Design, synthesis and molecular docking of novel diarylcyclohexenone and diarylindazole derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1080/14756366.2016.1244532

New target compounds were designed as inhibitors of tubulin polymerization relying on using two types of ring B models (cyclohexenone and indazole) to replace the central ring in colchicine. Different functional groups (R¹) were attached to man

Full text of DOI:10.1080/14756366.2016.1244532

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and molecular docking of
novel diarylcyclohexenone and diarylindazole
derivatives as tubulin polymerization inhibitors
Riham I. Ahmed, Essam Eldin A. Osman, Fadi M. Awadallah & Samir M. El-
Moghazy
To cite this article: Riham I. Ahmed, Essam Eldin A. Osman, Fadi M. Awadallah & Samir M. El-
Moghazy (2016): Design, synthesis and molecular docking of novel diarylcyclohexenone and
diarylindazole derivatives as tubulin polymerization inhibitors, Journal of Enzyme Inhibition
and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1244532
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1244532
Published online: 24 Oct 2016.
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Date: 29 October 2016, At: 12:36

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2016
http://dx.doi.org/10.1080/14756366.2016.1244532
RESEARCH ARTICLE
Design, synthesis and molecular docking of novel diarylcyclohexenone and
diarylindazole derivatives as tubulin polymerization inhibitors
Riham I. Ahmeda, Essam Eldin A. Osmanb, Fadi M. Awadallahb and Samir M. El-Moghazyb
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University in Beni Suef, Kornish Al Nile, Beni Suef, Egypt; Department
of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 31 May 2016
Revised 2 August 2016
Accepted 18 August 2016
Published online 20 October
2016
New target compounds were designed as inhibitors of tubulin polymerization relying on using two types
of ring B models (cyclohexenone and indazole) to replace the central ring in colchicine. Different functional
groups (R1) were attached to manipulate their physicochemical properties and/or their biological activity.
The designed compounds were assessed for their antitumor activity on HCT-116 and MCF-7 cancer cell
lines. Compounds 4b, 5e and 5f exhibited comparable or higher potency than colchicine against colon
HCT-116 and MCF-7 tumor cells. The mechanism of the antitumor activity was investigated through evalu-
ating the tubulin inhibition potential of the active compounds. Compounds 4b, 5e and 5f showed per-
centage inhibition of tubulin in both cell line homogenates ranging from 79.72% to 89.31%. Cell cycle
analysis of compounds 4b, 5e and 5f revealed cell cycle arrest at G2/M phase. Molecular docking revealed
the binding mode of these new compounds into the colchicine binding site of tubulin.
KEYWORDS
Antimitotic agent; cell cycle;
colchicine; docking; tubulin
Introduction
the three binding domains, colchicine binds with high affinity to
b-tubulin and forms entangled tubulin dimer, which inhibits the
microtubule assembly3. Literature revealed many colchicine site
inhibitors being evaluated under clinical investigation, and even
more in preclinical studies4. Colchicine I is a rigid molecule whose
rigidity is imparted by the B-ring which anchored rings A and C.
Rings A and C are aimed to fit with hydrophobic pockets in the
colchicine binding site. In addition, an H-bond acceptor (OCH3)
group on ring A is a key feature of these inhibitors. Therefore, in
the course of developing new more flexible colchicine analogs,
many trials were made to modify the bridge between rings A and
C5,6. More flexible derivatives involved the replacement of ring B
with an olefinic bridge as in combretastatin A-4 II7,8, insertion of a
carbonyl function9–11 or a variety of heterocyclic rings12–14 han-
dling rings A and C, as exemplified by the pyrazole derivative III15.
Drugs that disrupt microtubule/tubulin dynamics are widely used
in cancer chemotherapy. The vast majority of these molecules act
by binding to the protein tubulin, an a,b-heterodimer that forms
the core of the microtubules which play a crucial role in the main-
tenance of cell shape, signal transduction and chromosome segre-
gation during mitosis. Inhibitors of microtubules engage the cell
cycle surveillance mechanisms to arrest cell division in mitosis.
Microtubules targeting agents, also called antimitotic agents, per-
turb not only mitosis, but also arrest cells during interphase1,2
.
Microtubules targeting agents are known to interact with tubu-
lin through at least three binding sites: the paclitaxel domain,
vinca site and the colchicine domain. So far, tubulin binding
agents can be classified into two types based on their site of
action as microtubule destabilizing drugs (vinca site and the col- Alternatively, an alicyclic ring was used as demonstrated by the
cyclohexenone derivative IV16 (Figure 1).
chicine site) and microtubule-stabilizing drugs (taxane site). Out of
CONTACT Fadi M. Awadallah
fadi_mae@hotmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Eini Street, Cairo
11562, Egypt
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
R. I. AHMED ET AL.
Figure 1. Examples of colchicine binding site inhibitors.
shift values (d) were given downfield from TMS. Samples were dis-
solved in DMSO-d6, addition of D2O was used to confirm the
exchangeable protons. Compounds 1a,b were prepared according
to the previously reported procedure17.
General procedure for the preparation of 2a,b
Figure 2. General structures of target compounds.
A solution of ethyl acetoacetate (1.56 ml, 12 mmol) in sodium eth-
oxide solution (0.3 g sodium metal in 140 ml absolute ethanol) was
stirred at room temperature for 1 h. The propenone 1a,b
(12 mmol) was added to the above solution with stirring. The reac-
tion mixture was heated under reflux for 12 h and poured onto
cold hydrochloric acid. The obtained solid was filtered off, washed
with water, dried and crystallized from methanol.
With the goal of producing new antitumor agents targeting
the microtubules at the colchicine binding site, and based on the
aforementioned facts, the design of the new target compounds
relied on using two types of ring B models. The first involved the
cyclohexenone ring (General structure A) and the other involved
the indazole ring (General structure B) as linker moieties between
the two hydrophobic rings A and C. Different functional groups
(R1) were attached to ring B to manipulate their physicochemical
properties and/or their biological activity. While retaining the H-
bond acceptor methoxy group pendent on ring A, another
methoxy anchor group (R2) was introduced in ring C for compara-
tive reasons (Figure 2).
The designed compounds were assessed for their antitumor
activity through in vitro cytotoxicity study on selected human cancer
cell lines. The mechanism of the antitumor activity was investigated
through evaluating the tubulin inhibition potential of the active
compounds. Finally, a molecular docking study was carried out.
Ethyl 6-phenyl-4-(4-methoxyphenyl)-2-oxocyclohex-3-enecarboxy-
late (2a)
Compound 2a was prepared from compound 1a and ethylacetoa-
cetate. 61% yield, mp 70–73 ꢀC. 1H NMR d 0.96 (t, 3H, J ¼ 7.2 Hz,
CH3 ethyl), 2.98 (dd, 1H, J ¼ 4.6 Hz, J ¼ 17.8 Hz, H5cyclohex.ax.), 3.08
(ddd, 1H, J ¼ 2.1 Hz, J ¼ 11.2 Hz, J ¼ 17.68 Hz, H50cyclohex.eq.), 3.64 (m,
1H, H6cyclohex), 3.40 (s, 3H, OCH3), 3.93 (q, 2H, J ¼ 7.2, CH2 ethyl),
4.13 (d, 1H, J ¼ 3.9, H1cyclohex), 6.56 (d, 1H, J ¼ 1.9 Hz, H3cyclohex),
7.40–7.73 (m, 9H, Ar–Hs). Anal. calcd. for C22H24O4 (350.41): C,
75.41; H, 6.33. Found: C, 75.64; H, 6.42.
Materials and methods
Ethyl 4,6-(4-methoxyphenyl)-2-oxocyclohex-3-enecarboxylate (2b)
Compound 2b was prepared from compound 1b and ethylacetoa-
cetate. 64% yield, mp 78–81 ꢀC. 1H NMR d 0.95 (t, 3H, J ¼ 7.2 Hz,
CH3 ethyl), 3.02 (m, 2H, H5cyclohex.ax., H50cyclohex.eq.), 3.66 (m, 1H,
H6cyclohex), 3.80 (s, 6H, 2OCH3), 3.94 (q, 2H, J ¼ 7.2 Hz, CH2 ethyl),
4.11 (d, 1H, J ¼ 3.8 Hz, H1cyclohex), 6.52 (s, 1H, H3cyclohex), 6.98–7.72
(m, 8H, Ar–Hs). Anal. Calcd. for C23H24O5 (380.43): C, 72.61; H, 6.36.
Found: C, 72.88; H, 6.41.
Chemistry
Melting points were uncorrected and were detected by open
capillary tube using Electrothermal 9100 melting point apparatus
(Bibby Scientific Limited, Stone, UK). Thin layer chromatography
was performed using silica gel cards DC-Alufolien-Kiesel gel with
fluorescent indicator UV254 using chloroform or hexane–ethyl
acetate 8.5:1.5 as the eluting system and the spots were visualized
using Vilber Lourmet ultraviolet lamp at k ¼ 254 nm. Elemental
microanalyses were performed at the Regional Center for
Mycology and Biotechnology, Al-Azhar University. NMR spectra
were recorded at the Microanalytical unit, Faculty of pharmacy,
Cairo University on Bruker Avance III spectrometer (Zurich,
Switzerland) at 400 MHz for 1H and at 100 MHz for 13C. Chemical
General procedure for the preparation of 3a,b
To a solution of the ester 2a,b (10 mmol) in absolute ethanol
(30 ml), 98% hydrazine hydrate (0.64 ml, 20 mmol) was added. The
reaction mixture was stirred for 24 h. The precipitated solid was fil-
tered off and recrystallized from absolute ethanol.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
2-Hydroxy-4-(4-methoxyphenyl)-6-phenylcyclohexa-1,3-diene
carbohydrazide (3a)
General procedure for the preparation of 5a–f
A mixture of the corresponding hydrazide 4a,b (10 mmol) and the
appropriate isothiocyanate derivative (10 mmol) in ethanol (20 ml)
was heated under reflux for 3 h. The formed solid was filtered off,
washed with ethanol and crystallized from ethanol.
Compound 3b was prepared from compound 2b and 98% hydra-
1
zine hydrate by stirring at RT 67% yield, mp 124–127 ꢀC. H NMR d
2.69 (dd, 1H, J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 2.79 (ddd, 1H,
J ¼ 2.1 Hz, J ¼ 11.2 Hz, J ¼ 17.6 Hz, H50cyclohex.eq.), 3.72 (m, 1H,
H6cyclohex), 3.87 (s, 3H, OCH3), 6.30 (brs, 3H, NHNH2, D2O
exchange), 6.87 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), 6.98–7.50 (m, 9H,
Ar–Hs), 10.80 (brs, 1H, OH, D2O exchange). 13C NMR d 29.8
(C6cyclohex.), 34.8 (C5cyclohex.), 55.5 (OCH3), 114.2, 114.3, 123.7, 127.6,
128.6, 131.2, 133.0, 137.1, 138.6, 142.5 (Ar–Cs), 160.4 (C¼O). MS
(EI): m/z (%): 336.21 (14.91). Anal. calcd. For C20H20N2O3 (336.38):
C, 71.41; H, 5.99; N, 8.33. Found: C, 71.78; H, 6.07; N, 8.51.
3-Hydroxy-6-(4-methoxyphenyl)-4-phenyl-N-methyl-4,5-dihydroin-
dazole-1-carbothioamide (5a)
Compound 5a was prepared from compound 4a and methyl iso-
thiocyanate. 88% yield, mp 220–224 ꢀC. 1H NMR d 2.80, 2.84 (dd,
1H, J ¼ 3.2 Hz, J ¼ 16.8 Hz, H5indazol.eq), 3.00 (s, 3H, CH3), 3.11 (ddd,
1H, J ¼ 1.8, J ¼ 8.3 Hz, J ¼ 16.7 Hz, H50indazol.ax.), 3.79 (s, 3H, OCH3),
4.10, 4.14 (dd, 1H, J ¼ 3.0 Hz, J ¼ 8.1 Hz, H4indazol) 6.61 (d, 1H,
J ¼ 1.8 Hz, H7indazol), 7.10–8.30 (m, 9H, Ar–Hs), 10.36 (s, 1H, NH,
D2O exchange), 11.05 (s, 1H, OH, D2O exchange). 13C NMR d 31.1
(NHCH3), 34.7 (C4indazol), 36.3 (C5indazol), 55.6 (OCH3), 114.3, 114.6,
122.1, 126.9, 127.6, 128.6, 131.9, 132.1 (Ar–Cs), 137.1 (C7aindazol),
144.9 (C6indazol), 147.1, 147.7 (C4 of the 2 phenyl rings), 160.5
(C3indazol), 178.6 (C¼S). MS (EI): m/z (%): 391.24 (1.80). Anal. calcd.
for C22H21N3O2S (391.49): C, 67.50; H, 5.41; N, 10.73. Found: C,
67.84; H, 5.44; N, 10.49.
2-Hydroxy-4,6-bis(4-methoxyphenyl) cyclohexa-1,3-diene
carbohydrazide (3b)
Compound 3b was prepared from compound 2b and 98% hydra-
zine hydrate. 73% yield, mp 144–146 ꢀC. 1H NMR d 2.75 (dd, 1H,
J ¼ 4.7 Hz, J ¼ 17.8 Hz, H5cyclohex.ax.), 2.97 (ddd, 1H, J ¼ 2.2 Hz,
J ¼ 11.2 Hz, J ¼ 17.7 Hz, H50cyclohex.eq.), 3.72 (m, 1H, H6cyclohex), 3.87
(s, 6H, 2OCH3), 6.26 (brs, 3H, NHNH2, D2O exchange), 6.81 (d, 1H,
J ¼ 1.9 Hz, H3cyclohex), 6.91–7.45 (m, 9H, Ar–Hs and OH). 13C NMR d
30.0 (C6cyclohex.), 35.1 (C5cyclohex.), 55.5, 55.6 (2OCH3), 114.2, 114.3,
123.7, 128.5, 133.1, 137.1, 137.9, 146.4 (Ar–Cs), 159.0 (C¼O). MS
(EI): m/z (%): 336.48 (1.10). Anal. calcd For C21H22N2O4 (336.41): C,
68.84; H, 6.05; N, 7.65. Found: C, 68.97; H, 6.13; N, 7.69.
3-Hydroxy-4,6-bis(4-methoxyphenyl)-N-methyl-4,5-dihydroindazole-
1-carbothioamide (5b)
Compound 5b was prepared from compound 4b and methyl iso-
thiocyanate. 90% yield, mp 170–173 ꢀC. 1H NMR d 2.80, 2.85 (dd,
1H, J ¼ 3.2 Hz, J ¼ 16.8 Hz, H5indazol.eq), 2.99 (ddd, 1H, J ¼ 1.8,
J ¼ 8.3 Hz, J ¼ 16.7 Hz, H50indazol.ax.), 3.08 (s, 3H, CH3), 3.79 (s, 3H,
OCH3), 4.10, 4.14 (dd, 1H, J ¼ 3.0 Hz, J ¼ 8.1 Hz, H4indazol) 6.61 (d,
1H, J ¼ 1.8 Hz, H7indazol), 7.10–8.30 (m, 9H, Ar–Hs), 10.30 (s, 1H, NH,
D2O exchange), 11.01 (s, 1H, OH, D2O exchange). 13C NMR d 31.2
(NHCH3), 34.9 (C4indazol), 36.0 (C5indazol), 55.4, 55.7 (2OCH3), 114.2,
114.4, 122.0, 126.9, 128.4, 131.9, 132.1 (Ar–Cs), 137.1 (C7aindazol),
144.9 (C6indazol), 158.3, 159.9 (C4 of the two phenyl rings), 160.4
(C3indazol), 178.5 (C¼S). MS (EI): m/z (%): 421.48 (3.61). Anal. calcd.
for C23H23N3O3S (421.51): C, 65.54; H, 5.50; N, 9.97. Found: C,
65.81; H, 5.57; N, 10.04.
General procedure for the preparation of 4a,b
A mixture of 2a or 2b (10 mmol) and hydrazine hydrate (0.32 ml,
10 mmol) in ethanol (20 ml) was heated under reflux for 8 h. The
reaction mixture was evaporated under reduced pressure. After
cooling, the reaction mixture was poured onto crushed ice and
the solid thus obtained was filtered off, washed with water and
crystallized from ethanol to give 4a and 4b, respectively.
4-Phenyl-6-(4-methoxyphenyl)-4,5-dihydro-1H-indazol-3-ol (4a)
Compound 4a was prepared from compound 2a and 98% hydra-
zine hydrate under reflux. 76% yield, mp 107–110 ꢀC. 1H NMR d
2.86, 2.90 (dd, 1H, J ¼ 3.1 Hz, J ¼ 16.7 Hz, H5indazol.eq.), 3.12 (ddd,
1H, J ¼ 1.7 Hz, J ¼ 8.4 Hz, J ¼ 16.68 Hz, H50indazol.ax.), 3.79 (s, 3H,
OCH3), 4.15 (dd, 1H, J ¼ 3.0 Hz, J ¼ 8.2 Hz, H4indazol), 6.27 (s, 1H, NH,
3-Hydroxy-6-(4-methoxyphenyl)-4-phenyl-N-ethyl-4,5-dihydroinda-
zole-1-carbothioamide (5c)
D2O exchange), 6.66 (d, 1H, J ¼ 3.0 Hz, H7indazol), 7.00–7.70 (m, 9H, Compound 5c was prepared from compound 4a and ethyl isothio-
Ar–Hs), 10.80 (brs, 1H, OH, D2O exchange). 13C NMR d 29.8
cyanate. 83% yield, mp 202–205 ꢀC. 1H NMR d 1.01 (t, 3H, J ¼ 7.2,
(C4indazol), 34.8 (C5indazol), 55.5 (OCH3), 114.4, 114.6, 126.4, 127.7,
CH3 ethyl), 2.72, 2.74 (dd, 1H, J ¼ 3.2 Hz, J ¼ 16.8 Hz, H5indazol.eq),
2.83 (ddd, 1H, J ¼ 1.8 Hz, J ¼ 8.3 Hz, J ¼ 16.7 Hz, H50indazol.ax.), 3.50
128.5, 128.8, 129.6, 133.0 (Ar–Cs), 137.1 (C7indazol), 145.9, 146.3 (C4
of the 2 phenyl rings), 159.1 (C3indazol). MS (EI): m/z (%): 318.09
(q, 2H, J ¼ 7.2, CH2 ethyl), 3.78 (s, 3H, OCH3), 4.11,4.14 (dd, 1H,
(12.02). Anal. calcd. For C20H18N2O2 (318.37): C, 75.45; H, 5.70; N,
8.80. Found: C, 75.49; H, 5.76; N, 8.94.
J ¼ 3.0 Hz, J ¼ 8.1 Hz, H4indazol), 6.60 (d, 1H, J ¼ 1.8 Hz, H7indazol),
6.95–8.37 (m, 9H, Ar–Hs), 10.27 (s, 1H, NH, D2O exchange), 10.99
(s, 1H, OH, D2O exchange). 13C NMR d 15.0 (CH3 ethyl), 34.6
(C4indazol), 35.8 (CH2 ethyl), 38.1 (C5indazol), 55.6 (OCH3), 114.2, 122.1,
127.0, 127.4, 128.1, 128.6, 128.9, 131.9, 132.1 (Ar–Cs), 137.2
(C7aindazol), 144.9 (C6indazol), 159.9 (C4 of the two phenyl rings),
160.5 (C3indazol), 177.6 (C¼S). MS (EI): m/z (%): 405.17 (1.90). Anal.
calcd. for C23H23N3O2S (405.51): C, 68.12; H, 5.72; N, 10.36. Found:
C, 68.35; H, 5.81; N, 10.49.
4,6-Bis(4-methoxyphenyl)-4,5-dihydro-1H-indazol-3-ol (4b)
Compound 4b was prepared from compound 2b and 98% hydrazine
hydrate under reflux. 78% yield, mp 86–89 ꢀC. 1H NMR d 2.84, 2.88
(dd, 1H, J¼ 3.1 Hz, J¼ 16.8 Hz, H5indazol.eq.), 3.14 (ddd, 1H, J ¼ 1.7 Hz,
J¼ 8.4 Hz, J¼ 16.69 Hz, H50indazol.ax.), 3.80 (s, 6H, 2OCH3), 4.16,4.18 (dd,
1H, J¼ 3.0 Hz, J¼ 8.3 Hz, H4indazol), 6.20 (s, 1H, NH, D2O exchange),
6.89 (s, 1H, H3cyclohex), 7.00–7.70 (m, 8H, Ar–Hs), 10.79 (brs, 1H, OH,
D2O exchange). 13C NMR d 33.4 (C4indazol), 35.0 (C5indazol), 55.3, 55.6
3-Hydroxy-4,6-bis(4-methoxyphenyl)-N-ethyl-4,5-dihydroindazole-1-
(2OCH3), 114.2, 114.4, 128.2, 128.5, 129.6, 132.7 (Ar–Cs), 137.5 carbothioamide (5d)
(C7indazol), 157.9. 158.1 (C4 of the two phenyl rings), 160.4 (C3indazol).
MS (EI): m/z (%): 348.36 (2.81). Anal. calcd. for C21H20N2O3 (348.40): C,
72.40; H, 5.79; N, 8.04. Found: C, 72.53; H, 5.84; N, 8.17.
Compound 5d was prepared from compound 4b and ethyl iso-
thiocyanate. 86% yield, mp 210–213 ꢀC. 1H NMR d 1.11 (t, 3H,
J ¼ 7.2, CH3 ethyl), 2.71, 2.73 (dd, 1H, J ¼ 3.2 Hz, J ¼ 16.8 Hz,

4
R. I. AHMED ET AL.
H5indazol.eq), 2.85 (ddd, 1H, J ¼ 1.8 Hz, J ¼ 8.3 Hz, J ¼ 16.7 Hz, 5-[2-Hydroxy-4,6-bis(4-methoxyphenyl) cyclohexa-1,3-dienyl]-1,3,4
H50indazol.ax.), 3.50 (q, 2H, J ¼ 7.2, CH2 ethyl), 3.78 (s, 6H, 2OCH3),
oxadiazole-2-(3H)-thione (6b)
Compound 6b was prepared from compound 3b and carbon
4.12, 4.15 (dd, 1H, J ¼ 3.0 Hz, J ¼ 8.1 Hz, H4indazol), 6.60 (d, 1H,
disulfide. 89% yield, mp 278–281 ꢀC. 1H NMR d 2.73, 2.75 (dd, 1H,
J ¼ 1.8 Hz, H7indazol), 6.90–7.70 (m, 8H, Ar–Hs), 10.28 (s, 1H, NH,
D2O exchange), 10.99 (s, 1H, OH, D2O exchange). 13C NMR d 15.7
J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 2.82 (ddd, 1H, J ¼ 2.1 Hz, J ¼ 11.3 Hz,
J ¼ 17.8 Hz, H50cyclohex.eq.), 3.74 (m, 1H, H6cyclohex), 3.79 (s, 6H,
(NHCH2CH3), 31.7 (NHCH2CH3), 34.7 (C4indazol), 36.1 (C5indazol), 55.4,
55.6 (2OCH3), 114.2, 114.3, 114.5, 122.1, 126.8, 127.6, 128.6, 131.9,
2OCH3), 6.97 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), 7.00–7.60 (m, 8H, Ar–Hs),
132.1, 133.0 (Ar–Cs), 137.1 (C7aindazol), 145.0 (C6indazol), 157.8, 158.3
(C4 of the 2 phenyl rings), 160.5 (C3indazol), 177.6 (C¼S). MS (EI):
m/z (%): 435.20 (1.27). Anal. calcd. for C24H25N3O3S (435.54): C,
66.18; H, 5.79; N, 9.65. Found: C, 66.35; H, 5.82; N, 9.78.
10.09 (s, 1H, NH, D2O exchange), 11.47 (s, 1H, OH, D2O exchange).
13C NMR d 33.7 (C6cyclohex.), 40.6 (C5cyclohex.), 55.5, 55.7 (2OCH3),
113.9, 114.2, 114.7, 127.1, 127.4, 128.3, 128.5, 129.1, 134.5, 135.7
(Ar–Cs), 159.0 (C¼O), 163.9 (C2cyclohex.), 198.7 (C¼S). MS (EI): m/z
(%): 408.24 (0.29). Anal. calcd. for C22H20N2O4S (408.47): C, 64.69;
H, 4.94; N, 6.86. Found: C, 64.82; H, 4.98; N, 6.95.
3-Hydroxy-6-(4-methoxyphenyl)-4-phenyl-N-phenyl-4,5-dihydroin-
dazole-1-carbothioamide (5e)
Compound 5e was prepared from compound 4a and phenyl iso-
thiocyanate. 76% yield, mp 138–140 ꢀC. 1H NMR d 2.75, 2.78 (dd,
1H, J ¼ 3.2 Hz, J ¼ 16.8 Hz, H5indazol.eq), 2.87 (ddd, 1H, J ¼ 1.8 Hz,
J ¼ 8.3 Hz, J ¼ 16.7 Hz, H50indazol.ax.), 3.80 (s, 3H, OCH3), 4.11, 4.15
(dd, 1H, J ¼ 3.0 Hz, J ¼ 8.1 Hz, H4indazol), 6.70 (d, 1H, J ¼ 1.8 Hz,
H7indazol), 7.00–7.80 (m, 14H, Ar–Hs), 10.06 (s, 1H, NH, D2O
exchange), 10.77 (s, 1H, OH, D2O exchange). MS (EI): m/z (%):
453.11 (0.81). Anal. calcd. for C27H23N3O2S (453.56): C, 71.40; H,
5.11; N, 9.26. Found: C, 71.63; H, 5.14; N, 9.38.
General procedure for the preparation of 7a,b
A mixture of compound 3a,b (10 mmol) and acetylacetone (1 ml,
10 mmol) in a mixture of ethanol–acetic acid (100:10 v/v) was
heated under reflux for 10 h. The reaction mixture was cooled and
the precipitated solid was filtered off, washed with water, dried
and crystallized from ethanol.
(3,5-Dimethyl-1H-pyrazol-1-yl) [2-hydroxy-4-(4-methoxyphenyl)-6-
phenylcyclohexa-1,3-dienyl] methanone (7a)
Compound 7a was prepared from compound 3a and acetylace-
tone. 45% yield, mp 277–279 ꢀC. 1H NMR d 2.40 (s, 3H, CH3 at
C3pyrazole), 2.42 (s, 3H, CH3 at C5pyrazole), d 2.73, 2.77 (dd, 1H,
J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 2.98 (ddd, 1H, J ¼ 2.3 Hz,
J ¼ 11.3 Hz, J ¼ 17.8 Hz, H50cyclohex.eq.), 3.75 (m, 1H, H6cyclohex),
3.83 (s, 3H, OCH3), 6.43 (s, 1H, C4pyrazole), 6.71 (d, 1H, J ¼ 1.8 Hz,
H3cyclohex), 7.10–8.30 (m, 9H, Ar–Hs), 10.82 (s, 1H, OH, D2O
exchange). 13C NMR d 12.8, 13.7 (2 CH3), 35.8 (C6cyclohex.), 44.3
(C5cyclohex.), 55.5, 55.7 (2OCH3), 114.3, 114.8, 122.8, 128.4, 131.0,
132.1, 136.3, 139.9, 141.8, 148.4 (Ar–Cs), 159.7 (C¼O), 161.3
(C2cyclohex.). MS (EI): m/z (%): 400.23 (1.00). Anal. calcd. for
C25H24N2O3 (400.47): C, 74.98; H, 6.04; N, 7.00. Found: C, 75.12;
H, 6.13; N, 7.24.
3-Hydroxy-4,6-bis(4-methoxyphenyl)-N-phenyl-4,5-dihydroinda-
zole-1-carbothioamide (5f)
Compound 5f was prepared from compound 4b and phenyl iso-
thiocyanate. 78% yield, mp 155–159 ꢀC. 1H NMR d 2.78, 2.81 (dd,
1H, J ¼ 3.2 Hz, J ¼ 16.8 Hz, H5indazol.eq), 2.88 (ddd, 1H, J ¼ 1.8 Hz,
J ¼ 8.3 Hz, J ¼ 16.7 Hz, H50indazol.ax.), 3.80 (s, 6H, 2OCH3), 4.11, 4.14
(dd, 1H, J ¼ 3.0 Hz, J ¼ 8.1 Hz, H4indazol), 6.70 (d, 1H, J ¼ 1.8 Hz,
H7indazol), 7.10–7.80 (m, 13H, Ar–Hs), 10.06 (s, 1H, NH, D2O
exchange), 10.76 (s, 1H, OH, D2O exchange). MS (EI): m/z (%):
483.20 (2.22). Anal. calcd. for C28H25N3O3S (483.58): C, 69.54; H,
5.21; N, 8.69. Found: C, 69.78; H, 5.29; N, 8.94.
General procedure for the preparation of 6a,b
To a solution of the corresponding hydrazide 3a,b (10 mmol) and
potassium hydroxide (0.56 g, 10 mmol) in absolute ethanol (5 ml),
(3,5-Dimethyl-1H-pyrazol-1-yl) [2-hydroxy-4,6-bis(4-methoxy
carbon disulfide (0.95 ml, 15 mmol) was added. The reaction mix- phenyl)cyclohexa-1,3-dienyl] methanone (7b)
ture was heated under reflux for 5 h till the release of hydrogen
sulfide gas ceased. After dilution with water, the reaction mixture
was filtered. The filtrate was acidified with 1 N hydrochloric acid.
The precipitated solid was filtered off, washed with water and
crystallized from ethanol.
Compound 7b was prepared from compound 3b and acetylace-
tone. 48% yield, mp 268–270 ꢀC. 1H NMR d 2.39 (s, 3H, CH3 at
C3pyrazole), 2.45 (s, 3H, CH3 at C5pyrazole), d 2.72, 2.75 (dd, 1H,
J ¼ 4.8, J ¼ 17.7, H5cyclohex.ax.), 2.93 (ddd, 1H, J ¼ 2.3 Hz, J ¼ 11.3 Hz,
J ¼ 17.8 Hz, H50cyclohex.eq.), 3.73 (m, 1H, H6cyclohex), 3.79 (s, 6H,
2OCH3), 6.41 (s, 1H, C4pyrazole), 6.89 (d, 1H, J ¼ 1.6 Hz, H3cyclohex),
7.00–7.80 (m, 8H, Ar–Hs), 10.55 (s, 1H, OH, D2O exchange). 13C
NMR d 12.8,13.7 (2 CH3), 35.8 (C6cyclohex.), 44.3 (C5cyclohex.), 55.5,
55.7 (2OCH3), 114.3, 114.8, 122.8, 128.4, 131.0, 132.1, 136.3, 139.9,
141.8, 148.4 (Ar–Cs), 159.7 (C¼O), 161.3 (C2cyclohex.). MS (EI): m/z
(%): 430.14 (1.08). Anal. calcd. for C26H26N2O4 (430.50): C, 72.54; H,
6.09; N, 6.51. Found: C, 72.69; H, 6.08; N, 6.57.
5-[2-Hydroxy-4-(4-methoxyphenyl)-6-phenyl-cyclohexa-1,3-dienyl]-
1,3,4 oxadiazole-2–(3H)-thione (6a)
Compound 6a was prepared from compound 3a and carbon
disulfide. 86% yield, mp 282–284 ꢀC. 1H NMR d 2.73, 2.75 (dd,
1H, J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 2.82 (ddd, 1H, J ¼ 2.1 Hz,
J ¼ 11.2 Hz, J ¼ 17.6 Hz, H50cyclohex.eq.), 3.77 (m, 1H, H6cyclohex), 3.84
(s, 3H, OCH3), 6.95 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), 7.00–7.90 (m, 9H,
Ar–Hs), 13.09 (s, 1H, NH, D2O exchange), 14.05 (s, 1H, OH, D2O
exchange). 13C NMR d 30.8 (C6cyclohex.), 39.4 (C5cyclohex.), 55.8
(OCH3), 114.3, 114.4, 114.9, 123.9, 127.1, 127.4, 128.1, 128.6,
129.3, 134.5, 136.0 (Ar–Cs), 159.6 (C¼O), 161.1 (C2cyclohex.), 180.7
(C¼S). MS (EI): m/z (%): 378.19 (2.61). Anal. calcd. for
C21H18N2O3S (378.44): C, 66.65; H, 4.79; N, 7.40. Found: C, 66.82;
H, 4.846; N, 7.53.
General procedure for the preparation of 8a,b
A
mixture of compound 3a,b (1 mmol), ethyl acetoacetate
(0.13 ml, 1 mmol) and anhydrous potassium carbonate (0.21 g,
1.5 mmol) in ethanol (15 ml) was heated under reflux for 10 h. The
reaction mixture was poured on water and the precipitated solid
was filtrated off, washed with water, dried and crystallized from
ethanol.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
1-[2-Hydroxy-4-(4-methoxyphenyl)-6-phenyl
carbonyl]-3-methyl-1H-pyrazole-5(4H)-one (8a)
cyclohexa-1,3-diene- (C4cyclohex.), 158.3 (C4methoxyphenyl), 159.9 (C¼O), 160.5 (C2cyclohex.),
178.7 (C¼S). MS (EI): m/z (%): 439.25 (3.11). Anal. calcd. for
C23H25N3O4S (439.50): C, 62.85; H, 5.73; N, 9.56. Found: C, 63.04;
H, 5.76; N, 9.68.
Compound 8a was prepared from compound 3a and ethyl acetoa-
cetate. 51% yield, mp 249–252 ꢀC. 1H NMR d 1.66 (s, 3H, CH3 at
C3pyrazole), 1.84 (s, 2H, CH2pyrazolone), 2.81, 2.88 (dd, 1H, J ¼ 4.6 Hz,
J ¼ 17.6 Hz, H5cyclohex.ax.), 2.92 (ddd, 1H, J ¼ 2.1 Hz, J ¼ 11.2 Hz,
J ¼ 17.6 Hz, H50cyclohex.eq.), 3.72 (m, 1H, H6cyclohex), 3.81 (s, 3H,
OCH3), 6.90 (s, 1H, H3cyclohex), 7.00–7.90 (m, 9H, Ar–Hs), 10.30 (s,
1H, OH, D2O exchange). 13C NMR d 25.1 (CH3), 35.2 (C6cyclohex.),
39.3 (C5cyclohex.), 40.5 (CH2pyrazoline), 55.6 (OCH3), 69.7 (C3pyrazoline),
114.1, 114.7, 126.9, 127.4, 128.5, 128.9, 129.0, 132.1, 136.3, 139.9,
141.8, 144.5, 148.4 (Ar–Cs), 158.6 (C¼O), 175.7 (C2cyclohex.). MS (EI):
m/z (%): 402.17 (10.39). Anal. calcd. for C24H22N2O4 (402.44): C,
71.63; H, 5.51; N, 6.96. Found: C, 71.81; H, 5.58; N, 7.11.
1-[2-Hydroxy-4-(4-methoxyphenyl)-6-phenylcyclohexa-1,3-dienecar-
bonyl]-4-N-ethyl thiosemicarbazide (9c)
Compound 9c was prepared from compound 3a and ethyl isothio-
cyanate. 86% yield, mp 140–143 ꢀC. 1H NMR d 0.91 (t, 3H,
J ¼ 7.2 Hz, CH3 ethyl), 2.62 (dd, 1H, J ¼ 4.7 Hz, J ¼ 17.7 Hz,
H5cyclohex.ax.), 3.13 (ddd, 1H, J ¼ 2.1 Hz, J ¼ 11.2 Hz, J ¼ 17.7 Hz,
H50cyclohex.eq.), 3.40 (m, 1H, H6cyclohex), 3.50 (q, 2H, J ¼ 7.3 Hz, CH2
ethyl), 3.76 (s, 3H, OCH3), 6.60 (d, 1H, J ¼ 1.8 Hz, H3cyclohex),
6.90–7.40 (m, 9H, Ar–Hs), 9.80 (s, 3H, 3NHs, D2O exchange), 11.96
(s, 1H, OH, D2O exchange). Anal. calcd. for C23H25N3O3S (423.53):
C, 65.23; H, 5.95; N, 9.92. Found: C, 65.41; H, 6.02; N, 9.98.
1-[2-Hydroxy-4,6-bis(4-methoxyphenyl)cyclohexa-1,3-dienecar-
bonyl]-3-methyl-1H-pyrazole-5(4H)-one (8b)
Compound 8b was prepared from compound 3b and ethyl ace-
1-[2-Hydroxy-4,6-bis(4-methoxyphenyl)
bonyl]-4-N-ethyl thiosemicarbazide (9d)
cyclohexa-1,3-dienecar-
1
toacetate. 53% yield, mp 261–263 ꢀC. H NMR d 1.68 (s, 3H, CH3 at
C3 pyrazole), 1.85 (s, 2H, CH2pyrazolone), 2.75, 2.79 (dd, 1H, J ¼ 4.7 Hz,
J ¼ 17.7 Hz, H5cyclohex.ax.), 2.94 (ddd, 1H, J ¼ 2.2 Hz, J ¼ 11.3 Hz,
J ¼ 17.7 Hz, H50cyclohex.eq.), 3.72 (m, 1H, H6cyclohex), 3.81 (s, 6H,
2OCH3), 6.82 (s, 1H, H3cyclohex), 7.00–7.69 (m, 8H, Ar–Hs), 10.50 (s,
1H, OH, D2O exchange). MS (EI): m/z (%): 432.19 (5.24). Anal. calcd.
for C25H24N2O5 (432.47): C, 69.43; H, 5.59; N, 6.48. Found: C, 69.54;
H, 5.63; N, 6.61.
Compound 9d was prepared from compound 3b and ethyl iso-
thiocyanate. 87% yield, mp 155–157 ꢀC. 1H NMR d 1.10 (t, 3H,
J ¼ 7.22, CH3 ethyl), 2.69 (dd, 1H, J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 2.83
(ddd, 1H, J ¼ 2.1 Hz, J ¼ 11.4 Hz, J ¼ 17.7 Hz, H50cyclohex.eq.), 3.40 (m,
1H, H6cyclohex), 3.60 (q, 2H, J ¼ 7.23, CH2 ethyl), 3.79 (s, 6H, 2OCH3),
6.60 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), 6.90–7.50 (m, 8H, Ar–Hs), 10.20
(s, 3H, 3NHs, D2O exchange), 11.00 (s, 1H, OH, D2O exchange). 13C-
NMR d 15.0 (CH3 ethyl), 34.9 (C6cyclohex.), 36.0 (CH2ethyl), 38.3
(C5cyclohex.), 55.4, 56.5 (2OCH3), 112.8, 114.2,
General procedure for the preparation of 9a–f
114.5, 122.0, 127.0, 128.3, 128.5, 131.9, 132.1 (Ar–Cs), 144.9
(C4cyclohex.), 158.3 (C4methoxyphenyl), 159.9 (C¼O), 160.4 (C2cyclohex.),
177.4 (C¼S). MS (EI): m/z (%): 453.37 (0.45). Anal. calcd. for
C24H27N3O4S (453.55): C, 63.56; H, 6.00; N, 9.26. Found: C, 63.62; H,
6.09; N, 9.43.
A mixture of the corresponding hydrazide 3a,b (10 mmol) and the
appropriate isothiocyanate derivative (10 mmol) in ethanol (20 ml)
was heated under reflux for 5 h. The formed precipitate was fil-
tered off, washed with ethanol and crystallized from ethanol.
1-[2-Hydroxy-4-(4-methoxyphenyl)-6-phenylcyclohexa-1,3-dienecar-
bonyl]-4-N- methyl thiosemicarbazide (9a)
1-[2-Hydroxy-4-(4-methoxyphenyl)-6-phenyl
carbonyl]-4-N-phenyl thiosemicarbazide (9e)
Compound 9d was prepared from compound 3a and phenyl iso-
cyclohexa-1,3-diene-
Compound 9a was prepared from compound 3a and methyl iso-
thiocyanate. 83% yield, mp 136–139 ꢀC. 1H NMR d 2.86 (dd, 1H,
J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 3.10 (s, 3H, CH3), 3.18 (ddd, 1H, thiocyanate. 67% yield, mp 188–190 ꢀC. 1H NMR d 2.94 (dd, 1H,
J ¼ 2.1 Hz, J ¼ 11.2 Hz, J ¼ 17.6 Hz, H50cyclohex.eq.), 3.60 (m, 1H, J ¼ 4.8, J ¼ 17.6, H5cyclohex.ax.), 3.16 (ddd, 1H, J ¼ 2.3 Hz, J ¼ 11.2 Hz,
H6cyclohex), 3.79 (s, 3H, OCH3), 6.80 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), J ¼ 17.8 Hz, H50cyclohex.eq.), 3.74 (m, 1H, H6cyclohex), 3.80 (s, 3H,
7.10–8.3 (m, 9H, Ar–Hs), 10.80 (brs, 4H, 3NHs þ OH, D2O exchange). OCH3), 6.80 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), 7.00–7.80 (m, 14H, Ar–Hs),
13C NMR d 31.0 (CH3), 34.9 (C6cyclohex.), 38.3 (C5cyclohex.), 55.7 10.98 (s, 3H, 3NHs, D2O exchange), 11.00 (s, 1H, OH, D2O
(OCH3), 114.3, 114.6, 127.5, 127.6, 128.6, 128.7, 131.2, 138.7 exchange). 13C NMR d 34.7 (C6cyclohex.), 39.3 (C5cyclohex.), 55.7
(Ar–Cs), 145.6 (C4cyclohex.), 158.9 (C4methoxyphenyl), 159.9 (C¼O), (OCH3), 114.2, 114.6, 117.3, 121.5, 123.3, 124.9, 125.1, 127.5, 127.6,
160.4 (C2cyclohex.), 175.2 (C¼S). MS (EI): m/z (%): 409.32 (1.28). Anal. 128.4, 128.8, 129.4, 131.9, 138.9 (Ar–Cs), 145.4 (C4cyclohex.), 156.2
calcd. for C22H23N3O3S (409.50): C, 64.53; H, 5.66; N, 10.26. Found: (C4methoxyphenyl), 158.7 (C¼O), 160.1 (C2cyclohex.), 180.6 (C¼S). MS
C, 64.70; H, 5.72; N, 10.47.
(EI): m/z (%): 471.18 (0.71). Anal. calcd. for C27H25N3O3S (471.57): C,
68.77; H, 5.34; N, 8.91. Found: C, 68.94; H, 5.38; N, 9.02.
1-[2-Hydroxy-4,6-bis(4-methoxyphenyl)-6-phenyl cyclohexa-1,3-
1-[2-Hydroxy-4,6-bis(4-methoxyphenyl)
cyclohexa-1,3-dienecar-
dienecarbonyl]-4-N-methyl thiosemicarbazide (9b)
Compound 9b was prepared from compound 3b and methyl iso- bonyl]-4-N-phenyl thiosemicarbazide (9f)
thiocyanate. 80% yield, mp 147–150 ꢀC. 1H NMR d 2.69 (dd, 1H, Compound 9d was prepared from compound 3b and phenyl
J ¼ 4.6, J ¼ 17.5, H5cyclohex.ax.), 2.80 (ddd, 1H, J ¼ 2.2 Hz, J ¼ 11.3 Hz, isothiocyanate. 61% yield, mp 196–198 ꢀC. 1H NMR d 2.68 (dd,
J ¼ 17.8 Hz, H50cyclohex.eq.), 3.10 (s, 3H, CH3), 3.30 (m, 1H, 1H, J ¼ 4.7, J ¼ 17.6, H5cyclohex.ax.), 2.73 (ddd, 1H, J ¼ 2.2 Hz,
H6cyclohex), 3.78 (s, 6H, 2OCH3), 6.60 (d, 1H, J ¼ 1.7 Hz, H3cyclohex), J ¼ 11.1 Hz, J ¼ 17.6 Hz, H50cyclohex.eq.), 3.71 (m, 1H, H6cyclohex),
6.90–8.30 (m, 8H, Ar–Hs), 10.30 (s, 3H, 3NHs, D2O exchange), 3.84 (s, 6H, 2OCH3), 6.70 (d, 1H, J ¼ 1.8 Hz, H3cyclohex), 6.90–7.60
11.01 (s, 1H, OH, D2O exchange). 13C NMR d 31.1 (CH3), 34.9 (m, 13H, Ar–Hs), 9.80 (s, 3H, 3NHs, D2O exchange), 10.80 (s, 1H,
(C6cyclohex.), 36.1 (C5cyclohex.), 55.5, 55.7 (2OCH3), 112.9, 114.2, OH, D2O exchange). 13C NMR d 35.7 (C6cyclohex.), 38.6 (C5cyclohex.),
114.6, 122.1, 126.9, 128.2, 128.4, 128.7, 132.2, 137.1 (Ar–Cs), 144.9 55.5, 55.8 (2OCH3), 114.2, 114.5, 117.9, 122.0, 125.0, 125.1, 126.8,

6
R. I. AHMED ET AL.
127.8, 128.5, 129.1, 129.6, 132.0, 136.8 (Ar–Cs), 142.7 (C4cyclohex.), control and samples were averaged and subtracted from the aver-
age zero standard optical density. A standard curve was con-
structed by plotting the mean OD and concentration for each
156.2 (C4methoxyphenyl), 159.2 (C¼O), 161.7 (C2cyclohex.), 181.7
(C¼S). MS (EI): m/z (%): 501.67 (1.39). Anal. calcd. for
standard. A best fit curve was drawn through the points on the
graph, with concentration on the y-axis and absorbance on the x-
axis. In order to make the calculation easier, the OD values of the
standard (x-axis) were plotted against the known concentrations
of the standard (y-axis), although concentration is the independent
variable and OD value is the dependent variable.
C28H27N3O4S (501.60): C, 67.05; H, 5.43; N, 8.38. Found: C, 67.13;
H, 5.48; N, 8.49.
In vitro antitumor evaluation by MTT assay
Antiproliferative activity of the target compounds was determined
in cells treated with the different concentrations of the tested
compounds in comparison with untreated control using MTT assay
as following:
Sample preparation
The cell lysates obtained after incubation of MCF-7 and HCT-116
cells with the tested compounds at their IC50 concentration were
prepared according to the following:
1. Cells were grown as monolayer in media supplemented with
10% inactivated fetal bovine serum.
2. The monolayers of 10 000 cells were plated (104 cells/well)
in a 96-well tissue culture plate and incubated for 24 h at
37 ꢀC in a humidified incubator with 5% CO2 before treat-
ment with the compounds to allow attachment of cell to
the plate except blank wells without cells.
3. Different concentrations of 100, 10, 1.0, 0.1 and 0.01 mM of
each tested compound and positive control drug were
tested for cytotoxicity. Tetraplicate wells were prepared for
each concentration in addition to cell control (cell only with-
out compounds).
1. Adherent cells should be detached with trypsin and then col-
lected by centrifugation (suspension cells can be collected by
centrifugation directly).
2. Cells were washed three times in cold PBS.
3. Cells were resuspended in PBS (1ꢂ) and the cells was sub-
jected to ultrasonication for four times (or freeze cells
at ꢃ ꢁ20 ꢀC. Thaw cells with gentle mixing. Repeat the freeze/
thaw cycle for three times.)
4. Centrifugation was done at 1500g for 10 min at 2–8 ꢀC to
remove cellular debris.
4. Cells were incubated with the tested compounds for 48 h
into CO2 incubator at 37 ꢀC and 5% CO2.
5. Culture media containing different concentration of tested
compounds and dead cells were decanted leaving only
viable attached cells into the tissue culture plate.
6. The plate was washed twice with pre-warmed phosphate
buffered saline (PBS).
7. MTT reagent (40 ml) was added to each well including blank
and negative control wells.
8. After addition of MTT reagent 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.
Calculation of results
From the curve OD of each sample is converted to tubulin con-
centration, then percentage inhibition of tubulin polymerization
can be calculated by the following equation:
ꢀ
ꢁ
conc: of control ꢁ conc: of test
conc: of control
% inhibition ¼
ꢂ 100:
Cell cycle analysis by fluorescence-activated cell sorting analysis
9. DMSO (150 ml) was added to each well to solubilize the pur-
ple crystals of formazan.
10. Absorbance was measured at 570 nm with microplate reader
(ROBONIK TM P2000 Eliza plate reader; Robonik India Pvt.
Ltd, Maharashtra, India).
Fluorescence-activated cell sorting analysis following cell staining
with propidium iodide (PI) was used according to the following
protocol:
1. Approximately 106 cells (HCT-116 or MCF-7) were suspended
in 0.5 ml of PBS. The suspension was gently vortexed (5 s) or
gently aspirated several times with a Pasteur pipette to
obtain a mono-dispersed cell suspension, with minimal cell
aggregation.
2. Cells were fixed by transferring this suspension, with a Pasteur
pipette, into centrifuge tubes containing 4.5 ml of 70% etha-
nol, on ice. Cells were kept in ethanol for at least 2 h at 4 ꢀC.
Cells may be stored in 70% ethanol at 4 ꢀC for weeks.
3. The ethanol-suspended cells were centrifuged for 5 min at
300g. Ethanol was decanted thoroughly.
11. The percentage of cell survival was calculated by the follow-
ing equation:
ðAs ꢁ AbÞ
Survival rate % ¼
ꢂ 100;
ðAc ꢁ AbÞ
where As is the absorbance of sample, Ab is the absorbance
of blank and Ac is the absorbance of control.
12.
The inhibitory concentration 50 (IC50) was calculated from
the equation of the plot between molar concentration of
the tested compounds against survival rate percent.
4. The cell pellet was suspended in 5 ml of PBS, and after about
30 s it was centrifuged at 300g for 5 min.
5. The cell pellet was suspended in 1 ml of PI staining solution
and kept in the dark at room temperature for 30 min, or at
37 ꢀC for 10 min.
Tubulin polymerization assay
Standard curve construction
Seven different dilution of standard such as 2000, 1000, 500, 250,
125, 62.5, 31.2 pg/mL, and the last tubes with the blank 0 pg/mL
concentration were prepared, while test drugs were taken at their
IC50 concentration. The duplicate readings for each standard,
6. The sample was transferred to the flow cytometer, Becton
Dickinson Immunocytometry Systems and cell fluorescence
was measured. Maximum excitation of PI bound to DNA is at
536 nm, and emission is at 617 nm.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
7
7. Phoenix Flow Systems software (Phoenix Flow systems, Inc., Discovery Studio where hydrogen atoms were added at their
San Diego, CA) was used to deconvolute the DNA content fre-
quency histograms and to estimate the proportions of cells in
the respective phases of the cycle.
standard geometry, optical isomers and 3D conformations were
automatically generated.
8. The cell cycle progression was analyzed at a 10 mM concentra-
tion for 72 h.
Running docking
Docking was performed using CDOCKER protocol in Discovery
Studio keeping the parameters at default. The best scoring pose
of the docked compounds was recognized. Receptor–ligand inter-
actions of the complexes were examined in 2D and 3D styles.
Molecular docking procedure
X-ray crystal structure of tubulin in complex with DAMA-colchicine
and the stathmin-like domain (SLD) at 3.5 Å resolution (PDB: 1SA0)
was downloaded from protein data bank7. All molecular modeling
calculations and docking studies were carried out using Discovery
Studio software v4.0.0.1325918 running on a Windows7 PC.
Results and discussion
Chemistry
The designed compounds were synthesized adopting the chemical
pathways outlined in Schemes 1 and 2.
Binding site sphere determination
In the present work, the synthesis of the propenones 1a,b was
achieved by reacting benzaldehyde or 4-methoxybenzaldheyde
with 4-methoxy acetophenone in ethanol using aqueous NaOH as
a catalyst17. The cyclohexenone intermediates 2a,b were prepared
The protein–ligand complex obtained from the protein data bank
was prepared for docking as follows: Deletion of chains A, B and E
of the protein together with co-crystallized water molecules was
performed. Automatic protein preparation module was used
applying CHARMm forcefield. The binding site sphere has been via Michael addition through
a cyclo-condensation reaction
defined automatically by the software.
between the propenones 1a,b and the b-keto ester, ethyl acetoa-
cetate using sodium ethoxide as a catalyst. As the explored reac-
tion was not stereo selective, two chiral centers (C1 and C6) in the
structure of the cyclohexenones 2a,b were generated, which
would result in a mixture of diastereomers. No attempt to separate
Preparation of target compounds for docking
The docked compounds were prepared for docking by applying
the following protocol: 2D structures of the docked ligands were the diastereomeric cyclohexenones was undertaken, and the cyclo-
built using Marvin Sketch and copied to Discovery Studio 4.
Ligands were prepared using “Prepare Ligands” protocol in
condensation products were characterized in the form of the mix-
ture originated from the synthesis. The characteristic triplet-quartet
Scheme 1. Synthesis of compounds 3a,b, 4a,b and 5a–f.

8
R. I. AHMED ET AL.
Scheme 2. Synthesis of compounds 6a,b, 7a,b, 8a,b and 9a–f.
pattern confirmed the presence of the ethyl group of the ester.
1
The characteristic signal in the H NMR spectrum of 2a was, how-
ever, the two protons at C-5, being magnetically nonequivalent,
appeared as two different signals, the axial proton appeared as
double of doublet of doublets at around d 2.98 ppm showing gem-
inal coupling, vicinal coupling with H-6 and long range coupling
with vinyl proton H-3. The equatorial proton at C-4 appeared as
doublet of doublets at around d 3.08 ppm showing both germinal
and vicinal coupling. The vinyl proton, H-3, appeared as doublet at
d 6.56 ppm. Proton at C-6 appeared as multiplet due to vicinal cou-
pling to H-5 and H-1. H-1 proton appeared as doublet being
coupled to H-6. Reaction of the cyclohexenones 2a,b with 98%
hydrazine hydrate in ethanol at room temperature afforded deriva-
tives 3a,b, respectively. Proceeding the reaction under reflux condi-
tion resulted in cyclization with formation of indazole derivatives
4a,b. The appearance of the OH stretching band confirmed the
presence of the enol tautomer, which resulted in the loss of one of
the two chiral centers. According to a previous report on the tauto-
meric forms of indazole, the obtained compounds 4a,b could be
present in three tautomeric forms A, B and C (Figure 3). The
absence of carbonyl bands in the IR spectra of the products ruled
Figure 3. Tautomeric forms of 1H-indazol-3-ol.
Table 1. Antiproliferative activity against HCT-116 cell line and MCF-7.
IC50 (lM)
Compound number
R1
R2
(HCT-116)
(MCF-7)
3a
3b
4a
4b
H
OCH3
H
OCH3
H
–
–
–
–
55.35
28.04
19.21
6.78
>100
>100
>100
>100
6.71
11.05
58.46
59.80
30.25
88.83
85.07
27.31
60.60
>100
>100
34.40
>100
78.20
12.13
63.39
42.07
26.70
11.40
>100
60.90
>100
>100
5.50
11.55
44.70
>100
42.75
>100
58.45
60.41
>100
29.30
>100
>100
>100
>100
9.41
5a
CH3
CH3
CH2CH3
CH2CH3
C6H5
C6H5
–
5b
5c
5d
5e
5f
6a
OCH3
H
OCH3
H
OCH3
H
6b
OCH3
–
7a
7b
8a
H
OCH3
H
–
–
–
1
out lactam structures A and B19. The H NMR spectra of the inda-
zole derivatives exhibited three protons in the sp3 shift range (H-
8b
9a
9b
9c
9d
9e
9f
OCH3
H
OCH3
H
OCH3
H
–
5eq, H-5ax and H-4). H-5eq and H-4 appeared as doublet of doublets
CH3
CH3
CH2CH3
CH2CH3
C6H5
C6H5
–
at around d 2.90 and 4.18 ppm, respectively. While the signal for H-
5ax appeared as doublet of doublet of doublets at d 3.12 ppm
showing geminal coupling with H-5eq, vicinal coupling with H-4
and long range proton coupling with H-7. The vinylic proton at H-7
appeared as doublet at d 6.89 ppm with J¼ 1.7 Hz.
OCH3
–
Colchicine
Target compounds 5a–f were prepared by reacting compounds
4a,b with the appropriate substituted alkyl/aryl isothiocyanate in
absolute ethanol under reflux condition.
hydrazides 3a,b afforded the 3-methyl-1H-pyrazole-5-(4H)-ones
8a,b, respectively. Finally, reaction of isothiocyanates with hydra-
zides 3a,b in absolute ethanol under reflux furnished the corre-
sponding thiosemicarbazides 9a–f.
Substituted 1,3,4-oxadiazole-2(3H)-thione derivatives 6a,b were
synthesized by reacting the hydrazide derivatives 3a,b with carbon
disulfide in absolute ethanol in the presence of potassium hydrox-
ide. Pyrazole derivatives 7a,b were synthesized via cyclo-conden-
sation of acetyl acetone with the hydrazide derivatives 3a,b in a
mixture of ethanol and glacial acetic acid. The yield was found to
be solvent-dependent as cyclocondensation in a 10:1 (v/v) mixture
of ethanol–acetic acid afforded the corresponding pyrazole deriva-
tives in high yield, while the yield decreased upon using a mixture
of ethanol and triethyl amine. Using ethyl acetoacetate as a
The structures of all the synthesized compounds were con-
1
firmed using the EI MS, FT-IR, H and 13C NMR spectral analyses.
Biological screening
In vitro antitumor evaluation by MTT assay
The antiproliferative activity of the target compounds against
b-diketone, cyclocondensation reaction with the appropriate colon cancer HCT-116 and breast cancer MCF-7 cell lines was

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
9
measured at Vacsera, Egypt. The MTT method of assay was higher activity than the unsubstituted derivative 3a. Interestingly,
adopted and the IC50 values are listed in Table 1.
structure rigidification of 3a,b into the indazole derivatives 4a,b
resulted in increase in the antitumor activity especially for the 4-
methoxyphenyl derivative 4b (IC50¼ 6.78 and 11.40 mM against
HCT-116 and MCF-7 cells, respectively). Structure extension of the
indazole derivatives 4a,b with N-substituted carbothioamide moi-
ety was successful only with the N-phenyl derivatives 5e,f (IC50
ranging from 5.50 to 11.55 mM). The N-methyl (5a,b) and N-ethyl
derivatives (5c,d) were inactive. Unfortunately, introduction of het-
erocylic rings at position 1 of the cyclohexanol nucleus as in com-
pounds 6a,b, 7a,b, 8a,b did not reveal any advantage toward the
activity of the compounds, compared to their precursor less bulky
hydrazides 3a,b. In conclusion, it could be revealed that the bicylic
indazole derivatives 4a,b and 5e,f were the most potent of all
derivatives. The hydrazides 3a,b and the azacyclic related deriva-
tives 6a,b, 7a,b, 8a,b showed only moderate activity. The thiose-
micarbazides 9a–f and N-methyl 5a,b and N-ethyl 5c,d indazole-1-
carbothioamide derivatives were the least potent.
An overview of the results of MTT assay revealed that few com-
pounds exhibited IC50 values lower than or slightly higher than
colchicine. Concerning the antitumor activity against colon HCT-
116 tumor cell line, the obtained results showed that compounds
4b, 5e and 5f exhibited higher potency than colchicine.
Furthermore, compound 5f revealed comparable activity to doxo-
rubicin, while compounds 3b, 8b and 9d exerted moderate activ-
ity. Regarding antitumor activity against MCF-7 breast tumor cell
line, it can be revealed that compound 5e demonstrated higher
potency than colchicine. Meanwhile, compounds 4b and 5f were
less active than colchicine. The cyclohexenols 3a,b displayed IC50
of 55.35–63.39 mM, respectively. Regarding the effect of substitu-
tion on the phenyl ring (R1) the 4-methoxy derivative 3b showed
Table 2. Percentage inhibition of tubulin polymerization.
% Inhibition of tubulin polymerization
Compound
HCT-116
MCF-7
4b
5e
5f
86.96
89.31
84.33
82.24
84.53
79.72
86.30
86.84
Tubulin polymerization inhibition assay
Further investigation to assess the mechanism of action of the
most active compounds in the MTT assay as potential tubulin
polymerization inhibitors was carried out using tubulin polymeriza-
tion assay. The percentage inhibition of tubulin polymerization fol-
lowing sandwich enzyme immunoassay by ELISA method using
Enzyme-linked Immunosorbent Assay Kit was performed. Results
are summarized in Table 2. Percentage inhibition of tubulin poly-
merization was performed on the compounds with the highest
activity profile in the MTT assay, namely, 4b, 5e and 5f. The tested
compounds showed percentage inhibition of tubulin in both cell
line homogenates ranging from 79.72% to 89.31%. Compound 5e
was the most active on HCT-116 and 5f was the most active on
MCF-7 cells. It is noteworthy that activities of the tested
DAMA-colchicine
Table 3. Results of cell cycle analysis in HCT-116 and MCF-7 for compounds 4b,
5e and 5f.
% of cells in each phase
HCT-116
% of cells in each phase
MCF-7
G0-G1
S
G2-M
G0-G1
S
G2-M
Control
67.8
32.8
27.1
42.4
24
42
35.5
24.5
8.2
25.2
37.4
33.1
71.3
47.7
34.2
44.8
19.1
24.1
27.3
21.3
9.6
28.2
38.5
33.9
4b
5e
5f
Figure 4. Cell cycle analysis histograms for HCT-116 cells. (A) Control, (B) 4b, (C) 5e and (D) 5f.

10
R. I. AHMED ET AL.
compounds were comparable to that of colchicine or even higher lines (Table 3, Figures 4 and 5). Compared with the untreated
especially on HCT-116 cells homogenate.
control, tested compounds disturbed the cell cycle strongly at
G2/M phase, which was in agreement with the proposed mech-
anism of action.
Cell cycle analysis
It was hypothesized that the mechanism of action of compounds
4b, 5e and 5f involved arresting the process of mitosis.
Molecular modeling
Accordingly, cell cycle analysis was performed on HCT-116 and Based on the results of the tubulin polymerization assay, docking
MCF-7 cells after treatment with these compounds. Upon expos- of the most active compounds 4b, 5e and 5f was performed at X-
ure of the cells to the tested compounds, the percentages of ray crystal structure of tubulin in complex with (N-deacetyl-N-(2-
cells in the G0/G1 phase of the cell cycle in both cell lines, were mercaptoacetyl)colchicine) (DAMA-colchicine) and the SLD at 3.5 Å
markedly decreased, especially with compound 5e, while the per- resolution (PDB: 1SA0)7 using Discovery Studio 4 software pack-
centages in the G2/M phase of the cell cycle increased. age18 to shed light on their potential binding modes and investi-
Compound 5e had the highest effect on G2/M phase in both cell gate their similarity to the native ligand. Since the synthesized
Figure 5. Cell cycle analysis histograms for MCF-7 cells. (A) Control, (B) 4b, (C) 5e and (D) 5f.
Table 4. Results of the molecular docking study.
Compound
CDOCKER interaction energy
Type of interaction
Distance
Interacting moiety in the drug
Amino acid involved
DAMA–colchicine
ꢁ55.6986
H-Bonding
H-Bonding
H-Bonding
H-Bonding
Sigma-Pi
H-Bonding
H-Bonding
H-Bonding
Cation-Pi
H-Bonding
Sigma-Pi
H-Bonding
H-Bonding
Cation-Pi
2.1
2.3
1.9
2.4
2.8
2.5
2.3
2.1
6.3
2.3
2.8
2.1
2.7
4.9
6.3
2.3
2.7
2.2
SH
C:THR179
C:VAL181
D:CYS241
C:SER178
D:LYS352
D:THR353
C:SER178
C:THR179
D:LYS352
D:ALA250
C:LEU248
C:THR179
D:CYS241
D:LYS254
D:LYS352
D:CYS241
C:LEU248
D:ALA250
Carbonyl of tropone
OCH3
OH
Pyrazole ring
NH Pyrazole ring
OH
(R)-4b
ꢁ47.1318
(S)-4b
(R)-5e
ꢁ40.6168
ꢁ45.1529
OH
Pyrazole ring
OH
Pyrazole ring
OH
(S)-5e
(R)-5f
ꢁ48.6425
ꢁ51.0859
OCH3
Phenyl ring
Pyrazole ring
OCH3
Pyrazole ring
OH
Cation-Pi
H-Bonding
Sigma-Pi
(S)-5f
ꢁ52.3539
H-Bonding

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
11
Figure 6. 2D interaction diagram of the top docking pose of the R isomer of compound 4b.
Figure 7. Overlay of the top docking poses of R (green) and S (yellow) isomers
of 4b in the active site of tubulin (PDB: 1SA0).
Figure 8. 2D interaction diagram of the top docking pose of the R isomer of
compound 5e.
compounds are chiral, both isomers were enrolled in the docking
study. Table 4 illustrates the bonding and the nonbonding interac-
tions of the docked compounds with amino acids of the active
site. To ensure the validity of the docking protocol, re-docking of
the co-crystallized DAMA-colchicine into the active site of tublin
was performed. The coordinates of the best scoring docking pose
of DAMA-colchicine was compared with its coordinates in the co-
crystallized PDB file based on the binding mode and root mean
square deviation (rmsd). The docking validation showed a near
perfect alignment with the original ligand as obtained from the X-
ray resolved pdb file. The re-docked ligand showed an rmsd of
0.6995 Å with CDOCKER interaction energy of ꢁ55.6986 and the
same binding interactions. The binding site of DAMA-colchicine is
composed of two hydrophobic cavities accommodated by the
phenyl ring and tropone ring of DAMA-colchicine. Essential hydro-
gen bond of CYS241 with a methoxy group in DAMA-colchicine
was reported. Thiol and the carbonyl of tropone ring were
engaged in two hydrogen bonds with THR179 and VAL181,
respectively.
Figure 9. 2D interaction diagram of the top docking pose of the R isomer of
compound 5f.
or THR179, respectively (Figures 8 and 9), this hydrogen bond
was reported to increase the activity21. The S isomers of com-
pounds 5e and 5f were flipped in such a way that their OH
group was forming a hydrogen bond with Ala250 (Figures 10
and 11). Additional hydrogen bond interaction was observed at
Analysis of the docking results revealed that the docked com-
pounds showed comparable CDOCKER energy to the reference
ligand and they interacted with variable amino acids previously
reported in molecular modeling studies of CSIs20. A properly
positioned OH group at pyrazole ring of both isomers of com-
pound 4b (Figures 6 and 7) and R isomers of compounds 5e the methoxy group of compound 5f with CYS241 which was
and 5f was engaged in hydrogen bond interaction with SER178 reported to be crucial for CBSIs20. It is worth mentioning that

12
R. I. AHMED ET AL.
MCF-7 cell lines were analyzed revealing an increase of cell per-
centage at G2/M phase. Molecular docking was performed to
reveal the interaction of the active compounds into the colchicine
binding site of tubulin. Thereby, it could be claimed that the inda-
zole derivatives represented a promising starting point for further
study.
Disclosure statement
The authors report that that they have no conflicts of interest.
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