding site forming four hydrophobic interactions with
Leu255, Ala250, Ala316, and Cys241. The “B” ring with its side chain (2-
mercaptoacetamide moiety) occupied the second cavity of the colchi-
cine binding site forming a hydrogen bond with Leu254. Also, it formed
two hydrophobic interactions with Leu248 and Ala250. Furthermore,
the “C” ring (methoxytropone moiety) occupied the third pocket of the
receptor forming two hydrogen bonds with Ser187 (Fig. 8).
5a
5b
6j
DAMA-
colchicine
Colchicine
5c
5d
ꢀ 52.43
ꢀ 58.84
ꢀ 59.16
the third cavity of the colchicine binding site forming two hydrophobic
interactions with Ala316 and Lys352 (Fig. 9).
The binding modes of compounds 5i, 5j, 6i and 6j are depicted in
Supplementary Data.
Compound 4i showed a binding mode like that of the co-crystallized
ligand (DAMA-colchicine), with affinity value of ꢀ 64.54 kcal/mol. The
trimethoxy phenyl moiety (“A” ring) occupied the first cavity of the
colchicine binding site forming one hydrophobic interaction with
Lys254. Additionally, the 4,5-dihydro-1,2,4-triazin-3(2H)-one moiety
(“B” ring) and its ethyl propionate side chain occupied the second cavity
of the colchicine binding site forming two hydrogen bonds with Ala250
and Cys241. In addition, it formed two hydrophobic interactions with
Leu242 and Leu255. The p-methoxyphenyl moiety (“C” ring) occupied
2.3.3. In silico ADMET analysis
ADMET studies were carried out for the most cytotoxic members 4i,
4j, 5 g, 5i, and 6i. Colchicine was used as a reference drug. Discovery
studio 4.0 was used to predict ADMET descriptors for all compounds.
The predicted descriptors are listed in Table 7.
The results revealed that the tested compounds have low or very low
BBB penetration levels. Accordingly, such compounds were expected to
be safe to CNS. All the tested compounds showed good to optimal range
levels of ADMET aqueous solubility. Also, all compounds were appeared
to have good intestinal absorption levels [95]. All examined members
were predicted as non-inhibitors of CYP2D6. Consequently, liver
dysfunction side effect is not expected upon administration of these
compounds [96]. All compounds were expected to bind plasma protein
over than>90% (Fig. 10.).
(The predicted toxicity studies, Physico-chemical properties and
Lipinski rule of five are depicted in Supplementary Data)
2.4. Structure-Activity relationship (SAR)
Based on the aforementioned biological data, many structur-
e–activity relationships could be deduced. With respect to substitution
on “A” ring, the decreased IC50 values of compounds 4i, 5i, 6i, 4j, 5j,
and 6j than the corresponding members 4 h, 5 h, and 6 h, indicated that
substitution of “A” ring with 3,5-di methoxy or 3,4,5-trimethoxy is more
advantagous. For “B” ring, comparing the activity of compounds 3e (un-
substituted), 4i (ester substituted), 5i (acid substituted), and 6i (amide
substituted), indicated that substitution of 1,2,4-triazine ring at 2-posi-
tion with ester, acid, or amide is tolerated. Interestingly, ester (4f-j),
acid (5f-j), and amide (6f-j) derivatives with two carbon bridge
exhibited higher activities than the corresponding members with one
carbon bridge (4a-e), (5a-e), and (6a-e), hinting that elongation of the
linker enhances the antiproliferative activity. Furthermore, the
decreased IC50 values of compounds 3b, 4i, 5i, and 6i than the corre-
sponding members 3a, 4a, 5a, and 6a, suggesting that the substitution
in “C” ring is favorable (See Fig. 11).
Fig. 7. Flexible alignment of compounds 4i (carbon atoms in turquoise) with
the colchicine (carbon atoms in green). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of
this article.)
9

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
Fig. 8. A) 3D structure of co-crystallized ligand, DAMA-colchicine, docked into the colchicine binding site. B) Mapping surface showing DAMA-colchicine occupying
the active pocket of colchicine binding site.
Fig. 9. A) 3D structure of compound 4i docked into the colchicine binding site B) Mapping surface showing compound 4i occupying the active pocket of colchicine
binding site.
on the essential pharmacophoric features of the reported tubulin in-
Table 7
hibitors. The newly synthesized compounds were evaluated for their
Predicted ADMET for the designed compounds and reference drug.
anti-proliferative activities against three human cancer cell lines (MCF-
Comp.
BBB
Solubility
level b
Absorption
level c
CYP2D6
PPB
7, HepG-2, and HCT-116). Compounds 4i, 4j, 5i, and 6i were more
active than colchicine as cytotoxic agents and tubulin polymerization
inhibitors. SAR revealed that 3,5-dimethoxyphenyl and 3,4,5-trimethox-
yphenyl miety is more beneficial as a “A” ring, while 4-methoxyphenyl
moiety was more effective than phenyl ring as a “C” ring. Also, sub-
stitutions at the NH group of “B” ring with two carbon bridge acid, ester
or amide is highly favorable. In order to get an additional comprehen-
sion about the effect of the synthesized compounds on the inhibition of
cancer cell growth, the effect of compounds 3e and 5i on cell cycle
distribution and apoptosis induction was analyzed. The most active
compounds upregulated the level of active caspase 3 and Bax by five to
seven folds comparing to the control. To further understand and ratio-
nalize the promising inhibitory profile of the compounds, docking
studies were performed against the crystal structure of tubulin (PDB ID:
1SA0), which suggested that most of the synthesized compounds have
similar binding mode with the target crystal structure. In silico ADMET,
physico-chemical properties and toxicity studies were carried out for the
most active compounds. Most of them were predicted to have low
toxicity and good drug likeness. Further structural optimization of the
most active candidates may serve as useful lead compounds in search for
powerful and selective antineoplastic agents.
level
prediction d
prediction
a
e
4i
3
3
3
3
4
3
3
3
3
3
3
3
0
0
0
0
0
0
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
4j
5 g
5i
6i
Colchicine
a
Blood brain barrier (BBB) level, 0 = very high, 1 = high, 2 = medium, 3 =
low, 4 = very low.
b
c
Solubility level, 1 = very low, 2 = low, 3 = good, 4 = optimal.
Absorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor.
d
CYP2D6, cytochrome P2D6, TRUE = inhibitor, FALSE = non inhibitor.
e
PBB, plasma protein binding, FALSE means less than 90%, TRUE
means>90%
3. Conclusion
Clearly the present work highlights the usefulness of the 1,2,4-tri-
azines ring as a scaffold for synthesis of new colchicine analogues. We
have designed and synthesized thirty-five final 1,2,4-triazin-3(2H)-one
derivatives with different linkers. The design of these agents was based
10

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
Fig. 10. The expected ADMET study of compounds 4i, 4j, 5 g, 5i, and colchicine (These data were computationally generated using Discovery studio 4.0).
Ester, Acid and Amide
?
are tolerated
Two carbon bridge > One carbon bridge
O
n
N
O
N
B
N
3,4,5-Trimethoxyphenyl and
3,5-Di methoxyphenyl
are favorable
C
A
p-Methoxyphenyl > Phenyl
Fig. 11. Schematic diagram showing the SAR of the synthesized compounds.
4. Experimental
Nomenclature was determined using ChemBioDraw Ultra 14.0. The
purity of the compounds, peak area percentage, was checked by HPLC
analysis using Agilent 1200 Series instrument and Zorbax Rx-SIL col-
umn. The mobile phase was only prepared from HPLC grade solvents
and all samples were filtered via a syringe filter prior to injection.
4.1. Chemistry
All commercially available materials were purchased from com-
mercial sources and used without further purification⋅THF was passed
through an activated alumina column embedded in a solvent purifica-
tion system provided by LC Technology Solutions. All reactions were
performed in a temperature-controlled oil/wax bath. Reactions were
monitored by analytical thin layer chromatography (TLC), using
aluminum-backed plates, cut to size. TLC visualization was achieved by
UV and stain (such as KMnO4, ninhydrin and CAM). Flash column
4.1.1. General procedure for synthesis of 1,2,4-triazin-3(2H)-ones (3a-e).
In a dry microwave vial equipped with a stir bar, α-amino ketone
(1.05 equiv.) was added to phenyl carbazate (1.0 equiv.) and citric acid
(1.0 equiv.). The vial was then sealed and purged with argon followed by
the addition of purified THF (1.0 M). The resulting solution was left
stirring at rt for 12–16 h. The reaction mixture was concentrated under
reduced pressure, diluted with a saturated NaHCO3 solution (20 mL) and
extracted with EtOAc (3 × 10 mL). The organic layers were combined,
dried with NaSO4, filtered, and concentrated under reduced pressure.
The crude products were purified by column chromatography or
through recrystallization in MeOH.
chromatography was carried out using 40–63
μ
m Silicycle silica gel. 1H
NMR and 13C NMR spectra were recorded on Bruker AVANCE 300 MHz
and 400 MHz spectrometers at ambient temperature. Spectral data is
reported in ppm, coupling constants (J) in Hz. Infrared (IR) spectra were
obtained as neat solids on Thermo Scientific Nicolet 6700 FTIR (ATR
Diamond). High resolution mass spectroscopy (HRMS) was performed at
the Ottawa-Carleton Mass Spectroscopy Centre on the Kratos Concept
11A mass spectrometer for Electron Impact (EI) and Waters Micromass
Q-TOF I for electrospray ionization (ESI). All melting points were taken
on a Gallenkamp melting point apparatus and are uncorrected.
4.1.1.1. 4,6-Diphenyl-4,5-dihydro-1,2,4-triazin-3(2H)-one
3a.. White
solid (0.186 g, 74%). MP = 200–202 ◦C; IR (ATR diamond): 3216, 3097,
1662, 1625, 1593, 1195, 772 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm):
8.09 (s, 1H), 7.68 (ddd, J = 4.4, 2.5, 1.4 Hz, 2H), 7.44 (t, J = 2.7 Hz, 2H),
11

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
7.42 (dd, J = 2.7, 1.6 Hz, 4H), 7.40 (d, J = 3.1 Hz, 1H), 7.31 – 7.26 (m,
1H), 4.74 (s, 2H); 13C NMR (101 MHz, CDCl3) δ (ppm): 151.5, 143.3,
140.4, 133.4, 130.0, 129.2, 128.8, 126.6, 125.3, 124.5, 47.7; HRMS (EI):
Exact mass calcd for C15H13N3O [M]+: 251.1053. Found: 251.1056;
HPLC analysis showed purity > 99%.
CH2Cl2; MP = 121–123 ◦C; IR (ATR diamond): 2559, 1742, 1657, 1514,
1151, 750 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.67 – 7.62 (m,
2H), 7.39 – 7.35 (m, 7H), 7.25 – 7.19 (m, 1H), 4.74 (s, 2H), 4.61 (s, 2H),
4.23 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ (ppm): 169.3, 151.2, 142.9, 141.0, 133.4, 130.0, 129.0, 128.7,
126.3, 125.4, 124.6, 61.2, 51.8, 48.1, 14.3; HRMS (EI): Exact mass calcd
for C19H19N3O3 [M]+: 337.1421. Found: 337.1411; HPLC analysis
showed purity 97.4%.
4.1.1.2. 6-(4-Methoxyphenyl)-4-phenyl-4,5-dihydro-1,2,4-triazin-3(2H)
-one 3b.. White solid (0.149 g, 53%); TLC Rf = 0.14 in 5% EtOAc/
CH2Cl2; MP = 198–200 ◦C; IR (ATR diamond): 3191, 3110, 2922, 1692,
1451, 1190, 826 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.90 (s, 1H),
7.65 – 7.59 (m, 2H), 7.47 – 7.38 (m, 4H), 7.32 – 7.26 (m, 1H), 6.95 –
6.90 (m, 2H), 4.70 (s, 2H), 3.84 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 161.0, 151.6, 143.2, 140.5, 129.1, 126.8, 126.5, 126.0, 124.5,
114.1, 55.4, 47.6; HRMS (EI): Exact mass calcd for C16H15N3O2 [M]+:
281.1159. Found: 281.1165; HPLC analysis showed purity > 99%.
4.1.2.2. Ethyl 2-(6-(4-methoxyphenyl)-3-oxo-4-phenyl-4,5-dihydro-1,2,4-
triazin-2(3H)-yl)acetate 4b. Yellow viscous oil (0.349 g, 95%); TLC Rf =
0.30 in 10% EtOAc/CH2Cl2; IR (ATR diamond): 2980, 1750, 1669, 1514,
1173, 732 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.60 – 7.56 (m,
2H), 7.35 (d, J = 5.0 Hz, 4H), 7.26 – 7.15 (m, 1H), 6.90 – 6.85 (m, 2H),
4.68 (s, 2H), 4.58 (s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 1.27 (t, J
= 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 169.4, 161.1, 151.4,
142.9, 141.0, 129.0, 127.0, 126.2, 126.0, 124.5, 114.1, 61.2, 55.4, 51.8,
48.0, 14.3;
4.1.1.3. 4,6-Bis(4-methoxyphenyl)-4,5-dihydro-1,2,4-triazin-3(2H)-one
3c.. White solid (0.186 g, 74%); TLC Rf = 0.23 in 5% EtOAc/CH2Cl2;
MP = 195–197 ◦C; IR (ATR diamond): 3221, 3104, 1672, 1512, 1199,
802 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.53 (s, 1H), 7.60 – 7.56
(m, 2H), 7.31 – 7.27 (m, 2H), 6.93 – 6.87 (m, 4H), 4.61 (s, 2H), 3.80 (s,
3H), 3.79 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 160.9, 158.1,
152.1, 142.6, 133.5, 126.8, 126.3, 126.1, 114.5, 114.1, 55.5, 55.3, 48.2;
HRMS (EI): Exact mass calcd for C15H13N3O [M]+: 311.1264. Found:
311.1288; HPLC analysis showed purity > 99%.
HRMS (EI): Exact mass calcd for C20H21N3O4 [M]+: 367.1527.
Found: 367.1542; HPLC analysis showed purity 98.1%.
4.1.2.3. Ethyl 2-(4,6-bis(4-methoxyphenyl)-3-oxo-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl)acetate 4c.. White solid (0.377 g, 95%); TLC Rf = 0.30 in
10% EtOAc/CH2Cl2; MP = 140–142 ◦C; IR (ATR diamond): 2033, 1744,
1658, 1510, 1154, 731 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.58 –
7.54 (m, 2H), 7.27 – 7.23 (m, 2H), 6.86 (ddd, J = 8.9, 4.2, 2.2 Hz, 4H),
4.63 (dd, J = 3.6, 1.8 Hz, 2H), 4.55 (d, J = 1.5 Hz, 2H), 4.20 (qd, J = 7.1,
2.0 Hz, 2H), 3.77 (dd, J = 3.0, 1.5 Hz, 3H), 3.75 (dd, J = 3.1, 1.5 Hz,
3H), 1.26 (td, J = 7.1, 1.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm):
169.4, 161.0, 157.9, 151.5, 142.5, 134.0, 126.9, 126.2, 126.0, 114.3,
61.1, 55.5, 55.4, 51.8, 48.7, 14.2; HRMS (EI): Exact mass calcd for
4.1.1.4. 4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-4,5-dihydro-
1,2,4-triazin-3(2H)-one 3d.. White solid (0.186 g, 74%); TLC Rf = 0.23
in 5% EtOAc/CH2Cl2; MP = 187–189 ◦C; IR (ATR diamond): 3100,
2360, 1667, 1629, 1418, 1149, 850 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ
(ppm): 8.08 (s, 1H), 7.61 – 7.57 (m, 2H), 6.92 – 6.88 (m, 2H), 6.54 (d, J
= 2.2 Hz, 2H), 6.36 (t, J = 2.2 Hz, 1H), 4.63 (s, 2H), 3.82 (s, 3H), 3.78 (s,
6H); 13C NMR (101 MHz, CDCl3) δ (ppm): 161.1, 151.5, 143.2, 142.3,
126.8, 125.9, 114.1, 103.2, 98.7, 55.5, 55.4, 47.7; HRMS (EI): Exact
mass calcd for C15H13N3O [M]+: 341.1370. Found: 341.1403; HPLC
analysis showed purity > 99%.
C
21H23N3O5 [M]+: 397.1632. Found: 397.1631; HPLC analysis showed
purity 97.3%.
4.1.2.4. Ethyl 2-(4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl)-3-oxo-
4,5-dihydro-1,2,4-triazin-2(3H)-yl)acetate 4d. White solid (0.405 g,
95%); TLC Rf = 0.30 in 10% EtOAc/CH2Cl2; MP = 149–151 ◦C; IR (ATR
diamond): 2044, 1750, 1667, 1516, 1146, 741 cmꢀ 1
;
1H NMR (400
4.1.1.5. 6-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
1,2,4-triazin-3(2H)-one 3e.. White solid (0.186 g, 74%); TLC Rf = 0.23
in 5% EtOAc/CH2Cl2; MP = 203–205 ◦C; IR (ATR diamond): 3215,
3110, 2926, 1682, 1593, 1131, 822 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ
(ppm): (ppm): 8.45 – 8.40 (m, 1H), 7.58 (d, J = 8.8 Hz, 2H), 6.89 (d, J =
8.8 Hz, 2H), 6.58 (s, 2H), 4.62 (s, 2H), 3.83 (s, 6H), 3.82 (s, 3H), 3.80 (s,
3H); 13C NMR (101 MHz, CDCl3) δ 161.05, 153.56, 151.90, 142.85,
137.02, 136.38, 126.85, 126.00, 114.14, 103.20, 60.85, 56.28, 55.40,
48.43; HRMS (EI): Exact mass calcd for C15H13N3O [M]+: 371.1509.
Found: 371.1476; HPLC analysis showed purity > 99%.
MHz, CDCl3) δ (ppm): 7.59 – 7.55 (m, 2H), 6.89 – 6.85 (m, 2H), 6.52 (d,
J = 2.2 Hz, 2H), 6.32 (t, J = 2.2 Hz, 1H), 4.65 (s, 2H), 4.57 (s, 2H), 4.21
(q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.75 (s, 6H), 1.27 (t, J = 7.1 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ (ppm): 169.4, 161.1, 160.9, 151.2, 142.9,
142.8, 127.0, 125.9, 114.0, 103.2, 98.6, 61.1, 55.4, 51.7, 48.1, 14.2;
HRMS (EI): Exact mass calcd for C22H25N3O6 [M]+: 427.1738. Found:
427.1721; HPLC analysis showed purity 95.2%.
4.1.2.5. Ethyl 2-(6-(4-methoxyphenyl)-3-oxo-4-(3,4,5-trimethoxy-phenyl)-
4,5-dihydro-1,2,4-triazin-2(3H)-yl)acetate 4e. White solid (0.434 g, 95%);
TLC Rf = 0.30 in 10% EtOAc/CH2Cl2; MP = 137–139 ◦C; IR (ATR dia-
4.1.2. General procedure for synthesis of compounds (4a-j).
1
In a dry microwave vial equipped with a stir bar, 5 mL of dry DMF
was added to NaH (60% dispersion in mineral oil, 1.10 mmol). Com-
pound 3a-e (1.00 mmol) in 5 mL of DMF was slowly added to the slurry
with stirring. The reaction mixture was stirred until H2 evolution ceased
(10 min). The vial was then sealed and purged with argon followed by
slowly addition of ethyl bromoacetate (1.50 mmol) or ethyl 3-bromopro-
pionate (1.50 mmol). The reaction mixture was stirred at rt (for ethyl
bromoacetate) or reflux (for ethyl 3-bromopropionate), then allowed to
cool to rt. The reaction was quenched with 15 mL of water, then diluted
with EtOAc. The organic layer was washed several times with water and
brine. The organic layer was dried over Na2SO4 and concentrated. The
crude products were purified by column chromatography or through
recrystallization.
mond): 2940, 1731, 1666, 1594, 1144, 736 cmꢀ 1; H NMR (400 MHz,
CDCl3) δ (ppm): 7.59 – 7.55 (m, 2H), 6.89 – 6.85 (m, 2H), 6.56 (s, 2H),
4.67 (s, 2H), 4.56 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.81 (s, 6H), 3.79 (d, J
= 2.4 Hz, 6H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 169.4, 161.1, 153.4, 151.4, 142.7, 136.9, 127.0, 125.9, 114.0,
103.2, 61.2, 60.8, 56.3, 55.4, 51.7, 48.9, 14.2; HRMS (EI): Exact mass
calcd for C23H27N3O7 [M]+: 457.1844. Found: 457.1828; HPLC analysis
showed purity 96.3%.
4.1.2.6. Ethyl 3-(3-oxo-4,6-diphenyl-4,5-dihydro-1,2,4-triazin-2(3H)-yl)
propanoate 4f.. White solid (0.263 g, 75%); TLC Rf = 0.30 in 10%
EtOAc/CH2Cl2; MP = 145–147 ◦C;
IR (ATR diamond): 2189, 1731, 1649, 1495, 1199, 772 cmꢀ 1
;
1H
NMR (300 MHz, CDCl3) δ (ppm): 7.66 – 7.62 (m, 2H), 7.39 – 7.32 (m,
7H), 7.23 – 7.17 (m, 1H), 4.63 (s, 2H), 4.19 (t, J = 7.4 Hz, 2H), 4.12 (t, J
= 7.1 Hz, 2H), 2.80 (t, J = 7.1 Hz, 2H), 1.25 – 1.20 (m, 3H); 13C NMR
4.1.2.1. Ethyl 2-(3-oxo-4,6-diphenyl-4,5-dihydro-1,2,4-triazin-2(3H)-yl)
acetate 4a.. White solid (0.320 g, 95%); TLC Rf = 0.3 in 10% EtOAc/
12

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
(101 MHz, CDCl3) δ (ppm): 171.9, 151.0, 142.6, 141.1, 133.5, 129.9,
129.0, 128.7, 126.2, 125.3, 124.5, 60.5, 47.5, 45.9, 33.6, 14.3; HRMS
(EI): Exact mass calcd for C20H21N3O3 [M]+: 351.1577. Found:
351.1554; HPLC analysis showed purity 98.1%.
4.1.3.1. 2-(3-Oxo-4,6-diphenyl-4,5-dihydro-1,2,4-triazin-2(3H)-yl)acetic
acid 5a.. White solid (0.293 g, 95%); MP = 205–207 ◦C; IR (ATR dia-
mond): 3057, 2176, 1728, 1658, 1500, 1144, 772 cmꢀ 1; 1H NMR (400
MHz, CDCl3) δ (ppm): 11.10 (s, 1H), 7.67 – 7.62 (m, 2H), 7.38 (dd, J =
2.7, 6.2 Hz, 7H), 7.26 – 7.21 (m, 1H), 4.73 (s, 2H), 4.64 (s, 2H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 174.2, 151.5, 143.2, 140.7, 133.2, 130.1,
129.1, 128.7, 126.6, 125.5, 124.8, 51.6, 48.2; HRMS (EI): Exact mass
calcd for C17H15N3O3[M]+: 309. 1108. Found: 309.1105; HPLC analysis
showed purity > 99%.
4.1.2.7. Ethyl 3-(6-(4-methoxyphenyl)-3-oxo-4-phenyl-4,5-dihydro-1,2,4-
triazin-2(3H)-yl)propanoate 4 g. Yellow oil (0.247 g, 65%); TLC Rf =
0.30 in 10% EtOAc/CH2Cl2; IR (ATR diamond): 2997, 1727, 1666, 1572,
1173, 733 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.61 – 7.58 (m,
2H), 7.37 – 7.34 (m, 4H), 7.23 – 7.19 (m, 1H), 6.90 – 6.87 (m, 2H), 4.63
(s, 2H), 4.19 – 4.16 (m, 2H), 4.14 – 4.11 (m, 2H), 3.80 (s, 3H), 2.79 (t, J
= 7.2 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 171.9, 161.0, 151.2, 142.6, 141.2, 129.0, 126.9, 126.1, 124.5,
114.1, 60.5, 55.4, 47.5, 45.8, 33.6, 14.2; HRMS (ESI): Exact mass calcd
for C21H23N3O4Na [M + Na] +: 404.1581. Found: 404.1586; HPLC
analysis showed purity 96.4%.
4.1.3.2. 2-(6-(4-Methoxyphenyl)-3-oxo-4-phenyl-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl)acetic acid 5b.. White solid (0.3222 g, 95%); MP =
150–152 ◦C; IR (ATR diamond): 3060, 2501, 1742, 1632, 1514, 1147,
764 cmꢀ 1; 1H NMR (300 MHz, CDCl3) δ (ppm): 10.77 (s, 1H), 7.61 – 7.56
(m, 2H), 7.38 – 7.33 (m, 4H), 7.25 – 7.19 (m, 1H), 6.91 – 6.86 (m, 2H),
4.70 (s, 2H), 4.62 (s, 2H), 3.80 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 174.0, 161.1, 151.6, 143.1, 140.8, 129.0, 127.1, 126.5, 125.8,
124.7, 114.1, 65.9, 55.4, 51.6, 48.1, 15.2; HRMS (EI): Exact mass calcd
for C18H17N3O4 [M]+: 339. 1214.Found: 339.1236; HPLC analysis
showed purity > 99%.
4.1.2.8. Ethyl 3-(4,6-bis(4-methoxyphenyl)-3-oxo-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl)propanoate 4 h. White solid (0.337 g, 82%); TLC Rf = 0.30
in 10% EtOAc/CH2Cl2; MP = 146–148 ◦C; IR (ATR diamond): 2033,
1730, 1660, 1572, 1173, 733 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm):
7.54 – 7.50 (m, 2H), 7.18 (d, J = 8.9 Hz, 2H), 6.84 – 6.80 (m, 4H), 4.50
(s, 2H), 4.12 – 4.08 (m, 2H), 4.08 – 4.00 (m, 2H), 3.71 (s, 3H), 3.68 (s,
3H), 2.72 (t, J = 7.1 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H); 13C NMR (101
MHz, CDCl3) δ (ppm): 171.9, 160.9, 157.8, 151.3, 142.3, 134.2, 126.8,
126.1, 114.2, 114.0, 60.4, 55.4, 55.3, 48.1, 45.7, 33.6, 14.2; HRMS
(ESI): Exact mass calcd for C22H25N3O5Na [M + Na] +: 434.1687.
Found: 434.1692; HPLC analysis showed purity 97.3%.
4.1.3.3. 2-(4,6-Bis(4-methoxyphenyl)-3-oxo-4,5-dihydro-1,2,4-triazin-2
(3H)-yl)acetic acid 5c.. White solid (0.350 g, 95%); MP = 195–197 ◦C;
IR (ATR diamond): 3041, 2879, 1744, 1642, 1509, 1151, 733 cmꢀ 1; 1H
NMR (400 MHz, DMSO) δ (ppm): 12.73 (s, 1H), 7.68 – 7.64 (m, 2H),
7.32 – 7.28 (m, 2H), 6.96 – 6.91 (m, 4H), 4.75 (s, 2H), 4.37 (s, 2H), 3.75
(s, 3H), 3.73 (s, 3H); 13C NMR (101 MHz, DMSO) δ (ppm): 171.0, 161.0,
157.6, 151.2, 143.0, 134.5, 127.5, 126.7, 126.1, 114.4, 114.3, 55.8,
55.7, 51.8, 48.4; HRMS (EI): Exact mass calcd for C19H19N3O5 [M]+:
369.1319. Found:369.1339; HPLC analysis showed purity > 99%.
4.1.2.9. Ethyl 3-(4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl)-3-oxo-
4,5-dihydro-1,2,4-triazin-2(3H)-yl)propanoate 4i.. White solid (0.234 g,
55%); TLC Rf = 0.30 in 10% EtOAc/CH2Cl2; MP = 130–132 ◦C; IR (ATR
4.1.3.4. 2-(4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-3-oxo-4,5-
dihydro-1,2,4-triazin-2(3H)-yl)acetic acid 5d.. White solid (0.379 g,
95%); MP = 170–172 ◦C; IR (ATR diamond): 3078, 1747, 1631, 1516,
1149, 897 cmꢀ 1; 1H NMR (400 MHz, DMSO) δ (ppm): 12.76 (s, 1H), 7.71
– 7.66 (m, 2H), 6.97 – 6.93 (m, 2H), 6.58 (d, J = 2.2 Hz, 2H), 6.37 (t, J =
2.2 Hz, 1H), 4.78 (s, 2H), 4.38 (s, 2H), 3.76 (s, 3H), 3.71 (s, 6H); 13C
NMR (101 MHz, DMSO) δ (ppm): 171.0, 161.1, 160.7, 150.9, 143.5,
143.2, 127.6, 126.0, 114.5, 103.7, 98.2, 55.8, 55.8, 51.8, 47.8; HRMS
(EI): Exact mass calcd for C20H21N3O6 [M]+: 399.1425. Found:
399.1420; HPLC analysis showed purity > 99%.
diamond): 2043, 1713, 1661, 1513, 1149, 797 cmꢀ 1
;
1H NMR (400
MHz, CDCl3) δ (ppm): 7.59 – 7.55 (m, 2H), 6.88 – 6.84 (m, 2H), 6.49 (d,
J = 2.2 Hz, 2H), 6.30 (t, J = 2.2 Hz, 1H), 4.57 (s, 2H), 4.18 – 4.13 (m,
2H), 4.13 – 4.08 (m, 2H), 3.78 (s, 3H), 3.74 (s, 6H), 2.77 (t, J = 7.1 Hz,
2H), 1.21 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm):
171.9, 161.0, 160.9, 151.0, 143.0, 142.7, 126.9, 126.0, 114.1, 103.1,
98.4, 60.4, 55.5, 55.4, 47.5, 45.7, 33.6, 14.2; HRMS (ESI): Exact mass
calcd for C23H27N3O6Na [M + Na] +: 464.1792. Found:464.1796; HPLC
analysis showed purity 95%.
4.1.2.10. Ethyl
3-(6-(4-methoxyphenyl)-3-oxo-4-(3,4,5-trimethoxy-
4.1.3.5. 2-(6-(4-Methoxyphenyl)-3-oxo-4-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1,2,4-triazin-2(3H)-yl)acetic acid 5e.. White solid (0.407 g,
95%); MP = 235–237 ◦C; IR (ATR diamond): 2942, 2521, 1735, 1622,
1515, 1205, 769 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 9.32 (s, 1H),
7.55 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.56 (s, 2H), 4.65 (s,
2H), 4.58 (s, 2H), 3.80 (s, 9H), 3.78 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ (ppm): 173.5, 161.2, 153.4, 151.7, 143.0, 136.9, 136.6, 127.0, 125.7,
114.1, 103.3, 60.9, 56.3, 55.4, 51.5, 48.9; HRMS (EI):Exact mass calcd
for C21H23N3O7 [M]+: 429.1531. Found: 429.1561; HPLC analysis
showed purity > 99%.
phenyl)-4,5-dihydro-1,2,4-triazin-2(3H)-yl)propanoate 4j.. White solid
(0.259 g, 55%); TLC Rf = 0.30 in 10% EtOAc/CH2Cl2; MP = 128–130 ◦C;
IR (ATR diamond): 2833, 1728, 1662, 1592, 1122, 737 cmꢀ 1; 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.58 – 7.53 (m, 2H), 6.87 – 6.82 (m, 2H),
6.51 (s, 2H), 4.57 (s, 2H), 4.13 (d, J = 7.2 Hz, 2H), 4.10 – 4.06 (m, 2H),
3.79 (s, 6H), 3.77 (s, 3H), 3.76 (s, 3H), 2.74 (t, J = 7.1 Hz, 2H), 1.19 (t, J
= 7.2 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 171.9, 161.0, 153.4,
151.3, 142.4, 137.0, 136.6, 126.8, 126.0, 114.0, 103.0, 60.8, 60.4, 56.2,
55.4, 48.2, 45.7, 33.6, 14.2; HRMS (EI): Exact mass calcd for
C
24H29N3O7 [M]+: 471.2000. Found:471.2014; HPLC analysis showed
purity 95%.
4.1.3.6. 3-(3-Oxo-4,6-diphenyl-4,5-dihydro-1,2,4-triazin-2(3H)-yl) prop-
anoic acid 5f.. White solid (0.306 g, 95%); MP = 135–137 ◦C; IR (ATR
diamond): 3095, 2579, 1728, 1636, 1497, 1195, 752 cmꢀ 1
;
1H NMR
4.1.3. General procedure for synthesis of compounds (5a-5j).
In a dry microwave vial equipped with a stir bar, ester derivatives 4a-
j (1.0 equiv.) in sufficient amount of methanol, then a methanolic so-
lution of 2 N NaOH was added slowly. The resulting solution was left
stirring at reflux for 3–4 h. At this point, we formed the sodium salt, the
reaction mixture was concentrated under reduced pressure. After that,
the reaction was quenched with 1 N HCl and extracted with EtOAc (3 ×
10 mL). The organic layers were combined, dried with Na2SO4, filtered,
and concentrated under reduced pressure. The crude products were
purified by recrystallization in MeOH.
(400 MHz, CDCl3) δ (ppm): 7.66 – 7.63 (m, 2H), 7.39 – 7.34 (m, 7H),
7.25 – 7.22 (m, 1H), 4.68 (s, 2H), 4.20 (t, J = 6.9 Hz, 2H), 2.84 (t, J =
7.0 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ (ppm): 177.2, 151.3, 142.9,
141.0, 133.3, 130.0, 129.1, 128.7, 126.4, 125.4, 124.7, 47.7, 45.6, 33.4;
HRMS (EI): Exact mass calcd for C18H17N3O3 [M]+: 323.1264. Found:
323.1295; HPLC analysis showed purity > 99%.
4.1.3.7. 3-(6-(4-Methoxyphenyl)-3-oxo-4-phenyl-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl) propanoic acid 5 g.. White solid (0.320 g, 95%); MP =
13

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Bioorganic Chemistry 112 (2021) 104965
150–152 ◦C; IR (ATR diamond): 2942, 1703, 1656, 1512, 1170, 732
cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 10.57 (s, 1H), 7.61 – 7.56 (m,
2H), 7.39 – 7.31 (m, 4H), 7.24 – 7.18 (m, 1H), 6.90 – 6.87 (m, 2H), 4.61
125.5, 124.7, 53.5, 48.1; HRMS (EI): Exact mass calcd for C17H16N4O2
[M]+: 308. 1268.Found: 308.1268; HPLC analysis showed purity 99%.
(s, 2H), 4.17 (t, J = 7.0 Hz, 2H), 3.78 (s, 3H), 2.81 (t, J = 7.0 Hz, 2H); 13
C
4.1.4.2. 2-(6-(4-Methoxyphenyl)-3-oxo-4-phenyl-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl)acetamide 6b.. Yellow viscous oil (0.229 g, 68%); TLC Rf
= 0.30 in 3% MeOH/CH2Cl2; MP = 193–195 ◦C; IR (ATR diamond):
3056, 2543, 1728, 1640, 1146, 754 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.62 – 7.59 (m, 2H), 7.40 – 7.33 (m, 4H), 7.25 – 7.22 (m, 1H),
6.90 – 6.87 (m, 2H), 6.30 (s, 1H), 5.77 (s, 1H), 4.71 (s, 2H), 4.52 (s, 2H),
3.81 (s, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 171.6, 161.3, 151.7,
143.8, 140.8, 129.1, 127.1, 126.6, 125.5, 124.7, 114.2, 55.4, 53.7, 48.0;
HRMS (EI): Exact mass calcd for C18H18N4O3 [M]+: 338. 1373.Found:
338.1358; HPLC analysis showed purity 98.1%.
NMR (101 MHz, CDCl3) δ (ppm): 177.11, 161.06, 151.46, 142.92,
141.04, 129.01, 126.97, 126.32, 125.93, 124.68, 114.11, 55.39, 47.57,
45.51, 33.45; HRMS (EI):Exact mass calcd for C19H19N3O4 [M]+:
353.1370. Found: 353.1395; HPLC analysis showed purity > 99%.
4.1.3.8. 3-(4,6-Bis(4-methoxyphenyl)-3-oxo-4,5-dihydro-1,2,4-triazin-2
(3H)-yl) propanoic acid 5 h.. White solid (0.364 g, 95%); MP =
162–164 ◦C; IR (ATR diamond): 2942, 1703, 1656, 1512, 1170, 732
cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 10.61 (s, 1H), 7.59 – 7.54 (m,
2H), 7.25 – 7.21 (m, 2H), 6.90 – 6.86 (m, 4H), 4.59 (s, 2H), 4.15 (t, J =
6.9 Hz, 2H), 3.79 (s, 3H), 3.77 (s, 3H), 2.80 (t, J = 6.9 Hz, 2H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 177.1, 161.0, 158.0, 151.7, 142.6, 134.0,
126.9, 126.4, 126.0, 114.4, 114.1, 55.5, 55.4, 48.3, 45.5, 33.5; HRMS
(ESI): Exact mass calcd for C20H21N3O5Na [M + Na] +: 406.1374.
Found: 406.1379; HPLC analysis showed purity > 99%.
4.1.4.3. 2-(4,6-Bis(4-methoxyphenyl)-3-oxo-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl) acetamide 6c.. White solid (0.294 g, 80%); TLC Rf =
0.30 in 5% MeOH/CH2Cl2; MP = 195–197 ◦C; IR (ATR diamond): 3168,
2030, 1652, 1509, 1175, 828 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm):
7.59 – 7.54 (m, 2H), 7.25 – 7.21 (m, 2H), 6.89 – 6.85 (m, 4H), 6.44 (s,
1H), 5.92 (s, 1H), 4.64 (s, 2H), 4.47 (s, 2H), 3.79 (s, 3H), 3.75 (s, 3H);
13C NMR (101 MHz, CDCl3) δ (ppm): 171.9, 161.2, 158.1, 151.8, 143.4,
133.7, 127.0, 126.4, 125.6, 114.4, 114.1, 55.5, 55.4, 53.7, 48.7; HRMS
(EI): Exact mass calcd for C19H20N4O4 [M]+: 368. 1479. Found:
368.1484; HPLC analysis showed purity > 99%.
4.1.3.9. 3-(4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-3-oxo-4,5-
dihydro-1,2,4-triazin-2(3H)-yl) propanoic acid 5i.. White solid (0.392 g,
95%); MP = 172–174 ◦C; IR (ATR diamond): 3094, 2193, 1662, 1571,
1513, 1174, 799 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.58 (d, J =
8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.50 (d, J = 2.2 Hz, 2H), 6.33 (d, J
= 2.3 Hz, 1H), 4.59 (s, 2H), 4.16 (t, J = 6.9 Hz, 2H), 3.80 (s, 3H), 3.76 (s,
6H), 2.82 (t, J = 6.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ (ppm):
177.1, 161.1, 160.9, 151.3, 143.1, 142.8, 126.9, 125.8, 114.1, 103.3,
98.6, 55.5, 55.4, 47.7, 45.4, 33.5; HRMS (EI):Exact mass calcd for
4.1.4.4. 2-(4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-3-oxo-
4,5-dihydro-1,2,4-triazin-2(3H)-yl)acetamide
6d.. White
solid
(0.274 g, 69%); TLC Rf = 0.30 in 5% MeOH/CH2Cl2; MP = 205–207 ◦C;
IR (ATR diamond): 3459, 3301, 1678, 1639, 1414, 1147, 731 cmꢀ 1; 1H
NMR (400 MHz, CDCl3) δ (ppm): 7.61 – 7.58 (m, 2H), 6.90 – 6.87 (m,
2H), 6.50 (d, J = 2.2 Hz, 2H), 6.34 (t, J = 2.2 Hz, 1H), 6.29 (s, 1H), 5.71
(s, 1H), 4.66 (s, 2H), 4.51 (s, 2H), 3.81 (s, 3H), 3.76 (s, 6H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 171.5, 161.3, 161.0, 151.5, 143.9, 142.5,
127.1, 125.5, 114.1, 103.4, 98.8, 55.5, 55.4, 53.7, 48.1; HRMS (EI):
Exact mass calcd for C20H22N4O5 [M]+: 398. 1585.Found: 398.1583;
HPLC analysis showed purity > 99%.
C
21H23N3O6 [M]+: 413.1581. Found: 413.1607; HPLC analysis showed
purity > 99%.
4.1.3.10. 3-(6-(4-Methoxyphenyl)-3-oxo-4-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1,2,4-triazin-2(3H)-yl)propanoic acid 5j.. White solid (0.421 g,
95%); MP = 220–222 ◦C; IR (ATR diamond): 2942, 2360, 1717, 1643,
1594, 1126, 736 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.59 – 7.55
(m, 2H), 6.89 – 6.86 (m, 2H), 6.54 (s, 2H), 4.60 (s, 2H), 4.14 (t, J = 7.0
Hz, 2H), 3.82 (s, 6H), 3.80 (s, 3H), 3.79 (s, 3H), 2.79 (t, J = 7.0 Hz, 2H);
13C NMR (101 MHz, CDCl3) δ (ppm): 176.5, 161.1, 153.4, 151.5, 142.8,
136.8, 126.9, 125.9, 114.1, 103.2, 60.9, 56.3, 55.4, 50.5, 48.4, 45.5,
33.4; HRMS (EI):Exact mass calcd for C22H25N3O7 [M]+: 443.1687.
Found: 443.1712; HPLC analysis showed purity > 99%.
4.1.4.5. 2-(6-(4-Methoxyphenyl)-3-oxo-4-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1,2,4-triazin-2(3H)-yl)acetamide
6e.. White
solid
(0.308 g, 72%); TLC Rf = 0.30 in 3% MeOH/CH2Cl2; MP = 240–242 ◦C;
IR (ATR diamond): 3381, 3209, 1686, 1640, 1510, 1123, 740 cmꢀ 1; 1H
NMR (400 MHz, CDCl3) δ (ppm): 7.62 – 7.56 (m, 2H), 6.91 – 6.86 (m,
2H), 6.56 (s, 2H), 6.26 (s, 1H), 5.77 (s, 1H), 4.68 (s, 2H), 4.51 (s, 2H),
3.83 (s, 6H), 3.81 (d, J = 1.1 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 171.5, 161.3, 153.5, 151.7, 143.6, 137.1, 136.6, 127.0, 125.5,
114.2, 103.3, 60.9, 56.3, 55.4, 53.7, 48.9; HRMS (EI): Exact mass calcd
for C21H24N4O6 [M]+: 428. 1690.Found: 428.1688; HPLC analysis
showed purity 98.8%.
4.1.4. General procedure for synthesis of compounds (6a-6j).
In a dry microwave vial equipped with a stir bar, 5 mL of dry DMF
was added to NaH (60% dispersion in mineral oil, 1.10 mmol). Com-
pound 3a-e (1.00 mmol) in 5 mL of DMF was slowly added to the slurry
with stirring. The reaction mixture was stirred until H2 evolution ceased
(10 min). The vial was then sealed and purged with argon followed by
slowly addition of iodoacetamide (1.5 mmol) or 3-chloropropionamide
(1.5 mmol). The reaction mixture was stirred at rt (for iodoacetamide) or
reflux (for 3-chloropropionamide), then allowed to cool to rt. The re-
action was quenched with 15 mL of water, then diluted with EtOAc. The
organic layer was washed several times with water and brine. The
organic layer was dried over Na2SO4 and concentrated. The crude
products were purified by column chromatography or through
recrystallization.
4.1.4.6. 3-(3-Oxo-4,6-diphenyl-4,5-dihydro-1,2,4-triazin-2(3H)-yl) pro-
penamide 6f. White solid (0.229 g, 71%); TLC Rf = 0.30 in 3% MeOH/
CH2Cl2; MP = 127–129 ◦C; IR (ATR diamond): 3179, 2922, 1720, 1647,
1496, 1195, 751 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.68 – 7.63
(m, 2H), 7.39 (dtd, J = 2.0, 3.6, 7.2 Hz, 6H), 7.35 – 7.32 (m, 2H), 6.44 (s,
1H), 5.6 (s, 1H), 4.70 (s, 2H), 4.18 (t, J = 6.7 Hz, 2H), 2.75 (t, J = 6.7 Hz,
2H); 13C NMR (101 MHz, CDCl3) δ (ppm): 173.6, 151.5, 142.9, 140.9,
133.2, 130.1, 129.1, 128.8, 125.3, 124.7, 47.9, 46.3, 35.3, 29.7; HRMS
(EI): Exact mass calcd for C18H18N4O2 [M]+: 322. 1424.Found:
322.1441; HPLC analysis showed purity 97.9%.
4.1.4.1. 2-(3-Oxo-4,6-diphenyl-4,5-dihydro-1,2,4-triazin-2(3H)-yl)
acetamide 6a.. White solid (0.231 g, 75%); TLC Rf = 0.3 in 3% MeOH/
CH2Cl2; MP = 191–193 ◦C; IR (ATR diamond): 3185, 2178, 1651, 1593,
1498, 1146, 754 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.63 – 7.59
(m, 2H), 7.37 – 7.31 (m, 7H), 7.20 (tt, J = 2.1, 7.1 Hz, 1H), 6.52 (s, 1H),
6.10 (s, 1H), 4.68 (s, 2H), 4.48 (s, 2H); 13C NMR (101 MHz, CDCl3) δ
(ppm): 171.7, 151.5, 143.7, 140.8, 133.1, 130.2, 129.1, 128.7, 126.6,
4.1.4.7. 3-(6-(4-Methoxyphenyl)-3-oxo-4-phenyl-4,5-dihydro-1,2,4-
triazin-2(3H)-yl)propenamide 6 g.. Yellow crystal (0.354 g, 72%); TLC
Rf = 0.30 in 3% MeOH/CH2Cl2; MP = 190–192 ◦C; IR (ATR diamond):
1
3367, 3182, 1698, 1652, 1449, 1001, 759 cmꢀ 1; H NMR (400 MHz,
CDCl3) δ (ppm): 7.61 – 7.57 (m, 2H), 7.40 – 7.30 (m, 5H), 6.90 – 6.87 (m,
14

I.H. Eissa et al.
Bioorganic Chemistry 112 (2021) 104965
2H), 6.53 (s, 1H), 5.64 (s, 1H), 4.65 (s, 2H), 4.15 (t, J = 6.7 Hz, 2H), 3.81
(s, 3H), 2.73 (t, J = 6.7 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ (ppm):
173.7, 161.1, 151.6, 142.9, 141.0, 129.1, 126.9, 126.5, 125.8, 124.7,
114.1, 55.4, 47.8, 46.2, 35.3; HRMS (EI): Exact mass calcd for
absorbance of 570 nm using a plate reader (EXL 800, USA). The relative
cell viability in percentage was calculated as (A570 of treated samples/
A570 of untreated sample) X 100. Results for IC50values of the active
compounds were summarized in Table 1. The toxicities of the most
promising compounds against normal human cells (WI-38) were
assessed in the same procedure.
C
19H20N4O3 [M]+: 352. 1530.Found: 352.1540; HPLC analysis showed
purity > 99%.
4.2.2. Tubulin polymerization assay
4.1.4.8. 3-(4,6-Bis(4-methoxyphenyl)-3-oxo-4,5-dihydro-1,2,4-tri-
azin-2(3H)-yl) propenamide 6 h.. White solid (0.268 g, 70%); TLC Rf
The effect of the synthesized compounds on tubulin polymerization
was assessed turbidimetrically using a fluorescent plate reader method
[99]. At first, ten of the synthesized compounds and reference drug
(colchicine) were incubated in mixture of purified bovine tubulin (10
µM) and buffer system containing 20% glycerol and 1 mM ATP at 37 ◦C.
◦
= 0.30 in 10% EtOAc/CH2Cl2; MP = 140–142 C; IR (ATR diamond):
1
3392, 2029, 1697, 1652, 1515, 1173, 734 cmꢀ 1; H NMR (400 MHz,
CDCl3) δ (ppm): 7.59 – 7.54 (m, 2H), 7.24 – 7.19 (m, 2H), 6.90 – 6.86 (m,
4H), 6.61 (s, 1H), 5.68 (s, 1H), 4.59 (s, 2H), 4.12 (t, J = 6.7 Hz, 2H), 3.79
(s, 3H), 3.76 (s, 3H), 2.71 (t, J = 6.7 Hz, 2H); 13C NMR (101 MHz, CDCl3)
δ (ppm): 173.8, 161.1, 158.1, 151.8, 142.5, 133.9, 126.9, 126.4, 125.9,
114.4, 114.1, 55.5, 55.4, 48.5, 46.2, 35.3; HRMS (EI): Exact mass calcd
for C20H22N4O4 [M]+: 382. 1636.Found: 382.1636; HPLC analysis
showed purity > 99%.
◦
Then, the mixture cooled to 0 C. The IC50 value was defined as the
compound concentration that inhibited the extent of tubulin assembly
by 50%.
4.2.3. Cell cycle analysis
HepG-2 cells were seeded at density of 2 × 105 cells per well and
incubated for 24 h in six-well plates. Fetal bovine serum (FBS, 10%) was
added, after that cells were incubated at 37 ◦C and 5% CO2. The medium
was replaced with (DMSO 1% v/v) containing the tested compounds 3e
(67.7 µM) and 5i (8.2 µM) for HepG-2. Then, the cells were incubated for
48 h, washed with cold phosphate buffered saline (PBS), fixed with 70%
ethanol, rinsed with PBS then stained with the DNA fluorochrome PI,
kept for 15 min at 37 ◦C. Then samples were analyzed with a FACS
Caliber flow cytometer [88].
4.1.4.9. 3-(4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-3-oxo-
4,5-dihydro-1,2,4-triazin-2(3H)-yl)propenamide 6i.. White solid
(0.297 g, 72%); TLC Rf = 0.30 in 5% MeOH/CH2Cl2; MP = 170–172 ◦C;
IR (ATR diamond): 3421, 3303, 1651, 1605, 1513, 1170, 799 cmꢀ 1; 1H
NMR (400 MHz, CDCl3) δ (ppm): 7.61 – 7.57 (m, 2H), 6.91 – 6.87 (m,
2H), 6.50 (d, J = 2.2 Hz, 1H), 6.48 (d, J = 2.2 Hz, 2H), 6.34 (s, 1H), 5.52
(s, 1H), 4.61 (s, 2H), 4.16 (t, J = 6.7 Hz, 2H), 3.81 (s, 3H), 3.76 (s, 6H),
2.75 (t, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ (ppm): 173.6,
161.1, 161.0, 151.4, 143.0, 142.7, 126.9, 125.8, 114.1, 103.4, 98.6,
55.5, 55.4, 47.9, 46.1, 35.3; HRMS (EI): Exact mass calcd for
4.2.4. Annexin V-FITC apoptosis assay
The effect of compound 3e and 5i on apoptosis induction was
analyzed using Annexin V-FITC/PI apoptosis detection kit. In this test,
HepG-2 cells were stained with Annexin V fluorescein isothiocyanate
(FITC) and counterstained with propidium iodide (PI). Then, HepG-2
cells in densities of 2 × 105 per well were incubated with compounds
3e (67.7 µM) and 5i (8.2 µM) for HepG-2 for 48 h. Next, the cells were
trypsinized, washed with phosphate-buffered saline (PBS), and stained
for 15 min at 37 ◦C in the dark. Finally, FACS Caliber flow cytometer was
used in analysis process [91].
C
21H24N4O5 [M]+: 412. 1741.Found: 412.1761; HPLC analysis showed
purity > 99%.
4.1.4.10. 3-(6-(4-Methoxyphenyl)-3-oxo-4-(3,4,5-trimethox-
yphenyl)-4,5-dihydro-1,2,4-triazin-2(3H)-yl)propenamide 6j.. White
solid (0.309 g, 70%); TLC Rf = 0.30 in 5% MeOH/CH2Cl2; MP =
225–227 ◦C; IR (ATR diamond): 3394, 2937, 1663, 1629, 1595, 1124,
733 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.61 – 7.56 (m, 2H), 6.91
– 6.87 (m, 2H), 6.53 (s, 2H), 6.45 (s, 1H), 5.66 (s, 1H), 4.62 (s, 2H), 4.14
(t, J = 6.7 Hz, 2H), 3.82 (s, 6H), 3.80 (d, J = 0.7 Hz, 6H), 2.74 (t, J = 6.7
Hz, 2H); 13C NMR (101 MHz, CDCl3) δ (ppm): 173.6, 161.2, 153.5,
151.6, 142.7, 137.0, 136.8, 126.9, 125.8, 114.1, 103.2, 60.9, 56.3, 55.4,
48.6, 46.1, 35.3; HRMS (EI): Exact mass calcd for C22H26N4O6 [M]+:
442. 1847.Found: 442.1875; HPLC analysis showed purity 99%.
4.2.5. Caspase-3 determination
The percentage of caspase-3 activation was determined using the
Caspase- Invitrogen Caspase-3 ELISA Kit (KHO1091) following the
manufacturer’s instructions [100,101].
4.2.6. Bax determination (real-time PCR).
The effect of compounds 4i, 4j, 5 g, 5i and 6i on BAX expressions
was determined at concentrations of 14.4, 18.5, 23.2, 8.2, and 11.2 µM,
respectively after 48 h of treatment on HepG-2 cells, using real-time
PCR. The expression level of BAX was determined using a SYBR Green
qRT-PCR kit (Bioneer, Daejeon, Korea) according to the manufacturer’s
protocol. PCR reactions were performed using specific primers for BAX.
BAX forward primer: 5′-GCCCTTTTGCTTCAGGGTTT-3′; BAX Reverse
primer: 5′ TCCAATGTCCAGCCCATGAT-3′. The reactions were carried
out in triplicate using an Eco Real-Time PCR system (San Diego, CA,
USA) [102].
4.2. Biological evaluation
4.2.1. In vitro cytotoxic activity
Evaluation of cytotoxic activity of the synthesized compounds was
carried out using MTT assay protocol [81,97,98] against a group of
cancer cell lines namely; colorectal carcinoma (HCT-116), Hepatocel-
lular carcinoma (HepG-2) and breast cancer (MCF-7) and colchicine was
used as a standard drug. The cells were obtained from ATCC (American
Type Culture Collection) via the Holding company for biological prod-
ucts and vaccines (VACSERA) (Cairo, Egypt). The anti-cancer activity
was measured quantitatively as follows:
Into a medium of RPMI-1640 with 10% fetal bovine serum, the cell
lines were cultured. Then, penicillin (100 units/ml) and streptomycin
(100 µg/ml) were added at 37 ◦C in a 5% CO2 incubator. Next, seeding
the cell lines in a 96-well plate was achieved by a density of 1.0 × 104
cells / well at 37 ◦C for 48 h under 5% CO2. After incubation period, the
cell lines were treated with different concentration of the synthesized
compounds and incubated for 24 h. After treatment by 24 h, 20 µl of
MTT solution (5 mg/ml) was added and incubated for 4 h. Dimethyl
sulfoxide (100 µl) was added into each well to dissolve the formed
purple formazan. The colorimetric assay was measured and recorded at
4.3. In silico studies
4.3.1. Flexible alignment and docking
The crystal structure of tubulin heterodimers was downloaded from
the Protein Data Bank, http://www.rcsb.org/pdb (PDB ID: 1SA0, reso-
lution: 3.58 Å) using Discovery Studio 4.0 software. Water molecules
were removed from downloaded protein. Crystallographic disorders and
unfilled valence atoms were corrected using alternate conformations
and valence monitor options. Protein was subjected to energy minimi-
zation by applying CHARMm force fields for charge, and MMFF94 force
15

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Bioorganic Chemistry 112 (2021) 104965
[5] N. Dalbeth, T.J. Lauterio, H.R. Wolfe, Mechanism of action of colchicine in the
field for partial charge. Inflexibility of structure was obtained by
creating fixed atom constraint. The binding site of the protein was
defined and prepared for docking. DAMA-colchicine, colchicine and the
designed compounds 2D structures were sketched using ChemBioDraw
Ultra 14.0 and saved in MDL-SD file format. Next, the SD file was
opened, 3D structures were protonated, and energy was minimized by
applying CHARMm force fields for charge andMMFF94 force field for
partial charge and then prepared for docking by optimization of the
parameters. Docking was accomplished using CDOCKER-CHARMm-
based technique in the interface of Accelry’s Discovery Studio 4.0. A
maximum of 10 conformers was considered for each molecule in the
docking analysis. After that the docking scores (CDOCKER interaction
energy) of the best -fitted conformation of each of the docked molecules
with the amino acids at the tubulin heterodimers binding pocket were
recorded [103-105].
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4.3.2. In silico ADMET analysis
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autophagy and necrosis in human cancer cells, PLoS One 9 (1) (2014), e87064.
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Reviews 8 (1) (1988) 77–94.
ADMET descriptors (absorption, distribution, metabolism, excretion
and toxicity) of the compounds were determined using Discovery studio
4.0. At first, the CHARMM force field was applied then the compounds
were prepared and minimized according to the preparation of small
molecule protocol. Then ADMET descriptors protocol was applied to
carry out these studies [104,105].
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polymerization and PD-1/PD-L1 interaction for cancer treatment, European
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4.3.3. Toxicity studies
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Chemistry to Clinic, A Review. Pharmaceuticals (Basel) 13 (1) (2020).
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(2020) 8.
The toxicity parameters of the most promising members 4i, 4j, 5 g,
5i and 6i were calculated using Discovery studio 4.0. colchicine was
used as a reference drug. At first, the CHARMM force field was applied
then the compounds were prepared and minimized according to the
preparation of small molecule protocol. Then different parameters were
calculated from toxicity prediction (extensible) protocol.
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4.3.4. Physico-chemical properties and Lipinski rule of five
The physico-chemical properties of the selected compounds were
calculated using Discovery studio 4.0. At first, the CHARMM force field
was applied then the compounds were prepared and minimized ac-
cording to the preparation of small molecule protocol. Then different
parameters were calculated from calculate molecular properties from
small molecule protocol [103].
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Declaration of Competing Interest
[25] Y. Jin, et al., 3D-QSAR Study of Combretastatin A-4 Analogs Based on Molecular
Docking, Molecules 16 (8) (2011) 6684–6700.
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FEBS Lett 161 (1) (1983) 55–59.
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.
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derivatives bearing naphthalene moiety as novel tubulin polymerization
inhibitors, Bioorganic Chemistry 104 (2020), 104265.
[28] G. Li, et al., Design, synthesis, and bioevaluation of pyrazolo[1,5-a]pyrimidine
derivatives as tubulin polymerization inhibitors targeting the colchicine binding
site with potent anticancer activities, European Journal of Medicinal Chemistry
202 (2020), 112519.
Acknowledgment
We thank the University of Ottawa and NSERC for generous financial
support. M.A.D thanks the Egyptian Ministry of High Education and Al-
Azhar University, Cairo, Egypt for supporting the scholarship. The au-
thors extend their sincere appreciation to the Deanship of Scientific
Research and the Research Center, College of Pharmacy, King Saud
University for funding the biological testing and assay for the research
project.
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Arylpyridines as Antiproliferative Agent Targeting Tubulin, ACS Medicinal
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potent tubulin assembly inhibitors, Bioorganic & Medicinal Chemistry 28 (19)
(2020), 115684.
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