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This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021 New J. Chem.
Cite this: DOI: 10.1039/d1nj02885e
Design and synthesis of a new series of
3,5-disubstituted-1,2,4-oxadiazoles as potential
colchicine binding site inhibitors: antiproliferative
activity, molecular docking, and SAR studies†
Rana T. Diab,
a
Zakaria K. Abdel-Sami,
a
Eatedal H. Abdel-Aal,
a
Ahmed A. Al-Karmalawy *
b
and Nader E. Abo-Dya *
ac
The development of anticancer compounds targeting the colchicine-binding site of tubulin, termed
colchicine-binding site inhibitors (CBSIs) is a promising research area for pharmaceutical companies and
research institutes. A series of 3,5-disubstituted 1,2,4-oxadiazoles, sharing common structural features
with colchicine and combretastatins, was designed and synthesized for screening as antiproliferative
CBSIs. All targets were submitted to National Cancer Institute (NCI), USA for screening at 10 mM in full
NCI 60 cell panel. In addition, molecular docking studies were performed for the newly synthesized
oxadiazole derivatives as CBSIs in b-tubulin against the b-tubulin pocket of colchicine. Also, 5a – as the
most active member – was evaluated for its ability to inhibit b-tubulin polymerization against colchicine
6as a reference standard. The targets showed significant antiproliferative activities. Amongst the series,
target 5a demonstrated the broadest and most potent antiproliferative activity (positive cytotoxic effect
(PCE) = 33/60 and the mean growth inhibition (MGI) for the most sensitive cell lines (33) is 24.9%. In
addition, targets (5a–k) showed selective potency towards renal cancer in particular the A498 cell line.
The order of potency of the three most potent targets and the standard colchicine (retrieved from
NCI database) against A498 is: colchicine (GI = 84.5%) 45a (GI = 78%) 45d (GI = 51%) 45f (32%).
Compound 5a achieved superior binding affinity (8.06 kcal mol
1
) compared with that of colchicine 6
itself, which achieved (7.40 kcal mol
1
). Also, 5a showed a two-fold potent inhibitory activity of tubulin
polymerization (IC
50
: 1.18 mM) compared with that of colchicine (IC
50
: 2.37 mM). Thus, target 5a
represents a promising selective anticancer CBSI.
1. Introduction
Cancer is one of the top 10 leading causes of death globally.
It was responsible for about 10 million deaths in 2020.
1
Despite
the presence of multiple treatment strategies for cancer, che-
motherapy remains one of the most significant treatments in
cancer management.
2–4
Unfortunately, most current anticancer
drugs have side effects due to the lack of tumor specificity,
multidrug resistance, toxicity, and reduced bioavailability.
5–8
Thus, chemists are actively searching for new drugs with higher
efficiency and lower side effects.
Microtubule-binding agents (MTAs) inhibit cell proliferation
by halting microtubule assembly/disassembly dynamics during
themitoticphaseofthecellcycle.
9
MTAs are classified into
two major groups, the microtubule-stabilizing agents, and
microtubule-destabilizing agents (Fig. 1). The later ‘‘destabilizing’’
agents prevent tubulin polymerization and promote depolymeri-
zation when present at high concentrations.
10
Most of these
agents bind to either the ‘‘vinca’’ domain near the exchangeable
GTP-binding site (E-site) or the ‘‘colchicine’’ domain at the
intradimer interface between a-andb-tubulin. Examples of vinca
site binders include vinblastine, vincristine, dolastatins, eribulin,
and tasidotin. Colchicine-site binders include colchicine and
its analogs, combretastatins, 2-methoxyestradiol, phenylahistins
(diketopiperazine), steganacins, and curacins.
10
Most of ‘‘micro-
tubule-stabilizing’’ agents bind to the taxoid binding site on
b-tubulin, which is located on the inside surface of the micro-
tubule and enhances its polymerization at high drug concen-
trations, e.g., taxol (paclitaxel, Taxolt), docetaxel (Taxoteret),
epothilones, ixabepilone (Ixemprat), and patupilone.
11
a
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Zagazig University, Zagazig 44519, Egypt. E-mail: nader_elmaghry88@yahoo.com
b
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Horus
University-Egypt, New Damietta 34518, Egypt. E-mail: akarmalawy@horus.edu.eg
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Tabuk, Tabuk 71491, Saudi Arabia
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1nj02885e
Received 12th June 2021,
Accepted 27th October 2021
DOI: 10.1039/d1nj02885e
rsc.li/njc
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Tubulin inhibitors that bind to the colchicine binding site
have therapeutic merit over other MTAs, where they inhibit
angiogenesis and usually overcome multidrug resistance
(MDR). Colchicine (Fig. 2) is a very cheap alkaloid isolated
from natural sources. Colchicine has a well-defined binding
site to microtubule (CBS) and effectively arrests mitosis. Unfor-
tunately, its narrow therapeutic index (as it binds to tubulin in
non-cancerous cells) and multiple mechanisms of action led
to systemic toxicities and multiorgan dysfunction in a clinical
trial and thus was not approved by the FDA for cancer
treatment.
12,13
Encouragingly, colchicine served as a template
for the development of advantageous MTAs targeting the CBS,
which are often: (i) effective in P-glycoprotein, MRP1, and
MRP2 overexpression-mediated multidrug resistance, (ii) effec-
tive as a vasculature disrupting agents, and (iii) cost-effective
compared to biologicals, such as monoclonal antibodies.
13–15
Accordingly, the development of novel analogs of colchicine
binding site inhibitors (CBSIs) with lower side effects is a goal
of many former and current research endeavors.
16
Combretastatin A-4 (CA-4) is structurally similar to colchi-
cine and has been extensively studied to improve its activity
and pharmacokinetics properties. Molecules that fall into the
combretastatin family generally share three common structure
features: a trimethoxy A ring, a B-ring containing substituents
often at C30and C40, and an ethene bridge between the two
rings which provides necessary structural rigidity (Fig. 2).
Structure–activity relationship studies found that the cis-
configuration of the double bond and the 3,4,5-trimethoxy
group on ring A is essential for the anti-tubulin activity of
CA-4.
17
However, this is not always the case with its analogs.
The trans-configuration of a-methyl chalcone Iinhibited
cancer cell proliferation in K562 cells at an IC
50
=0.21nM.
18,19
The clinical application of CA-4 as an anticancer CBSI was
hampered by its poor water solubility, short half-life, isomeriza-
tion of the ethene bond, as well as its toxic side effects.
20
To overcome these limitations, many CA-4 analogs have been
developed that retained the biological effects of the parent
molecule with enhanced water solubility and pharmacokinetic
properties (Fig. 2).
The common structural features in several reported potent
colchicine and CA-4 analogs (Fig. 2):
(1) Two aryl groups separated by a 2-3 carbons spacer e.g.:
(a) cis-double bond,
(b) a,b-unsaturated ketone,
(c) a,g-disubstituted five-membered heterocycle such as
imidazole II,
21
thiazole III,
22
and 1,3,4-oxadiazole IV.
23
Fig. 1 Tubulin binding sites.
Fig. 2 Colchicine, combretastatin A-4, examples of reported CBSIs (I,II,andIII), and the novel targets 5a–k.
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(2) One of the two aryl groups is always a trimethoxy
phenyl ring.
Targets 5a–k are structurally similar to compounds I,II, and
III, where they contain a a,g-disubstituted five-membered
heterocycle (1,2,4-oxadiazole) and one substituent is 3,4,5-
trimethoxyphenyl group and the other one is aryl or aralkyl
groups (Fig. 2).
1.1. Rational of molecular design
CA-4 binds to the colchicine binding site in tubulin, inhibits
tubulin aggregation in microtubules, alters microtubule dynamics
and this has been confirmed by biochemical experiments and
X-ray crystallography.
24
Docking studies and molecular
dynamics simulations examined the binding models of struc-
turally diverse CBSIs and concluded seven common pharma-
cophoric points: three hydrogen bond acceptors (A1, A2, and
A3), one hydrogen bond donor (D1), two hydrophobic centers
(H1 and H2), and one planar group (R1).
25,26
The seven points
of the pharmacophoric model are partitioned into two planes.
Plane A consists of points A2, A3, and H2, and plane B consists
of points (A1, D1, H1, and R1), Fig. 3.
26,27
In the light of these studies, the molecules that will have
these seven pharmacophoric features will be considered as
promising tubulin inhibitors. Consequently, we could predict
the essential pharmacophoric features for our newly designed
compounds to synthesize promising anticancer agents acting
as CBSIs compared to colchicine as a reference standard
(Fig. 4).
25,28
2. Results and discussion
2.1. Chemistry
3,4,5-Trimethoxybenzamidoxime 2was synthesized by heating
under reflux 3,4,5-trimethoxybenzonitrile 1,withhydroxylamine
HCl in the presence of equivalent sodium carbonate (Scheme 1).
29
N-Acylbenzotriazoles 4a–k were synthesized from commercially
available carboxylic acids 3a–k using two different procedures
Fig. 3 Seven pharmacophoric features: 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. 4 Schematic representation showing the applied pharmacophoric features for the design of the newly synthesized compounds as CBSIs in respect
to colchicine as a standard reference.
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depending on the chemical nature of the functional groups
present in the acid. First, N-acylbenzotriazoles 4a and 4e–k were
synthesized from commercially available carboxylic acids 3a and
3e–k, respectively, using the thionyl chloride procedure.
30
On the
other hand, N-acyl benzotriazoles containing free amino group
4b–d were synthesized using the tosyl chloride route.
31
Intermediate
N-acylbenzotriazoles 4a–k were crystallized from CH
2
Cl
2
/hexanes
and their melting points were compared to reported values and
were taken to the next step without further identification.
31–36
Targets 5a–k were characterized using
1
HNMR,
13
CNMR, as
well as IR spectroscopy. The purity of targets was confirmed by
elemental analyses.
2.1.1. Synthesis of 1,2,4-oxadiazoles 5a–k. 1,2,4-Oxadi-
azoles 5a–k were synthesized by the reaction of N-acylbenzo-
triazole 4a–k with 3,4,5-trimethoxybenzamidoxime 2in the
presence of triethylamine under reflux conditions (Scheme 1).
37
Targets 5a–k were characterized using
1
HNMR,
13
CNMR, as well
as IR spectroscopy, and their purity was confirmed by elemental
analyses. The mechanism of the reaction involved initial depro-
tonation of the hydroxyl group of amidoxime by adding triethyl-
amine to give the nitroxide anion of amidoxime. The anion was
acylated by the electron-deficient carbonyl group of N-acylbenzo-
triazoles to form the N-O-acyl amidoxime derivative 7.Intra-
molecular cyclodehydration of intermediate N-O-acyl amidoxime
Scheme 1 Reagents and conditions; (a) NH
2
OHHCl, Na
2
CO
3
, ethanol, reflux; (b) 1-H-benzotriazole, SOCl
2
,CH
2
Cl
2
, stirring for 2–3 h at r.t.;
(c) (1) DMAP, TEA, CH
2
Cl
2
, 0.5 h at 25 1C, (2) 1-H-benzotriazole, 1.5 h; (d) TEA, ethanol, reflux.
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derivatives produced the corresponding 1,2,4-oxadiazoles
(Scheme 2).
38
2.2. Biological evaluation
2.2.1. Anti-proliferative activities towards 60 subpanel
tumor cell lines. All the target compounds 5a–k were submitted
to the Developmental Therapeutics Program hosted by the
National Cancer Institute, USA. Their antiproliferative activities
were tested against 60 diverse human cancer cell lines (the
NCI-60) by single-dose assay (concentration 10
5
M). The com-
plete NCI-60 results are represented in the ESI.†The percentages
of growth inhibition were significant and the most potent
compounds were 5a–e and 5k (Table 1).
2.2.2. Tubulin polymerization inhibitory activity. Tubulin
polymerization inhibitory activity was also evaluated for the
most active compound (5a) using ELISA assay for unpolymerized
Tubulin Beta (TUBb) to confirm its proposed mechanism of action.
The polymerization was monitored through the fluorescence
intensity change after their incubation. Compound (5a)showed
superior inhibitory activity (IC
50
=1.18mM) against the colchicine
binding site in b-tubulin compared to colchicine as a reference
standard (IC
50
=2.37mM) as depicted in Table 2.
2.3. Molecular docking studies
At first, we studied the colchicine binding site in b-tubulin, the
co-crystallized native inhibitor (N-[(7S)-1,2,3,10-tetramethoxy-9-
oxo-5,7-dihydro-5H-benzo[d]heptalen-7-yl]ethanamide) was found
to be stabilized inside its binding pocket of the CBS receptor
through the formation of one hydrogen bond with Lys350
amino acid.
Molecular docking of the tested oxadiazole derivatives 5a–k,
colchicine 6, and the co-crystallized native inhibitor (N-[(7S)-
1,2,3,10-tetramethoxy-9-oxo-5,7-dihydro-5H-benzo[d]heptalen-7-yl]
ethanamide 7inside the active site of the CBS in b-tubulin was
performed.
All of the newly tested synthesized compounds 5a–k were got
stabilized inside the CBS in b-tubulin by variable scores and
binding interactions with the amino acids of the receptor
pocket. They showed promising binding scores (from 6.41
to 8.06 kcal mol
1
), compared to both colchicine (6,7.40),
and the docked co-crystallized inhibitor (7,8.13). Also, the
RMSD_refine values of the selected poses were within the
acceptable range (from 0.93 to 2.26).
The docked co-crystallized inhibitor (7) fitted inside the CBS
in the b-tubulin pocket through a hydrogen bond formation
with Lys350 amino acid at 2.72 Å. Also, on the other hand,
colchicine (6) – as the first known tubulin destabilizing agent –
got stabilized inside the colchicine binding site by a pi–H bond
formation with Lys350 amino acid at 3.67 Å (Table 3). Accord-
ingly, this indicates the very crucial role of Lys350 amino acid to
stabilize ligands inside the pocket of the CBS in b-tubulin.
It is worth mentioning that all of the newly synthesized
derivatives 5a–k achieved nearly a fingerprint binding mode to
both colchicine (6) and the co-crystallized inhibitor in binding
to the same essential amino acid (Lys350) required for the
inhibitory activity of the CBS in b-tubulin, indicating an
expected affinity and subsequent intrinsic activity to the newly
synthesized derivatives as promising CBSIs.
Surprisingly, the target compound (5a) of the newly synthesized
derivatives achieved superior binding affinity (8.06 kcal mol
1
)to
that of colchicine (6) itself, which achieved (7.40 kcal mol
1
).
Also, their RMSD_refine values were 1.85 and 0.95, respectively,
indicating highly valid selected poses as well. On the other
hand, target derivative (5e) showed a very promising binding
score (7.38 kcal mol
1
) with an RMSD_refine value of 1.60
compared to colchicine (6) as mentioned before. At the same
Scheme 2 Mechanism of the reaction of 3,4,5-trimethoxybenzamidoxime (2)andN-acylbenzotriazoles 4a–k to form oxadiazoles 5a–k.
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time, the obtained binding scores of compounds 5a and 5e
were very close to those of the co-crystallized inhibitor (7) with a
binding score of 8.13 kcal mol
1
.
Moreover, both 5a and 5e targets as the most two promising
derivatives bound Lys350 amino acid at 3.62 and 3.65 Å,
respectively, by a pi–H bond formation. Also, compound 5a
formed an extra pi-H bond with Leu253 at 3.92 Å and com-
pound 5e bound Met257 by a second H-bond at 3.52 Å (Table 4).
By considering the docking findings of the newly synthe-
sized compounds 5a–k against the binding pocket of the CBS in
b-tubulin compared to its co-crystallized inhibitor 7and colchi-
cine 6as a second promising reference standard, together with
the great matching with the biological cytotoxicity studies per-
formed as well, we can conclude a very promising idea concerning
their recommended mechanism of action as CBSIs.
2.4. Structure–activity relationship (SAR) study
SAR studies based on the biological activities (Tables 1 and 2) of
the newly synthesized series of 3,5 disubstituted 1,2,4-oxadi-
azoles regarding their pharmacophoric features (represented in
Fig. 3 and 4) together with the previously discussed docking
results (Tables 3 and 4), we can conclude the following inter-
esting results:
(a) The most potent designed target (5a), which possesses all
the required pharmacophoric points for binding to the CBS has
the highest antiproliferative activity. It was found to be the only
member containing a benzyl moiety as a planar pharmacophoric
group which may explain its superior anti-proliferative and
CBSI biological activities. Also, it may clarify the reason behind
its attractive fitting and binding score observed through the
Table 1 Percentage growth inhibition (GI%) of compounds 5a–k over 60 subpanel tumor cell lines
Comp.
no.
60 cell lines assay in one dose 10.0 mM concentration: GI%
MGI% MGI-S PCE Most sensitive cell lines
5a 15.5 24.9 33/60 Leukemia (K-562 : 30%, RPMI-8226: 20%, SR:23.%), NSC Lung Cancer (A549/ATCC:11%, EKVX: 23%, HOP-92:
61%, NCI-H226: 15%, NCI-H23: 13%, NCI-H322M:13%, NCI-H522: 22%), Colon Cancer (HCT-15: 19%,
HCT-116: 13%, HT29: 11%), CNS Cancer (SF-268: 14%, SF-295: 11%, SNB-75: 27%), Melanoma (LOX IMVI: 17%,
UACC-62: 35%, SK-MEL-5: 12%), Ovarian Cancer (IGROV1: 34%, OVCAR-4: 23%), Renal Cancer (A498: 78%,
CAKI-1: 43%, UO-31: 46%, 786-0: 15%, ACHN:15%, RXF 393: 12%, SN12C:19%) Prostate Cancer (PC-3: 37%),
Breast Cancer (MCF7: 23%,MDA-MB-231/ATCC: 29%, HS 578T: 26%, T-47D: 34%).
5b 8.1 17.9 23/60 Leukemia (HL-60(TB):21%, RPMI-8226: 23%), NSC Lung Cancer (EKVX: 25%,NCI-H226: 21%,A549/ATCC:11%,
NCI-H23: 12%, NCI-H522: 14%), Colon Cancer (HCT-15: 14%), CNS Cancer (SF-268: 13%, SF-539: 14%),
Melanoma (M14: 12%, UACC-62: 13%), Ovarian Cancer (IGROV1: 15% SK-OV-3: 11%), Renal Cancer (RXF 393:
15%, CAKI-1: 14%, UO-31: 22%), Prostate Cancer (PC-3: 31%), Breast Cancer (MCF7: 22%,MDA-MB-231/ATCC:
20%, T-47D:37%, MDA-MB-468: 23%, HS 578T:10%)
5c 8.1 19.8 16/60 Leukemia (CCRF-CEM:16%, HL-60(TB): 30%, K-562: 17%, RPMI-8226: 14%, MOLT-4: 12.%), NSC Lung Cancer
(A549/ATCC:17%, NCI-H522: 13%), Colon Cancer (HCT-15: 16%, KM12: 16%), Renal Cancer (SN12C:19%, CAKI-
1: 24%, UO-31: 40%), Prostate Cancer (PC-3: 33%), Breast Cancer (MCF7: 12%, BT-549: 12%, T-47D: 27%).
5d 9.4 21.3 21/60 Leukemia (K-562: 17%, CCRF-CEM:11%, SR:27.%, HL-60(TB):40%, MOLT-4: 14%,RPMI-8226: 21%), NSC Lung
Cancer (EKVX: 13%, NCI-H226: 14%, NCI-H522: 19%), Colon Cancer (HCT-15: 12%, KM12: 20%), CNS Cancer
(SNB-75: 32%), Melanoma (LOX IMVI: 14%, UACC-62: 15%), Renal Cancer (A498: 51%, CAKI-1: 24%, UO-31:
33%, SN12C:17%) Prostate Cancer (PC-3: 30%), Breast Cancer (MDA-MB-231/ATCC: 16%, T-47D: 16%)
5e 10.2 19.6 27/60 Leukemia (K-562: 24%, CCRF-CEM:11%, SR:11%, HL-60(TB):17%,RPMI-8226: 23%), NSC Lung Cancer (HOP-62:
19%, NCI-H23: 13%, NCI-H522: 25%), Colon Cancer (HCT-116: 13%), CNS Cancer (SF-539: 15%, SNB-19: 17%,
SNB-75: 59%), Melanoma (LOX IMVI: 13%, UACC-62: 22%, M14: 15%,K-MEL-2: 12%), Ovarian Cancer (OVCAR-
8: 12%), Renal Cancer (CAKI-1: 28%, UO-31: 25%, 786-0: 15%, ACHN:18%, SN12C:16%) Prostate Cancer (PC-3:
16%), Breast Cancer (MCF7: 12%,MDA-MB-231/ATCC: 31%, HS 578T: 34%, T-47D: 15%)
5f 2.2 19.8 9/60 NSC Lung Cancer (HOP-92: 16%, NCI-H226: 11%), CNS Cancer (SNB-75: 26%), Melanoma (UACC-62: 17%),
Renal Cancer (CAKI-1: 19%, UO-31: 31%, A498: 32%) Prostate Cancer (PC-3: 14%), Breast Cancer (T-47D: 13%)
5g 4.9 16.5 10/60 Leukemia (HL-60(TB):33%), NSC Lung Cancer (NCI-H226: 12%), Colon Cancer (HCT-15: 11%) Melanoma
(UACC-62: 19%), Renal Cancer (UO-31: 13%, SN12C:11%) Prostate Cancer (PC-3: 19%), Breast Cancer
(MCF7: 11%,MDA-MB-231/ATCC: 11%, T-47D: 25%)
5h 2.3 20.8 6/60 Leukemia (SR:19%), NSC Lung Cancer (HOP-92: 39%), CNS Cancer (SNB-75: 23%), Renal Cancer (UO-31: 20%)
Prostate Cancer (PC-3: 13%), Breast Cancer (HS 578T: 11%)
5i 2.5 14.4 10/60 Leukemia (K-562: 13%, MOLT-4: 13%), NSC Lung Cancer (NCI-H522: 12%), CNS Cancer (SNB-75: 17%,
SNB-19: 11%), Melanoma (UACC-62: 23%), Renal Cancer (UO-31: 11%, SN12C:12%) Breast Cancer
(MCF7: 15%, T-47D: 17%)
5j 1.9 13.6 7/60 NSC Lung Cancer (HOP-92: 12%), CNS fCancer (SNB-75: 24%, SNB-19: 11%), Melanoma (UACC-62: 12%),
Renal Cancer (UO-31: 11%) Breast Cancer (MCF7: 13%, MDA-MB-468: 12%)
5k 8.1 19.7 18/60 Leukemia (HL-60(TB):29%), NSC Lung Cancer (EKVX: 14%, HOP-92: 35%, NCI-H522: 19%), Colon Cancer (HCT-
15: 19%, HCT-116: 13%, HT29: 11%), CNS Cancer (SNB-19: 14%SNB-75: 30%), Melanoma (UACC-62: 28%),
Ovarian Cancer (IGROV1: 23%), Renal Cancer (CAKI-1: 17%, UO-31: 26%, 786-0: 14%, SN12C:15%) Prostate
Cancer (PC-3: 17%), Breast Cancer (MCF7: 18%,MDA-MB-231/ATCC: 20%, HS 578T: 11%, T-47D: 19%)
PCE: positive cytotoxic effect, which is the ratio between some cell lines with percentage growth inhibition 410% and the total number of cell
lines, MGI%: mean growth inhibition percentage, MGI-S%: mean growth inhibition percentage for the most sensitive cell lines (with percentage
growth inhibition 410%).
Table 2 IC
50
values for (TUBb) inhibition of compound 5a and colchicine
Compound Tubulin binding, IC
50
,uM
5a 1.18 0.037
Colchicine 2.37 0.075
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molecular docking study especially its binding to the crucial
amino acid (Lys350) that is mainly responsible for the inhibi-
tory effect of the CBS as discussed before. The positive cytotoxic
effect (PCE) of 5a is 33/60 and the mean growth inhibition
(MGI) for the most sensitive cell lines (33) is 24.9%, while MGI
for the 60 cell lines = 15.5%.
(b) Target (5e) has a second 3,4,5-trimethoxyphenyl group
(a phenyl moiety was added as the planar pharmacophoric
group attached to a trimethoxy group to introduce a second
3,4,5-trimethoxyphenyl group in addition to the first permanent
hydrophobic one). This allowed 5e to be the second most active
target of the whole series, PCE = 27/60 and MGI for 60 cell lines =
10.2%, MGI for 27 cell lines = 19.6. Besides, it is worth mention-
ing that 5e was also able to form a pi-H bond to the crucial
amino acid (Lys350) as described earlier, which may explain its
better anti-proliferative activity as the second most promising
candidate.
(c) The absence of benzyl side chain (planar group: R1)
in (5f) dramatically reduced its PEC to 9/60 and MGI for 60
cell lines to 2.2 and MGI for the nine cell lines to 19.8.
Table 3 3D representations showing the binding interactions and positioning of colchicine (6) and the docked co-crystallized inhibitor (7) into the CBS
in b-tubulin. The red dash represents H-bonds and the black dash represents H–pi interactions
Comp. 3D binding interactions 3D pocket positioning
*Col. (6)
*Inh. (7)
*Col. = colchicine; *Inh. = co-crystallized inhibitor of CBS.
Table 4 3D representations showing the binding interactions and positioning between the most two promising newly synthesized compounds
(5a and 5e) inside the CBS in b-tubulin. The red dash represents H-bonds and the black dash represents H–pi interactions
Comp. 3D binding interactions 3D pocket positioning
5a
5e
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(d) Replacement of the benzyl group of (5a), with a branched
nonplanar hydrophobic group (isopropyl group) in (5j), gave
the least active target with PCE = 7/60 and the MGI for 60 cell
lines to = 1.9%.
(e) Insertion of a methylene group between the isopropyl
group and the main chain gave a slightly more active compound
(5k) (PCE = 18/60, MGI = 8.07%, and MGI for 18 cell lines =
19.7%).
(f) Replacement of the Cbz–Phe–OH side chain with o-, m-,
and p-aminophenyl groups gave (5b), (5c), and (5d), respec-
tively. The three compounds are of comparable PCEs (16–21/60)
and MGI for 60 cell lines = 8.1–9.4%. The order of activity was
p-(5d)4m-(5c)4o-(5b).
(g) Replacement of the p-amino-group of (5d)byp-methoxy
(5g) reduced the activity, PCE = 10/60 and MGI for 60 cell lines =
4.9%.
(h) Replacement of the aryl group at C5 by n-propyl in (5h)
gave the lowest activity (PCE = 6/60 and MGI for 60 cell lines =
2.3%). This indicates the great importance of the presence of
the second aryl moiety for the receptor fitting and the subse-
quent enhanced anti-proliferative activity of the studied mem-
bers as CBSIs.
(i) Finally, the presence of long-chain unsaturated alkyl
group (5i) did not significantly improve the activity (PCE = 10/
60 and MGI for 60 cell lines = 2.5%).
It is noteworthy that targets (5a–k) showed selective potency
towards renal cancer in a particular A498 cell line, which
represents renal cell carcinoma (RCC). RCC is one of the most
common kidney cancers and is highly resistant to chemo-
therapy.
39
The order of potency of the most potent targets
against A498 is 5a (GI = 78%) 45d (GI = 51%) 45f (32%).
Moreover, targets 5a–f, k inhibited the growth of CAKI-1-
clear cell renal cell carcinoma (ccRCC) cell line – with GI%
ranging from 14 – to 43%. ccRCC Is the most common and
lethal form of urological cancer diagnosed globally.
40
In addi-
tion, all the designed targets (5a–k) showed significant growth
inhibition against UO-31 (GI = 11–46%).
3. Conclusion
In conclusion, we have designed and synthesized eleven 3,5-
disubstituted oxadiazole derivatives (5a–k). The targets were
evaluated for their antiproliferative activity at 10
5
M in full
NCI 60 cell panel and showed moderate antiproliferative
activities. In addition, molecular docking studies revealed that
targets 5a–k showed promising affinities towards the colchicine
binding site in b-tubulin using. Target 5a demonstrated the
most potent antiproliferative activity in terms of positive cyto-
toxic effect (PCE = 33/60) and growth inhibition (MGI for 33 cell
lines = 24.9%) and showed a binding affinity more than that of
the docked Colchicine (6), and very near to that of the co-
crystallized inhibitor (7). Furthermore, 5a inhibited b-tubulin
polymerization in vitro with IC
50
= 1.18 mM, which is twofold the
potency of Colchicine (6), IC
50
= 2.37 mM. In the light of these
findings, 5a is a promising candidate for further investigation
as an anticancer CBSI and as a lead for future development of
more potent anticancer CBSIs to combat multidrug resistance
(MDR) cancers.
4. Experimental section
4.1. Chemistry
4.1.1. General. All reagents were purchased from common
commercial sources and used without further purification.
Melting points (m.p.) were measured on melting point appara-
tus SMP3, Stuart Scientific (UK), and were uncorrected.
1
H NMR
(400 MHz) and
13
C NMR (100 MHz) spectra were determined on
a Bruker 400 MHz FT-NMR spectrometer using DMSO-d
6
. The
chemical shift (d) was reported on ppm relative to the internal
tetramethylsilane (TMS) standard and the coupling constant ( J)
was calculated in Hz (Hertz). IR was determined using Bruker
Alpha ft IR spectrometer. TLC-MASS was determined using
Advion compact mass spectrometer (CMS) NY|USA using elec-
trospray (ES) ionization mode. Reactions were monitored
by TLC (Thin Layer Chromatography) on silica gel 50 GF254
(E-Merck, Germany) and using a UV lamp for visualization at a
wavelength (l) of 254 nm.
4.1.1. Synthesis of 3,4,5-trimethoxybenzamidoxime (2).
3,4,5-Trimethoxy benzonitrile (1) (5 mmol, 950 mg) was reacted
with hydroxylamine hydrochloride (5 mmol, 410 mg) and
sodium carbonate (5 mmol, 540 mg) in ethanol (25 ml). The
mixture was heated under reflux for 5 h. To drive the reaction to
completion, additional hydroxylamine hydrochloride (5 mmol,
410 mg) and sodium carbonate (5 mmol, 540 mg) were added
and the reaction was heated under reflux for another 14 hrs.
The mixture was filtered while hot. The product was then
crystallized from ethanol giving amidoxime as pure white
crystals, 95% yield, m.p. 155–157 1C (as reported).
41
1
H NMR (DMSO-d
6
, 400 MHz, dppm): 9.55 (s, 1H, OH), 5.79
(s, 2H, aromatic H), 5.80 (s, 2H, NH
2
), 3.78 (s, 5H, 3,5
di-methoxy), 3.55 (s, 3H, 4-methoxy).
4.1.2. Synthesis of N-acyl benzotriazoles (4a, e–k). A mix-
ture of benzotriazole (4 mmol, 480 mg) in methylene chloride
and SOCl
2
(1.2 mmol, 140 mg) was stirred for 20–30 m at
(20 1C), then appropriate acid (1 mmol) was added. The stirring
was continued for 2–3 hr at room temperature, then acidic
workup using 5 N hydrochloric acid (3 20 ml) or basic workup
using sodium carbonate (3 20 ml) was performed. The
methylene chloride layer was dried over anhydrous sodium
sulfate. The solid was crystallized from hexane/methylene chloride
to give the corresponding N-acyl benzotriazole (4a, e–k).
Benzyl (S)-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-
2-yl)carbamate (4a). White microcrystals, yield (98%), m.p. 160–
162 1C (ref. 32, 162–163 1C).
(2-Aminophenyl)(1H-benzo[d][1,2,3]triazol-1-yl)methanone (4b).
Pale yellow microcrystals, yield (60%), m.p. 130–132 1C(ref.33,
130–132 1C).
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(3-Aminophenyl)(1H-benzo[d][1,2,3]triazol-1-yl)methanone (4c).
Pale yellow microcrystals, yield (75%), m.p. 171–172 1C(ref.31,
171–172 1C).
(4-Aminophenyl)(1H-benzo[d][1,2,3]triazol-1-yl)methanone (4d).
Pale yellow microcrystals, yield (80%), m.p. 178–180 1C(ref.31,
178–180 1C).
(1H-Benzo[d][1,2,3]triazol-1-yl)(3,4,5-trimethoxyphenyl)methanone
(4e). White microcrystals, yield (95%), m.p. 125–127 1C(ref.33,
126–128 1C).
Benzyl (2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)carbamate
(4f). White microcrystals, yield (93%), m.p. 110–112 1C (ref. 33,
107–109 1C).
(1H-Benzo[d][1,2,3]triazol-1-yl)(4-methoxyphenyl)methanone (4g).
White microcrystals, yield (90%), m.p. 103–105 1C (ref. 33, 102–
104 1C).
1-(1H-Benzo[d][1,2,3]triazol-1-yl)butan-1-one (4h). White
microcrystals, yield (88%), m.p. 57–59 1C (ref. 34, 59–60 1C).
(9Z,12Z)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)octadeca-9,12-dien-1-
one (4i). Colorless oil, yield (90%), (ref. 35 oil).
Benzyl (S)-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-3-methyl-1-oxobutan-
2-yl)carbamate (4j). White microcrystals, yield (87%), m.p. 107–
108 1C (ref. 36, 107–108 1C).
Benzyl (S)-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-4-methyl-1-oxopentan-
2-yl)carbamate (4k). White microcrystals, yield (80%), m.p. 150–
151 1C (ref. 32, 150–152 1C).
4.1.3. Synthesis of N-acyl benzotriazole from m,p-amino-
benzoic acid (4b–d). A mixture of p-toluene sulfonyl chloride
(1 mmol, 190 mg) and DMAP (0.13 mmol, 15 mg) was stirred in
methylene chloride (5 ml) for 10 min. o,m, and p-aminobenzoic
acids (1 mmol, 0.14 gm) were dissolved in methylene chloride
(5 ml) containing TEA (1.5 mmol, 151 mg), then were added
to the reaction mixture. After 20 m, benzotriazole (1.2 mmol,
142 mg) was added and the reaction was stirred for another
1.5 h at 25 1C. When the reaction was complete, methylene
chloride (50 ml) was added and the organic layer was washed
with saturated sodium carbonate (3 10 ml), water (2 10 ml),
and brine (1 10 ml). The methylene chloride layer was dried
over anhydrous sodium sulfate, then hexane (20 ml) was added.
The precipitated solid was filtered and dried under vacuum to
give the target N-acylbenzotriazoles (4b–d).
4.1.4. Synthesis of 3,5-disubstituted-1,2,4-oxadiazoles
(5a–k). 3,4,5-Trimethoxy benzamidoxime (1 mmol, 230 mg)
was reacted with N-acyl benzotriazoles (1 mmol) in ethanol
(20 ml) containing TEA (1 mmol, 100 mg). The mixture was
stirred for 10–20 m, then it was heated under reflux for (2–30 h)
for different acyl benzotriazoles. The product was obtained by
precipitation from ethanol. The reaction mixture was filtered,
then the product was washed on a filter paper with water,
followed by ethanol, and dried under vacuum and recrystallized
from ethyl acetate to obtain pure oxadiazoles. The oily oxadia-
zoles (5i–k) were obtained by evaporation of ethanol under
vacuum, then the residue was dissolved in ethyl acetate (15 ml)
and washed with sodium carbonate (3 5 ml) to remove
benzotriazole, then dried over anhydrous sodium sulfate, ethyl
acetate was evaporated under vacuum to give the product.
4.1.4.1. Benzyl (S)-(2-phenyl-1-(3-(3,4,5-trimethoxyphenyl)-
1,2,4-oxadiazol-5-yl)ethyl) carbamate (5a). White solid. Yield:
90%, m.p. 112–115 1C.
1
H NMR (DMSO-d
6
, 400 MHz, d, ppm):
8.36 (d, 1H, J= 8 Hz, N
H), 7.30–7.25 (m, 12H, C
2
–
H, C
6
–
Hof
trimethoxy phenyl ring and 10 Ar–
H of phenyl rings), 5.20 (d,
1H, J= 6 Hz, C
H–NH), 5.00 (s, 2
H, CH
2
–O), 3.86 (s, 6H, 3,5-di
(OC
H
3
) of 3,4,5-trimethoxyphenyl), 3.75 (s, 3H, 4-OC
H
3
of 3,4,5-
trimethoxyphenyl), 3.36 (s, 2H, C
H
2
-phenyl).
13
C NMR (DMSO-
d
6
, 100 MHz, d, ppm): 179.81 (
C
3
-1,2,4-oxadiazole), 167.44 (
C
5
of
1,2,4-oxadiazole), 155.76 (
CQO), 153.40 (
C
3
and
C
5
of 3,4,5-
trimethoxyphenyl), 140.22 (
C
4
of 3,4,5-trimethoxyphenyl),
136.74 (
C
1
of phenyl of Cbz), 136.55 (
C
1
of phenyl of Phe),
129.28 (
C
3
and
C
5
of phenyl of Cbz and
C
3
and
C
5
of phenyl of
Phe), 128.31 (
C
4
of phenyl of Cbz and Phe), 127.83 (
C
2
and
C
6
of phenyl of Phe), 127.57 (
C
2
and
C
6
of phenyl of Cbz), 121.27
(
C
1
of 3,4,5-trimethoxyphenyl), 104.25 (
C
2
and
C
6
of 3,4,5-
trimethoxyphenyl), 65.64 (
CH–O), 60.1 (4-O
CH
3
), 56.05 (3,5-di-
O
CH
3
), 49.91 (
CH
2
–N), 37.63 (
CH
2
– of Phe). Anal. calcd for
C
27
H
27
N
3
O
5
: C, 66.25, H, 5.56, N, 8.58%; found; C, 66.43; H,
5.79; N, 8.81%.
4.1.4.2. 2-(3-(3,4,5-Trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)-
aniline (5b). White solid. Yield: 85%, m.p. 152–155 1C.
1
HNMR
(DMSO-d
6
, 400 MHz, d, ppm): 7.857.83 (d, 1H, J=7.2Hz,C
3
–
Hof
aniline), 7.39–7.35 (m, 3H, C
5
–
H of aniline and NH
2
), 6.97–6.93
(m, 3H, C
6
–
H of aniline and C
2
–
H, C
6
–
Hoftrimethoxyphenyl),
6.69 (s, 1H, C
4
–
H of aniline), 3.90 (s, 6H, 3,5 di (OC
H
3
)of
3,4,5-trimethoxy phenyl), 3.75 (s, 3H, 4-OC
H
3
of 3,4,5-tri-
methoxyphenyl).
13
C NMR (DMSO-d
6
, 100 MHz, d,ppm):
174.64 (
C
5
-1,2,4-oxadiazole), 167.23 (
C
3
-1,2,4-oxadiazole), 153.38
(
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 148.74 (
C
1
of aniline),
140.24 (
C
4
of 3,4,5-trimethoxyphenyl), 134.05 (
C
3
of aniline),
128.49 (
C
5
of aniline), 121.49 (
C
1
of 3,4,5-trimethoxyphenyl),
116.59 (
C
2
of aniline), 115.63(
C
6
of aniline), 104.64 (
C
2
and
C
6
of 3,4,5-trimethoxyphenyl), 103.46 (
C
4
of aniline), 60.18 (3,5 di-
CH
3
O), 56.16 (4-
CH
3
O). Anal. calcd for C
17
H
17
N
3
O
4
:C,62.38;H,
5.23; N, 12.84%; found; C, 62.16; H, 5.37; N, 13.12%.
4.1.4.3. 3-(3-(3,4,5-Trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)-
aniline (5c). White solid. Yield: 40%, m.p. 112–114 1C.
1
HNMR
(DMSO-d
6
, 400 MHz, d, ppm): 7.40–7.27 (m, 5H, C
5
,C
4
and C
2
–H
of aniline, C
2
–H, C
6
–H of trimethoxy phenyl), 6.88 (d, 1H, J=6.8Hz,
C
6
–H of aniline), 5.60 (s, 2H, NH
2
), 3.89 (s, 6H, 3,5-di(OCH
3
)
of 3,4,5-trimethoxy phenyl), 3.75 (s, 3H,4-OCH
3
of 3,4,5-
trimethoxyphenyl).
13
C NMR (DMSO-d
6
, 100 MHz, d,ppm):
176.04 (
C
5
-1,2,4-oxadiazole), 167.99 (
C
3
-1,2,4-oxadiazole), 153.39
(
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 149.65 (
C
1
of aniline),
140.16 (
C
4
of 3,4,5-trimethoxyphenyl), 130.03 (
C
5
of aniline),
123.73 (
C
3
of aniline), 121.57 (
C
1
of 3,4,5-trimethoxyphenyl),
118.46 (
C
6
of aniline), 114.93 (
C
4
of aniline), 112.22 (
C
2
of
aniline), 104.28 (
C
2
and
C
6
of 3,4,5-trimethoxyphenyl), 60.19
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(3,5 di-
CH
3
O), 56.05 (4-
CH
3
O). Anal. calcd for C
17
H
17
N
3
O
4
:C,
62.38; H, 5.23; N, 12.84%; found; C, 62.50; H, 5.41; N, 13.10%.
4.1.4.4. 4-(3-(3,4,5-Trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)-
aniline (5d). White solid. Yield: 51%, m.p. 155–158 1C. IR (KBr)
cm
1
: 3300 (NH
2
), 2928, 2855 (CH aliphatic), 1559 (CQN), 1512
(CQC), 1258, 1125(C–O).
1
H NMR (DMSO-d
6
, 400 MHz, d, ppm):
7.85 (d, 2H, J= 8.8 Hz, C
3
–
H, C
5
–
H of aniline), 7.31 (s, 2H, C
2
–
H,
C
6
–
H of trimethoxy phenyl), 6.71 (d, 2H, J= 8.4 Hz, C
2
–
H, C
6
–
H
of aniline), 6.18(s, 2H, N
H
2
), 3.88 (s, 6H, 3,5-di (OC
H
3
)of
3,4,5-trimethoxy phenyl), 3.74 (s, 3H, 4-OC
H
3
of 3,4,5-tri-
methoxyphenyl).
13
C NMR (DMSO-d
6
, 100 MHz, d, ppm):
176.55 (
C
5
-1,2,4-oxadiazole), 168.17 (
C
3
-1,2,4-oxadiazole),
153.92 (
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 153.82 (
C
1
of
aniline), 140.45 (
C
4
of 3,4,5-trimethoxyphenyl), 130.32 (
C
3
and
C
5
of aniline), 122.39 (
C
1
of 3,4,5-trimethoxyphenyl), 114.07 (
C
2
and
C
6
of aniline), 110.04 (
C
1
of aniline), 104.78 (
C
2
and
C
6
of
3,4,5-trimethoxyphenyl), 60.81 (3,5 di-
CH
3
O), 56.58 (4-
CH
3
O).
Anal. calcd for C
17
H
17
N
3
O
4
: C, 62.38; H, 5.23; N, 12.84%; found;
C, 62.19; H, 5.40; N, 13.08%.
4.1.4.5. 3,5-Bis(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (5e).
White solid. Yield: 52%, m.p. 152–155 1C.
1
H NMR (DMSO-d
6
,
400 MHz, d, ppm): 7.43 (s, 2H, C
2
-
H, C
6
–
H of trimethoxy phenyl
on C
5
), 7.35 (s, 2H, C
2
–
H, C
6
–
H of trimethoxy phenyl on C
3
),
3.92 (s, 12H, 2 [3,5-di(OC
H
3
) of 3,4,5 - trimethoxy phenyl]),
3.78 (s, 6H, 2 [4-O
C
H
3
of 3,4,5-trimethoxyphenyl]).
13
C NMR
(DMSO-d
6
, 100 MHz, d, ppm): 175.19 (
C
5
-1,2,4-oxadiazole),
167.97 (
C
3
-1,2,4-oxadiazole), 153.46 (
C
3
and
C
5
of 3,4,5-
trimethoxyphenyl on C
5
-1,2,4-oxadiazole), 153.38 (
C
3
and
C
5
of 3,4,5-trimethoxyphenyl on C
3
-1,2,4-oxadiazole), 140.16 (
C
4
of
3,4,5-trimethoxyphenyl-1,2,4-oxadiazole), 129.99 (
C
1
of 3,4,5-
trimethoxyphenyl), 121.58 (
C
1
of 3,4,5-trimethoxyphenyl),
104.56 (
C
2
and
C
5
of 3,4,5-trimethoxyphenyl), 104.47 (
C
2
and
C
6
of 3,4,5-trimethoxyphenyl), 60.30 (4-
CH
3
O of 3,4,5-
trimethoxyphenyl), 60.19 (4-
CH
3
O of 3,4,5-trimethoxyphenyl),
56.29 (3,5 di-
CH
3
O of 3,4,5-trimethoxyphenyl), 56.06 (3,5 di-
CH
3
O of 3,4,5-trimethoxyphenyl). Anal. calcd for C
20
H
22
N
2
O
7
:C,
59.70; H, 5.51; N, 6.96%; found; C, 59.89; H, 5.63; N, 7.22%.
4.1.4.6. Benzyl (3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazol-5-
yl)methylcarbamate (5f). White solid. Yield: 70%, m.p. 102–
104 1C.
1
H NMR (DMSO-d
6
, 400 MHz, d, ppm): 8.18 (s, 1H,
N
H), 7.35 (m, 5H, C
H of phenyl-CH
2
ring), 7.23 (s,2H, C
2
–
H,
C
6
–
H of trimethoxy phenyl), 5.07 (s, 2H, C
H
2
O), 4.58(s, 2H,
CH
2
N
H), 3.84 (s, 6H, 3,5-di(OC
H
3
) of 3,4,5-trimethoxy phenyl),
3.73 (s, 3H, 4-OC
H
3
of 3,4,5-trimethoxyphenyl).
13
C NMR
(DMSO-d
6
, 100 MHz, d, ppm): 177.83 (
C
5
-1,2,4-oxadiazole),
167.54 (
C
3
-1,2,4-oxadiazole), 156.45 (CQO), 153.44 (
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 140.22 (
C
4
of 3,4,5-trimethoxy-
phenyl), 136.77 (
C
1
of phenyl of Cbz), 128.44 (
C
3
and
C
5
of
phenyl of Cbz), 127.87 (
C
2
and
C
4
and
C
6
of phenyl of Cbz),
121.33 (
C
1
of 3,4,5-trimethoxyphenyl), 104.27 (
C
2
and
C
6
of
3,4,5-trimethoxyphenyl), 65.97 (
CH–O), 60.23 (4-
CH
3
Oof
3,4,5-trimethoxyphenyl), 56.09 (3,5 di-
CH
3
O of 3,4,5-trimethoxy-
phenyl), 37.05 (
CH
2
–N). Anal. calcd for C
20
H
21
N
3
O
5
: C, 60.14, H,
5.30, N, 10.52%; found; C, 60.39; H, 5.52; N, 10.79%.
4.1.4.7. 4-(Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxa-
diazole (5g). White solid. Yield: 40%, m.p. 148–150 1C; IR (KBr)
cm
1
: 2943, 2930 (CH aliphatic), 1583 (CQN), 1502 (CQC), 1230,
1128 (C–O). Anal. calcd for C
20
H
22
N
2
O
7
: C, 63.15; H, 5.30; N,
8.18%; found; 63.38; H, 5.43; N, 8.40%.
4.1.4.8. 5-Propyl-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole
(5h). White solid. Yield: 50%, m.p. 53–55 1C.
1
H NMR (DMSO-
d
6
, 400 MHz, d, ppm): 7.26 (s, 2H, C
2
–
H, C
6
–
Hoftrimethoxy
phenyl), 3.85 (s, 6H,3,5-di (OC
H
3
) of 3,4,5-trimethoxy phenyl),
3.74 (s, 3H, 4-OC
H
3
of 3,4,5-trimethoxyphenyl), 3.00 (t, 2H,
J=7.4Hz,C
1
–
H of propyl), 1.84–1.80 (m, 2H, C
2
–
H of propyl),
1.00 (t, 3H, J=7.4Hz,C
3
–
Hofpropyl).
13
CNMR(DMSO-d
6
,
100 MHz, d, ppm): 180.17 (
C
5
-1,2,4-oxadiazole), 167.34
(
C
3
-1,2,4-oxadiazole), 153.36 (
C
3
and
C
5
of 3,4,5-trimethoxy-
phenyl), 140.03 (
C
4
of 3,4,5-trimethoxyphenyl), 121.5733
(
C
1
of 3,4,5-trimethoxyphenyl), 104.17 (
C
2
and
C
6
of 3,4,5-
trimethoxyphenyl), 60.15 (4-
CH
3
O of 3,4,5-trimethoxyphenyl),
56.02 (3,5 di-
CH
3
O of 3,4,5-trimethoxyphenyl), 27.58 (
C
1
of
propyl), 19.63 (
C
2
of propyl), 13.36 (
C
3
of propyl). Anal. calcd
for C
14
H
18
N
2
O
4
: C, 60.42; H,6.52%; N, 10.07; found; C, 60.71;
H, 6.68; N, 10.28%.
4.1.4.9. 5-((9Z,12Z)-Heptadeca-9,12-dien-1-yl)-3-(3,4,5-trimethoxy-
phenyl)-1,2,4-oxadiazole (5i). Orange oil. Yield: 50%.
1
H NMR
(DMSO-d
6
, 400 MHz, d, ppm): 7.25 (s, 2H, C
2
–
H, C
6
–
Hof
trimethoxy phenyl), 5.32–5.30 (m, 4H, 2 (C
HQC
H)), 3.85
(s, 6H, 3,5-di (OC
H
3
) of 3,4,5-trimethoxy phenyl), 3.73 (s, 3H,
4-OC
H
3
of 3,4,5-trimethoxyphenyl), 3.00 (t, 2H, J= 7.6 Hz, C
Hof
(CHQC–CH–C = CH)), 2.73 (t, 2H, J= 6 Hz, C
1
–
H of hepta-
decadienyl), 2.0 (dd, 4H, J= 6.4, 6 Hz, 2 [C
H
2
of (CH
2
–CQCH)]),
1.78 (q, 2H, C
2
–
H of heptadecadienyl), 1.32–1.23 (m, 14H, C
3
to
C
7
–
H and C
15
,C
16
–H of heptadecadienyl), 0.83 (t, 3H,J= 6.8 Hz,
C
17
–
H of heptadecadienyl).
13
C NMR (DMSO-d
6
, 100 MHz, d,
ppm): 180.38 (
C
5
-1,2,4-oxadiazole), 167.15 (
C
3
-1,2,4-oxadiazole),
153.51 (
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 140.49 (
C
4
of 3,4,5-
trimethoxyphenyl), 130.40 (
C
9
and
C
13
of heptadecadienyl),
128.82 (
C
12
of heptadecadienyl), 127.21 (
C
10
of heptadeca-
dienyl), 121.63 (
C
1
of 3,4,5-trimethoxyphenyl), 104.20 (
C
2
and
C
6
of 3,4,5-trimethoxyphenyl), 60.18 (4-
CH
3
O of 3,4,5-trimeth-
oxyphenyl), 56.03 (3,5 di-
CH
3
O of 3,4,5-trimethoxyphenyl), 28.13
(
C
15
of heptadecadienyl), 25.66 (
C
11
of heptadecadienyl), 22.105
(
C
16
of heptadecadienyl) 13.83 (
C
17
of heptadecadienyl). Anal.
calcd for C
28
H
42
N
2
O
4
: C, 71.46; H, 9.00; N, 5.95%; found; C,
71.32; H, 8.95; N, 6.17%.
4.1.4.10. Benzyl(S)-(2-methyl-1-(3-(3,4,5-trimethoxyphenyl)-1,2,4-
oxadiazol-5-yl)propyl)carbamate (5j). Colorlessoil,yield:55%;IR
(KBr) cm
1
: 3301 (NH), 2954, 2938 (CH aliphatic), 1702 (CQO),
1570 (CQN), 1525 (CQC), 1231, 1125 (C–O);
1
HNMR(DMSO-d
6
,
400 MHz, d, ppm): 8.30 (d, 1H, J=8Hz,N
H), 7.35–7.25 (m, 7H,
C
2
–
H, C
6
–
H of trimethoxy phenyl and 5
HofphenylofCbz),5.07
(s, 2H, C
H
2
O), 4.80 (t,1H, J=7.6Hz,C
H-(CH(CH
3
)
2
)), 3.86 (s, 6H,
3,5-di (OC
H
3
) of 3,4,5-trimethoxy phenyl), 3.74 (s, 3H, 4-OC
H
3
of
3,4,5-trimethoxyphenyl), 2.28 (m, 1H, C
H–(CH
3
)
2
), 0.89 (d, 6H, J=
6.8 Hz, CH-(C
H
3
)
2
).
13
CNMR(DMSO-d
6
,100MHz,d, ppm):
179.80 (
C
5
-1,2,4-oxadiazole), 167.44 (
C
3
-1,2,4-oxadiazole), 156.24
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(
CQO), 153.40 (
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 140.22
(
C
4
of 3,4,5-trimethoxyphenyl), 136.74 55 (
C
1
of phenyl of Cbz),
128.37 (
C
3
and
C
5
of phenyl of Cbz), 127.93 (
C
2
,
C
4
and
C
6
of
phenyl of Cbz), 121.33 (
C
1
of 3,4,5-trimethoxyphenyl), 104.46
(
C
2
and
C
6
of 3,4,5-trimethoxyphenyl), 65.84 (
CH
2
-O), 60.19
(4-
CH
3
O of 3,4,5-trimethoxyphenyl), 56.05 (3,5 di-
CH
3
O of 3,4,5-
trimethoxyphenyl), 31.07 (
CH-(CH
3
)
2
), 18.21 (CH-(
CH
3
)
2
). Anal.
calcd for C
23
H
27
N
3
O
5
: C, 62.57; H, 6.16; N, 9.52; found; C, 62.75;
H, 6.42; N, 9.73%.
4.1.4.11. Benzyl (S)-(3-methyl-1-(3-(3,4,5-trimethoxyphenyl)-
1,2,4-oxadiazol-5-yl)butyl)carbamate (5k). Colorless oil, yield:
55%.
1
H NMR (DMSO-d
6
, 400 MHz, d, ppm): 8.26 (d, 1H, J=
7.6 Hz, N
H), 7.36–7.30 (m, 5H, 5
H of phenyl of Cbz), 7.24 (s, 2H,
C
2
–H, C
6
–H of trimethoxy phenyl ring) 5.07 (s, 2H, C
H
2
O),
4.96 (app s,1H, CH–N
H), 3.85 (s, 6H, 3,5-di (OC
H
3
) of 3,4,5-
trimethoxy phenyl), 3.74 (s, 3H, 4-OC
H
3
of 3,4,5-trimethoxy-
phenyl), 1.73 (d, 2H, J= 8.4 Hz, C
H
2
–CH(CH
3
)
2
), 1.70–1.67
(m, 1H, C
H(CH
3
)
2
), 0.93 (d, 6H, J= 6.4 Hz, CH(C
H
3
)
2
).
13
C
NMR (DMSO-d
6
100 MHz, d, ppm): 180.60 (
C
5
-1,2,4-oxadiazole,
167.52 (
C
3
-1,2,4-oxadiazole), 156.01 (
CQO), 153.42 (
C
3
and
C
5
of 3,4,5-trimethoxyphenyl), 140.23 (
C
4
of 3,4,5-trimeth-
oxyphenyl), 136.78 (
C
1
of phenyl of Cbz), 128.41 (
C
3
and
C
5
of phenyl of Cbz), 127.72 (
C
2
,
C
4
and
C
6
of phenyl of Cbz),
121.33 (
C
1
of 3,4,5-trimethoxyphenyl), 104.30 (
C
2
and
C
6
of 3,4,5-trimethoxyphenyl), 65.86 (
CH
2
–O), 60.22 (4-
CH
3
Oof
3,4,5-trimethoxyphenyl), 56.09 (3,5 di-
CH
3
O of 3,4,5-trimeth-
oxyphenyl), 46.89 (
CH
2
–NH), 24.18 (
CH(CH
3
)
2
, 22.66 (CH(
CH
3
)
2
.
Anal. calcd for C
24
H
29
N
3
O
6
: C, 63.28 H, 6.42; N, 9.23%,; found; C,
63.39; H, 6.48; N, 9.47%.
4.2. Biological evaluation
4.2.1. Antiproliferative screening. Antiproliferative effects
of compounds 5a–k were screened over about 60 human tumor
cell lines derived from nine different cancer types, according to
the protocol of the Developmental Therapeutics Program,
National Cancer Institute, Bethesda, MD.
42
4.2.2. Evaluation of in vitro tubulin polymerization inhibi-
tion. The effect of compound 5a – as the most promising
candidate on tubulin polymerization was determined using
an ELISA kit for Tubulin b(TUBb), SEB870Hu (Cloud-Clone
Corp., TX, USA). The microtiter plate provided in kits was pre-
coated with a biotin-conjugated antibody specific to Tubulin
Beta (TUBb). Standards or samples were then added to the
appropriate microtiter plate wells with a biotin-conjugated
antibody. Avidin protein conjugated to horseradish peroxidase
(HRP) enzyme was added to microplates to bind the biotin-
labeled antibody and incubated. After the addition of TMB
(3,30,5,500 -tetramethylbenzidine) substrate solution, only wells
that contained the complex gave a characteristic color change.
This color was measured spectrophotometrically after the addi-
tion of the sulfuric acid solution to stop the enzyme–substrate
reaction. The decrease of color intensity was used as an
indicator for tubulin inhibition.
4.2.3. Tubulin polymerization assay. The assay was made
using a highly purified porcine tubulin. The kit used was
(cytoskeleton, cat. BK011P). At first, 95 well plates were warmed
in the fluorimeter at 37 1C for 10 min. After the preparation of
the tested compound in solution 5a, 1.5 mL of the buffer 1 and
20 mL of GTP stock were defrosted and put in ice. Tubulin
glycerol buffer was then removed from 4 1C and placed on the
ice. 88 ml of tubulin was defrosted in a room temperature water
bath, then instantly placed on the ice. Assay components were
immediately mixed, then 5 ml of the control buffer was pipetted
into the first duplicate wells A1 and B1 as well as, 5 mL of
colchicine, followed by 5 mL of the tested compound 5a. The
plate was placed back into a warm plate reader for 1 min, then
50 mL of the tubulin reaction mixture was added into each of the
eight wells and the plate reader was turned on. The intensity of
fluorescence was determined every 50 s for 90 min.
43
After polymerization of tubulin, an increase in fluorescence
emission was measured at 410–450 nm over a 50 min period at
37 1C to the first duplicate wells. 50 mL of the tubulin reaction
was added into each of the eight wells using a single channel
pipette. The fluorescence intensity was recorded every 50 s for
90 min in a multifunctional microplate reader. The area under
the curve was used to determine the concentration that inhi-
bited tubulin polymerization by 50% (IC
50
) (Table 2).
4.3. Molecular docking
Molecular docking studies using MOE 2019.012 suite
44,45
were
performed to propose the expected mechanism of the action for
the tested compounds 5a–k as CBSIs by evaluating their bind-
ing scores and interaction modes at the b-tubulin active site of
the CBS. The co-crystallized native inhibitor (N-[(7S)-1,2,3,10-
tetramethoxy-9-oxo-5,7-dihydro-5H-benzo[d]heptalen-7-yl]ethan-
amide, 7) was isolated from the protein complex and inserted in
the database together with colchicine (colchicine, 6) as the two
reference standards.
4.3.1. Preparation of the newly synthesized oxadiazole
tested compounds (5a–k) and colchicine (6). Each derivative
of the aforementioned newly synthesized compounds (5a–k)
and colchicine (6) as well was built using the MOE builder, and
then prepared for the docking process as described earlier.
46–48
Moreover, all of the prepared synthesized compounds (5a–k)
and the prepared colchicine molecules (6) were imported in
the same database together with the co-crystallized native
inhibitor ((N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,7-dihydro-5H-
benzo[d]heptalen-7-yl]ethanamide (7) and saved as an MDB file
to be suitable for importing in the docking process.
4.3.2. Preparation of the active site of colchicine binding
site in b-tubulin. The Protein Data Bank website (https://www.
rcsb.org/) was used to download the X-ray structure of the CBS
receptor in b-tubulin (PDB ID: 5XIW, Resolution: 2.90 Å).
49
Then, the CBS complex was subjected to the default preparation
steps described before in detail.
50–52
4.3.3. Validation of the applied docking process using
the MOE program. A validation process for the MOE docking
procedure was performed at first and before the docking
process by redocking the co-crystallized native inhibitor (N-[(7S)-
1,2,3,10-tetramethoxy-9-oxo-5,7-dihydro-5H-benzo[d]heptalen-7-yl]-
ethanamide) inside its active site of the colchicine binding site
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in b-tubulin. It is worth mentioning that a very small difference
was observed between both the co-crystallized (native) and the
docked inhibitor molecules (RMSD = 0.54) as represented in the
ESI,†indicating a highly valid performance of the applied MOE
program.
53–55
4.3.4. Docking of the database to the binding pocket of
CBS in b-tubulin. A general docking process was performed
using the aforementioned prepared database, which was
inserted as an MDB file at the ligand site. The default specifica-
tions of the applied docking process were adjusted as repre-
sented before in detail.
56–58
Furthermore, we selected the best
pose for each studied compound according to its binding
score, RMSD value, and similar binding interactions with the
co-crystallized ligand for more considerations.
Conflicts of interest
The authors declare no conflict of interest.
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NJC Paper
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