Novel cyanocombretastatins as potent tubulin polymerisation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.089

A series of novel cyanocombretastatins bearing a 3,4,5-trimethoxyphenyl moiety combined with a variety of substituted phenyl rings, were synthesised and their antitumour activity was evaluated. The Z-cyanocombretastatins were synthesised in a one-step pro

Full text of DOI:10.1016/j.bmcl.2012.08.089

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6731–6734
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel cyanocombretastatins as potent tubulin polymerisation inhibitors
⇑
Pouria H. Jalily, John A. Hadfield, Nicholas Hirst , Steven B. Rossington
KidsCan Research Laboratories, Centre for Biochemistry, Drug Design and Cancer Research, School of Environment and Life Sciences, University of Salford, M5 4WT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel cyanocombretastatins bearing a 3,4,5-trimethoxyphenyl moiety combined with a
variety of substituted phenyl rings, were synthesised and their antitumour activity was evaluated. The
Z-cyanocombretastatins were synthesised in a one-step protocol in high purity and yield. Fluoro, bromo,
iodo, and derivatives with boronic acid and an ethyne function at meta position of the B ring were
synthesised. In vitro MTT bioassays against human chronic myelogenous leukaemia (K562) and transfec-
ted breast adenocarcinoma (MDA NQO1) cell lines, revealed promising IC50 inhibitory values in nanomo-
lar range (<50 nM). Introduction of a nitrile function on the olefinic bond not only increased the
cytotoxicity of the less active Z-isomers but rendered the analogues as moderate to potent inhibitors
Received 25 July 2012
Revised 20 August 2012
Accepted 23 August 2012
Available online 7 September 2012
Keywords:
Cyanocombretastatins
Combretastatins
CA-4 analogues
Tubulin polymerisation inhibitor
Antiproliferative activity
of tubulin polymerisation comparable to that of CA-4 (IC50 = 2.2 lM).
Ó 2012 Elsevier Ltd. All rights reserved.
There is a continuing effort in cancer research to discover and
develop new drugs that are capable of rapidly destroying tumour
vasculature leading to tumour necrosis and antitumour efficacy.
Such vasculature disrupting agents are promising anticancer
drugs.1,2 The combretastatins are a class of cis-stilbinoid natural
products isolated from the genus Combretum that comprises
numerous trees and shrubs. Whilst being structurally simple these
materials are exceptionally potent inhibitors of cell division.3 One
of the most prominent lead compound is Combretastatin A-4 1.
Stilbene 1 was first isolated from the African Willow Tree, Combre-
tum caffrum, by Pettit et al.4 Stilbene 1 has been found to be a
potent inhibitor of tubulin polymerisation and a cytotoxic agent
against a wide variety of human cancer cell lines, including
multi-drug resistant cell lines.5 As a result of its capacity to dock
in the colchicine binding site of tubulin, it displays a potent antimi-
totic property that has led into its development as an anti-vascular
agent.6
potential antimitotic drug, several hundred analogues have been
synthesised that retain the biological activity of the parent mole-
cule but increase solubility and improve other pharmacokinetic
properties. The water-soluble prodrugs, combretastatin A-4 phos-
phate (CA-4P 2), combretastatin A-1 diphosphate (CA-1P 4),
AC-7739 5, and AVE8062 6 are such promising candidates for the
treatment of cancer (Fig. 1).9 Recent clinical trials demonstrated
that the phosphate prodrug 2 prolonged the survival of patients
with advanced anaplastic thyroid cancer without any adverse side
effects, but failed to reduce the growth of tumour in murine CT-26
adenocarcinoma colon cancer model.10 However, neither of candi-
dates has entered into clinical use and there remains a need to de-
sign and develop new vascular disrupting agents to treat solid
tumours.11
Reversible high affinity binding to the colchicine site of tubulin
appears to be the major mechanism of action of stilbene 1. Poor
aqueous solubility, bioavailability, short biological half life,
isomerisation of the olefinic bond and a number of detrimental ad-
verse side effects are amongst the disadvantages of CA-4 as an
efficient therapeutic agent.7 Acute coronary and other thromboph-
lebitic syndromes, alterations in blood pressure, heart rate, and
ventricular conduction, as well as tumour pain have raised a ques-
tion regarding the safety aspect of vascular disrupting agents in
anticancer therapy.8 To improve the therapeutic index of 1 as a
Figure 1. Structures of naturally-occurring CA-4 1 & CA-1 3, their corresponding
phosphate prodrugs 2 and 4, along with other candidates (5 and 6) currently under
clinical investigation.
⇑
Corresponding author. Tel.: +44 161 295 5465; fax: +44 161 295 5999.
E-mail address: n.hirst@salford.ac.uk (N. Hirst).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.089

6732
P. H. Jalily et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6731–6734
Figure 2. E and Z-cyanocombretastatins.
Scheme 2. Synthesis of analogues 14a and 15a. Reagents and conditions: (i)
NaOMe/MeOH 45–65 °C.
The SAR studies12–14 of 1 revealed that 30,40,50-trimethoxy A ring
and cis-configuration of the olefinic bond is essential for biological
activity. The key structural factor is the presence of an ethylene
bridge connecting the two rings which holds them at an appropri-
ate distance and maintains the correct dihedral angle to maximise
the interactions with protein tubulin. McGown and Fox15 showed
that in order to obtain a compound capable of binding to colchicine
site on tubulin the two planar rings should be tilted at 50–60° to
each other.
The work herein stated is based on the paper published by Ohs-
umi et al.12 which was the first group to report the synthesis of ni-
trile bearing CA-4 analogues. As a part of our ongoing research
towards characterising novel antitumour agents, we have synthes-
ised a series of nitrile bearing E and Z-combretastatins as shown in
Figure 2. Introduction of a heteroatom via a moderately polar ni-
trile function is believed to have a net effect on polarity of the ana-
logues and increase their water solubility in return.
13 and substituted aldehydes (Scheme 2). Similarly stilbenes 18,
20, and 24a were prepared in good yield (Schemes 3–5). The syn-
thesis of the alkyne 20 was achieved by condensation of benzalde-
hyde 19 and phenylacetonitrile 13 as well as concomitant
desilylation (Scheme 4). Based on the previous findings5,18–21 it
was decided to synthesise an analogue bearing a boronic acid func-
tionality at the meta position of the ring B of the parent structure
24a.
Acetal-protection of aldehyde 12 was achieved using triethyl
orthoformate, tetrabutylammonium tribromide, and 1,3-propane-
diol.22 The aldehyde-functionalised boronic acid 22 was synthes-
ised using nBuLi/trimethyl borate in high yield (86%) (Scheme 5).
Owing to the Lewis acidity of boronic acids and the hydrogen
bond donating capability of their hydroxyl groups, it was thought
that its insertion into CA-4 analogues would bring about a change
in biological activity. It is predictable that boron may interact with
tubulin not only through hydrogen bonding but also by forming
covalent bonds, and this interaction would have an impact on
the kinetics of ligand–protein binding.11 Therefore, it was decided
to synthesise an analogue containing a boronic acid at meta posi-
tion of the B ring 24a. Combretastatins possessing an ethyne unit
have shown potential as tubulin-disrupting agents and stilbene
20 was prepared in order to test its biochemical efficacy.16
Cinammic acid 9 was synthesised by the published procedure
from 3,4,5-trimethoxyphenylacetic acid 8, and 3-fluoro-4-
methoxybenzaldehyde 7 (Scheme 1).17 The cinnamic acid 9 was
converted to the amide 10 using EDCI/HOBt and ammonium
hydroxide which was dehydrated to the nitrile 11a in good yield.
The aldehydes used in this study are either purchased from com-
mercial sources or synthesised as shown in Schemes 3–5. The Z-
isomers of the cyanocombretastatin were synthesised by base-cat-
alysed Perkin condensation of 3,4,5-trimethoxyphenylacetonitrile
Scheme 3. Synthesis of analogue 18. Reagents and conditions: (i) I2, CF3COOAg RT,
53%; (ii) 3,4,5-trimethoxyphenylacetonitrile; (iii) NaOMe/MeOH 45 °C, 93%.
Scheme 4. Synthesis of analogue 20. Reagents and conditions: (i) ethynyltrimeth-
ylsilane, TPP, TEA, Pd(OAc)2 85-90 °C, 79%; (ii) 3,4,5-trimethoxyphenylacetonitrile;
(iii) NaOMe/MeOH 55 °C, 50%.
Scheme 5. Synthesis of analogue 24a. Reagents and conditions: (i) triethyl
orthoformate, TBATB/propanediol RT, 98%; (ii) (CH3O)3B, n-BuLi/THF ꢀ78 °C, HCl,
86%; (iii) ethylene glycol, MgSO4 RT, 75%; (iv) 3,4,5-trimethoxyphenylacetonitrile;
(iv) NaOMe/MeOH 50 °C, 66%.
Scheme 1. Synthesis of cyanocombretastatin 11a. Reagents and conditions: (i)
(CH3CO)2O, TEA, HCl 100 °C, 70%; (ii) EDCI, HOBt, N,N-diisopropylethylamine,
NH4OH/THF 35 °C, 55%; (iii) SOCl2/Pyridine RT, 67%.

P. H. Jalily et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6731–6734
6733
Table 1
Table 3
13C GD NMR experiment
Introduction of nitrile function and its effect on antiproliferative activity
Compound
R
R0
J values (Hz)⁄
Configuration
Compound
R
R0
Configuration IC50
(K562) nMa
IC50 (MCF-
7)b
lM
7
8
COOH
CONH2
CN
CN
CN
CN
CN
CN
F
F
F
F
6.6
6.9
9.4
14.4
14.3
13.7
14.9
18.2
E
E
E
Z
Z
Z
Z
Z
1
H
CN
H
CN
H
OH
F
F
F
F
Z
E
Z
Z
E
Z
E
Z
E
2.3 ( 1.6)
6.7 ( 2.8)
727
18.7 ( 10.5)
>2500
—
—
—
—
—
—
—
—
9a
12a
13a
16
18
22a
11a
11b
14a
14b
15a
15b
24a
24b
Br
I
C„C–H
B(OH)2
CN Br
Br
CN B(OH)2
B(OH)2
49.5( 4.8)
H
783028
20.4 ( 12.6)
—
H
470 ( 140)20
Table 2
Antiproliferative activity, and tubulin polymerisation inhibition
a
Values are means of at least three experiments; standard deviation is given in
parentheses.
b
IC50 SD nMa
IC50 SD l
M*
Human breast carcinoma Cells.
Compound
K562
MDA NQO1
Tubulin assembly assay
1
2.3 ( 1.6)
6.7 ( 2.8)
3.1 ( 1.8)
4.1 ( 1.5)
2.2 ( 1.2)
4.2 ( 0.5)
18.5 ( 0.7)
35.6 ( 1.6)
43.8 ( 2.3)
25.3 ( 2.8)
17.4 ( 1.1)
11a
14a
15a
18
20
24a
the Z-isomers (11a and 11b where aryl rings are cis), insertion of
a nitrile group to the Z-isomers (aryl rings trans) significantly in-
creased the antiproliferative activity as compared to the corre-
sponding counterpart (14a, 15a, 24a with 14b, 15b and 24b).
Although the data (24a and 24b) is derived from different cell lines
and precaution is required when comparing, the large difference in
IC50 values proves that nitrile addition to the double is tolerated
and enhances the biological activity.
18.7 ( 10.5)
49.5 ( 4.8)
42.6 ( 7.2)
30.2 ( 6.3)
20.4 ( 12.6)
20.3 ( 11.8)
70.6 ( 9.6)
85.3 ( 10.2)
46.5 ( 7.7)
35.7 ( 13.2)
a
Values are means of at least three experiments; standard deviation is given in
parentheses.
Following a similar method23 the boronic acid was then converted
into the dioxaborolane 23. Subsequent reaction with phenylaceto-
nitrile 13 yielded cyanocombretastatin 24a (Scheme 5). The stereo-
chemistry of the analogues was elucidated using 13C Gated
Decoupling NMR. Presence of a doublet at about d 118 ppm
showed the coupling between the olefinic proton and the nitrile
carbon. In the E-configuration we observed a lower J value (typi-
cally 6–9 Hz) compared to the Z-configuration (>12 Hz) (Table 1).
All the analogues were tested for their ability to inhibit the pro-
liferation of MDA NQO124 and K56225 cancer cell lines. Many of the
analogues exhibited excellent antiproliferative activity (Table 2).
The cell lines were mycoplasma-free and proliferation tests were
carried out using the MTT bioassay described previously.26 The
IC50 value was calculated by reference to a standard curve con-
structed for control cells. The isolation of the tubulin and the
assembly assay were carried out with minor modifications to the
methods described previously.14,15,27 The results enabled the cal-
culation of the drug dose required to inhibit the assembly of tubu-
lin by 50% (IC50 value), determined by graphical means as
percentage of the control assembly (Table 2).
The antiproliferative activity of compound 11a rivals that of CA-
4 itself. Selected analogues that exhibited significant antiprolifera-
tive activities were also tested for their ability to inhibit the poly-
merisation of tubulin. The Z-isomers of the combretastatin are
more cytotoxic than their E-counterparts by about a factor of
1000. In these instances the Z-isomers are where the aryl rings
are cis. The addition of a cyano group on the double bond changes
the E/Z nomenclature so that for E-compounds the aryl rings are cis
and for Z-compounds the aryl rings are trans.
It is unusual that these Z-isomers (aryl rings trans) show such
potent cytotoxicities compared to their cis-counterparts.29,30 This
may arise because the cyano group is large compared to a proton
and may twist these Z-isomers so that they can adopt a conforma-
tion where the aryl rings are twisted from a flat trans arrangement
to a more twisted cis arrangement. This configurational change oc-
curs when an ethyl or methyl group is attached to an olefinic bond
on a combretastatin.30 However, the agent with the most potent
ability to inhibit the assembly of tubulin is fluoro stilbene 11a.
As expected, this molecule has the aryl rings in a cis arrangement.
In the course of this study we have synthesised a series of com-
bretastatin analogues bearing a nitrile function on the olefinic
bridge, and have found many of these agents to be potent in vitro
inhibitors of the proliferation of two cancer cell lines, viz. K562
and MDA NQO1. Compounds 11a, 14a, and 24a exhibited moderate
to potent tubulin-binding activity. In contrast with the general be-
lief that the thermodynamically more stable trans-isomers are bio-
logically inactive,6 the Z-analogues reported herein have exhibited
significantly high antiproliferative activity against MDA NQO1 and
K562 cell lines. The results herein published suggest that replacing
the olefinic hydrogen with a nitrile function is well-tolerated and
does not diminish the ability of appropriately substituted ana-
logues to prevent microtubule assembly. We identify 11a as a lead
candidate for further evaluation.
Acknowledgment
The authors would like to thank Mr. Kirit Amin and Mr. Patrick
Killoran for their technical assistance throughout this project.
The double bond bearing a nitrile would be an effective replace-
ment for the Z-olefin of CA-4 analogues by comparing the activities
of compounds 14a with that of 14b, and 15a with that of 15b (Ta-
ble 3). It can be clearly seen that although the presence of the ni-
trile function did not make a significant change in the activity of
Supplementary data
Supplementary data (copies of 1H, 13C NMR, IR and mass spec-
tra) associated with this article can be found, in the online version,

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P. H. Jalily et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6731–6734
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clude MOL files and InChiKeys of the most important compounds
described in this article.
17. Burja, B.; Cimbora-Zovko, T.; Tomic´, S.; Jelusic´, T.; Kocevar, M.; Polanc, S.;
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