Discovery of potential anticancer multi-targeted ligustrazine based cyclohexanone and oxime analogs overcoming the cancer multidrug resistance

Article abstract of DOI:10.1016/j.ejmech.2017.04.025

The drug research and development nowadays is focusing on multi-target drugs. In the treatment of cancer, therapies using drugs inhibiting one numerous targets signify a novel viewpoint. In comparison with traditional therapy, multi-targeted drugs directly aim cell subpopulations which are involved in progression of tumor. The current study comprises the synthesis of 34 novel ligustrazine-containing α, β-unsaturated carbonyl-based compounds and oximes. The growth of 5 various cancer cell types was strongly inhibited by ligustrazine-containing oximes as revealed by biological evaluation. A strong SAR was provided by the antiproliferative activity. The mechanistic effects of most active antiproliferative compounds on tubulin polymerization, EGFR TK kinases, KAF and BRAF^V600E were investigated, followed by in?vitro investigation of reversal of efflux-based resistance developed by cancer cells. EGFR was strongly inhibited by two oximes 7e and 8o. Out of all linkers including positive control, 1-isopropyl-piperidin-4-one linker-bearing compounds showed best inhibition of FAK. The strongest inhibitory activity of BRAF^V600E was showed by compound 5e with an IC₅₀ of 0.7?μM. Analogs such as 5 and 7 (b,e,f) exhibited a dual role as anticancer as well as MDR reversal agents. For understanding the target protein integrations with new compounds, molecular docking studies were also carried out.

Full text of DOI:10.1016/j.ejmech.2017.04.025

Accepted Manuscript
Discovery of potential anticancer multi-targeted ligustrazine based cyclohexanone
and oxime analogs overcoming the cancer multidrug resistance
Gao-Feng Zha, Hua-Li Qin, Bahaa G.M. Youssif, Muhammad Wahab Amjad, Maria
Abdul Ghafoor Raja, Ahmed H. Abdelazeem, Syed Nasir Abbas Bukhari
PII:
S0223-5234(17)30282-9
10.1016/j.ejmech.2017.04.025
EJMECH 9372
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2017
Revised Date: 6 April 2017
Accepted Date: 11 April 2017
Please cite this article as: G.-F. Zha, H.-L. Qin, B.G.M. Youssif, M.W. Amjad, M.A.G. Raja, A.H.
Abdelazeem, S.N.A. Bukhari, Discovery of potential anticancer multi-targeted ligustrazine based
cyclohexanone and oxime analogs overcoming the cancer multidrug resistance, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.025.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Discovery of potential anticancer multi-targeted ligustrazine based cyclohexanone
and oxime analogs overcoming the cancer multidrug resistance
Gao-Feng Zha, Hua-Li Qin, Bahaa G. M. Youssif, Muhammad Wahab Amjad, Maria Abdul
Ghafoor Raja, Ahmed H. Abdelazeem, Syed Nasir Abbas Bukhari

ACCEPTED MANUSCRIPT
Discovery of potential anticancer multi-targeted ligustrazine based
cyclohexanone and oxime analogs overcoming the cancer multidrug resistance
a
a
b,c,
d
Gao-Feng Zha ‡, Hua-Li Qin* , Bahaa G. M. Youssif
Muhammad Wahab Amjad , Maria
d
e,f
a,b
Abdul Ghafoor Raja , Ahmed H. Abdelazeem , Syed Nasir Abbas Bukhari* ‡
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 Chemistry, College of Pharmacy, Aljouf University, Aljouf,
Sakaka2014, Saudi Arabia.
c
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt.
d
College of Pharmacy, Northern Border University, King Abdullah Road, Rafha, Saudia
Arab
e
Department of Medicinal Chemistry, Faculty of pharmacy, Beni-Suef University, Beni-Suef
6
2514, Egypt.
f
Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif
1974, Saudi Arabia.
2
‡
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;
*

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Abstract
The drug research and development nowadays is focusing on multi-target drugs. In the treatment
of cancer, therapies using drugs inhibiting one numerous targets signify a novel viewpoint. In
comparison with traditional therapy, multi-targeted drugs directly aim cell subpopulations which
are involved in progression of tumor. The current study comprises the synthesis of 34 novel
ligustrazine-containing α, β-unsaturated carbonyl-based compounds and oximes. The growth of 5
various cancer cell types was strongly inhibited by ligustrazine-containing oximes as revealed by
biological evaluation. A strong SAR was provided by the antiproliferative activity. The
mechanistic effects of most active antiproliferative compounds on tubulin polymerization, EGFR
V600E
TK kinases, KAF and BRAF
were investigated, followed by in vitro investigation of
reversal of efflux-based resistance developed by cancer cells. EGFR was strongly inhibited by
two oximes 7e and 8o. Out of all linkers including positive control, 1-Isopropyl-piperidin-4-one
linker-bearing compounds showed best inhibition of FAK. The strongest inhibitory activity of
V600E
BRAF
was showed by compound 5e with an IC of 0.7 µM. Analogs such as 5 and 7 (b,e,f)
50
exhibited a dual role as anticancer as well as MDR reversal agents. For understanding the target
protein integrations with new compounds, molecular docking studies were also carried out.
Keywords: Epidermal growth factor receptor (EGFR), BRAF, Focal adhesion kinase (FAK),
Tubulin polymerization, Cancer cell lines.

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ABBREVIATIONS
MDR , multidrug resistance; EGFR , epidermal growth factor receptor; European Medicines
Evaluation Agency (EMEA); US Food and Drug Administration (FDA); NSCLC, non-small cell
lung; focal adhesion kinase (FAK); RTK , receptor tyrosine kinases; MAPK, mitogen-activated
protein kinase; ABC, ATP-binding cassette, NF-kB, NF-kappaB; Four-point-one, ezrin, radixin,
moesin (FERM); Ligustrazine or tetramethylpyrazine (TMP); Adriamycin (Adr); DRS, death
receptors; tumor necrosis factor (TFN), PI, propidium iodide; TKIs, tyrosine kinase inhibitors;
FAR , fluorescence activity ratio; HRMS, high resolution mass spectra; TLC, thin layer
chromatography; UV, ultraviolet; DMEM, Dulbecco's modified Eagle's medium; DMSO,
dimethyl sulfoxide; 2-iodoxybenzoic acid (IBX).
INTRODUCTION
After heart disease, cancer is the second most common cause of death, as per statistics [1]. The
past few decades have witnessed cancer as a global killer [2]. The antiangiogenic, pro-apoptotic,
anti-proliferative and antioxidant properties mediated by multiple signaling pathways are the
primary mechanisms of tumor development and progression [3]. To selectively target tubulin
polymerization and oncogenically-activated kinases, considerable efforts have been made to
discover small molecule inhibitors. To treat HER-2, EGFR and BRAF-driven cancers, a range of
inhibitors has been developed such as lapatinib, gefitinib, erlotinib and vemurafenib. Up till now,
their effectiveness is restricted to subcategories of oncogenic alterations and majority of kinase
inhibitors might not be active against all activating mutations for a particular target.

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It has been discovered lately that nearly all patients suffering from hairy-cell leukemia carry the
V600E
BRAF
mutation and the enzyme inhibition resulted in substantial reduction of disease [4].
These discoveries have motivated the researchers to search for small molecule inhibitors aiming
V600E
the kinase activity of BRAF mutants (particularly BRAF
) [5, 6]. For the treatment of BRAF-
mutated metastatic melanoma, European Medicines Evaluation Agency (EMEA) and US Food
and Drug Administration (FDA) have recently approved BRAF-specific inhibitors including
dabrafenib (GSK2118436, GSK) and vemurafenib (Zelboraf, Roche), and numerous other BRAF
inhibitors are undergoing development stages [7]. Epidermal growth factor receptor (EGFR), a
trans-membrane glycoprotein, belongs to a family (erbB) of closely related cell membrane
receptors such as EGFR (erbB-1 or HER1), erbB-2 (HER2), erbB-3 (HER3), and erbB-4 (HER4)
[
8-12]. The dysregulation, expression or over expression of EGFR has been discovered to be
associated with numerous human solid tumors such as breast, ovarian, non-small cell lung
NSCLC), colorectal, head and neck [13-17]. EGFR activation can enhance tumor growth by
(
increasing cell proliferation, motility [18], adhesion and invasive capacity [19], and by apoptosis
inhibition [20]. The strategy of regulating the signaling of EGFR in the treatment of cancer has
been revealed lately by a range of molecular inhibitors which have been developed and are going
through clinical trials.
Alternatively, a 125 KD non-receptor protein tyrosine kinase (focal adhesion kinase, FAK),
controls cell proliferation, survival and migration in reaction to extracellular signals [21, 22]. The
triggering of FAK is initiated by the binding of integrin to extra cellular matrix (ECM), followed
by FAK autophosphorylation at Tyr-397, which is vital for majority of cell functions [23]. The
FAK phosphorylation is vital in stimulating FAK-dependent transduction of signal as its state of
phosphorylation can control the capability of these molecules to interact with

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phosphatidylinositol 3-kinase and Src, and/or it controls FAK catalytic activity which impacts
these molecules’ ability to become phosphorylated [24]. FAK’s amino-terminal domain holds an
area which shares sequence correspondence to Four-point-one, ezrin, radixin, moesin (FERM)
domains, which might be an indication in controlling the phosphorylation state and activity of
FAK in cells [25]. Several tumors such as thyroid, brain, liver, prostate, breast, colon, head and
neck have been discovered to overexpress FAK. Moreover, this over-expression is associated
with an invasive phenotype in such tumors [26-28]. The invasion of ovarian cancer cells and
glioblastomas was found to be reduced by FAK signaling inhibition via over-expression of
dominant-negative FAK fragments [29]. Additionally, the involvement of FAK in survival,
angiogenesis, metastasis and invasion of cancer cell has been also discovered [30]. Therefore, the
search for FAK inhibitor offers a new understanding of the anti-tumor ability.
Along with DNA, microtubules are one of the most strategic and logical molecular targets
among all the existing ones for chemotherapy. Owing to better biological relevance and
understanding of microtubule as a target for cancer therapy, tubulin inhibitors have been under
the spotlight as cancer therapeutics for the past few years leading to comprehensive preclinical
and clinical studies of several agents. Nonetheless, owing to the failure of numerous agents to
exhibit effectiveness in phase III studies, certain novel inhibitors together with formerly studied
agents (in various combinations and malignancies) are being clinically evaluated.
The main hindrance in the successful tumor chemotherapy includes the development of
resistance in tumor cells against cytotoxic agents and the failure in discovery of novel anticancer
agents possessing multi target approach. Due to the structural irrelativeness of this resistance to
cytostatics, it was termed multidrug resistance (MDR), which is categorized by a reduced
responsiveness to cytotoxic agents. So far, researchers have been struggling to overcome or

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circumvent multidrug resistance (MDR) in cancer [31, 32]. Hence, MDR remains one of the
main complications for efficacious treatment of tumor. The most discussed mechanism of MDR
states that the efflux of chemotherapeutic drugs from cells takes place by ‘drug pumps’ [33].
Chemotherapeutics which have not been found to be the substrates of drug efflux pumps have
gained attention in current drug development approaches to overcome MDR. Furthermore, in
clinical trials, efforts to overcome resistance to chemotherapeutics by using MDR modulators for
the past 20 years have not yielded promising results [34, 35]. Drugs which efficiently target
drug-resistant tumors are the need of the hour. In spite of massive research endeavours and rapid
progress made, there is an increasing demand for new therapies as ever. It is vital to find new
targets and novel multifunctional agents for cancer treatment.
On the basis of our earlier findings, we have strived to find novel compounds with multiple
functions and in doing so we have expanded our previously reported work [36-38] by
synthesizing 34 new α, β-unsaturated carbonyl-based compounds. The novelty of this work lies
in the incorporation of ligustrazine chemically known as tetramethylpyrazine (TMP) in new
compounds as it has been recently reported to be a beneficial adjuvant in the reversal of MDR in
tumor cells. For instance, in response to Adr TMP was discovered to improve the
chemosensitivity of human hepatocellular carcinoma cells [39]. TMP is a vital component of
Chinese conventional medicinal herb chuanxiong (Ligusticum chuanxiong Hort). In current
study, a series of novel α, β-unsaturated carbonyl-based compounds with TMP moiety was
designed, synthesized and assessed for its anticancer potential on human cancer cell lines.
Among these, the 6 most active compounds were selected as precursors for additional synthesis
to their oxime derivatives. The 12 most potent among all 34 compounds were designated for

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V600E
anticancer mechanistic studies for their effects on BRAF
, EGFR TK kinases, FAK and
tubulin polymerization and were tested in vitro to reverse MDR developed by cancer cells.
RESULTS AND DISCUSSION
Chemistry
In this study, 34 novel cyclohexanone compounds and a few with cyclohexanone-extended
oxime backbone were synthesized as previously reported[38, 40] (Table 1), however tetra methyl
pyrazine (TMP) was introduced in synthesis as new modification. Starting from ligustrazine (1),
2
-hydroxymethyl-3,5,6-trimethylpyrazine (2) was first synthesized by Boekelheide reaction. An
aldehyde group-containing intermediate (3) was produced after 2-iodoxybenzoic acid (IBX)
oxidation of (2) (Scheme 1). Claisen Schmidt condensation was used between suitable aryl
aldehydes including TMP-based aldehyde (3) and various cyclohexanone ketone linkers (4a-g) at
a molar ratio 2:1 for the synthesis of desired α, β-unsaturated carbonyl-based compounds (5a-g)
and (6a-u), (Scheme 1). Of cyclohexanone backbone, all novel compounds contained
ligustrazine (TMP)-based aldehyde either on one side (6a-6u) or on both sides (5a-g). The
derivatives of cyclohexanone bearing TMP-based aldehyde on one side contained different
aldehyde types with various substitution patterns on the other as previously reported. To
synthesize oxime analogs, specific cyclohexanone derivatives were used as precursors. For
yielding respective oximes, hydroxylamine hydrochloride was allowed to react with each
selected compound. HPLC analysis revealed that all newly synthesized compounds were more
1
3
1
than 95% pure. All compounds were subjected to microanalysis (CHNS), HRMS, C NMR, H
NMR and melting point (MP) analyses and their details are provided in experimental part.

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Cell viability assay
The rule of three (activity-exposure-toxicity) is the most difficult challenge faced by potential
new drugs in the phase of design and development. In drug discovery, absorption, distribution,
metabolism and excretion (ADME) studies are frequently performed to convert lead compounds
into drugs which are both effective and safe [41]. To perform in vitro cell viability assay, human
mammary gland epithelial cells (MCF-10A) were used. The MCF-10A cells were exposed to
synthesized compounds for 96h and MTT assay was used to investigate cell viability. The results
are shown as percentage toxicity (Table 2). All compounds exhibited a cell viability percentage
of more than 88% and were found to be safe.
Antiproliferative effects of synthetic compounds
Propidium iodide (PI) fluorescence assay was carried out to assess the compounds’ cytotoxicity
to the A-549 (epithelial cancer cell line), PC-3 (prostate cancer cell line), MCF-7 (breast cancer
cell line), PaCa-2 (pancreatic carcinoma cell line) and HT-29 (colon cancer cell line). Erlotinib
hydrochloride (Tarceva), a drug used to treat pancreatic and non-small cell lung cancer (NSCLC)
cells is a receptor tyrosine kinase inhibitor which acts on the epidermal growth factor receptor
(EGFR) resulting in blocking the signals within the cancer cells and inhibiting EGFR activity
followed by cell death. Erlotinib was used as a positive control in this study.
The results of cell viability assays (on different cell lines) of all compounds were almost the
same (antiproliferative) and the difference in their inhibitory activities was lesser than 11%.
Among all compounds (including erlotinib and precursor α, β-unsaturated carbonyl-based
compounds), oximes 7(b,e,f) and 8(f,o,r) exhibited best antiproliferative activity with the values
of IC50 in the range of 0.01±0.01 µM to 0.1±0.08 µM (Table 2). Compound 7e was found to be

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the best antiproliferative agent against all cancer cells. As reported earlier, the compounds were
found to be active against only one or two cell lines.
The structural variations can be correlated with the activity of compounds. Owing to the presence of
cyclohexanone linkers, oximes were found to be more active than precursor α, β-unsaturated
carbonyl-based compounds. Compounds bearing same linker but substituted aldehyde on one
side and TMP on the other side of linker were found to be less potent as compared to those
containing the dual incorporation of TMP moiety on both sides of linkers. Better tumor growth
inhibitory potential of compound 5a in comparison to compounds 6a-c could be attributed to the
presence of double TMP moiety in 5a. Similar activity pattern was seen among other compounds
with the same linker. Wang et al. previously reported a new derivative of ligustrazine which
showed extremely low toxicity and potent antitumor activity by COX-2 and NF-κB/p65
suppression in S180 mice [42].
Compounds (5e and 6o) bearing were discovered to be most potent in comparison to compounds
containing other linkers. After 1-methyl-piperidin-4-one, 1-Isopropyl-piperidin-4-one linker
turned out to be most active with 4-methyl-cyclohexanone linker (5b, 6d and 6f) also showing
noteworthy effects on the growth of cancer cells.
The substitution patterns at R , R , and R of second arm aldehydes for synthesizing 6a-u also
2
3
4
showed activity difference. The derivatives of cyclohexanone bearing single methoxy groups at
position 3 were discovered to be less active in comparison to those containing two methoxy
groups at positions 2 and 3. On the benzene ring of all compounds, halogens (Cl or Br) were
present at position 4. As no compound from this sequence of compounds was free from halogen,
the halogen influence on compound activity could not be detected. The mechanistic effects of
most active antiproliferative compounds on tubulin polymerization, EGFR TK kinases, KAF and

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V600E
BRAF
were investigated, followed by in vitro investigation of reversal of efflux-based
resistance developed by cancer cells
EGFR inhibitory activity
EGFR might get activated due to mutations, over-expression leading to autocrine expression or
constitutive activation of ligand[43, 44]. Ras expression in cells over-expressing EGFR also
leads to substantial activation of EGFR-induced apoptosis signifying that activation of Ras is an
important survival signal produced by EGFR. So, for developing novel therapies for cancer
treatment, EGFR kinase is an attractive target. Apart from other classes of inhibitors, 4-
(
phenylamino) quinazolines have been discovered to be most potent [45-47].
To evaluate the EGFR inhibitory potential of compounds, EGFR-TK assay was performed
Table 3). The findings of the cancer cell-based assays have been complemented by the results
from this assay. All compounds showed potent EGFR inhibition with IC50 in the range of
.02±0.01 to 4.1±1.0 µM. The compounds 7e and 8o displayed comparable and most potent
activities (IC = 0.02±0.01 µM) among all compounds (including positive control). After 7e and
(
0
5
0
8
o, compound 5e was also found to be more effective than positive control. Similar to erlotinib,
-methyl-piperidin-4-one-bearing compound 6o inhibited the activity of EGFR (IC = 0.06±0.05
1
5
0
µM). Focusing on the chemical structure, compound 6o bears an aldehyde on one side of the
linker whereas a TMP-based aldehyde lies on the other side with substitutions of methoxy groups
2 3 4
at R and R and halogen at R position of aromatic ring. Xu and coworkers reported the
synthesis of a similar series of α, β-unsaturated cyclohexanone analogues of curcumin, they
investigated their antiproliferative effect using two cancer cell lines and also determined their
inhibitory activity against EGFR TK kinases. For both studies the effects of compounds were
found to be remarkable[48]. The incorporation of different linkers and TMP moiety improved the

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activity of compounds in comparison to the data reported previously. This study shows the
compounds to be strong inhibitors of EGFR and can probably be used as anticancer agents.
V600E
BRAF
inhibitory activity
In the BRAF activation segment, a valine to glutamate substitution at position 600 (V600E) has
V600E
been discovered to be responsible for 90% of cancer-related mutations of BRAF, and BRAF
displays enhanced transformation and increased activity of kinases in melanocytes and
fibroblasts in vitro [5, 49-52]. A comprehensive cancer genome mutation mapping has revealed
BRAF mutation in 7% of human cancers, and this finding signifies BRAF as a key oncogene,
predominantly in melanomas. These collective findings render BRAF kinase as a promising
target for the development of anticancer drugs.
V600E
Ten most active compounds were subjected to in vitro BRAF
inhibitory assay. All
compounds exhibited IC ranging from 0.7±0.4-3.1±0.4 µM (Table 3). Compounds possessing
5
0
V600E
TMP moiety on both sides of the linker were found to be strong inhibitors of BRAF
strongest activity was shown by compound 5e bearing TMP dual moiety and 1-methyl-piperidin-
-one linker. In comparison to parent cyclohexanone derivatives, oximes were discovered to be
. The
4
less potent. In comparison to compounds bearing TMP core on one side and methoxy-substituted
aldehydes on the other, compounds possessing TMP backbone on both sides of linker were
found to be more active. The compounds were discovered to be effective inhibitors of BRAF
enzyme and can be referred to as prospective anticancer agents. From a library of 23000
compounds, 2-(3,4,5-trimethoxyphenylamino)-6-(3-acetamidophenyl)-pyrazine was discovered
to be a potent inhibitor (IC 3.5 µM) of BRAF[53]. Lately, a 2,6-disubstituted pyrazine scaffold
50
V600E
designated from a high output screening has yielded a new BRAF
inhibitor series. Pyrazine
inhibitors which were potent (<500 nM) and selective (5-86-fold) against BRAF in comparison

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to CRAF were obtained [53].
In comparison to previously reported 2-(3,4,5-
Trimethoxyphenylamino)-6-(3-acetamidophenyl)-pyrazine,
synthesized in this study were discovered to be more active.
FAK inhibitory assay
TMP-containing
compounds
Through effects on the stromal cells of the tumor microenvironment and on cancer cells, FAK
stimulates metastasis and tumor progression. Cancer stem cell self-renewal, gene expression,
survival, invasion and cell movement are regulated by FAK’s kinase-dependent and independent
functions. Small-molecule inhibitors of FAK have been discovered to decrease metastasis and
tumor growth in numerous preclinical models with lesser adverse effects. The FAK inhibitory
potency of compounds was investigated and the findings revealed most of the compounds to be
active against FAK (Table 3). Comparable to erlotinib (IC50 7.4±1.5 µM), compound 7f
exhibited the best inhibitory activity (IC50 1.0±0.2 µM) among all compounds. Best FAK
inhibitory effects were exhibited by 1-Isopropyl-piperidin-4-one linker-bearing compounds (5f,
6
8
r, 7f and 8r) followed by 1-methyl-piperidin-4-one linker-bearing compounds (5e, 6o, 7e and
o), whereas compounds bearing 4-methyl-cyclohexanone linker (5b, 6f, 7b and 8f) were
discovered to be least active. In comparison to precursor α, β-unsaturated carbonyl-based
compounds (5f and 6r), oximes (7f and 8r) were found to be more active FAK inhibitors.
Effects on tubulin polymerization
Microtubule-targeting agents interfere with microtubule function and stop mitosis, resulting in
necrosis and apoptosis and ultimately cell death [54, 55]. The effect of twelve selected synthetic
compounds on tubulin polymerization was investigated. Most compounds inhibited the assembly
of tubulin with compounds 5b and 6f turning out to be most potent inhibitors. On the other hand,
compounds (5e and 8o) did not inhibit the assembly of tubulin suggesting a mechanism of action

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other than tubulin inhibition for the observed cytotoxicity. In comparison to model drug
docetaxel, no compound exhibited promising microtubule-stabilizing action [56]. After analysis,
it was observed that 4-methyl-cyclohexanone moiety played key role for the inhibition of tubulin
polymerization while 1-methyl-piperidin-4-one core-bearing compounds (5e, 6o, 7e and 8o)
were found to be potent inhibitors of EGFR; however, they did not show the inhibition of tubulin
assembly.
MDR reversal activity
MDR of tumor cells is a main hurdle to the efficacy of chemotherapy [57]. Rhodamine
accumulation experiment was carried out to investigate the effect of compounds at nontoxic
concentration on the accumulation of drugs in MDR cancer cells (Table 3). Trypan blue
experiment was carried out to study the toxic effects of new synthetic compounds. The
compounds were nontoxic to cells at 50 µg/mL concentration. On R123 accumulation, most of
compounds were ineffective at 5 µg/ml concentration. After an incubation of 30 min, the
compounds reversed the MDR on mouse lymphoma cells at a concentration of 50 µg/mL. The
positive control used in the experiment was verapamil. The cyclohexanone derivatives (5b, e and
f) bearing TMP arms on both sides and oximes 7(b, e and f) were found to be most active. The
fluorescence activity ratio (FAR) of 1 or more signified that the compounds were able to reverse
MDR. The MDR modulators bind to Pgp trans-membrane domains, bringing about Pgp
structural change resulting in the prevention of ABC transporter activity [58]. All compounds
were discovered to be active against human mdr1 gene-transfected cells of mouse lymphoma.
The sensitivity of K562/A02 cells to Adriamycin (Adr) and subsequent cytotoxicity was found to
be enhanced using a ligustrazine derivate as it enhanced Adr intracellular accumulation. The

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TMP derivative-induced Adr accumulation was linked with down regulation of GSTp expression
and GST-associated enzyme activity [59]. MDR of cancer cells can be reversed using a
combination of resistance modifiers and traditional chemotherapeutics. Compounds possessing
double properties as MDR modulators and as strong anticancer agents can be potential
candidates for the treatment of cancer.
Molecular Docking Study
In order to gain some structural insights into both FAK and EGFR inhibitory activities of the
newly synthesized compounds, a molecular docking simulation was performed using
LIGANDFIT embedded in the Discovery Studio software (Accelrys® software corporation, San
Diego, USA). The 3D crystal structure of FAK (PDB IDs: 2JKO) in complex with bis-anilino
pyrimidine inhibitor, TAF089, and 3D crystal structure of EGFR (PDB ID: 1M17) in complex
with erlotinib were used for this docking study [60, 61]. The most active compounds 5f and 7f
compared with the least active one 6f were selected to be docked into the FAK active site where
their interactions and binding patterns were investigated. Meanwhile, the active compounds 8o
and 6o in addition to the least active one 8r were docked into EGFR binding site using the same
docking protocol. The superimposition of the active docked poses inside both proteins binding
pockets were presented in Figure 1. Initially, the docking protocol settings were validated
through the redocking of the extracted co-crystallized ligand TAF089 from the 3D structure
2
JKO using the same protocol for compounds 5f, 6f and 7f. Interestingly, the used docking
protocol closely reproduced the bound structure with RMSD value of 0.37 Å confirming the
confidence in our docking study. The docked pose of TAF089 and the experimental co-
crystallized structure have been shown in Figure 2.

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The inspection of docking results revealed that all the three docked ligands 5f, 6f and 7f adopted
a nearly similar disposition inside the ATP binding pocket of the FAK kinase, whereas the
common core, piperidinone or cycohexanone, was leaned between Ile428, Gly429 and Glu506
residues within the kinase hinge region, Figure 3 (A,B,D). Meanwhile, the pyrazine ring bearing
three methyl groups made hydrophobic contacts with Gly429, Glu500, Asp564 and Leu567
located near the gatekeeper residue Met499 and DFG motif of FAK activation loop. Obviously,
compounds 5f and 7f exhibited mostly an identical orientation with a hydrophobic interaction
between the isopropyl moiety and Thr503 residue. The slight increase in potency of 7f (IC50
=
1
.0 µM) compared to 5f (IC50 = 1.2 µM) may be attributed to the additional H-bond interaction
formed between the hydroxyl amine moiety in compound 7f with Ile428 residue in the hinge
region of FAK kinase, Figure 3(C). On the contrary, the 2-bromo-4,5-dimethoxybenzylidene
moiety in compound 6f seems to considerably protrude outside the FAK active site owing to its
inferior inhibitory activity, Figure 3(D). The superimposition of the two active poses 5f and 7f in
addition to TAF089 into the active site FAK kinase was shown in Figure 3.
On the other hand, the docking results of the most active compound 8o against EGFR indicated
that it fits well inside the ATP-active site forming a network of hydrogen bonds with Lys721,
Thr766 and Met769 residues. Also, it was found that the 2-bromo-4,5-dimethoxybenzylidene
moiety aligns near the DFG motif which plays a pivotal role in the regulation of kinase activity.
The trimethyl pyrazine moiety was oriented towards the hinge region making hydrophobic
interactions with Leu694, Gly722 and Phe771 residue. Moreover, the methyl piperidin-4-one
core bearing an oxime group was located in the middle of the active site where the oxime was
directed towards Met769 and the methyl group directed to the bottom forming a hydrophobic
interaction with Leu694 and Val702 residues, Figure 4(A). However, compound 6o adopted a

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similar binding pattern to that of compound 8o, there was a slight decrease in its EGFR
inhibitory activity. The absence of the hydroxyl amine moiety in compound 6o, which forms an
important hydrogen bond with Met769 residue, may greatly contribute to this change in potency,
Figure 4(B). Conversely, compound 8r with the least potency against EGFR adopted a
completely reversal orientation and binding pattern compared to compound 8o where the 2-
bromo-4,5-dimethoxybenzylidene moiety was oriented, to some extent, away from the hinge
region and the trimethyl pyrazine was located near the DFG motif. Also, the hydrozyl amine
moiety was pushed towards the hydrophobic residue Leu694 forming no hydrogen bonds,
Figure 4(C,D). This opposite dispositioning may be the reason behind the low potency of
compound 8r against EGFR kinase.
Conclusion
A series of α, β-unsaturated cyclohexanone derivatives bearing different substitutions and
ligustrazine incorporation were synthesized and investigated in vitro for their antiproliferative
effect on 5 different human cancer cell lines. The compounds exhibiting the strongest
antiproliferative activities were selected for successive synthesis of oximes and their subsequent
assessment through mechanistic studies. New oxime analogs as inhibitors of tubulin
V600E
polymerization, BRAF
, FAK and EGFR-TK have been discovered. MDR reversal activity
was exhibited by most of the compounds. In all studies, compounds 5e and 7e exhibited potent
activities. Hence, we have recognized compounds possessing double properties as MDR
modulators and as strong anticancer agents, and have collected some useful SAR evidence which
may turn out to be beneficial in the synthesis of additional MDR reversal agents with inherent
antitumor activity.

ACCEPTED MANUSCRIPT
EXPERIMENTAL SECTION
Chemistry General Information:
Acros Organics, Merck and Sigma-Aldrich were the manufacturers whose analytical grade
13
1
reagents and chemicals were used in this project. C and H NMR spectra were recorded on
JEOL ECP spectrometer (500 MHz) using CDCl and dimethyl sulfoxide (DMSO-d ) as solvents
3
6
while the internal standard used was Me Si. High resolution mass spectra (HRMS) were
4
recorded on MicroTOF-Q mass spectrometer (Bruker). Fison EA 1108 elemental analyzer was
used to obtain the microanalyses data. Silica gel 60 (230-400 mesh, Merck) was used to carry out
flash column chromatography whereas thin layer chromatography (TLC) was performed using
pre-coated silica plates (kiesel gel 60 F254, BDH). The melting points of compounds were
determined using an electrothermal instrument. After burning on a hotplate, the compounds were
examined using vanillin stain or under UV light (254 nm). To determine the purity of
compounds, Beckmann system Gold HPLC equipped with Kinetex C18 Coloumn (250mmX
mm, 5µm) was used with a linear gradient from 0% to 100% solvent B (0.1% TFA/ 40% water/
6
0% acetonitrile). Compounds 3 and 4 were synthesized as reported previously [62]. All the
compounds were discovered to be more than 95% pure.
Synthesis of ligustrazine containing α, β-unsaturated carbonyl based compounds
New α, β-unsaturated carbonyl-based compounds (5a-g and 6a-u) were synthesized using direct
coupling technique [40] (Scheme 1). The reaction was carried out using base-catalyzed Claisen-
Schmidt condensation reaction, by reacting different types of ketones with appropriate aromatic
aldehyde at molar ratio 2:1 to synthesize new compounds (5a-g) and at molar ratio 1:1 for (6a-
u). For synthesis of 6a-u first 5a-g intermediates were synthesized and in second step appropriate

ACCEPTED MANUSCRIPT
aldehydes were reacted with intermediates. The detailed method of synthesis has already been
reported by us previously [38, 40]. Scheme 1 shows the highlights of synthesis of compound 3, 4
and α, β-unsaturated carbonyl-based compounds along with oxime derivatives. 15 mL ethanol
was taken in a round bottom flask and aromatic aldehyde (20 mmol, 2 equivalant) and specific
ketone (10 mmol, 1 equivalent) were added and dissolved using a stirrer for 2-3 min at 5°C. Into
the above solution, 40% NaOH solution in ethanol was added drop wise and the mixture was
stirred for 1-24 h at 27°C. The color change and precipitate formation in the reaction mixture
showed product formation. TLC was used to monitor the reaction and acidified ice was added to
quench the reaction once completed. The isolation of compounds was done by recrystallization
and/or by using column chromatography.
2
,6-Bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-cyclohexanone (5a )
1
Yield: 74%; Mp: 112-113 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.87 (s, 2H), 2.55 (s, 6H); 2.51 (s,
1
3
6
1
3
1
H)
;
2.48 (s, 6H)
;
2.37 (t, J=8.0 Hz, 4H), 1.85 (m, 2H); C NMR (500 MHz, CDCl
3
) δ: 181.2,
52.7, 150.4, 149.6, 147.9, 146.2, 141.4, 27.9, 27.1, 17.1, 16.8, 16.1; HRMS (ESI) m/z:
+
63.4754 [M+H] , Microanalysis calculated for C H N O (362.47), C: 72.90%, H: 7.23%, N:
22
26
4
5.46%. Found C: 72.96%, H: 7.25%, N: 15.42%.
2
,6-bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-4-methyl-cyclohexanone (5b )
1
Yield: 69%; Mp: 108-109 ˚C; H NMR (500 MHz, CDCl ) δ: 7.89 (s, 2H), 2.59 (s, 6H)
;
2.54 (s,
3
1
3
6
H); 2.50 (s, 6H); 2.07 (d, J=8.5 Hz, 4H), 1.72 (m, H); 1.19 (d, J=8.5 Hz, 3H); C NMR (500
MHz, CDCl ) δ: 180.5, 152.5, 151.1, 149.4, 147.2, 146.1, 140.9, 32.5, 29.2, 22.8, 17.9, 17.0,
3
+
1
7
6.2; HRMS (ESI) m/z: 377.5029 [M+H] , Microanalysis calculated for C H N O (376.49), C:
2
3
28
4
3.37%, H: 7.50%, N: 14.88%. Found C: 73.45%, H: 7.52%, N: 14.67%.

ACCEPTED MANUSCRIPT
2
,6-bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)- 4-isopropyl-cyclohexanone (5c )
1
Yield: 61%; Mp: 121-122 ˚C; H NMR (500 MHz, CDCl ) δ: 7.92 (s, 2H), 2.55 (s, 12H)
;
2.51
3
1
3
(
s, 6H); 2.09 (d, J=8 Hz, 4H), 1.75(m, H); 1.65(m, H);1.14 (d, J=7 Hz, 6H); C NMR (500 MHz,
CDCl ) δ: 182.2, 151.8, 150.2, 149.1, 147.0, 146.4, 140.5, 38.5, 30.9, 29.7, 22.1, 17.2, 16.4,
3
+
1
7
5.5; HRMS (ESI) m/z: 405.5541 [M+H] , Microanalysis calculated for C H N O (404.55), C:
2
5
32
4
4.22%, H: 7.97%, N: 13.85%. Found C: 74.28%, H: 7.82%, N: 13.81%.
3
,5-Bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-piperidin-4-one (5d )
1
Yield: 65%; Mp: 147-148 ˚C; H NMR (500 MHz, CDCl ) δ: 7.91 (s, 2H), 2.61 (s, 6H)
;
2.57 (s,
3
1
3
6
1
H)
;
2.50 (s, 6H)
;
2.68 (s, 4H); C NMR (500 MHz, CDCl ) δ: 182.1, 152.5, 150.6, 149.2,
3
+
47.8, 146.1, 142.5, 47.2, 17.8, 17.1, 16.2; HRMS (ESI) m/z: 364.4677 [M+H] , Microanalysis
25 5
calculated for C21H N O (363.46), C: 69.40%, H: 6.93%, N: 19.27%. Found C: 69.49%, H:
6
.98%, N: 19.01%.
3
,5-bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-1-methyl-piperidin-4-one (5e )
1
Yield: 55%; Mp: 149-151 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.88 (s, 2H), 2.72 (s, 4H); 2.59 (s,
1
3
6
1
H)
;
2.54 (s, 6H)
;
2.50 (s, 6H)
;
2.14 (s, 3H); C NMR (500 MHz, CDCl
3
) δ: 182.5, 152.2, 151.1,
+
49.4, 147.2, 146.0, 142.7, 49.5, 32.7, 17.9, 17.0, 16.3; HRMS (ESI) m/z: 378.4019 [M+H] ,
Microanalysis calculated for C H N O (377.48), C: 70.00%, H: 7.21%, N: 18.55%. Found C:
2
2
27
5
7
0.29%, H: 7.54%, N: 18.46%.
3
,5-bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)- 1-Isopropyl-piperidin-4-one (5f )
1
Yield: 59%; Mp: 142-143 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.81 (s, 2H), 2.57 (s, 6H)
;
2.52 (s,
1
3
6
H); 2.47 (s, 6H); 2.27 (s, 4H), 1.60(m, H);1.12 (d, J=7 Hz, 6H); C NMR (500 MHz, CDCl ) δ:
3

ACCEPTED MANUSCRIPT
1
84.1, 152.4, 150.6, 148.1, 147.4, 146.2, 142.9, 49.2, 32.8, 22.1, 18.0, 17.2, 16.6; HRMS (ESI)
+
m/z: 406.5432 [M+H] , Microanalysis calculated for C H N O (405.54), C: 71.08%, H: 7.70%,
2
4
31
5
N: 17.27%. Found C: 71.12%, H: 7.96%, N: 17.22%.
3
,5-Bis-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-tetrahydro-pyran-4-one (5g )
1
Yield: 69%; Mp: 127-129 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.79 (s, 2H), 2.65 (s, 4H), 2.52 (s,
1
3
6
1
H)
;
2.47 (s, 6H)
;
2.44 (s, 6H)
;
C NMR (500 MHz, CDCl
3
) δ: 182.5, 152.5, 150.4, 149.0,
+
47.6, 146.4, 142.2, 52.5, 17.9, 17.4, 16.5; HRMS (ESI) m/z: 365.4482 [M+H] , Microanalysis
calculated for C H N O (364.44), C: 69.21%, H: 6.64%, N: 15.37%. Found C: 69.29%, H:
2
1
24
4
2
6
.68%, N: 15.27%.
2
-(2-Chloro-benzylidene)-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-cyclohexanone (6a )
1
Yield: 68%; Mp: 124-125 ˚C; H NMR (500 MHz, CDCl ) δ: 7.91 (s, H), 7.77 (s, H), 7.39 (d,
3
J=7.0 Hz, H), 7.19 (d, J=7.0 Hz, H), 6.80 (t, J=7.5 Hz, H), 6.62 (t, J=7.5 Hz, H), 2.61 (s, 9H)
;
1
3
2
1
.39 (t, J=8.0 Hz, 4H), 1.87 (m, 2H); C NMR (500 MHz, CDCl ) δ: 181.7, 152.4, 150.1, 149.5,
3
47.2, 146.4, 145.2, 141.7, 135.5, 132.2, 130.4, 128.9, 128.1, 126.5, 27.6, 27.0, 17.9, 16.9, 16.5;
+
HRMS (ESI) m/z: 353.8659 [M+H] , Microanalysis calculated for C H ClN O (352.86), C:
2
1
21
2
7
1.48%, H: 6.00%, N: 7.94%. Found C: 71.59%, H: 6.28%, N: 7.65%.
2
-(2-Chloro-4-methoxy-benzylidene)-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-cyclohexanone
(6b )
1
Yield: 72%; Mp: 133-134 ˚C; H NMR (500 MHz, CDCl ) δ: 7.87 (s, H), 7.62 (s, H), 7.35 (d,
3
J=7.0 Hz, H), 7.20 (d, J=7.0 Hz, H), 6.57 (s, H), 3.51(s, 3H)
;
2.67 (s, 6H); 2.61 (s, 3H); 2.35 (t,
1
3
J=8.0 Hz, 4H), 1.82 (m, 2H); C NMR (500 MHz, CDCl
3
) δ: 182.5, 162.2, 157.4, 149.1, 147.5,

ACCEPTED MANUSCRIPT
1
46.2, 145.3, 141.5, 134.7, 132.6, 130.8, 126.2, 118.6, 116.9, 55.7, 27.2, 26.4, 15.9, 15.1, 14.2;
+
HRMS (ESI) m/z: 383.8917 [M+H] , Microanalysis calculated for C H ClN O (382.88), C:
2
2
23
2
2
6
9.01%, H: 6.05%, N: 7.32%. Found C: 69.15%, H: 6.25%, N: 7.21%.
2
-(2-Bromo-4,5-dimethoxy-benzylidene)-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
cyclohexanone (6c)
1
Yield: 74%; Mp: 132-133 ˚C; H NMR (500 MHz, CDCl
H), 6.29 (s, H), 3.59(s, 6H) 2.61 (s, 3H) 2.54 (s, 3H) 2.49 (s, 3H)
m, 2H); C NMR (500 MHz, CDCl ) δ: 182.0, 162.8, 156.6, 148.6, 147.2, 146.0, 145.5, 141.2,
3
) δ: 7.88 (s, H), 7.42 (s, H), 7.15 (s,
;
;
;
;
2.36 (t, J=8.0 Hz, 4H), 1.89
1
3
(
3
1
35.1, 132.9, 130.2, 126.4, 118.2, 117.1, 55.7,55.0, 27.9, 26.5, 15.6, 15.0, 14.4; HRMS (ESI)
+
m/z: 458.3671 [M+H] , Microanalysis calculated for C H BrN O (457.36), C: 60.40%, H:
2
3
25
2
3
5
.51%, N: 6.13%. Found C: 60.49%, H: 5.89%, N: 6.01%.
2
-(2-Chloro-benzylidene)-4-methyl-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-cyclohexanone
(6d)
1
Yield: 68%; Mp: 141-142 ˚C; H NMR (500 MHz, CDCl ) δ: 7.99 (s, H), 7.76 (s, H), 7.32 (d,
3
J=7.0 Hz, H), 7.14 (d, J=7.0 Hz, H), 6.87 (t, J=7.0 Hz, H), 6.60 (t, J=7.0 Hz, H), 2.62 (s, 6H)
;
1
3
2
.57 (s, 3H); 2.05 (d, J=8.5 Hz, 4H), 1.70 (m, H); 1.14 (d, J=8.5 Hz, 3H); C NMR (500 MHz,
CDCl
3
) δ: 181.2, 152.7, 150.8, 149.1, 147.6, 146.2, 145.3, 141.2, 135.6, 132.4, 130.9, 128.6,
+
1
27.4, 126.2, 32.2, 29.5, 22.6, 17.3, 16.9, 16.0; HRMS (ESI) m/z: 367.8925 [M+H] ,
Microanalysis calculated for C H ClN O (366.88), C: 72.02%, H: 6.32%, N: 7.64%. Found C:
22
23
2
7
2.25%, H: 6.57%, N: 7.58%.
2
-(2-Chloro-4-methoxy-benzylidene)-4-methyl-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
cyclohexanone (6e)

ACCEPTED MANUSCRIPT
1
Yield: 62%; Mp: 129-130 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.97 (s, H), 7.68 (s, H), 7.41 (d,
2.64 (s, 3H) 2.54 (s, 3H) 2.49 (s,
2.10 (d, J=8.5 Hz, 4H), 1.79 (m, H); 1.21 (d, J=8.5 Hz, 3H); C NMR (500 MHz, CDCl3)
J=7.0 Hz, H), 7.24 (d, J=7.0 Hz, H), 6.52 (s, H), 3.54(s, 3H)
;
;
;
13
3
H)
;
δ: 181.6, 159.6, 152.2, 149.4, 148.1, 146.7, 145.2, 141.5, 135.4, 132.2, 130.6, 128.1, 127.2,
+
1
26.3, 57.5, 32.9, 28.1, 22.4, 18.1, 17.2, 16.5; HRMS (ESI) m/z: 397.9172 [M+H] ,
Microanalysis calculated for C H ClN O (396.91), C: 69.60%, H: 6.35%, N: 7.06%. Found C:
2
3
25
2
2
6
9.90%, H: 6.52%, N: 6.89%.
2
-(2-Bromo-4,5-dimethoxy-benzylidene)-4-methyl-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
cyclohexanone (6f)
1
Yield: 71%; Mp: 147-148 ˚C; H NMR (500 MHz, CDCl ) δ: 7.92 (s, H), 7.59 (s, H), 7.07 (s, H),
3
6
.27 (s, H), 3.64(s, 6H)
;
2.60 (s, 3H)
;
2.55 (s, 3H)
;
2.49 (s, 3H)
) δ: 182.1, 160.2, 152.8, 150.7, 148.6,
46.3, 145.1, 141.2, 136.3, 132.7, 130.8, 128.6, 127.0, 126.2, 57.7, 56.9, 32.4, 28.6, 21.9, 18.7,
; 2.12 (d, J=8.5 Hz, 4H), 1.76 (m,
1
3
H); 1.14 (d, J=8.5 Hz, 3H); C NMR (500 MHz, CDCl
3
1
1
+
7.9, 17.2; HRMS (ESI) m/z: 472.3952 [M+H] , Microanalysis calculated for C H BrN O
3
24
27
2
(471.39), C: 61.15%, H: 5.77%, N: 5.94%. Found C: 61.29%, H: 5.90%, N: 5.82%.
2
-(2-Chloro-benzylidene)-4-isopropyl-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-cyclohexanone
(6g)
1
Yield: 59%; Mp: 119-120 ˚C; H NMR (500 MHz, CDCl ) δ: 7.90 (s, H), 7.66 (s, H), 7.24 (d,
3
J=7.0 Hz, H), 7.04 (d, J=7.0 Hz, H), 6.81 (t, J=7.0 Hz, H), 6.59 (t, J=7.0 Hz, H), 2.57 (s, 9H)
;
1
3
2
.07 (d, J=8 Hz, 4H), 1.88(m, H); 1.71(m, H); 1.11(d, J=7 Hz, 6H); C NMR (500 MHz, CDCl )
3
δ: 184.6, 152.8, 150.6, 149.8, 147.4, 146.1, 145.0, 140.2, 135.9, 131.1, 130.2, 128.3, 127.4,
+
1
26.1, 38.2, 30.6, 29.2, 22.5, 18.4, 17.8, 16.5; HRMS (ESI) m/z: 395.9451 [M+H] ,

ACCEPTED MANUSCRIPT
2
Microanalysis calculated for C24H27ClN O (394.94), C: 72.99%, H: 6.89%, N: 7.09%. Found C:
7
3.21%, H: 6.99%, N: 7.02%.
2
-(2-Chloro-4-methoxy-benzylidene)-4-isopropyl-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
cyclohexanone (6h)
1
Yield: 57%; Mp: 113-114 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.94 (s, H), 7.70 (s, H), 7.37 (d,
2.59 (s, 9H) 2.05 (d, J=8 Hz, 4H),
J=7.0 Hz, H), 7.14 (d, J=7.0 Hz, H), 6.50 (s, H), 3.59(s, 3H)
;
;
1
3
1
1
3
.80 (m, H); 1.72 (m, H); 1.10 (d, J=7 Hz, 6H); C NMR (500 MHz, CDCl ) δ: 182.9, 159.3,
3
52.4, 149.2, 147.3, 146.5, 144.9, 140.5, 134.8, 132.6, 130.1, 128.7, 126.9, 125.2, 58.2, 39.5,
+
2.8, 28.7, 22.9, 17.9, 17.1, 16.3; HRMS (ESI) m/z: 425.9705 [M+H] , Microanalysis calculated
for C H ClN O (424.96), C: 70.66%, H: 6.88%, N: 6.59%. Found C: 70.72%, H: 6.93%, N:
2
5
29
2
2
6
.27%.
2
-(2-Bromo-4,5-dimethoxy-benzylidene)-4-isopropyl-6-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
cyclohexanone (6i)
1
Yield: 67%; Mp: 128-129 ˚C; H NMR (500 MHz, CDCl ) δ: 7.91 (s, H), 7.64 (s, H), 7.05 (s, H),
3
6
.22 (s, H), 3.61(s, 6H)
;
2.52 (s, 9H)
Hz, 6H); C NMR (500 MHz, CDCl
33.5, 132.2, 130.5, 128.9, 127.2, 125.1, 58.9, 57.4, 39.6, 32.3, 28.4, 23.1, 17.8, 17.4, 16.8;
;
2.06 (d, J=8 Hz, 4H), 1.77(m, H); 1.69 (m, H); 1.12 (d, J=7
13
3
) δ: 182.5, 158.6, 153.5, 148.7, 147.1, 146.4, 143.2, 140.1,
1
+
HRMS (ESI) m/z: 500.4480 [M+H] , Microanalysis calculated for C H BrN O (499.44), C:
2
6
31
2
3
6
2.53%, H: 6.26%, N: 5.61%. Found C: 62.79%, H: 6.29%, N: 5.55%.

ACCEPTED MANUSCRIPT
3
-(2-Chloro-benzylidene)-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-piperidin-4-one (6j)
1
Yield: 62%; Mp: 121-121 ˚C; H NMR (500 MHz, CDCl ) δ: 7.90 (s, H), 7.70 (s, H), 7.31 (d,
3
J=7.0 Hz, H), 7.12 (d, J=7.0 Hz, H), 6.85 (t, J=7.0 Hz, H), 6.64 (t, J=7.0 Hz, H), 2.92 (s, 4H);
1
3
2
1
1
.60 (s, 3H)
;
2.55 (s, 3H)
;
2.50 (s, 3H)
;
C NMR (500 MHz, CDCl ) δ: 182.1, 152.5, 150.6,
3
49.2, 147.8, 146.1, 144.9, 142.5, 135.9, 131.1, 130.2, 128.3, 127.4, 126.1, 44.6, 18.1, 17.9,
+
6.8; HRMS (ESI) m/z: 354.8535 [M+H] , Microanalysis calculated for C20
H20ClN
3
O (353.84),
C: 67.89%, H: 5.70%, N: 11.88%. Found C: 67.91%, H: 5.72%, N: 11.85%.
3
-(2-Chloro-4-methoxy-benzylidene)-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-piperidin-4-one
(6k)
1
Yield: 75%; Mp: 141-142 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.89 (s, H), 7.67 (s, H), 7.29 (d,
2.97 (s, 4H); 2.59 (s, 3H) 2.54 (s,
C NMR (500 MHz, CDCl ) δ: 182.4, 160.7, 154.4, 148.5, 147.3, 146.7,
J=7.0 Hz, H), 7.15 (d, J=7.0 Hz, H), 6.48 (s, H), 3.56 (s, 3H)
;
;
1
3
3
1
H); 2.49 (s, 3H);
3
44.5, 141.3, 134.6, 131.4, 130.6, 128.7, 127.8, 126.4, 57.7, 48.3, 18.2, 17.5, 16.8; HRMS (ESI)
+
m/z: 384.8797 [M+H] , Microanalysis calculated for C H ClN O (383.87), C: 65.71%, H:
2
1
22
3
2
5
.78%, N: 10.95%. Found C: 65.92%, H: 5.79%, N: 10.82%.
3
-(2-Bromo-4,5-dimethoxy-benzylidene)-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-piperidin-4-
one (6l)
1
Yield: 79%; Mp: 139-140 ˚C; H NMR (500 MHz, CDCl ) δ: 7.85 (s, H), 7.62 (s, H), 7.07 (s, H),
3
1
3
6
.25 (s, H), 3.62(s, 6H)
;
2.95 (s, 4H); 2.52 (s, 3H)
;
2.47 (s, 3H)
;
2.41 (s, 3H)
;
C NMR (500
MHz, CDCl ) δ: 183.5, 160.1, 155.2, 148.1, 147.2, 146.5, 144.6, 141.5, 133.9, 131.6, 130.0,
3
+
1
28.8, 127.4, 126.5, 58.2, 57.3, 48.7, 18.6, 17.8, 17.0; HRMS (ESI) m/z: 459.3572 [M+H] ,

ACCEPTED MANUSCRIPT
3 3
Microanalysis calculated for C22H24BrN O (458.34), C: 57.65%, H: 5.28%, N: 9.17%. Found C:
5
7.69%, H: 5.35%, N: 9.05%.
3
-(2-Chloro-benzylidene)-1-methyl-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-piperidin-4-one
(6m)
1
Yield: 84%; Mp: 119-120 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.86 (s, H), 7.61 (s, H), 7.27 (d,
J=7.0 Hz, H), 7.10 (d, J=7.0 Hz, H), 6.87 (t, J=7.0 Hz, H), 6.61 (t, J=7.0 Hz, H), 2.70 (s, 4H);
1
3
2
1
3
.59 (s, 3H); 2.52 (s, 3H); 2.49 (s, 3H); 2.15 (s, 3H); C NMR (500 MHz, CDCl ) δ: 184.1,
3
52.4, 150.3, 149.2, 147.1, 146.2, 145.3, 142.5, 136.2, 132.4, 130.5, 128.2, 127.1, 126.7, 48.4,
+
2.5, 17.6, 17.1, 16.5; HRMS (ESI) m/z: 368.8795 [M+H] , Microanalysis calculated for
C H ClN O (367.87), C: 68.56%, H: 6.03%, N: 11.42%. Found C: 68.61%, H: 6.11%, N:
21
22
3
1
1.22%.
3
-(2-Chloro-4-methoxy-benzylidene)-1-methyl-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
piperidin-4-one (6n)
1
Yield: 79%; Mp: 117-118 ˚C; H NMR (500 MHz, CDCl ) δ: 7.88 (s, H), 7.67 (s, H), 7.26 (d,
3
J=7.0 Hz, H), 7.14 (d, J=7.0 Hz, H), 6.43 (s, H), 3.55 (s, 3H)
;
2.78 (s, 4H); 2.57 (s, 3H)
; 2.51 (s,
1
3
3
1
1
H)
;
2.49 (s, 3H)
;
2.17 (s, 3H); C NMR (500 MHz, CDCl ) δ: 182.9, 162.1, 156.2, 149.5,
3
47.6, 146.0, 145.2, 142.2, 135.6, 132.5, 129.8, 128.5, 127.4, 126.2, 58.3, 49.2, 32.7, 17.9, 17.0,
+
6.4; HRMS (ESI) m/z: 398.9055 [M+H] , Microanalysis calculated for C H ClN O (397.89),
2
2
24
3
2
C: 66.41%, H: 6.08%, N: 10.56%. Found C: 66.57%, H: 6.29%, N: 10.29%.

ACCEPTED MANUSCRIPT
3
-(2-Bromo-4,5-dimethoxy-benzylidene)-1-methyl-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
piperidin-4-one (6o)
1
Yield: 74%; Mp: 134-135 ˚C; H NMR (500 MHz, CDCl ) δ: 7.84 (s, H), 7.67 (s, H), 7.09 (s, H),
3
1
3
6
.21 (s, H), 3.65(s, 6H)
; 2.75 (s, 4H); 2.59 (s, 3H); 2.55 (s, 3H); 2.50 (s, 3H); 2.14 (s, 3H); C
NMR (500 MHz, CDCl ) δ: 182.6, 161.9, 156.5, 150.2, 147.3, 146.1, 145.7, 141.6, 135.2, 131.8,
3
1
29.2, 128.7, 127.2, 126.4, 58.9, 57.6, 50.0, 32.6, 17.4, 16.9, 16.3; HRMS (ESI) m/z: 473.3825
+
[
M+H] , Microanalysis calculated for C23
H26BrN O
3 3
(472.37), C: 58.48%, H: 5.55%, N: 8.90%.
Found C: 58.52%, H: 5.67%, N: 8.87%.
3
-(2-Chloro-benzylidene)-1-isopropyl-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-piperidin-4-one
(6p)
1
Yield: 79%; Mp: 128-129 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.91 (s, H), 7.65 (s, H), 7.26 (d,
J=7.0 Hz, H), 7.14 (d, J=7.0 Hz, H), 6.80 (t, J=7.0 Hz, H), 6.57 (t, J=7.0 Hz, H), 2.69 (s, 3H)
;
13
2
.64 (s, 3H); 2.57 (s, 3H); 2.24 (s, 4H), 1.61(m, H);1.09 (d, J=7 Hz, 6H); C NMR (500 MHz,
CDCl ) δ: 185.0, 154.6, 150.2, 148.7, 147.6, 146.4, 144.0, 142.8, 136.7, 132.3, 130.0, 128.7,
3
+
1
27.6, 126.4, 48.4, 31.6, 23.4, 18.7, 17.9, 17.0; HRMS (ESI) m/z: 396.9329 [M+H] ,
Microanalysis calculated for C H ClN O (395.92), C: 69.77%, H: 6.62%, N: 10.61%. Found C:
23
26
3
6
9.79%, H: 6.69%, N: 10.55%.
3
-(2-Chloro-4-methoxy-benzylidene)-1-isopropyl-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
piperidin-4-one (6q)
1
Yield: 61%; Mp: 132-133 ˚C; H NMR (500 MHz, CDCl ) δ: 7.92 (s, H), 7.63 (s, H), 7.22 (d,
3
J=7.0 Hz, H), 7.19 (d, J=7.0 Hz, H), 6.41 (s, H), 3.58 (s, 3H)
;
2.62 (s, 3H)
;
2.58 (s, 3H); 2.54 (s,
1
3
3
H)
;
2.27 (s, 4H), 1.52(m, H);1.07 (d, J=7 Hz, 6H); C NMR (500 MHz, CDCl
3
) δ: 184.8,

ACCEPTED MANUSCRIPT
1
4
60.2, 154.1, 148.5, 147.9, 146.3, 144.2, 141.4, 135.4, 131.9, 130.2, 129.1, 128.1, 127.4, 59.2,
+
9.9, 32.8, 24.6, 18.1, 17.5, 16.9; HRMS (ESI) m/z: 426.9592 [M+H] , Microanalysis calculated
for C H ClN O (425.95), C: 67.67%, H: 6.63%, N: 9.87%. Found C: 67.72%, H: 6.65%, N:
2
4
28
3
2
9
.82%.
3
-(2-Bromo-4,5-dimethoxy-benzylidene)-1-isopropyl-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-
piperidin-4-one (6r)
1
Yield: 62%; Mp: 126-127 ˚C; H NMR (500 MHz, CDCl ) δ: 7.97 (s, H), 7.71 (s, H), 7.10 (s, H),
3
6
.27 (s, H), 3.69 (s, 6H)
;
2.60 (s, 3H); 2.53 (s, 3H); 2.49 (s, 3H); 2.26 (s, 4H), 1.50(m, H);1.05 (d,
1
3
J=7 Hz, 6H); C NMR (500 MHz, CDCl ) δ: 184.0, 160.4, 154.5, 148.7, 147.6, 146.2, 143.8,
3
1
41.8, 134.2, 131.8, 130.7, 129.4, 128.6, 127.2, 58.7,58.0, 48.4, 32.9, 24.5, 18.6, 17.8, 17.0;
+
HRMS (ESI) m/z: 501.4355 [M+H] , Microanalysis calculated for C H BrN O (500.42), C:
2
5
30
3
3
6
0.00%, H: 6.04%, N: 8.40%. Found C: 60.22%, H: 6.15%, N: 8.41%.
3
-(2-Chloro-benzylidene)-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-tetrahydro-pyran-4-one (6s)
1
Yield: 54%; Mp: 112-113 ˚C; H NMR (500 MHz, CDCl ) δ: 7.78 (s, H), 7.63 (s, H), 7.22 (d,
3
J=7.0 Hz, H), 7.09 (d, J=7.0 Hz, H), 6.73 (t, J=7.0 Hz, H), 6.55 (t, J=7.0 Hz, H), 2.61 (s, 4H),
1
3
2
.50 (s, 3H)
;
2.47 (s, 3H)
;
2.43 (s, 3H)
;
3
C NMR (500 MHz, CDCl ) δ: 182.6, 152.4, 150.7,
1
49.8, 148.1, 147.2, 146.1, 141.9, 134.6, 131.7, 131.0, 129.8, 128.2, 127.3, 54.7, 17.7, 17.0,
+
1
6.6; HRMS (ESI) m/z: 355.8385 [M+H] , Microanalysis calculated for C H ClN O (354.83),
2
0
19
2
2
C: 67.70%, H: 5.40%, N: 7.89%. Found C: 67.84%, H: 5.45%, N: 7.95%.

ACCEPTED MANUSCRIPT
3
-(2-Chloro-4-methoxy-benzylidene)-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-tetrahydro-
pyran-4-one (6t)
1
Yield: 58%; Mp: 119-120 ˚C; H NMR (500 MHz, CDCl ) δ: 7.86 (s, H), 7.69 (s, H), 7.27 (d,
3
J=7.0 Hz, H), 7.04 (d, J=7.0 Hz, H), 6.52 (s, H), 3.61 (s, 3H)
;
2.72 (s, 4H), 2.56 (s, 3H)
; 2.52 (s,
1
3
3
1
H); 2.48 (s, 3H);
C NMR (500 MHz, CDCl ) δ: 182.9, 159.6, 154.2, 149.7, 148.4, 147.2,
3
46.7, 141.5, 133.9, 131.2, 130.2, 129.3, 128.4, 127.5, 58.8, 54.3, 17.9, 17.2, 16.5; HRMS (ESI)
+
m/z: 385.8640 [M+H] , Microanalysis calculated for C21
H21ClN
2
O
3
(384.85), C: 65.54%, H:
5
.50%, N: 7.28%. Found C: 65.59%, H: 5.72%, N: 7.15%.
3
-(2-Bromo-4,5-dimethoxy-benzylidene)-5-(3,5,6-trimethyl-pyrazin-2-ylmethylene)-tetrahydro-
pyran-4-one (6u)
1
Yield: 59%; Mp: 121-122 ˚C; H NMR (500 MHz, CDCl
3
) δ: 7.89 (s, H), 7.67 (s, H), 7.12 (s, H),
1
3
6
.24 (s, H), 3.69 (s, 6H) 2.78 (s, 4H), 2.55 (s, 3H) 2.51 (s, 3H)
;
;
;
2.48 (s, 3H)
;
C NMR (500
MHz, CDCl ) δ: 182.3, 159.8, 153.6, 148.9, 148.0, 147.1, 146.4, 142.2, 132.7, 131.6, 130.9,
3
+
1
29.0, 128.5, 127.3, 58.2, 57.7, 54.2, 17.5, 17.0, 16.6; HRMS (ESI) m/z: 460.3412 [M+H] ,
Microanalysis calculated for C H BrN O (459.33), C: 57.53%, H: 5.05%, N: 6.10%. Found C:
2
2
23
2
4
5
7.58%, H: 5.12%, N: 6.05%.
Synthesis of oxime analogs
Most potent inhibitors of cancer cells growth from ligustrazine containing α, β-
unsaturated carbonyl based compounds were selected as precursors for the synthesis of oxime
analogs. For oxime synthesis, hydroxylamine hydrochloride (2 mmol) was reacted with each
selected compound (1 mmol) in 10 mL ethanol to yield the respective oxime (7b, 7e, 7f, 8f, 8o
and 8r). The average completion time of a reaction was around 6-8 h, as determined by TLC.