Synthesis and mechanistic studies of curcumin analog-based oximes as potential anticancer agents

Article abstract of DOI:10.1111/cbdd.12964

The incidence of cancer can be decreased by chemoprevention using either natural or synthetic agents. Apart from synthetic compounds, numerous natural products have exhibited promising potential to inhibit carcinogenesis in vivo. In this study, α, β-unsaturated carbonyl-based anticancer compounds were used as starting materials to synthesize new oxime analogs. The findings from the antiproliferative assay using seven different human cancer cell lines provided a clear picture of structure–activity relationship. The oxime analogs namely 7a and 8a showed strong antiproliferative activity against the cell lines. The mechanistic effects of compounds on EGFR-TK kinases and tubulin polymerization and BRAF^{V 600E} were investigated. In addition, the efficacy of compounds in reversing the efflux-mediated resistance developed by cancer cells was also studied. The compounds 5a and 6a displayed potent activity on various targets such as BRAF^{V 600E} and EGFR-TK kinases and also exhibited strong antiproliferative activity against different cell lines hence showing potential of multifunctional anticancer agents.

Full text of DOI:10.1111/cbdd.12964

DR. BAHAA YOUSSIF (Orcid ID : 0000-0002-6834-6548)
DR. SYED NASIR ABBAS BUKHARI (Orcid ID : 0000-0001-8125-7972)
Received Date : 10-Oct-2016
Revised Date : 22-Jan-2017
Accepted Date : 28-Jan-2017
Article type : Research Article
Synthesis and mechanistic studies of curcumin analogs based
oximes as potential anticancer agents
a
Hua-Li Qin*a‡, Jing Leng , Bahaa G. M. Youssifb,c, Muhammad Wahab Amjadd, Maria Abdul
Ghafoor Rajad, Muhammad Ajaz Hussaine, Zahid Hussainf, Syeda Naveed Kazmig, Syed Nasir
Abbas Bukhari*a,c‡
a. Department of Pharmaceutical Engineering, School of chemistry, chemical engineering
and life science, Wuhan University of Technology, 205 Luoshi Road, Wuhan,430070,
P.R.China.
b. Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut
University, Assiut, 71526, Egypt.
c. Department of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University,
Aljouf, Sakaka2014, Saudi Arabia
d. College of Pharmacy, Northern Border University, King Abdullah Road, Rafha, Saudia
Arabia..
e. Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan.
f. Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar
Puncak Alam 42300, Selangor, Malaysia.
g. Department of Mathematics, Capital University of Science and Technology (CUST),
Islamabad Expressway, Kahuta Road Zone-V, Islamabad, Pakistan.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12964
This article is protected by copyright. All rights reserved.

‡These authors contributed equally
* Authors to whom correspondence should be addressed;
Hua-Li Qin: qinhuali@whut.edu.cn; Syed Nasir Abbas Bukhari: snab_hussaini@yahoo.com;
Abstract
The incidence of cancer can be decreased by chemoprevention using either natural or synthetic
agents. Apart from synthetic compounds, numerous natural products have exhibited promising
potential to inhibit carcinogenesis in vivo. In this study, α, β-unsaturated carbonyl based
anticancer compounds were used as starting materials to synthesize new oxime analogs. The
findings from the antiproliferative assay using seven different human cancer cell lines provided a
clear picture of structure-activity relationship. The oxime analogs namely 7a and 8a showed
strong antiproliferative activity against the cell lines. The mechanistic effects of compounds on
EGFR TK kinases and tubulin polymerization, and BRAFV600E were investigated. In addition, the
efficacy of compounds in reversing the efflux-mediated resistance developed by cancer cells was
also studied. The compounds 5a and 6a displayed potent activity on various targets such as
BRAFV600E and EGFR TK kinases and also exhibited strong antiproliferative activity against
different cell lines hence showing potential of multifunctional anticancer agents.
Keywords: Natural compounds; α, β-unsaturated carbonyl; tubulin polymerization; epidermal
growth factor receptor (EGFR); multidrug resistance (MDR).
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INTRODUCTION
Cancer is one of the main causes of mortality around the globe. The careful estimation and
statistics of World Health Organization (WHO) show that the number of new cases of cancer is
likely to increase by around 70% in next 2 decades. Some aspects such as virus infection, chronic
inflammation, genetic variation and life style may affect the vulnerability to cancer. Apart from
orthodox treatments including radiotherapy and chemotherapy, molecular targeted therapy is
evolving to be a prospective technique for cancer therapeutics (1). For the individuals who are at
high risk of developing cancer, chemoprevention might be an alternate interference to delay or
inhibit carcinogenesis (2). Although several chemotherapeutic agents have been in the clinic for
years, still, there is a long way for chemopreventive agents to be safely administered to humans.
The recognition of biomarkers and targets that could help monitor the efficacy of
chemopreventive agents is an enormous task (1).
The synthesis of novel compounds or alteration or modification in the structure of natural
compounds on the basis of designs of a natural compound scaffolding, have delivered us lots of
vital new drugs within the agriculture, medicine and food spheres (3, 4). Due to their acceptable
safety profiles, plant-based natural compounds have continuously gathered the attention of
scientists around the world on the basis of being biocompatible. Plant-based compounds are
considered as potentially safe and effective anticancer and skin lightening agents (5). Curcumin
is a natural compound derived from turmeric, a spice that has been used for decades for its
several health benefits. The anticancer characteristics of curcumin have been under the spotlight
from the past 3-4 decades. Lately, we reported a series of thirty compounds bearing α, β-
carbonyl moiety as present in curcumin for potential use in agriculture, food, cosmetics and
medicine industry (6). The effect of synthesized compounds on seven different human cancer
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cell lines was assessed. Some compounds were found to be strong anticancer agents exhibiting
81-82% cytotoxicity. In another study, we found that the oxime analogs showed strong
antiproliferative activity against cancer cell lines as compared to curcumin-like oxime ether,
cyclohexanone and tetralone analogs (7).
In the present study, we extended our previously reported work (6) by synthesizing new oxime
derivatives of most potent curcumin-like α, β-unsaturated carbonyl-based compounds reported in
an earlier study. The anticancer activity of all new oxime derivatives was assessed and the
derivatives were subjected to further studies to investigate their effects on tubulin
polymerization, EGFR TK kinases and BRAFV600E and were tested in vitro for the reversal of
efflux-mediated resistance developed by the cancer cells.
EXPERIMENTAL SECTION
Synthesis of new oxime analogs
The previously reported method (7) by our group was used to synthesize new oxime analogs
using most potent α, β-unsaturated carbonyl based compounds (1-8)(6) as precursors. Each
selected compound (1 mmol) was reacted to hydroxylamine hydrochloride (2 mmol) in 10 mL
ethanol to produce the corresponding oxime (1a-8a). The reaction mixture was filtered upon
completion and rotary evaporator was used to evaporate the solvent. Distilled water (15 mL) and
dichloromethane (15 mL x 3) were added to the mixture for the extraction of organic
compounds. To dry the organic extracts, anhydrous MgSO4 was added, followed by filtration.
TLC was used to check the purity of product. Ethylacetate was used to recrystallize the product
yielding solid powder. Some products were purified by column chromatography using
ethylacetate: hexane (70:30 v/v) as eluent.
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2,6-Bis[4-(diethoxymethyl)benzylidene]cyclohexanone oxime (1a)
Yield (41%). mp: 117-118 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.42 (s, H), 7.47 (s, 2H), 6.99 (d,
J=8Hz, 4H), 6.55 (d, J=8Hz, 4H), 5.85 (s, 2H), 3.49 (q, J=7, 8H), 2.29 (t, J=7 Hz, 4H), 1.78 (m,
2H), 1.25 (t, J=7.5, 12H); 13C NMR (125 MHz, CDCl3) δ: 166.5, 152.7, 145.2, 135.5, 131.2,
127.5, 125.2, 100.7, 55.5, 28.5, 26.2, 16.8 ; HRMS (ESI) m/z: [M+H]+ calculated 494.6423,
found 494.6525, Microanalysis calculated for C30H39NO5 (493.63), C: 72.99%, H: 7.96%. N:
2.84%. Found C: 72.98%, H: 8.15%, N: 2.75%.
2,6-Bis-(4-dimethylamino-2-nitro-benzylidene)-cyclohexanone oxime (2a)
Yield (50%). mp: 141-142 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.37 (s, H), 7.64 (s, 2H), 7.22 (d,
J=8Hz, 2H), 6.97 (d, J=8Hz, 2H), 6.82 (s, 2H), 3.15 (s, 12H), 2.55 (t, J=7 Hz, 4H), 1.82 (m, 2H)
13
; C NMR (125 MHz, CDCl3) δ: 169.0, 148.5, 145.2, 144.2, 140.5, 128.5, 117.1, 115.2, 105.1,
46.9, 29.4, 27.1; HRMS (ESI) m/z: [M+H]+ calculated 466.5096, found
466.5037,
Microanalysis calculated for C24H27N5O5 (465.50), C: 61.92%, H: 5.85%, N: 15.04%. Found C:
62.10%, H: 5.87%, N: 15.00%.
3,5-Bis-(4-diethoxymethyl-benzylidene)-tetrahydro-pyran-4-one oxime (3a)
Yield (51%). mp: 109-110 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.41 (s, H), 7.65 (s, 2H), 7.12 (d,
J=8Hz, 4H), 6.65 (d, J=8Hz, 4H), 5.55 (s, 2H), 3.69 (q, J=7, 8H), 2.67 (s, 4H), 1.21 (t, J=8,
12H); 13C NMR (125 MHz, CDCl3) δ: 167.9, 150.5, 143.4, 135.1, 130.7, 126.4, 125.3, 100.5,
62.5, 55.2, 16.7; HRMS (ESI) m/z: [M+H]+ calculated 496.6151, found 496.6255, Microanalysis
calculated for C29H37NO6 (495.61), C: 70.28%, H: 7.52%, N: 2.83%. Found C: 70.46%, H:
7.57%, N: 2.80%.
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3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]tetrahydro-pyran-4-one oxime (4a)
Yield (45%). mp: 185- 186 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.49 (s, H), 7.61 (s, 2H), 7.26 (d,
J=8Hz, 2H), 6.94 (d, J=8Hz, 2H), 6.55 (s, 2H), 3.12 (s, 12H), 2.90 (s, 4H); 13C NMR (125 MHz,
CDCl3) δ: 169.5, 148.1, 146.5, 144.9, 139.2, 128.8, 118.6, 116.2, 102.3, 65.9, 46.8; HRMS (ESI)
m/z: [M+H]+ calculated 468.4824, found 468.4729, Microanalysis calculated for C23H25N5O6
(467.47), C: 59.09%, H: 5.39%, N: 14.98%. Found C: 59.27%, H: 5.52%, N: 14.62%.
3,5-Bis[4-(diethoxymethyl)benzylidene]-1-methyl-piperidin-4-one oxime (5a)
Yield (39%). mp: 144-145 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.45 (s, H), 7.23 (s, 2H), 7.02 (d,
J=8Hz, 4H), 6.91 (d, J=8Hz, 4H), 5.57 (s, 2H), 3.20 (q, J=7.5, 8H), 2.72 (s, 4H), 2.14 (s, 3H),
13
1.18 (t, J=7.0, 12H); C NMR (125 MHz, CDCl3) δ: 169.0, 148.5, 146.2, 139.3, 135.7, 128.4,
126.5, 100.9, 56.5, 50.9, 40.5, 16.2 ; HRMS (ESI) m/z: [M+H]+ calculated 509.6569, found
509.6452 [M+H]+, Microanalysis calculated for C30H40N2O5 (508.65), C: 70.84%, H: 7.93%, N:
5.51%. Found C: 70.99%, H: 8.05%, N: 5.49%.
3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]-1-methyl-piperidin-4-one oxime (6a)
Yield (42%). mp: 176- 177 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.40 (s, H), 7.16 (s, 2H), 7.05 (d,
13
J=8Hz, 2H), 6.87 (d, J=8Hz, 2H), 6.72 (s, 2H), 3.18 (s, 12H), 2.99 (s, 4H), 2.17 (s, 3H); C
NMR (125 MHz, CDCl3) δ: 165.6, 148.7, 146.3, 142.1, 141.5, 126.4, 118.0, 115.4, 106.7, 45.2,
42.8, 37.5; HRMS (ESI) m/z: [M+H]+ calculated 481.5243, found 481.5210, Microanalysis
calculated for C24H28N6O5 (480.52), C: 59.99%, H: 5.87%, N: 17.49%. Found C: 60.24%, H:
5.95%, N: 17.25%.
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3,5-Bis[4-(diethoxymethyl)benzylidene]piperidin-4-one oxime (7a)
Yield (51%). mp: 103-105 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.35 (s, H), 7.29 (s, 2H), 6.97 (d,
J=7.5Hz, 4H), 6.77 (d, J=7.5Hz, 4H), 5.62 (s, 2H), 3.29 (q, J=7.5, 8H), 2.62 (s, 4H), 1.20 (t,
J=7.0, 12H); 13C NMR (125 MHz, CDCl3) δ: 161.3, 144.2, 142.7, 138.0, 136.5, 128.7, 125.2,
101.4, 55.5, 48.2, 17.1 ; HRMS (ESI) m/z: [M+H]+ calculated 495.6304, found 495.6329
[M+H]+, Microanalysis calculated for C29H38N2O5 (494.62), C: 70.42%, H: 7.74%, N: 5.66%.
Found C: 70.59%, H: 7.85%, N: 5.52%.
3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]piperidin-4-one oxime (8a)
Yield (40%). mp: 181- 182 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.39 (s, H), 7.22 (s, 2H), 7.05 (d,
J=8Hz, 2H), 6.89 (d, J=8Hz, 2H), 6.72 (s, 2H), 3.15 (s, 12H), 3.07 (s, 4H); 13C NMR (125 MHz,
CDCl3) δ: 169.4, 148.5, 146.3, 142.0, 140.9, 126.4, 117.2, 114.5, 102.4, 48.5, 46.7; HRMS (ESI)
m/z: [M+H]+ calculated 467.4977, found 467.5014 [M+H]+, Microanalysis calculated for
C23H26N6O5 (466.49), C: 59.22%, H: 5.62%, N: 18.02%. Found C: 59.29%, H: 5.65%, N:
17.95%.
Biological evaluation
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to
determine the effect of new oxime compounds on mammary epithelial cells (MCF-10A) (6, 7).
Different types of cell lines including HT-29 (colon cancer), PC-3 (prostate cancer), A-549
(epithelial), Panc-1(pancreas cancer), MCF-7 (breast cancer), H-460 (lung cancer) and PaCa-2
(pancreatic carcinoma) were used to perform PI fluorescence assay (6, 7) to study the
antiproliferative efficacy of compounds. Tubulin Polymerization Assay Kit (Cytoskeleton Inc.,
Denver, CO, USA), which works on the principle of fluorescent reporter enhancement (8), was
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used to investigate the effect of compounds on tubulin polymerization. EGFR and BRAF and
MDR-reversal assays were performed as reported previously (7). Detailed methodologies are
provided in supplementary materials for this article.
Statistical analysis
All studies were carried out thrice and data are presented as the mean standard error of mean
(SEM). Graph Pad Prism 5 software was used to calculate IC50 values. One-way analysis of
variance (ANOVA) for multiple comparisons was used to analyze the data.
RESULTS AND DISCUSSION
Synthesis of oximes
Previously reported method was used to synthesize new oxime analogs in this study (7).
Respective oximes (1a-8a) were synthesized by reacting selected α, β-unsaturated carbonyl
based compound (1 mmol) with hydroxylamine hydrochloride (2 mmol) in 10 mL ethanol
1
(Scheme 1). Different techniques such as microanalysis (CHNS), HRMS, H NMR, 13C NMR
and melting point (MP) analysis were used to characterize all synthesized compounds and the
data are provided in experimental part. The purity grade of all compounds is ≥95%.
Cell viability
The cell viability assay was performed using human mammary gland epithelial cell line (MCF-
10A). The synthesized compounds were treated with MCF-10A cells for 96 h and MTT assay
was used to determine cell viability. The results are shown as toxicity (%) in Table 1. All
compounds exhibited a cell viability of more than 88% and hence were nontoxic.
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Antiproliferative activity
Different types of cell lines including HT-29 (colon cancer), PC-3 (prostate cancer), A-549
(epithelial), Panc-1(pancreas cancer), MCF-7 (breast cancer), H-460 (lung cancer) and PaCa-2
(pancreatic carcinoma) were used to perform PI fluorescence assay to study the antiproliferative
efficacy of compounds. The trend of cell growth inhibition by individual compound was similar
against various cell lines. The IC50 for all compounds was calculated using GraphPad Prism
software (GraphPad Software, San Diego, CA, USA). According to IC50 data, substantial
association was observed among compounds in relation to their chemical structures.
The compound 8a exhibited strongest anticancer activity with IC50 of 0.02 µM for Panc-1 cells,
PaCa-2, PC-3 and MCF-7, followed by 7a with IC50 of 0.03 µM for MCF-7 cell line. The activity
of these two compounds was similar to that of positive control erlotinib. Following 7a and 8a
were oxime compounds 5a and 6a which also exhibited strong anticancer potency (Table 1).
Next to follow were two compounds 3a and 4a which also displayed potent cancer cell growth
inhibition however less in comparison to 5a and 6a.
All oxime analogs exhibited strong antiproliferative activity against 7 different cell lines in
comparison to parent α, β-unsaturated carbonyl-based compounds (6), with IC50 in range of 0.02
to 2.7 µM as shown in Table 1. Among oxime analogs same behavior regarding substitution
patterns on aromatic rings was witnessed as by parent curcumin-like compounds in previous
study (6). The anticancer activity of curcumin-related compounds was enhanced by the
introduction of 2-nitro-4-dimethylamine combination on aromatic rings. Amongst the
investigated compounds, two substitution patterns were present and the results showed that
compounds having combination of 4-dimethylamine and 2-nitro possessed extremely potent
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anticancer activity in comparison to those having diethoxymethyl group substitution at position 4
of rings.
Previously (6), curcumin-like compounds that are used in this study to synthesize new oximes
showed unlike behavior as far as linkers were concerned. Amongst curcumin-like compounds (1-
8), tetrahydropyran-4-one (3-4) linker-containing compounds showed the most powerful
activities, temperate activity was exhibited by N-Methyl-4-piperidone-containing compounds (5-
6), whereas compounds 7-8 bearing 4-piperidone moiety exhibited strong inhibition potential,
nonetheless it was weaker as compared to that of compounds having N-Methyl-4-piperidone.
Among newly synthesized oxime analogs (1a-8a), 4-piperidone (7a-8a) linker-containing
compounds showed the most potent activities, strong cell proliferation inhibitory potential was
shown by N-Methyl-4-piperidone-bearing compounds (5a-6a), though compounds 3-4
containing tetrahydropyran-4-one linker displayed potent inhibitory potential nevertheless it was
weaker to that of compounds having N-Methyl-4-piperidone. The resultant difference in the
activity of compounds could be attributed to the molecular interactions owing to the presence of
oxime moiety in newly synthesized compounds.
Anticancer mechanistic studies such as BRAFV600E, tubulin polymerization and EGFR-TK were
performed to check the activity of all new compounds, in addition to the in vitro investigation of
multidrug resistance reversal potential of the compounds.
Tubulin polymerization assay
Tubulin is a globular protein which has appeared to be an important molecular target in drug
discovery against cancer. Tubulin has important roles in numerous cell processes including
segregation of chromosomes in mitosis, cell signaling and maintenance of skeletal integrity of
cell (9, 10). A number of compounds have been identified which attach to various parts of the
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tubulin protein and thus inhibit the polymerization or depolymerization of microtubules leading
to mitotic spindle arrest (11). Compounds which are capable of disturbing cellular microtubule
tubulin equilibrium are beneficial in the treatment of diseases (12, 13). The accomplishment of
inhibitors of tubulin polymerization as anticancer agents has inspired interest to recognize new
compounds which could be more effective in tumors or targeted tissues.
Figure 1 summarizes the effect of new synthetic compounds on tubulin polymerization. Most
compounds exhibited influence on the assembly of tubulin with compounds 3a and 4a proving to
be strongest inhibitors of tubulin assembly. As per PI fluorescence assay, compounds 5a and 6a
did not inhibit tubulin assembly suggesting a different mechanism behind the observed
cytotoxicity rather than tubulin inhibition. As compared to model antimitotic chemotherapeutic
drug docetaxel, none of the compound showed potent microtubule-stabilizing potential (14).
EGFR inhibition
The epidermal growth factor receptor (EGFR) belongs to the erbB family of closely linked cell
membrane receptors including EGFR (erbB-1 or HER1), erbB-2 (HER2), erbB-3 (HER3), and
erbB-4 (HER4) (15, 16). EGFR expression, overexpression, or dysregulation is witnessed in
many human solid tumors, including colorectal, non-small cell lung (NSCLC), ovarian, breast
and head and neck cancers (17-20). The activation of EGFR might enhance tumor growth by
increasing invasive capacity, by blocking apoptosis, cell adhesion, motility and proliferation
(19). Small molecule EGFR inhibitors are known to be potent antitumor moieties (17). Erlotinib
has exhibited potential against numerous human cancer types in clinical trials and has been
approved for the treatment of colon cancers and NSCLC (21).
Table 2 shows the findings of EGFR-TK assay performed to evaluate the EGFR
inhibitory potential of new compounds. The findings of the cancer cell-based assays mentioned
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before were complemented by the results from this assay. Potent EGFR inhibition was shown by
all compounds with IC50 in the range of 0.04 to 2.5 µM. According to Table 2, compounds 5a
and 6a were discovered to be most active and they inhibited EGFR similar to that of positive
control erlotinib (IC50 = 0.05 0.02 µM). The activity of compounds 5a and 6a were found to be
enhanced by the addition of N-Methyl-4-piperidone moiety. Although, they did not inhibit
tubulin polymerization nonetheless were found to be potent inhibitors of EGFR. The compounds
7a-8a bearing 4-piperidone moiety also exhibited potent inhibition of EGFR however the activity
was marginally weaker in contrast to compounds having N-Methyl-4-piperidone linker. The
study showed that α, β-unsaturated carbonyl-based oxime compounds are strong inhibitors of
EGFR and possess potential to be used as anticancer agents.
BRAFV600E inhibitory activity
High occurrence of BRAF mutations (particularly, the replacement of glutamic acid for valine at
position 600, V600E) have been observed in melanomas. This replacement which lies in the
kinase activation loop, principally prevents the requirement for phosphorylation and leads to
constitutively active BRAF that shows almost a 500-fold upsurge in activity of kinase over wild
type isoform (22-24). Oncogenic BRAFV600E avoids the necessity for upstream regulation by
phosphorylation that leads to a MAPK signaling pathway which is activated in the deficiency of
extracellular growth factor signals, bringing augmented survival of cell, progression of tumor
and unrestrained proliferation of cell. Mutant BRAF signaling inhibition, by either inhibition of
MEK or by direct inhibition of the enzyme, has been revealed to stop the growth of tumor (25,
26). Lately, BRAF mutant melanoma treatment with selective BRAF inhibitor has exhibited
antitumor activity (27), authenticating mutant BRAF as therapeutic target and providing
prospects for the development of anti- melanoma drugs. So, inhibitors (having small molecules)
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which target V600E mutant BRAF protein kinase are being designed and developed for cancer
treatment.
The potential of newly synthesized compounds against BRAFV600E was investigated using an in
vitro experiment. Table 2 shows the IC50 (ranging from 1.3-3.1 µM) of all investigated
compounds. All α, β-unsaturated carbonyl-based oxime compounds strongly inhibited
BRAFV600E. The compounds 5a and 6a showed almost same BRAF inhibitory activity and were
discovered to be potent inhibitors of cancer cell proliferation and were also observed to be strong
EGFR inhibitors. The results from this study show that the compounds have potential to be used
as anticancer agents and they also inhibit BRAF enzyme efficiently.
MDR reversal effects
A key factor in the unresponsiveness of several types of chemotherapy is multidrug resistance,
the mechanism by which numerous cancers become resistant to chemotherapeutic drugs. The
patients are affected with a range of solid tumors and blood cancers such as lower GIT, lung,
ovarian and breast cancers. Tumors comprise two different types of malignant cells, some of
which are drug-resistant whereas others are drug-sensitive. The cells which are sensitive to drugs
are killed by chemotherapeutic agents, but most of drug-resistant cells remain alive. There are
chances of chemotherapy to fail once the tumor regrows as the residual tumor cells become
resistant. This resistance is considered to be attributed to the occurrence of at least two molecular
‘pumps’ in plasma membranes of tumor cells that vigorously eject chemotherapeutic agents from
the interior of cell. The effects of drug or molecular pathways in the cytoplasm or nucleus are
thus evaded by cancer cells. The multidrug resistance-associated protein (MRP) and P-
glycoprotein are the two pumps which cause chemoresistance in cancer. These pumps are the
targets of numerous anticancer therapeutic regimens due to their importance and function(28).
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The effect of compounds on drug accumulation in MDR cancer cells was investigated using
rhodamine accumulation experiment (Table 2). To examine the toxicity of these compounds,
trypan blue assay was performed. At a concentration of 50µg/mL, the compounds were found to
be non-toxic to cells. The compounds were not effective on R123 accumulation at a
concentration of 5 µg/ml concentration. However, at a concentration of 50µg/mL and 30 min
incubation, the compounds were found to revert the multidrug resistance in mouse lymphoma
cells. The positive control used in the experiment was verapamil. The results of experiments
related to MDR are presented in Table 2. The fluorescence activity ratio (FAR) of 1 or more
designated the reversal of MDR by compounds. The MDR mediators link to the transmembrane
portion of Pgp, hence inducing a change in structure of Pgp, which then stops the activity of
ABC transporters (29). The oxime analogs 4a and 8a were found to be most potent while
compounds 1a and 2a did not possess beneficial activity. Against human mdr1 gene-transfected
mouse lymphoma cells, all compounds were found to be active. Resistance modifiers and
traditional chemotherapeutics can be collectively used in the treatment of MDR cancer cells.
Those chemical moieties having both MDR modulatory potential as well as potent anticancer
activity can be used for treatment of cancer.
Conclusion
To summarize, a series of α, β-unsaturated cyclohexanone derivatives-based oxime analogs with
different substitutions were synthesized, and investigated in vitro using human cancer cell lines
for their antiproliferative potential. Mechanistic assays were also performed to assess the
anticancer potential of new compounds. This study has discovered some new oxime analogs as
tubulin polymerization, BRAFV600E and EGFR-TK inhibitors. The compounds 5a and 6a were
discovered to be most potent in all trials. Thus, we have identified potent anticancer agents and
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have collected some useful SAR information that may turn out to be beneficial in synthesizing
additional MDR reversal agents with antitumor activity.
Acknowledgements
SNAB and HQ are grateful to Wuhan University of Technology for financial support.
Notes
“No author has Competing Financial Interest”
FIGURE LEGENDS
Figure 1: Effect of selected synthetic compounds on tubulin polymerization activity at
concentration of 25 µM. Results are the mean values of three experiments, n=3.While
vincristine and docetaxel (3 µM) were used as reference compounds. The y-axis
demonstrates tubulin polymerization activity measured at 15 min (arbitrary units) in
the growth phase of the tubulin polymerization curve.
Scheme 1: Synthesis scheme of α, β-unsaturated carbonyl based compounds and oxime analogs.
Reagents and conditions: (i) NaOH, EtOH, Room temperature (ii) NH2OH.HCl,
pyridine, ethanol, anhyd., reflux.
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Table 1: Inhibitory effects of new oxime analogues on the growth of normal (MCF-10A)
mammary epithelial cells (cell viability) and different types of human cancer cells.
Cell
viability
%
Antiproliferative activity IC50 (µM)
Comp.
MCF-7
HT-29
PC-3
A-549
PaCa-2
H-460
Panc-1
1a
2a
90
92
94
91
89
95
95
94
-
2.4 0.5
2.0 0.9
2.6 0.2
2.1 0.6
1.2 0.9
0.5 0.1
0.4 0.08
2.5 0.1
1.9 0.7
1.7 0.2
0.8 0.2
0.2 0.1
2.7 1.1
2.0 0.5
1.8 0.7
0.9 0.4
0.3 0.1
2.4 0.4
1.8 0.6
1.6 0.5
0.7 0.2
0.4 0.1
2.5 0.5
1.9 0.3
1.3 0.5
0.9 0.6
0.2 0.5
2.6 0.8
2.1 0.6
1.8 0.8
0.5 0.1
0.4 0.07
3a
1.4 0.2
4a
0.8 0.4
5a
0.2 0.09
0.09 0.04
0.03 0.02
0.02 0.01
0.03 0.02
6a
0.05 0.04 0.08 0.02 0.09 0.02 0.07 0.05 0.08 0.04 0.1 0.05
0.04 0.02 0.04 0.02 0.05 0.02 0.04 0.03 0.03 0.01 0.04 0.02
0.03 0.01 0.02 0.01 0.04 0.03 0.02 0.01 0.04 0.02 0.02 0.01
0.02 0.02 0.02 0.01 0.03 0.01 0.04 0.01 0.02 0.01 0.02 0.01
7a
8a
Erlotinib
MCF-7 (breast cancer cell line): HT-29 (colon cancer cell line): PC-3 (prostate cancer cell line):
A-549 (epithelial): PaCa-2 (pancreatic carcinoma cell line): H-460 (lung cancer cell line): Panc-1
(pancreas cancer cell line).
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Table 2: Effects of selected synthetic compounds on EGFR, BRAFV600E and MDR.
EGFR
inhibition
IC50 (µM)
BRAF
inhibition
IC50 (µM)
Fluorescence
activity ratio
(FAR)
Comp.
1a
2.5 0.8
2.0 0.5
1.9 0.5
1.6 0.9
0.05 0.02
0.04 0.01
0.9 0.1
0.7 0.4
0.05 0.02
-
2.9 0.5
2.2 1.0
3.1 0.5
2.9 0.9
1.5 0.7
1.3 0.5
2.1 0.9
1.9 0.2
0.07 0.02
-
6.5
2a
9.8
3a
24.6
27.5
17.9
21.5
22.9
28.5
25.5
12.5
4a
5a
6a
7a
8a
Erlotinib
Verapamil
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