Hydrothiolation of benzyl mercaptan to arylacetylene: Application to the synthesis of (E) and (Z)-isomers of on 01910·Na (Rigosertib), a phase III clinical stage anti-cancer agent

Article abstract of DOI:10.1039/c3ob27220f

A stereoselective and efficient method for free radical addition of benzyl thiol to aryl acetylene in the presence of Et₃B-hexane has been developed for the synthesis of (Z) and (E)-styryl benzyl sulfides where base catalyzed hydrothiolations h

Full text of DOI:10.1039/c3ob27220f

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Hydrothiolation of benzyl mercaptan to arylacetylene:
application to the synthesis of (E) and (Z)-isomers of
ON 01910·Na (Rigosertib®), a phase III clinical stage
anti-cancer agent†
Cite this: Org. Biomol. Chem., 2013, 11,
1964
Venkat R. Pallela,‡a Muralidhar R. Mallireddigari,‡a Stephen C. Cosenza,b
Balaiah Akula,a D. R. C. Venkata Subbaiah,b E. Premkumar Reddyb and
M. V. Ramana Reddy*b
A stereoselective and efficient method for free radical addition of benzyl thiol to aryl acetylene in the
presence of Et3B-hexane has been developed for the synthesis of (Z) and (E)-styryl benzyl sulfides where
base catalyzed hydrothiolations have failed. The scope of this reaction was successfully extended for the
synthesis of (E)-ON 01910·Na, a phase III clinical stage anti-cancer agent and its inactive geometrical
isomer (Z)-ON 01910·Na. It is interesting to note that all the E-isomers synthesized have shown better
cytotoxicity profile on cancer cells compared to the Z-isomers.
Received 14th November 2012,
Accepted 18th January 2013
DOI: 10.1039/c3ob27220f
www.rsc.org/obc
activity studies of (E)-ON 01910·Na confirmed that the nature,
Introduction
number, and position of substituents on both aromatic rings
ON 01910·Na (Rigosertib®)1 is a styryl benzyl sulfone and it is of the molecule along with geometry of the styryl group are
biologically active only as an (E)-isomer. It is a novel non-ATP critical for activity.9
competitive anticancer agent that inhibits mitotic progression,
(E)-ON 01910·Na was synthesized by Knoevenagel conden-
promotes G2/M arrest and induces apoptosis in a number of sation as described in our earlier publication.9 It exists as a
cancer cells while leaving nonmalignant cells virtually stable E-isomer, which is the preferred form of this molecule.
unaffected.2 (E)-ON 01910·Na has exhibited both antitumor The double bond in (E)-ON 01910·Na is capable of exhibiting
activity and anti-angiogenic activity with a low toxicity profile geometrical isomerism resulting in both E- and Z-isomers.
in various preclinical tumor xenograft models. Unlike hypo- Stability studies indicate that over a period of time (E)-ON
methylating agents, (E)-ON 01910·Na exerts potent antitumor 01910·Na, when exposed to light, accumulates traces of the
activity against leukemic and pre-leukemic cells by inhibition Z-isomer in both solid and solution form. Similar kinds of
of the PI/3K/Akt/mTOR pathway and down regulation of cyclin photo-induced geometric isomerism changes have been
D1 translation.3,4 Cyclin D proteins are elevated in many hema- reported previously10 and have resulted in the formation of
tologic malignancies, including the myelodysplastic syn- mixed geometrical isomers11 from a single isomer. Surpris-
dromes (MDS).5 A phase III pivotal trial of (E)-ON 01910·Na in ingly, neither thermal exposure of (E)-ON 01910·Na in the
MDS patients has been underway.6 This compound has also absence of light nor storage in amber colored vials resulted in
shown promising therapeutic effects and drug tolerance in the accumulation of Z-isomer. This indicates that photo iso-
patients with advanced solid tumors including pancreatic merization is taking place resulting in the formation of traces
(phase II/III) and ovarian (phase II) cancer, both as a single of (Z)-ON 01910·Na (Fig. 1), which has the same molecular
agent and in combination therapy protocols.7,8 The structure weight and molecular formula but different retention time in
HPLC. The objective of the present study is to synthesize the
aDepartment of Medicinal Chemistry, Onconova Therapeutics Inc., 375 Pheasant
Run, Newtown, PA 18940-3423, USA
bDepartment of Oncological Sciences, Mount Sinai School of Medicine, Icahn
Medical Institute, 1425 Madison Ave., New York, NY 10029-6514, USA.
E-mail: r.reddy@mssm.edu; Fax: +1 215 893 6989; Tel: +1 212 659 6875
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of all new compounds. See DOI: 10.1039/c3ob27220f
‡These authors contributed equally.
Fig. 1 Photoisomerization of ON 01910·Na.
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presence of Et3B-hexane leading to stereoselective formation of
(Z)-styryl benzyl sulfides. Thus, a general and simple method
for the synthesis of stereoselective (Z)-styryl benzyl sulfides
from alkynes remains a challenge to be addressed. Hence,
herein we report the hydrothiolation of aryl acetylenes by
benzyl thiols in the presence of Et3B-hexane as a radical
initiator, resulting in stereoselective formation of (Z)-styryl
benzyl sulfides under mild reaction conditions.
Fig. 2 Radical and catalytic alkyne hydrothiolations.
Z-isomer of ON 01910·Na in sufficient quantities to character-
ize and compare the pharmacokinetic and biological activity of
this compound with that of the E-isomer. As the geometry of a
molecule is known to play an important role in SAR, the syn-
thesis of the Z-isomer provides an opportunity to compare the
biological and pharmacological properties of both isomers.
This study also allows us to design alternate methods for exclu-
sive synthesis of the E-isomer, processes for purification and
stabilization, and to minimize or totally eliminate the un-
desired product(s).
Alkyne hydrothiolation (Fig. 2) is a challenging reaction
associated with an atom-economical method for C–S bond for-
mation, Markovnikov and (or) anti-Markovnikov addition, and
stereoselectivity. Hydrothiolation is also of great importance as
it provides an attractive method for the formation of vinyl sul-
fides, which can be used as complementary building blocks to
carbonyl compounds12 and Michael acceptors13 for synthesis
of many polymeric materials,14 natural products,15 and syn-
thetic reagents.16 Conventional approaches to the synthesis of
vinyl sulfides include the addition of thiols to alkynes under
free-radical17 or transition metal catalyzed conditions,18 Wittig
olefination,19 and direct nucleophilic substitution through use
of vinyl halides.20 Several metal catalysts have proven effective
in hydrothiolation of unsaturated compounds, but the stereo-
and regioselectivity control still remains an important
challenge.21
Results and discussion
Synthesis of key starting materials
4-Methoxy-3-nitrobenzyl thiol (3) was synthesized as shown in
Scheme 1. 4-Methoxy-3-nitro toluene (1) was brominated with
NBS in the presence of a catalytic amount of benzoyl peroxide
in CCl4 to obtain 4-methoxy-3-nitrobenzyl bromide (2) which
on subsequent reaction with thiourea gave an intermediate iso-
thiouronium salt which on further reduction with ammonia
yielded 3 along with its dimer 1,2-bis(4-methoxy-3-nitrobenzyl)-
disulfane (4) as a byproduct. 2,4,6-Trimethoxyphenyl acetylene
(7) and its propiolic acid derivative 7a were prepared as shown
in Scheme 2. Reaction of 2,4,6-trimethoxybenzaldehyde (5)
with bromomethyltriphenylphosphonium bromide in the pres-
ence of potassium t-butoxide gave 2-(2-bromovinyl)-1,3,5-tri-
methoxybenzene (6) which on dehydrobromination with
potassium t-butoxide afforded 7 in moderate yields. Com-
pound 7a was synthesized starting from 5 and CBr4 in the pres-
ence of Ph3P and the resulting 2′,2′-dibromovinyl-1,3,5-
trimethoxybenzene (8) was treated with methyl chloroformate
and n-BuLi in THF at −78 °C to get methyl ester 9 which on
subsequent hydrolysis with lithium hydroxide afforded 7a in
low yields.
Numerous methods have been developed for the stereo-
selective synthesis of (E)-vinyl sulfides.22 In contrast, it has
been challenging to prepare Z-isomers stereoselectively.23 The
synthesis of (Z)-vinyl sulfides utilizing transition metal cata-
lysts has been limited to neutral and cationic complexes of
rhodium and iridium. These methods suffer from limited sub-
strate scope and low stereoselectivity.24 Base-mediated hydro-
thiolations were first reported in 1956 by Truce et al.,25 and,
more recently, a cesium carbonate catalyzed process to syn-
thesize (Z)-vinyl sulfides was reported.26 This process requires
a radical inhibitor, and the substrate scope was limited to only
alkyl thiols. Alkyne hydrothiolation via a radical mechanism
was first reported in 1970, whereby a free radical process
yielded anti-Markovnikov products with typically low E/Z selec-
tivity.27,28,17a,b Synthesis of vinyl sulfides by the addition of
thiols to acetylenes in the presence of Et3B was reported29 in
1987, but the scope is limited to alkyl acetylenes only.
Although the free-radical addition of aryl and alkyl thiols with
various phenyl and alkyl acetylenes in the presence of AIBN
has been reported,17b,30 we have noticed no reports concerning
Scheme 1 Synthesis of 4-methoxy-3-nitrobenzyl thiol (3).
Scheme 2 Synthesis of 2,4,6-trimethoxyphenyl acetylene (7) and its propiolic
the addition of benzyl mercaptans to aryl acetylenes in the acid derivative (2,4,6-trimethoxyphenyl)propynoic acid (7a).
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Table 1 Addition of 4-methoxy-3-nitrobenzyl thiol (3) to 2,4,6-trimethoxyphenyl acetylene (7) or 2,4,6-trimethoxyphenyl propynoic acid (7a) under different
conditionsa
Entry
R
Base
Solvent
Catalyst
Temp (°C)
Time (h)
Yieldb (%)
Z/Ec
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
COOH
COOH
H
H
H
H
Na metal
NaOH
—
—
K2CO3
EtOH
EtOH
Toluene
Water
NMP
NMP
NMP
DMSO
NMP
DMSO
Benzene
Benzene
Benzene
Benzene
—
—
80
80
60
100
80
80
80
85
90
90
80
80
25
25
24
12
2
Traces
Ndd
—
0
<10
40
Traces
0
Traces
10
0
<10
62
0
82
0
Al2O3/KF
—
CuI
CuI
CuI
—
CuI
—
—
56 : 44
79 : 21
Ndd
—
2
24
24
24
24
24
4
5
5
2
2
Et3N
Cs2CO3
Ndd
70 : 30
—
Cs2CO3 + TEMPOe
Cs2CO3
9
10
11
12
13
14
Cs2CO3 + TEMPOe
AIBN
Ndd
79 : 21
—
AIBN + TEMPOe
Et3B-hexanef
Et3B-hexanef + TEMPOe
—
—
—
86 : 14
—
a Unless noted, all the reactions were carried out with 3 (1.0 mmol), 7 or 7a (1.0 mmol), base (1.2 mmol), catalyst (1.2 mmol) in 10 mL of solvent
at indicated time and temperature. b Yield of isolated product. c Z/E ratio was determined by the 1H NMR spectrum of crude products. d Not
determined. e 0.2 mmol was added along with the base. f 1.0 M solution from Sigma (Cat # 195030).
In our first attempt, we tried different conventional
As base-catalyzed reactions resulted either in low yields or
approaches to make 1,3,5-trimethoxy-2-[2-(4-methoxy-3-nitro- poor selectivity, we moved to a strategy employing free radical
benzylsulfanyl)vinyl]benzene (10) by the reaction of 4-methoxy- initiators. Addition of 3 to 7 in the presence of AIBN in
3-nitro benzyl thiol (3) and 2,4,6-trimethoxyphenyl acetylene (7) benzene at 80 °C afforded a mixture of addition products
in the presence of a number of bases and catalysts (Table 1). (Z/E = 79 : 21) in 62% yield (entry 11). Encouraged by this
Initially, we tested the effect of base on this hydrothiolation result, we attempted the same reaction with Et3B-hexane as the
reaction. As we have used metallic sodium in ethanol or radical initiator which further improved the overall yield and
sodium hydroxide in ethanol in our earlier attempts to make selectivity (Z/E = 86 : 14) (entry 13). This reaction provided very
(Z)-vinyl sulfides,31 the same approach was tried for this reac- good yields, better stereoselectivity, shorter reaction times and
tion. Both reactions (entries 1 and 2) did not proceed at reflux the cleanest product of all the reactions tried. Next, we exam-
temperatures for more than 24 h. Alternatively, with a solid ined whether this reaction would proceed if free radical for-
supported Al2O3/KF catalyst at 60 °C, this reaction proceeded to mation were suppressed by the addition of 20 mol% of
give a mixture of isomers (Z/E = 56 : 44) with yields of less than TEMPO. In the presence of TEMPO, the reaction is completely
10%. We then attempted addition of the thiol with acetylene inhibited both in AIBN and in Et3B initiated reactions (entries
with a base and catalyst free reaction in the presence of water 12 and 14, respectively), demonstrating that this reaction is
as the solvent.32 The reaction did not proceed at room tempera- mediated by a radical mechanism. We varied the solvents and
tures, but at reflux temperatures the reaction produced moder- reaction temperatures in order to establish the optimal con-
ate yields of a mixture of isomers (Z/E = 79 : 21) (entry 4). ditions for this reaction (Table 2). Among them, non-polar sol-
Reactions with bases such as K2CO3, Et3N and Cs2CO3 in NMP vents such as benzene and toluene yielded addition products
solvent and CuI as catalyst did not yield the addition product with high yields and greater selectivity at room temperatures.
(entries 5–7). Recently, coupling reactions initiated by the de- Addition of polar solvents such as methanol as an additive to
carboxylation of carboxylic acids catalyzed by copper have either benzene (entry 3) or toluene (entry 7) completely
shown great promise in the synthesis of stereoselective (Z)-vinyl reversed the stereoselectivity towards the formation of
sulfides.33 The reaction of 2,4,6-trimethoxyphenyl acetylene (7) E-isomers with higher overall yields.22d It is known that in
(entry 7) or its propiolic acid derivative (7a) (entry 9) and 3 in polar solvents the proton abstraction from thiol is slow and the
the presence of CuI and cesium carbonate did not proceed formed Z-vinyl radical (10a) (Scheme 3) can easily isomerize to
even after stirring for 24 h at 80–90 °C. Addition of a radical a thermodynamically more stable E-vinyl radical (10b) and
inhibitor such as TEMPO (2,2,6,6-tetramethyl-piperidine- then abstracts the hydrogen atom leading to the formation of
N-oxyl) (entries 8 and 10) resulted only in very poor yields of the E-isomer. This is also supported by the fact that the bond
the product. As hydrothiolation reactions with transition metal dissociation energy of RS–H would be higher in polar solvents
catalysts produce predominantly E-isomers, we have not made and the formed Z-radical would have enough time to convert
any attempts to synthesize Z-isomers using these catalysts.
to a more stable E-radical.34 The same phenomenon was also
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to −30 °C (Z/E = 91 : 9) to −78 °C (Z/E = 100 : 0). As hydrothiola-
tion reactions are solvent, temperature and catalyst dependent,
a temperature effect was found to be operative in this reaction.
The results suggest that the trans addition process giving a cis
adduct is presumably a kinetically controlled process in
aprotic solvents such as DCM with a high dipole moment
leading to the formation of a cis addition product.34b,c,35
Although the reaction proceeded more slowly at lower tempera-
tures, its selectivity improved, resulting in the formation of a
single isomer and thereby avoiding the need for separation of
isomers. Hence, the addition reaction in DCM at −30 °C or at
−78 °C in the presence of Et3B-hexane was chosen as the best
method for the preparation of (Z)-styryl benzyl sulfides 10.
A possible mechanism for the formation of styryl benzyl
sulfides by free radical addition of the thiol to acetylene is out-
lined in Scheme 3. At the initiation step, an ethyl radical gen-
erated from triethylborane in the presence of molecular
oxygen abstracts a hydrogen atom from thiol (3) to form a thiyl
radical (3a). As the thiyl radicals are known to behave as elec-
tron deficient species, they immediately react with a more elec-
tron rich SP-hybridized carbon of the acetylene (7), to furnish
a β-sulfanyl radical (10a). Hydrogen abstraction from the start-
ing thiol 3 afforded a styryl benzyl sulfide (10) and a new thiyl
radical 3a is regenerated to complete the radical chain pro-
cess.34b,36 Although, the carbon–sulfur bond formation occurs
regioselectively at the β-position of the acetylene where higher
electron density resides, there is a possibility of the formation
of a mixture of E- and Z-stereoisomers. Though the isomeriza-
tion between Z- and E-sulfanyl radicals (10a and 10b) is faster
than the abstraction of the proton from thiol 3, the rate of
interconversion would be largely determined by steric hin-
drance between their cis-2-substituents (i.e. RS or H) and a
radical scavenger rather than by their equilibrium posi-
tion.30,34c As the cis-2-substituent H of the Z-sulfanyl radical
(10a) is smaller than the cis-2 substituent RS of the E-sulfanyl
radical (10b), 10a preferentially abstracts hydrogen from 3
leading to the formation of Z-styryl benzyl sulfide (10).30,36 In
light of the proposed mechanism and based on the experimen-
tal results from Tables 1 and 2, it may be concluded that the
addition of 3 to 7 under radical reaction conditions proceeds
preferably in trans fashion to afford Z-styryl benzyl sulfides,
although the exact stereo chemical course of these addition
reactions remains uncertain due to the influence of reaction
conditions (e.g. temperature and solvent).
Table 2 Optimization reaction conditions of Et3B-hexane induced radical
addition of 4-methoxy-3-nitrobenzyl thiol (3) to 2,4,6-trimethoxyphenyl acety-
lene (7)a
Temp
(°C)
Time
(h)
Yieldb
(%)
Entry Solvent
Z/Ec
1
2
3
Benzene
Benzene
Benzene + 4 eq.
methanold
Toluene
Toluene
Toluene
Toluene + 4 eq.
methanold
Acetonitrile
1,4-Dioxane
DCM
DCM
DCM
THF
NMP
DMSO
DMF
25
5
25
2
2
2
82
75
92
86 : 14
78 : 22
0 : 100
4
5
6
7
25
0
−20
0
2
3
4
2
85
80
75
95
88 : 12
79 : 21
86 : 14
0 : 100
8
9
25
25
25
−30
−78
25
25
25
25
3
3
0.5
1
8
4
2
2
2
82
60
85
88
68
80
85
60
72
72 : 28
48 : 52
58 : 42
91 : 9
100 : 0
62 : 38
63 : 37
8 : 92
10
11
12
13
14
15
16
28 : 72
a Unless noted, all the reactions were carried out with 3 (1.2 mmol), 7
(1.0 mmol), base (1.2 mmol) in 10 mL of solvent at indicated time and
temperature. b Yield of isolated product. c Z/E ratio was determined by
the 1H NMR spectrum of crude products. d Methanol was added to the
reaction mixture before Et3B-hexane addition.
Scheme 3 A plausible reaction mechanism of radical addition of the thiol to
acetylene.
Oxidation of sulfides to sulfoxides and sulfones
observed in reactions with other polar aprotic solvents such as
DMSO (entry 15) and DMF (entry 16) where selectivity towards
the E-isomer was high with reasonably good yields. Reactions
in acetonitrile, 1,4-dioxane, NMP and THF resulted in poor
selectivity towards Z-isomer formation, prolonged reaction
times and low to moderate yields. When we tried reactions in
DCM at 25 °C (entry 10) and at −30 °C (entry 11), the reactions
proceeded very rapidly, resulting in very good yields. The selec-
tivity profile of the product formation has changed completely
as the reaction temperature changed from 25 °C (Z/E = 58 : 42)
After separating the mixture of Z/E-styryl benzyl sulfide 10 to
pure (Z)-styryl benzyl sulfide (11) and (E)-styryl benzyl sulfide
(12) by column chromatography, we oxidized them to their cor-
responding sulfoxides and sulfones. Several oxidizing agents
were tried to optimize the oxidation process to achieve better
yields and selectivity without isomerization (Table 3) of the
product. Among the reagents used, oxidation of 11 with 30%
hydrogen peroxide in glacial acetic acid at room temperature
resulted in a decomposed product (entry 1), whereas at 5 °C
both 11 and 12 afforded corresponding sulfoxides 13 and 14,
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Table 3 Oxidation of (Z) and (E)-1,3,5-trimethoxy-2-[2-(4-methoxy-3-nitrobenzylsulfanyl)vinyl]benzene (11 and 12)
Entry
Compd
Oxidizing agent (eq.)
Solvent
Temp (°C)
Time (h)
Producta
Yieldb (%)
c
1
2
3
4
5
6
7
8
9
11
12
11
12
12
11
12
11
11
11
H2O2
H2O2
H2O2
H2O2
AcOH
AcOH
AcOH
AcOH
AcOH
MeOH
MeOH
THF/MeOH (2 : 1)
THF/MeOH (2 : 1)
Hexafluoro-2-propanol
rt
rt
5
8
24
6
6
6
12
24
24
24
2
—
16
13
14
16
—
16
—
40
55
75
85
5
H2O2
60
rt
rt
rt
rt
rt
c
m-CPBA (2.5)
m-CPBA (2.5)
Oxonee (4)
Oxonee (6)
H2O2
—
50
55
65
40
13 & 15d
15
10
13
a Starting compound geometry was retained in the product. b Yield of isolated product. c Starting compound decomposed. d Partial oxidation
leading to mixture of sulfoxide and sulfone (ratio:15/85). e Oxone was dissolved in water (2 mL g−1) and then added to the reaction mixture.
respectively, in good yields (entries 3 and 4). Oxidation of 12
with hydrogen peroxide in glacial acetic acid at room tempera-
ture for 24 h gave the sulfone (16) in moderate yields (entry 2),
whereas when the same reaction was conducted at 60 °C for
6 h sulfone (16) was obtained in much higher yields in shorter
reaction times (entry 5). Oxidation of the Z-isomer 11, with
m-chloroperoxybenzoic acid (m-CPBA) at room temperature,
resulted in decomposition of the product (entry 6) whereas oxi-
dation of the E-isomer 12 under the same conditions gave
sulfone 16 in moderate yields (entry 7). Due to poor yields and
sensitivity to harsh reaction conditions, oxidation of the
Z-isomer was attempted under mild reaction conditions using
Oxone in THF/MeOH. With 4 equivalents of Oxone, the
Z-sulfide 11 underwent partial oxidation to afford a mixture of
sulfoxide (13) and sulfone (15) in a 15/85 ratio (entry 8),
whereas the reaction with 6 equivalents of Oxone resulted in
complete oxidation to sulfone 15 in relatively good yields
(entry 9). When 1,1,1,3,3,3-hexafluoro-2-propanol and 30%
H2O2 in THF was used for oxidation of 11, only sulfoxide (13)
was formed in moderate yields (entry 10). Thus, from these
observations, we conclude that the use of Oxone at room temp-
erature for the oxidation of 11 to Z-sulfones and the use of
H2O2 in AcOH at 5 °C for making Z-sulfoxides are the best
methods of oxidation for making the sulfones and sulfoxides,
respectively. Similarly, oxidations with H2O2 in AcOH at 60 °C
and H2O2/AcOH at 5 °C are the best methods for converting
(E)-vinyl sulfides (12) into their corresponding sulfones and
sulfoxides, respectively. In all these reactions the starting com-
pound geometry was not affected by the oxidation.
Table 4 Reduction of 3-nitro (E)-styryl benzyl sulfone (16) to 3-amino (E)-styryl
benzyl sulfone (22)
Temp Time Yielda
Entry Catalyst
Solvent
(°C) (h)
(%)
1
2
3
4
Na2S2O4
Zn
RANEY® Ni/NH2NH2 MeOH
Indium/NH4Cl
SnCl2
Fe(0)
Acetone/water (2 : 1) 50
1
3
4
5
4
3
3
20
40
50
50
40
95
74
AcOH
25
40
80
25
EtOH
EtOH
5
7
AcOH/MeOH (1 : 2) 80
8b
3% Pt/C (sulfided)/H2 CH3CN 60
a Yield of isolated product. b Pt catalyst purchased from Evonik
Degussa Corporation, KY, USA.
Optimization of the reaction conditions for reduction of the
nitro compounds was initiated using compound 16. Reduction
of the nitro group in 16 with sodium hydrosulfite in acetone/
water (2 : 1) at 50 °C gave a clean reduced product 22 with very
low yields (entry 1) whereas the reduction with Zn/AcOH at
room temperature afforded moderate yields of the amino com-
pound 22 (entry 2). Reduction of 16 with RANEY® Ni/NH2NH2
in MeOH at 40 °C or indium metal/NH4Cl in EtOH at 80 °C or
SnCl2 in EtOH at room temperature resulted in the formation
22 in moderate to good yields (entries 3–5). Reduction of the
nitro compound 16 with metallic iron powder in AcOH/MeOH
(1 : 2) at reflux temperatures gave corresponding amine 22 in
very high yields (entry 7). Reduction with 3% Pt/C in aceto-
nitrile at 60 °C in the presence of H2 also resulted in the for-
mation of the corresponding amine compound with yields
comparable to that of the iron method (entry 8). Based on the
cost and effectiveness of all the reducing agents, the use of
Reduction of nitro styryl benzyl sulfides, sulfoxides and
sulfones into their corresponding amino compounds
The reduction of nitro styryl benzyl sulfides, sulfoxides and
sulfones to their corresponding amino compounds was tried
with various reducing agents to optimize the product yields
and to reduce the formation of byproduct impurities (Table 4).
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iron powder for the reduction of the nitro group in sulfides, geometry in the biological activity, E-isomers are more potent
sulfoxides and sulfones was considered to be the economically than their corresponding Z-isomers.
best choice among the reducing agents used. We extended this
The compounds 21 and 22, which have high potency, also
optimized reduction condition with the iron catalyst for redu- have very low solubility in aqueous buffers and solutions. In
cing the nitro sulfides 11 and 12 to their corresponding amino order to enhance their water solubility and bioavailability,
sulfides 17 and 18, nitro sulfoxides 13 and 14 to their corres- amino groups in 21 and 22 were converted to glycine deriva-
ponding amino sulfoxides 19 and 20 and nitro sulfone 15 to tives 23 and 24. As shown in Table 5, glycyl esters of the
amino sulfone 21. The starting compound geometry was not Z-isomers are less active than those of the E-isomers. Similarly
affected by the reduction conditions.
the sodium salt of (Z)-ON 01910·Na (25) is approximately 5000
times less potent in killing K562 cells and 437 times less
potent in killing DU145 cells than the corresponding salt of
(E)-ON 01910·Na (26). From this study, it is evident that apart
from the nature and position of the substituents in a given
molecule, the geometry of the molecule also contributes to its
biological activity. As the biological activity of the molecule
depends upon its ability to bind to a cellular target, the orien-
tation of the groups within the molecule ultimately determines
binding affinity. The trimethoxy styryl group in the trans-
configuration may have a better orientation with which to
interact with a given target than the cis-isomer. Co-crystal
studies of these molecules with their targets would demon-
strate these interactions more clearly and also explain the
superior biological activity of the E-isomer over the Z-isomer.
As the E-configuration of the molecule proved to be essential
for biological activity, the inactive Z-isomer could be used as a
negative control in the biological assays for determining the
mechanism of action of these molecules.
Synthesis of (Z) and (E)-isomers of ON 01910·Na (25 and 26)
In order to enhance the solubility and bio-availability of these
molecules, the (Z) and (E)-amino sulfones 21 and 22 were
alkylated to their corresponding glycyl esters 23 and 24 with
methyl 2-bromoacetate in the presence of a mild base sodium
acetate, which on subsequent hydrolysis afforded the corres-
ponding highly water soluble sodium salt of acids 25 (Z-ON
01910·Na) and 26 (E-ON 01910·Na) (Scheme 4).
In vitro cytotoxicity assay
The potency of the (Z) and (E)-isomers synthesized was
measured in cell viability studies using two different human
tumor cell lines: DU-145 prostate cancer cells and K562 leuke-
mic cells. These cell lines were selected as a benchmark for
the initial routine screening of all compounds synthesized in
our laboratory. In a recent paper,9 we described in detail the
synthesis, structure–activity relationship and biological activity
of some (E)-styryl benzyl sulfones. These studies confirmed
that the nature, number, and position of substituents on the
two aromatic rings of the parent molecule are the determining
Conclusions
factors for the biological activity of these compounds. In the In this communication, we described the Et3B-hexane cata-
present report, we investigated the effect of the geometry on lyzed radical hydrothiolation of 2,4,6-trimethoxyphenyl acety-
the cytotoxicity of these molecules, as the geometrical isomer- lene as a highly efficient method for the synthesis of (Z)-styryl
ism in a molecule plays a crucial role in the biological activity benzyl sulfides where base catalyzed hydrothiolations have
of the compound. The results presented in Table 5 showed failed. Of the various methods tried, only reactions performed
that in general all E-isomers showed superior cytotoxic activity in the presence of radical initiators such as AIBN and Et3B
compared to Z-isomers. The data from the table show that all offered good yields with better Z-selectivity. Among these,
of the 3-nitro substituted Z and E-isomers are much less Et3B-hexane in DCM afforded the best yields with very good
potent than their corresponding 3-amino analogs. Next we selectivity towards the Z-isomer. Optimum reaction conditions
examined the contribution of the oxidative state of sulfur in for the stereoselective synthesis of (Z)-styryl benzyl sulfides in
the molecules with regard to biological activity. Analysis of the the presence of Et3B-hexane were established with different
biological data presented in the table reveals that the sulfides solvents, temperatures and reaction times. We observed that
are less active than the sulfoxides and the sulfoxides are, in lower temperatures favor Z-isomer formation. The conditions
turn, less active than the sulfones, indicating that the com- for oxidation of sulfides to sulfoxides and sulfones and
plete oxidative state of sulfur is required for attaining the reduction of nitro compounds to amines were optimized by
optimum activity in these molecules. In terms of the role of trying various reagents under different reaction conditions.
Scheme 4 Synthesis of glycine esters and sodium salt of acids 25 and 26: (a) BrCH2COOCH3, MeOH, CH3COONa, 4 h; (b) NaOH, EtOH, 60–65 °C, EtOAc, H2O, rt,
2–3 h.
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Table 5 In vitro cytotoxicity of (Z) and (E)-styryl benzyl sulfides, sulfoxides and sulfones
K562b
DU145c
Compd
Structure
Isomer
IC50a (μ M)
IC50a (μ M)
11
Z
15 0.9
35 2.1
12
E
5
0.2
5
0.2.8
13
14
Z
E
75 5.0
15 0.7
>100
17 0.6
15
16
Z
E
15 0.6
15 0.75
2.5 0.1
2.5 0.15
17
18
Z
E
0.25 0.01
0.75 0.04
0.03 0.002
0.05 0.003
19
20
Z
E
5
0.3
5
0.2
0.02 0.0015
0.04 0.002
21
22
Z
E
0.15 0.006
0.15 0.005
0.003 0.0002
0.003 0.0001
23
24
Z
E
40 2.0
38 2.2
0.1 0.004
0.1 0.002
25
Z
37 1.5
35 2.0
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Table 5 (Contd.)
K562b
DU145c
Compd
Structure
Isomer
IC50a (μ M)
IC50a (μ M)
26
E
0.0075 0.0003
0.08 0.004
a IC50: represents the compound concentration (μM) that is lethal to survival of 50% of tumor cells following 96 h treatment with the tested
compound; values represent the average SD of two experiments. b K562: myelogenous leukemia. c DU145: human prostate cancer.
Among the various reducing agents tried, reduction with iron reaction mixture was heated at reflux for 18 h and after com-
powder was found to be more cost effective producing the pletion of the reaction (TLC monitoring, hexane/ethyl acetate,
product in greater than 90% yield. To increase the solubility 9 : 1 on silica gel plate), the contents were cooled to room
and bio-availability of 21 and 22, they were converted to temperature, water was added, and the product was isolated by
glycine analogs 25 and 26. In the biological assay, all the extraction with dichloromethane. The organic phase was
E-isomers have shown superior biological efficacy compared to washed with water, brine, dried over anhydrous sodium
Z-isomers, suggesting that the geometry of the molecule also sulfate, filtered, and concentrated under vacuum to obtain the
contributes to potency apart from the nature of the substitu- crude product which on purification by silica gel flash column
ents on the aromatic ring.
chromatography afforded pure 2. The yield of this reaction was
60%, giving a light yellow solid with a melting point of
1
106–108 °C; H NMR (300 MHz, CDCl3) δ (ppm): 3.99 (s, 3H,
OCH3), 4.49 (s, 2H, CH2), 7.09 (d, J = 8.7 Hz, 1H, Ar-H), 7.60
(dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.91 (d, J = 2.4 Hz, 1H, Ar-H);
13C NMR (75 MHz, CDCl3) δ (ppm): 31.3, 56.7, 113.9, 126.3,
130.2, 134.8, 139.4, 152.9; HRMS (ESI) m/z calcd for
C8H8BrNO3 [M + H] 245.9688, found 245.9682.
4-Methoxy-3-nitrobenzyl thiol (3) and 1,2-bis(4-methoxy-3-
nitrobenzyl)disulfane (4). A solution of 4-methoxy-3-nitroben-
zyl bromide (2, 10.0 g, 41 mmol) and thiourea (10.0 g,
131 mmol) in 50 mL water was heated under reflux for 2 h.
The reaction mixture was cooled and stirred at room tempera-
ture for 2 h and the solid was filtered. The resultant isothio-
uronium salt was decomposed by boiling several times with
concentrated ammonia and hexane (100 mL, 15 : 85). Concen-
tration of the combined hexane extracts provided a mixture
which on further purification with silica gel flash column
chromatography (hexane/ethyl acetate, 4 : 1) yielded pure 3 and
4. The yield of this reaction was 78%.
Experimental
General procedures
All reagents and solvents were obtained from commercial sup-
pliers and used without further purification unless otherwise
stated. Solvents were dried using standard procedures and
reactions requiring anhydrous conditions were performed
under a N2 atmosphere. Reactions were monitored by Thin
Layer Chromatography (TLC) on pre-coated silica gel F254
plates (Sigma-Aldrich) with a UV indicator. Column chromato-
graphy was performed with Merck 70–230 mesh silica gel 60 Å.
Yields were of purified product and were not optimized.
Melting points were determined using an Electrothermal
Mel-Temp® 3.0 micro melting point apparatus and are un-
corrected. Nuclear magnetic resonance spectra were recorded on
a Bruker Avance III (300 MHz, 1H NMR and 75 MHz, 13C NMR)
Spectrometer. The chemical shift values are expressed in ppm
(parts per million) relative to tetramethylsilane as an internal
standard: s, singlet; d, doublet; dd, doublet of a doublet; t,
triplet; m, multiplet; br s, broad singlet. Coupling constants
(J values) were measured in hertz (Hz). Compound purity
was determined by LC/MS analysis and was confirmed to
be >95% for all compounds. All LC/MS data were gathered on
an Agilent 1200 LC with Agilent 6410 triple quadrupole
mass spectrometer detectors. The compound solution was
infused into the electrospray ionization source operating
positive and negative modes in methanol/water/trifluoroacetic
acid (50 : 50 : 0.1% v/v) at 0.4 mL min−1. The sample cone
(declustering) voltage was set at 100 V. The instrument was
externally calibrated for the mass range m/z 100 to m/z 1000.
4-Methoxy-3-nitrobenzyl bromide (2). To a stirred solution
of 4-methoxy-3-nitro toluene (1, 10 g, 60 mmol) in carbon
tetrachloride (200 mL) were added benzoyl peroxide (1.45 g,
6.0 mmol) and N-bromosuccinimide (12.8 g, 72 mmol). The
(3) A yellow solid with a melting point of 48–49 °C; 1H NMR
(300 MHz, CDCl3) δ (ppm): 1.74 (t, J = 7.8 Hz, SH), 3.67 (d, J =
7.8 Hz, 2H, CH2), 3.89 (s, 3H, OCH3), 6.98 (d, J = 8.7 Hz, 1H,
Ar-H), 7.45 (dd, J = 8.7, 2.4 Hz, 1H, Ar-H), 7.76 (d, J = 2.1 Hz,
1H, Ar-H); 13C NMR (75 MHz, CDCl3) δ (ppm): 27.7, 56.7,
113.8, 125.2, 133.5, 133.8, 139.4, 152.0; HRMS (ESI) m/z calcd
for C8H9NO3S [M − H] 198.0303, found 198.0320.
(4) A yellow solid with a melting point of 108–110 °C;
1H NMR (300 MHz, CDCl3) δ (ppm): 3.64 (s, 4H, (CH2)2), 3.99
(s, 6H, (OCH3)2), 7.08 (d, J = 8.4 Hz, 2H, Ar-H), 7.43 (dd, J = 8.7,
2.1 Hz, 2H, Ar-H), 7.71 (d, J = 2.4 Hz, 2H, Ar-H); 13C NMR
(75 MHz, CDCl3) δ (ppm): 41.6, 56.6, 113.7, 126.4, 129.7, 135.0,
139.2, 152.4; HRMS (ESI) m/z calcd for C16H16N2O6S2 [M − H]
395.0450, found 395.0446.
2-(2-Bromovinyl)-1,3,5-trimethoxybenzene (6). To a solution
of 2,4,6-trimethoxybenzaldehyde (5, 5.0 g, 25.5 mmol) and bro-
momethyltriphenylphosphonium bromide (13 g, 30.6 mmol)
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in dry tetrahydrofuran (THF) (100 mL) at 0 °C potassium t-but- this temperature for 15 min and then the temperature was
oxide (32 mL of 1 M solution in THF, 33 mmol) was added raised to room temperature and the solution was stirred for an
slowly and the resulting mixture was continuously stirred at additional 1 h. Methyl chloroformate (0.32 g, 3.4 mmol) was
this temperature for 1 h and at room temperature for 8 h. After then added to the reaction mixture at −78 °C drop wise and
completion of the reaction, THF was removed under reduced the mixture was continuously stirred at room temperature for
pressure and the residue was extracted with ethyl acetate. The 3 h. After completion of the reaction, the contents were
organic phase was washed with water and brine, dried over quenched with saturated ammonium chloride solution and
anhydrous sodium sulfate, and concentrated under vacuum to extracted with ethyl acetate. The combined organic layers were
obtain the crude product which on purification by silica gel dried and the solvent was removed under reduced pressure.
flash column chromatography afforded pure 6. The yield of The crude product obtained was purified by flash chromato-
this reaction was 68%, giving a light yellow solid with a graphy to afford pure 9. The yield of this reaction was 42%,
melting point of 104–105 °C; 1H NMR (300 MHz, CDCl3) giving a white solid with a melting point of 85–86 °C; 1H NMR
δ (ppm): 3.84 (s, 3H, OCH3), 3.85 (s, 6H, 2X OCH3), 6.13 (s, 2H, (300 MHz, CDCl3) δ (ppm): 3.83 (s, 3H, COOCH3), 3.86 (s, 3H,
Ar-H), 7.10 (d, J = 13.8 Hz, 1H, vCH), 7.41 (d, J = 13.8 Hz, 1H, OCH3), 3.88 (s, 6H, 2× OCH3), 6.08 (s, 2H, Ar-H); 13C NMR
vCH); 13C NMR (75 MHz, CDCl3) δ (ppm): 55.3, 55.7, 90.6, (75 MHz, CDCl3) δ (ppm): 29.7, 52.5, 55.5, 56.1, 56.6, 81.7,
106.6, 108.0, 127.4, 158.9, 160.7; HRMS (ESI) m/z calcd for 87.8, 90.3, 90.4, 91.0, 155.0, 164.0, 164.3; HRMS (ESI) m/z calcd
C11H13BrO3 [M + H] 274.1231, found 274.1244.
for C13H14O5 [M + H] 251.0841, found 251.0845.
2,4,6-Trimethoxyphenyl acetylene (7). To
a
solution of
(2,4,6-Trimethoxyphenyl)propynoic acid (7a). To a solution
2-(2-bromovinyl)-1,3,5-trimethoxy-benzene (6, 4.6 g, 17 mmol) of 2,4,6-trimethoxyphenyl-propynoic methyl ester (9, 0.2 g,
dissolved in THF (100 mL), potassium t-butoxide (35 mL of 0.8 mmol) in 5 mL of THF/water/methanol (4 : 1 : 1), lithium
1 M solution in THF, 34 mmol) was added slowly and the reac- hydroxide monohydrate (50 mg, 1.2 mmol) was added at room
tion mixture was maintained at reflux for 2 h. After completion temperature and the resulting mixture was continuously
of the reaction, the potassium bromide salts were filtered and stirred for 12 h. After completion of the reaction, 10 mL of
washed with an additional 20 mL of THF. All filtrates were ethyl acetate was added and the pH was adjusted to 4 with 1 N
then combined, concentrated under reduced pressure, and the hydrochloric acid. The reaction contents were extracted with
crude product was purified by flash column chromatography ethyl acetate, dried over anhydrous sodium sulfate, and filtered
(hexane/ethyl acetate, 9 : 1) to afford pure 7. The yield of this and the solvent was removed under reduced pressure. Flash
reaction was 65%, giving a white solid with a melting point of column chromatography of the crude product gave pure 7a.
1
119–122 °C; H NMR (300 MHz, CDCl3) δ (ppm): 3.52 (s, 1H, The yield of this reaction was 55%, giving a white solid with a
CH), 3.85 (s, 3H, OCH3), 3.90 (s, 6H, 2×OCH3), 6.12 (s, 2H, Ar- melting point of 128–130 °C; 1H NMR (300 MHz, DMSO-d6)
H); 13C NMR (75 MHz, CDCl3) δ (ppm): 55.4, 56.1, 83.8, 90.4, δ (ppm): 3.75 (s, 9H, 3× OCH3), 6.28 (s, 2H, Ar-H), 13.19 (br s,
93.1, 161.9, 163.0; HRMS (ESI) m/z calcd for C11H12O3 [M + H] 1H, COOH); 13C NMR (75 MHz, CDCl3) δ (ppm): 32.5, 55.4,
193.0786, found 193.0764.
55.5, 55.8, 56.0, 82.1, 90.6, 91.0, 158.4, 159.4, 162.7, 164.3,
2-(2′,2′-Dibromovinyl)-1,3,5-trimethoxybenzene (8). To a sol- 201.8; HRMS (ESI) m/z calcd for C12H12O5 [M − H] 235.0685,
ution of 2,4,6-trimethoxybenzaldehyde (5, 5.0 g, 25.5 mmol) found 235.0689.
and triphenyl phosphine (13.37 g, 51.0 mmol) in anhydrous
Et3B-hexane mediated addition of 4-methoxy-3-nitrobenzyl
dichloromethane (60 mL) was added a solution of tetrabromo- thiol (3) to 2,4,6-trimethoxyphenyl acetylene (7): a typical
methane (9.8 g, 30.0 mmol) in dichloromethane (10 mL), procedure for the synthesis of 10. A hexane solution of
keeping the temperature below 5 °C. The reaction mixture was triethylborane (0.18 mL, 1.2 mmol) was added to a solution of
stirred for an additional 30 min at this temperature. After com- 2,4,6-trimethoxyphenyl acetylene (7, 0.2 g, 1.0 mmol) and
pletion of the reaction, monitored by TLC (hexane/ethyl 4-methoxy-3-nitrobenzyl thiol (3, 0.24 g, 1.2 mmol) in dichloro-
acetate, 9 : 1 on silica gel plate), the reaction mixture was fil- methane (10 mL) at −30 °C. The resulting mixture was stirred
tered and concentrated in vacuo. The residue was purified by for 1 h at −30 °C. After completion of the reaction monitored
flash column chromatography (hexane/ethyl acetate, 9 : 1) to by TLC (hexane/ethyl acetate, 9 : 1 on a silica gel plate), the
afford pure 8. The yield of this reaction was 72%, giving a reaction mixture was quenched with 1 molar ammonium
white solid with a melting point of 128–130 °C; 1H NMR chloride solution and extracted with dichloromethane. The
(300 MHz, CDCl3) δ (ppm): 3.83 (s, 6H, 2× OCH3), 3.85 (s, 3H, organic phase was washed with water, dried over anhydrous
OCH3), 6.13 (s, 2H, Ar-H), 7.21 (s, 1H, CHv); 13C NMR sodium sulfate, filtered and concentrated in vacuo and the
(75 MHz, CDCl3) δ (ppm): 55.4, 55.7, 90.5, 92.7, 106.8, 130.9, residue was purified by column chromatography (hexane/ethyl
158.0, 161.7; HRMS (ESI) m/z calcd for C11H12Br2O3 [M + H] acetate, 9 : 1) resulting in a stereo isomeric mixture (10, 0.34 g,
352.9133, found 352.9112.
Z/E = 91 : 9) with 88% yield as a white solid with m.p.:
(2,4,6-Trimethoxyphenyl)propynoic acid methyl ester (9). To 98–99 °C; the Z/E ratio was determined by 1H NMR spec-
1
a solution of 2-(2-bromovinyl)-1,3,5-trimethoxybenzene (8, 1 g, troscopy; H NMR (300 MHz, CDCl3) δ (ppm): 3.84 (s, 12H, 4×
2.8 mmol) in dry THF (10 mL) cooled to −78 °C, n-butyllithium OCH3), 3.96 (s, 2H, CH2S), 6.12 (s, 2H, Ar-H), 6.15 (s, 2H,
(0.6 mL of 2.5 M solution in hexane, 6.2 mmol) was added Ar-H), 6.22 (d, J = 10.2 Hz, 1H, CHv), 6.48 (d, J = 10.5 Hz,
drop wise. The resulting mixture was continuously stirred at 1H,vCH), 6.84 (d, J = 15.6 Hz, CHv), 6.97–7.08 (m, Ar-H and
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vCH), 7.51–7.62 (m, Ar-H), 7.83–7.89 (m, Ar-H); 13C NMR disappearance of the sulfide (2 h), the excess hydrogen per-
(75 MHz, CDCl3) δ (ppm): 37.0, 55.3, 55.6, 56.6, 90.4, 106.7, oxide was quenched with saturated sodium sulfite (Na2SO3)
113.6, 120.7, 126.0, 126.0, 131.0, 134.5, 152.0, 158.2, 161.2; solution (5.0 mL) and the fluorous organic phase containing
HRMS (ESI) m/z calcd for C19H21NO6S [M + H] 392.1090, found the sulfoxide was separated. After removal of the solvent
392.1099.
in vacuo, sulfoxide 13 was obtained as a semisolid. Flash
(Z)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfide column chromatography (chloroform) gave pure 13. The yield
(11). This compound was prepared from mixture 10 by of this reaction was 40%. The analytical data of the compound
column chromatographic separation as a white solid with a are in accord with the compound synthesized by method A.
melting point of 102–104 °C; 1H NMR (300 MHz, CDCl3)
(E)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfox-
δ (ppm): 3.84 (s, 12H, 4× OCH3), 3.96 (s, 2H, CH2S), 6.15 (s, ide (14). The title compound was obtained by the oxidation of
2H, Ar-H), 6.21 (d, J = 10.5 Hz, 1H, CHv), 6.47 (d, J = 10.5 Hz, 12 following the procedure as described in compound 13
1H, vCH), 7.03 (d, J = 8.4 Hz, 1H, Ar-H), 7.52 (dd, J = 8.7, 2.4 (Method A). Yield, 75%; white solid, m.p.: 148–150 °C;
Hz, 1H, Ar-H), 7.83 (d, J = 2.4 Hz, 1H, Ar-H); 13C NMR 1H NMR (300 MHz, CDCl3) δ (ppm): 3.83 (s, 6H, 2× OCH3),
(75 MHz, CDCl3) δ (ppm): 37.0, 55.3, 55.6, 56.6, 90.4, 106.8, 3.84 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.05 (d, J = 12.9 Hz, 2H,
113.6, 120.7, 125.9, 126.1, 131.0, 134.5, 139.5, 152.0, 158.2, CH2S), 6.10 (s, 2H, Ar-H), 7.04 (d, J = 15.6 Hz, 1H, CHv), 7.09
161.2; HRMS (ESI) m/z calcd for C19H21NO6S [M + H] 392.1090, (s, 1H, Ar-H), 7.41 (d, J = 15.9 Hz, 1H, vCH), 7.53 (dd, J = 8.7,
found 392.1084.
2.4 Hz, 1H, Ar-H), 7.78 (d, J = 2.4 Hz, 1H, Ar-H); 13C NMR
(E)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfide (75 MHz, CDCl3) δ (ppm): 55.4, 55.7, 56.6, 58.9, 90.4, 104.7,
(12). This compound was prepared from mixture 10 by 113.6, 122.4, 127.5, 128.5, 129.4, 136.5, 139.2, 152.8, 160.7,
column chromatographic separation as a white solid with a 162.8; HRMS (ESI) m/z calcd for C19H21NO7S [M + H] 408.1039,
melting point of 121–123 °C; 1H NMR (300 MHz, CDCl3) δ found 408.1032.
(ppm): 3.82 (s, 9H, 3× OCH3), 3.94 (s, 3H, OCH3), 3.96 (s, 2H,
(Z)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfone
CH2S), 6.12 (s, 2H, Ar-H), 6.84 (d, J = 15.6 Hz, 1H, CHv), 6.99 (15). Compound (Z)-2′,4′,6′-trimethoxystyryl-4-methoxy-3-nitro-
(d, J = 15.6 Hz, 1H, vCH), 7.06 (d, J = 8.7 Hz, 1H, Ar-H), 7.59 benzyl sulfide (11, 0.4 g, 1.0 mmol) was dissolved in methanol/
(dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 7.89 (d, J = 2.1 Hz, 1H, Ar-H); THF (1 : 1, 20 mL) and cooled to 0 °C. A solution of Oxone®
13C NMR (75 MHz, CDCl3) δ (ppm): 36.2, 55.3, 55.7, 56.6, 90.7, (1.84 g, 6 mmol in 10 mL of water) was added drop wise over
107.3, 113.8, 121.0, 123.1, 125.9, 131.0, 134.7, 139.2, 152.0, 10–15 min at 0 °C. After stirring for 24 h at rt, the reaction
158.8, 160.0; HRMS (ESI) m/z calcd for C19H21NO6S [M + H] mixture was partitioned between dichloromethane (DCM) and
392.1090, found 392.1088.
water. The aqueous layer was back-extracted, and the organic
(Z)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfox- layers were combined, dried over anhydrous sodium sulfate
ide (13). Method A: A solution of (Z)-2′,4′,6′-trimethoxystyryl- and concentrated in vacuo. The crude product was purified by
4-methoxy-3-nitrobenzyl sulfide (11, 0.5 g, 1.3 mmol) in 40 mL column chromatography (hexane/ethyl acetate) to obtain the
of glacial acetic acid was cooled to 5 °C on an ice bath. Hydro- title compound 15 as a white solid (0.27 g, 65% yield) with
1
gen peroxide 30% w/v (0.20 mL, 2.0 mmol) was added and the m.p.: 154–156 °C; H NMR (300 MHz, CDCl3) δ (ppm): 3.74 (s,
mixture was stirred at 5 °C for 6 h. After completion of the 6H, 2× OCH3), 3.76 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.12 (s,
reaction, 10% sodium hydrogen carbonate solution (25 mL) 2H, CH2S), 6.05 (s, 2H, Ar-H), 6.09 (d, J = 11.4 Hz, 1H, CHv),
was added slowly and stirred for 10 min and extracted with 7.00 (d, J = 8.7 Hz, 1H, Ar-H), 7.02 (d, J = 11.7 Hz, 1H, vCH),
ethyl acetate. The organic layer was washed with water (2 × 7.54 (dd, J = 8.7, 2.4 Hz, 1H, Ar-H), 7.78 (d, J = 2.1 Hz, 1H,
25 mL), dried over sodium sulfate, filtered, and concentrated Ar-H); 13C NMR (75 MHz, CDCl3) δ (ppm): 55.4, 55.6, 56.7,
under reduced pressure. The resulting residue was purified by 58.9, 90.3, 103.5, 113.9, 120.7, 125.8, 128.1, 135.7, 136.8, 139.4,
column chromatography (hexane/ethyl acetate) to obtain the 153.3, 158.8, 162.9; HRMS (ESI) m/z calcd for C19H21NO8S
title compound 13 as a white solid (0.29 g, 55% yield) with m.p.: [M + H] 424.0988, found 424.0994.
1
121–123 °C; H NMR (300 MHz, CDCl3) δ (ppm): 3.76 (s, 6H,
(E)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfone
2× OCH3), 3.78 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.90 (d, 2H, (16). A solution of (E)-2′,4′,6′-trimethoxystyryl-4-methoxy-3-
CH2S), 5.97 (d, J = 10.8 Hz, 1H, CHv), 6.05 (s, 2H, Ar-H), 6.84 nitrobenzyl sulfide (12, 0.5 g, 1.3 mmol) in glacial acetic acid
(d, J = 10.8 Hz, 1H, vCH), 6.98 (d, J = 8.7 Hz, 1H, Ar-H), 7.45 (40 mL) was taken in a 100 mL round-bottomed flask and
(dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 7.66 (d, J = 2.1 Hz, 1H, Ar-H); hydrogen peroxide 30% w/v (0.20 mL, 2.0 mmol) was added in
13C NMR (75 MHz, CDCl3) δ (ppm): 55.4, 55.7, 56.6, 90.5, portions at frequent intervals. Then the reaction mixture was
104.8, 113.6, 123.0, 127.4, 130.1, 133.7, 136.4, 139.2, 152.9, stirred at 60 °C for 6 h. After completion of the reaction moni-
158.3, 162.5; HRMS (ESI) m/z calcd for C19H21NO7S [M + H] tored by TLC (chloroform/methanol, 9 : 1 on silica gel plate),
408.1039, found 408.1046.
the reaction mixture was concentrated under vacuum and
Method B: An aqueous 30% hydrogen peroxide (0.28 mL, poured into ice water. The solid formed was filtered, washed
2.6 mmol) was added to a stirred solution of (Z)-2′,4′,6′-tri- with water and dried and the resulting crude product was puri-
methoxystyryl-4-methoxy-3-nitrobenzyl sulfide 11 (0.50 g, fied by column chromatography (chloroform/methanol, 9 : 1)
1.3 mmol) in 1,1,1,3,3,3-hexafluoro-2-propanol (5 mL) at rt. to obtain the title compound 16 as a white solid (0.46 g, 85%
The reaction was monitored by TLC. After the complete yield) with m.p.: 184–186 °C; 1H NMR (300 MHz, CDCl3)
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δ (ppm): 3.84 (s, 6H, 2× OCH3), 3.86 (s, 3H, OCH3), 3.98 (s, 3H, CDCl3) δ (ppm): 3.84 (br s, 12H, 4× OCH3), 3.99 (d, J = 12.6 Hz,
OCH3), 4.23 (s, 2H, CH2), 6.09 (s, 2H, Ar-H), 7.03 (d, J = 2H, CH2S), 6.11 (s, 2H, Ar-H), 6.67–6.76 (m, 3H, Ar-H), 7.15 (d,
15.6 Hz, 1H, vCH), 7.10 (d, J = 8.7 Hz, 1H, Ar-H), 7.63 (dd, J = J = 15.6 Hz, 1H, CHv), 7.50 (d, J = 15.6 Hz, 1H, vCH);
8.7, 2.4 Hz, 1H, Ar-H), 7.80 (d, J = 15.6 Hz, 1H, CHv), 7.85 (d, 13C NMR (75 MHz, CDCl3): δ (ppm): 55.4, 55.5, 55.7, 61.4,
J = 2.1 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) δ (ppm): 55.5, 90.4, 105.3, 110.4, 116.7, 120.4, 123.0, 123.3, 127.9, 130.2,
55.8, 56.7, 60.5, 90.4, 103.6, 113.8, 121.4, 121.4, 128.0, 136.8, 136.3, 147.3, 160.5, 162.2; HRMS (ESI) m/z calcd for
136.9, 139.3, 153.2, 161.6, 164.2; HRMS (ESI) m/z calcd for C19H23NO5S [M + H] 378.1297, found 378.1292.
C19H21NO8S [M + H] 424.0988, found 424.0982.
(Z)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-aminobenzyl sulfone
(Z)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-aminobenzyl sulfide (21). The title compound was obtained by the reduction of 15
(17). A solution of (Z)-2′,4′,6′-trimethoxystyryl-4-methoxy-3- following the procedure as described in compound 17. Yield,
nitrobenzyl sulfide (11, 0.5 g, 1.3 mmol) in methanol/acetic 90%; white solid; m.p.: 142–144 °C; 1H NMR (300 MHz, CDCl3)
acid (2 : 1, 30 mL) was heated to 60 °C and iron powder (0.36 g, δ (ppm); 3.84 (s, 9H, 3× OCH3), 3.87 (s, 3H, OCH3), 4.14 (s, 2H,
6.5 mmol) was added and the resulting mixture was continu- CH2), 6.13 (s, 2H, Ar-H), 6.20 (d, J = 11.7 Hz, 1H, vCH),
ously stirring at 80 °C for 3 h. After completion of the reaction, 6.78–6.83 (m, 3H, Ar-H), 7.02 (d, J = 11.7 Hz, 1H, CHv);
the contents were cooled to 0 °C, DCM (30 mL) was added and 13C NMR (75 MHz, CDCl3) δ (ppm): 55.3, 55.5, 55.6, 55.7, 60.3,
the acetic acid was neutralized with 10% sodium hydroxide 90.3, 103.0, 110.4, 117.2, 120.7, 121.2, 134.2, 136.4, 147.7,
solution (10 mL). The contents were stirred for 15 min and the 158.8, 162.6; HRMS (ESI) m/z calcd for C19H23NO6S [M + H]
organic phase was separated, washed with water and brine, 394.1246, found 394.1240.
and dried over anhydrous sodium sulfate. Removal of the
(E)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-aminobenzyl sulfone
solvent under reduced pressure gave the crude product which (22). The title compound was obtained by the reduction of 16
on purification by silica gel flash column chromatography following the procedure as described in compound 17. Yield,
(ethyl acetate/hexane) afforded pure 17 as a white solid (0.44 g, 95%; white solid, m.p.: 184–186 °C; 1H NMR (300 MHz, CDCl3)
1
95% yield) with m.p.: 90–92 °C; H NMR (300 MHz, CDCl3) δ δ (ppm): 3.83 (s, 6H, 2× OCH3), 3.84 (s, 3H, OCH3), 3.85 (s, 3H,
(ppm): 3.67 (s, 2H, CH2), 3.74 (s, 6H, 2× OCH3), 3.75 (s, 6H, 2× OCH3), 4.13 (s, 2H, CH2), 6.09 (s, 2H, Ar-H), 6.67–6.76 (m, 3H,
OCH3), 6.06 (s, 2H, Ar-H), 6.25 (d, J = 10.5 Hz, 1H, CHv), 6.32 Ar-H), 7.06 (d, J = 15.6 Hz, 1H, vCH), 7.85 (d, J = 15.9 Hz, 1H,
(d, J = 10.5 Hz, 1H, vCH), 6.61 (d, J = 1.8 Hz, 2H, Ar-H), 6.65 CHv); 13C NMR (75 MHz, CDCl3) δ (ppm): 55.5, 55.5, 55.7,
(d, J = 1.5 Hz, 1H, Ar-H); 13C NMR (75 MHz, CDCl3) δ (ppm): 61.7, 90.3, 103.8, 110.2, 117.2, 121.0, 121.1, 122.8, 135.1, 136.2,
38.2, 55.3, 55.6, 90.4, 107.2, 110.2, 115.7, 118.7, 119.1, 127.7, 147.6, 161.4, 163.7; HRMS (ESI) m/z calcd for C19H23NO6S
130.6, 136.1, 146.5, 158.2, 160.9; HRMS (ESI) m/z calcd for [M + H] 394.1246, found 394.1252.
C19H23NO4S [M + H] 362.1348, found 362.1357.
(Z)-Methyl 2-(2-methoxy-5-((2′,4′,6′-trimethoxystyrylsulfonyl)-
(E)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-aminobenzyl sulfide methyl)phenylamino)acetate (23). Sodium acetate (0.25 g,
(18). The title compound was obtained by the reduction of 12 3.1 mmol) was dissolved in methanol (15 mL). Methyl 2-bro-
following the procedure as described in compound 17. Yield, moacetate (0.67 g, 4 mmol) was added to the above solution
94%; white solid, m.p.: 106–108 °C; 1H NMR (300 MHz, CDCl3) and refluxed for 10 min. To the cooled reaction mixture com-
δ (ppm): 3.73 (s, 9H, 3× OCH3), 3.76 (s, 3H, OCH3), 3.80 (s, 2H, pound (Z)-2′,4′,6′-trimethoxystyryl-4-methoxy-3-aminobenzyl-
CH2), 6.03 (s, 2H, Ar-H), 6.65–6.76 (m, 3H, Ar-H and vCH), sulfone (21, 0.43 g, 1 mmol) was added and then refluxed for
7.00 (d, J = 15.9 Hz, 1H, CHv), 7.19 (s, 1H, Ar-H); 13C NMR 4 h. The reaction mixture was concentrated under vacuum and
(75 MHz, CDCl3) δ (ppm): 22.7, 29.7, 31.9, 37.2, 55.3, 55.6, poured into ice water. The formed precipitate was filtered,
55.7, 90.8, 107.9, 110.2, 115.6, 118.6, 119.0, 125.5, 130.5, 136.2, washed with water and dried under vacuum. The crude
146.5, 158.6, 159.5; HRMS (ESI) m/z calcd for C19H23NO4S product on purification by silica gel flash column chromato-
[M + H] 362.1348, found 362.1345.
graphy (ethyl acetate/hexane) afforded pure 23. Yield, 74%;
(Z)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-aminobenzyl sulfox- white solid, m.p.: 157–158 °C; 1H NMR (300 MHz, CDCl3) δ
ide (19). The title compound was obtained by the reduction of (ppm): 3.73 (s, 2H, CH2), 3.80 (s, 3H, OCH3), 3.83 (s, 9H, 3×
13 following the procedure as described in compound 17. OCH3), 3.86 (s, 3H, OCH3), 3.95 (d, J = 5.4 Hz, 2H, NH-CH2),
1
Yield, 95%; white solid, m.p.: 108–109 °C; H NMR (300 MHz, 4.82 (br s, 1H, NH), 6.15 (s, 2H, Ar-H), 6.34 (d, J = 10.5 Hz, 1H,
CDCl3) δ (ppm): 3.69 (s, 3H, OCH3), 3.74 (s, 6H, 2× OCH3), vCH), 6.41 (d, J = 10.5 Hz, 1H, CHv), 6.48 (s, 1H, Ar-H),
3.76 (s, 3H, OCH3), 3.83 (d, 2H, CH2), 6.04 (s, 2H, Ar-H), 6.13 6.66–6.72 (m, 2H, Ar-H); 13C NMR (75 MHz, CDCl3) δ (ppm):
(d, J = 10.5 Hz, 1H, CHv), 6.57–6.68 (m, 3H, Ar-H), 6.79 (d, J = 38.6, 45.5, 52.2, 55.4, 55.6, 90.4, 107.2, 109.3, 110.7, 118.0,
10.8 Hz, 1H, vCH); 13C NMR (75 MHz, CDCl3) δ (ppm): 55.4, 118.7, 127.7, 130.7, 137.0, 146.3, 158.2, 160.9, 171.6; HRMS
55.5, 55.6, 58.4, 90.4, 105.2, 110.3, 116.7, 120.5, 123.3, 128.3, (ESI) m/z calcd for C22H27NO8S [M + H] 466.1457, found
134.8, 136.2, 147.3, 158.4, 162.1; HRMS (ESI) m/z calcd for 466.1453.
C19H23NO5S [M + H] 378.1297, found 378.1280.
(E)-Methyl 2-(2-methoxy-5-((2′,4′,6′-trimethoxystyrylsulfonyl)-
(E)-2′,4′,6′-Trimethoxystyryl-4-methoxy-3-aminobenzyl sulfox- methyl)phenylamino)acetate (24). The title compound was
ide (20). The title compound was obtained by the reduction of obtained by the alkylation of (E)-2′,4′,6′-trimethoxystyryl-4-
14 following the procedure as described in compound 17. methoxy-3-aminobenzylsulfone 22 with methyl 2-bromoacetate
1
Yield, 94%; white solid, m.p.: 131–133 °C; H NMR (300 MHz, following the procedure as described in compound 23. Yield,
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70%; white solid, m.p.: 150–152 °C; 1H NMR (300 MHz, CDCl3) maintained at 37 °C under 5% CO2 in either DMEM or RPMI
δ (ppm): 3.69 (s, 3H, OCH3), 3.75 (s, 6H, 2× OCH3), 3.78 (s, 3H, supplemented with 10% fetal bovine serum and 1 unit per mL
OCH3), 3.79 (s, 3H, OCH3), 3.81 (d, J = 5.4 Hz, 2H, NH-CH2), penicillin–streptomycin solution. The tumor cells were plated
4.09 (s, 2H, CH2), 4.74 (t, J = 5.4 Hz, 1H, NH), 6.01 (s, 2H, into 6 well dishes at a cell density of 1.0 × 105 cells per mL per
Ar-H), 6.41 (d, J = 1.8 Hz, 1H, Ar-H), 6.61–6.68 (m, 2H, Ar-H), well and compounds were added 24 h later at various concen-
6.97 (d, J = 15.6 Hz, 1H, vCH), 7.73 (d, J = 15.6 Hz, 1H, CHv); trations. DMSO was used as a negative control. Cell counts
13C NMR (75 MHz, CDCl3) δ (ppm): 45.3, 52.2, 55.4, 55.5, 55.7, were determined from duplicate wells after 96 h of treatment.
62.1, 90.4, 104.0, 109.4, 112.3, 120.3, 121.3, 122.9, 135.3, 137.0, The total number of viable cells was determined by trypan
147.4, 161.5, 163.7, 171.4; HRMS (ESI) m/z calcd for blue exclusion.
C22H27NO8S [M + H] 466.1457, found 466.1456.
Sodium (Z)-2-(2-methoxy-5-((2′,4′,6′-trimethoxystyrylsulfonyl)-
methyl)phenylamino)acetate (25). The title compound was
obtained by the direct hydrolysis of (Z)-methyl 2-(2-methoxy-5-
Conflict of interest
((2′,4′,6′-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate
23. To a solution of sodium hydroxide (0.036 g, 0.9 mmol) in
ethanol (10 mL) was added (Z)-methyl 2-(2-methoxy-5-((2′,4′,6′-
Dr E. P. Reddy is a stockholder, board member, grant recipient
and paid consultant of Onconova Therapeutics Inc. Dr M. V. R.
Reddy is a stock holder and paid consultant of Onconova Inc.
Dr S. Cosenza is a paid consultant of Onconova Therapeutics
Inc.
trimethoxystyrylsulfonyl)methyl)phenylamino)acetate
23
(0.35 g, 0.75 mmol) and the resulting mixture was stirred at
60 °C for 30 min. After completion of the reaction monitored
by TLC (chloroform/methanol, 9 : 1 on silica gel plate), the
resulting mixture was cooled to room temperature and stirred
further for 2 h. The precipitated white solid was filtered and Acknowledgements
dried. This compound was taken into 20 mL of anhydrous
The authors are thankful to Onconova Therapeutics Inc.,
ethyl acetate and stirred for 2 h at room temperature and the
solid was filtered, washed with anhydrous ethyl acetate and
dried to get the title compound 25. Yield, 85%; white solid,
m.p. 157–158 °C; 1H NMR (300 MHz, D2O) δ (ppm): 3.59 (s,
2H, NH-CH2), 3.66 (s, 6H, 2× OCH3), 3.74 (s, 3H, OCH3), 3.77
(s, 3H, OCH3), 4.24 (s, 2H, CH2), 6.16 (s, 2H, Ar-H), 6.38 (d, J =
11.7 Hz, 1H, vCH), 6.44 (s, 1H, Ar-H), 6.63 (d, J = 7.8 Hz, 1H,
Ar-H), 6.81 (d, J = 8.4 Hz, 1H, Ar-H), 7.03 (d, J = 11.4 Hz, 1H,
CHv); 13C NMR (75 MHz, DMSO-d6,) δ (ppm): 47.8, 55.2, 55.3,
55.5, 59.2, 90.4, 103.5, 108.9, 111.7, 117.5, 120.8, 128.1, 133.4,
138.0, 146.2, 158.2, 161.9, 171.12; HRMS (ESI) m/z calcd for
C21H24NNaO8S [M + H] 474.1120, found 474.1126.
Newtown, PA and Mount Sinai School of Medicine, New York
for the financial assistance and interest in this project. We are
also thankful to Dr Ramana Tantravahi for editorial
assistance.
Notes and references
1 For more information, see: http://www. onconova.com,
http://www. mdsclinicaltrial.com and http://www.clinicaltrials.
gov
Sodium (E)-2-(2-methoxy-5-((2′,4′,6′-trimethoxystyrylsulfonyl)-
methyl)phenylamino)acetate (26). The title compound was
obtained by the hydrolysis of (E)-methyl 2-(2-methoxy-5-
((2′,4′,6′-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate
(24) following the procedure as described in compound 25.
2 K. Gumireddy, M. V. R. Reddy, S. C. Cosenza,
R. Boominathan, S. J. Baker, N. Papathi, J. Jiang, J. Holland
and E. P. Reddy, Cancer Cell, 2005, 7, 275.
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1
Yield, 80%; white solid; m.p.: 174–178 °C: H NMR (300 MHz,
DMSO-d6) δ (ppm): 3.11 (d, J = 3.9 Hz, 2H, NH-CH2), 3.78 (s,
3H, OCH3), 3.84 (s, 6H, 2× OCH3), 3.85 (s, 3H, OCH3), 4.26 (s,
2H, CH2), 5.18 (t, J = 3.9 Hz, NH), 6.28 (s, 2H, Ar-H), 6.32 (d, J =
1.8 Hz, 1H, Ar-H), 6.47 (dd, J = 8.1, 2.1 Hz, 1H, Ar-H), 6.72 (d,
J = 8.1 Hz, 1H, Ar-H), 7.10 (d, J = 15.9 Hz, 1H, vCH), 7.56 (d,
J = 15.9 Hz, 1H, CHv); 13C NMR (75 MHz, DMSO-d6) δ (ppm):
47.6, 55.2, 55.6, 56.0, 60.6, 90.9, 102.6, 108.8, 111.8, 117.5,
121.5, 123.6, 132.5, 137.8, 146.1, 160.8, 163.5, 171.8; HRMS
(ESI) m/z calcd for C21H24NNaO8S [M + H] 474.1120, found
474.1116.
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Cytotoxicity assay. We have tested growth inhibition assay
in K562 and Du145 tumor cell lines using a dose response end
point assay system. The cells were purchased from ATCC and
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