Tubulin inhibitors: Discovery of a new scaffold targeting extra-binding residues within the colchicine site through anchoring substituents properly adapted to their pocket by a semi-flexible linker

Article abstract of DOI:10.1016/j.bioorg.2020.103767

Bis-hydrazides 13a-h were designed and synthesized as potential tubulin inhibitors selectively targeting the colchicine site between α- and β-tubulin subunits. The newly designed ring-B substituents were assisted at their ends by ‘anchor groups’ which are expected to exert binding interaction(s) with new additional amino acid residues in the colchicine site (beyond those amino acids previously reported to interact with reference inhibitors as CA-4 and colchicine). Conformational flexibility of bis-hydrazide linker assisted these ‘extra-binding’ properties through reliving ligands’ strains in the final ligand-receptor complexes. Compound 13f displayed the most promising computational and biological study results in the series: MM/GBSA binding energy of ?62.362 kcal/mol (extra-binding to Arg α:221, Thr β:353 & Lys β:254); 34% NCI-H522 cells’ death (at 10 μM), IC₅₀ = 0.073 μM (MTT assay); significant cell cycle arrest at G2/M phase; 11.6% preG1 apoptosis induction and 83.1% in vitro tubulin inhibition (at concentration = IC₅₀). Future researchers in bis-hydrazide tubulin inhibitors are advised to consider the 2-chloro-N-(4-substituted-phenyl)acetamide derivatives as compound 13f due to extra-binding properties of their ring B.

Full text of DOI:10.1016/j.bioorg.2020.103767

Journal Pre-proofs
Tubulin Inhibitors: Discovery of a New Scaffold Targeting Extra-binding
Residues within the Colchicine Site through Anchoring Substituents Properly
Adapted to their Pocket by a Semi-flexible Linker
Raed M. Maklad, El-Shimaa M.N. AbdelHafez, Dalia Abdelhamid, Omar M.
Aly
PII:
S0045-2068(19)31218-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.103767
YBIOO 103767
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
28 July 2019
6 March 2020
15 March 2020
Please cite this article as: R.M. Maklad, E.M.N. AbdelHafez, D. Abdelhamid, O.M. Aly, Tubulin Inhibitors:
Discovery of a New Scaffold Targeting Extra-binding Residues within the Colchicine Site through Anchoring
Substituents Properly Adapted to their Pocket by a Semi-flexible Linker, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.103767
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Tubulin Inhibitors: Discovery of a New Scaffold Targeting Extra-binding
Residues within the Colchicine Site through Anchoring Substituents
Properly Adapted to their Pocket by a Semi-flexible Linker
Raed M. Maklad a,b,c,*, El-Shimaa M. N. AbdelHafez d, Dalia Abdelhamid d, Omar M. Aly d
a Institute of Drug Discovery and Development, Kafrelsheikh University, Kafrelsheikh, Egypt.
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh 33516, Egypt.
c Zewail City of Science and Technology, 6th of October, Giza, Egypt.
d Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia, Egypt.
* Corresponding author:
Raed M. Maklad
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Kafrelsheikh University, Kafrelsheikh 33516, Egypt
E-mail address: raed_mostafa2010@yahoo.com; rmostafa@zewailcity.edu.eg
Abstract
Bis-hydrazides 13a-h were designed and synthesized as potential tubulin inhibitors selectively
targeting the colchicine site between α- and β-tubulin subunits. The newly designed ring-B
substituents were assisted at their ends by ‘anchor groups’ which are expected to exert binding
interaction(s) with new additional amino acid residues in the colchicine site (beyond those amino
acids previously reported to interact with reference inhibitors as CA-4 and colchicine).
Conformational flexibility of bis-hydrazide linker assisted these ‘extra-binding’ properties
through reliving ligands’ strains in the final ligand-receptor complexes. Compound 13f displayed
the most promising computational and biological study results in the series: MM/GBSA binding
energy of -62.362 kcal/mol (extra-binding to Arg α:221, Thr β:353 & Lys β:254); 34% NCI-
H522 cells’ death (at 10 µM), IC50= 0.073 µM (MTT assay); significant cell cycle arrest at G2/M
phase; 11.6% preG1 apoptosis induction and 83.1% in vitro tubulin inhibition (at concentration =
IC50). Future researchers in bis-hydrazide tubulin inhibitors are advised to consider the 2-chloro-
N-(4-substituted-phenyl)acetamide derivatives as compound 13f due to extra-binding properties
of their ring B.
Keywords: Antiproliferative agent; Anticancer; Hydrazide; Tubulin inhibitor; Combretastatin A-
4 analog; Molecular modeling; Colchicine binding site; Docking; Drug design; In silico study.
1

1. Introduction
Tubulin targeting agents [1–9] were historically classified [10,11] according to their binding
sites within tubulin protein [4,12–21]. Owing to their structure simplicity, colchicine-binding site
(CBS) inhibitors [17] especially combretastatin A-4 (CA-4) derivatives were subjected to full
SAR study [22–25] (Fig. 1 “Supplementary Material”). Several reported experimental results
proved that the 3,4,5-trimethoxyaryl ring (ring A) of CA-4 (1) and its analogues is essential for
the activity [26,27] while ring B has been amenable to modifications [22–25] (Fig. 1), especially
at C3' and C4'. The C3'-substituted analogues with hydrogen bond acceptors (HBAs) e.g. Cl
atom (compounds 2f,g [28]), NH2 group (compound 3 [24]) and OH group (compounds 1 [29], 4
[30] and 6a,b [31]) displayed the highest biological activities. Meanwhile, OMe and OEt groups
[26,28] remained the most common C4'-substitutions which preserve the activity of the parent
nucleus. Higher sterically-hindered alkyl substitution usually leads to loss of activity [26,28].
Regarding the olefinic linker, cis-geometry was necessary for maximal activity [26].
Accordingly, cis-restricted heterocyclic linkers were reported [32–35], tetrazole analogues 2a-g
[28] (Fig. 1) serve a representative example. However, open-chain linker analogues (ranging
from one- to four-atom length) [24,30,31,36] also preserved the activity (compounds 2-6, Fig. 1).
Target compounds (13a-h) were designed so as to preserve the general structural features of
the CBS tubulin inhibitors (Fig. 1 “Supplementary Material”) while introducing a new linker as
well as a new para-substitution pattern for the ring B. Designing new linkers have been relatively
encouraged so as to overcome some specific possible limitations concerning the classical cis-
olefinic linker. The liability to metabolic isomerization to the trans-isomer was reported as a
possible metabolic pathway of CA-4 in rat and human liver microsomes [37]. Moreover, the
linker chain stereochemistry played an interesting role. It is believed that the linker may not be
involved into interaction with the receptor. However, it preserves an optimum mean dihedral
angle between both aromatic rings (for maximal receptor interaction) beyond which narrow
range of deviation is allowed. Thus, if an open-chain linker was introduced with limited
conformational changes, an optimum receptor fitting and higher activity may be reached. For
example, the (1Z,3E)-isomer of the conjugated diene analogue 5 [36] and the (E)-isomer of the
chalcone analogue 6 [31] displayed optimal activities among all possible geometrical isomers.
Compound 5b (Fig. 1) has a conjugated diene linker with an inner C2-C3 single bond which
allows, to some extent, free rotation. Similarly, compound 6b (Fig. 1) has some free rotation
2

around the inner C-C single bond of the α,β-unsaturated ketone linker. Despite this partial
flexibility of the linker, both compounds were potent. Thus, complete rigidity of the linker was
not a must. The design of the linker chain in compounds (13a-h) was based on preserving some
structural rigidity of the lead (CA-4), and some similar flexibility to that of compounds 5b and
6b, at the same time. Both rigidity and flexibility are expected to be maintained in balance by the
effect of resonance as well as free N-N single bond rotation, respectively (for more discussion,
see section 3.1.1).
Fig. 1. Comparative cytotoxicity and antitubulin activity study of CA-4 (1) [24,28,29,31,36]
against some of its reported potent analogues (2 [28], 3 [24], 4 [30], 5 [36] and 6 [31]).
Formerly, similar linker chains like N-acylhydrazones [38,39] were versatile in several
drugs. Regarding the anticancer drug design, several structurally-related linker chains were
designed in potential antiproliferative agents; hydrazone [40,41], hydrazide-hydrazone [42–50],
bis-hydrazide [45,51–53], ketohydrazide-hydrazone [54], ketoamide [55], thiosemicarbazone
[42] and aroylhydrazone [49] were all reported examples of possible open-chain linker
functionalities. Regarding the bis-hydrazide scaffold, it was more frequently reported as a
possible intermediate for the synthesis of heterocyclic pharmaceutical agents [56–60].
Meanwhile, only a few studies (to the best of our knowledge) have targeted the design of potent
antiproliferative anti-tubulin agents having bis-hydrazide linkers [45,51,53] or linkers which are
structurally-related to bis-hydrazide [54,55,61].
The little number of research articles covering this point, as well as the large number of
those discussing anticancer drug candidates structurally-related to bis-hydrazide scaffold [38–55]
have increased our curiosity about the expected biological activity of the new scaffold. In
addition, the narrow range of studied ring-B para-substituents [26,28] has created our motivation
to optimize C4'-substituent chain length with incorporation of new terminal groups. Varying the
degrees of electronic and/or hydrophobic parameters of these C4'-substituents was utilized as a
helpful technique for finding an extra-binding site within the receptor pocket (section 3.1.
provides more details about the design of target bis-hydrazides 13a-h as potential tubulin binding
agents).
2. Results:
2.1. Molecular Modeling (In Silico) Study:
3

2.1.1. Receptor-based Pharmacophore Hypothesis:
Since the exact X-ray structure of tubulin was frequently reported with several co-
crystallized ligands [62–67], we utilized Phase [68] to generate a receptor-based pharmacophore
hypothesis. After, a PDB survey since 2004 to present, all the reported PDB files of tubulin co-
crystallized with ligands inside the CBS were downloaded. They are listed as follows: 1SA0
[62], 1SA1 [62], 4O2B [64], 5GON [65], 5JVD [66], 5LP6 [63] and 5LYJ [67]. Then, all the
ligand-receptor interactions were defined and used directly to generate a 15-feature
pharmacophore (Fig. 2A). After ligand-pharmacophore alignment, a validation step was
performed via calculation of "Phase Screen Score" for reference and target compounds. Among
the series 13a-h, compounds 13a, 13g and 13f displayed the best fitting (Fig. 2B), giving "Phase
Screen Score" values of 0.58, 0.54 and 0.60, respectively. Interestingly, both compounds 13a &
13f displayed protrusion of the 4’-substitution outside the pharmacophore (Fig. 2B). Can the
C=C and/or the NHCOCH2Cl groups (in compounds 13a & 13f, respectively) target different
amino acid residue(s) within the CBS other than those interacting with reference co-crystallized
tubulin ligands [62–67] (Fig. 2)?? Section 3.1.2 serves potential answers to this question.
Fig. 2. A) Proposed receptor-based pharmacophore hypothesis having 15 features: Hydrophobic
(Hp) interactions (in green); hydrogen bond acceptor (HBA) interactions (in red); hydrogen bond
donor (HBD) interactions (in blue). Close receptor amino acid residues (wire representation) are
colored by atom (C: grey, N: blue, O: red). Amino acid labels (Cys 241, Asn 258, Thr 179 ….)
are shown in blue. Feature-feature distances are represented as dashed lines in yellow and their
values (in angstrom) are shown in yellow. B) Ligand alignments of compounds 13a, 13f and 13g
(atom style: thick-tube representation; color {by atom: C: grey, H: white, N: blue, O: red, Cl:
green}) to the proposed Pharmacophore: H-bond (dashed line in yellow); π-cation interaction
(dashed line in blue).
2.1.2. Docking Study, Post-docking Energy Minimization & Scoring:
Docking study of target compounds 13a-h inside the CBS of tubulin (PDB code 1SA0) [62]
was carried out using Maestro 9.0 (Maestro, version 9.0 Schrödinger, LLC, New York, USA,
2009. www.schrodinger.com). The results revealed superior "docking scores" for compounds
13b, 13d, 13f and 13h (-6.454, -6.409, -5.935 and -5.835, respectively) against colchicine (-
5.008) and higher than or comparable to CA-4 (-6.062). This represents a rough measure of the
affinity of the ligand to the rigid receptor. However, more reliable results were obtained after
refinement of the overall ligand-receptor complex using Prime [70]. Now, minimization of ligand
strain was considered, with respect to several acceptable low energy receptor conformations
4

(based on the Induced-fit theory by Daniel Koshland in 1958 [71]). Finally, an accurate
MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) energy scoring function was
employed to evaluate the ligand strain and the binding interactions in the final refined receptor-
ligand complex.
Fig. 3. Docking study of top three compounds 13f, 13e and 13h inside CBS of tubulin.
The detailed docking results including the receptor interactions (amino acid residue, type,
length, per-residue score and clashes) as well as the ligand strain energy, the docking score and
the MM/GBSA binding energy of all target compounds vs. colchicine and CA-4 in addition to
the detailed procedure are found in the “Supplementary Material”.
Among the series 13a-h, compound 13f displayed the highest binding energy (-62.362
kcal/mol). This recommends compound 13f as the most efficient ligand in the series, with eight
H-bonding interactions with the receptor and one hydrophobic one (Fig. 3). The most important
amino acid is Thr β:353 (per-residue score= -8.019) due to the formation of two H-bonds of 1.73
and 2.09 Ǻ length. The 4-chloroacetamido group is involved into three H-bonding interactions
with Lys β:254, Cys β:241& Leu β:252 and one hydrophobic one with Leu β:255.
Compounds 13e & 13h displayed considerably high values of binding energy (-61.072 and -
59.947 kcal/mol, respectively), as well. Furthermore, the 4-azidoacetamido group in compound
13h displayed one H-bond with Tyr α:210 (1.82 Ǻ) and another one with Gln α:176 (1.88 Ǻ) as
well as an additional H-bond with Leu β:252 (2.58 Ǻ) in addition to other multiple hydrophobic
interactions as shown in Fig. 3.
2.2. Chemistry:
2.2.1. Synthesis of target compounds 13a-h:
Scheme 1. Synthesis of bis-hydrazides 13a-ha
aReagents and conditions: i) NaOH/Me2SO4, reflux 2 h, then NaOH, reflux 2 h, then HCl; ii)
SOCl2, reflux 2 h then NaOMe/ MeOH, stirring at r.t, 2 h; iii) NH2NH2.H2O/EtOH, reflux, 48 h;
iv) SOCl2/benzene, reflux 2 h; v) TEA, DMF, stirring at r.t, 2 h; vi) Na2CO3(aq.), EtOH, stirring,
80 °C, 8 h; vii) NaN3(aq.), DMF, stirring, 75°C, overnight.
1
Bis-hydrazides 13a-h were prepared as illustrated in Scheme 1, and characterized using H
13
NMR, DEPT-Q C NMR and elemental analysis (see the “Supplementary Material” for more
details). Firstly, compound 8 [72] was prepared starting from gallic acid (7) via Williamson's
5

ether synthesis. Then, the ester 9 [73] was prepared in high yield by activating the acid 8 with
thionyl chloride [74] followed by methanolysis of the produced crude acid chloride. After that,
the hydrazide 10 [73] was prepared by treatment of the ester 9 with hydrazine hydrate. Acyl
chlorides 12a [75], 12b [76], 12c [77,78], 12d [79], 12e and 12f [80] were prepared from their
corresponding carboxylic acids 11a [81], 11b [82,83], 11c [84], 11d [85], 11e [86] & 11f [80,87]
via reaction with thionyl chloride [74]. Acylation of hydrazide 10 with crude acyl chlorides 12a-f
afforded bis-hydrazides 13a-f, the procedure was previously reported [88,89]. Compounds 13g
and 13h were prepared from compounds 13e and 13f via ester hydrolysis and SN2 reaction with
sodium azide, respectively.
1
Compound 13g was previously reported [90], so only H NMR (Supplementary Material)
and elemental analysis for this compound (“Experimental” section) was found to be sufficient for
structure confirmation.
2.3. Biological evaluation of anticancer activity:
2.3.1. In vitro NCI-60 cell line panel cytotoxicity assay:
According to the protocol of the drug evaluation branch of the National Cancer Institute
(NCI), Bethesda, USA, compounds 13a and 13d-h were selected for in vitro NCI-60 cell line
panel cytotoxicity assay. The detailed growth inhibition percent for these bis-hydrazides against
the NCI-60 cell line panel is found in Table 3 (Supplementary Material). As shown in Fig. 4,
compound 13f displayed comparable antiproliferative activity to colchicine [91] and sometimes
even higher (as in U251 cells). The NCI results revealed also that compounds 13a and 13h (4’-
substituted ring-B derivatives) exhibited moderate cytotoxic activity against the NCI-H522 cell
line (growth inhibition percent of 34.23 and 39.51%, respectively). Additionally, compound 13a
displayed moderate growth inhibitory activity against A549/ATCC and UACC-257 cells (growth
inhibition 31.94 and 37.61%, respectively). Similar results were observed for compound 13h
against UACC-257 cells (30.26% growth inhibition). These results appreciate the effect of 4-O-
allyl and 4-NHCOCH2N3 substitutions of ring B.
Regarding the 3’,4’-disubstituted-ring-B derivatives (13d,e and 13g): in most cell lines, 13d
has higher activity than 13g. Moreover, it seems from the NCI results (Table 3, Supplementary
Material) that compound 13e has higher activity than compound 13g (“33.65 vs. 10.08%” as well
as “32.64 vs. 6.41%” growth inhibition for T-47D and SNB-75 cells, respectively). For detailed
comments on the NCI results as well as the overall experimental results, see the “Discussion and
6

Conclusion” section. Unfortunately, no one of the members of bis-hydrazide series 13a-h was
selected for further five-dose screening (at 1, 0.1, 0.01, 0.001 and 0.0001 µM) by the NCI,
including the potent derivative 13f. More information about the NCI compound selection criteria
are found in the “Supplementary Material”.
Fig. 4. NCI results of in vitro growth inhibition percent of 13f vs. colchicine [91] against the
most sensitive cell lines at 10 µM.
2.3.2. MTT assay:
To determinate their IC50, all the synthesized bis-hydrazides 13a-h were further analyzed
using the MTT assay [92,93] against NCI-H522 cells, using CA-4 reference. The choice of this
cell line was based on its high sensitivity to most tested bis-hydrazide derivatives in the NCI-60
cell line assay (Fig. 4). The results confirmed the promising activity of 13f (IC50= 0.073 µM vs.
0.001 µM for CA-4, see Table 1). Moderate activities of bis-hydrazides 13a, 13c, 13e and 13g,h
were also observed (Table 1).
2.3.3. In vitro tubulin inhibitory activity assay:
In order to prove the mechanism of their antiproliferative activities, and confirm the validity
of the molecular modeling results, bis-hydrazides 13a-h were subjected to in vitro tubulin
polymerization inhibitory activity assay against NCI-H522 cells using ELISA for β-tubulin
(taking CA-4 as a positive control). The detailed results of tubulin assay are found in the
“Supplementary Material”. The highest antitubulin activity was displayed by compound 13f
(83.1% inhibition), which intimately approaches the value of CA-4 (83.9%). Similarly,
compounds 13h and 13e displayed remarkable antitubulin activities (73.9 & 73.3% inhibition,
respectively as in Table 1).
Table 1. Calculated IC50 (µM) from linear equation of dose-response curve of compounds 13a-h
in MTT assay vs. their tubulin inhibition percent against NCI-H522 cells.
7

Cpd R=
MTT assay
Tubulin assay
Residual Tub Tub. Inhib. % b
Conc (µM)
a
Mean IC50 (µM)
Log IC50 (µM)
4-O-allyl
31.72
55.41
38.61
262.1
20.16
0.073
31.91
16.38
0.001
1.50 ± 0.10
1.74 ± 0.10
1.59 ± 0.10
2.42 ± 0.07
1.30 ± 0.10
-1.14 ± 0.06
1.50 ± 0.10
1.21 ± 0.10
-2.98 ± 0.04
547.41
575.94
521.98
652.86
453.06
286.90
512.44
442.56
273.36
1694.22
67.7
66.0
69.2
61.5
73.3
83.1
69.8
73.9
83.9
13a
13b
13c
13d
13e
13f
3-NO2, 4-OCH3
4-OCH3
3-Cl, 4-OEt
3-OAc, 4-OCH3
4-NHCOCH2Cl
3-OH, 4-OCH3
4-NH-CO-CH2-N3
CA-4 (ref)
13g
13h
Control
a Log IC50: Log10 concentration required to inhibit NCI-H522 cells' viability by 50% (mean ± SE
from the dose-response curves of three experiments); b tubulin inhibition percent was calculated
at concentration= IC50 (from MTT assay) against the same cell line (NCI-H522).
2.3.4. Correlation between results of docking study and tubulin inhibitory activity assay:
To judge the significance of the docking study results, illustrated previously, the correlation
between the computational study results (calculated binding energies) and the results of the anti-
tubulin activity assay was studied. A graphical representation is represented in the
“Supplementary Material, S39” correlating between the "-MM/GBSA dG Bind" and the "tubulin
inhibition percent" of compounds 13a-h. The plot shows a direct linear relationship with r
(correlation coefficient) value of 0.904.
2.3.5. Cell cycle analysis by flow cytometry:
Furthermore, the most potent derivative (13f) was subjected to NCI-H522 cell cycle analysis
via flow cytometry (Supplementary Material, S40) at 0.073 µM (the IC50 value). The results
8

showed that compound 13f produced a marked decrease in the cell population at G1 and S
phases as compared to the control (13f: 54.13% and 54.45% versus control: 45.79% and 92.08%,
respectively). Whereas, it caused significant increase of cell population in the G2/M phase
compared to untreated control cells (92.42% vs. 59.13%, respectively). These results
demonstrated that compound 13f exerts its cytotoxic effect in NCI-H522 cells via arresting the
cell growth in G2/M phase. Detailed results are provided in the “Supplementary Material”.
2.3.6. Annexin-V FITC apoptotic study:
Successful antiproliferative agents mostly have the ability to induce cancer cell apoptosis
[94]. The annexin V apoptosis detection method is based on the observation that soon after
initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face
of the plasma membrane to the cell surface [95]. Once on the cell surface, PS can be easily
detected by staining with a fluorescent conjugate of annexin V protein which has a high affinity
for PS. Furthermore, the addition of Propidium Iodide (PI) stain (which cannot cross the
membrane of live cells) facilitates the differentiation among necrotic, apoptotic and healthy cells
[96]. Detection can be analyzed by flow cytometry which indicates whether the cell death is
achieved through programed apoptosis or nonspecific necrosis [97].
Therefore, the most active compound (13f) was subjected to “Annexin-V FITC” apoptotic
study. The results displayed a marked increase in the percent of Annexin V-FITC positive
apoptotic cells (including both the early and late apoptotic phases) from 7.03% to 11.61% (15.9
times increase compared to the untreated NCI-H522 cells (Fig. 5). Moreover, only 2.42% of the
treated cells showed non-specific necrosis as compared to the control (0.68%). These findings
propound undoubtedly that compound 13f exhibits an apoptotic induction activity contributing to
its mechanism of action as an antiproliferative agent.
Fig. 5. Representative dot plots of NCI-H522 cells treated with compound 13f at its IC50 (0.073
µM) for 24 h analyzed by flow cytometry after double cells’ staining with annexin-V FITC and
PI. The blue color represents the stained Annexin V cells: a) results of control; b) results of 13f.
3. Discussion and Conclusion:
3.1. Design of target bis-hydrazides 13a-h:
3.1.1. Rationally-directed design:
9

As mentioned earlier in the “Introduction”, this study has been mainly devoted to the design
of a proposed semi-flexible linker which maintains an optimum dihedral angle between both
aromatic rings involved in receptor binding. The bis-hydrazide linker “CONHNHCO” is such an
ideal one as it is capable of assuming narrow range of possible conformations inside the active site
so that the molecule could adapt its structure easily to the receptor. Such conformational changes
are brought about via free rotation around N-N single bond. Nevertheless, the conformation in bis-
hyrdazide scaffold is not completely free, due to C-N partial double bond character developed by
resonance (Fig. 6) which restricts bond rotation to some extent. Again, this conformational
restriction is also helpful in the design, as this maintains (to some extent) the rigidity of the bis-
hydrazide linker chain (resembling the rigid olefinic linker in CA-4 which is a principle structural
moiety in the SAR study). Four main possible conformers of the bis-hydrazide scaffold were
proposed (Fig. 6) showing two possible resonating structures for each conformer. Hydrogen
bonding may help assist the stability of a given conformer, thus contributing to structural rigidity.
Reynolds et al [98] reported a theoretical basis for the study of the structure and the rotational
barriers of diacylhydrazine conformations. For summary, the bis-hydrazide linker introduced in
this work is expected to maintain balance between two useful structural features: a] rigidity
(rotationally restricted C-N bond due to double bond character resulting from resonance); besides
b] narrow range of conformational flexibility (due to N-N single bond free rotation). This balance
allows facile adaptation of the bis-hydrazide structure to preserve an expected optimum distance
between both substituted phenyl rings for maximal receptor binding.
Fig. 6: Four proposed conformational isomers for the bis-hydrazide scaffold.
Regarding the design of ring B substitution pattern: bis-hydrazides 13a, 13f & 13h were
substituted only at C4’ while bis-hydrazides 13d, 13e & 13g were substituted at C3’ and C4’.
Concerning the 3’,4’-disubstituted analogues, common groups which preserve the activity as
OMe [22–25] and OEt [23,25,28] were introduced. Similarly, the C3’-substituent was designed
to maintain H-bond acceptors which again preserve the activity (13g was designed to retain the
3’-OH of CA-4 while 13d preserves the 3’-Cl atom analogous to other potent reported 3’-Cl
analogues [28,99]). Although it was previously reported that the 4’-O-acetyl substitution at the
ring B leads to a remarkable decrease in the activity [22,25], the 3’-O-acetyl analogues are less
frequent in the literature. Nevertheless, several N-acetyloxadiazoline analogues were reported to
preserve the potency of the lead CA-4 [57,100,101]. One member of this series had the 3’-OAc-
10

4’-OMe phenyl as the ring B and still preserves the antiproliferative activity (even more active
than the corresponding 4’-OMe phenyl analogue [101]). This result has created our motivation to
discover the new role of the 3’-O-acetyl group in the bis-hydrazide series through designing
compound 13e. The corresponding 3’-OH derivative (13g) was designed to afford a reference for
reliable comparison with 13e (See the NCI-60 assay results in the “Supplementary Material”).
Regarding compound 13b, the 3’-NO2 group was introduced to mimic some of the reported
potent 3’-nitro-substituted CA-4 analogues [24,102].
Beyond this classical design, the 4’-substituted bis-hydrazides were subjected to major
modification in their design. Firstly, compound 13c was designed with the classical 4’-OMe
group [22–25] as a standard bis-hydrazide derivative for comparison. Then, novel C4’-
substituents (e.g. OCH2CH=CH2, NHCOCH2Cl and NHCOCH2N3) were introduced in
compounds 13a, 13f and 13h, respectively. This was just a way of increasing the chain lengths of
the C4’-substituents and assisting their free ends with anchor groups (as C=C, Cl and N3,
respectively) having different polarities, lipophilicities and steric effects (C=C is the most
lipophilic, while N3 is polar with negatively charged nitrogen and Cl quite polar and bulky).
Moreover, a literature survey was carried out to enhance this theoretical design by actually
reported tubulin inhibitors containing similar anchor groups. The survey revealed some
interesting examples of similarity e.g. the chloroacetyl derivatives of colchicine [103,104] as
well as the N-chloroacetyl oxadiazole derivative reported elsewhere [57]. Although the latter was
not subjected to biological evaluation, it was instead utilized as a synthetic precursor to the
corresponding 2-acetoxyacetyl, 2-azidoacetyl, 2-(dimethylamino)acetyl derivatives which were
all biologically active [57]. Among these compounds, the 2-azidoacetyl derivative displayed
potent antiproliferative activity against HCT-15 & NCI-H460 cells (IC50 in the nanomolar scale)
[57]. Herein, the same 2-azidoacetyl group was introduced in compound 13h in addition to its
synthetic precursor “2-chloroacetyl group” in compound 13f. Both functional groups were
expected to act as H-bond acceptors targeting the close receptor amino acid residues. For reliable
comparison, another poor H-bond acceptor group (preferably capable of hydrophobic binding)
had better be designed in parallel. The 4’-allyloxy group in 13a was thought to be sufficient. By
that time, we had three new C-4’ substituents at ring B in compounds 13a, 13f and 13h designed
to target an “extra-binding” site in the receptor pocket.
3.1.2. Computer-aided design:
11

As mentioned earlier (section 2.1.1.), a pharmacophore hypothesis (Fig. 2) was generated
from seven reported X-ray structures of tubulin individually co-crystallized with CBS ligands.
Careful selection of the closest amino acid residues (3ºA around each ligand) afforded the 13
amino acids showing 15 interaction features (10 Hp, 3 HBD and 2 HBA features, Fig 3A), which
constitute the apparent shape of the receptor pocket. Nevertheless, other amino acid residues
within the CBS (e.g. Tyr α:210, Arg α:221, Asp β:251…etc) haven’t been targeted by reported
ligand molecules. This may be due to strict adherence to the lead structure during the design of
its analogues (e.g. compounds 2-6 in Fig. 1 are so closely related to CA-4 that there is hardly any
expected different binding interaction). However, beyond those 13 amino acids shown in Fig. 2,
the “anchor groups” in this study (C=C, Cl and N3 in compounds 13a, 13f and 13h, respectively)
have successfully targeted new amino acid residues within the CBS as shown by the following
docking study results (Table 1, “Supplementary Material”): 13a (extra-Van-der-Waals interaction
of C=C with Asp β:251 [per-residue score -0.434]); 13f (extra-H-bonding of Cl atom with Lys
β:254 [2.25ºA] in addition to other extra-H-bonding with Arg α:221 [2.34ºA] and Thr β:353
[1.73 & 2.09ºA] with per-residue scores of -3.947, -3.451 and -8.019, respectively); and 13h
(extra-H-bonding of N3 group with Tyr α:210 [1.82ºA] as well as other extra-H-bonding with Gln
α:176 [1.88ºA] and Leu β:252 [2.58ºA] with per-residue scores of -0.22, -4.381 and -1.794,
respectively), see detailed results in Table 1 “Supplementary Material”. Generally, targeting new
amino acid residue(s) (“extra-binding” site) within the CBS by the newly designed “anchor
groups” usually adds further contribution to the binding energy (“extra-binding” properties). The
final MM/GBSA scoring function has accurately ranked the series 13a-h in terms of receptor
binding energy and ligand strain. The best results were displayed by compound 13f (MM/GBSA
dG bind -62.362 and ligand strain +3.949 kcal/mol, Table 1 “Supplementary Material”).
3.2. Synthesis and biological evaluation of target compounds (13a-h):
3.2.1. Synthesis:
Chemical synthesis and characterization of target compounds 13a-h was successfully carried
out using simple and straightforward synthetic methodology (Scheme 1).
3.2.2. Antiproliferative activity studies:
The cytotoxicity investigation of bis-hydrazides 13a-h passed into multiple stages. Firstly,
the NCI-60 cell line panel SRB assay served a preliminary screening of cytotoxicity against wide
12

range of cancer cell lines derived from different types of cancer (leukemia, non-small cell lung
cancer, breast cancer …. etc). The results of growth inhibition percent (measured at 10 µM
concentration) of the NCI-60 cell line panel were interpreted so as to evaluate the series 13a-h
versus colchicine (reference data, 6-2016 [91], Table 3 “Supplementary Material”). Similarly,
the comparison in Fig. 4 was held between the growth inhibition results of the most potent
derivative 13f and colchicine against the most sensitive cell line (which was NCI-H522 in our
case). Unfortunately, none of compounds 13a-h passed the selection criteria for the next step of
five-dose assay including the most potent one 13f. Thus, IC50 values had better be calculated by
further multiple-dose cytotoxicity assay. Based on the results shown in Table 3 and Fig. 4, we
selected the NCI-H522 cell line for the next steps of biological evaluation. The MTT assay was
considered the most common well-established technique available to us for cell viability
measurement which can provide an accurate measure of cytotoxicity [92,93]. The new cell
viability data obtained from the MTT assay were used in calculation of the dose-response curves
for compounds 13a-h and CA-4 against NCI-H522 cells, and then the IC50 values were
calculated from the curves. Table 1 provides a reliable comparison of the antiproliferative
activities of compounds 13a-h versus CA-4 in terms of their IC50 values against NCI-H522 cells.
The NCI-60 cell line panel assay as well as the MTT cytotoxicity assay revealed that
compound 13f displayed the maximal antiproliferative activity in the whole series (Table 1) and
in some cell lines higher than colchicine (Fig. 4). This provided a potential proof of the
previously illustrated extra-binding role of 4’-NHCOCH2Cl group. Moderate activities of 13a
and 13h against NCI-H522 confirmed the same hypothesis for 4’-allyloxy and 4’-NHCOCH2N3
groups, respectively. Regarding the 3’,4’-disubstituted-ring-B derivatives; compound 13d has a
comparable to higher activity than 13g against several cell lines especially HCT-116 and BT-549
(despite lower activity “IC50=262.1µM” against NCI-H522 cells, see Table 1). This enhances the
recommendation of the 3’-Cl substituent as a better pharmacophoric moiety than the ordinary 3’-
OH in this bis-hydrazide series. The promising results displayed by the previously reported 3’-
chloro CA-4 analogues [28,99] enhance this proposal. Compound 13d is expected to penetrate
the cancer cell membrane more rapidly than 13g due to higher log P and membrane dG insert
values (more details are found in the "Supplementary Material”). Concerning compound 13e (the
3’-O-acetyl derivative), it displayed higher activity when compared to 13g (the corresponding 3’-
OH derivative) as shown in SNB-75 and NCI-H522 cell lines (Table 3, Supplementary
13

Material). This provides a strong practical evidence to the theoretical docking study results of
13e (acetyl group in 13e exerts H-bonding with Cys β:241 (1.98 ºA), as in Fig. 3). This may lead
us to reevaluate the role of ester group in 3-O-acetyl CA-4 bis-hydrazide analogues as an extra-
binding group when compared to the classical 3-OH group.
3.2.3. Insights into the mechanism of action of the most potent analogues:
Anti-tubulin activity proved the mechanism of action of this series especially compounds
13e, 13f and 13h. Again 13f was the most potent one (83.1% inhibition vs. 83.9% for CA-4).
Linear relationship between anti-tubulin activity and the calculated binding energy in the
“Supplementary Material” indicates the reliability of the docking results in bis-hydrazide
derivatives. The linear equation can be used for rough prediction of the expected anti-tubulin
activity of any proposed member of this series with different substitution pattern, prior to
chemical synthesis.
Returning back to the most potent compound (13f), an additional insight into its mechanism of
action was obtained from the results of NCI-H522 cell cycle analysis, which displayed cell growth
arrest at the G2/M phase at the IC50 value (0.073 µM). Further contribution to the cytotoxicity of
13f results from 11.61% apoptosis induction (15.9 times of control) at the preG1 phase with only
2.42% non-specific necrosis.
4. Experimental:
4.1. Chemistry
All substituted benzoic acids carboxylic acids 11a [81], 11b [82,83], 11c [84], 11d [85], 11e
[86] & 11f [80,87], as well as gallic acid (7), the reagents and analytical or HPLC grade solvents
were purchased from Sigma-Aldrich®. All melting points were determined on an electro-thermal
melting point apparatus (Stuart Scientific, Model SMP1, U.K). Pre-coated silica gel plates (TLC
aluminum sheets, silica gel 60 F254, thickness 0.2 mm, Merck, Germany) were used.
Visualization of the spots was effected by UV-lamp at = 254nm. IR spectra were recorded on
Nicolet IS5 FT-IR spectrometer. Unless otherwise stated, NMR spectrometric analysis was
carried out in DMSO-d6 solvent using Brucker Advance spectrometer (400 and 100 MHz for 1H
NMR and DEPT-Q 13C NMR experiments, respectively). Chemical shifts () values are given in
parts per million (ppm), relative to DMSO-d6 (2.54 for proton and 40.45 ppm for carbon) and
14

coupling constants (J) in Hertz. Splitting patterns are designated as follows: s, singlet; d, doublet;
t, triplet; q, quartet; dd, doublet of doublet; m, multiplet, brs; broad singlet. Elemental analysis
was performed on Vario El Elementar CHN Elemental analyzer.
4.1.1. Preparation of 3,4,5-trimethoxybenzoic acid (8) [72]
The detailed procedure for preparation of this compound is included in the “Supplementary
Material”. The identity of this compound was confirmed by its melting point 165–167°C
(reported [72] 167-169°C). IR; 1681 cm-1 (C=O).
4.1.2. General procedure [74] for synthesis of substituted benzoyl chlorides
A mixture of the aromatic acid (2.500 mmol), thionyl chloride (0.894 g, 7.511 mmol) and
benzene (7 mL) was refluxed for 3 h. Then, benzene and the excess of thionyl chloride were
distilled off. The crude acid chloride was weighed by difference and used directly in the next
step without any further purification. For further purification, recrystallization from hexane may
be performed. Crude yields of acyl chlorides 12a [75], 12b [76], 12c [77,78], 12d [79], 12e and
12f [80] were as high as 70-95%.
4.1.3. Preparation of methyl 3,4,5-trimethoxybenzoate (9) [73]
The detailed procedure for preparation of compound (9) starting from 3,4,5-
trimethoxybenzoyl chloride (prepared by the previously mentioned general method [74]) is
included in the “Supplementary Material”. The identity of this compound was confirmed by its
melting point 83°C (reported 82–83ºC) [73] and by IR: 1717 cm-1 (C=O).
4.1.4. Preparation of 3,4,5-trimethoxybenzohydrazide (10) [73]
The detailed procedure for preparation of this compound is included in the “Supplementary
Material”. The identity of this compound was confirmed by its melting point 166 ºC (reported
168°C [73]). IR: 3323-3201 cm-1 (NHNH2), 1702 cm-1 (C=O).
4.1.5. General procedure [88,89] for synthesis of bis-hydrazides 13a-f
To a stirred solution of hydrazide (10) (2.002 mmol, 0.453 g) and triethylamine (2.283
mmol, 0.231 g) in DMF (15 mL), a solution of the appropriate acid chloride (2.100 mmol) in
DMF (10 mL) was added dropwise. After stirring for 72 h at ambient temperature, the reaction
15

mixture was poured into 150 mL of ice water while vigorous stirring for 15 min. The precipitate
was filtered under suction, washed several times with water, air-dried, then dried in the oven
below 110°C for several hours, and finally recrystallized from hot saturated acetonitrile solution
or (if insoluble in hot acetonitrile) washed with boiling ethyl acetate, then filtered and dried. In
certain cases and whenever needed, the product may be further recrystallized to give the final
bis-hydrazide. Theoretical yields percent were calculated for all derivatives as relative to
compound 10.
N'-(4-(Allyloxy)benzoyl)-3,4,5-trimethoxybenzohydrazide (13a)
Yellowish white powder; (Yield: 0.707 g, 91%); m.p= 100-103°C; IR (neat): two broad
1
bands 3426, 3195 cm-1 (NHNH), 1669, 1654 cm-1 (two C=O hydrazide); H NMR (400 MHz,
DMSO-d6): δ 10.44 (1H, brs, NH), 10.40 (1H, brs, NH), 7.95 (2H, d, J = 8.9 Hz, ArH), 7.31 (2H,
s, ArH), 7.11 (2H, d, J = 8.9 Hz, ArH), 6.10 (1H, ddt, Jtrans, cis, vic = 17.2, 10.5, 5.3 Hz, olefinic
H), 5.46 (1H, dd, Jtrans, gem = 17.3, 1.5 Hz, olefinic H), 5.33 (1H, dd, Jcis, gem = 10.5, 1.3 Hz,
olefinic H), 4.70 (2H, d, Jvic = 5.3 Hz, allylic CH2), 3.89 (6H, s, 3,5-di(OCH3), 3.77 (3H, s, 4-
OCH3); 13C NMR (100 MHz, DMSO-d6): δ 166.55, 166.42, 162.03, 153.75, 141.44, 134.32,
130.43, 128.71, 125.86, 118.89, 115.49, 106.02, 69.40, 61.20, 57.08. Anal. Calcd. for
C20H22N2O6 (386.15): C, 62.17; H, 5.74; N, 7.25, Found: C, 62.39; H, 5.95; N, 7.12.
3,4,5-Trimethoxy-N'-(4-methoxy-3-nitrobenzoyl)benzohydrazide (13b)
Greenish white powder; (Yield: 0.619 g, 76%); m.p=118-120°C; IR (neat): two broad bands
1
at 3223, 3003cm-1 (NHNH), 1674, 1617 cm-1 (two C=O hydrazide), 1489cm-1 (NO2); H NMR
(400 MHz, DMSO-d6) δ 10.75 (1H, s, NH), 10.59 (1H, s, NH), 8.51 (1H, d, Jmeta = 1.9 Hz, ArH),
8.29 (1H, dd, Jortho, meta = 8.8, 1.9 Hz, ArH), 7.58 (1H, d, Jortho = 8.9 Hz, ArH), 7.32 (2H, s, ArH),
4.06 (3H, s, 4-OCH3 ring B), 3.89 (6H, s, 3,5-di(OCH3) ring A), 3.77 (3H, s, 4-OCH3 ring A);
13C NMR (100 MHz, DMSO-d6) δ 166.20, 164.62, 155.58, 153.70, 141.47, 139.74, 134.57,
128.41, 125.47, 125. 36, 115.58, 106.00, 61.11, 58.17, 57.02. Anal. Calcd. for C18H19N3O8
(405.12): C, 53.33; H, 4.72; N, 10.37, Found: C, 53.35; H, 4.79; N, 10.43.
N'-(4-methoxybenzoyl)-3,4,5-trimethoxybenzohydrazide (13c)
Dark yellow powder; (Yield: 0.398 g, 55%); mp = 154-156°C; IR (neat) 1667,1651 cm-1
1
(two C=O hydrazide); H NMR (400 MHz, DMSO-d6) δ 10.44 (1H, s, NH), 10.40 (1H, s, NH),
16

7.96 (2H, d, Jortho = 8.8 Hz), 7.31 (2H, s, ArH), 7.10 (2H, d, Jortho = 8.8 Hz), 3.89 (6H, s, 3,5-
di(OCH3)), 3.87 (3H, s, 4-OCH3), 3.77 (3H, s, 4-OCH3); 13C NMR (100 MHz, DMSO-d6) δ
166.63, 166.49, 163.16, 153.79, 141.50, 130.49, 128.75, 125.78, 114.87, 106.09, 61.26, 57.14,
56.52. Anal. Calcd. For C18H20N2O6 (360.36): C, 59.99; H, 5.59; N, 7.77. Found: C, 60.25; H,
6.07; N, 7.82.
N'-(3-Chloro-4-ethoxybenzoyl)-3,4,5-trimethoxybenzohydrazide (13d)
White powder (m.p = 177-181ºC, yield = 0.393 g, 48%); IR (neat) 1670, 1650 cm-1 (two
C=O hydrazide);1H NMR (400 MHz, DMSO-d6): δ 10.48 (2H, brs, NHNH), 8.04 (1H, d, Jmeta
=
2.0 Hz), 7.95 (1H, dd, Jortho,meta = 8.6, 2.0 Hz), 7.32 (1H, d, Jortho = 8.8 Hz,), 7.30 (2H, s), 4.26
(2H, q, Jvic = 6.9 Hz, OCH2CH3), 3.89 (6H, s, 3,5-di(OCH3)), 3.77 (3H, s, 4-OCH3), 1.43 (3H, t,
13
Jvic = 7.0 Hz, OCH2CH3). C NMR (100 MHz, DMSO-d6): δ 166.13, 165.21, 157.50, 153.62,
141.36, 130.01, 129.03, 128.46, 126.20, 122.10, 114.20, 105.92, 65.64, 61.05, 56.96, 15.36.
Anal. Calcd. For C19H21ClN2O6 (408.83): C, 57.59; H, 5.64; N, 11.19. Found: C, 57.60; H, 5.70;
N, 11.23.
2-Methoxy-5-(2-(3,4,5-trimethoxybenzoyl)hydrazinecarbonyl)phenyl acetate (13e)
1
Brown powder, m.p = 135-138ºC, yield: 0.385 g, 46%; H NMR (400 MHz, DMSO-d6): δ
10.46 (2H, brs, NHNH), 7.925 (1H, dd, Jortho,meta = 8.6, 2.0 Hz), 7.71 (1H, d, Jmeta = 2.0 Hz), 7.32
(1H, d, Jortho =8.6, ArH) overlapped with 7.30 (2H, s, ArH), 3.90 (3H, s, 4-OCH3, ring B), 3.89
(6H, s, 3,5-di(OCH3)), 3.77 (3H, s, 4-OCH3, ring A), 2.34 (3H, s, CH3C=O). 13C NMR (100
MHz, DMSO-d6): δ 169.62, 166.31, 165.73, 154.85, 153.72, 141.46, 139.93, 128.57, 127.72,
125.79, 123.36, 113.49, 106.01, 61.16, 57.18, 57.05, 49.62, 21.36. Anal. Calcd. for C20H22N2O8
(418.40): C, 57.41; H, 5.30; N, 6.70. Found: C, 57.48; H, 5.70; N, 6.76.
2-Chloro-N-(4-(2-(3,4,5-trimethoxybenzoyl)hydrazinecarbonyl)phenyl)acetamide (13f)
Beyond the general methodology for synthesis of bis-hydrazides 13a-f (in part 4.1.5),
compound 13f was further purified by several washings with boiling DCM, ethyl acetate and
acetonitrile. Then, the remaining residue was loaded on a flash column using silica gel (80-120
mesh size) as stationary phase and a mixture of acetonitrile and methanol (1:1) as mobile phase.
The appropriate fraction (having Rf =0.23) was left undisturbed at ambient temperature to
crystallize. The produced yellow crystals had m.p= 210-213ºC (single spot on TLC). These
crystals were used for the final NMR analysis without further purifications (Yield = 0.380 g,
45%). 1H NMR (500 MHz, DMSO-d6): δ 10.75 (1H, s, NH amide), 10.455, 10.453 (2H, two brs,
17

NHNH hydrazide), 7.95 (2H, d, Jortho = 8.5 Hz), 7.76 (2H, d, Jortho = 8.8 Hz), 7.30 (2H, s, ArH),
13
4.35 (2H, s, CH2Cl), 3.88 (6H, s, 3,5-di(OCH3)), 3.77 (3H, s, 4-OCH3). C NMR (125 MHz,
DMSO-d6): δ 167.12, 166.18, 165.91, 153.63, 142.54, 141.37, 129.45, 128.57, 128.56, 119.68,
105.95, 61.06, 56.97, 44.54. Anal. Calcd. for C19H20ClN3O6 (421.83): C, 54.10; H, 4.78; N, 9.96.
Found: C, 54.19; H, 4.83; N, 10.01.
4.1.6. Preparation of N'-(3-Hydroxy-4-methoxybenzoyl)-3,4,5-trimethoxybenzohydrazide
(13g) [90]
Compound 13e (99.2 mg, 0.237 mmol) was dissolved in 3 mL absolute ethanol, then added
dropwise to a solution of sodium carbonate (0.053 g, 0.500 mmol) in 5 mL water. The mixture
was heated while stirring at 80°C for 3 h, then solvent evaporated and the residue washed with
water, filtered and finally recrystallized from acetonitrile to afford the final product as white
1
crystals (m.p = 193-195ºC); (Yield: 0.039 g, 44%); H NMR (400 MHz, DMSO-d6): δ 10.37
(1H, brs, NH), 10.30 (1H, brs, NH), 9.34 (1H, brs, OH), 7.45 (1H, dd, Jortho,meta = 8.4, 1.7 Hz),
7.40 (1H, d, Jmeta = 1.8 Hz), 7.30 (2H, s, ArH), 7.06 (1H, d, Jortho = 8.5 Hz), 3.88 (6H, s, 3,5-
di(OCH3)), 3.88 (3H, s, 4-OCH3), 3.77 (3H, s, 4-OCH3). Anal. Calcd. for C18H20N2O7 (376.36):
C, 57.44; H, 5.36; N, 7.44. Found: C, 57.49; H, 5.40; N, 7.45.
4.1.7. Preparation of 2-Azido-N-(4-(2-(3,4,5-trimethoxybenzoyl)hydrazinecarbonyl)
phenyl) acetamide (13h)
A mixture of compound 13f (0.100 g, 0.237 mmol), sodium azide (0.033 g, 0.508 mmol) and
5 mL DMF were heated while stirring at 75°C overnight and then poured into 30 mL water,
filtered and finally recrystallized from acetonitrile to afford the final product as white crystals
1
(m.p = 188-190ºC; yield = 0.045 g, 44%); H NMR (400 MHz, DMSO-d6): δ 10.62 (1H, brs,
NH), 10.48 (1H, brs, NH), 10.46 (1H, brs, NH), 7.94 (2H, d, Jortho = 8.5 Hz), 7.77 (2H, d, Jortho
=
8.4 Hz), 7.29 (2H, s, ArH), 4.15 (2H, s, CH2-N3), 3.88 (6H, s, 3,5-di(OCH3)), 3.76 (3H, s, 4-
OCH3). 13C NMR (100 MHz, DMSO-d6): δ 168.12, 166.62, 166.59, 153.91, 142.70, 141.58,
129.71, 128.96, 128.94, 120.00, 109.82, 106.19, 61.40, 57.25, 52.57. Anal. Calcd. for
C19H20N6O6 (428.14): C, 53.27; H, 4.71; N, 19.62. Found: C, 53.37; H, 4.79; N, 19.71.
4.2. Biological evaluation of anticancer activity:
4.2.1. NCI-60 cell line panel anticancer screening:
18

The NCI anticancer screening methodology has been described elsewhere in detail at
https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm. A summary of the
experimental assay methodology is found in the “Supplementary Material”.
4.2.2. In vitro anti-proliferative activity using MTT assay: [92,93]
Cell culture:
NCI-H522 cancer cell lines were purchased from American Type Cell Culture Collection
(ATCC, Manassas, USA) and grown on Dulbecco's modified Eagle's medium (DMEM) or
Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 100 mg/mL of
streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum.
Measurement of cytotoxicity using MTT assay:
Cytotoxicity was determined through morphological changes in NCI-H522 cells treated with
compounds 13a-h in comparison with untreated control cells. Firstly, cells were grown as
monolayer (104 cells/well in 96-well tissue culture plate) in media supplemented with 10%
inactivated fetal bovine serum. Cells were incubated for 24 h at 37°C in a humidified incubator
with 5% CO2 before treatment to allow cells’ attachment to the plate.
Then, NCI-H522 cells were treated individually with different concentrations (100 to 0.01 µM)
of compounds 13a-h & CA-4 (positive control). Eight wells were prepared for each
concentration in addition to eight other wells without the test compounds as cell control. Cells
were incubated with the tested compounds for 48 h into CO2 incubator at 37°C and 5% CO2.
After 48 h, the control and treated cells were observed under inverted microscope (before
completing the assay) for any morphology differences. Subsequently, culture media containing
tested compound and dead cells were decanted leaving only viable attached cells into the plate.
The plate was then washed twice with pre-warmed PBS (phosphate buffered saline). MTT
reagent (40 μL) was added to each well including blank and negative control wells. After that,
the plates were incubated in dark for 4 h for the reduction of MTT into formazan (purple needle
color) by dehydrogenase activity in mitochondria of viable cells. DMSO (150 μL) was added to
each well to solubilize the purple crystals of formazan. Finally, the absorbance was measured at
570 nm with microplate reader (ROBONIK TM P2000 Eliza plate reader).
The percentage of cell survival relative to control was calculated by the equation below:
19

The viability percent was plotted against the logarithm of sample concentration. Then the
concentration that induces 50% of maximum inhibition of cell proliferation (IC50) was calculated
for each compound from their dose-response curves using Graph Pad Prism 7.00 software
(Graph Pad software Inc, CA®).
4.2.3. In vitro tubulin polymerization inhibitory assay:
Cell culture:
NCI-H522 cancer cells were obtained from American Type Culture Collection, cells were
cultured using DMEM (Invitrogen/Life Technologies) supplemented with 10% Fetal Bovine
Serum (FBS) (Hyclone), 10 µg/mL of insulin (Sigma), and 1% penicillin-streptomycin. All of
the other chemicals and reagents were from Sigma, or Invitrogen. Cells were plated (cells density
1.2–1.8 × 10,000 cells/well) in a volume of 100 µL complete growth medium + 100 µL of the
tested compound per well in a 96-well plate for 18–24 h prior to β-tubulin assay.
NCI-H522 cells were lysed before the assay according to the following directions: Adherent cells
were detached with trypsin and then collected by centrifugation (suspended cells can be collected
by centrifugation directly). Cells were washed three times in cold PBS followed by resuspension
again in PBS and finally ultrasonication 4 times. A final centrifugation at 1500 rpm for 10 min at
2-8 oC was needed to remove cellular debris.
Tubulin assay procedure:
In vitro anti-tubulin activities of compounds 13a-h was measured using ELISA kit for β-
tubulin (Cloud Clone. Corp.) on NCI-H522 cell line. Growing NCI-H522 cells were trypsinized,
counted and seeded at the appropriate densities into 96-well microtiter plates. Cells then were
incubated in a humidified atmosphere at 37˚C for 24 h. The standards (NCI-H522 cell lysates
containing untreated β-tubulin), the tested compounds, and the control (CA-4) were diluted to
designated concentrations. On the 96-well microtiter plates 100 µL of each one of tested
compounds, standard as well as control was added to each well, and incubated at 37°C for 2 h.
20

The solution was aspirated and 100 µL of prepared "Detection Reagent A" (biotin-conjugated
anti-β-tubulin antibody) was added to each well. Incubation was done at 37°C for 2 h. After
washing 100 µL of prepared Detection Reagent B (Avidin conjugated to HRP “Horseradish
Peroxidase”) was added and incubation was continued at 37 °C for 30 min. Five washings were
done, then 90 µL of substrate solution of 3,3',5,5'-tetramethylbenzidine (“TMB”, a chromogenic
visualizing agent acting as a hydrogen donor for the reduction of liberated hydrogen peroxide by
HRP) was added and incubated at 37 °C for 15-25 min. Stop solution (sulphuric acid) was added
in 50 µL. Optical density (O.D.) of the reaction product (diimine of TMB) was measured at 450
nm using microplate reader (Spectromax Plus 96 well plate spectrophotometer). O.D. was
measured for each compound at a concentration (µM) equaling the IC50 value of this compound
against the same cell line (NCI-H522), in the previously mentioned MTT assay. A standard
curve was plotted (using Graph Pad Prism 7.00 software, Graph Pad software Inc, CA®) which
describes a relationship between blank-corrected optical density and the logarithm of tubulin
concentration. The software was used to calculate the best-fit curve which was firstly used to
restandardize the tubulin standards prepared by serial dilution. And then by interpolation, the
curve was used to determine unknown logarithm of residual tubulin concentrations in cell lysates
treated with known concentrations of samples (NCI-H522 cell lysates treated with compounds
13a-h) and reference (NCI-H522 cell lysates treated with CA-4). Finally, tubulin inhibition
percent was calculated relative to untreated control cells by the following equation:
4.2.4. Cell cycle analysis:
The procedure was previously reported [105]. For summary, compound 13f (at conc = IC50
= 0.073 µM) was incubated with NCI-H522 cells (2 x 105 cells/well) for 24 h. Then, cells were
washed with ice-cold phosphate buffer saline (PBS) twice and then centrifuged. After that, cells
were fixed in ethanol 70% (v/v) at 4 ºC for 30 min. then washed again with PBS for another 30-
min. period but now at 37 ºC. Again, cells were collected by 2000-rpm centrifugation for 5 min.,
stained with propidium iodide (PI) buffer, gently and homogenously mixed, and left in darkness
21

at ambient temperature for 20 min. BD FACS CALIBER flow cytometer was used for analysis
of DNA content of the cells.
4.2.5. Annexin-V FITC apoptotic study:
Generally, Annexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide) Apoptosis
Detection Kit [ab#139418] (BioVision Research Products, 980 Linda Vista Avenue, Mountain
View, CA 94043 USA) was used for apoptosis assay according to the manufacturer's instruction.
Firstly, NCI-H522 cells (4 x 106 cells/well) were incubated with compound 13f (at conc = IC50 =
0.073 µM) for 24 h. Control experiment (with untreated NCI-H522 cells) was carried out for
comparison. Cells were washed three times with ice-cold PBS, re-suspended in PBS and then
stained for 15 min with 5 mL Annexin V-FITC and 5 mL PI binding buffer in dark place at
ambient temperature. Finally, BD FACS CALIBER flow cytometer was used for analysis of
stained cells and measurement of extent of apoptosis.
Acknowledgements
The IR spectroscopic analyses were funded by and carried out at the Faculty of Pharmacy,
Minia University, Egypt. The authors acknowledge the National Cancer Institute, Bethesda,
USA, for performing the in vitro single-dose NCI-60 cell line panel cytotoxicity assay.
Acknowledgements are also extended to the Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science & Technology, Shanghai, China for
providing access to the licensed software used in the molecular modeling studies. Deep thanks to
Dr. Esam Rashwan, at Serum and Vaccine Research Institute, Dokki, Giza, Egypt for performing
the cell cycle analysis, apoptotic study, MTT and tubulin assays. We acknowledge Dr. Ayman M.
Ibrahim at Organic Chemistry Department, Faculty of Pharmacy, Deraya University, Minia,
Egypt for appreciable efforts in the figures' editing. Unless otherwise stated, all chemical
reagents, solvents, NMR analysis and biological studies were self-funded by the first author.
Finally, the authors pay special thanks of gratitude to Dr. Minna M. Abdel-Fattah for her time
and effort spent during revision of grammar and sentence structure in the whole manuscript.
Conflict of Interest
The authors declare that they have no conflict of interest.
22

Author Contributions
R.M. Maklad designed target compounds, carried out the molecular modeling studies, performed
the chemical synthesis, literature survey, statistical analysis, data collection, analyzed the results
wrote the manuscript. ES.M.N. AbdelHafez discussed the synthetic methodology, contacted NCI
for cytotoxicity study, discussed biological results & revised the manuscript. D. Abdelhamid
discussed the synthetic methodology, discussed the biological results, the conclusion & revised
the manuscript. O.M. Aly put the research point (tubulin inhibitors), designed the biological
evaluation tests, discussed the synthetic methodology & revised the manuscript.
Supplementary Material
SAR of CA-4 analogues; Synthetic methods for compounds 8-10; Characterization of
1
compounds 13a-h using H NMR, DEPT-Q 13C NMR; detailed discussion on the docking study
results and procedure; calculated log P and membrane permeability for compounds 13a-h; NCI-
60 cell line one-dose assay results of compounds 13a, 13d-h & colchicine; Principle of MTT
assay; Correlation between results of antitubulin activity assay & docking study; Results of cell
cycle analysis by flow cytometry; detailed results of cytotoxicity and ELISA anti-tubulin activity
assays.
References:
[1] D.M. Bollag, P.A. McQueney, J. Zhu, O. Hensens, L. Koupal, J. Liesch, M. Goetz, E.
Lazarides, C.M. Woods, Epothilones, a new class of microtubule-stabilizing agents with a
taxol-like mechanism of action, Cancer Res. 55 (1995) 2325–2333.
[2] M. Harrison, C. Swanton, Epothilones and new analogues of the microtubule modulators in
taxane-resistant disease, Expert Opin. Investig. Drugs. 17 (2008) 523–546.
https://doi.org/10.1517/13543784.17.4.523
[3] K.H. Altmann, B. Pfeiffer, S. Arseniyadis, B.A. Pratt, K.C. Nicolaou, The Chemistry and
Biology of Epothilones—The Wheel Keeps Turning, ChemMedChem. 2 (2007) 396–423.
https://doi.org/10.1002/cmdc.200600206.
[4] R.M. Buey, I. Barasoain, E. Jackson, A. Meyer, P. Giannakakou, I. Paterson, S. Mooberry,
J.M. Andreu, J.F. Díaz, Microtubule Interactions with Chemically Diverse Stabilizing
Agents: Thermodynamics of Binding to the Paclitaxel Site Predicts Cytotoxicity, Chem.
Biol. 12 (2005) 1269–1279. https://doi.org/10.1016/j.chembiol.2005.09.010.
23

[5] R.L. Bai, G.R. Pettit, E. Hamel, Binding of dolastatin 10 to tubulin at a distinct site for
peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites, J. Biol.
Chem. 265 (1990) 17141–17149.
[6] A. Brossi, H.J.C. Yeh, M. Chrzanowska, J. Wolff, E. Hamel, C.M. Lin, F. Quin, M.
Suffness, J. Silverton, Colchicine and its analogues: Recent findings, Med. Res. Rev. 8
(1988) 77–94. https://doi.org/10.1002/med.2610080105.
[7] L.H. Zhang, L. Wu, H.K. Raymon, R.S. Chen, L. Corral, M.A. Shirley, R. Krishna Narla, J.
Gamez, G.W. Muller, D.I. Stirling, J.B. Bartlett, P.H. Schafer, F. Payvandi, The Synthetic
Compound CC-5079 Is a Potent Inhibitor of Tubulin Polymerization and Tumor Necrosis
Factor-α Production with Antitumor Activity, Cancer Res. 66 (2006) 951–959.
https://doi.org/10.1158/0008-5472.CAN-05-2083.
[8] S.M. Kupchan, R.W. Britton, M.F. Ziegler, C.J. Gilmore, R.J. Restivo, R.F. Bryan, Tumor
inhibitors. LXXXX. Steganacin and steganangin, novel antileukemic lignan lactones from
Steganotaenia
araliacea,
J.
Am.
Chem.
Soc.
95
(1973)
1335–1336.
https://doi.org/10.1021/ja00785a054.
[9] E. Hamel, Antimitotic natural products and their interactions with tubulin, Med. Res. Rev.
16 (1996) 207–231.
[10] T. Carlomagno, Tubulin-Binding Agents, Springer Berlin Heidelberg, Berlin, Heidelberg,
2009. https://doi.org/10.1007/978-3-540-69039-9.
[11] S. Iwasaki, Antimitotic agents: Chemistry and recognition of tubulin molecule, Med. Res.
Rev. 13 (1993) 183–198. https://doi.org/10.1002/med.2610130205.
[12] M. Zweifel, G.C. Jayson, N.S. Reed, R. Osborne, B. Hassan, J. Ledermann, G. Shreeves, L.
Poupard, S.P. Lu, J. Balkissoon, D.J. Chaplin, G.J.S. Rustin, Phase II trial of combretastatin
A4 phosphate, carboplatin, and paclitaxel in patients with platinum-resistant ovarian cancer,
Ann. Oncol. 22 (2011) 2036–2041. https://doi.org/10.1093/annonc/mdq708.
[13] K.A. Lyseng-Williamson, C. Fenton, Docetaxel: A Review of its Use in Metastatic Breast
Cancer, Drugs. 65 (2005) 2513–2531. https://doi.org/10.2165/00003495-200565170-00007.
[14] H.A. Burris, Single-agent docetaxel (Taxotere) in randomized phase III trials, Semin.
Oncol. 26 (1999) 1–6.
[15] R.L. Noble, The discovery of the vinca alkaloids--chemotherapeutic agents against cancer,
Biochem. Cell Biol. Biochim. Biol. Cell. 68 (1990) 1344–1351.
[16] B. Gigant, C. Wang, R.B.G. Ravelli, F. Roussi, M.O. Steinmetz, P.A. Curmi, A. Sobel, M.
Knossow, Structural basis for the regulation of tubulin by vinblastine, Nature. 435 (2005)
519–522. doi:10.1038/nature03566.
[17] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An Overview of Tubulin Inhibitors That
Interact with the Colchicine Binding Site, Pharm. Res. 29 (2012) 2943–2971.
doi:10.1007/s11095-012-0828-z.
[18] T.J. Kim, M. Ravoori, C.N. Landen, A.A. Kamat, L.Y. Han, C. Lu, Y.G. Lin, W.M.
Merritt, N. Jennings, W.A. Spannuth, R. Langley, D.M. Gershenson, R.L. Coleman, V.
Kundra, A.K. Sood, Antitumor and Antivascular Effects of AVE8062 in Ovarian
24

Carcinoma, Cancer Res. 67 (2007) 9337–9345. https://doi.org/10.1158/0008-5472.CAN-
06-4018.
[19] L. Bohlin, B. Rosen, Podophyllotoxin derivatives: drug discovery and development, Drug
Discov. Today. 1 (1996) 343–351. https://doi.org/10.1016/1359-6446(96)10028-3.
[20] H. Goto, S. Yano, H. Zhang, Y. Matsumori, H. Ogawa, D.C. Blakey, S. Sone, Activity of a
new vascular targeting agent, ZD6126, in pulmonary metastases by human lung
adenocarcinoma in nude mice, Cancer Res. 62 (2002) 3711–3715.
[21] J.W. Lippert, Vascular disrupting agents, Bioorg. Med. Chem. 15 (2007) 605–615.
https://doi.org/10.1016/j.bmc.2006.10.020.
[22] M. Cushman, D. Nagarathnam, D. Gopal, A.K. Chakraborti, C.M. Lin, E. Hamel, Synthesis
and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that
inhibit tubulin polymerization, J. Med. Chem. 34 (1991) 2579–2588.
https://doi.org/10.1021/jm00112a036.
[23] M. Cushman, D. Nagarathnam, D. Gopal, H.M. He, C.M. Lin, E. Hamel, Synthesis and
evaluation of analogs of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene as
potential cytotoxic and antimitotic agents, J. Med. Chem. 35 (1992) 2293–2306.
https://doi.org/10.1021/jm00090a021.
[24] K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, Y. Morinaga, Y. Nihei, K. Ohishi, Y.
Suga, Y. Akiyama, T. Tsuji, Novel Combretastatin Analogues Effective against Murine
Solid Tumors: Design and Structure-Activity Relationships, J. Med. Chem. 41 (1998)
3022–3032. https://doi.org/10.1021/jm980101w.
[25] N.H. Nam, Combretastatin A-4 Analogues as Antimitotic Antitumor Agents, Curr. Med.
Chem. 10 (2003) 1697–1722. https://doi.org/10.2174/0929867033457151.
[26] M. Cushman, D. Nagarathnam, D. Gopal, H.M. He, C.M. Lin, E. Hamel, Synthesis and
evaluation of analogues of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene as
potential cytotoxic and antimitotic agents, J. Med. Chem. 35 (1992) 2293–2306.
[27] K. Gaukroger, J.A. Hadfield, N.J. Lawrence, S. Nolan, A.T. McGown, Structural
requirements for the interaction of combretastatins with tubulin: how important is the
trimethoxy
unit?,
Org
Biomol
Chem.
1
(2003)
3033–3037.
https://doi.org/10.1039/B306878A.
[28] R. Romagnoli, P.G. Baraldi, M.K. Salvador, D. Preti, M. Aghazadeh Tabrizi, A. Brancale,
X.H. Fu, J. Li, S.Z. Zhang, E. Hamel, R. Bortolozzi, G. Basso, G. Viola, Synthesis and
Evaluation of 1,5-Disubstituted Tetrazoles as Rigid Analogues of Combretastatin A-4 with
Potent Antiproliferative and Antitumor Activity, J. Med. Chem. 55 (2012) 475–488.
https://doi.org/10.1021/jm2013979.
[29] Data for in vitro cytotoxicity assay of combretastatin A-4, NCI database, internet website:
https://dtp.cancer.gov/dtpstandard/dwindex/index.jsp, NSC 613729, (updated 6-2016), DTP
60 cell/5 dose data, GI50 Mean Graph of 6 Tests.
25

[30] G.R. Pettit, B. Toki, D.L. Herald, P. Verdier-Pinard, M.R. Boyd, E. Hamel, R.K. Pettit,
Antineoplastic agents. 379. Synthesis of phenstatin phosphate, J. Med. Chem. 41 (1998)
1688–1695. https://doi.org/10.1021/jm970644q.
[31] S. Ducki, R. Forrest, J.A. Hadfield, A. Kendall, N.J. Lawrence, A.T. McGown, D.
Rennison, Potent antimitotic and cell growth inhibitory properties of substituted chalcones,
Bioorg. Med. Chem. Lett. 8 (1998) 1051–1056.
[32] L. Wang, K.W. Woods, Q. Li, K.J. Barr, R.W. McCroskey, S.M. Hannick, L. Gherke, R.B.
Credo, Y.H. Hui, K. Marsh, R. Warner, J.Y. Lee, N. Zielinski-Mozng, D. Frost, S.H.
Rosenberg, H.L. Sham, Potent, orally active heterocycle-based combretastatin A-4
analogues: synthesis, structure-activity relationship, pharmacokinetics, and in vivo
antitumor activity evaluation, J. Med. Chem. 45 (2002) 1697–1711.
[33] K. Mahal, B. Biersack, H. Caysa, R. Schobert, T. Mueller, Combretastatin A-4 derived
imidazoles show cytotoxic, antivascular, and antimetastatic effects based on cytoskeletal
reorganisation, Invest. New Drugs. 33 (2015) 541–554. https://doi.org/10.1007/s10637-
015-0215-9.
[34] A. Brancale, R. Silvestri, Indole, a core nucleus for potent inhibitors of tubulin
polymerization, Med. Res. Rev. 27 (2007) 209–238. https://doi.org/10.1002/med.20080.
[35] O.M. Aly, E.A. Beshr, R.M. Maklad, M. Mustafa, A.M. Gamal-Eldeen, Synthesis,
Cytotoxicity, Docking Study, and Tubulin Polymerization Inhibitory Activity of Novel 1-
(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1 H -1,2,4-triazole-3-carboxanilides: 1-
(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1 H -1,2,4-triazole-3-carboxanilides,
Arch. Pharm. (Weinheim). 347 (2014) 658–667. https://doi.org/10.1002/ardp.201400096.
[36] J. Kaffy, R. Pontikis, J.C. Florent, C. Monneret, Synthesis and biological evaluation of
vinylogous combretastatin A-4 derivatives, Org. Biomol. Chem. 3 (2005) 2657–2660.
https://doi.org/10.1039/b505955k.
[37] S. Aprile, E. Del Grosso, G.C. Tron, G. Grosa, In Vitro Metabolism Study of
Combretastatin A-4 in Rat and Human Liver Microsomes, Drug Metab. Dispos. 35 (2007)
2252–2261. https://doi.org/10.1124/dmd.107.016998.
[38] S. Thota, D.A. Rodrigues, P. de S.M. Pinheiro, L.M. Lima, C.A.M. Fraga, E.J. Barreiro, N-
Acylhydrazones as drugs, Bioorg. Med. Chem. Lett. 28 (2018) 2797–2806.
https://doi.org/10.1016/j.bmcl.2018.07.015.
[39] R. do C. Maia, R. Tesch, C.A.M. Fraga, Acylhydrazone derivatives: a patent review, Expert
Opin. Ther. Pat. 24 (2014) 1161–1170. https://doi.org/10.1517/13543776.2014.959491.
[40] A. Sabt, O.M. Abdelhafez, R.S. El-Haggar, H.M.F. Madkour, W.M. Eldehna, E.E.-D.A.M.
El-Khrisy, M.A. Abdel-Rahman, Laila.A. Rashed, Novel coumarin-6-sulfonamides as
apoptotic anti-proliferative agents: synthesis, in vitro biological evaluation, and QSAR
studies,
J.
Enzyme
Inhib.
Med.
Chem.
33
(2018)
1095–1107.
https://doi.org/10.1080/14756366.2018.1477137.
[41] W.M. Eldehna, R.I. Al-Wabli, M.S. Almutairi, A.B. Keeton, G.A. Piazza, H.A. Abdel-
Aziz, M.I. Attia, Synthesis and biological evaluation of certain hydrazonoindolin-2-one
26

derivatives as new potent anti-proliferative agents, J. Enzyme Inhib. Med. Chem. 33 (2018)
867–878. https://doi.org/10.1080/14756366.2018.1462802.
[42] N. Trotsko, A. Przekora, J. Zalewska, G. Ginalska, A. Paneth, M. Wujec, Synthesis and in
vitro antiproliferative and antibacterial activity of new thiazolidine-2,4-dione derivatives, J.
Enzyme
Inhib.
Med.
Chem.
33
(2018)
17–24.
https://doi.org/10.1080/14756366.2017.1387543.
[43] D. Das Mukherjee, N.M. Kumar, M.P. Tantak, A. Das, A. Ganguli, S. Datta, D. Kumar, G.
Chakrabarti, Development of Novel Bis(indolyl)-hydrazide–Hydrazone Derivatives as
Potent Microtubule-Targeting Cytotoxic Agents against A549 Lung Cancer Cells,
Biochemistry. 55 (2016) 3020–3035. https://doi.org/10.1021/acs.biochem.5b01127.
[44] R. Sreenivasulu, K.T. Reddy, P. Sujitha, C.G. Kumar, R.R. Raju, Synthesis,
antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-
hydrazone
derivatives,
Bioorg.
Med.
Chem.
27
(2019)
1043–1055.
https://doi.org/10.1016/j.bmc.2019.02.002.
[45] M. de Freitas Silva, L.F. Coelho, I.M. Guirelli, R.M. Pereira, G.Á. Ferreira-Silva, G.Y.
Graravelli, R. de O. Horvath, E.S. Caixeta, M. Ionta, C. Viegas, Synthetic resveratrol-
curcumin hybrid derivative inhibits mitosis progression in estrogen positive MCF-7 breast
cancer cells, Toxicol. In Vitro. 50 (2018) 75–85. https://doi.org/10.1016/j.tiv.2018.02.020.
[46] H. He, X. Wang, L. Shi, W. Yin, Z. Yang, H. He, Y. Liang, Synthesis, antitumor activity
and mechanism of action of novel 1,3-thiazole derivatives containing hydrazide–hydrazone
and carboxamide moiety, Bioorg. Med. Chem. Lett. 26 (2016) 3263–3270.
https://doi.org/10.1016/j.bmcl.2016.05.059.
[47] B. Yadagiri, U.D. Holagunda, R. Bantu, L. Nagarapu, V. Guguloth, S. Polepally, N. Jain,
Rational design, synthesis and anti-proliferative evaluation of novel benzosuberone tethered
with hydrazide–hydrazones, Bioorg. Med. Chem. Lett. 24 (2014) 5041–5044.
https://doi.org/10.1016/j.bmcl.2014.09.018.
[48] D. Kumar, N. Maruthi Kumar, S. Ghosh, K. Shah, Novel bis(indolyl)hydrazide–hydrazones
as potent cytotoxic agents, Bioorg. Med. Chem. Lett. 22 (2012) 212–215.
https://doi.org/10.1016/j.bmcl.2011.11.031.
[49] S. Vogel, D. Kaufmann, M. Pojarová, C. Müller, T. Pfaller, S. Kühne, P.J. Bednarski, E.
von Angerer, Aroyl hydrazones of 2-phenylindole-3-carbaldehydes as novel antimitotic
agents,
Bioorg.
Med.
Chem.
16
(2008)
6436–6447.
https://doi.org/10.1016/j.bmc.2008.04.071.
[50] H.-Z. Zhang, J. Drewe, B. Tseng, S. Kasibhatla, S.X. Cai, Discovery and SAR of indole-2-
carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a
cell-based
HTS
assay,
Bioorg.
Med.
Chem.
12
(2004)
3649–3655.
https://doi.org/10.1016/j.bmc.2004.04.017.
[51] R. Sreenivasulu, R. Durgesh, S.S. Jadav, P. Sujitha, C. Ganesh Kumar, R.R. Raju,
Synthesis, anticancer evaluation and molecular docking studies of bis(indolyl) triazinones,
27

Nortopsentin analogs, Chem. Pap. 72 (2018) 1369–1378. https://doi.org/10.1007/s11696-
017-0372-8.
[52] S. Wang, H.Y. Liu, R.F. Xu, J. Sun, Synthesis, biological evaluation, and molecular
docking studies of diacylhydrazine derivatives possessing 1,4-benzodioxan moiety as
potential anticancer agents, Russ. J. Gen. Chem. 87 (2017) 2671–2677.
https://doi.org/10.1134/S1070363217110238.
[53] L.B. Salum, A. Mascarello, R.R. Canevarolo, W.F. Altei, A.B.A. Laranjeira, P.D.
Neuenfeldt, T.R. Stumpf, L.D. Chiaradia-Delatorre, L.L. Vollmer, H.N. Daghestani, C.P.
de Souza Melo, A.B. Silveira, P.C. Leal, M.J.S. Frederico, L.F. do Nascimento, A.R.S.
Santos, A.D. Andricopulo, B.W. Day, R.A. Yunes, A. Vogt, J.A. Yunes, R.J. Nunes, N-(1′-
naphthyl)-3,4,5-trimethoxybenzohydrazide as microtubule destabilizer: Synthesis,
cytotoxicity, inhibition of cell migration and in vivo activity against acute lymphoblastic
leukemia,
Eur.
J.
Med.
Chem.
96
(2015)
504–518.
https://doi.org/10.1016/j.ejmech.2015.02.041.
[54] M.P. Tantak, L. Klingler, V. Arun, A. Kumar, R. Sadana, D. Kumar, Design and synthesis
of bis(indolyl)ketohydrazide-hydrazones: Identification of potent and selective novel
tubulin
inhibitors,
Eur.
J.
Med.
Chem.
136
(2017)
184–194.
https://doi.org/10.1016/j.ejmech.2017.04.078.
[55] G. Bacher, B. Nickel, P. Emig, U. Vanhoefer, S. Seeber, A. Shandra, T. Klenner, T.
Beckers, D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral
activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks
neurotoxicity, Cancer Res. 61 (2001) 392–399.
[56] B. Burja, T. Čimbora-Zovko, S. Tomić, T. Jelušić, M. Kočevar, S. Polanc, M. Osmak,
Pyrazolone-fused combretastatins and their precursors: synthesis, cytotoxicity, antitubulin
activity and molecular modeling studies, Bioorg. Med. Chem. 18 (2010) 2375–2387.
https://doi.org/10.1016/j.bmc.2010.03.006.
[57] B.G. Szczepankiewicz, G. Liu, H.S. Jae, A.S. Tasker, I.W. Gunawardana, T.W. von
Geldern, S.L. Gwaltney, J.R. Wu-Wong, L. Gehrke, W.J. Chiou, R.B. Credo, J.D. Alder,
M.A. Nukkala, N.A. Zielinski, K. Jarvis, K.W. Mollison, D.J. Frost, J.L. Bauch, Y.H. Hui,
A.K. Claiborne, Q. Li, S.H. Rosenberg, New Antimitotic Agents with Activity in Multi-
Drug-Resistant Cell Lines and in Vivo Efficacy in Murine Tumor Models, J. Med. Chem.
44 (2001) 4416–4430. https://doi.org/10.1021/jm010231w.
[58] Y. Hu, C.Y. Li, X.M. Wang, Y.H. Yang, H.L. Zhu, 1,3,4-Thiadiazole: Synthesis, Reactions,
and Applications in Medicinal, Agricultural, and Materials Chemistry, Chem. Rev. 114
(2014) 5572–5610. https://doi.org/10.1021/cr400131u.
[59] D. Kumar, N. Maruthi Kumar, K.H. Chang, K. Shah, Synthesis and anticancer activity of 5-
(3-indolyl)-1,3,4-thiadiazoles, Eur. J. Med. Chem. 45 (2010) 4664–4668.
https://doi.org/10.1016/j.ejmech.2010.07.023.
[60] A. Kamal, D. Dastagiri, M.J. Ramaiah, E.V. Bharathi, J.S. Reddy, G. Balakishan, P. Sarma,
S.N.C.V.L. Pushpavalli, M. Pal-Bhadra, A. Juvekar, S. Sen, S. Zingde, Synthesis,
28

anticancer activity and mitochondrial mediated apoptosis inducing ability of 2,5-
diaryloxadiazole–pyrrolobenzodiazepine conjugates, Bioorg. Med. Chem. 18 (2010) 6666–
6677. https://doi.org/10.1016/j.bmc.2010.07.067.
[61] N. Sirisoma, A. Pervin, J. Drewe, B. Tseng, S.X. Cai, Discovery of substituted N′-(2-
oxoindolin-3-ylidene)benzohydrazides as new apoptosis inducers using a cell- and caspase-
based HTS assay, Bioorg. Med. Chem. Lett. 19 (2009) 2710–2713.
https://doi.org/10.1016/j.bmcl.2009.03.121.
[62] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, M. Knossow,
Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain,
Nature. 428 (2004) 198–202. https://doi.org/10.1038/nature02393.
[63] J. Marangon, M.S. Christodoulou, F.V.M. Casagrande, G. Tiana, L. Dalla Via, A. Aliverti,
D. Passarella, G. Cappelletti, S. Ricagno, Tools for the rational design of bivalent
microtubule-targeting drugs, Biochem. Biophys. Res. Commun. 479 (2016) 48–53.
https://doi.org/10.1016/j.bbrc.2016.09.022.
[64] A.E. Prota, F. Danel, F. Bachmann, K. Bargsten, R.M. Buey, J. Pohlmann, S. Reinelt, H.
Lane, M.O. Steinmetz, The novel microtubule-destabilizing drug BAL27862 binds to the
colchicine site of tubulin with distinct effects on microtubule organization, J. Mol. Biol.
426 (2014) 1848–1860. https://doi.org/10.1016/j.jmb.2014.02.005.
[65] P. Zhou, Y. Liu, L. Zhou, K. Zhu, K. Feng, H. Zhang, Y. Liang, H. Jiang, C. Luo, M. Liu,
Y. Wang, Potent Antitumor Activities and Structure Basis of the Chiral β-Lactam Bridged
Analogue of Combretastatin A-4 Binding to Tubulin, J. Med. Chem. 59 (2016) 10329–
10334. https://doi.org/10.1021/acs.jmedchem.6b01268.
[66] M.D. Canela, S. Noppen, O. Bueno, A.E. Prota, K. Bargsten, G. Sáez-Calvo, M.L. Jimeno,
M. Benkheil, D. Ribatti, S. Velázquez, M.J. Camarasa, J.F. Díaz, M.O. Steinmetz, E.M.
Priego, M.J. Pérez-Pérez, S. Liekens, Antivascular and antitumor properties of the tubulin-
binding
https://doi.org/10.18632/oncotarget.9527.
[67] R. Gaspari, A.E. Prota, K. Bargsten, A. Cavalli, M.O. Steinmetz, Structural Basis of cis -
chalcone
TUB091,
Oncotarget.
8
(2017)
14325–14342.
and trans -Combretastatin Binding to Tubulin, Chem.
https://doi.org/10.1016/j.chempr.2016.12.005.
2
(2017) 102–113.
[68] S.L. Dixon, A.M. Smondyrev, S.N. Rao, PHASE: A Novel Approach to Pharmacophore
Modeling and 3D Database Searching, Chem. Biol. Drug Des. 67 (2006) 370–372.
https://doi.org/10.1111/j.1747-0285.2006.00384.x.
[69] Maestro, version 9.0, Schrödinger, LLC, New York, USA, 2009. www.schrodinger.com.
[70] M.P. Jacobson, D.L. Pincus, C.S. Rapp, T.J.F. Day, B. Honig, D.E. Shaw, R.A. Friesner, A
hierarchical approach to all-atom protein loop prediction, Proteins. 55 (2004) 351–367.
https://doi.org/10.1002/prot.10613.
[71] D.E. Koshland, Application of a Theory of Enzyme Specificity to Protein Synthesis, Proc.
Natl. Acad. Sci. U. S. A. 44 (1958) 98–104.
29