Antitumor evaluation and molecular docking study of substituted 2-benzylidenebutane-1,3-dione, 2-hydrazonobutane-1,3-dione and trifluoromethyl-1H-pyrazole analogues

Article abstract of DOI:10.3109/14756366.2014.960863

A series of 2-(arylidene)-1-(4-chlorophenyl)-4,4,4-trifluorobutane-1,3-diones (2-4), 4-(arylidene)-3-(4-chlorophenyl)-5-(trifluoromethyl)-4H-pyrazoles (5-7), 1-(4-chlorophenyl)-4,4,4-trifluoro-2-(2-(aryl)hydrazono)butane-1,3-diones (8, 9), 3-(4-chlorophen

Full text of DOI:10.3109/14756366.2014.960863

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J Enzyme Inhib Med Chem, Early Online: 1–9
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.960863
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RESEARCH ARTICLE
Antitumor evaluation and molecular docking study of substituted
2-benzylidenebutane-1,3-dione, 2-hydrazonobutane-1,3-dione and
trifluoromethyl-1H-pyrazole analogues
Ibrahim A. Al-Suwaidan1, Naglaa I. Abdel-Aziz2, Adel S. El-Azab1,3, Magda A.-A. El-Sayed4, Amer M. Alanazi1,
Mahmoud B. El-Ashmawy2, and Alaa A.-M. Abdel-Aziz1,2
2
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, Department of Medicinal
3
Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt, Department of Organic Chemistry, Faculty of Pharmacy,
4
Al-Azhar University, Cairo, Egypt, and Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, University of
Mansoura, Mansoura, Egypt
Abstract
Keywords
A series of 2-(arylidene)-1-(4-chlorophenyl)-4,4,4-trifluorobutane-1,3-diones (2–4), 4-(arylidene)-
3-(4-chlorophenyl)-5-(trifluoromethyl)-4H-pyrazoles (5–7), 1-(4-chlorophenyl)-4,4,4-trifluoro-2-
(2-(aryl)hydrazono)butane-1,3-diones (8, 9), 3-(4-chlorophenyl)-4-(2-(aryl)hydrazono)-5-(trifluor-
omethyl)-4H-pyrazoles (10, 11), 2-((3-(4-chlorophenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazol-
4-yl)methylene)malononitrile (13), 2-((5-(4-chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-pyra-
zol-4-yl)methylene)cycloalkan-1-ones (14, 15) and 1-(aryl)-3-(5-(4-chlorophenyl)-1-phenyl-3-
(trifluoromethyl)-1H-pyrazol-4-yl)prop-2-en-1-ones (16, 17) were designed, synthesized and
evaluated for their in vitro antitumor activity. 1-(4-Chlorophenyl)-4,4,4-trifluoro-2-(2-(4-
methoxyphenyl)hydrazono)butane-1,3-dione (8) showed potential and broad spectrum
antitumor activity compared to the known drug 5-FU with GI50, (6.61 and 22.60 mM),
TGI (42.66 and5100 mM) and LC50 (93.33 and5100 mM) values, respectively. On the other hand,
compound 8 yielded selective activities toward melanoma, colon, non-small lung and breast
cancer cell lines compared with erlotinib and gefitinib. Molecular docking methodology was
performed for compound 8 into binding site of B-RAFV600E and EGFR kinases which showed
similar binding mode to vemurafenib (PLX4032) and erlotinib, respectively.
Antitumor activity, chalcones, hydrazones,
molecular docking, pyrazoles
History
Received 11 May 2014
Revised 29 August 2014
Accepted 29 August 2014
Published online 28 November 2014
Introduction
A number of small molecule EGFR kinase inhibitors have
been evaluated in cancer clinical trials7–13, such as gefitinib
and erlotinib7–13. Moreover, B-RAF mutations are found in all
melanoma subtypes but are most common in melanomas derived
from skin without chronic sun-induced damage. Melanoma is the
most aggressive type of skin cancer in which B-RAFV600E
Cancer is a disease characterized by a shift in the controlled
mechanisms that govern cell proliferation and differentiation. It is
considered as a major health problem worldwide to treat and is the
leading cause of human mortality exceeded only by cardiovascu-
lar diseases1–6. It may require treatment with compounds that
could target multiple intracellular components1–6. Many of
chemotherapeutic agents currently used in cancer therapy are
agents which inhibit tumor growth by inhibiting the replication
and transcription of DNA. On the other hand, the epidermal
growth factor receptor (EGFR) is a cellular trans-membrane
tyrosine kinase that is over-expressed in a significant number of
kinase is over-expressed in 63% of all malignant melanomas14–18
.
Vemurafenib (PLX4032) is a drug which targets inhibition of
B-RAFV600E kinase14–18. It was approved by the U.S. Food and
Drug Administration (FDA) for treatment of late-stage melanoma,
so inhibition of B-RAFV600E kinase is a very potential avenue
for the treatment of melanoma14–18
.
On the other hand, several heterocyclic compounds
are intensively studied to enhance the range of anticancer
agents17–24. During the last decade, lots of pyrazole anticancer
agents were discovered, and such pyrazole core has increasingly
human tumors (e.g. breast, ovarian, colon, renal and prostate)7–13
Over-expression of EGFR family receptors has always been
observed in these tumors, approximately in 60% of all tumors.
.
attracted the attention of synthetic medicinal chemists17,18,20–24
.
Some small chemical molecules containing pyrazole ring
system have been exhibited potent anticancer activities and
identified as selective inhibitors of both B-RAFV600E and EGFR
kinases17,18,24. Moreover, chalcones and hydrazones are an
important class of molecules and speculated as promising
Address for correspondence: Alaa A.-M. Abdel-Aziz, Department of
Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia. Tel: +966-53-5991127. E-mail:
alaa_moenes@yahoo.com
candidates as anticancer agents25,26
.

2
I. A. Al-Suwaidan et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Following these findings, as part of our efforts to design novel Methodology of the in vitro cancer screen
antitumor agents, we thus initiated a screening program to search
for novel utility of a group of chalcone, hydrazone and pyrazole
Full in vitro five-dose antitumor assay
derivatives as potential antitumor. Moreover, molecular docking A primary anticancer assay was performed for an approximately
methodology was used to identify the structural features required 60 human tumor cell line panel derived from nine neoplastic
for the antitumor properties of these series. These models are diseases, in accordance with the protocol of the Drug Evaluation
necessary to obtain a consistent and more precise picture of the Branch, National Cancer Institute, Bethesda, MD31,32. Tested
biological active molecules at the atomic level which can be used compounds were added to the culture at a single concentration
to design novel therapeutic agents27–30. However, the results of (10ꢀ5 M), and the cultures were incubated for 48 h. End point
this molecular docking could support the postulation that our determinations were made with a protein binding dye, sulforho-
active compounds may act on the same enzyme target where damine B (SRB). The results for each tested compound were
EGFR and B-RAFV600E inhibitor act confirming the molecular reported as the growth percentage of the treated cells when
design of the reported class of antitumor agents.
compared to that of the untreated control cells. The percentage
growth was evaluated spectrophotometrically versus controls not
treated with test agents. The cytotoxic and/or growth inhibitory
effects of the most active selected compounds were tested in vitro
against the full panel of about 60 human tumor cell lines at 10-fold
Experimental
Synthesis
The target compounds in Scheme 1 were synthesized. The full dilutions of five concentrations ranging from 10ꢀ4 to 10ꢀ8 M.
details of their synthesis, spectroscopic and elemental character- A 48-h continuous drug exposure protocol was followed, and an
ization have been published recently according to our previous SRB protein assay was used to estimate cell viability or growth.
reports28,29
.
By use of the seven absorbance measurements [time zero, (Tz),
O
F C
3
N
Cl
Cl
Cl
Ar
R
N
HN
2
O
O
ArCHO
O
R
O
CF
EtOH
3
O
CF
NaOH/EtOH
3
Cl
Ar= 4-methoxyphenyl(2)
R= MeO (8), Cl (9)
Ar= 3,4-dimethoxyphenyl(3)
Ar= 2-furyl(4)
1
NH NH
2
2
NH NH
2
2
EtOH
EtOH
NHNH
2
Cl
/DMF
POCl
3
C H OH/H SO
N
2
5
2
4
R
N
CF
3
N
Ar
N
H
N
Cl
N
F C
3
N
N
Ar= 4-methoxyphenyl(5)
Cl
R= MeO (10), Cl (11)
Ar= 3,4-dimethoxyphenyl(6)
Ar= 2-furyl(7)
CF
3
OHC
12
N
N
CH (CN)
2
2
EtOH/NaOH
Cyclicketone
N
N
CF
O
3
CF
3
Cl
DMF/NaOAc
CH
CN
O
CN
Cl
R
n
13
CF
N
3
n= NCH (15)
3
N
R
O
R= MeO (16), Cl (17)
Scheme 1. Synthesis of the designed chalcone, hydrazone and pyrazole analogues.
Cl

DOI: 10.3109/14756366.2014.960863
Antitumor evaluation and molecular docking
3
control growth in the absence of drug (C) and test growth in the erlotinib in the PDB file 1M17 and vemurafenib in the PDB file
presence of drug at the five concentration levels (Ti)], the 3OG7, respectively, after which erlotinib and vemurafenib were
percentage growth was calculated at each of the drug concentra- deleted. The default Triangle Matcher placement method was
tions levels. Percentage growth inhibition was calculated as:
used for docking. GBVI/WSA dG scoring function which
estimates the free energy of binding of the ligand from a given
pose was used to rank the final poses. The ligand–enzyme
complex with lowest S_score was selected.
½ðTi ꢀ TzÞ=ðC ꢀ TzÞ ꢁ 100ꢂ for concentration of which Ti ꢃ Tz
ð1Þ
½ðTi ꢀ TzÞ=TzÞ ꢁ 100ꢂ for concentration of which Ti 5 Tz:
ð2Þ
Results and discussion
Chemistry
Three dose–response parameters (GI50, TGI and LC50) were
calculated for each compound. Growth inhibition of 50% (GI50)
was calculated from [(Ti ꢀ Tz)/(C ꢀ Tz)]ꢁ100 ¼ 50, which was
the drug concentration resulting in a 50% lower net protein
increase in the treated cells (measured by SRB staining) compared
to the net protein increase seen in the control cells. The drug
concentration resulting in total growth inhibition (TGI) was
calculated from Ti ¼ Tz. The LC50 (concentration of drug resulting
in a 50% reduction in the measured protein at the end of the drug
treatment compared to that at the beginning) indicating a net
loss of cells following treatment was calculated from
[(Ti ꢀ Tz)/Tz]ꢁ100 ¼ ꢀ50. Values were calculated for each of
these three parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded, the value
for that parameter was expressed as more or less for the maximum
or minimum concentration tested. The lowest values are
obtained with the most sensitive cell lines. The compounds
having GI50 ꢄ 100 lM were declared to be active.
The target compounds in Scheme 1 were synthesized in our
laboratories and showed significant anti-inflammatory with
COX-2 inhibitory activities. The full details of their synthesis,
spectroscopic and elemental characterization were published
recently28,29
.
Antitumor activity
Twelve compounds 2–7, 8, 10 and 13–16 were selected by
National Cancer Institute, Bethesda, MD (Tables 1 and 2) on the
basis of degree of the structure variation and computer modeling
techniques for evaluation of their antitumor activity31,32. The
selected compounds were subjected to in vitro anticancer assay
against tumor cells in a full panel of 60-cell lines taken from nine
different organs (lung, colon, breast, ovary, blood, kidney, skin,
prostate and brain). The compounds 2–7, 8, 10 and 13–16 were
evaluated at a single dose concentration of 10 mM, the percentages
of growth inhibitions over the 60 tested cell lines were determined
and the results were compared with 5-fluorouracil as reference
drug (Tables 1 and 2). In the screening methodology, each cell
line was inoculated and incubated for 24–48 h on a microtiter
plate. Test molecules were then added at a single concentration,
and the culture was incubated for further 48 h. The end point
determinations were made with a protein binding dye, sulforho-
damine (SRB). The results for each evaluated molecule were
reported as the percentage growth of the treated cells comparing
Docking methodology
Molecular modeling studies were performed33 with MOE
2008.10, software available from Chemical Computing Group
Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, QC.
Selection of protein crystal structures
Ligand-bound crystallographic structures of EGFR and to the untreated control cells. The preliminary screening results
B-RAFV600E kinases are available in the Protein Data and percentages of growth inhibition over sensitive cell lines are
Bank34,35. In this study, EGFR kinase crystal structure 1M17 shown in Tables 1 and 2.
and B-RAFV600E kinase were evaluated and selected for
The synthesized compounds 2–7, 8, 10 and 13–16 (Tables 1
docking34,35. The errors of the protein were corrected by and 2) displayed significant activity in the in vitro screening on
the structure preparation process in MOE. The first step in the the tested cell lines in 10 mM concentration with positive cytotoxic
generation of suitable protein structures is the assignment of effect (PCE) of 3/53–46/53. These molecules showed a cytotoxic
hydrogen positions on the basis of default rules. Water molecules effect on the most of the cancer cell lines especially compounds 3,
contained in the PDB file have been removed. Finally, partial 5, 7, 8 and 16 with mean growth inhibition percentages (MGI%)
charges (the Gasteiger methodology) were calculated, and the of 15.68, 16.95, 16.79, 37.13 and 9.92%, respectively. Derivatives
active site of the ensemble has been defined as the collection of based on hydrazone 8 were more active compared with the
˚
residues within 10.0 A of the bound inhibitor and comprised the corresponding chalcone 2 and pyrazole 10 analogues as indicated
union of all ligands of the ensemble. All atoms located less than by mean growth inhibition percentages of 37.13, 3.75 and
˚
2.11%, respectively. Moreover, the dimethoxy chalcone 3
10.0 A from any ligand atom were considered.
(MGI% ¼ 15.68) showed higher antitumor activity than the
Preparation of the ligand
corresponding mono-methoxy derivative
2
(MGI% ¼ 3.75).
Replacement of the 4-methoxybenzylidene with furylidene as in
compounds 5 and 7 leads to maintain the antitumor activity with
mean growth inhibition percentages of 16.95 and 16.79%,
respectively. On the other hand, conversion of compounds 3 and
8 to the corresponding pyrazoles 6 and 10 led to sharp decrease of
the antitumor activity with mean growth inhibition percentages of
15.68, 37.13, 0.25, and 2.11%, respectively (Table 1), which may
attributed to restricted conformation of compounds 6 and 10 on
the receptor binding site. Interestingly, trisubstituted pyrazoles 5
and 7 showed higher activity compared with the tetrasubstituted
derivatives 13, 14, 15, and 16 with mean growth inhibition
The ligand coordinates were built using the builder tool of the
MOE program33. Next, the correct atom types (including
hybridization states) and correct bond types were defined,
hydrogen atoms were added, charges were assigned to each
atom, and finally the structures were energy minimized
(MMFF94x, gradient: 0.01)36. The energies of ligand structures
were previously minimized using the semiempirical AM1
method37 with MOE program33.
Docking experiment
The docking experiment on EGFR and B-RAFV600E kinass was percentages of 16.95, 16.79, 0.81, 2.56, ꢀ2.99 and 9.92%,
carried out by superimposing the energy minimized ligand on respectively. Moreover, chalcone 16 is more active than the

4
I. A. Al-Suwaidan et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Table 1. Antitumor activity of chalcone, hydrazone and pyrazole derivatives 2–7, 8, 10 and 13–16 presented as growth inhibition percentages (GI%)
over 60 subpanel tumor cell lines.
60 cell lines assay in one dose 10.0 mM concentration: GI%
Compd. No.
MGI%
3.75
PCE
Most sensitive cell lines
2
11/59
Leukemia (CCRF-CEM: 10.3%, MOLT-4: 14.2%, SR: 23.8%), NSC Lung Cancer
(NCI-H322M: 22.3%, NCI-H522: 25.3%), Colon Cancer (HT29: 17.9%), Melanoma
(SK-MEL-2: 13.6%), Renal Cancer (A498: 17.6%, TK-10: 24.7%, UO-31: 21.4%),
Breast Cancer (MCF7: 22.9%).
3
15.68
32/59
Leukemia (CCRF-CEM: 34.5%, HL-60(TB): 14.4%, K-562: 53.5%, MOLT-4: 18.8%,
PRMI-8226: 70.5%, SR: 70.5%), NSC Lung Cancer (NCI-H226: 13.8%, HOP-92: 18.9%,
NCI-H23: 14.4%, NCI-H522: 32.6%), Colon Cancer (COLO 205: 14.9%, HCC-2998:
11.2%, HCT-116: 66.9%, HCT-15: 22.1%, HT29: 36.8%, KM12: 25.6%, SW-620: 47.3%),
CNS Cancer (SF-539: 13.3%, SNB-19: 11.6%, U251: 18.7%), Melanoma (LOX IMVI:
32.5%), Ovarian Cancer (OVCAR-3: 10.9%, OVCAR-4: 17.1%, OVCAR-8: 10.6%,
NCI/ADR-RES: 10.6%), Renal Cancer (CAKI-1: 13.6%, UO-31: 25.7%), Prostate Cancer
(PC-3: 10.2%), Breast Cancer (MCF7: 80.4%, MDA-MB-231/ATCC: 12.1%, T-47D:
25.3%, MDA-MB-468: 16.7%).
4
5
0.63
8/58
Leukemia (HL-60(TB): 12.5%, MOLT-4: 17.4%, SR: 12.8%), CNS Cancer (SF-295: 12.3%,
SNB-75: 11.6%), Melanoma (M14: 41.9%) Renal Cancer (UO-31: 35%), Prostate Cancer
(PC-3: 10.3%).
16.95
33/58
Leukemia (CCRF-CEM: 73.4%, HL-60(TB): 21.7%, K-562: 51.1%, MOLT-4: 29.4%,
PRMI-8226: 18.4%, SR: 59.8%), NSC Lung Cancer (EKVX: 28.7%, HOP-62: 10.9%,
NCI-H23: 18.5%, NCI-H226: 12.1%, NCI-H522: 27.7%), Colon Cancer (HCT-116: 70.9%,
HCT-15: 15.9%, KM12: 32.8%, SW-620: 31.5%), CNS Cancer (SF-539: 13.7%, SNB-19:
10.1%, SNB-75: 20%, U251: 16.4%), Melanoma (LOX IMVI: 28.4%, UACC-62: 12.6%),
Ovarian Cancer (IGROV1: 50.7%, OVCAR-3: 16.4%, OVCAR-8: 14%), Renal Cancer
(ACAKI-1: 13.7%, SN12C: 11.3%, UO-31: 56%), Prostate Cancer (PC-3: 13.1%),
Breast Cancer (MCF7: 81.5%, MDA-MB-231/ATCC: 20.4%, T-47D: 27.5%, HS 578T:
19.9%, MDA-MB-468: 11.2%).
6
7
ꢀ0.25
9/57
Leukemia (MOLT-4: 20.4%, SR: 13.9%), NSC Lung Cancer (EKVX: 14.3%, NCI-H23: 11%,
NCI-H522: 20.1%), Colon Cancer (HCT-15: 10.3%), Renal Cancer (UO-31: 21.4%),
Breast Cancer (MCF7: 24.1%, MDA-MB-468: 12%).
16.79
33/57
Leukemia (CCRF-CEM: 26.6%, HL-60(TB): 23.8%, K-562: 54.4%, MOLT-4: 16.2%,
SR: 58.7%), NSC Lung Cancer (A549/ATCC: 14.7%, NCI-H23: 18.2%, NCI-H460: 13.9%,
NCI-H522: 31.2%), Colon Cancer (COLO 205: 11.8%, HCC-2998: 32%, HCT-116: 13.4%,
HCT-15: 34.8%, HT29: 39.9%, KM12: 58.6%, SW-620: 31.7%), CNS Cancer (SF-268:
21.4%, SF-295: 12.8%, SNB-75: 12.5%, U251: 27.4%), Melanoma (LOX IMVI: 22.3%,
M14: 18.8%, SK-MEL-2: 41.8%, SK-MEL-5: 12.9%, UACC-257: 14.9%, UACC-62: 12%),
Ovarian Cancer (OVCAR-3: 17.1%, NCI/ADR-RES: 30.8%), Renal Cancer (CAKI-1:
14.2%, UO-31: 22.2%), Breast Cancer (MCF7: 40.5%, MDA-MB-231/ATCC: 22.3%,
MDA-MB-468: 59.2%).
8
37.13
46/53
Leukemia (CCRF-CEM: 58.9%), NSC Lung Cancer (A549/ATCC: 48.8%, HOP-92: 34.8%,
NCI-H23: 38.9%, NCI-H322M: 20.1%, NCI-H460: 43.15%, NCI-H522: 51.3%), Colon
Cancer (HCC-2998: 30.7%, HCT-116: 42.4%, HCT-15: 61.6%, HT29: 50.9%, KM12:
61.9%, SW-620: 56.7%), CNS Cancer (SF-268: 36.9%, SF-295: 88%, SF-539: 34.1%,
SNB-19: 16.6%, SNB-75: 14.7%, U251: 66.4%), Melanoma (LOX IMVI: 75.9%,
MALME-3M: 14.0%, M14: 65.9%, MDA-MB-435: 85.0%, SK-MEL-2: 42.7%,
SK-MEL-28: 16.3%, SK-MEL-5: 18.1%, UACC-257: 23.0%, UACC-62: 85.1%),
Ovarian Cancer (IGROV1: 33.8%, OVCAR-3: 35.7%, OVCAR-5: 59.5%, OVCAR-8:
29.9%, NCI/ADR-RES: 75.2%), Renal Cancer (786-0: 28.3%, A498: 26.4%, ACHN: 20.5%,
CAKI-1: 45.1%, SN12C: 20.2%, UO-31: 36.9%), Prostate Cancer (PC-3: 49.2%, DU-145:
26.7%), Breast Cancer (MCF7: 34.8%, MDA-MB-231/ATCC: 51.9%, BT-549: 21.7%,
T-47D: 29.6%, HS 578T: 29.0%).
10
13
14
2.11
0.81
2.56
8/51
7/58
12/53
NSC Lung Cancer (NCI-H522: 14%), Colon Cancer (KM12: 16.4%), Melanoma
(MDA-MB-435: 41.4%), Renal Cancer (A498: 44%, UO-31: 19.1%), Breast Cancer
(MCF7: 15.6%, T-47D: 16.9%, BT-549: 14.4%).
Leukemia (MOLT-4: 18.3%), CNS Cancer (SNB-75: 13.1%), Melanoma (MALME-3M:
12.9%, SK-MEL-2: 15.2%), Renal Cancer (TK-10: 11.2%, UO-31: 24.2%), Breast Cancer
(T-47D: 10.2%).
Leukemia (CCRF-CEM: 21.1%), NSC Lung Cancer (A549/ATCC: 17.5%, EKVX: 21.7%),
Colon Cancer (HCT-116: 13.6%), Melanoma (UACC-62: 13.2%), Renal Cancer (A498:
21.3%, CAKI-1: 16.1%, UO-31: 18.4%), Breast Cancer (MCF7: 13.8%, MDA-MB-231/
ATCC: 12.7%, HS 578T: 12.2%, T-47D: 23.6%).
15
16
ꢀ2.99
3/53
27/54
Leukemia (CCRF-CEM: 11%), Renal Cancer (A498: 24.2%, UO-31: 10.1%).
Leukemia (CCRF-CEM: 14.5%), NSC Lung Cancer (A549/ATCC: 18.7%, HOP-92: 34.9%,
NCI-H23: 19.2%, NCI-H460: 17.7%, NCI-H522: 21.1%), Colon Cancer (HCT-116: 36.6%,
HCT-15: 22.3%, HT29: 10.1%, KM12: 15.2%), CNS Cancer (SNB-75: 13.9%, U251:
13.7%), Melanoma (LOX IMVI: 25%, SK-MEL-2: 11%, SK-MEL-5: 14.7%, UACC-62:
17.5%), Ovarian Cancer (OVCAR-4: 11%, OVCAR-8: 22.2%, SK-OV-3: 17.3%), Renal
Cancer (A498: 12.9%, CAKI-1: 10%, RXF 393: 11.6%, SN12C: 11.9%, UO-31: 25.9%),
Breast Cancer (MCF7: 38.2%, MDA-MB-231/ATCC: 20.5%, T-47D: 35.2%).
9.92
PCE positive cytotoxic effect in the ratio between number of cell lines with percentage growth inhibition 510% and total number of cell lines.
MGI% mean growth inhibition percentage.

DOI: 10.3109/14756366.2014.960863
Antitumor evaluation and molecular docking
5
Table 2. Antitumor activity of the most active chalcone, hydrazone
and pyrazole 3, 5, 7, 8 and 16 with GI% over the most sensitive tumor
cell line.
Tables 1 and 2) and possessed a considerable cytotoxic activity
against cell lines of leukemia (GI% ¼ 34.5–73.4), non-small cell
lung (GI% ¼ 31.2–51.3), colon (GI% ¼ 30.7–70.9), CNS
(GI% ¼ 34.1–88.0), melanoma (GI% ¼–32.5–85.1), ovarian
% Growth
(GI% ¼ 30.8–75.2),
renal
(GI% ¼ 36.9–56)
and
breast
60 Subpanel
tumor cell lines
inhibition
(GI%)*
3
(GI% ¼ 34.8–81.5). This great inhibition at the mentioned con-
centration indicates a great potency for the compounds 3, 5, 7, 8
and 16 with a strong lethal effect over cancer cells.
5
7
8
16
5-Flu
Leukemia
CCRF-CEM
K-562
PRMI-8226
SR
Regarding the activity toward individual cell lines (Tables 1
and 2), compounds 3 and 5 showed selective activity against
leukemia cell lines CCRF-CEM, K-562, SR and PRMI-8226 with
GI% values of 34.5, 53.5, 70.5, 70.5, 73.4, 51.1, 59.8 and 18.4,
respectively. Non-small cell lung NCI-H522 cell line proved to be
selectively sensitive to 3, 5, 7, 8 and 16 with GI% values of 32.6,
27.7, 31.2, 51.3 and 21.1, respectively. In addition, compounds 8
and 16 proved to have equal susceptibility to the HOP-92 cell line
with GI% values of 34.8 and 34.9, respectively. Compound 8 have
broad spectrum activity against non-small cell lung A549/ATCC,
HOP-92, NCI-H23, NCI-H322M, NCI-H522 and NCI-H460 cell
lines with GI% values of 48.8, 34.8, 38.9, 20.1, 51.3 and 43.1,
respectively. Concerning colon cancer, compounds 3, 5, 7, 8 and
16 showed GI% values of 25.6, 32.0, 58.6, 61.9, 15.2, 66.9, 70.9,
13.4, 42.4 and 36.8 with colon KM12 and HCT-116 cell lines,
respectively. On the other hand, compounds 3, 5, 7, 8 and 16
verified sensitivity in 22.1, 15.9, 34.8, 61.6 and 22.3% to colon
HCT-15 cancer cells. Respecting melanoma, compounds 3, 5, 7, 8
and 16 are active against LOX IMVI cell lines with GI% values of
32.5, 28.4, 22.3, 75.9 and 25.0, respectively. Compound 8 showed
broad spectrum activities against melanoma LOX IMVI,
M14, MDA-MB-435, SK-MEL-28, SK-MEL-2, SK-MEL-5,
UACC-257 and UACC-62 cell lines with GI% values of 75.9,
65.9, 85.0, 16.3, 42.7, 18.1, 23.0 and 85.1. Pertaining to renal
cancer, compounds 3, 5, 7, 8 and 16 were active against UO-31
cell line with GI% values of 25.7, 56.0, 22.2, 36.9 and 25.9,
respectively. Renal CAKI-1 cell line sensitive to compound 8
with GI% value of 45.1. Relating to breast cancer, breast MCF7,
MDA-MB-231/ATCC, T-47D and MDA-MB-468 cell lines
possess convinced responsive to compounds 3, 5, 7, 8 and 16
with GI% values from 16 to 81%. Compounds 5 and 8 showed
GI% effectiveness against breast HS 578T cell line with values of
19.9 and 29.0, respectively. On the other hand, ovarian IGROV1,
OVCAR-3, OVCAR-5 and NCI/ADR-RES cell lines receptive to
compound 8 with GI% values of 33.8, 35.7, 59.5 and 75.2,
respectively. Additionally, compound 5 demonstrated remarkable
activities to IGROV1 with GI% value of 50.7, while NCI/
ADR-RES cell line sensitive to compound 7 with GI% value 30.8.
Finally, compound 8 was selected in advanced assay against a
panel of approximately 60 tumor cell lines at 10-fold dilution of
five concentrations (100, 10, 1, 0.1 and 0.01 mM), and the results
were compared with 5-fluorouracil, gefitinib and erlotinib as
reference drugs (Tables 3 and 4; Figure 1). Based on the
cytotoxicity assays, three antitumor activity dose–response par-
ameters were calculated for experimental molecule against each
34.5
53.5
70.5
70.5
73.4
51.1 54.4
58.9
57.1
42.3
41.4
59.8 58.7
Non-small cell lung cancer
A549/ATCC
HOP-92
48.8
34.8
38.9
43.15
34.2
50.6
39.0
34.9
36.6
NCI-H23
NCI-H460
NCI-H522
32.6
31.2 51.3
58.0
Colon cancer
HCC-2998
HCT-116
HCT-15
HT29
32
30.7
42.4
34.8 61.6
39.9 50.9
4100
17.8
66.9
36.8
47.3
70.9
KM12
SW-620
32.8 58.6 61.9
31.5 31.7 56.7
40.7
50.1
CNS cancer
SF-268
SF-295
SF-539
U251
36.9
88
34.1
66.4
59.0
69.1
4100
50.3
Melanoma
LOX IMVI
M14
32.5
75.9
65.9
30.4
Nt
MDA-MB-435
SK-MEL-2
UACC-62
85.0
41.8 42.7
85.1
36.6
95.5
39.7
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-5
NCI/ADR-RES
50.7
33.8
35.7
59.5
30.8 75.2
51.2
47.4
44.3
47.6
Renal cancer
CAKI-1
UO-31
45.1
36.9
39.4
41.3
56
Prostate cancer
PC-3
49.2
58.2
Breast cancer
MCF7
80.4
81.5 40.5 34.8
51.9
38.2
35.2
MDA-MB-231/
ATCC
T-47D
78.1
56.7
Nt
MDA-MB-468
59.2
5-Flu, 5-fluorouracil; Nt, not tested
*Only GI % higher than 30% are shown.
corresponding cyclic analogues 14 and 15 with mean growth cell lines: GI50, molar concentration of the compound that inhibits
50% net cell growth; TGI, molar concentration of the compound
inhibition percentages of 9.92, 2.56 and ꢀ2.99%, respectively.
By investigating the variation in selectivity and broad leading to total inhibition; LC50, molar concentration of the
spectrum of the tested compounds over the full panel of cell compounds leading to 50% net cell death (Tables 3 and 4;
lines, it was revealed that nearly five of the tested derivatives Figure 1). Furthermore, mean graph midpoints (MG_MID) were
(Tables 1 and 2) showed significant inhibition for the most cell calculated for each of the parameters, giving an average
lines used in this assay with growth inhibition reached to 88% and activity parameter over all cell lines for the tested molecules
are mainly active against leukemia, non-small cell lung (NSCLC), (Tables 3 and 4; Figure 1).
colon, CNS, melanoma, ovarian, renal and breast cell lines with
growth inhibition reached to 73.4, 51.3, 70.9, 88.0, 85.1, 75.2, 56, (Tables 3 and 4; Figure 1) possessed potent in vitro antitumor
and 81.5%, respectively. activity, with mean GI50 values across the 60 cell lines ranging
The above screening data analysis indicated that compound 8
Compounds 3, 5, 7, 8 and 16 showed broad spectrum from 1.84 to 11.00 mM. Mean GI50, TGI and LC50 of compound 8
and significant inhibition for cancer cells (59 cell lines; in comparison with standard anticancer agent 5-fluorouracil

6
I. A. Al-Suwaidan et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Table 3. Compounds 8 and 5-Flu median growth inhibitory (GI50, mM), total growth inhibitory (TGI, mM) and median lethal (LC50, mM) concentration
of in-vitro subpanel tumor cell lines.
Subpanel tumor cell lines
NSC lung
cancer
Colon
cancer
CNS
cancer
Ovarian
cancer
Renal
cancer
Prostate
cancer
Breast
cancer
Compd.
Activity
Leukemia
Melanoma
MG-MID*
8
GI50
TGI
LC50
GI50
TGI
5.08
60.73
4100
15.1
4100
4100
9.90
37.09
85.91
4100
4100
4100
5.46
46.20
84.4
8.4
4100
4100
8.73
52.85
4100
72.1
4100
4100
5.20
43.33
93.69
70.6
4100
4100
11.40
66.31
98.1
61.4
4100
4100
10.43
60.86
94.04
45.6
4100
4100
12.5
6.55
49.75
>100
76.4
4100
4100
6.61
42.66
93.33
22.60
4100
4100
85.45
>100
22.7
4100
4100
5-FU
LC50
*Full panel mean-graph midpoint (mM).
Table 4. GI50 values (mM) of compound 8, gefitinib and erlotinib over the
most sensitive cell lines of non-small lung cancer, colon cancer,
melanoma, breast cancer, CNS cancer and leukemia subpanels.
(LOX IMVI, M14, MDA-MB-435, SK-MEL-2 and SK-MEL-5)
cell line expresses high levels of B-RAFV600E kinase (Tables 3
and 4; Figure 1). Results indicated that compound 8 is more active
than gefitinib and erlotinib against most selected cell lines except
non-small lung (NCI-H522) and breast cancer (MDA-MB-468)
cell lines. Moreover, compound 8 is more potent than gefitinib
and erlotinib against all selected melanoma cell lines and showed
the lowest GI50 values.
The SAR study revealed that (1) potent antitumor activity of
tested compounds depended on the presence of a hydrazone and
chalcone scaffolds which were more active in comparison with
pyrazole analogue; (2) attachment of trisubstituent to pyrazole
ring system increases the activity in comparison with tetrasub-
stitent pyrazole; (3) introduction of two methoxy groups at the
chalcone part led to the enhancement of activity; (4) one methoxy
group favors the activity of pyrazole core, while two methoxy
moieties favor the activity of chalcone core. This would indicate
that increase of electron density on chalcone moiety is important
for the inhibitory activity; (5) replacement of the arylidene
attached to pyrazole with steric bulky chalcone fragment led to
decrease of the antitumor activity.
GI50 (mM)
Subpanel tumor cell lines
8
Gefitinib
Erlotinib
Non-small cell lung cancer
A549/ATCC
NCI-H460
NCI-H23
NCI-H522
3.83
3.77
11.0
4.93
7.9
6.3
15.9
6.3
7.9
5.0
20.0
1.0
Colon cancer
KM12
HT29
HCC-2998
HCT-15
SW-620
2.61
4.60
4.01
4.68
4.36
7.9
4.0
10.0
5.0
63.1
50.1
79.4
3.2
7.9
50.1
Ovarian cancer
IGROV1
OVCAR-4
OVCAR-8
NCI/ADR-RES
SK-OV-3
3.69
4.54
4.58
8.87
2.89
0.20
7.94
10.0
12.59
0.63
0.25
19.95
7.94
6.31
0.40
Melanoma
LOX IMVI
M14
MDA-MB-435
SK-MEL-2
SK-MEL-5
Molecular modeling and computational studies
3.29
2.84
2.49
2.78
3.63
7.9
5.0
3.2
12.6
4.0
5.0
6.3
15.9
12.6
15.8
The level of antitumor activities of the compound 8 over breast
cancer, colon, renal, small lung and melanoma cancer cells, in
which epidermal growth factor receptor (EGFR) and
B-RAFV600E kinases are highly expressed, prompted us to
perform molecular docking into the ATP binding site of EGFR
and B-RAFV600E kinase to predict if this compound has
analogous binding mode to the EGFR and B-RAFV600E kinase
inhibitors. We assumed that the active target compound 8 might
demonstrate antiproliferative activity against breast cancer, colon,
renal, small lung and melanoma cell lines through inhibition of
EGFR and B-RAFV600E.
Breast cancer
MCF7
T-47D
4.11
5.98
1.84
10
6.3
0.01
100
3.2
0.2
MDA-MB-468
CNS cancer
SF-295
SF-539
2.67
6.33
4.55
2.0
10
10
15.9
12.6
20.0
U251
Compound 8 was docked into receptor active site of EGFR
along with their inhibitor (Figure 2). All calculations were
performed using MOE 2008.10 software33 installed on 2.4G Core
(TM) i7. The crystal structure of epidermal growth factor receptor
with erlotinib (TarcevaÔ) (PDB code: 1M17)34 was obtained
from protein data bank (PDB). The automated docking program
Leukemia
CCRF-CEM
K-562
4.61
3.47
4.26
5.0
2.5
3.2
15.9
15.9
6.3
SR
(5-Flu) are given in Table 3. This compound 8 ranks as nearly of MOE 2008.10 was used to dock compound 8 along with the
3-fold more potent compared with the reference standard 5-Flu as inhibitor erlotinib into ATP binding site of EGFR. The complexes
indicated by mean GI50 of 6.61 and 22.6 mM, respectively.
were energy-minimized with a MMFF94 force field36 till the
Moreover, comparison of the antitumor activity presented as gradient convergence 0.01 kcal/mol was reached. The binding
GI50 (mM) values of compound 8 with the activity of gefitinib and energies of compound 8 and erlotinib docked into the active site
erlotinib was performed against non-small lung cancer (A549/ of EGFR were ꢀ24.99 and ꢀ27.03 kcal/mol, respectively
ATCC, NCI-H460, NCI-H23 and NCI-H522), colon cancer (Figure 2). These docking studies have revealed that a water
(KM12, HT29, HCC-2998, HCT-15 and SW-620), breast cancer (HOH-10) molecule-mediated hydrogen bonding interaction is
(MCF7, T-47D and MDA-MB-468) cell lines in which EGFR was observed between the methoxy group of 4-methoxyphenyl
over-expressed (Table 4; Figure 1). In addition, melanoma fragment and the Thr-766 side chain. This interaction revealed

DOI: 10.3109/14756366.2014.960863
Antitumor evaluation and molecular docking
7
National Cancer Institute Developmental Therapeutics Program
Dose Response Curves
NSC: 763019 / 1
SSPL: 0XIV
EXP. ID: 1201NS94
Report Date: October 02, 2013
Test Date: January 17, 2012
Colon Cancer
Leukemia
Non-Small Cell Lung Cancer
100
50
100
50
100
50
0
0
0
−50
−100
−50
−100
−50
−100
−9
−8
−7
−6
−5
−4
−4
−4
−9
−8
−7
−6
−5
−4
−4
−4
−9
−8
−7
−6
−5
−4
−4
−4
Log of Sample Concentration (Molar)
Log of Sample Concentration (Molar)
Log of Sample Concentration (Molar)
10
10
10
CCRF-CEM
MOLT-4
HL-60(TB)
RPMI-8226
K-562
SR
A549/ATCC
NCI-H226
NCI-H522
EKVX
NCI-H23
HOP-62
NCI-H460
COLO205
HCT-15
SW-620
HCC-2998
HT29
HCT-116
KM12
CNS Cancer
Melanoma
Ovarian Cancer
100
50
100
50
100
50
0
0
0
−50
−50
−50
−100
−9
−100
−9
−100
−9
−8
−7
−6
−5
−8
−7
−6
−5
−8
−7
−6
−5
Log of Sample Concentration (Molar)
Log of Sample Concentration (Molar)
Log of Sample Concentration (Molar)
10
10
10
SF-268
SNB-19
SF-295
SNB-75
SF-539
U251
LOXIMVI
MDA-MB-435
SK-MEL-5
MALME-3M
SK-MEL-2
UACC-257
M14
IGROV1
OVCAR-5
SK-OV-3
OVCAR-3
OVCAR-8
OVCAR-4
NCI/ADR-RES
SK-MEL-28
UACC-62
Renal Cancer
Prostate Cancer
Breast Cancer
100
50
100
50
100
50
0
0
0
−50
−50
−50
−100
−9
−100
−9
−100
−9
−8
−7
−6
−5
−8
−7
−6
−5
−8
−7
−6
−5
Log of Sample Concentration (Molar)
Log of Sample Concentration (Molar)
Log of Sample Concentration (Molar)
10
10
10
786-0
CAKI-1
TK-10
A498
RXF393
UO-31
ACHN
SN12C
PC-3
DU-145
MCF7
BT-549
MDA-MB-231/
T-47D
HS578T
MDA-MB-468
Figure 1. Dose-response curves (% growth versus sample concentration at NCI fixed protocol, mM) obtained from the NCIs in-vitro disease-oriented
human tumor cells line of compound 8 on nine cancer disease.
Figure 2. Right panel shows docking studies on compound 8 with active site of epidermal growth factor receptor and left panel shows superimposition
of compound 8 (red colored) on erlotinib (yellow colored) inside the pockets of active site. Hydrogen bonds are shown in green.
the importance of 4-methoxyphenyl fragment for binding and the fashion similar to erlotinib and showed the occurrence of three
˚
subsequent inhibitory capacity. Similarly, the NH atom of hydrogen bonds with and HOH-10 (3.19 A)-mediated hydrogen
hydrazone part and trifluoromethyl fragment interact with the bonding interaction with Thr-766 side chain. Moreover, the
amino acid Asp-831. Compound 8 complexed with EGFR-TK in a hydrazone NH atom and CF3 fragment showed two additional

8
I. A. Al-Suwaidan et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 3. Right panel shows docking studies on compound 8 with B-RAFV600E kinase domain and left panel shows superimposition of compound 8
(red colored) on PLX4032 inhibitor (green colored) inside the pockets of active site. Hydrogen bonds are shown in green.
˚
hydrogen bonds with Asp-831 (2.91 and 2.96 A, respectively). Declaration of interest
In short, Figure 2 demonstrates binding model of hydrazone 8 in
the ATP binding site, and the results of this molecular docking
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for funding this
could support the postulation that our active compound may act
on the same enzyme target where EGFR inhibitor acts confirming
the molecular design of the reported class of antitumor
research group No. (RG-1435-046).
agents11,12,18,24
.
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compound 8 over melanoma cancer cells, in which B-RAFV600E
kinase is highly expressed14–17,35, prompted us to perform
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kinase to predict if this compound 8 has analogous binding mode
to the V600E-B-RAF kinase inhibitors (Figure 3). The crystal
structure of B-RAFV600E kinase in complex with vemurafenib
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