Design, synthesis and evaluation of (E)-α-benzylthio chalcones as novel inhibitors of BCR-ABL kinase

Article abstract of DOI:10.1016/j.bmc.2010.01.051

Novel (E)-α-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells.

Full text of DOI:10.1016/j.bmc.2010.01.051

Bioorganic & Medicinal Chemistry 18 (2010) 2317–2326
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of (E)-a-benzylthio chalcones as novel
inhibitors of BCR-ABL kinase
a,
M. V. Ramana Reddy a, , Venkat R. Pallela , Stephen C. Cosenza a, , Muralidhar R. Mallireddigari b,
*
Revathi Patti b, Marie Bonagura a, May Truongcao a, Balaiah Akula b, Shashidhar S. Jatiani a,
E. Premkumar Reddy a,
*
a Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, PA 19140-5101, United States
b Onconova Therapeutics Inc., 375 Pheasant Run, Newtown, PA 18940, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel (E)-a-benzylthio chalcones are reported with preliminary in vitro activity data indicating that sev-
Received 11 December 2009
Revised 19 January 2010
Accepted 20 January 2010
Available online 25 January 2010
eral of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phos-
phorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such
compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising
lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leuke-
mic cells.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
a-Benzylthio chalcones
BCR-ABL kinase
Chronic myelogenous leukemia
1. Introduction
CML cells acquire imatinib resistance.8–10 Results from these stud-
ies indicate that the mechanism that accounts for a majority of
The Philadelphia chromosome (Ph), discovered in 1960 by Now-
ell and Hungerford,1 results from a reciprocal translocation be-
tween chromosomes 9 at band q34 and chromosome 22 at band
q11.2,3 This translocation fuses the breakpoint cluster region
(BCR) and the ABL genes and creates the BCR-ABL oncogene.4 Be-
cause the BCR-ABL protein is active in greater than 90% of chronic
myelogenous leukemia (CML) cases, it has been possible to synthe-
size small molecules that inhibit BCR-ABL kinase activity in leuke-
mic cells without adversely affecting the normal cell population.
Imatinib (STI571, GleevecÒ)5 is a small molecule inhibitor that
binds to the kinase domain of BCR-ABL when the protein is in its
closed, inactive conformation,5 thereby inhibiting its activity, and
is now considered as a first-line therapy for the majority of CML
cases due to its high efficacy and relatively mild side effects.6 In
spite of the fact that the majority of patients receiving imatinib re-
spond to treatment at both the hematological and cytogenetic lev-
els, relapse occurs in a large percentage of patients.7 Several
studies have attempted to address the mechanism(s) by which
imatinib-resistant leukemias, in vivo, is mutation of the BCR-ABL
gene itself. Mutation within the kinase domain is the most com-
mon and to date, more than 50 different clinically relevant point
mutations within this domain have been identified. It is believed
that certain amino acid substitutions interfere with the ability of
imatinib to interact directly with the BCR-ABL kinase domain
whereas others destroy or hinder the ability of the BCR-ABL kinase
domain to adopt a conformation that is required for imatinib
binding.7,11
The challenges of mutational relapse in CML patients undergo-
ing imatinib therapy has paved the way for the development of
second generation BCR-ABL inhibitors such as PD180970,12
CGP76030,13 BMS-354825,14 AMN 107 or Nilotinib15 and more re-
cently AP24534 (Chart 1).16 These new BCR-ABL inhibitors are all
ATP-competitive agents and therefore will potentially encounter
challenges similar to imatinib via accumulation of kinase domain
mutations.
Because of the frequency of mutations within the ATP-binding
site, efforts are now focused on the identification of novel inhibi-
tors that inhibit the BCR-ABL signaling pathway by mechanisms
other than competing with ATP. Different approaches have re-
cently been described to overcome this resistance in at least some
CML cases. Farnesyltransferase inhibitors, such as SCH66336, and
the proteasome inhibitor Bortezomib have been shown to have
growth inhibitory effects on certain imatinib-resistant leukemias.17
Abbreviations: SAR, structure–activity relationship; FBS, fetal bovine serum.
* Corresponding authors. Tel.: +1 215 707 7336; fax: +1 215 893 6989 (M.V.R.R.);
tel.: +1 215 707 4307; fax: +1 215 707 1454 (E.P.R.).
E-mail addresses: rreddy@temple.edu (M.V. Ramana Reddy), reddy@temple.edu
(E.P. Reddy).
Contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.051

2318
M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
It has also been argued that non-ATP-competitive kinase inhibitors
might constitute better drug candidates.18 Since there is a limited
number of chemotypes that act as non-ATP competitive inhibitors,
we undertook the synthesis and characterization of new chemo-
types that are unrelated to ATP and other purine and pyrimidine
nucleosides but still possess kinase inhibitory activity.
In Scheme 2, a method for the synthesis of new (E)-a-benzylsul-
fonyl chalcones is described. Oxidation of phenacyl benzylsulfides
4 with 30% hydrogen peroxide in the presence of glacial acetic acid
gave phenacyl benzylsulfones 7. Condensation of phenacyl benzyl-
sulfones 7 with aldehydes 5 in glacial acetic acid and ammonium
acetate provided a-benzylsulfonyl chalcones 8. Alternatively, these
Here we report the discovery of (E)-
a
-benzylthio chalcones as a
sulfonyl chalcones 8 were also prepared by the oxidation of ben-
new class of inhibitors that potently inhibit the growth of cells har-
boring the activated BCR-ABL protein by inhibiting the phosphory-
lation of the kinase.
zylthio chalcones 6 with an excess of m-chloroperbenzoic acid
(m-CPBA) in chloroform. Compound 6t,
a-benzylsulfoxide chal-
cone was prepared by the controlled oxidation of 6c with 1 equiv
of m-CPBA in chloroform.
To delineate the role of benzylthio moiety in the biological
activity of 6, we synthesized a few molecules replacing the benzyl
moiety with aryl group. In Scheme 3 we have outlined the synthe-
2. Chemistry
The general method for the synthesis of novel benzylthio chal-
cones is outlined in Scheme 1. For the synthesis of compounds 6a–
6t and 6aa–6ag, phenacyl bromides 2 were synthesized from ace-
tophenones 1 and then reacted with benzylmercaptans 3 to pro-
duce phenacyl benzyl sulfides 4.19 Knoevenagel condensation of
4 with aromatic aldehydes 5 in glacial acetic acid in the presence
sis of a-arylthio chalcones starting from 2. Condensation of 2 with
aryl mercaptans 9 in methanolic sodium hydroxide solution pro-
duced phenacyl arylsulfides 10, which on Knoevenagel type con-
densation with aromatic aldehydes 5 in the presence of a base
yielded (E)-
To enhance the solubility and bioavailability of the active (E)-
a
-arylthio chalcones 11.21
of ammonium acetate yielded a
-benzylthio chalcones 6.20 Alterna-
tively, the condensation between 4 and 5 was also carried out in
glacial acetic acid in the presence of a catalytic amount of benzyl-
amine to obtain 6.21
a
-
benzylthio chalcones, we modified the carboxylic acid group
located at the para position of the benzoyl aromatic ring of 6 as
amide substituted piperazine groups. In Scheme 4 benzylthio
F
F
F
N
O
H
N
H
N
H
N
N
N
N
N
H
N
N
N
N
O
N
N
(STI 571, Imatinib)
(AMN 107, Nilotinib)
F
F
F
OH
N
N
H
N
N
S
O
N
N
N
HN
N
N
N
N
N
H
O
Cl
(AP24534)
Chart 1. Structures of known BCR-ABL kinase inhibitors.
(BMS-354825, Dasatinib)
O
HS
O
Br
b
a
+
R2
R1
R1
1
2
3
R3
R2
R2
O
H
R1
R1
c or d
+
R3
S
S
O
O
6
5
4
Scheme 1. General method for the synthesis of (E)-
70 °C, 3 h; (d) PhCH2NH2, AcOH, reflux, 8 h.
a-benzylthio chalcones. Reagents and conditions: (a) Br2, AcOH, 70 °C, 3 h; (b) NaOH, MeOH, rt, 2 h; (c) NH4OAc, AcOH,

M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
2319
R2
R2
R1
R1
a
S
S
O2
O
O
7
4
b
5
+
R3
R3
R2
R2
R1
R1
c
S
S
O2
O
O
6
8
d
R3
R2
R1
S
O
O
6t
Scheme 2. Synthesis of (E)-a-benzyl sulfoxide/sulfonyl chalcones. Reagents and conditions: (a) 30% H2O2, AcOH, 70 °C, 2 h; (b) NH4OAc, AcOH, reflux, 8 h; (c) m-CPBA
(3 equiv), CHCl3, rt, 2 h; (d) m-CPBA (1 equiv), CHCl3, 0 °C to rt, 2 h.
O
O
SH
b
a
Br
+
R2
R1
R1
1
9
2
R3
R1
O
H
S
c or d
O
S
R1
+
R3
O
R2
10
11
R2
5
Scheme 3. Method for the synthesis of (E)-a-arylthio chalcones. Reagents and conditions: (a) Br2, AcOH, 70 °C, 3 h; (b) NaOH, MeOH, rt, 2 h; (c) NH4OAc, AcOH, reflux, 8 h; (d)
PhCH2NH2, AcOH, reflux, 8 h.
chalcones, 6c and 6h were treated either with primary or cyclic
secondary amines to produce amide substituted piperazines 12a–
12d (Scheme 4).
the thio group, (ii) the oxidation state of the sulfur atom in the
molecule and (iii) the nature and site of the substituents in the aro-
matic rings. During our screening of about 600
sulfonyl chalcones, we found that some of these compounds
showed a very good cytotoxicity (IC50 = 0.3–0.9 M) in K562 cells
a-benzylthio and
l
3. Results and discussion
(Table 1). To analyze the effect of the substituents on the aromatic
rings of 6, we synthesized a number of analogs containing meth-
oxy, halo, methyl, nitro, carboxy and hydroxy groups on these
rings. Cytotoxicity analyses of these analogues on K562 cells
showed that the compounds with a fluoro atom at the fourth posi-
tion and a nitro group at the third position on the styryl aromatic
ring exhibited the best activity in these series. To further assess the
significance of the 3-nitro and 4-fluoro groups on the styryl ring in
6, we replaced these groups in 6c with bromo at the site of nitro
and hydroxyl at the position of fluoro atom (6ab). Both these
replacements resulted in total loss of activity. Since double
3.1. Structure–activity relationships (SAR)
After the synthesis of these compounds, their in vitro cytotoxic-
ity was assessed using K562, a BCR-ABL positive leukemic cell line.
As some of these compounds are highly potent against K562 cells,
their ability to inhibit the phosphorylation of BCR-ABL was as-
sessed by western blotting of the drug treated cell lysates with
phospho anti BCR-ABL antibodies. The results of this study are pre-
sented in Tables 1–3 and Figure 1. These studies show that the
cytotoxicity of these compounds depends on (i) the position of

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M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
O2N
O2N
Br
Br
F
F
N
N
O
N
COOH
H2N
N
EDCI,
N
H
S
HOBt, DIPEA, THF, rt, 2h
S
O
O
6c
12a
O2N
Br
F
OH
N
O
EDCI, HN
6c
N
HOBt, DIPEA, THF, rt, 2h
N
S
OH
O
12b
O2N
Br
F
O
N
EDCI, H2N
N
6c
N
H
HOBt, DIPEA, THF, rt, 2h
S
O
12c
O2N
F
O2N
F
F
F
OH
N
O
COOH
EDCI, HN
N
HOBt, DIPEA, THF, rt, 2h
N
S
S
O
OH
O
12d
6h
Scheme 4. Synthesis of compounds 12a–12d.
replacement of the 3-nitro and 4-fluoro substituents on styryl ring
in 6c caused a moderate loss of activity, we then made single
changes either at 3-position keeping 4-position intact or vice versa
and studied the effect of these changes in cytotoxic assay in tumor
cells. Modifying the fluorine atom alone on the 4-position of styryl
ring in 6c with a hydroxyl group resulted in the loss of the activity
(6ac). The activity of 6c was found to be unaltered when fluorine at
4-position of styryl ring was replaced with chloro (6a) or bromo
(6b) atoms. This confirms that a halogen atom at that position is
essential for the activity of the molecule. Any modifications replac-
ing fluorine with atoms or groups other than halogen atoms results
in total loss of activity. To understand the role of the 3-position ni-
tro group on the cytotoxicity of the molecule, the nitro group of 6c
was replaced with a methoxy group (6aa). This substitution led to
a drastic reduction in the cytotoxicity of the resulting molecule
(6aa). Since both the nitro group and the fluorine atom on the ring
seem to be critical for the cytotoxicity of the molecule, we tested
whether their distribution on the ring other than at 3 and 4-posi-
tions has any effect on the tumor cell killing activity. To analyze
the role of these two substituent locations on the ring, we synthe-
sized 6ad, 6ae, 6af and 6ag where fluoro and nitro substituents are
distributed at 2, 5 (6ad), 3, 4 (6ae), 4, 2 (6af) and 2, 4 (6ag) posi-
tions. Cytotoxicity data from K562 cells treated with these com-
pounds showed that these alterations are drastic leading to the
loss of cytotoxicity in the resulting molecules (Table 2). From this
analysis it is evident that a nitro group at the third position and a
fluorine atom at the fourth position on the styryl aromatic ring are
critical for the anti-tumor activity of these molecules. It is also
clear from Table 1 that the nature and position of the substituents
on the benzoyl and benzylthio rings do not affect the cytotoxicity
of the molecules as long as 3-nitro and 4-fluoro substituents are
on the styryl ring. To determine the significance of sulfur oxidation
state, we oxidized the sulfur atom in benzylthio group to sulfoxide
and sulfone and then assayed the resulting molecules for their
cytotoxicity. In both the cases, the sulfoxide (6t) or the sulfones
(8a and 8b) produced by the oxidation of 6c were found to have
significantly reduced cytotoxicity.
These results show that these molecules exhibit much higher
potency towards tumor cells when an un-oxidized sulfur atom is
present in their structure. To understand the role of the benzyl
methylene group in structure–activity relationship, we replaced
the benzylthio moiety with phenylthio group where the sulfur
atom is directly connected to the aromatic ring instead of having
a methylene bridge between the ring and the sulfur atom (11). This
modification seems to have an adverse effect on the molecule
resulting in a fivefold reduction in cytotoxicity.
We next focused our attention on enhancing the aqueous solubil-
ity and bioavailability of the active molecules. As all the active mol-
ecules synthesized have very poor aqueous solubility (<1 mg/mL),
these were dissolved in dimethyl sulfoxide (DMSO) to treat the cells
or administer to the mice by intraperitoneal (IP) injection. Bioavail-
ability and pharmacokinetic data from the mice injected with 6c in
DMSO showed very low levels of this compound in serum. To im-
prove the solubility and bioavailability of these compounds, we con-
verted the carboxylic acid on the benzoyl ring of 6c and 6h in to
compounds 12a–12d having secondary and tertiary amino groups.
As shown in Table 3, secondary and tertiary amine salts of 6c and
6h were made and tested for their solubility and cytotoxicity. The

M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
2321
Table 1
Table 3
In vitro cytotoxicity of
a
-aryl and benzyl thio chalcones
R3
In vitro cytotoxicity of carboxamide derivatives of
a
-benzylthio chalcones
Solubility (mg/mL)
Compd
IC50
(
lM)
K562
pBCR/ABL
R1
12a
12b
12c
12d
0.6
0.2
0.75
0.3
2.5
0.5
0.5
0.5
>10.0
>10.0
>10.0
>10.0
R2
X
O
Compd R2
X
R1
R3
IC50
(lM)
K562 pBCR/ABL
6aa
6ba
6ca
6da
6ea
6fa
4-Br
4-Br
4-Br
4-Cl
4-Cl
4-Cl
4-F
4-F
4-F
4-F
2-F
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
CH2S
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-F
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
3-NO2, 4-Cl
3-NO2, 4-Br
3-NO2, 4-F
3-NO2, 4-F
3-NO2, 4-Cl
3-NO2, 4-Br
3-NO2, 4-Cl
3-NO2, 4-F
3-NO2, 4-Br
3-NO2, 4-F
3-NO2, 4-Cl
3-NO2, 4-F
3-NO2, 4-Cl
3-NO2, 4-F
3-NO2, 4-Br
3-NO2, 4-F
3-NO2, 4-F
0.7
0.9
0.3
0.5
0.5
0.6
0.5
0.4
0.4
0.6
0.8
0.5
0.6
0.4
0.6
0.3
0.4
0.3
0.3
10
10
0.5
2.5
10
2.5
2.5
2.5
15
2.5
10
10
2.5
2.5
2.5
2.5
0.2
0.75
2.5
>20
>20
>20
20
6ga
6ha
6ia
6jb
6ka
6la
2-Cl
6ma
6na
6oa
6pa
6qa
6rb
6sa
6ta
8a
2,4-Cl2 CH2S
2,4-Cl2 CH2S
2,4-Cl2 CH2S
4-CH3
4-CF3
4-Br
H
4-Br
4-Br
4-Br
4-Br
CH2S
CH2S
CH2S
CH2S
CH2SO
CH2SO2 4-COOH
CH2SO2 4-COOH
S
4-COOCH3 3-NO2, 4-F
4-COOH
4-COOH
3-NO2, 4-F
3-NO2, 4-F
3-NO2, 4-F
3-NO2, 4-Cl
3- NO2, 4-F
75
5
25
1.5
8b
11c
4-COOH
a
b
c
These compounds were synthesized by utilizing method A.
These compounds were synthesized by utilizing method B.
These compounds were synthesized by utilizing method A and method B.
Figure 1. BCR-ABL kinase inhibition in human chronic myelogenous leukemic cells.
K562 Cells expressing BCR-ABL p210, were treated with the indicated micromolar
concentrations of each compound, 1 lM Gleevec(G) or Vehicle(DMSO) for 2 h. Total
cellular proteins were harvested and resolved by 10%-SDS–PAGE. The gel was
transferred and hybridized against antibodies specific for P-BCR-ABL or BCR-ABL.
The western blot was treated with secondary antibodies conjugated with infrared
dyes (LiCor) and scanned using Odyssey (LiCor) scanner. Percent inhibition was
determined by quantifying each band using the software provided by LiCor, then
normalizing the P-BCR-ABL signal to the parental BCR-ABL signal and determining%
inhibition based on the vehicle control signal.
Table 2
In vitro cytotoxicity of
a
-benzyl thio chalcones
R3
R2
R1
tern blot analysis with anti-phospho BCR-ABL antibody (Fig. 1).
These results showed that while all the compounds, in general,
inhibited the phosphorylation of BCR-ABL, compounds 6c, 6q, 6r,
12a, 12b and 12d were most effective in this assay. Imatinib5
was used as a positive control while DMSO was used as the nega-
tive control. The results of this study showed that some of the
S
O
Compda
R2
R1
R3
IC50 (lM)
K562
pBCR/ABL
6aa
6ab
6ac
6ad
6ae
6af
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
3-OCH3, 4-F
3-Br, 4-OH
3-NO2, 4-OH
2-F, 5-NO2
3-F, 4-NO2
2-NO2, 4-F
2-F, 4-NO2
20
35
75
7.5
5.0
20
7.5
>20
>20
>20
>20
>20
>20
>20
a-benzylthio chalcones (6c, 6q, 6r, 12b, 12c and 12d) inhibited
the phosphorylation of BCR-ABL with an IC50 (0.2–0.75 M)
l
comparable to that of imatinib (0.75
3.3. Discussion
lM).
6ag
a
Our current study shows that (E)-a-benzylthio chalcones exert
All the compounds were synthesized by utilizing method A.
a cytotoxic effect by inhibiting BCR-ABL phosphorylation in leuke-
mic cell lines that over express this protein, thereby causing
growth arrest and cell death. We describe the development of no-
vel small molecules that inhibit autophosphorylation of BCR-ABL at
amino hydrochloride salts (12a–12d) made from 6c and 6h dis-
played excellent aqueous solubility and retained their cytotoxicity
in K562 cells. The bioavailability of these compounds is being tested.
a concentration of 0.2–0.75 lM. While assessing the kinase inhib-
itory profile of 6c against other kinases implicated in cancer, we
found 6c to be a specific inhibitor of BCR-ABL. 6c exhibited 10–
15-fold higher selectivity for BCR-ABL over other tyrosine kinases
and more than 40-fold higher selectivity for BCR-ABL over serine/
threonine kinases (Table 4). The optimization of 6c to improve its
bioavailability and pharmacokinetics is ongoing. The in vivo effi-
cacy of the optimized version of 6c needs to be tested in xenograft
mouse models of leukemia and ex vivo on patient samples.
3.2. Inhibition of BCR-ABL kinase activity
The cell based cytotoxicity assays reported above demonstrate
that (E)-a-benzylthio chalcones exhibit nanomolar cytotoxicity
against K562 cells. To assess whether these compounds directly in-
hibit the phosphorylation of BCR-ABL, we tested the cell lysates
prepared from K562 cells treated with these compounds in wes-

2322
M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
Table 4
Kinase inhibition profile of 6c
wise stated. Solvents were dried using standard procedures
and reactions requiring anhydrous conditions were performed
under N2 atmosphere. Reactions were monitored by Thin Layer
Chromatography (TLC) on precoated Silica Gel F254 plates (Sig-
ma–Aldrich) with a UV indicator. Column chromatography 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 Electro thermal Mel-TempÒ
3.0 micro melting point apparatus and are uncorrected. 1H
Kinase
IC50 (lM)
6c
BCR-ABL
Lyn B
Src
ErbB2
EGFR
Plk-1
Cdk-1
0.75
17.0
13.2
>10.0
>20.0
>50.0
>50.0
NMR spectra were obtained with
a Bruker AM 300 and
400 MHz spectrometer. The chemical shifts are reported in
parts per million (d) downfield using tetramethyl silane (Me4Si)
as internal standard and. Spin multiplicities are given as s (sin-
glet), d (doublet), br s (broad singlet), m (multiplet), and q
(quartet). Coupling constants (J values) were measured in hertz
(Hz). The purity of the final compounds was determined by
HPLC and is 95% or higher unless specified otherwise. Phena-
cylbromides were prepared according to the procedure
reported in the literature.34
Studies with kinase inhibitors have identified three general
mechanisms for pharmacological inhibition of kinase activity: (1)
direct binding in the ATP-binding site, (2) binding in the sub-
strate-binding site, and (3) engagement of an allosteric site which
results in the altered conformation of the kinase causing a block in
proper substrate phosphorylation. The first kinase inhibitor to
reach the market was Imatinib (GleevecÒ),5 an inhibitor of BCR-
ABL tyrosine kinase that has been a remarkable success for the
treatment of Philadelphia chromosome positive (Ph+) chronic
myelogenous leukemia’s (CMLs). This was followed by the devel-
opment of a series of kinase inhibitors, which include gefitinib
(Iressa),22 erlotinib (OSI-1774; Tarceva),23 lapatinib (GW-572016),24
canertinib (CI 1033),25 semaxinib (SU5416),26 vatalanib (PTK787/
ZK222584),27 sorafenib (BAY 43-9006),28 sutent (SU11248),29 and
leflunomide (SU101).30 All of these compounds are ATP-competi-
tive inhibitors and an understanding of the pharmacological prop-
erties and anticancer activities of these compounds have resulted
in a rapid advance to our understanding of the advantages and lim-
itations associated with this class of therapeutic agents. One of the
important facts that has emerged in the past few years has been
the realization that tumor cells often develop resistance to ATP-
competitive kinase inhibitors as a result of accumulating muta-
tions in the ATP-binding site of the kinase, which has been
observed in patients undergoing treatment with imatinib31 as well
as gefitinib32 and erlotinib.33
5.1.2. General procedure for the preparation of 2-(benzyl/aryl
thio)-1-arylethanone (4 and 10)
To a solution of sodium hydroxide (20 mmol) in methanol
(50 mL) benzylthiol 3 (20 mmol) or arylthiol 9 (20 mmol) was
added and the contents were stirred at room temperature for
10 min. To this reaction mixture phenacylbromide 2 (20 mmol)
was added and stirred for further 1–2 h. After completion of the
reaction, the contents were cooled, poured on to ice-water and
the solid obtained was collected by filtration. The crude product
was recrystallized from 2-propanol to get pure 4 or 10. The follow-
ing 2-(benzyl/aryl thio)-1-arylethanones 4 and 10 were prepared
using the above procedure.
5.1.2.1. 4-(2-(4-Bromobenzylthio)acetyl)benzoic acid (4a). Yield:
95%; white solid, mp 195–197 °C. 1H NMR (CDCl3, 300 MHz): d 3.72
(s, 2H, CH2), 3.95 (s, 2H, CH2), 7.27–8.05 (m, 8H, Ar–H), 13.40 (br s,
1H, COOH). HRMS found m/z 364.78. Calcd for C16H13BrO3S (M+H)+
m/z 364.98.
Compound6cand its analogsdo notresemble typicalATP mimet-
ics in structure. Hence 6c and its analogs are expected to target
regions outside the ATP-binding site of their target kinases and offer
the potential to be unaffected by mutations in the kinase domain
that make tumor cells resistant to ATP-competitive inhibitors.
5.1.2.2. 4-(2-(4-Chlorobenzylthio)acetyl)benzoic acid (4b). Yield:
92%; white solid, mp 194–196 °C. 1H NMR (CDCl3, 300 MHz): d
3.78 (s, 2H, CH2), 4.05 (s, 2H, CH2), 7.28–8.20 (m, 8H, Ar–H),
13.35 (br s, 1H, COOH). HRMS found m/z 321.08. Calcd for
C16H13ClO3S (M+H)+ m/z 321.04.
4. Conclusion
5.1.2.3. 4-(2-(4-Fluorobenzylthio)acetyl)benzoic acid (4c). Yield:
88%; white solid, mp 200–202 °C. 1H NMR (CDCl3, 300 MHz): d 3.67
(s, 2H, CH2), 3.92 (s, 2H, CH2), 7.21–8.05 (m, 8H, Ar–H), 13.38 (br s,
1H, COOH). HRMS found m/z 305.10. Calcd for C16H13FO3S (M+H)+
m/z 305.06.
In conclusion, we describe here, for the first time, the discovery
and synthesis of a novel class of compounds, (E)-a-benzylthio chal-
cones, which possess potent kinase inhibitory activity and exhibit
cytotoxicity against human tumor cells that express the oncogenic
kinase BCR-ABL. While these compounds are comparable to
imatinib in their in vitro efficacy they do not resemble typical
ATP-mimetics. Hence, they offer the potential to be unaffected by
mutations in the kinase domain that make tumor cells resistant
to ATP-competitive inhibitors. These compounds possess a simple
molecular structure and are easy to synthesize which makes them
very attractive for further exploration as kinase inhibitors with
application in cancer therapy.
5.1.2.4. 2-(4-Fluorobenzylthio)-1-(4-fluorophenyl)ethanone
(4d). Yield: 84%; white solid, mp 68–70 °C. 1H NMR (CDCl3,
300 MHz): d 3.60 (s, 2H, CH2), 3.63 (s, 2H, CH2), 7.18–7.85 (m,
8H, Ar–H). HRMS found m/z 279.06. Calcd for C15H12F2OS
(M+H)+ m/z 279.07.
5.1.2.5. 4-(2-(2-Fluorobenzylthio)acetyl)benzoic acid (4e). Yield:
72%; white solid, mp 189–190 °C. 1H NMR (CDCl3, 300 MHz):d 3.82 (s,
2H, CH2), 4.02 (s, 2H, CH2), 7.15–8.14 (m, 8H, Ar–H), 13.35 (br s, 1H,
COOH). HRMS found m/z 304.58. Calcd for C16H13FO3S (M+H)+ m/z
304.58.
5. Experimental
5.1. Chemistry
5.1.1. General information
All reagents and solvents were obtained from commercial
suppliers and used without further purification unless other-
5.1.2.6. 4-(2-(2-Chlorobenzylthio)acetyl)benzoic acid (4f). Yield:
84%; white solid, mp 190–192 °C. 1H NMR (CDCl3, 300 MHz):d 3.71 (s,
2H, CH2), 3.93 (s, 2H, CH2), 7.11–8.10 (m, 8H, Ar–H), 13.25 (br s, 1H,

M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
2323
COOH). HRMS found m/z 321.02. Calcd for C16H13ClO3S (M+H)+ m/z
321.04.
Method B: A mixture of 2-(benzyl/aryl thio)-1-arylethanone (4
or 10) (10 mmol), araldehyde (5) (10 mmol), glacial acetic acid
(5 mL), and a catalytic amount (100 lL) of benzyl amine was re-
5.1.2.7. 4-(2-(2,4-Dichlorobenzylthio)acetyl)benzoic acid (4g).
Yield: 89%; white solid, mp 215–217 °C. 1H NMR (CDCl3,
300 MHz): d 3.88 (s, 2H, CH2), 4.12 (s, 2H, CH2), 7.28–8.21 (m,
7H, Ar–H), 13.55 (br s, 1H, COOH). HRMS found m/z 354.97. Calcd
for C16H12Cl2O3S (M+H)+ m/z 354.99.
fluxed for 5–8 h. After completion of the reaction (TLC monitoring,
CHCl3/MeOH on silica gel plate), the contents were cooled to room
temperature, the precipitated product was filtered, washed with 2-
propanol and petroleum ether and dried. If solid was not formed,
the reaction mixture was poured on to ice-water and extracted
with ethyl acetate. The organic layer was dried over sodium sul-
fate, filtered and concentrated under vacuum to obtain the crude
6 or 11. The crude product was recrystallized form 2-propanol to
yield an analytically pure sample of 6 or 11.
5.1.2.8. 4-(2-(4-Methylbenzylthio)acetyl)benzoic acid (4h). Yield:
1
93%; white solid, mp 196–198 °C. H NMR (CDCl3, 300 MHz): d 2.27
(s, 3H, CH3), 3.68 (s, 2H, CH2), 3.91 (s, 2H, CH2), 7.10–8.04 (m, 8H, Ar–
H), 13.25 (br s, 1H, COOH). HRMS found m/z 301.07. Calcd for
C17H16O3S (M+H)+ m/z 301.09.
5.1.5.1. (E)-4-(2-(4-Bromobenzylthio)-3-(4-chloro-3-nitro-
phenyl)acryloyl)benzoic acid (6a).
Yield: 78%; light yellow
solid, mp 148–150 °C. 1H NMR (CDCl3, 400 MHz): d 3.92 (s, 2H,
CH2), 6.84–6.94 (m, 3H, Ar–H), 7.17–7.23 (m, 5H, Ar–H), 7.22 (s,
1H, CH), 7.55–7.57 (dd, 2H, Ar–H), 7.58–7.62 (m, 1H, Ar–H),
7.85–7.87 (d, 2H, J = 8.0 Hz, Ar–H), 8.04–8.15 (dd, 1H, Ar–H),
13.50 (br s, 1H, COOH). HRMS found m/z 533.98. Calcd for
C23H15BrClNO5S (M+H)+ m/z 533.96.
5.1.2.9. 4-(2-(4-(Trifluoromethyl)benzylthio)acetyl)benzoic acid
(4i).
300 MHz): d 3.68 (s, 2H, CH2), 3.95 (s, 2H, CH2), 7.11–8.10 (m, 8H, Ar–
H), 13.35 (br s, 1H, COOH). HRMS found m/z 355.04. Calcd for
C17H13F3O3S (M+H)+ m/z 355.06.
Yield: 78%; white solid, mp 162–164 °C. 1H NMR (CDCl3,
5.1.2.10. Methyl 4-(2-(4-bromobenzylthio)acetyl)benzoate (4j).
Yield: 90%; white solid, mp 75–77 °C. 1H NMR (CDCl3, 300 MHz):
d 3.72 (s, 2H, CH2), 3.92 (s, 3H, OCH3), 4.05 (s, 2H, CH2), 7.26–
8.15 (m, 8H, Ar–H). HRMS found m/z 379.00. Calcd for C17H15BrO3S
(M+H)+ m/z 379.00.
5.1.5.2. (E)-4-(2-(4-Bromobenzylthio)-3-(4-bromo-3-nitro-
phenyl)acryloyl)benzoic acid (6b).
Yield: 80%; light yellow
solid, mp 168–170 °C. 1H NMR (CDCl3, 400 MHz): d 3.90 (s, 2H,
CH2), 6.85–6.92 (m, 3H, Ar–H), 7.22–7.28 (m, 5H, Ar–H), 7.25 (s,
1H, CH), 7.62–7.65 (dd, 2H, Ar–H), 7.60–7.62 (m, 1H, Ar–H),
7.82–7.84 (d, 2H, J = 8.0 Hz, Ar–H), 8.02–8.09 (dd, 1H, Ar–H),
13.40 (br s, 1H, COOH). HRMS found m/z 577.92. Calcd for
C23H15Br2NO5S (M+H)+ m/z 577.91.
5.1.2.11. 4-(2-(Benzylthio)acetyl)benzoic acid (4k).
Yield:
93%; white solid, mp 182–184 °C. 1H NMR (CDCl3, 300 MHz): d
3.88 (s, 2H, CH2), 4.02 (s, 2H, CH2), 6.98–8.04 (m, 9H, Ar–H),
13.50 (br s, 1H, COOH). HRMS found m/z 287.07. Calcd for
C16H14O3S (M+H)+ m/z 287.07.
5.1.5.3. (E)-4-(2-(4-Bromobenzylthio)-3-(4-fluoro-3-nitro-
phenyl)acryloyl)benzoic acid (6c).
Yield: 72%; light yellow
solid, mp 170–172 °C. 1H NMR (CDCl3, 400 MHz): d 3.72 (s, 2H,
CH2), 6.68–6.75 (m, 3H, Ar–H), 6.98–7.06 (m, 5H, Ar–H), 7.01 (s,
1H, CH), 7.30–7.37 (dd, 2H, Ar–H), 7.54–7.58 (m, 1H, Ar–H),
7.87–7.89 (d, 2H, J = 8.3 Hz, Ar–H), 8.13–8.16 (dd, 1H, Ar–H),
13.33 (br s, 1H, COOH). HRMS found m/z 518.10. Calcd for
C23H15BrFNO5S (M+H)+ m/z 517.99.
5.1.3. 4-(2-(4-Bromobenzylsulfonyl)acetyl)benzoic acid (7)
To a mixture of 4a (50 mmol) in glacial acetic acid (100 mL),
30% hydrogen peroxide (60 mL) was added and the contents were
refluxed for 1 h. After completion of the reaction, the cooled reac-
tion contents were poured on to ice-water and the separated solid
was filtered and dried. The crude product was recrystallized from
methanol to obtain pure sample of 7.
Yield: 80%; white solid, mp 270–272 °C. 1H NMR (CDCl3,
300 MHz): d 3.82 (s, 2H, CH2), 4.05 (s, 2H, CH2), 7.36–8.15 (m,
8H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 396.98. Calcd
for C16H13BrO5S (M+H)+ m/z 396.98.
5.1.5.4. (E)-4-(2-(4-Chlorobenzylthio)-3-(4-fluoro-3-nitrophenyl)-
acryloyl)benzoic acid (6d). Yield: 82%; light yellow solid, mp 165–
168 °C. 1H NMR (CDCl3, 400 MHz): d 3.75 (s, 2H, CH2), 6.71–6.76 (m,
3H, Ar–H), 6.95–7.06 (m, 5H, Ar–H), 7.05 (s, 1H, CH), 7.28–7.35 (dd,
2H, Ar–H), 7.48–7.55 (m, 1H, Ar–H), 7.78–7.85 (d, 2H, J = 8.2 Hz,
Ar–H), 8.10–8.14 (dd, 1H, Ar–H), 13.36 (br s, 1H, COOH). HRMS found
m/z 472.05. Calcd for C23H15ClFNO5S (M+H)+ m/z 472.04.
5.1.4. 4-(2-(4-Bromophenylthio)acetyl)benzoic acid (10a)
Yield: 94%; white solid, mp 188–190 °C. 1H NMR (CDCl3,
300 MHz): d 3.85 (s, 2H, CH2), 7.21–8.15 (m, 8H, Ar–H), 13.50 (br
s, 1H, COOH). HRMS found m/z 350.98. Calcd for C15H11BrO3S
(M+H)+ m/z 350.97.
5.1.5.5. (E)-4-(2-(4-Chlorobenzylthio)-3-(4-chloro-3-nitro-
phenyl)acryloyl)benzoic acid (6e). Yield: 77%; light yellow so-
lid, mp 170–162 °C. 1H NMR (CDCl3, 400 MHz):d 3.82 (s, 2H,
CH2), 6.69–6.71 (m, 3H, Ar–H), 6.82–7.08 (m, 5H, Ar–H), 7.01 (s,
1H, CH), 7.18–7.24 (dd, 2H, Ar–H), 7.35–7.55 (m, 1H, Ar–H),
7.79–7.91 (d, 2H, J = 8.2 Hz, Ar–H), 8.04–8.10 (dd, 1H, Ar–H),
13.40 (br s, 1H, COOH). HRMS found 488.05. Calcd for
C23H15Cl2NO5S (M+H)+ m/z 488.01.
5.1.5. General procedure for the preparation of (E)-2-(benzyl-
thio)-1,3-diphenylprop-2-en-1-one (6) and (E)-1,3-diphenyl-2-
(phenylthio)prop-2-en-1-one (11)
Method A: A mixture of 2-(benzyl/aryl thio)-1-arylethanone (4
or 10) (10 mmol), araldehyde (5) (12.5 mmol) and ammonium
acetate (25 mmol) in glacial acetic acid (10 mL) was refluxed
for 5–8 h. After completion of the reaction, reaction mixture
was cooled and separated product was filtered, washed with 2-
propanol and petroleum ether and dried. If solid was not formed,
the reaction mixture was poured on to crushed ice, extracted
with ethyl acetate, washed with water, brine and dried over so-
dium sulfate. After concentration, crude product obtained was
recrystallized in 2-propanol to yield analytically pure sample of
6 or 11.
5.1.5.6. (E)-4-(2-(4-Chlorobenzylthio)-3-(4-bromo-3-nitro-
phenyl)acryloyl)benzoic acid (6f).
Yield: 85%; light yellow
solid, mp 189–191 °C. 1H NMR (CDCl3, 400 MHz): d 3.80 (s, 2H,
CH2), 6.68–6.75 (m, 3H, Ar–H), 6.82–7.08 (m, 5H, Ar–H), 7.11 (s,
1H, CH), 7.22–7.28 (dd, 2H, Ar–H), 7.41–7.56 (m, 1H, Ar–H),
7.81–7.91 (d, 2H, J = 8.0 Hz, Ar–H), 8.15–8.22 (dd, 1H, Ar–H),
13.50 (br s, 1H, COOH). HRMS found m/z 533.95. Calcd for
C23H15BrClNO5S (M+H)+ m/z 533.96.

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M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
5.1.5.7. (E)-4-(2-(4-Fluorobenzylthio)-3-(4-chloro-3-nitro-
phenyl)acryloyl)benzoic acid (6g). Yield: 68%; light yellow
6.92–6.98 (m, 2H, Ar–H), 7.12–7.19 (m, 2H, Ar–H), 7.20 (s, 1H,
CH), 7.25–7.33 (dd, 2H, Ar–H), 7.52–7.59 (d, 1H, J = 8.1 Hz, Ar–H),
7.68–7.73 (d, 2H, J = 8.0 Hz, Ar–H), 7.81–7.88 (dd, 1H, Ar–H),
8.10–8.16 (dd, 1H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found
m/z 506.00. Calcd for C23H14Cl2FNO5S (M+H)+ m/z 506.00.
solid, mp 164–166 °C. 1H NMR (CDCl3, 400 MHz): d 3.69 (s, 2H,
CH2), 6.65–6.75 (m, 3H, Ar–H), 6.80–7.09 (m, 5H, Ar–H), 7.08 (s,
1H, CH), 7.15–7.30 (dd, 2H, Ar–H), 7.35–7.48 (m, 1H, Ar–H),
7.78–7.89 (d, 2H, J = 8.1 Hz, Ar–H), 8.05–8.18 (dd, 1H, Ar–H),
13.50 (br s, 1H, COOH). HRMS found m/z 472.05. Calcd for
C23H15ClFNO5S (M+H)+ m/z 472.04.
5.1.5.15. (E)-4-(3-(4-Bromo-3-nitrophenyl)-2-(2,4-dichloroben-
zylthio)acryloyl)benzoic acid (6o).
Yield: 74%; white solid,
mp 160–162 °C. 1H NMR (CDCl3, 400 MHz): d 3.95 (s, 2H, CH2),
6.90–6.98 (m, 2H, Ar–H), 7.15–7.21 (m, 2H, Ar–H), 7.25 (s, 1H,
CH), 7.28–7.32 (dd, 2H, Ar–H), 7.49–7.53 (d, 1H, J = 8.1 Hz, Ar–H),
7.62–7.70 (d, 2H, J = 8.0 Hz, Ar–H), 7.78–7.84 (dd, 1H, Ar–H),
8.15–8.20 (dd, 1H, Ar–H), 13.48 (br s, 1H, COOH). HRMS found
m/z 567.92. Calcd for C23H14BrCl2NO5S (M+H)+ m/z 567.92.
5.1.5.8. (E)-4-(2-(4-Fluorobenzylthio)-3-(4-fluoro-3-nitro-
phenyl)acryloyl)benzoic acid (6h).
Yield: 82%; light yellow
solid, mp 160–162 °C. 1H NMR (CDCl3, 400 MHz): d 3.97 (s, 2H,
CH2), 6.93–6.97 (m, 3H, Ar–H), 7.11–7.15 (m, 5H, Ar–H), 7.21 (s,
1H, CH), 7.67–7.69 (dd, 2H, Ar–H), 7.94–7.99 (m, 1H, Ar–H),
8.03–8.07 (d, 2H, J = 8.1 Hz, Ar–H), 8.48–8.50 (dd, 1H, Ar–H),
13.50 (br s, 1H, COOH). HRMS found m/z 456.05. Calcd for
C23H15F2NO5S (M+H)+ m/z 456.07.
5.1.5.16. (E)-4-(3-(4-Fluoro-3-nitrophenyl)-2-(4-methylbenzyl-
thio)acryloyl)benzoic acid (6p).
Yield: 85%; white solid, mp
148–150 °C. 1H NMR (CDCl3, 400 MHz): d 2.25 (s, 3H, CH3), 3.90
(s, 2H, CH2), 6.83–6.92 (m, 2H, Ar–H), 6.98–7.12 (m, 2H, Ar–H),
7.20 (s, 1H, CH), 7.25–7.30 (dd, 2H, Ar–H), 7.38–7.43 (d, 1H,
J = 8.1 Hz, Ar–H), 7.67–7.70 (d, 2H, J = 8.0 Hz, Ar–H), 7.85–7.89
(dd, 1H, Ar–H), 8.22–8.33 (dd, 1H, Ar–H), 13.50 (br s, 1H, COOH).
HRMS found m/z 452.11. Calcd for C24H18FNO5S (M+H)+ m/z
452.10
5.1.5.9. (E)-4-(2-(4-Fluorobenzylthio)-3-(4-bromo-3-nitro-
phenyl)acryloyl)benzoic acid (6i).
Yield: 75%; light yellow
solid, mp 185–187 °C. 1H NMR (CDCl3, 400 MHz): d 3.93 (s, 2H,
CH2), 6.88–6.99 (m, 3H, Ar–H), 7.18–7.25 (m, 5H, Ar–H), 7.26 (s,
1H, CH), 7.55–7.68 (dd, 2H, Ar–H), 7.87–7.89 (m, 1H, Ar–H),
7.98–8.02 (d, 2H, J = 8.1 Hz, Ar–H), 8.35–8.40 (dd, 1H, Ar–H),
13.40 (br s, 1H, COOH). HRMS found m/z 517.98. Calcd for
C23H15FNO5S (M+H)+ m/z 517.99.
5.1.5.17. (E)-4-(3-(4-Fluoro-3-nitrophenyl)-2-(4-(trifluoro-
methyl)benzylthio)acryloyl)benzoic acid (6q).
Yield: 68%;
5.1.5.10. (E)-3-(4-Fluoro-3-nitrophenyl)-2-(4-fluorobenzyl-
white solid, mp 183–186 °C. 1H NMR (CDCl3, 400 MHz): d 3.87 (s,
2H, CH2), 6.73 (s, 1H, CH), 7.03–7.11 (m, 2H, Ar–H), 7.17–7.19 (d,
2H, Ar–H), 7.30–7.37 (d, 1H, J = 8.1 Hz, Ar–H), 7.59–7.65 (dd, 2H,
J = 8.0 Hz, Ar–H), 7.88–7.98 (dd, 1H, Ar–H), 8.19–8.21 (dd, 1H,
Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 506.07. Calcd for
C24H15F4NO5S (M+H)+ m/z 506.07.
thio)-1-(4-fluorophenyl)prop-2-en-1-one (6j).
Yield: 79%;
light yellow solid, mp 88–90 °C. 1H NMR (CDCl3, 400 MHz): d
4.02 (s, 2H, CH2), 6.98–7.02 (m, 2H, Ar–H), 7.15–7.19 (m, 2H, Ar–
H), 7.25 (s, 1H, CH), 7.62–7.67 (dd, 2H, Ar–H), 7.71–7.73 (d, 1H,
J = 8.0 Hz, Ar–H), 7.98–8.03 (d, 2H, J = 8.1 Hz, Ar–H), 8.07–8.11
(dd, 1H, Ar–H), 8.52–8.54 (dd, 1H, Ar–H). HRMS found m/z
430.06. Calcd for C22H14F3NO3S (M+H)+ m/z 430.07.
5.1.5.18. (E)-Methyl4-(2-(4-bromobenzylthio)-3-(4-fluoro-3-
nitrophenyl)acryloyl)benzoate (6r).
Yield: 76%; white solid,
5.1.5.11. (E)-4-(3-(4-Chloro-3-nitrophenyl)-2-(2-fluorobenzyl-
mp 143–145 °C. 1H NMR (CDCl3, 400 MHz): d 3.83 (s, 3H, OCH3),
3.92 (s, 2H, CH2), 6.83 (s, 1H, CH), 7.06–7.12 (m, 2H, Ar–H), 7.20–
7.28 (d, 2H, Ar–H), 7.30–7.37 (d, 1H, J = 8.1 Hz, Ar–H), 7.55–7.65
(dd, 2H, J = 8.0 Hz, Ar–H), 7.78–7.88 (dd, 1H, Ar–H), 8.12–8.20
(dd, 1H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 532.01.
Calcd for C24H17BrFNO5S (M+H)+ m/z 532.01.
thio)acryloyl)benzoic acid (6k).
Yield: 66%; white solid,
yield: 78%; mp 138–140 °C. 1H NMR (CDCl3, 400 MHz): d 4.04 (s,
2H, CH2), 6.95–7.02 (m, 2H, Ar–H), 7.09–7.18 (m, 2H, Ar–H), 7.20
(s, 1H, CH), 7.34–7.32 (dd, 2H, Ar–H), 7.73–7.80 (m, 2H, Ar–H),
7.95–8.06 (d, 2H, J = 8.1 Hz, Ar–H), 8.16–8.22 (dd, 1H, Ar–H),
8.44–8.59 (dd, 1H, Ar–H), 13.57 (br s, 1H, COOH). HRMS found
m/z 472.01. Calcd for C23H15Cl FNO5S (M+H)+ 472.03.
5.1.5.19. (E)-4-(2-(Benzylthio)-3-(4-fluoro-3-nitrophenyl)acry-
loyl)benzoic acid (6s).
Yield: 85%; white solid, mp 148–
5.1.5.12. (E)-4-(2-(2-Chlorobenzylthio)-3-(4-fluoro-3-nitro-
150 °C. 1H NMR (CDCl3, 400 MHz): d 3.87 (s, 2H, CH2), 6.76 (s,
1H, CH), 6.96–7.00 (m, 4H, Ar–H), 7.08–7.14 (m, 2H, Ar–H), 7.40–
7.43 (d, 2H, J = 12 Hz, Ar–H), 7.67–7.71 (m, 1H, Ar–H), 7.93–7.95
(d, 2H, J = 8 Hz, Ar–H), 8.20–8.22 (dd, 1H, J = 8.0 Hz, Ar–H), 13.50
(br s, 1H, COOH). HRMS found m/z 438.08. Calcd for C23H16FNO5S
(M+H)+ m/z 438.08.
phenyl)acryloyl)benzoic acid (6l).
Yield: 76%; white solid,
mp 198–200 °C. 1H NMR (CDCl3, 400 MHz): d 4.15 (s, 2H, CH2),
7.03–7.14 (m, 2H, Ar–H), 7.22–7.28 (m, 2H, Ar–H), 7.20 (s, 1H,
CH), 7.58–7.62 (dd, 2H, Ar–H), 7.78–7.83 (d, 1H, J = 8.0 Hz, Ar–H),
7.98–8.03 (d, 2H, J = 8.0 Hz, Ar–H), 8.05–8.10 (dd, 1H, Ar–H),
8.43–8.54 (dd, 1H, Ar–H), 13.47 (br s, 1H, COOH). HRMS found
472.04. Calcd for C23H15ClFNO5S (M+H)+ m/z 472.04.
5.1.5.20. (E)-4-(2-(4-Bromobenzylsulfinyl)-3-(4-fluoro-3-nitro-
phenyl)acryloyl)benzoic acid (6t).
To a mixture of 6c (0.51 g,
5.1.5.13. (E)-4-(3-(4-Chloro-3-nitrophenyl)-2-(2,4-dichloroben-
10 mmol) in dry chloroform (10 mL), m-chloroperoxybenzoic acid
(0.22 g, 10 mmol) in 5 mL dry chloroform was added drop wise
at 0 °C. After the addition, the reaction was continued at this tem-
perature for 1 h and at room temperature for an additional 1 h. The
separated solid was filtered and recrystallized from chloroform to
obtain a pure sample of 6t. Yield: 78%; white solid, mp 213–
215 °C. 1H NMR (DMSO-d6, 300 MHz): d 4.47–4.63 (q, 2H, CH2),
7.22–7.24 (d, 2H, Ar–H), 7.36–7.39 (d, 2H, Ar–H), 7.47–7.56 (m,
2H, Ar–H), 7.63–7.67 (m, 1H, CH), 7.81–7.98 (m, 1H, Ar–H), 8.02–
8.05 (dd, 1H, Ar–H), 8.11–8.24 (m, 3H, Ar–H), 13.50 (br s, 1H,
COOH). HRMS found m/z 531.90. Calcd for C23H15BrFNO6S
(M+H)+ m/z 531.99.
zylthio)acryloyl)benzoic acid (6m).
Yield: 85%; white solid,
mp 126–130 °C. 1HNMR (CDCl3, 400 MHz): d 4.10 (s, 2H, CH2),
6.93–7.12 (m, 2H, Ar–H), 7.24–7.29 (m, 2H, Ar–H), 7.26 (s, 1H,
CH), 7.55–7.60 (dd, 2H, Ar–H), 7.82–7.89 (d, 1H, J = 8.1 Hz, Ar–H),
8.03–8.08 (d, 2H, J = 8.0 Hz, Ar–H), 8.11–8.18 (dd, 1H, Ar–H),
8.51–8.59 (dd, 1H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found
m/z 523.97. Calcd for C23H14Cl3NO5S (M+H)+ m/z 523.97.
5.1.5.14. (E)-4-(3-(4-Fluoro-3-nitrophenyl)-2-(2,4-dichloroben-
zylthio)acryloyl)benzoic acid (6n).
Yield: 80%; white solid,
mp 124–128 °C. 1H NMR (CDCl3, 400 MHz): d 3.92 (s, 2H, CH2),

M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
2325
5.1.6. (E)-4-(2-(4-Bromobenzylsulfonyl)-3-(4-fluoro-3-nitro-
phenyl)acryloyl)benzoic acid (8a)
5.1.7.5. (E)-4-(2-(4-Bromobenzylthio)-3-(3-fluoro-4-nitrophenyl)-
acryloyl)benzoic acid (6ae).
Yield: 90%; yellow solid, mp 198–
This compound was prepared from 7 and 3-nitro-4-fluorobenz-
aldehyde in 62% yield as described method A. Alternatively, this
compound was also prepared by the oxidation of 6c with excess
(3 equiv) of m-chloroperoxybenzoic acid as described in 6t. Yield:
62%; white solid, mp 178–180 °C. 1H NMR (DMSO-d6, 300 MHz):
d 4.82 (s, 2H, CH2), 7.39–7.49 (m, 2H, Ar–H), 7.51–7.67 (m, 4H,
Ar–H), 7.81 (s, 1H, CH), 7.91–7.99 (m, 3H, Ar–H), 8.10–8.14 (m,
1H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 549.98. Calcd
for C23H15BrFNO7S (M+H)+ m/z 549.98.
200 °C. 1H NMR (CDCl3, 300 MHz): d 4.01 (s, 2H, CH2), 6.52 (s,
1H, CH), 6.92–7.09 (m, 2H, Ar–H), 7.16–7.27 (m, 4H, Ar–H), 7.38–
7.46 (m, 1H, Ar–H), 7.62–7.67 (m, 2H, Ar–H), 8.11–8.36 (m, 2H,
Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 517.98. Calcd for
C23H15BrFNO5S (M+H)+ m/z 517.99.
5.1.7.6. (E)-4-(2-(4-Bromobenzylthio)-3-(4-fluoro-2-nitro-
phenyl)acryloyl)benzoic acid (6af).
Yield: 86%; yellow solid:
mp 194–196 °C. 1H NMR (DMSO-d6, 300 MHz): d 3.89 (s, 2H, CH2),
6.59 (s, 1H, CH), 6.91–7.06 (m, 2H, Ar–H), 7.16–7.27 (m, 4H, Ar–H),
7.40–7.49 (m, 1H, Ar–H), 7.68–7.74 (m, 2H, Ar–H), 8.16–8.36 (m,
2H, Ar–H), 13.49 (br s, 1H, COOH). HRMS found m/z 517.97. Calcd
for C23H15BrFNO5S (M+H)+ m/z 517.99.
5.1.6.1. (E)-4-(2-(4-Bromobenzylsulfonyl)-3-(4-chloro-3-nitro-
phenyl)acryloyl)benzoic acid (8b).
Yield: 48%; white solid,
mp 148–150 °C. 1H NMR (DMSO-d6, 400 MHz): d 4.53 (s, 2H,
CH2), 7.20–7.24 (m, 3H, Ar–H), 7.30–7.36 (m, 5H, Ar–H), 7.38 (s,
1H, CH), 7.43–7.55 (dd, 2H, Ar–H), 7.51–7.59 (m, 1H, Ar–H),
7.95–7.98 (d, 2H, J = 8.6 Hz, Ar–H), 8.09–8.21 (dd, 1H, Ar–H),
13.50 (br s, 1H, COOH). HRMS found m/z 565.92. Calcd for
C23H15BrClNO7S (M+H)+ m/z 565.95.
5.1.7.7. (E)-4-(2-(4-Bromobenzylthio)-3-(2-fluoro-4-nitro-
phenyl)acryloyl)benzoic acid (6ag).
Yield: 87%; yellow solid,
mp 196–199 °C. 1H NMR (DMSO-d6, 300 MHz): d 3.89 (s, 2H, CH2),
6.52 (s, 1H, CH), 6.92–7.09 (m, 2H, Ar–H), 7.16–7.27 (m, 4H, Ar–H),
7.38–7.46 (m, 1H, Ar–H), 7.62–7.67 (m, 2H, Ar–H), 8.11–8.36 (m,
2H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 517.98. Calcd
for C23H15BrFNO5S (M+H)+ m/z 517.99.
5.1.7. (E)-4-(2-(4-Bromophenylthio)-3-(4-fluoro-3-nitrophenyl)-
acryloyl)benzoic acid (11)
Yield: 89%; white solid, mp 145–147 °C. 1H NMR (DMSO-d6,
400 MHz): d 7.12–7.25 (m, 2H, Ar–H), 7.28–7.31 (d, 2H, Ar–H),
7.65 (s, 1H, CH), 7.88–7.90 (d, 1H, J = 8.1 Hz, Ar–H), 8.00–8.03
(dd, 2H, J = 8.0 Hz, Ar–H), 8.24–8.27 (dd, 1H, Ar–H), 8.65–8.67
(dd, 1H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 502.97.
Calcd for C22H13BrFNO5S (M+H)+ m/z 502.97.
5.1.8. (E)-4-(2-(4-Bromobenzylthio)-3-(4-fluoro-3-nitrophenyl)
acryloyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide (12a)
A mixture of 6c (0.155 g, 3 mmol), 1-hydroxybenzotriazole hy-
drate (0.040 g, 3 mmol), N,N-diisopropylethylamine (157
9 mmol),
lL,
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (0.064 g, 3.3 mmol) in dry tetrahydrofuran (10 mL)
were stirred at room temperature under nitrogen atmosphere for
30 min. To this reaction mixture, 2-(4-methylpiperazin-1-yl)eth-
anamine (0.039 g, 3 mmol) in THF (2 mL) was added drop wise
and continued stirring for additional 2 h. The progress of the reac-
tion was monitored by TLC (9:1 chloroform/methanol on silica gel
plate). After completion of the reaction, THF was removed under
reduced pressure, diluted with water and extracted with ethyl ace-
tate. The ethyl acetate layer was washed with saturated sodium
bicarbonate solution, water and brine and dried over anhydrous
sodium sulfate. Removal of the solvent under reduced pressure
afforded a crude product which was purified by silica gel column
chromatography, eluting with chloroform/methanol (9:0.5), to
yield compound 12a. Yield: 75%; light orange solid, mp 60–62 °C.
1H NMR (CDCl3, 400 MHz): d 2.19 (s, 3H, CH3), 2.25–2.54 (m,
10H), 3.45–346, (d, 2H, NH–CH2), 3.98 (s, 2H, CH2), 6.80–6.95 (m,
2H, Ar–H), 7.12–7.25 (m, 3H, Ar–H), 7.39–7.49 (dd, 2H, Ar–H),
7.63–7.78 (m, 2H, Ar–H), 8.28–7.29 (d, 2H, J = 8.0 Hz, Ar–H), 8.58
(br s, 1H, NH). HRMS found m/z 643.12. Calcd for C30H30BrFN4O4S
(M+H)+ m/z 643.12.
5.1.7.1. (E)-4-(2-(4-Bromobenzylthio)-3-(4-fluoro-3-methoxy-
phenyl)acryloyl)benzoic acid (6aa).
Yield: 65%; white solid,
mp 72–74 °C. 1H NMR (DMSO-d6, 400 MHz): d 3.85 (s, 3H, OCH3),
4.04 (s, 2H, CH2), 6.84–6.94 (m, 2H, Ar–H), 7.18–7.27 (m, 5H, Ar–
H), 7.29 (s, 1H, CH), 7.55–7.57 (dd, 2H, Ar–H), 7.58–7.62 (m, 1H,
Ar–H), 7.85–7.87 (d, 2H, J = 8.0 Hz, Ar–H), 8.04–8.15 (dd, 1H, Ar–
H), 13.50 (br s, 1H, COOH). HRMS found m/z 502.34. Calcd for
C24H18BrFO4S (M+H)+ m/z 502.36.
5.1.7.2. (E)-4-(2-(4-Bromobenzylthio)-3-(4-hydroxy-3-bromo)
acryloyl)benzoic acid (6ab).
Yield: 60%; yellow solid: mp
158–160 °C. 1H NMR (DMSO-d6, 400 MHz): d 3.45 (br s, 1H, OH),
3.98 (s, 2H, CH2), 7.20–7.26 (m, 4H, Ar–H), 7.30–7.37 (m, 2H, Ar–
H), 7.62–7.69 (m, 4H, Ar–H), 7.95–8.04 (dd, 1H, J = 12.0, 8.0 Hz,
Ar–H), 8.52–8.61 (m, 1H, Ar–H), 13.48 (br s, 1H, COOH). HRMS
found m/z 549.30. Calcd for C23H16Br2O4S (M+H)+ m/z 549.25.
5.1.7.3. (E)-4-(2-(4-Bromobenzylthio)-3-(4-hydroxy-3-nitro-
phenyl)acryloyl)benzoic acid (6ac).
Yield: 78%; yellow solid:
5.1.8.1. (E)-2-(4-Bromobenzylthio)-3-(4-fluoro-3-nitrophenyl)-
mp 200–202 °C. 1H NMR (DMSO-d6, 400 MHz): d 3.35 (br s, 1H,
OH), 4.05 (s, 2H, CH2), 7.00–7.04 (m, 2H, Ar–H), 7.11–7.21 (m,
4H, Ar–H), 7.63–7.65 (d, 2H, J = 8.0, Ar–H), 7.90–7.93 (dd, 1H,
J = 12.0, 8.0 Hz, Ar–H), 8.00–8.02 (d, 2H, J = 8.0, Ar–H), 8.38–8.39
(d, 1H, Ar–H), 13.50 (br s, 1H, COOH). HRMS found m/z 515.32.
Calcd for C23H16BrNO6S (M+H)+ m/z 515.36.
1-(4-(4-(2-hydroxy-ethyl)piperazine-1-carbonyl)phenyl)prop-
2-en-1-one (12b).
Yield: 70%; white solid, mp 117–119 °C. 1H
NMR (CDCl3, 400 MHz): d 2.44–2.54 (m, 6H), 2.76 (br s, 1H, OH),
3.34–3.37 (d, 2H), 3.57–3.61 (m, 2H), 3.74–3.79 (m, 2H,), 3.89 (s,
2H, CH2), 6.84–6.85 (s, 1H, Ar–H), 6.92–6.94 (d, 2H, Ar–H), 7.14–
7.16 (d, 2H, Ar–H), 7.20–7.26 (m, 2H, Ar–H), 7.34–7.36 (d, 2H,
Ar–H), 7.50–7.52 (d, 2H, Ar–H), 7.74–7.77 (m, 1H, Ar–H). HRMS
found m/z 630.08. Calcd for C29H27BrFN3O5S (M+H)+ m/z 630.09.
5.1.7.4. (E)-4-(2-(4-Bromobenzylthio)-3-(2-fluoro-5-nitro-
phenyl)acryloyl)benzoic acid (6ad).
Yield: 68%; yellow solid:
mp 223–225 °C. 1H NMR (CDCl3, 400 MHz): d 4.00 (s, 2H, CH2),
6.95–7.02 (m, 2H, Ar–H), 7.12–7.19 (m, 2H, Ar–H), 7.29–7.35 (m,
2H, Ar–H), 7.38–7.43 (d, 1H, J = 8.1 Hz, Ar–H), 7.61–7.70 (d, 2H,
J = 8.0 Hz, Ar–H), 7.92–7.98 (m, 2H, Ar–H), 8.28–8.33 (dd, 1H, Ar–
H), 13.50 (br s, 1H, COOH). HRMS found m/z 517.32. calcd for
C23H15BrFNO5S (M+H)+ m/z 517.35.
5.1.8.2. (E)-4-(2-(4-Bromobenzylthio)-3-(4-fluoro-3-nitrophenyl)-
acryloyl)-N-(2-(diethylamino)ethyl)benzamide (12c).
Yield:
72%; white solid, mp 60–61 °C. 1H NMR (CDCl3, 400 MHz): d
2.44–2.54 (m, 6H), 2.76 (br s, 1H, OH), 3.34–3.37 (d, 2H), 3.57–
3.61 (m, 2H), 3.74–3.79 (m, 2H,), 3.89 (s, 2H, CH2), 6.84–6.85 (s,
1H, Ar–H), 6.92–6.94 (d, 2H, Ar–H), 7.14–7.16 (d, 2H, Ar–H),

2326
M. V. Ramana Reddy et al. / Bioorg. Med. Chem. 18 (2010) 2317–2326
7.20–7.26 (m, 2H, Ar–H), 7.34–7.36 (d, 2H, Ar–H), 7.50–7.52 (d, 2H,
Ar–H), 7.74–7.77 (m, 1H, Ar–H). HRMS found m/z 616.10. Calcd for
C29H29BrFN3O4S (M+H)+ m/z 616.11.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.051.
5.1.8.3. (E)-3-(4-Fluoro-3-nitrophenyl)-2-(4-fluorobenzylthio)-
1-(4-(4-(2-hydroxyethyl)piperazine-1-carbonyl)phenyl)prop-2-
References and notes
en-1-one (12d).
Yield: 75%; light yellow solid, mp 68–70 °C.
1H NMR (CDCl3, 400 MHz): d 2.21 (br s, 2H), 2.28–2.33 (m, 6H),
3.13 (br s, 1H, OH), 3.35–3.38 (t, 2H), 3.54 (br s, 2H,), 3.71 (s, 2H,
CH2), 6.51–6.55 (m, 2H, Ar–H), 6.65 (s, 1H, Ar–H), 6.80–6.86 (m,
2H, Ar–H), 6.98–7.03 (dd, 2H, Ar–H), 7.10–7.15 (m, 2H, Ar–H),
7.33–7.35 (d, 2H, Ar–H), 7.50–7.53 (m, 1H, Ar–H), 8.12–8.14 (dd,
1H, Ar–H). HRMS found m/z 568.15. Calcd for C29H27F2N3O5S
(M+H)+ m/z 568.16.
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ated by the addition of 1
lg recombinant substrate, 20 lM ATP
and 20 Ci -32P-ATP. The reactions were incubated at 30 °C for
l
c
20 min, terminated by the addition of 2ꢀ Laemmli sample buffer,
boiled for 2 min, resolved by 12% acrylamide SDS–PAGE and sub-
jected to autoradiography. The autoradiograms were scanned and
the band corresponding to phosphorylation of substrate was quan-
titated using MacBas software. The densitometric values obtained
were plotted as a function of log drug concentration using Prism
4 Graphpad software and IC50 values determined by plotting sig-
moidal non-linear regression curves with variable slope. ErbB2
and EGFR assays were performed using cell based immunoprecip-
itation assays like those performed by us earlier.35
Acknowledgments
This work was supported by grants from NIH (R01CA-109820),
DOD (W81XWtt-06–1-0267) and Onconova Therapeutics Inc.