A diaryl sulfide, sulfoxide, and sulfone bearing structural similarities to combretastatin A-4

Article abstract of DOI:10.1016/j.ejmech.2008.12.018

Studies examining various spacer groups that link the two aromatic rings of combretastatin A-4 (CA4) have shown that the biological activity of analogs does not require the cis-stilbene configuration of CA4. Oxygen or nitrogen, carbonyl, methylene and eth

Full text of DOI:10.1016/j.ejmech.2008.12.018

European Journal of Medicinal Chemistry 44 (2009) 2685–2688
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
A diaryl sulfide, sulfoxide, and sulfone bearing structural similarities to
combretastatin A-4
a
a
a
b
b
´
Euzebio G. Barbosa , Luis A.S. Bega , Adilson Beatriz , Taradas Sarkar , Ernest Hamel ,
c
a,
*
ˆ
Marcos S. do Amaral , Denis Pires de Lima
a Universidade Federal de Mato Grosso do Sul, Departamento de Qu´ımica-CCET, Laborato´rio LP4, CP 549, 79070-900 Campo Grande (MS), Brazil
b Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
c Universidade Federal de Mato Grosso do Sul, Departamento de F´ısica-CCET, LAB2M, CP 549, 79070-900 Campo Grande (MS), Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Studies examining various spacer groups that link the two aromatic rings of combretastatin A-4 (CA4)
have shown that the biological activity of analogs does not require the cis-stilbene configuration of CA4.
Oxygen or nitrogen, carbonyl, methylene and ethylene spacers, for example, are present in CA4 analogs
that show good activity. Up to now sulfur was not tested for this purpose. In this article we describe the
synthesis of sulfide, sulfoxide and sulfone spacers between two aromatic rings comparable to those of
CA4. We also compared them with CA4 for inhibitory effects on cell growth, tubulin polymerization, and
the binding of [3H]colchicine to tubulin. We found that the sulfide is highly active and may be a lead
compound for the preparation of antitumor compounds.
Received 26 July 2008
Received in revised form
13 November 2008
Accepted 15 December 2008
Available online 25 December 2008
Keywords:
Diaryl sulfide
Diaryl sulfoxide
Diaryl sulfone
Ó 2008 Elsevier Masson SAS. All rights reserved.
Combretastatin A-4
Cytotoxicity
Tubulin polymerization
1. Introduction
shown that the two phenyl rings can be separated by cyclic and
non-cyclic linkers. Phenstatin, for example, is highly active and has
Microtubules play an important role in cell division and struc-
ture. Their critical role in the mitotic spindle is exploited in the use
of antitubulin agents as chemotherapeutic drugs. One of the most
successful of these compounds clinically is taxol [1], and the
colchicine site compound combretastatin A-4 (CA4) [2] (Fig. 1) has
attracted a great deal of experimental attention. While agents that
bind at the taxoid site of tubulin promote increased microtubule
assembly [1], colchicine site compounds cause microtubule depo-
lymerization. The cis-stilbene CA4 was discovered by Pettit and co-
workers as the most potent of numerous related compounds
produced by the African plant Combretum caffrum. CA4, in its high
affinity for the colchicine site of tubulin, causes significant cyto-
toxicity and, moreover, in vivo shows marked inhibitory effects on
angiogenesis [3].
a carbonyl moiety rather than the 2-carbon stilbene bridge of CA4
between the aromatic rings [5]. Oxygen and nitrogen linkers were
also tested. Some ether analogs are comparable to CA4 as inhibitors
of tubulin polymerization, but they are much less cytotoxic. Amines
are significantly less active than their ether counterparts [6].
Double bond reduction of CA4 yields compounds with moderately
reduced activity, as does a methylene spacer group [7–9]. More-
over, if the ring B hydroxyl group is missing in the cis-stilbene
structure, activity is only modestly reduced relative to the activity
of CA4 [7]. In this communication, we report our exploration of
sulfur bridges as biosteric replacements for other active atoms
between the two phenyl rings of combretastatin A-4. We found
high activity with a sulfide bridge, while compounds with a sulfone
or a sulfoxide bridge were inactive.
Because of the structural simplicity of CA4, numerous analogs
have been synthesized, and many of these analogs exhibit similar
biological activity. The literature demonstrates the possibilities for
structural variability in analogs in this class [4]. SAR studies have
2. Results and discussion
2.1. Chemistry
Since the potential activity of CA4 analogs with a sulfur atom
linker has not yet been explored, we devised a synthetic route to
* Corresponding author. Tel.: þ55 67 3345 3578; fax: þ55 67 3345 3552.
E-mail address: dlima@nin.ufms.br (D.P. de Lima).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.12.018

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E.G. Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 2685–2688
methylene spacers, but less active than the analog with the cis-
stilbene linker.
MeO
MeO
AcO
OH
NHCOPh
O
O
OH
Ph
Sulfur oxidized compounds 2 and 3 displayed greatly reduced
activity when compared with sulfide 1. This could derive from
conformational issues. Crystallographic structures show that
planes formed by rings in diphenyl sulfoxides and sulfones tend to
be almost orthogonal [12,13], unlike diphenyl sulfides [14], Z-
diphenylethenes [15], and benzophenones [5]. Unfortunately,
however, we have not yet been able to crystallize compound 1 to
confirm this hypothesis.
HO
O
OMe
OH
O
H
OAc
OMe
Combretastatin A-4
OCOPh
Taxol
Fig. 1. Structures of of taxol and CA4.
the sulfide 1, which was then oxidized to the sulfoxide 2 and
sulfone 3 (Scheme 1).
3. Conclusions
Compound 1 was obtained in high yield by an efficient coupling
reaction in which an iodobenzene and a thiophenol were mixed
and refluxed in an oxygen free environment with neocuproine–Cuþ
catalyst and the base Na-tert-butoxide [10]. Sulfoxide 2 and sulfone
3 were obtained in high yield reactions using m-CPBA, one equiv-
alent resulting in sulfoxide 2 and two equivalents in sulfone 3 [11].
The success of the coupling and oxidation reactions was confirmed
by spectral analysis (see Material and methods).
Three new compounds with structural similarity to CA4 were
prepared using reported methodologies. Surveying the data pre-
sented in Table 1, one can conclude that the hydroxyl group in the B
ring generally enhanced the activity of this family of diaryl
compounds, making it worthwhile to consider synthesis of
a hydroxyl analog of compound 1, which may exhibit even better
biological activity than 1. However, activity was not entirely
predictable. The additional hydroxyl group in the B ring seemed to
disproportionately enhance activity with a carbonyl spacer, as in
phenstatin, while having only negligible effect with a cis-stilbene
linker. The conformational status of the sulfoxide (2) and sulfone
(3) probably results in a poor interaction with key amino acids
residues in the colchicine site, and this in turn results in poor
cytotoxicity.
2.2. Bioevaluations
As shown in Table 1, we examined compounds 1–3 for activity as
inhibitors of the growth of MCF-7 human breast cancer cells, the
binding of colchicine to tubulin, and tubulin assembly, in compar-
ison with CA4, phenstatin, and other available, closely related
analogs [2,5,7–9] (we thank Drs. G.R. Pettit of Arizona State
University and M.S. Cushman of Purdue University for supplying
most of these compounds). Only the sulfide 1 had substantial
activity. The relative antiproliferative activities of compounds 1–3
obtained in the MCF-7 cells were also observed in four additional
human cancer cell lines (Table 2).
4. Material and methods
4.1. Chemistry
In comparison with CA4, 1 was similar as an inhibitor of tubulin
assembly and over half as active as an inhibitor of colchicine
binding, but it was about 10-fold less active as an inhibitor of MCF-7
cell growth. Comparing 1 only with the tetramethoxy series
summarized in Table 1, it was more active in all assays as compared
with the compounds with dihydrostilbene, carbonyl, and
All melting points were determined using Uniscience of Brazil
Mod. 498 equipment. Absorption FT-IR spectra were obtained using
the KBr pellet method or in chloroform solution performed with
a Perkin Elmer Mod. 783-FT spectrometer. NMR spectra were
recorded on Bruker DPX-300 spectrometer, the chemical shifts
were presented in parts per million (
d
) relative to TMS (
d
¼ 0.0), and
O
MeO
MeO
S
OMe
2
OMe
iii
NH2
I
MeO
MeO
S
HS
i
ii
+
OMe
OMe
MeO
OMe
MeO
OMe
OMe
1
OMe
OMe
iv
O
O
MeO
MeO
S
OMe
3
OMe
Scheme 1. Reagents and conditions (i) NaNO2, 1:1 conc. HCl/H2O, 5 ꢀC, KI (85%); (ii) neocuproine and CuI (10 mol%), 1.5 equiv. Na-t-butoxide, toluene, 110 ꢀC (98%); (iii) 1 equiv. m-
CPBA, CH2Cl2 (96%); (iv) 2 equiv. m-CPBA, CHCl3 (100%).

E.G. Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 2685–2688
2687
Table 1
Inhibitory effects of compounds 1–3, CA4, and structurally related analogs on tubulin assembly, colchicine binding to tubulin, and the growth of MCF-7 human breast cancer
cells.a
Drug
Tubulin polymerization IC50
(
m
M) ꢂ SD
Colchicine binding % inhibition ꢂ SD
M Drug M Drug
MCF-7 cell growth IC50 (nM) ꢂ SD
R
G
MeO
1
m
5
m
50
mM Drug
A
B
MeO
OMe
OMe
G: S, R: H (1)
G: S]O, R: H (2)
G:O]S]O, R: H (3)
G: CH2CH2, R: OH
G: CH2CH2, R: H
Phenstatin
1.2 ꢂ 0.1
31 ꢂ 3
56 ꢂ 7
89 ꢂ 1
16 ꢂ 7
18 ꢂ 7
67 ꢂ 5
50 ꢂ 6
–
16 ꢂ 5
–
41 ꢂ 6
53 ꢂ 9
–
89 ꢂ 0.1
2200 ꢂ 500
2600 ꢂ 700
13 ꢂ 3
>40
–
1.6 ꢂ 0.03
49 ꢂ 3
2.6 ꢂ 0.3
–
78 ꢂ 10
G: C]O, R: OH
G: C]O, R: H
G: CH2 , R: OH
G: CH2 , R: H
0.73 ꢂ 0.02
2.2 ꢂ 0.03
2.9 ꢂ 0.04
4.0 ꢂ 0.3
70 ꢂ 9
78 ꢂ 4
50 ꢂ 6
44 ꢂ 3
36 ꢂ 5
–
34 ꢂ 20
160 ꢂ 70
59 ꢂ 30
130 ꢂ 40
–
–
–
95 ꢂ 6
91 ꢂ 9
83 ꢂ 3
CA4
G: CH]CH, R: OH
G: CH]CH, R: H
1.1 ꢂ 0.1
1.1 ꢂ 0.2
85 ꢂ 5
74 ꢂ 5
99 ꢂ 0.5
97 ꢂ 2
–
–
1.4 ꢂ 0.1
4.8 ꢂ 1
a
All data presented were obtained in contemporaneous experiments.
CDCl3 was employed as a solvent. High resolution electrospray
ionization–mass spectrometry (ESI–MS) analyses were performed
using an UltroTOF-Q – Electrospray Quadrupole Time-of-Flight
Mass Spectrometer with electronspray ionizer APOLLO as the
interface in positive ion mode. Internal patterns: coumarin (147 and
169) and monensin (693).
The solvents employed in the reactions and silica gel column
chromatography were previously purified and dried according to
procedures found in the literature [16]. Thin-layer chromatography
(TLC) was carried out on silica gel plates with a fluorescence indi-
cator of F254 (0.2 mm, E. Merck); the spots were visualized in UV
light and by spraying with a 1% ethanol solution of vanillin or by
using a charring reagent. Purification of compounds was performed
by column chromatography, the stationary phase was silica gel 60
(80–230 mesh) from ACROS (Brazil), silica gel 60 (230–400 mesh)
from Merck and celite. All reagents used in the present study were
of analytical grade.
4.1.2. Synthesis of 1,2,3-trimethoxy-5-
[(4-methoxyphenyl)thio]benzene (1)
Into a round-bottomed flask, stirred magnetically, were placed
0.186 g (1.94 mmol) of sodium tert-butoxide and 0.025 g of
copper iodide (10 mol% of 5-iodo-1,2,3-trimethoxybenzene). After
the reaction vessel was sealed, 0.17 mL (1.33 mmol) of 4-
methoxybenzenethiol and 0.38 g (1.29 mmol) of 5-iodo-1,2,3-
trimethoxybenzene in 6.0 mL of toluene were injected through
the septum. The reaction mixture was heated for 24 h at 110 ꢀC.
Purification was performed by flash chromatography, and an
amorphous solid was obtained (92% yield). M.p. ¼ 119–120 ꢀC. 1H
NMR (300 MHz, CDCl3):
6.47 (s, 2H),
6.98 (d, J ¼ 8.85 Hz, 2H),
2H). 13C NMR (75 MHz, CDCl3),
55.35, 60.87,
134.37, 136.84, d 153.61,
d 3.76 (s, 6H), d 3.81 (s, 3H), d 3.82 (s, 3H),
d
d
d
7.39 (d, J ¼ 8.85 Hz,
d
d
56.11,
d
d
d
106.61,
159.61.
d
114.88,
d
125.18,
d
134.64,
d
d
HRMS [ESI (þ) – MS]: C16H18O4S [M þ H]þ m/z, calc. 307.1004,
found 307.1097.
4.1.1. General procedure for the ipso iodination of 3,4,5-
4.1.3. Synthesis of 1,2,3-trimethoxy-5-
trimethoxyaniline: synthesis of 5-iodo-1,2,3-trimethoxybenzene (i)
A solution of 3,4,5-trimethoxyaniline (Aldrich Chemical Co., 1.0
g, 2.3 mmol) in 2.2 mL of a 1:1 mixture of conc. HCl and H2O was
added dropwise into a solution of sodium nitrite (0.70 g, 2.3 mmol)
over 15 min, taking care to keep the mixture below 5 ꢀC. Sodium
iodine was then added very slowly, keeping the solution temper-
ature below 0 ꢀC. After being stirred overnight at room tempera-
ture, the mixture was extracted with EtOAc (3 ꢁ 50 mL). The
organic extracts were combined and dried over MgSO4. The puri-
fication was carried out by flash chromatography, yielding an
amorphous solid (85% yield). M.p. ¼ 83–85 ꢀC. The product has
been well described in the literature [17].
[(4-methoxyphenyl)sulfinyl]benzene (2)
To a solution of 30 mg (9.2 mmol) of compound 1 and 5 mL of
dichloromethane was added very slowly 1 equiv. of m-CPBA over
3 h. Sulfoxide formation was monitored by thin-layer chromatog-
raphy. Purification was performed with a flash chromatographic
column, and an amorphous powder was obtained (87% yield).
M.p. ¼ 107.0 ꢀC. 1H NMR (300 MHz, CDCl3):
d 3.85 (s, 3H), d 3.86 (s,
3H),
d
3.89 (s, 6H),
d
6.98 (d, J ¼ 9.04 Hz, 2H), 7.14 (s, 2H), 7.87 (d,
J ¼ 9.04 Hz, 2H). 13C NMR (75 MHz, CDCl3),
d
55.62, 60.89,
d
56.45,
d
d
d
104.73,
d
114.5,
d
128.27,
d
129.6,
d
133.72,
d
136.84, d 153.46,
163.29. HRMS [ESI (þ) – MS]: C16H18O5S [M þ H]þ m/z, calc.
323.0953, found 323.0976.
Table 2
Cytotoxic activity of the compounds 1–3 observed in four additional human tumor cell lines.
Drug
IC50
MDA/MB-435
[mg/mL (m
M)]a
HCT-8
SF-295
HL-60
1
2
3
0.001 (0.003) 0.001–0.004
0.09 (0.28) 0.07–0.12
0.75 (2.32) 0.64–0.89
0.01 (0.04) 0.01–0.02
0.15 (0.47) 0.10–0.26
3.95(12.25) 1.13–3.71
0.01 (0.03) 0.01–0.02
0.47 (1.46) 0.28–0.80
2.61 (8.10) 0.62–0.94
0.001 (0.003) 0.001–0.002
0.11 (0.34) 0.09–0.15
1.53 (4.75) 1.03–2.25
a
Data are presented as IC50 values and 95% confidence intervals obtained by non-linear regression for leukemia (HL-60), breast (MDA/MB-435), colon (HCT-8) and glyo-
blastoma (SF-295) cells from two independent experiments.

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E.G. Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 2685–2688
4.1.4. Synthesis of 1,2,3-trimethoxy-5-
[(4-methoxyphenyl)sulfonyl]benzene (3)
stabilizing buffer system [22] and 1.0
5.0
M [3H]colchicine (from Perkin–Elmer), compound at 1.0, 5.0,
or 50 M in a final dimethyl sulfoxide concentration of 5% (v/v). The
0.1 mL reaction mixtures were incubated for 10 min at 37 ꢀC, at
which time the reaction is about 40–50% complete in the absence of
compound. The reactions were stopped with 3 mL of ice water, and
each diluted reaction mixture was filtered through a stack of two
DEAE-cellulose filters from Whatman. The amount of radiolabeled
colchicine bound to the tubulin adsorbed to the filters was deter-
mined in a scintillation counter.
mM (0.1 mg/mL) tubulin,
m
To a solution of 30 mg (9.2 mmol) of compound 1 and 5 mL of
dichloromethane was added very slowly 2 equiv. of m-CPBA over 3
h. The reaction was monitored by thin-layer chromatography.
Purification was performed by flash chromatography, and an
amorphous powder was obtained (90% yield). M.p. ¼111–112 ꢀC. 1H
m
NMR (300 MHz, CDCl3):
d
d 3.85 (s, 3H), d 3.86 (s, 3H), d 3.89 (s, 6H),
6.98 (d, J ¼ 9.04 Hz, 2H), 7.14 (s, 2H), 7.87 (d, J ¼ 9.04 Hz, 2H). 13
C
NMR (75 MHz, CDCl3),
d
55.62,
d
56.45,
d
60.89,
d
104.73,
d
114.5,
d
128.27,
d
129.6,
d
133.72,
d
136.84,
d
153.46, d 163.29. HRMS [ESI
(þ) – MS]: C16H18O6S [M þ H]þ m/z, calc. 339.0902, found 339.0993.
Acknowledgements
4.2. Antiproliferative and antitubulin activities
The authors owe a debt of gratitude to the following Brazilian
research agencies: FUNDECT-MS, CAPES, PROPP-UFMS, FINEP for
financial support and scholarships. We are also grateful to Kardol
MCF-7 human breast cancer cells were obtained from the
National Cancer Institute drug screening program. They were
grown in monolayer culture in RPMI 1640 medium supplemented
with 5% fetal bovine serum in a humidified 5% CO2 atmosphere at
37 ꢀC. Compounds, dissolved in dimethyl sulfoxide, were added at
varying concentrations, with a final dimethyl sulfoxide concentra-
tion of 1% (v/v). Compounds were added to cells that had been
seeded in 96-well plates 24 h earlier, and incubation continued for
48 h. Cells were fixed with 5% (w/v) trichloroacetic acid, and cell
protein was stained with sulforhodamine B [18]. The plates were
read in a Molecular Devices plate reader (Spectra Max 340). The
IC50 was the compound concentration that reduced the increase in
cell protein after 48 h by 50%.
´
´
Industria Quımica Ltda. (Brazil) for providing some reagents and,
LOE-UFC (Brazil) for carrying out the additional cytotoxic tests.
´
Euzebio thanks and dedicates this work to Nazinha.
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tubulin [20]. The assembly assay [21] was performed with 10 mM
(1.0 mg/mL) tubulin in 0.8 M monosodium glutamate (pH 6.6 with
HCl in a 2.0 M stock solution), 0.4 mM GTP, and 4% (v/v) dimethyl
sulfoxide (as compound solvent). Tubulin and varying compound
concentrations were preincubated without GTP for 15 min at 30 ꢀC,
samples were placed on ice, and GTP was added. The samples were
transferred to 0 ꢀC cuvettes in Beckman DU7400 and DU7500
recording spectrophotometers equipped with electronic tempera-
ture controllers. After baselines were established at 350 nm, the
temperature was jumped to 30 ꢀC (less than 1 min), and sample
turbidity was followed for 20 min. The IC50 was the compound
concentration that reduced the turbidity reading at 20 min by 50%
relative to a control reaction mixture without compound.
The colchicine binding assay was described in detail by Verdier-
Pinard et al. [22]. The reaction mixtures contained a tubulin