Synthesis and biological evaluation of heteroaryl styryl sulfone derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.10.062

Herein we disclose a series of novel heteroaryl styryl sulfone derivatives as potential anticancer agents. Structure-activity relationships of these newly synthesised compounds were explored with respect to the significance of the position and number of nitrogen atom of the heteroaryl ring for anti-proliferative activity in human cancer cell lines. A lead compound 14f was tested against a panel of cancerous and untransformed cell lines, and found to be highly potent against cancer cells with minimal toxicity in the untransformed cells. Further mechanistic studies uncovered that 14f caused cell-cycle arrest at the G2/M phase and induced apoptosis by targeting CDC25C and Mcl-1 proteins in A2780 ovarian cells.

Full text of DOI:10.1016/j.bmcl.2016.10.062

Accepted Manuscript
Synthesis and biological evaluation of heteroaryl styryl sulfone derivatives as
anticancer agents
Yi Long, Mingfeng Yu, Peng Li, Saiful Islam, Aik Wye Goh, Malika
Kumarasiri, Shudong Wang
PII:
S0960-894X(16)31100-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.062
BMCL 24365
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2016
20 October 2016
21 October 2016
Please cite this article as: Long, Y., Yu, M., Li, P., Islam, S., Wye Goh, A., Kumarasiri, M., Wang, S., Synthesis
and biological evaluation of heteroaryl styryl sulfone derivatives as anticancer agents, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.062
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Graphical Abstract

Bioorganic & Medicinal Chemistry Letters
Synthesis and biological evaluation of heteroaryl styryl sulfone derivatives as
anticancer agents
Yi Long a, Mingfeng Yu a, Peng Lia, Saiful Islam a, Aik Wye Goha, Malika Kumarasiri a, Shudong Wang a
a
Centre for Drug Discovery and Development, Sansom Institute for Health Research and School of Pharmacy and Medical Sciences, University of South
Australia, Adelaide, South Australia 5001, Australia
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Herein we disclose a series of novel heteroaryl styryl sulfone derivatives as potential
anticancer agents. Structure-activity relationships of these newly synthesised compounds were
explored with respect to the significance of the position and number of nitrogen atom of the
heteroaryl ring for anti-proliferative activity in human cancer cell lines. A lead compound 14f
was tested against a panel of cancerous and untransformed cell lines, and found to be highly
potent against cancer cells with minimal toxicity in the untransformed cells. Further
mechanistic studies uncovered that 14f caused cell-cycle arrest at the G2/M phase and induced
apoptosis by targeting CDC25C and Mcl-1 proteins in A2780 ovarian cells.
Keywords:
Kinase inhibitor
Polo-like kinase 1
Ras-driven signaling pathways
Non-ATP competitive inhibition
Anti-cancer agents
2016 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +61 8 8302 2372; E-mail: shudong.wang@unisa.edu.au (S. Wang).

Rigosertib (ON01910.Na, Fig. 1) is a synthetic benzyl styryl
sulfone that is currently in different phases of clinical trials for
the treatment of myelodysplastic syndrome (MDS) and various
other cancers.1-5 It has shown efficacy with good tolerability
through a range of dosing schedules by intravenous infusion.6,7
However, it has poor oral bioavailability and ambiguous
pharmacokinetics.8 We have identified a new series of (E)-2/3-
((styrylsulfonyl)methyl)pyridine derivatives as mechanistic
mimetics of rigosertib. The lead drug candidates 8 and 18 (Fig.
1) showed potent anti-proliferative activity against various cancer
cell lines and significant efficacy in xenografted tumour models.9
Both compounds 8 and 18 possess superior cell permeability,
metabolic stability and pharmacokinetic properties with good
oral bioavailability to rigosertib.9,10
cancer cells: (1) abnormal cell division containing irregular
chromosomal segregation and cytokinesis; (2) mitotic (G2/M
phase) arrest and apoptosis; and (3) reduced expression levels of
CDC25C and cyclin D1.11,16
Figure 1. Structures of rigosertib and (E)-2/3-((styrylsulfonyl)methyl)
pyridines 8 and 18.
There are currently two plausible mechanisms of action
proposed for rigosertib and its analogues. Firstly, rigosertib was
found to target Polo-like kinase 1 (Plk1) in a substrate-dependent
and ATP-independent manner.11,12 Plk1 is an essential regulator
of cell cycle progression as it regulates the activation of cyclin
B1 and CDC25C phosphatase.11,13 Secondly, rigosertib acts as a
Ras mimetic. This is believed to be mediated by the binding of
rigosertib to the Ras-binding domain (RBD) found in many Ras
effector proteins, including the Raf and PI3K kinases.14,15 As
such, rigosertib can inhibit multiple Ras-driven signalling
pathways. Previous data showed that rigosertib as well its
mechanistic mimetics 8 and 18 gave rise to three major effects on
In an attempt to discover further novel series of styryl
sulfones for potential drug development, we designed and
synthesised a series of styrylsulfonyl derivatives with different N-
containing heteroaryl systems. Introduction of additional nitrogen
atom(s) into the methylpyridinyl moiety of molecules may result
in not only favourable pharmacological properties, but also new
intellectual property. Herein, we report the synthesis of these
novel compounds, their SAR analysis with respect to the
alterations of heteroaryl rings and the adjacent methylene group,
and insights into the mechanism of action. These data provide
valuable clues for further optimising this class as potential
anticancer agents.

Scheme 1. General synthetic routes to (E)-2-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)pyrazine derivatives (A), (E)-2,3-dimethoxy-5-(((2,4,6-trimethoxystyryl)
sulfonyl)methyl)pyridine (B), (E)-N-(hetero)aryl-2-(2,4,6-trialkoxyphenyl)ethene-1-sulfonamide derivatives (C) and (E)-5-methoxy-4-(((2,4,6-trimethoxystyryl)
sulfonyl)methyl)pyrimidine derivatives (D). Reagents and conditions: (a) NaBH4, MeOH, 0 °C, o/n, 66%; (b) SOCl2, DCM, rt, 1 h, 81%; (c) methyl
thioglycolate, Na2CO3, MeOH, rt, 12 h, 62-87%; (d) H2O2, acetic acid, 60 °C, 12 h, 78-93%; (e) Na2CO3, H2O/MeOH (1:1), rt, 12 h, 61-94%; (f) 2,4,6-
trimethoxybenzaldehyde or 2-ethoxy-4,6-dimethoxybenzaldehyde, benzoic acid, piperidine, toluene, reflux, 3 h, 38-47%; (g) MeONa (or EtONa), MeOH (or
EtOH), 90 °C, 2 h, 68-72%; (h) NBS, AIBN, CH2Cl2, reflux, 18 h, 36-47%; (i) MeI, K2CO3, DMF, rt, 12 h, 65%; (j) methyl 2-(chlorosulfonyl)acetate, Et3N,
.
DCM, rt, 3 h, 74-82%; (k) 32% aqueous ammonia, reflux, o/n, 10%; (l) MeNH2 HCl, Et3N, 2-methoxyethanol, microwave, 150 °C, 1 h, 79%.
The general synthetic route to designed derivatives is outlined
in Scheme 1. The synthesis of 7 started with the reduction of
ester 1, and the subsequent chlorination gave 2-chloro-5-
(chloromethyl)pyrazine 2. Replacement of chlorine of the methyl
group with methyl thioglycolate yielded thioether 3, which was
further oxidised to give sulfone 4. After hydrolysis, Doebner
modification of Knoevenagel condensation between 5 and 2,4,6-
trimethoxybenzaldehyde was carried out to generate 6, which
was reacted with sodium alkoxide to yield 7a and 7b.
These newly synthesised molecules were tested for their anti-
proliferative activity against two tumour cell lines derived from
ovarian carcinoma A2780 and colorectal carcinoma HCT-116
using a 72 h MTT cell viability assay. Rigosertib served as a
positive control, and the results are presented in Table 1.
Pyrazine derivative 6 displayed moderate to weak anti-
proliferative activity against A2780 and HCT-116 cells (GI50
=
4.76 μM and > 10 μM, respectively). Replacement of the bridging
methylene between the pyrazine and sulfone moieties of 6 (Z =
CH2) with a secondary amino group (14g, Z = NH) abolished the
activity (GI50 > 10 μM for both cell lines), while conversion of
the chloride on 6 (R1 = Cl) into an alkyloxy group (7a, R1 = OMe;
7b, R1 = OEt) dramatically boosted the potency against both cell
lines with the GI50 values in the range of 0.49-1.28 μM.
Replacement of the pyrazine ring of 7a (Y = N) with a pyridine
ring (11, Y = CH) reduced the potency by approximately 2 folds
against both cell lines. On the other hand, replacement of the R3
Above synthetic route was employed to prepare 10 and 16
following the bromination of the methyl group of 8 and 15 with
N-bromosuccinimide. 3-Pyridinol 10 was methylated with
iodomethane to give 11; chloride 16 was reacted with aqueous
ammonia or methylamine hydrochloride to produce 17a and 17b
respectively. Preparation of 14a-g started with the coupling of an
appropriate amine 12 with methyl 2-(chlorosulfonyl)acetate to
give the corresponding sulfonamide 13, which was followed by
saponification and condensation to yield 14.
of the comparator
8
(R3
=
NH2) with methoxy

Table 1.
Anti-proliferative activity of the synthesised compounds against human tumour cancer cells
Structure
72 h MTT growth inhibition, GI50 (µM)a
Compd
X
N
Y
N
Z
R1
R2
R3
H
A2780
4.76 ± 1.91
0.49 ± 0.01
0.54 ± 0.12
0.93 ± 0.11
2.60 ± 0.53
0.38 ± 0.04
0.47 ± 0.12
> 10
HCT-116
> 10
6
7a
CH2
CH2
CH2
CH2
NH
NH
NH
NH
NH
NH
NH
-
Cl
OMe
OMe
OMe
OMe
OMe
OMe
OEt
N
N
OMe
OEt
OMe
Cl
H
0.95 ± 0.16
1.28 ± 0.24
2.81 ± 0.30
5.64 ± 0.68
1.00 ± 0.10
0.42 ± 0.07
> 10
7b
N
N
H
11
N
CH
CH
CH
CH
CH
CH
CH
N
OMe
H
14a
14b
14c
14d
14e
14f
N
N
OMe
OMe
Cl
H
N
H
CH
CH
CH
N
OMe
OMe
OMe
OMe
-
H
OMe
OEt
Cl
H
0.01 ± 0.01
0.31 ± 0.02
> 10
0.01 ± 0.01
0.35 ± 0.03
> 10
H
14g
16
H
-
-
-
Cl
> 10
> 10
17a
17b
Rigosertib
8
-
-
-
-
-
-
-
-
-
-
NH2
NHMe
> 10
> 10
7.54 ± 0.80
0.04 ± 0.01
0.01 ± 0.01
> 10
CH
CH
CH2
OMe
OMe
NHCH2COONa
0.06 ± 0.01
0.01 ± 0.01
NH2
N
CH
CH2
OMe
OMe
aGI50 values are presented as mean ± standard deviation derived from at least two replicates.
(11, R3 = OMe) significantly decreased anti-proliferative activity
by ~100 folds, indicating the vital role of the amino group. These
results indicate that the position and number of the heteroaryl
nitrogen atom significantly affect the anti-proliferative activities,
and that alkyloxy substitutions at R1 position also contribute to
the potency of pyrazine analogues. Surprisingly, all pyrimidine
derivatives (16, 17a and 17b) were not active against both A2780
and HCT-116 cells regardless of the nature of substitutions on the
pyrimidine ring (R3 = Cl, NH2 or NHMe).
Human cell line
Origin Destinatio
72 h MTT assay, GI50 (μM)a
14f
Breast
Colon
MCF-7
HCT-116
HT-29
0.53 ± 0.05
0.35 ± 0.03
0.58 ± 0.02
0.59 ± 0.03
0.34 ± 0.00
0.31 ± 0.02
0.30 ± 0.01
0.60 ± 0.01
0.52 ± 0.15
0.45 ± 0.01
0.53 ± 0.03
0.52 ± 0.16
0.34 ± 0.01
> 10
Colo 205
KM12
Ovarian
Pancreatic
Prostate
A2780
PANC-1
PC3
(E)-N-Phenyl-2-(2,4,6-trimethoxyphenyl)ethene-1-sulfon-
amide and (E)-N-(pyridin-3-yl)-2-(2,4,6-trimethoxyphenyl)-
ethene-1-sulfonamide series i.e. 14a-14f, where Z = NH, show
similar SAR profiles. 14a (R1 = Cl) exhibited moderate
cytotoxicity (GI50 = 2.60 μM against A2780 and GI50 = 5.64 μM
against HCT-116). Replacement of chloride with methoxy
resulting in 14b (R1 = OMe) dramatically enhanced cellular
potency with GI50 values of 0.38 μM (A2780) and 1.00 μM
(HCT-116), which are approximately 5-7 folds more potent than
14a. The potency was retained when the R2 position of the styryl
ring of 14b (R2 = OMe) was altered to the ethoxy (14c, R2 =
OEt), suggesting that the less bulky alkyloxy group is tolerated at
R2 position. Amongst all the heteroaryl styryl sulfones
synthesised, aniline derivative 14e (X = Y = CH, Z = NH, R1 =
OMe) displayed the highest potency with a GI50 value of 0.01 μM
against both cell lines, and is 38-fold and 100-fold more potent
against A2780 and HCT-116 cells respectively than its pyridinyl
LNCAP
C4-2B
22-RV1
DU145
DAOY
WI-38
Medulloblastoma
Untransformed
MRC-5
> 10
aGI50 values are presented as mean ± standard deviation derived from at least
two replicates.
counterpart 14b, suggesting the favorable carbon atom over
nitrogen atom at the X position. As expected, the corresponding
ethoxy molecule 14f had a slightly reduced activity (GI50 = 0.31
μM against A2780 and GI50 = 0.35 μM against HCT-116)
Table 2.
Anti-proliferative activity of 14f against a panel of human cell lines

whereas the chloride counterpart 14d (R1 = Cl) was inactive
(GI50 > 10 μM for both cell lines).
Taken together, alkyloxy substitutions at R1 position of
pyrazine, pyridinylamine and aniline derivatives significantly
improved toxicity against A2780 and HCT-116 cells. The
position and number of the nitrogen atom in the heterocyclic ring
play an essential role in the cytotoxicity.
To further assess anti-proliferative activities and cellular mode
of action, 14f, one of the most active compounds, was tested
against a panel of 12 human cancer cell lines and 2
untransformed cell lines. The results are summarised in Table 2.
14f exhibited potent activity against all cancer cell lines with GI50
values ranging from 0.30 to 0.60 μM. It was particularly active
against pancreatic cancer cells (PANC-1) and ovarian cancer
cells (A2780) with a GI50 value of 0.30 and 0.31 μM respectively.
The GI50 values of 14f for untransformed cell lines (WI-38 and
MRC-5) were greater than 10 μM, showing high selectivity of
this compound against cancer cell lines over normal cell lines.
Figure 3. Induction of apoptosis of 14f in A2780 cells after treatment
with 14f (or rigosertib) for 24 h by annexin V/PI assay.
phosphorylated CDC25C directs dephosphorylation of CDC2/
cyclin B1 complex, leading to entry into mitosis.17,18 We have
previously reported that (E)-2/3-((styrylsulfonyl)methyl) pyridine
derivatives 8 and 18 inhibited CDC25C, and that there was a
good correlation between the level of CDC25C and growth
inhibition of cancer cells treated with these compounds.9 As
shown in Fig. 4, the level of p-CDC25C was significantly
reduced by 14f, confirming its cellular inhibition of Plk1. The
reduction in CDC25C expression might be modulated by CHK2
and p53 activities due to Plk1 inhibition.19,20 These two proteins
can promote the down-regulation of CDC25C.21,22 Significantly,
14f was capable of down-regulating anti-apoptotic protein
myeloid cell leukaemia 1 (Mcl-1) and of causing cleavage of
poly (ADP-ribose) polymerase (PARP), both of which are
associated with the apoptosis observed in A2780 cells. These
results clearly demonstrated the mode of apoptotic cell death
induced by 14f. Rigosertib exerted similar effects on these
proteins.
In order to examine whether the anti-proliferative effects of 14f
were a consequence of inhibition of cell-cycle progression,
A2780 cells were exposed to 14f (or rigosertib) for 24 h at the
concentrations of GI50 and 5×GI50 M and analysed by flow
cytometry. As shown in Fig. 2, significant disturbance of cell-
cycle progression was observed. 14f caused a marked increase in
the population of G2/M-phase cells and a large accumulation of
sub-G1-phase cells at 5×GI50 concentration, which was
companied by a substantial loss of G1-phase cells and a minor
decrease of S-phase cells. Rigosertib showed similar cell-cycle
effects, but to a lesser extent.
Figure 4. Cellular mode of action investigation of 14f in A2780 cells
after treatment with 14f for 24 h by western blot analysis.
Figure 2. Cell cycle analysis of A2780 cells after treatment with 14f or
rigosertib for 24 h.
In summary, we have identified a series of new N-containing
heteroaryl styryl sulfone derivatives and their SARs were
analysed. We have demonstrated that the position and number of
heteroaryl nitrogen atom influenced their anti-proliferative
activities. 14f exhibited high potency and was selectively
cytotoxic against human cancer cells with minimal effects on
untransformed cells. Cellular mechanistic studies showed that 14f
arrested cells at the G2/M phase, down-regulated Mcl-1 and
caused PARP cleavage, thus inducing apoptosis in cancer cells.
This work offers a valuable direction for further modification on
these heteroaryl styryl sulfone scaffolds for potential drug
development.
Induction of apoptosis was further confirmed using annexin V/
propidium iodide (PI) staining assay following treatment of
A2780 cells with 14f or rigosertib for 24 h. As illustrated in Fig.
3, both compounds induced apoptotic cell death (annexin V+/PI-
and annexin V+/PI+) in comparison with the control. Again, 14f
was at least equal or more effective in inducing apoptosis
compared to rigosertib.
Given that 14f caused G2/M arrest and induced apoptosis, the
cellular mechanism of action was further evaluated using
Western blotting. CDC25C is an essential substrate of Plk1, and
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This work was supported by the Beat Cancer Project grant, South
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