Spirohydantoins and 1,2,4-triazole-3-carboxamide derivatives as inhibitors of histone deacetylase: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.01.021

Two structurally novel series of histone deacetylase inhibitors (HDACIs) involving two potential surface recognition moieties; 3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione (in series I) and 1-(3-methoxyphenyl)-5-(3,4,5-trimethoxypheny

Full text of DOI:10.1016/j.ejmech.2018.01.021

Accepted Manuscript
Spirohydantoins and 1,2,4-triazole-3-carboxamide derivatives as inhibitors of histone
deacetylase: Design, synthesis, and biological evaluation
Alshimaa M.A. Aboeldahab, Eman A.M. Beshr, Mai E. Shoman, Safwat M. Rabea,
Omar M. Aly
PII:
S0223-5234(18)30021-7
10.1016/j.ejmech.2018.01.021
EJMECH 10098
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 August 2017
Revised Date: 22 December 2017
Accepted Date: 8 January 2018
Please cite this article as: A.M.A. Aboeldahab, E.A.M. Beshr, M.E. Shoman, S.M. Rabea, O.M. Aly,
Spirohydantoins and 1,2,4-triazole-3-carboxamide derivatives as inhibitors of histone deacetylase:
Design, synthesis, and biological evaluation, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.01.021.
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Graphical abstract
O
NH
N
HOHN
O
O
Compound 5f (Docked into HDLP active site) showed comparable
cytotoxicity and HDAC inhibitory activity to SAHA

ACCEPTED MANUSCRIPT
Spirohydantoins and 1,2,4-Triazole-3-Carboxamide Derivatives as Inhibitors of Histone
Deacetylase: Design, Synthesis, and Biological Evaluation
a
a
a
b
Alshimaa M. A. Aboeldahab , Eman A. M. Beshr , Mai E. Shoman , Safwat M. Rabea , Omar M. Aly
a,c
*.
a
Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt.
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC V6T 1Z3
b
Canada.
C
College of Clinical Pharmacy, Albaha University, Kingdom of Saudi Arabia
*Corresponding author
E-mail addresses: omarsokkar@yahoo.com; omarsokkar@mu.edu.eg
Abstract
Two structurally novel series of histone deacetylase inhibitors (HDACIs) involving two potential surface
recognition moieties; 3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione (in series I) and
1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide (in series II) were
designed, synthesized, and evaluated for their anti-proliferative activities, HDAC inhibitory activities,
and their binding modes to HDAC protein. Compounds 5f and 10e showed comparable HDAC
inhibitory activity to SAHA.
Series II have been also demonstrated as potential HDAC-tubulin dual inhibitors, promoted with
structural similarities between (1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamide) nucleus, of series II, and Combretastatin A4.
The tubulin inhibitory activities of series II members, together with their docking into colchicine binding
site of β-tubulin were performed. Compound 9a showed remarkable cytotoxicity. Hybrid 10e behaved as
potent HDAC-tubulin dual inhibitor. It showed better tubulin inhibition than CA4 as well as its
effectiveness against HDAC.
Key words: HDAC; Epigenetics; Spirohydantoin; Triazole.
1

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1. Introduction
Cancer is mainly a disease of tissue growth regulation failure, which its initiation and progression is
controlled by genetic (by mutation) and epigenetic (by deregulation) processes [1, 2]. Among the
epigenetic events is acetylation/deacetylation of histone N-terminal lysine, which obviously leads to
changes in gene expression through change of chromatin structure without changing gene sequence [3]
.
The turnover of the acetyl groups on histone tail occurs normally in the cells and the level of acetylation
is controlled by the equilibrium between activities of two enzymes; histone acetyl transferases (HATs)
and histone deacetylases (HDACs) [4]. HATs neutralize the positive charge of lysine residues in the N-
terminal tail of histone, by adding an acetyl group, allowing the phosphate backbone of the DNA to open
up and relax, thus, facilitates the accessibility of a variety of factors to DNA and the transcriptional
activation of many genes including tumor suppressor genes silenced in cancer [5, 6]. HDACs, however, do
exactly the reverse; leading to a protonated histone tail, by deacetylation of lysine amino group, that
interact strongly with negatively charged DNA phosphate backbone, resulting in abnormal closed
chromatin structure and transcriptional repression. The overexpression of HDACs, found in various
cancers, leads to histone hypoacetylation which silences the expression of onco-suppressor genes (p53,
p21, p27) [7]. HDACs, being localized in various organelles in addition to the nucleus, they regulate the
chromatin structure by controlling the acetylation state of, not only histone protein, but also non-histone
proteins such as tubulin, ERα, p53, HSP90, NF-γA, and GATA-1[8-13]
.
HDACs, thus, have a crucial role on cell cycle, cell growth, differentiation, apoptosis, immunogenicity,
and angiogenesis [14] and their deregulated expression has been observed in neurological disorders [15]
,
viral infection [16], inflammation [17] and in malignancies [18-20]. Therefore, HDACs have been identified
as attractive targets for various human disorders, especially malignancies [8, 21, 22]. Many studies show
that inhibition of HDAC elicits anticancer activities in several tumor cell lines [23]. HDAC inhibitors
show great potential for the treatment of cancer by induction of cell cycle arrest, differentiation, and
apoptosis [24-27]. To date Four HDAC-targeting drugs have been approved by FDA for treatment of
2

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hematological malignancies. Vorinostat (soberoylanilide hydroxamic acid; SAHA, Merck, 2006) [28, 29]
and Romidepsin (FK228, Gloucester Pharmaceuticals now Celgene, 2009) have been approved by FDA
for treatment of cutaneous T-cell lymphoma (CTCL), Belinostat (PDX-101, TopoTarget now Onxeo,
2014) [30] has been approved for treatment of peripheral T-cell lymphoma (PTCL), and Panobinostat
(
LBH-589, Novartis, 2015) has been approved for multiple myeloma (MM) [29, 31-33]. In 2015, Chinese
Food and Drug Administration have approved a fifth HDAC: Chidamide (Shenzhen Chipscreen
Biosciences, 2015) for the treatment of PTCL. The FDA approval of these HDACIs has encouraged the
development of new active molecules of the same category. Over twenty HDACIs are currently in
clinical trials for targeting different cancers [34, 35]. Comparing the structures of known HDACIs like
trichostatin A (TSA) and SAHA, it clearly appeared that, despite the variety of structural motifs, they
share a common pharmacophore model: a metal binding functionality; in another word, zinc binding
2
+
group (ZBG) acts as a chelating group for Zn in the active site of HDAC, a spacer that efficiently
presents the ZBG to the active site by filling the narrow tubular pocket of the enzyme. This spacer is
attached to a hydrophobic terminal residue acting as surface recognition moiety called Cap via a polar
connecting unit [36-41] (Fig. 1). Hydroxamic acid derivatives are the broadest class of HDACIs with high
2
+
affinity to HDACs, owing to the perfect chelation between hydroxamate group and Zn [42]. They
inhibit HDAC at considerably lower concentrations than those considered being cytotoxic [43]. Three out
of four FDA approved HDACIs are hydroxamates. This has encouraged efforts aiming at developing
novel potent hydroxamate derived HDACIs with better pharmacokinetic profiles.
Figure 1.
Encouraged by the aforementioned aspects, herein, we reported the synthesis, HDAC enzyme inhibition,
and anti-proliferative activities against some tumor cell lines of two series of novel spirohydantoins and
1,2,4-triazole-3-carboxamides with the objective of designing novel and potent HDACIs (Fig. 2). Two
novel nuclei were introduced; 3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione (in
3

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series I) and 1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide (series
II), to serve as two surface recognition moieties (Caps) to make contacts with amino acid residues at the
external entrance of the HDAC active site, with the objective of comparing their effect on cytotoxicity
and HDAC inhibitory activity of the synthesized compounds. To probe the impact of the linker length [44-
4
6]
, six aliphatic chain spacers of variable lengths were introduced (in series I) and five spacers (in series
II). Two different ZBGs; hydroxamic acid and carboxylic groups were also provided.
The structural similarities between members of series II and a series of tubulin polymerization inhibitors
in our previous work [47, 48], together with the surprising and potent anti-proliferative activity of
compound 9a, have pushed us toward study the tubulin polymerization inhibitory activity of all members
of series II, to demonstrate their potential activity as promising HDAC-tubulin dual inhibitors. Owing to
structural similarities between (1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamide) nucleus and Combretastatin A4 (CA4) (Fig. 2), in series II, the 1,2,4-triazole-3-
carboxamide nucleus which was supposed to serve as a potential Cap for HDAC inhibition, was also
introduced as a potential bioisostere to the cis-double bond of the CA4 to arrest the bioactive cisoid
conformation.
Combretastatin A4 (CA4) is a cytotoxic agent belonging to microtubule-targeted agents. Microtubule-
targeted agents (MTAs) are those agents that bind tubulin binding sites, thus, interfere with the dynamic
stability of microtubules via stabilizing or destabilizing microtubule assembly. These agents act as
spindle poisons leading to mitotic catastrophe, cell cycle arrest, and eventually, apoptotic cell death.
CA4, in turn, belongs to the class of MTAs that destabilizes microtubules and inhibits tubulin
polymerization by binding the colchicine domain of tubulin [49]. It is a potent natural anti-mitotic cis-
stilbene derivative that was isolated by Pettit and co-workers [50-52]. It exhibits strong anti-tubulin activity
and potent cytotoxicity against a variety of human cancer cell lines including those that are multidrug
resistant [53, 54]. Most importantly, it has demonstrated powerful anti-angiogenesis properties [55] by
altering endothelial cell structure and causing vascular permeability and rapid destruction to tumor
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vasculature . It is able to elicit irreversible vascular shutdown within solid tumors leading to central
tumor necrosis, leaving normal vasculature intact [56, 57]. However, its poor water solubility, low
bioavailability, dose dependent toxicity [58-60], and possibility to in vivo convert into the more stable
inactive trans-configuration [61] restrict its clinical application [62]. Owing to its potent anti-proliferative
activity and relatively simple chemical structure, CA4 has attracted significant interest as a lead
compound in the development of new anticancer analogues. Significant efforts have been done aiming at
improving the pharmacokinetic profile and stabilizing the active cis-configuration. A prodrug of CA4;
combretastatin A4 phosphate (CA4P) [63] together with a wide range of more water soluble cis-restricted
analogues of CA4 has been identified as colchicine binding site inhibitors (CBSIs) with potent antitumor
activity [64-71]. The structure-activity studies of a wide variety of CA4 analogues have demonstrated a
clear view of the anticancer pharmacophore. Three preferred constituents include: a 3,4,5-trimethoxyaryl
motif in ring-A, a cis-configured stilbene or analogous bridge retains the cisoid configuration linking
ring-A to a 3,4-disubstituted ring-B [69, 72]. Because modifications of ring A reduce the bioactivity in
most analogues of CA4 [73], ring B and the olefinic bridge offer better prospects for modification. The
ring B was found to be tolerant to wide ranging of structural modification as well as the olefinic bridge
which also has been subjected to substitution with different bridges in order to arrest the conformation in
the cisoid form [74-76]
.
Figure 2.
2.
Results and Discussion
2.1. Chemistry
The designed ɛ-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-hydroxyl
alkanamides 5a-f were prepared starting with one putt Bucherer-Bergs reaction [77] to afford the parent
spirohydantoin (3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione) 2, from α-tetralone 1,
Scheme 1, followed by alkylating the amidic amino group of the spirohydantoin 2 with various
5

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ethylbromo esters to give six ethyl ester derivatives 3a-f. Ester 3a-f hydrolysis yielded the free acid
derivatives
ɛ-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)alkanoic
acids
4a-f, which then stirred with hydroxylamine hydrochloride in presence of N,N'-carbonyldiimidazole
(CDI) [78, 79] to afford the hydroxamic acid derivatives 5a-f.
Scheme 1.
Procedure employed to synthesize series II involved the reaction of glycine with 3,4,5-
trimethoxybenzoyl chloride in 10% sodium hydroxide to yield 2-(3,4,5-trimethoxybenzamido)acetic acid
6
[80]. Heating 6 with acetic anhydride afforded the corresponding cyclic form 7, Scheme 2. The
synthesis of intermediate 8 was carried out through coupling of the diazonium salt of 3-methoxyaniline
with the active methylene of 7 [81]. Synthesis of the targeted compounds ɛ-(1-(3-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)alkanoic
acids
9a-e
and
-N-
hydroxyalkanamides 10a-e was achieved by Sawdey rearrangement reaction of 8 with five terminal
amino acids to afford the acids 9a-e, followed by stirring the acid derivatives 9a-e with hydroxylamine
hydrochloride in presence of CDI to afford the hydroxamic acid derivatives 10a-e.
Scheme 2.
2
.2. Biological evaluation
.2.1. In vitro anti-proliferative activity
2
In vitro anti-proliferative activity of compounds 4a-f, 5a-f, 9a-e, and 10a-e, were tested using MTT
assay against two cancer cell lines as shown in Table 1. Results were reported as IC values (in µM
5
0
concentrations). Voriostat (SAHA) and CA4 were used as positive references. In series I (spirohydantoin
derivatives), compounds 4a-f, having carboxylic group as a potential ZBG, showed no anti-proliferative
activity against the two tested cancer cell lines. The corresponding hydroxamic acid derivatives 5a-f
were found to be good growth inhibitors of MCF-7 cells, Table 1. The potency of the compounds
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increased with increasing linker length n, which made compound 5f the most potent growth inhibitor of
MCF-7 cells in this series having IC value very close to SAHA against this cancer cell line (IC = 2.56
5
0
50
µM and 2.18 µM, for 5f and SAHA respectively). Meanwhile, compounds 5a-f showed low activity
against HepG2 cell line. Furthermore, changing length of aliphatic linker didn’t show a uniform pattern
in inhibiting HepG2 cell line like that seen in case of MCF-7 cell line. For 1,2,4-triazole derivatives
(series II), compounds 9a-e, having carboxylic group as a potential ZBG, showed no anti-proliferative
activity against the two tested cancer cell lines, except for the carboxylic derivative 9a which
demonstrated potent inhibition of HepG2 cells growth, with IC value comparable to that of SAHA
5
0
against the same cell line, and it is actually the most potent derivative of series II, Table 1. It also
showed a moderate cytotoxic activity against MCF-7 cell line, with IC value slightly lower than that of
5
0
CA4. The hydroxamic acid derivatives 10a-e showed a different pattern of anti-proliferative activity
from the corresponding spirohydantoin derivatives against the two different cancer cell lines. For MCF-7
cell line they demonstrated good anti-proliferative activity, compared to CA4, but lower than SAHA.
The pattern of increasing activity of 10a-e against MCF-7 cell line was in order of decreasing linker
length n, with the most potent derivative 10a having 1 methylene linker. For HepG2 cell line, 10a-e also
showed good to moderate inhibitory activity, but potency increased in the order of increasing the linker
length n and in this case the most potent derivative is 10e having 5 methylene linker.
Table 1.
2
2
.2.2.
In vitro enzyme inhibition
.2.2.1.
In vitro HDAC inhibitory activity
To gain insight into the mechanism of action of the synthesized compounds, compounds 5f,
1
0a, and 10e were tested for in vitro HDAC inhibitory activity, IC values for compounds 5f,
50
1
0a, and 10e were determined against HDAC1, HDAC2, HDAC4, and HDAC6 isoforms, and
SAHA was provided as a positive reference, Table 2. Results revealed that all tested
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compounds 5f, 10a, and 10e showed significant HDAC inhibitory activities against the four
tested HDAC isoforms. It could be noticed that compound 5f with spirohydantoin Cap and
hexamethylene linker afforded the highest HDAC inhibitory activity with IC50 value
comparable to SAHA against HDAC4 and even lower IC value in case of HDAC1. It is worth
5
0
noting that, surprisingly, 10a with one methylene bridge showed considerably high HDAC
inhibitory activity.
Table 2.
2.2.2.2.
In vitro tubulin polymerization inhibitory activity
Three members of series II; 9a, 10a and 10e were tested for in vitro tubulin polymerization
inhibitory activity in MCF-7 cells using ELISA for β-tubulin (TUBb). CA4 was employed as a
positive reference, and results were reported as IC values (in µM concentrations), Table 3.
50
From table 3 it is noticeable that compound 9a and CA4 afforded very close IC values for
5
0
TUBb inhibition. A noteworthy addition is that compound 10e with pentamethylene bridge
potently inhibited TUBb polymerization and, surprisingly, showed higher potency than CA4
(
IC values 4.82 and 5.73 for 10e and CA4; respectively).
50
Table 3.
2.3. Docking studies
Molecular modeling study was performed to investigate the mode of binding of some of the synthesized
compounds. The flexible docking experiment was performed using the multistep docking protocol
introduced by Koska et al. [82] and implemented in the Accelrys Discovery Studio 2.5 software. The
−
1
obtained docked poses were ranked according to their CDocker energy (kcal·mol ) which was
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calculated at the final stage of the flexible docking protocol and was used as indication for the binding
strength of the ligands [83, 84]
.
2.3.1.
Docking at the vorinostat binding site in HDLP (Histone Deacetylase-Like Protein)
Molecular docking simulation of compounds 5e, 5f, 10a, and 10e into HDLP active site was done. They
got stabilized at the vorinostat binding site by hydrophobic and hydrogen bond interactions, Table 4. It
could be noticed that all the docked compounds 5e, 5f, 10a and 10e showed similar binding to vorinostat
at the HDLP active site. They all showed hydrogen bonding with TYR297 as shown in vorinostat, in
addition to additional bonding, listed in Table 4. The results of binding energies, as well as, interaction
energies with HDLP were listed in Table 4, and revealed high CDocker energies (used as indication for
the binding strength of the ligands) and CDocker interaction energies of the docked compounds.
Compound 5f showed higher CDocker energy than vorinostat which means better binding at HDLP
active site (Fig. 3). It should be noticed that compounds 5e and 5f (spirohydantoin Cap) afforded much
higher CDocker energies than 10a and 10e (1,2,4-triazole-3-carboxamide Cap), suggesting that
spirohydantoin nucleus may be a better Cap for HDAC binding than 1,2,4-triazole-3-carboxamide
nucleus, and these findings look consistent with HDAC inhibitory assay findings (Section (2.2.2.1)).
Table 4.
Figure 3.
2.3.2.
Docking at the colchicine binding site in tubulin protein
Compounds 9a, 10a, 10e and CA4 were docked into the colchicine binding site buried between α-tubulin and β-
tubulin mainly in the β subunit. The X-ray crystallographic structure of DAMA colchicine-tubulin complex (PDB
(www.rcsb.org) code 1SA0) was used as the tubulin protein template. Molecular docking simulation of compounds
9
a, 10a and 10e into tubulin protein binding site was done. They got stabilized at the colchicine-binding site of
tubulin by hydrophobic and hydrogen bond interactions (types of interactions were listed in Table 5). The results of
9

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binding energies, as well as, interaction energies with tubulin protein were listed in Table 5, and revealed high
CDocker energies and CDocker interaction energies of the docked compounds. Compound 9a and 10a afforded
almost the same CDocker energies as CA4. Meanwhile, compound 10e afforded higher CDocker energy than CA4
indicating better binding of 10e at the colchicine binding site. It should be noticed that there is an extra hydrogen
bond between hydroxamate moiety of 10e and GTP in the binding site, and these findings are in agreement with the
results of tubulin inhibition assay (Section 2.2.2.2). These results reflect the importance of the carboxylate or
hydroxamate groups separated from the 1,2,4-triazole group by one or more methylene group in holding the
molecule at place (binding) at the active site by extra hydrogen bonds and justifies the experimental findings
(Section 2.2.2). These findings may be fruitful in designing compounds of better tubulin polymerization inhibition
than CA4. [85]
Table 5.
2.4. Drug likeliness and oral bioavailability
The increase in molecular weight of the deigned compounds is still in an acceptable range.
ꢀ
Reports suggest that oral bioavailability is mainly affected by 3 different criteria; 1- no more
than 10 rotatable bonds in the specified compound 2- 12 or fewer H bond donors and acceptors, 3-
2
the compound have a polar surface area (PSA) of 140 A or less [86]
.
Table 6.
From the above table we can suggest that the designed compounds are orally available. They have
2
only 4 HBA and 3 donors. The polar surface area measured did not exceed 140 A . Only compound
10e exceeded the 500 dalton rule in Lipiniski’s but this is the only violation to the rule, also the 500
molecular weight cutoff has been questioned. Polar surface area and the number of rotatable bonds
has been found to better discriminate between compounds that are orally active and those that are
1
0

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not for a large data set of compounds in the ratio with number of HBD and acceptors (12) and PSA
(124). Log P, all compounds in an acceptable range (the accepted range is 0.4-5.6).
3. Conclusion
Two novel series of spirohydantoins and 1,2,4-triazole-3-carboxamide derivatives were successfully
designed, synthesized, evaluated for their anti-proliferative activity as well as enzyme inhibitory activity.
Molecular modeling study was performed to assess the potential binding ability of some compounds to
HDAC enzyme as well as the colchicine binding site of β-tubulin. Results of anti-proliferative activity,
tubulin and HDAC enzyme inhibitory activities, as well as docking simulation results of tested
compounds were found to be consistent with each other in a reasonable way, and they confirmed the
following; For series I, introduction of the spirohydantoin nucleus (3',4'-dihydro-2'H-
spiro[imidazolidine-4,1'-naphthalene]-2,5-dione) as a new Cap for HDAC binding is successful. Linking
this Cap to a hydroxamic moiety as a ZBG looks essential for activity of these derivatives, as the
corresponding carboxylic derivatives devoided from any cytotoxic activity against the tested cell lines.
Aliphatic linkers of various lengths affect the anti-proliferative activity together with HDAC inhibitory
activity of compounds; the potency against MCF-7 cell line increase, in an ascending pattern, with
increasing methylene bridge length n, reaching the highest activity with hexamethylene bridge in
compound 5f, which also showed a remarkable inhibition of HDAC comparable to SAHA (Sections
(2.2.2) and (2.3.1)). The involvement of HDAC inhibition as a possible mechanism of action is
confirmed from in vitro HDAC inhibitory assay as well as docking at HDLP active site. For series II, the
nucleus (1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide) is found to
be a successful Cap for HDAC binding, as well as, a good CA4 analogous moiety for tubulin inhibition
(sections (2.2.2) and (2.3.2)). The lack of anti-proliferative activity in compounds 9b-e suggests the
importance of a small linker (one methylene bridge) for the activity of carboxylic derivatives as seen
from the great variation between the activity of 9a and the rest of members 9b-e against HepG2 cell line.
For hydroxamic acid derivatives 10a-e, aliphatic linkers of variable lengths affect the anti-proliferative
1
1

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activity as follows; for MCF-7 cell line, the pattern of increasing activity of 10a-e was in order of
decreasing linker length (n). These results are in agreement with results of previous studies introducing
HDAC-tubulin dual acting entities on the same cell line (MCF-7) [44]. Meanwhile, for HepG2 cell line,
potency of 10a-e increased in the order of increasing the linker length (n), which goes in line with
HDAC and tubulin inhibitory findings, as well as docking results. The previous findings suggests that,
the possible mechanisms explaining the cytotoxic actions of the series II derivatives 10a-e might involve
both HDAC inhibition, in addition to tubulin polymerization inhibition activities. Comparing the results
of spirohydantoin derivatives 5a-f versus 1,2,4-triazole derivatives 10a-e, the higher HDAC inhibition
shown for spirohydantoin series 5a-f suggests that spirohydantoin nucleus may act as a better Cap for
HDAC binding than 1,2,4-triazole-3-carboxamide nucleus. Meanwhile, the higher anti-proliferative
activities of 10a-e than 5a-e suggest involvement of another mechanism of action for 1,2,4-triazole
derivatives, which might be tubulin polymerization inhibition.
4. Experimental
4.1. Chemistry
Chemicals and solvents used were of analytical grade. Reaction progression was routinely monitored by
thin-layer chromatography (TLC) on Merck 9385 pre-coated aluminum plate silica gel (Kieselgel 60) 5 x
20 cm plates with a layer thickness of 0.2 mm, and spots were visualized by exposure to UV-lamp at
λ =
254 nm. Melting points were determined on Stuart electro-thermal melting point apparatus and were
uncorrected. IR spectra were recorded on Nicolet iS5 (ATR) FT-IR spectrometer, Faculty of Pharmacy,
1
Minia University. H NMR spectra were carried out using Bruker apparatus 400 MHz spectrometer,
Faculty of Pharmaceutical sciences, The University of British Columbia, Canada, using DMSO-d (2.5)
6
13
as a solvent. C NMR spectra were carried out using Bruker apparatus 100 MHz spectrometer, Faculty
of Science, Western University, Canada, using DMSO-d (39.51) as a solvent. High resolution mass
6
spectra (HRMS) were obtained on a Thermo Scientific Q Exactive™ Orbitrap mass spectrometer,
Faculty of Pharmaceutical sciences, The University of British Columbia, Canada.
1
2

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4.1.1.
3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione 2 [87]. Yellowish white
solid (1.44 g, 66.67% yield); mp 239-41 °C; reported 241-2.5 °C.
4.1.2.
General procedure for the synthesis of compounds 3a-f. To a solution of the spirohydantoin
2
(0.01 mol, 2.16 g) in dry acetone (90 mL), anhydrous potassium carbonate (0.02 mol, 2.76 g) and the
appropriate bromoester (0.01 mol) were added. The reaction mixture was stirred under reflux for 6 h, the
solvent was then distilled off under reduced pressure, and the reaction mixture was diluted with ice
water. The precipitate formed was filtered off, washed with water, and dried to give the corresponding
ester.
4.1.2.1.
Ethyl 2-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl) acetate 3a
[
88]. Reaction of 2 with ethyl bromoacetate yielded pale yellow powder (1.96 g, 64.90% yield); mp 130-
2
4
3
2
°C; reported 133-135°C.
.1.2.2.
Ethyl 3-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl) propionate
b. Reaction of 2 with ethyl 3-bromopropionate yielded pale yellow powder (1.92 g, 60.76%); mp 161-
-1
1
°C; IR (cm ): 3317 (NH), 1771 (C=O), 1751 (C=O), 1704 (C=O), 1190 (C-O); H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.18 (t, 3H, J = 7.20 Hz, CH -CH ), 1.82-1.93 (m, 2H, CH -cyclohexane), 2.06 (t,
6
2
3
2
2
H, J = 8.01 Hz, CH -cyclohexane), 2.64 (t, 2H, J = 6.80 Hz, CH -CO), 2.79 (t, 2H, J = 8.01 Hz, CH -
2
2
2
cyclohexane), 3.69 (t, 2H, J = 6.80 Hz, N-CH ), 4.05 (q, 2H, J = 7.20 Hz, O-CH -CH ), 7.04 (d, 1H, J =
2
2
3
8.00 Hz, Ar-H), 7.17 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.24 (d, 1H, J = 8.00 Hz, Ar-H), 8.86 (s, 1H, NH);
+
HRMS: m/z calculated for C H N O [M-H] : 315.13503, found: 315.13538.
1
7
20
2
4
4.1.2.3.
Ethyl 4-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl) butyrate 3c.
Reaction of 2 with ethyl 4-bromobutyrate yielded pale yellow powder (2.24 g, 67.87%); mp 172-4°C; IR
-1
1
(
cm ): 3309 (NH), 1771 (C=O), 1748 (C=O), 1707 (C=O), 1190 (C-O); H NMR (400 MHz, DMSO-d )
6
δ (ppm): 1.19 (t, 3H, J = 7.20 Hz, CH -CH ), 1.80-1.84 (m, 2H, CH -CH -CO), 1.92-1.94 (m, 2H, CH -
2
3
2
2
2
cyclohexane), 2.08 (t, 2H, J = 7.20 Hz, CH -cyclohexane), 2.34 (t, 2H, J = 6.80 Hz, CH -CO), 2.79 (t,
2
2
2
H, J = 7.20 Hz, CH -cyclohexane), 3.47 (t, 2H, J = 6.80 Hz, N-CH ), 4.06 (q, 2H, J = 7.20 Hz, O-CH -
2
2
2
1
3

ACCEPTED MANUSCRIPT
CH ), 6.99 (d, 1H, J = 8.00 Hz, Ar-H), 7.19 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.24 (d, 1H, J = 8.00 Hz, Ar-
3
+
H), 8.83 (br s, 1H, NH); HRMS: m/z calculated for C H N O [M-H] : 329.15068, found: 329.15054.
1
8
22
2
4
4.1.2.4.
Ethyl 5-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl) pentanoate
3d. Reaction of 2 with ethyl 5-bromovalerate yielded pale yellow powder (2.12 g, 61.62%); mp 156-7°C;
-1
1
IR (cm ): 3306 (NH), 1771 (C=O), 1753 (C=O), 1700 (C=O), 1207 (C-O); H NMR (400 MHz, DMSO-
d ) δ (ppm): 1.17 (t, 3H, J = 6.80 Hz, CH -CH ), 1.52-1.57 (m, 4H, CH -CH -CH -CO), 1.82-1.94 (m,
6
2
3
2
2
2
2
2
H, CH -cyclohexane), 2.07 (t, 2H, J = 6.40 Hz, CH -cyclohexane), 2.34 (t, 2H, J = 7.20 Hz, CH -CO),
2 2 2
.79 (t, 2H, J = 6.40 Hz, CH -cyclohexane), 3.43 (t, 2H, J = 7.20 Hz, N-CH ), 4.05 (q, 2H, J = 6.80 Hz,
2
2
O-CH -CH ), 6.99 (d, 1H, J = 8.00 Hz, Ar-H), 7.18 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.25 (d, 1H, J = 8.00
2
3
+
Hz, Ar-H), 8.83 (br s, 1H, NH); HRMS: m/z calculated for C H N O [M-H] : 343.16633, found:
1
9
24
2
4
3
4
3
5
43.16595.
.1.2.5.
Ethyl 6-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl) hexanoate
e. Reaction of 2 with ethyl 6-bromohexanoate yielded pale yellow powder (2.20 g, 61.45%); mp 143-
-1
1
°C; IR ν (cm ): 3306 (NH), 1771 (C=O), 1753 (C=O), 1700 (C=O), 1177 (C-O); H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.16 (t, 3H, J = 7.20 Hz, CH -CH ), 1.25-1.30 (m, 2H, CH -CH -CH -CO), 1.56-
6
2
3
2
2
2
1
.59 (m, 4H, CH -CH -CH -CH -CO), 1.80-1.93 (m, 2H, CH -cyclohexane), 2.08 (t, 2H, J = 6.40 Hz,
2 2 2 2 2
CH -cyclohexane), 2.29 (t, 2H, J = 7.20 Hz, CH -CO), 2.79 (t, 2H, J = 6.40 Hz, CH -cyclohexane), 3.41
2
2
2
(
t, 2H, J = 7.20 Hz, N-CH ), 4.06 (q, 2H, J = 7.20 Hz, O-CH -CH ), 6.97 (d, 1H, J = 7.20 Hz, Ar-H),
2 2 3
7.19 (d, 2H, J = 7.20 Hz, 2 Ar-H), 7.24 (d, 1H, J = 7.20 Hz, Ar-H), 8.80 (br s, 1H, NH); HRMS: m/z
+
calculated for C H N O [M-H] : 357.18198, found: 357.18216.
2
0
26
2
4
4
3
3
.1.2.6.
Ethyl 7-(2,5-dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl) heptanoate
f. Reaction of 2 with ethyl 7-bromoheptanoate yielded pale yellow powder (2.30 g, 61.82%); mp 162-
-1
1
°C; IR ν (cm ): 3306 (NH), 1772 (C=O), 1751 (C=O), 1703 (C=O), 1189 (C-O); H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.15-1.17 (m, 2H, CH -CH -CH -CO), 1.20 (t, 3H, J = 6.80 Hz, CH -CH ), 1.25-
6
2
2
2
2
3
1
.29 (m, 2H, N-CH -CH -CH ), 1.56-1.59 (m, 4H, CH -CH -CH -CH -CH -CO), 1.82-1.93 (m, 2H,
2 2 2 2 2 2 2 2
1
4

ACCEPTED MANUSCRIPT
CH -cyclohexane), 2.08 (t, 2H, J = 7.20 Hz, CH -cyclohexane), 2.28 (t, 2H, J = 7.20 Hz, CH -CO), 2.79
2
2
2
(
t, 2H, J = 7.20 Hz, CH -cyclohexane), 3.41 (t, 2H, J = 7.20 Hz, N-CH ), 4.06 (q, 2H, J = 6.80 Hz, O-
2
2
CH -CH ), 6.97 (d, 1H, J = 7.80 Hz, Ar-H), 7.20 (d, 2H, J = 7.80 Hz, 2 Ar-H), 7.24 (d, 1H, J = 7.80 Hz,
2
3
+
Ar-H), 8.82 (br s, 1H, NH); HRMS: m/z calculated for C H N O [M-H] : 371.19887, found:
2
1
28
2
4
371.19977.
4.1.3.
General procedure for the synthesis of compounds 4a-f
General procedure for the synthesis of compounds 4a-f [89]
.1.3.1. 2-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)acetic acid 4a [88]
Yellowish white powder (3.50 g, 51.10% yield); mp 228-29°C; reported 231-3°C.
.1.3.2. 3-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)propionic acid 4b.
.
4
.
4
-1
Yellowish white powder (3.70 g, 51.40% yield); mp 240-1°C; IR ν (cm ): 3675-2570 (OH), 3306 (NH),
1
1
769 (C=O), 1706 (C=O), 1690 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.83-1.93 (m, 2H,
6
CH -cyclohexane), 2.06 (t, 2H, J = 7.20 Hz, CH -cyclohexane), 2.58 (t, 2H, J = 8.01 Hz, CH -CO), 2.79
2
2
2
(
t, 2H, J = 7.20 Hz, CH -cyclohexane), 3.66 (t, 2H, J = 8.01 Hz, N-CH ), 7.06 (d, 1H, J = 7.80 Hz, Ar-
2
2
H), 7.18 (d, 2H, J = 7.80 Hz, 2 Ar-H), 7.23 (d, 1H, J = 7.80 Hz, Ar-H), 8.84 (s, 1H, NH), 12.39 (br s, 1H,
1
3
OH); C NMR (100 MHz, DMSO-d ) δ (ppm): 176.4, 162.3, 155.9, 138.2, 134.5., 129.6, 128.5, 127.3,
6
+
1
26.9, 62.3, 34.6, 33.9, 32.5, 28.9, 18.8; HRMS: m/z calculated for C H N O [M-H] : 287.10373,
1
5
16
2
4
found: 287.10397.
4.1.3.3.
4-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)butyric acid 4c.
-1
Yellowish white powder (3.85 g, 51% yield); mp 249-51°C; IR ν (cm ): 3670-2550 (OH), 3322 (NH),
1
1
765 (C=O), 1705 (C=O), 1690 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.81-1.85 (m, 2H,
6
CH -CH -CO), 1.89-1.94 (m, 2H, CH -cyclohexane), 2.09 (t, 2H, J = 7.20 Hz, CH -cyclohexane), 2.26
2
2
2
2
(
t, 2H, J = 6.80 Hz, CH -CO), 2.79 (t, 2H, J = 7.20 Hz, CH -cyclohexane), 3.46 (t, 2H, J = 6.80 Hz, N-
2
2
CH ), 6.99 (d, 1H, J = 6.40 Hz, Ar-H), 7.19 (d, 2H, J = 6.40 Hz, 2 Ar-H), 7.25 (d, 1H, J = 6.40 Hz, Ar-
2
13
H), 8.83 (s, 1H, NH),12.11 (br s, 1H, OH); C NMR (100 MHz, DMSO-d ) δ (ppm): 176.7, 174.1,
6
1
5

ACCEPTED MANUSCRIPT
156.3, 138.3, 134.5, 129.7, 128.5, 127.0, 126.9, 62.3, 37.8, 33.9, 31.3, 28.9, 23.6, 18.8; HRMS: m/z
+
calculated for C H N O [M-H] : 301.11938, found: 301.11938.
1
6
18
2
4
4.1.3.4.
5-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)pentanoic acid 4d.
-1
Yellowish white powder (4.02 g, 50.88% yield); mp 260-1°C; IR (cm ): 3665-2560 (OH), 3400 (NH),
1
1
766 (C=O), 1711 (C=O), 1700 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.56-1.58 (m, 4H,
6
CH -CH -CH -CO), 1.88-1.93 (m, 2H, CH -cyclohexane), 2.08 (t, 2H, J = 6.80 Hz, CH -cyclohexane),
2
2
2
2
2
2
.26 (t, 2H, J = 7.80 Hz, CH -CO), 2.78 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 3.42 (t, 2H, J = 7.80
2
2
Hz, N-CH ), 6.99 (d, 1H, J = 8.00 Hz, Ar-H), 7.18 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.24 (d, 1H, J = 8.00
2
1
3
Hz, Ar-H), 8.83 (s, 1H, NH), 12.03 (br s, 1H, OH); C NMR (100 MHz, DMSO-d ) δ (ppm): 177.9,
6
174.3, 157.5, 139.5, 135.8, 131.0, 129.7, 128.2, 63.5, 39.1, 35.3, 34.5, 30.1, 28.7, 23.3, 15.8; HRMS: m/z
+
calculated for C H N O [M-H] : 315.13503, found: 315.13531.
1
7
20
2
4
4.1.3.5.
6-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)hexanoic acid 4e.
-1
Yellowish white powder (4.20 g, 50.90% yield); mp 275-7°C; IR (cm ): 3678-2575 (OH), 3348 (NH),
1
1
764 (C=O), 1711 (C=O), 1698 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.24-1.29 (m, 2H,
6
CH -CH -CH -CO), 1.51-1.59 (m, 4H, CH -CH -CH -CH -CO), 1.82-1.94 (m, 2H, CH -cyclohexane),
2
2
2
2
2
2
2
2
2
.07 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 2.21 (t, 2H, J = 7.80 Hz, CH -CO), 2.78 (t, 2H, J = 6.80
2
2
Hz, CH -cyclohexane), 3.42 (t, 2H, J = 7.20 Hz, N-CH ), 6.98 (d, 1H, J = 7.80 Hz, Ar-H), 7.20 (d, 2H,
2
2
1
3
J = 7.80 Hz, 2 Ar-H), 7.24 (d, 1H, J = 7.80 Hz, Ar-H), 8.82 (s, 1H, NH), 11.94 (br s, 1H, OH); C NMR
(
100 MHz, DMSO-d ) δ (ppm): 176.7, 174.7, 156.3, 138.2, 134.5, 129.7, 128.4, 127.3, 126.9, 62.2, 38.1,
6
+
3
4.0, 33.9, 28.9, 27.7, 26.1, 24.5, 18.9; HRMS: m/z calculated for C H N O [M-H] : 329.15068,
1
8
22
2
4
found: 329.15088.
4.1.3.6.
7-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)heptanoic acid 4f.
-1
Yellowish white powder (4.43 g, 51.51% yield); mp 284-6°C; IR (cm ): 3675-2550 (OH), 3335 (NH),
1
1
769 (C=O), 1708 (C=O), 1698 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.18-1.22 (m, 2H,
6
CH -CH -CH -CO), 1.27-1.30 (m, 2H, N-CH -CH -CH ), 1.52-1.59 (m, 4H, CH -CH -CH -CH -CH -
2
2
2
2
2
2
2
2
2
2
2
1
6

ACCEPTED MANUSCRIPT
CO), 1.82-1.94 (m, 2H, CH -cyclohexane), 2.08 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 2.23 (t, 2H, J =
2
2
7
.20 Hz, CH -CO), 2.78 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 3.41 (t, 2H, J = 7.20 Hz, N-CH ), 6.97
2
2
2
(d, 1H, J = 7.20 Hz, Ar-H), 7.19 (d, 2H, J = 7.20 Hz, 2 Ar-H), 7.25 (d, 1H, J = 7.20 Hz, Ar-H) 8.83 (s,
1
3
1
H, NH), 11.96 (br s, 1H, OH); C NMR (100 MHz, DMSO-d ) δ (ppm): 177.9, 174.2, 156.3, 138.2,
6
135.2, 129.7, 128.4, 126.9 126.8, 62.2, 38.2, 34.0, 28.8, 28.5, 27.8, 26.2, 25.6, 25.5, 18.6; HRMS: m/z
+
calculated for C H N O [M-H] : 343.17088, found: 343.17090.
1
9
24
2
4
4
.1.4.
in tetrahydrofuran (THF, 10 mL), N,N'-carbonyldiimidazole (CDI, 0.04 mol, 6.48 g) was added at 25-
0°C and stirred for 1 h. Hydroxylamine hydrochloride (0.04 mol, 2.78 g) was added, and the stirring
General procedure for the synthesis of compounds 5a-f. The acid 4 (0.01 mol) was dissolved
3
was continued for another 1 h. The solvent was distilled off, ethylacetate (10 mL) was added, washed
with water (2 x 10 mL), and the organic layer was collected, dried over anhydrous sodium sulphate,
filtered, and evaporated under vacuum. The product was collected as oily droplets.
4.1.4.1.
2-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-
-1
hydroxyacetamide 5a. Yellowish brown oil (1.46 g, 50.52% yield); IR (cm ): 3401 (OH), 3360 (NH),
1
3
306 (NH), 1777 (C=O), 1706 (C=O), 1698 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.95-1.97
6
(
m, 2H, CH -cyclohexane), 2.09 (t, 2H, J = 7.20 Hz, CH -cyclohexane), 2.79 (t, 2H, J = 7.20 Hz, CH -
2
2
2
cyclohexane), 4.14 (s, 2H, N- CH -CO), 7.07 (d, 1H, J = 8.00 Hz, Ar-H), 7.19 (d, 2H, J = 8.00 Hz, 2 Ar-
2
1
3
H), 7.25 (d, 1H, J = 8.00 Hz, Ar-H), 8.99 (s, 1H, NH), 11.20 (br s, 2H, NHOH); C NMR (100 MHz,
DMSO-d ) δ (ppm): 176.4, 169.3, 155.5, 138.2, 134.4, 129.6, 128.5, 127.6, 126.9, 62.8, 40.8, 34.0, 28.8,
6
+
1
8.8; HRMS: m/z calculated for C H N O [M-H] : 288.10923, found: 288.10935.
1
4
15
3
4
4.1.4.2.
3-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-
-1
hydroxypropionamide 5b. Yellowish brown oil (1.51 g, 49.83% yield); IR (cm ): 3400 (OH), 3362
1
(
NH), 3305 (NH), 1778 (C=O), 1705 (C=O), 1697 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm):
6
1
.85-1.93 (m, 2H, CH -cyclohexane), 2.07 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 2.58 (t, 2H, J = 7.20
2
2
Hz, CH -CO), 2.78 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 3.66 (t, 2H, J = 7.20 Hz, N-CH ), 7.06 (d,
2
2
2
1
7

ACCEPTED MANUSCRIPT
1
1
1
H, J = 6.40 Hz, Ar-H), 7.17 (d, 2H, , J = 6.40 Hz, 2 Ar-H), 7.23 (d, 1H, , J = 6.40 Hz, Ar-H), 8.84 (s,
1
3
H, NH), 11.50 (br s, 2H, NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 176.4, 169.7, 155.9, 138.2,
6
34.5., 129.6, 128.5, 127.3, 126.9, 63.5, 34.6, 33.9, 32.5, 28.9, 18.8;
HRMS: m/z calculated
+
for C H N O [M-H] : 302.11963, found: 302.11985.
1
5
17
3
4
4.1.4.3.
4-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-
-1
hydroxybutyramide 5c. Pale brown oil (1.53 g, 48.26% yield); IR (cm ): 3402 (OH), 3365 (NH), 3302
1
(
NH), 1777 (C=O), 1707 (C=O), 1697 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.81-1.85 (m,
6
2H, CH -CH -CO), 1.89-1.94 (m, 2H, CH -cyclohexane), 2.09 (t, 2H, J = 6.80 Hz, CH -cyclohexane),
2 2 2 2
2
.26 (t, 2H, J = 7.20 Hz, CH -CO), 2.80 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 3.46 (t, 2H, J = 7.20 Hz,
2
2
N-CH ), 6.99 (d, 1H, J = 8.00 Hz, Ar-H), 7.19 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.25 (d, 1H, J = 8.00 Hz,
2
1
3
Ar-H), 8.83 (s, 1H, NH), 11.30 (br s, 2H, NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 174.1,
6
162.3, 156.3, 138.3, 134.5, 129.7, 128.5, 127.0, 126.9, 62.4, 37.8, 33.9, 31.4, 28.9, 23.6, 18.8; HRMS:
+
m/z calculated for C H N O [M-H] : 316.13811, found: 316.13902.
1
6
19
3
4
4.1.4.4.
5-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-
-1
hydroxypentanamide 5d. Pale brown oil (1.71 g, 51.66% yield); IR (cm ): 3404 (OH), 3367 (NH), 3306
1
(
NH), 1778 (C=O), 1707 (C=O), 1699 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.56-1.58 (m,
6
4
H, CH -CH -CH -CO), 1.88-1.93 (m, 2H, CH -cyclohexane), 2.07 (t, 2H, J = 6.80 Hz, CH2-
2 2 2 2
cyclohexane), 2.26 (t, 2H, J = 7.20 Hz, CH -CO), 2.79 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 3.43 (t,
2
2
2
1
H, J = 7.20 Hz, N-CH ), 6.99 (d, 1H, J = 8.00 Hz, Ar-H), 7.18 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.24 (d,
2
1
3
H, J = 8.00 Hz, Ar-H), 8.83 (s, 1H, NH), 11.25 (br s, 2H, NHOH); C NMR (100MHz, DMSO-d ) δ
6
(ppm): 174.4, 169.3, 157.4, 139.5, 135.7, 131.0, 129.7, 128.2, 63.5, 38.9, 35.3, 34.5, 30.1, 28.8, 23.3,
+
1
5.9; HRMS: m/z calculated for C H N O [M-H] : 330.14883, found: 330.14903.
1
7
21
3
4
4.1.4.5.
6-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-
-1
hydroxyhexanamide 5e. Pale brown oil (1.75 g, 50.72% yield); IR (cm ): 3407 (OH), 3367 (NH), 3306
1
(
NH), 1778 (C=O), 1708 (C=O), 1699 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.26-1.29 (m,
6
1
8

ACCEPTED MANUSCRIPT
2
2
H, CH -CH -CH -CO), 1.53-1.60 (m, 4H, CH -CH -CH -CH -CO), 1.82-1.94 (m, CH -cyclohexane),
2
2
2
2
2
2
2
2
.07 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 2.26 (t, 2H, J = 7.20 Hz, CH -CO), 2.79 (t, 2H, J = 6.80
2
2
Hz, CH -cyclohexane), 3.41 (t, 2H, J = 7.20 Hz, N-CH ), 6.97 (d, 1H, J = 7.00 Hz, Ar-H), 7.19 (d, 2H, J
2
2
1
3
=
7.00 Hz, 2 Ar-H), 7.23 (d, 1H, J = 7.00 Hz, Ar-H), 8.82 (s, 1H, NH), 11.46 (br s, 2H, NHOH); C
NMR (100 MHz, DMSO-d ) δ (ppm): 174.7, 169.4, 156.5, 138.4, 134.5, 129.7, 128.4, 127.3, 126.9,
6
+
6
3
4
2.3, 38.3, 34.0, 33.9, 28.8, 27.7, 26.1, 24.3, 18.9; HRMS: m/z calculated for C H N O [M-H] :
1
8
23
3
4
44.16158, found: 344.16301.
.1.4.6. 7-(2,5-Dioxo-3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalen]-1-yl)-N-
-1
hydroxyheptanamide 5f. Pale brown oil (4.43 g, 51.51% yield); IR (cm ): 3402 (OH), 3365 (NH), 3302
1
(
NH), 1777 (C=O), 1707 (C=O), 1697 (C=O); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.19-1.22 (m,
6
2
H, CH -CH -CH -CO), 1.27-1.30 (m, 2H, N-CH -CH -CH ), 1.52-1.60 (m, 4H, CH -CH -CH -CH -
2 2 2 2 2 2 2 2 2 2
CH -CO), 1.82-1.94 (m, 2H, CH -cyclohexane), 2.07 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 2.21 (t,
2
2
2
2
H, J = 7.20 Hz, CH -CO), 2.78 (t, 2H, J = 6.80 Hz, CH -cyclohexane), 3.42 (t, 2H, J = 7.20 Hz, N-
2
2
CH ), 6.98 (d, 1H, J = 6.40 Hz, Ar-H), 7.20 (d, 2H, J = 6.40 Hz, 2 Ar-H), 7.24 (d, 1H, J = 6.40 Hz, Ar-
2
1
3
H) 8.82 (s, 1H, NH), 11.17 (br s, 2H, NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 176.7, 169.7,
6
156.3, 138.3, 135.1, 129.7, 128.4, 126.9 126.8, 62.2, 38.1, 34.0, 28.8, 28.5, 27.8, 26.3, 25.6, 25.5, 18.8;
+
HRMS: m/z calculated for C H N O [M-H] : 358.17723, found: 358.17920.
1
9
25
3
4
4
.1.5.
Orange solid (3.50 g, 70.01% yield); mp 88-90 °C; reported 90-91 °C.
.1.6. General procedure for the synthesis of compounds 9a-e. A mixture of 8 (0.01 mol, 3.85 g),
4-(2-(3-methoxyphenyl)hydrazono)-2-(3,4,5-trimethoxyphenyl)oxazol-5(4H)-one 8 [89].
4
the appropriate amino acid (0.012 mol), and anhydrous sodium acetate (0.75 g) were dissolved in glacial
acetic acid (25 mL). The reaction mixture was stirred under reflux for 2 h, and was left to cool, and was
diluted with ice water. The precipitate formed was filtered off, washed with water, and re-crystallized
from aqueous methanol.
1
9

ACCEPTED MANUSCRIPT
4.1.6.1.
2-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)acetic
-
acid 9a. Reaction of 8 with glycine yielded pale yellow powder (2.10 g, 47.50%); mp 177-8°C; IR (cm
1
1
)
: 3675-2595 (OH), 3302 (NH), 1701 (C=O), 1669 (C=O), 1589 (C=N); H NMR (400 MHz, DMSO-
d ) δ (ppm): 3.61 (s, 6H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 3.95 (d, 2H, J = 7.20 Hz,
6
3
3
3
HN-CH -CO), 6.81 (s, 2H, 2 Ar-H), 7.07 (t, 1H, J = 7.80 Hz, Ar-H), 7.16 (d, 2H, J = 7.80 Hz, 2 Ar-H),
2
1
3
7
.48 (s, 1H, Ar-H), 8.87 (t, 1H, J = 7.20 Hz, NH), 12.66 (br s, 1H, OH); C NMR (100 MHz, DMSO-d )
6
δ (ppm): 171.3, 162.3, 160.3, 159.3, 156.1, 154.8, 153.1, 139.6, 139.1, 130.8, 122.3, 118.8, 118.1, 116.1,
+
1
12.4, 106.8, 60.6, 56.4, 56.2, 56.1, 41.4; HRMS: m/z calculated for C H N O [M-H] : 441.14157,
21
22
4
7
found: 441.14221.
4.1.6.2.
3-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamido)propionic acid 9b. Reaction of 8 with β-alanine yielded yellowish white powder (2.25 g,
-1
4
9.34%); mp 189-91°C; IR (cm ): 3640-2540 (OH), 3301 (NH), 1698 (C=O), 1669 (C=O), 1592 (C=N);
1
H NMR (400 MHz, DMSO-d ) δ (ppm): 2.54 (t, 2H, J = 6.80 Hz, CH -CO), 3.48-3.53 (m, 2H, HN-
6
2
CH ), 3.61 (s, 6H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.77 (s, 3H, OCH ), 6.80 (s, 2H, 2 Ar-H), 7.05 (t, 1H, J
2
3
3
3
=
7.80 Hz, Ar-H), 7.14 (d, 2H, J = 7.80 Hz, 2 Ar-H), 7.47 (s, 1H, Ar-H), 8.65 (t, 1H, J = 6.80 Hz, NH),
1
3
1
2.30 (br s, 1H, OH); C NMR (100 MHz, DMSO-d ) δ (ppm): 173.3, 160.3, 159.0, 156.4, 154.7, 153.1,
6
139.6, 139.1, 130.8, 122.3, 118.8, 116.1, 112.4, 106.8, 60.6, 56.2, 56.1, 35.4, 34.1; HRMS: m/z
+
calculated for C H N O [M-H] : 455.15722, found: 455.15796.
2
2
24
4
7
4.1.6.3.
4-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamido)butyric acid 9c. Reaction of 8 with 4-aminobutyric acid yielded yellowish white powder
-1
(
2.30 g, 48.93%); mp 198-200°C; IR (cm ): 3640-2550 (OH), 3303 (NH), 1700 (C=O), 1667 (C=O),
1
1
588 (C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.77-1.83 (m, 2H, CH -CH -CO), 2.55 (t, 2H, J
6
2
2
=
6.80 Hz, CH -CO), 3.55-3.58 (m, 2H, HN-CH ), 3.62 (s, 6H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.79 (s,
2 2 3 3
3
7
H, OCH ), 6.83 (s, 2H, 2 Ar-H), 7.05 (t, 1H, J = 8.00 Hz, Ar-H), 7.16 (d, 2H, J = 8.00 Hz, 2 Ar-H),
3
1
3
.47 (s, 1H, Ar-H), 8.63 (t, 1H, J = 6.80 Hz, NH), 12.39 (br s, 1H, OH); C NMR (100 MHz, DMSO-d )
6
2
0

ACCEPTED MANUSCRIPT
δ (ppm): 174.7, 160.3, 159.2, 156.6, 154.6, 153.1, 139.5, 139.2, 130.8, 122.4, 118.8, 116.1, 112.4, 106.8,
+
6
4
4
0.6, 56.2, 56.1, 38.6, 31.6, 25.0; HRMS: m/z calculated for C H N O [M-H] : 469.17287, found:
2
3
26
4
7
69.17322.
.1.6.4.
5-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamido)pentanoic acid 9d. Reaction of 8 with 5-aminovaleric acid yielded yellowish white powder
-1
(
2.61 g, 53.73%); mp 211-3°C; IR (cm ): 3672-2569 (OH), 3299 (NH), 1701 (C=O), 1659 (C=O), 1594
1
(
C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.54-1.59 (m, 4H, CH -CH -CH -CO), 2.34 (t, 2H, J =
6
2
2
2
6
.80 Hz, CH -CO), 3.48-3.52 (m, 2H, HN-CH ), 3.61 (s, 6H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.77 (s, 3H,
2 2 3 3
OCH ), 6.80 (s, 2H, 2 Ar-H), 7.05 (t, 1H, J = 7.20 Hz, Ar-H), 7.15 (d, 2H, J = 7.20 Hz, 2 Ar-H), 7.47 (s,
3
13
1
H, Ar-H), 8.66 (t, 1H, J = 6.80 Hz, NH), 12.44 (br s, 1H, OH); C NMR (100 MHz, DMSO-d ) δ
6
(ppm): 174.8, 162.3, 160.3, 159.0, 156.6, 154.6, 153.1, 139.5, 139.2, 130.8, 122.4, 118.8, 116.0, 112.4,
+
1
4
4
06.9, 60.6, 56.2, 56.1, 38.7, 33.8, 29.0, 22.3; HRMS: m/z calculated for C H N O [M-H] :
2
4
28
4
7
83.19157, found: 483.19215.
.1.6.5. 6-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-
carboxamido)hexanoic acid 9e. Reaction of 8 with 6-aminohexanoic acid yielded pale yellow powder
-1
(
2.35 g, 47.20%); mp 223-5°C; IR (cm ): 3629-2570 (OH), 3303 (NH), 1700 (C=O), 1666 (C=O), 1598
1
(
C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.23-1.26 (m, 2H, CH -CH -CH -CO), 1.54-1.62 (m,
6
2
2
2
4
6
H, CH -CH -CH -CH -CO), 2.58 (t, 2H, J = 6.20 Hz, CH -CO), 3.56-3.58 (m, 2H, HN-CH ), 3.61 (s,
2 2 2 2 2 2
H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 6.81 (s, 2H, 2 Ar-H), 7.07 (t, 1H, J = 8.00 Hz,
3
3
3
Ar-H), 7.16 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.48 (s, 1H, Ar-H), 8.87 (t, 1H, J = 6.40 Hz, NH), 12.51 (br s,
1
3
1
1
2
4
H, OH); C NMR (100 MHz, DMSO-d ) δ (ppm): 174.9, 162.3, 160.3, 159.0, 156.6, 154.6, 153.1,
6
39.5, 139.2, 130.8, 128.6, 122.4, 116.0, 112.4, 111.9, 106.8, 60.6, 56.3, 56.2, 56.1, 38.5, 34.0, 29.3,
+
6.4, 24.7; HRMS: m/z calculated for C H N O [M-H] : 497.20417, found: 497.20513.
2
5
30
4
7
.1.7.
General procedure for the synthesis of compounds 10a-e. The acid 9 (0.01 mol) was
dissolved in tetrahydrofuran (THF, 10 mL), N,N'-carbonyldiimidazole (CDI) (0.04 mol, 6.48 g) was
2
1

ACCEPTED MANUSCRIPT
added at 25-30°C and stirred for 1 h. Hydroxylamine hydrochloride (0.04 mol, 2.78 g) was added, and
the stirring was continued for another 1 h. The solvent was distilled off, ethylacetate (10 mL) was added,
washed with water (2 x 10 mL), and the organic layer was collected, dried over anhydrous sodium
sulphate, filtered, and evaporated under vacuum. The product was collected as oily droplets.
4.1.7.1.
2-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)-N-
-1
hydroxylacetamide 10a. Pale brown oil (2.24 g, 49.01% yield); IR (cm ): 3402 (OH), 3365 (NH), 3302
1
(
NH), 1707 (C=O), 1697 (C=O), 1589 (C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 3.61 (s, 6H, 2
6
OCH ), 3.69 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 3.95 (d, 2H, J = 7.20 Hz, HN-CH -CO), 6.81 (s, 2H, 2
3
3
3
2
Ar-H), 7.07 (t, 1H, J = 8.00 Hz, Ar-H), 7.16 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.48 (s, 1H, Ar-H), 8.87 (t,
1
3
1
1
5
4
H, J = 7.20 Hz, NH), 11.51 (br s, 2H, NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 169.3, 162.3,
6
60.3, 159.2, 156.1, 154.8, 153.1, 139.6, 139.2, 130.8, 122.4, 118.8, 118.1, 116.1, 112.4, 106.8, 60.6,
+
6.4, 56.2, 56.1, 41.4; HRMS: m/z calculated for C H N O [M-H] : 456.15247, found: 456.15311.
21
23
5
7
.1.7.2.
3-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)-N-
-1
hydroxypropionamide 10b. Pale brown oil (2.31 g, 49.04% yield); IR ν (cm ): 3404 (OH), 3366 (NH),
1
3
302 (NH), 1706 (C=O), 1698 (C=O), 1592 (C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 2.53 (t,
6
2
H, J = 6.80 Hz, CH -CO), 3.51-3.56 (m, 2H, HN-CH ), 3.61 (s, 6H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.78
2
2
3
3
(
s, 3H, OCH ), 6.80 (s, 2H, 2 Ar-H), 7.05 (t, J = 8.00 Hz, 1H, Ar-H), 7.14 (d, 2H, J = 8.00 Hz, 2 Ar-H),
3
1
3
7
.47 (s, 1H, Ar-H), 8.66 (t, 1H, J = 6.80 Hz, NH), 11.46 (br s, 2H, NHOH); C NMR (100 MHz,
DMSO-d ) δ (ppm): 169.6, 160.3, 159.0, 156.4, 154.7, 153.1, 139.6, 139.1, 130.8, 122.3, 118.8, 116.1,
6
+
1
12.4, 106.8, 60.6, 56.2, 56.1, 35.4, 34.1; HRMS: m/z calculated for C H N O [M-H] : 470.18124,
22
25
5
7
found: 470.18213.
4.1.7.3.
4-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)-N-
-1
hydroxybutyramide 10c. Brown oil (2.42 g, 49.89% yield); IR ν (cm ): 3402 (OH), 3366 (NH), 3301
1
(
NH), 1705 (C=O), 1699 (C=O), 1588 (C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.77-1.83 (m,
6
2
H, CH -CH -CO), 2.55 (t, 2H, J = 6.80 Hz, CH -CO), 3.55-3.58 (m, 2H, HN-CH ), 3.62 (s, 6H, 2
2 2 2 2
2
2

ACCEPTED MANUSCRIPT
OCH ), 3.69 (s, 3H, OCH ), 3.79 (s, 3H, OCH ), 6.83 (s, 2H, 2 Ar-H), 7.05 (t, 1H, J = 8.00 Hz, Ar-H),
3
3
3
7.16 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.47 (s, 1H, Ar-H), 8.63 (t, 1H, J = 6.80 Hz, NH), 11.30 (br s, 2H,
1
3
NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 169.3, 162.3, 160.3, 159.2, 156.6, 154.6, 153.1,
6
139.6, 139.2, 130.8, 122.4, 118.8, 116.1, 112.4, 106.8, 60.6, 56.2, 56.1, 38.6, 30.6, 24.8; HRMS: m/z
+
calculated for C H N O [M-H] : 484.19117, found: 484.19194.
2
3
27
5
7
4.1.7.4.
5-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)-N-
-1
hydroxypentanamide 10d. Brown oil (2.61 g, 52.3% yield); IR ν (cm ): 3402 (OH), 3365 (NH), 3302
1
(
NH), 1707 (C=O), 1697 (C=O), 1594 (C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.54-1.59 (m,
6
4
H, CH -CH -CH -CO), 2.34 (t, 2H, J = 6.80 Hz, CH -CO), 3.48-3.52 (m, 2H, HN-CH2), 3.61 (s, 6H, 2
2 2 2 2
OCH ), 3.69 (s, 3H, OCH ), 3.77 (s, 3H, OCH ), 6.80 (s, 2H, 2 Ar-H), 7.05 (t, 1H, J = 8.00 Hz, Ar-H),
3
3
3
7.15 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.47 (s, 1H, Ar-H), 8.65 (t, 1H, J = 6.80 Hz, NH), 11.22 (br s, 2H,
1
3
NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 167.0, 162.3, 160.3, 159.0, 156.6, 154.6, 153.1,
6
139.5, 139.2, 130.8, 122.4, 118.8, 116.0, 112.4, 106.9, 60.6, 56.2, 56.1, 38.7, 32.8, 29.0, 22.3; HRMS:
+
m/z calculated for C H N O [M-H] : 498.19882, found: 498.19916.
2
4
29
5
7
4.1.7.5.
6-(1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamido)-N-
-1
hydroxyhexanamide 10e. Pale brown oil (2.57 g, 50.09% yield); IR (cm ): 3405 (OH), 3365 (NH), 3306
1
(
NH), 1707 (C=O), 1699 (C=O), 1598 (C=N); H NMR (400 MHz, DMSO-d ) δ (ppm): 1.23-1.26 (m,
6
2
H, CH -CH -CH -CO), 1.54-1.62 (m, 4H, CH -CH -CH -CH -CO), 2.58 (t, 2H, J = 6.20 Hz, CH -CO),
2 2 2 2 2 2 2 2
3
.56-3.58 (m, 2H, HN-CH ), 3.61 (s, 6H, 2 OCH ), 3.69 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 6.81 (s, 2H,
2 3 3 3
2
Ar-H), 7.07 (t, 1H, J = 8.00 Hz, Ar-H), 7.16 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.48 (s, 1H, Ar-H), 8.86 (t,
13
1
H, J = 6.80 Hz, NH), 11.32 (br s, 2H, NHOH); C NMR (100 MHz, DMSO-d ) δ (ppm): 169.6, 162.3,
6
160.3, 159.0, 156.6, 154.6, 153.1, 139.6, 139.2, 130.8, 128.6, 122.4, 116.0, 112.4, 106.8, 60.6, 56.3,
+
5
6.2, 56.1, 38.5, 33.3, 29.3, 26.4, 24.3; HRMS: m/z calculated for C H N O [M-H] : 512.21507,
25
31
5
7
found: 512.21304.
4.2. Biological evaluation
2
3

ACCEPTED MANUSCRIPT
4.2.1.
In vitro anti-proliferative activity assay
Cancer cells from different cancer cell lines; human breast adenocarcinoma (MCF7) and human
hepatocellular carcinoma (HepG2) 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. The cell lines were kept in a
humidified, 5% (v/v) CO atmosphere at 37 ºC. Exponentially growing cells from the two cancer cell
2
lines were trypsinized (for detachment), counted and seeded at the appropriate densities (2000-1000
2
cells/0.33 cm well) into 96-well microtiter plates. Cells then were incubated in a humidified atmosphere
at 37˚C for 24 hours. Then, cells were exposed to different concentrations of compounds 4a-f, 5a-f, 9a-e,
1
0a-e, SAHA, and CA4 (0.1,1, 10,100,1000 µM) for 72 h. The viability of treated cells was determined
using MTT technique. Media were removed, cells were incubated with MTT (5%, 200 ꢀL) solution/well
Sigma Aldrich, MO, USA), and were allowed to metabolize the dye into a colored-insoluble formazan
(
crystals for 2 h. The remaining MTT solution were discarded from the wells and the formazan crystals
were dissolved in 200 µL/well acidified isopropanol for 30 min, covered with aluminum foil and
continuously shaken using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc, MI, USA) at room
R
temperature. Absorbances were measured at 570 nm using a Stat Fax 4200 plate reader (Awareness
Technology, Inc., FL, USA). The cell viability were expressed as percentage of control and the
concentration that induces 50% of maximum inhibition of cell proliferation (IC ) were determined using
5
0
Graph Pad Prism 5 software (Graph Pad software Inc, CA) [90, 91]
.2.2. In vitro enzyme inhibitory assays
.
4
HeLa cells and MCF-7 cell lines were obtained from American Type Culture Collection, cells were
cultured using Dulbecco's Modified Eagle's Medium (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
2
4

ACCEPTED MANUSCRIPT
were plated (cells density 1.2 – 1.8 × 100,000 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 before the enzyme
assay for HDAC and Tubulin.
4
.2.2.1.
The in vitro HDAC inhibitory activities of compounds 5f, 10a and 10e against four HDAC isoforms
HDAC1, 2, 4, 6) were measured using ELISA assay kits (Mybiosource, Inc.) according to
HDAC inhibitory assay
(
manufacturer’s directions. Briefly, HeLa 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, the tested compounds, and the positive reference SAHA were diluted to designated
concentrations. On the 96-well microtiter plates standard or sample was added to each well in 100 µL,
and incubated at 37 °C for 2 h. The solution was aspirated and 100 µL of prepared Detection Reagent A
was added to each well. Incubation was done at 37°C for 2 h. After washing 100 µL of prepared
Detection Reagent B was added and incubation was continued at 37°C for 30 min. Five washings were
done, then 90 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added and incubated at
3
7°C for 15-25 min. Stop solution was added in 50 µL. Cells were exposed to different concentrations of
compounds 5f, 10a, 10e and SAHA (0.01,0.1, 1,10 µM) for 72 h Optical density (O.D.) was measured at
50 nm using microplate reader (Spectromax Plus 96 well plate spectrophotometer), and the
concentration that induces 50% of maximum inhibition of HDAC isoforms (IC ) were determined using
.
4
5
0
Graph Pad Prism 5 software (Graph Pad software Inc, CA).
.2.2.2. Tubulin polymerization inhibitory assay
4
In vitro kinetics of microtubule assembly were measured using ELISA kit for TUBb (Cloud-Clone.
Corp.) on MCF-7 cell line. The compounds tested were 9a, 10a, 10e, and CA4. Briefly, growing cells
from MCF-7 cell lines 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,
the tested compounds, and the control CA4 were diluted to designated concentrations. On the 96-well
2
5

ACCEPTED MANUSCRIPT
microtiter plates standard or sample was added to each well in 100 µL, and incubated at 37°C for 2 h.
The solution was aspirated and 100 µL of prepared Detection Reagent A was added to each well.
Incubation was done at 37°C for 2 h. After washing 100 µL of prepared Detection Reagent B was added
and incubation was continued at 37°C for 30 min. Five washings were done, then 90 µL of 3,3',5,5'-
tetramethylbenzidine (TMB) substrate solution was added and incubated at 37°C for 15-25 min. Stop
solution was added in 50 µL. Cells were exposed to different concentrations of compounds 9a, 10a, 10e
and CA4 (0.4,2, 10,50 µM) for 72 h. Optical density (O.D.) was measured at 450 nm using microplate
reader (Spectromax Plus 96 well plate spectrophotometer), and the concentration that induces 50% of
maximum inhibition of TUBb (IC ) were determined using Graph Pad Prism 5 software (Graph Pad
5
0
software Inc, CA) [90, 91]
.
Docking studies
Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA) was used for docking analysis.
Fully automated docking tool using “Dock ligands (CDocker)” protocol running on Intel(R) core(TM)
i32370 CPU @ 2.4 GHz 2.4 GHz, RAM Memory 2 GB under the Windows 7.0 system. The receptor
protein is prepared. The force field applied is CharmM to the receptor and the hydrogens are minimized.
By selecting only the ligand part and clicking on “Define sphere from selection” so that the crystal
ligand is used to define the binding site of 13, 11 Angstroms for tubulin and HDAC, respectively, on the
receptor molecule. Now the above prepared receptor is given as input for “input receptor molecule”
parameter in the CDocker protocol parameter explorer. Force fields are applied on compounds 5f, 5e, 9a,
10a and 10e to get the minimum lowest energy structure. The obtained poses were studied and the poses
showing best ligand–tubulin or HDAC interactions were selected and used for CDocker energy (protein-
ligand interaction energies) calculations.
4.2.2.3.
Docking of the target molecules to HDLP in vorinostat binding site.
Compounds 5f, 5e, 10a, 10e and vorinostat were docked into the empty vorinostat binding site of HDLP
Histone Deacetylase Like Protein) a homologue from hyperthermophilic bacterium Aquifex aeolicus,
(
2
6

ACCEPTED MANUSCRIPT
that shares 35.2% identity with human HDAC 1 over 375 residues. Docking of the conformation
database of the target compound was done using Discovery Studio 2.5 software program. The X-ray
crystallographic structure of HDLP complexed with vorinostat was obtained from the Protein Data Bank
(http://www. rcsb.org/, PDB code 1C3S) [36]
.
4.2.2.4.
Docking of the target molecules to tubulin protein in the colchicine binding site
Compounds 9a, 10a, 10e and CA4 were docked into the empty colchicine binding site of tubulin.
Docking of the conformation database of the target compound was done using Discovery Studio 2.5
software program. The X-ray crystallographic structure of tubulin complexed with DAMA-colchicine
was obtained from the Protein Data Bank (http://www. rcsb.org/, PDB code 1SA0) [85]
.
Acknowledgments
The authors are thankful to Dr. Tamer Abdelghany, Faculty of Pharmacy, Al-Azhar University, Cairo,
for performing the anti-proliferative activity of the compounds. The help rendered by Dr. Esam
Rashwan, at Serum and Vaccine Research Institute, for performing the tubulin and HDAC inhibitory
activity assays is gratefully acknowledged.
5
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