Synthesis and cellular pharmacology studies of a series of 2-amino-3-aroyl-4-substituted thiophene derivatives

Article abstract of DOI:10.2174/157340610793563974

Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an important target for cancer therapy. In search of new compounds with strong antiproliferative activity and simple molecular structure, we have synthesized four different series of compounds in which different substituents were linked to the 4- or 5-position of the 2-amino-3-(3',4',5'-trimethoxybenzoyl) thiophene system. When these compounds were analyzed in vitro for their inhibition of cell proliferation, the 4-aryl substituted derivatives had little activity. In contrast, the presence of a methylene, oxymethyl, aminomethyl or methylenepiperazino moiety between the aryl and the 4-position of the thiophene ring resulted in statistically significant improvement in activity relative to the 4-aryl substituted derivatives. It is noteworthy that the antiproliferative effects of the synthesized compounds were more pronounced against human Molt/4 and CEM as compared with murine L1210 and FM3A cells. The effects of a selected series of compounds on cell cycleprogression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found that the antiproliferative effects of the most active compounds were associated with increase of the proportion of cells in the G2/M and sub-G1 phases of the cell cycle. The structure-activity relationships observed in the series of compounds described here should permit the design of more active molecules.

Full text of DOI:10.2174/157340610793563974

Medicinal Chemistry, 2010, 6, 329-343
329
Synthesis and Cellular Pharmacology Studies of a Series of
2-amino-3-aroyl-4-substituted Thiophene Derivatives
Romeo Romagnoli*,1, Pier Giovanni Baraldi1, Maria Giovanna Pavani1, Olga Cruz-Lopez1, Ernest Hamel2,
Jan Balzarini3, Eleonora Brognara4, Cristina Zuccato4 and Roberto Gambari4
1Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy
2Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, USA
3Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Minderbroedersstraat 10, B-3000
Leuven, Belgium
4BioPharmaNet, Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, 44100 Ferrara, Italy
Abstract: Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an impor-
tant target for cancer therapy. In search of new compounds with strong antiproliferative activity and simple molecular
structure, we have synthesized four different series of compounds in which different substituents were linked to the 4- or
5-position of the 2-amino-3-(3’,4’,5’-trimethoxybenzoyl) thiophene system. When these compounds were analyzed in vi-
tro for their inhibition of cell proliferation, the 4-aryl substituted derivatives had little activity. In contrast, the presence of
a methylene, oxymethyl, aminomethyl or methylenepiperazino moiety between the aryl and the 4-position of the thio-
phene ring resulted in statistically significant improvement in activity relative to the 4-aryl substituted derivatives. It is
noteworthy that the antiproliferative effects of the synthesized compounds were more pronounced against human Molt/4
and CEM as compared with murine L1210 and FM3A cells. The effects of a selected series of compounds on cell cycle
progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found
that the antiproliferative effects of the most active compounds were associated with increase of the proportion of cells in
the G2/M and sub-G1 phases of the cell cycle. The structure-activity relationships observed in the series of compounds
described here should permit the design of more active molecules.
Keywords: Thiophene, inhibition of tubulin polymerization, inhibition of tumor cell growth, antiproliferative agents.
1. INTRODUCTION
concentrations [10], and its structural simplicity makes CA-4
of great interest from the medicinal chemistry viewpoint.
The microtubule system of eukaryotic cells is essential in
a variety of fundamental cellular processes, including mito-
sis, formation and maintenance of cell shape, regulation of
motility, cell signaling, secretion and intracellular transport
[1, 2]. There has long been considerable interest in the dis-
covery and development of novel small molecules, many of
which are natural products, able to inhibit tubulin polymeri-
zation for the treatment of cancer [3-5]. Combretastatin A-4
(CA-4, 1), isolated from an African willow, Combretum caf-
frum (Combretaceae), shows interesting anticancer potential
due to its antitubulin properties [6]. Previous studies have
found that the 3’,4’,5’-trimethoxy group on the A ring was
essential for strong activity, while the 3’-hydroxy group on
the B-ring is not necessary for potent activity [7-9]. CA-4
strongly binds to the colchicine site of tubulin, and this bind-
ing prevents tubulin polymerization and causes an antimitotic
effect. CA-4 inhibits cell growth at low to mid nanomolar
For these reasons, a large number of synthetic CA-4 ana-
logues have been synthesized and evaluated in structure-
activity relationship (SAR) studies [7-12]. Replacing the
double bound of CA-4 with a carbonyl group furnished a
benzophenone-type CA-4 analogue named phenstatin (2),
which was found to be a very strong cytotoxic agent with the
same characteristics as CA-4 [13]. The 2-amino benzophe-
none derivative 3 showed significantly increased cytotoxicity
against many human cancer cell lines as compared with
phenstatin 2, but 3 was nevertheless slightly less potent than
CA-4 [14].
The classical bioisosteric equivalence between benzene
and thiophene prompted us to synthesize a series of thio-
phene derivatives with general formula 4, in which a 2-
aminothiophene system replaced the 2-aminobenzene moiety
in the 2-amino phenstatin analogue 3 [15]. Many of the 2-
amino-3-(3’,4’,5’-trimethoxybenzoyl)-5-phenyl
thiophene
molecules with general structure 4 are potent inhibitors of
tubulin polymerization and show strong antiproliferative
activities against two leukemic cell lines, L1210 and K562,
with accumulation of cells in the G2/M phase of the cell cy-
*Address correspondence to this author at the Dipartimento di Scienze
Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17-19, I-44121
Ferrara, Italy; Tel: +39-(0)532-455303; Fax: +39-(0)532-455953;
E-mail: rmr@unife.it
1573-4064/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

330 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
cle. The most active compounds (such as 4a) had activities
comparable with those of CA-4.
the synthesis of molecules 5e-f, we investigated the effect on
antiproliferative activity of the 3’,4’,5’-trimethoxybenzoyl
group in the 2-aminothiophene derivative 5a.
On the basis of previous encouraging results on the anti-
tumor activity of 2-amino-3-(3’,4’,5’-trimethoxybenzoyl)-5-
aryl thiophenes, we have now extended this study by the
design, synthesis and biological evaluation of a related series
of compounds characterized by a common 2-amino-3-
(3’,4’,5’-trimethoxybenzoyl) thiophene core but with differ-
ent substituents at the 4- or 5-positions of the thiophene ring.
The 3’,4’,5’-trimethoxyphenyl of the 2-benzoyl moiety was
kept unchanged because it is the characteristic structural re-
quirement for activity in a large series of inhibitors of tubulin
polymerization, such as colchicine, combretastatin A-4 and
podophyllotoxin [7, 16]. This furnished four different series
of compounds.
The second series was represented by compounds 6a-h,
characterized by a flexible benzyl moiety at the 4-position of
the thiophene ring. The substituents examined at the 5-
position included hydrogen (6a-d) or phenyl (6e-h). We ex-
plored the SAR by examining various substitutions with
electron-withdrawing (F and Cl) and electron-releasing (Me
and MeO) groups (ERG’s and EWG’s, respectively) on the
phenyl of the 2-benzyl moiety.
In the third series of derivatives 7a-h, the methylene
spacer between the aryl moiety and the 4-position of the
thiophene ring was replaced with a more flexible and longer
oxymethyl or aminomethyl tether, to furnish derivatives 7a-d
and 7e-h, respectively.
In the first series of compounds 5a-d, which is structur-
ally related to derivatives with general formula 4, we evalu-
ated the effect on biological activity on moving the aryl
group from the 5- to the 4-position of the thiophene ring. By
The fourth and final series of molecules 8a-d was charac-
terized by the presence of an aryl piperazine moiety as tether
to the 4-position of the thiophene ring attached to the meth-
H3CO
O
R1
H3CO
H3CO
R2
R3
A
H3CO
B
CH3O
OR
CH3O
OCH3
R1=H, R2=OH, R3=OCH3, Phenstatin, 2
R1=NH2, R2=H, R3=OCH3, 3
R=H, Combretastatin A-4 (CA-4), 1
R4
R1
O
O
NH2
H3CO
S
R2
H3CO
CH3O
4
R3
5a-g
NH2
S
R
R=H, halogen, Me,
CF3, OMe
R=H, 4a
R1-3=OMe, R4=H, 5a
R1-3=OMe, R4=F, 5b
R1-3=OMe, R4=Cl, 5c
R1-3=OMe, R4=OMe, 5d
R1-2=OMe, R3-4=H, 5e
R1=OMe, R2-4=H, 5f
OMe
O
OMe
R1,3=OMe, R2=OH, R4=H, 5g
R1
OMe
6a-h
OMe
NH2
R2
R1-2=H, 6a
S
R
O
OMe
X
R1=p-Cl, R2=H, 6b
R1=p-Me, R2=H, 6c
R1=m-OMe, R2=H, 6d
R1=H, R2=Ph, 6e
R1=p-F, R2=Ph, 6f
R1=p-Cl, R2=Ph, 6g
R1=p-OMe, R2=Ph, 6h
OMe
7a-h
NH2
S
R=H, X=O, 7a
R=p-Cl, X=O, 7b
R=p-Me, X=O, 7c
OMe
OMe
O
R=m, m'-OMe, X=O, 7d
R=H, X=NH, 7e
N
N
R=m-F, X=NH, 7f
R=p-Cl, X=NH, 7g
R=m-OMe, X=NH, 7h
R
OMe
8a-d
R=H, 8a
R=m-F, 8b
R=p-Cl, 8c
NH2
S
R=m-OMe, 8d
Chart 1. General chemical structures of compounds 1-8.

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 331
ylene spacer. Piperazine is a basic hydrophilic functionality
able to increase water solubility.
d. Removal of the N-protected phthaloyl group was accom-
plished with hydrazine in refluxing ethanol, to afford 6a-d.
Compound 12 was employed as intermediate for the syn-
thesis of 4-benzyl-5-aryl derivatives 6e-h. Compound 12 was
brominated in the 5-position with NBS in benzene to give
the 5-bromo thiophene derivative 15. The subsequent intro-
duction of a phenyl moiety at the 5-position of the thiophene
ring by a standard Suzuki cross-coupling reaction (phenylbo-
ronic acid, Pd(PPh3)4, K2CO3), followed by reaction with
NBS in CCl4 resulted in the formation of the 4-
bromomethyl-5-phenyl thiophene analogue 17. The arylation
by Suzuki reaction with the appropriate arylboronic acid and
the subsequent treatment with hydrazine in refluxing ethanol,
afforded 6e-h.
2. MATERIALS AND METHODS
2.1. Chemistry
The 2-amino-3-(3’,4’,5’-trimethoxyphenyl)-4-arylthiop-
henes 5a-f were synthesized by a two-step procedure de-
scribed in Scheme 1. The Knoevenagel reaction of ꢀ-
ketonitriles 9a-c and the appropriate acetophenone in ben-
zene in the presence of ꢀ-alanine and acetic acid furnished
compounds 10a-f as a mixture of the E- and Z-olefin iso-
mers. The subsequent cyclization in ethanol and sulfur in the
presence of TEA yielded thiophene derivatives 5a-f. Deriva-
tive 5g was obtained by treatment of 5a with neat sulfuric
acid.
Derivatives 7a-k and 8a-d were synthesized following
the strategy reported in Scheme 3. The condensation of 4-
bromomethyl-5-bromo thiophene 13 with the appropriate
phenol, aniline or arylpiperazine, in the presence of K2CO3
or TEA as base, provided the derivatives 19a-d, 19e-h and
20a-d, respectively. Finally, the treatment of 19a-h and 20a-d
with ethanolic hydrazine afforded 7a-h and 8a-d, respectively.
The approach taken for the preparation of compounds 6a-
h is shown in Scheme 2. The treatment of the 2,5-dimethyl-
(1,4)dithiane-2,5-diol (the dimer of ꢁ-mercaptoacetone) in
ethanol with triethylamine (TEA) (2.2 equiv.) followed by
the addition of 3-(3’,4’,5’-trimethoxyphenyl)-3-oxopropan-
enitrile 9a, furnished the 2-amino-3-(3’,4’,5’-trimethoxy-
benzoyl)-4-methylthiophene 11. Compound 11 was con-
verted almost quantitatively to the phthalimido derivative 12
using phthalic anhydride in acetic acid, and the subsequent
reaction with N-bromosuccinimide (NBS) in CCl4 resulted in
side-chain bromination and the formation of the 4-
bromomethyl-thiophene analogue 13, used as a common
intermediate for the synthesis of final compounds 6a-d, 7a-h
and 8a-d by a three-steps synthesis. The 4-benzyl derivatives
6a-d were synthesized from the key intermediate 13 by a
standard Suzuki cross-coupling reaction with appropriate
aryl boronic acids under heterogeneous conditions [Pd(PPh3)4,
K2CO3] in refluxing toluene [17], to furnish derivatives 14a-
2.2. Chemistry Experimental section
1
H NMR spectra were recorded on a Bruker AC 200
spectrometer. Chemical shifts (ꢂ) are given in ppm upfield
from tetramethylsilane as internal standard, and the spectra
were recorded in appropriate deuterated solvents, as indi-
cated. Melting points (mp) were determined on a Buchi-
Tottoli apparatus and are uncorrected. All products reported
1
showed H NMR spectra in agreement with the assigned
structures. All reactions were carried out under an inert at-
mosphere of dry nitrogen, unless otherwise indicated. Stan-
dard syringe techniques were applied for transferring dry
R4
R1
R1
R2
R3
R2
R3
a
R1-3=OMe, R4=H, 10a
R1-3=OMe, R4=F, 10b
CN
CN
R
1-3=OMe, R4=Cl, 10c
R
1-3=OMe, R4=OMe,10d
9a-c
O
O
O
10a-f
R1-2=OMe, R3-4=H, 10e
R1=OMe, R2-4=H, 10f
R1-3=OMe, 9a
R1,2=OMe, R3=H, 9b
R1=OMe, R2,3=H, 9c
b
R4
R1
R2
R3
5a-f
NH2
S
R1-3=OMe, R4=H, 5a
R
R
1-3=OMe, R4=F, 5b
1-3=OMe, R4=Cl, 5c
c
R1-3=OMe, R4=OMe, 5d
1-2=OMe, R3-4=H, 5e
R1=OMe, R2-4=H, 5f
R1,3=OMe, R2=OH, R4=H, 5g
R
Reagents and conditions. a: Ketone, AcOH, ꢀ-alanine, toluene, rx; b: S8, TEA, EtOH, 70 °C; c: H2SO4 neat, rt.
Scheme 1. Synthesis of compounds 5a-g.

332 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
MeO
OMe
MeO
OMe
MeO
MeO
e
O
O
R1
R1
R1-2=H, 14a, 6a
R1=p-Cl, R2=H, 14b, 6b
R1=p-Me, R2=H, 14c, 6c
R1=m-OMe, R2=H, 14d, 6d
O
6a-d
N
S
NH2
S
14a-d
O
d
MeO
OMe
MeO
MeO
OMe
MeO
OMe
MeO
MeO
Br
O
O
O
c
b
a
O
9a
O
N
NH2
S
S
N
S
11
12
13
O
O
f
MeO
OMe
MeO
OMe
MeO
MeO
OMe
MeO
MeO
d
c
O
Br
O
O
O
O
O
N
S
Br
N
N
S
S
16
15
17
O
O
O
d
MeO
MeO
OMe
MeO
OMe
R1=H, 18e, 6e
R1=p-F, 18f, 6f
MeO
R1=p-Cl, 18g, 6g
R1=p-OMe, 18h, 6h
e
O
O
R1
R1
O
NH2
S
N
S
O
18e-h
6e-h
Reagents. a: 2,5-Dimethyl-[1,4]dithiane-2,5-diol, TEA, EtOH; b: phthalic anhydride, AcOH; c: NBS, CCl4;
d: ArB(OH)2, Pd(PPh3)4, Na2CO3, PhMe; e: NH2NH2, EtOH; f: NBS, C6H6, rx.
Scheme 2. Synthesis of compounds 6a-h.

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 333
OMe
OMe
OMe
R
R
O
O
O
OMe
OMe
O
b
O
OMe
N
S
NH2
S
R=H, 19a, 7a
7a-d
19a-d
R=p-Cl, 19b, 7b
R=p-Me, 19c, 7c
R=m, m'-OMe, 19d, 7d
O
a
MeO
OMe
OMe
OMe
R
R
O
O
O
MeO
Br
OMe
OMe
HN
HN
b
c
O
OMe
OMe
O
N
NH2
S
S
O
7e-h
N
S
R=H, 19e, 7e
R=m-F, 19f, 7f
19e-h
13
R=p-Cl, 19g, 7g
R=m-OMe, 19h, 7h
O
d
MeO
OMe
MeO
OMe
MeO
N
MeO
N
b
R=H, 20a, 8a
R=m-F, 20b, 8b
R=p-Cl, 20c, 8c
R=m-OMe, 20d, 8d
N
N
O
O
R
R
O
8a-d
NH2
N
S
S
20a-d
O
Reagents. a: ArOH, K2CO3, DMF, rt; b: NH2NH2, EtOH; c: ArNH, TEA, DCM, rt; d: aryl piperazine, K2CO3, DCM, rt.
Scheme 3. Synthesis of compounds 7a-h and 8a-g.
solvents. Reaction courses and product mixtures were rou-
Synthesis of (2E/Z)-2-(3,4,5-trimethoxybenzoyl)-3-phenyl-
tinely monitored by TLC on silica gel (precoated F
but-2-enenitrile (10a)
254
Merck plates) and visualized with aqueous KMnO4. Flash
chromatography was performed using 230-400 mesh silica
gel and the indicated solvent system. Organic solutions were
dried over anhydrous Na2SO4.
Following general procedure (A) for the condensation be-
tween 3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile and
acetophenone, the crude residue was purified by column
chromatography on silica gel (EtOAc-petroleum ether 2-8
1
v/v), and 10a was obtained as a yellow oil (yield: 40%). H
General Procedure (A) for the Synthesis of Knoeve-
nagel’s Adduct between the Appropriate ꢀ-ketonitrile
and Different Acetophenones
NMR (CDCl ): ꢁ 2.35 (s, 3H), 2.66 (s, 3H), 3.85 (s, 6H),
3
3.88 (s, 3H), 3.93 (s, 6H), 3.96 (s, 3H), 7.00 (s, 2H), 7.17 (m,
2H), 7.23 (m, 2H), 7.24 (m, 2H), 7.28 (s, 2H), 7.48 (m, 2H),
7.63 (m, 2H).
A mixture of the appropriate aroylacetonitrile (5 mmol),
acetophenone (5.5 mmol), ꢀ-alanine (89.1 mg, 1 mmol), ace-
tic acid (1.2 mL) and benzene (40 mL) was refluxed under
Dean-Stark conditions for 24 h. The mixture was cooled and
diluted with ethyl acetate (40 mL), and the resulting mixture
was washed with water (10 mL) and brine (10 mL) and dried
over Na2SO4 and the organic solvent evaporated under re-
duced pressure. The residue was purified by column chroma-
tography on silica gel (EtOAc-petroleum ether as eluent) to
furnish the E/Z mixture of the Knoevenagel’s adduct.
Synthesis of (2E/Z)-2-(3,4,5-trimethoxybenzoyl)-3-(4-
fluorophenyl)but-2-enenitrile (10b)
Following general procedure (A) for the condensation be-
tween 3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile and p-
fluoroacetophenone, the crude residue was purified by col-
umn chromatography on silica gel (EtOAc-petroleum ether
2-8 v/v), and 10b was obtained as a white solid. Yield: 52%,
1
mp 143-144 °C H NMR (CDCl ): ꢁ 1.62 (s, 3H), 2.34 (s,
3
3H), 3.86 (s, 6H), 3.89 (s, 3H), 3.91 (s, 6H), 3.94 (s, 3H),

334 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
7.22 (t, J=9.2 Hz, 2H), 7.32 (s, 2H), 7.34 (s, 2H), 7.64 (m,
6H).
solved in dichloromethane (DCM) (15 mL), washed succes-
sively with an aqueous solution of HCl (0.1N, 5 mL), a satu-
rated solution of NaHCO3 (5 mL), water (5 mL), and brine (5
mL). The organic phase was dried and evaporated at reduced
pressure. The crude residue was purified by column chroma-
tography on silica gel (EtOAc-petroleum ether as eluent).
Synthesis of (2E/Z)-2-(3,4,5-trimethoxybenzoyl)-3-(4-
chlorophenyl)but-2-enenitrile (10c)
Following general procedure (A) for the condensation be-
tween 3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile and p-
chloroacetophenone, the crude residue was purified by col-
umn chromatography on silica gel (EtOAc-petroleum ether
Synthesis of (2-amino-4-phenylthiophen-3-yl)-(3,4,5-trim-
ethoxyphenyl)-methanone (5a)
2-8 v/v), and 10c was obtained as a yellow solid. Yield:
Following general procedure (B), the crude residue was
purified by column chromatography (EtOAc-petroleum ether
1
42%, mp 52-54 °C H NMR (CDCl ): ꢀ 2.33 (s, 3H), 2.63
3
(s, 3H), 3.87 (s, 6H), 3.91 (s, 3H), 3.93 (s, 6H), 3.96 (s, 3H),
7.08 (s, 2H), 7.12 (d, J=9.2 Hz, 2H), 7.22 (d, J=9.2 Hz, 2H),
7.26 (s, 2H), 7.49 (d, J=9.2 Hz, 2H), 7.52 (d, J=9.2 Hz, 2H).
3:7) and furnished 5a as a yellow solid. Yield: 55%, mp 154-
1
156 °C. H NMR (CDCl ): ꢀ 3.69 (s, 3H), 3.71 (s, 6H), 6.22
3
(bs, 2H), 6.59 (s, 2H), 7.02 (m, 6H).
Synthesis of (2E/Z)-2-(3,4,5-trimethoxybenzoyl)-3-(4-
methoxyphenyl)but-2-enenitrile (10 d)
Synthesis of (2-amino-4-(4-fluorophenyl)thiophen-3-yl)(3,
4,5-trimethoxyphenyl)methanone (5b)
Following general procedure (A) for condensation be-
tween 3-oxo-3-(3,4,5-trimethoxyphenyl)-propionitrile and p-
methoxyacetophenone, the crude residue was purified by
column chromatography on silica gel (EtOAc-petroleum
Following general procedure (B), the crude residue was
purified by column chromatography (EtOAc-petroleum ether
3:7) and furnished 5b as a yellow solid. Yield: 53%, mp 167-
170 °C. 1H NMR (CDCl3): ꢀ 3.76 (m, 6H), 3.78(s, 3H), 6.21
(s, 1H), 6.60 (s, 2H), 6.62 (bs, 2H), 6.78 (t, J=9.2 Hz, 2H),
7.04 (m, 2H).
ether 3-7 v/v), and 10d was obtained as a yellow oil. Yield:
1
49%. H NMR (CDCl ): ꢀ 2.30 (s, 3H), 2.62 (s, 3H), 3.84
3
(s, 6H), 3.87 (s, 3H), 3.89 (s, 3H), 3.93 (s, 6H), 3.94 (s, 3H),
3.96 (s, 3H), 7.06 (s, 2H), 7.10 (d, J=9.2 Hz, 2H), 7.20 (d,
J=9.2 Hz, 2H), 7.26 (s, 2H), 7.50 (d, J=9.2 Hz, 2H), 7.54 (d,
J=9.2 Hz, 2H).
Synthesis of (2-amino-4-(4-chlorophenyl)thiophen-3-yl)
(3,4,5-trimethoxyphenyl)methanone (5c)
Following general procedure (B), the crude residue was
purified by column chromatography (EtOAc-petroleum ether
2:8) and furnished 5c as a yellow solid. Yield: 47%, mp 163-
165 °C. 1H NMR (CDCl3): ꢀ 3.73 (m, 6H), 3.75 (s, 3H), 6.20
(s, 1H), 6.59 (s, 2H), 6.68 (bs, 2H), 7.02 (d, J=9.2 Hz, 2H),
7.09 (d, J=9.2 Hz, 2H).
Synthesis of (2E/Z)-2-(3,4-dimethoxybenzoyl)-3-phenyl-
but-2-enenitrile (10e)
Following general procedure (A) for the condensation be-
tween 3-(3,4-dimethoxyphenyl)-3-oxopropanenitrile and
acetophenone, the crude residue was purified by column
chromatography on silica gel (EtOAc-petroleum ether 2-8
Synthesis of (2-amino-4-(4-methoxyphenyl)thiophen-3-
yl)(3,4,5-trimethoxyphenyl)methanone (5d)
1
v/v), and 10e was obtained as a yellow oil (yield: 47%). H
NMR (CDCl ): ꢀ 2.30 (s, 3H), 2.64 (s, 3H), 3.87 (s, 3H),
3
Following general procedure (B), the crude residue was
purified by column chromatography (EtOAc-petroleum ether
3:7) and furnished 5d as a yellow solid. Yield: 44%, mp 138-
140 °C.
1H NMR (CDCl3): ꢀ 3.70 (m, 6H), 3.72 (s, 3H), 3,74 (s,
3H), 6.20 (s, 1H), 6.58 (s, 2H), 6.63 (bs, 2H), 6.85 (d, J=8.8
Hz, 2H), 7,20 (d, J=8.8 Hz, 2H).
3.91 (s, 3H), 3.97 (s, 3H), 3.98 (s, 3H), 6.81 (d, J=9.2 Hz,
1H), 6.94 (d, J=9.2 Hz, 1H), 7.18 (m, 4H), 7.24 (d, J=9.2 Hz,
1H), 7.28 (s, 1H), 7.36 (s, 1H), 7.48 (m, 3H), 7.57 (m, 3H),
7.74 (d, J=9.2 Hz, 1H).
Synthesis of (2E/Z)-2-(4-methoxybenzoyl)-3-phenylbut-2-
enenitrile (10f)
Following general procedure (A) for the condensation be-
tween 3-(4-methoxyphenyl)-3-oxopropanenitrile and aceto-
phenone, the crude residue was purified by column chroma-
tography on silica gel (EtOAc-petroleum ether 2-8 v/v), and
Synthesis of (2-amino-4-phenylthiophen-3-yl)(3, 4-dim-
ethoxyphenyl)methanone (5e)
Following general procedure (B), the crude residue was
purified by column chromatography on silica gel (EtOAc-
1
10f was obtained as a yellow oil (yield: 52%). H NMR
(CDCl ): ꢀ 2.30 (s, 3H), 2.63 (s, 3H), 3.83 (s, 3H), 3.90 (s,
petroleum ether 3-7 v/v) and afforded 5e as a yellow solid.
3
1
3H), 6.81 (d, J=9.2 Hz, 2H), 6.99 (d, J=9.2 Hz, 2H), 7.17 (m,
3H), 7.20 (m, 3H), 7.48 (m, 2H), 7.57 (m, 2H), 7.77 (d,
J=9.2 Hz, 2H), 8.00 (d, J=9.2 Hz, 2H).
Yield: 64%, mp 134-136 °C. H NMR (CDCl ): 3.74 (s,
3
3H), 3.76 (s, 3H), 6.21 (s, 1H), 6.36 (bs, 2H), 6.46 (d, J=8.4
Hz, 2H), 6.95 (s, 1H), 7.04 (m, 5H).
General Procedure (B) for the Synthesis of 2-amino-3-
aroyl-4-aryl Thiophenes
Synthesis of (2-amino-4-phenylthiophen-3-yl)(4-methoxy-
phenyl)methanone (5f)
Knoevenagel’s adduct (2 mmol) was dissolved in EtOH
(20 mL), and sulfur (71 mg, 2.2 mmol) and TEA (300 μL,
2.3 mmol) were added. The mixture was stirred for 2 h at
reflux and evaporated in vacuo, and the residue was dis-
Following general procedure (B), the crude residue was
purified by column chromatography on silica gel (EtOAc-
petroleum ether 3-7 v/v) and afforded 5f as a yellow solid.
1
Yield: 72%, mp 155-158 °C. H NMR (CDCl ): 3.67 (s,
3

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 335
3H), 6.21 (s, 1H), 6.34 (bs, 2H), 6.49 (d, J=8.4 Hz, 2H), 6.99
(m, 5H), 7.33 (d, J=9.2 Hz, 2H).
to the filtrate. The organic phase was subsequently washed a
saturated solution of NaHCO3 (20 mL), water (20 mL), and
brine (20 mL), dried (Na2SO4) and concentrated. The crude
product was purified by column chromatography on silica
gel (EtOAc-petroleum ether 2-8 as eluent) to afford 13 as a
yellow solid. Yield 68%, mp 174-176 °C. 1H NMR (CDCl3):
ꢀ 3.58 (s, 3H), 3.78 (s, 6H), 4.73 (s, 2H), 6.90 (s, 2H), 7.48
(s, 1H), 7.72 (m, 2H), 7.74 (m, 2H).
Synthesis of (2-amino-4-phenylthiophen-3-yl)(4-hydroxy-
3,5-dimethoxyphenyl)methanone (5g)
The thiophene derivative 5a (184 mg, 0.5 mmol) was
added to concentrated sulfuric acid (98%, 0.5 mL, 1
mmol/mL), and the resulting green mixture was stirred at
room temperature for 6 days. The mixture was slowly poured
over ice water and then neutralized with sodium carbonate
(pH=9). The mixture was extracted with EtOAc (2 x10 mL)
and the organic phase washed with water (5 mL) and brine (5
mL), dried over Na2SO4 and evaporated. The residue, puri-
fied by column chromatography on silica gel (EtOAc-
Synthesis of 2-[4-methyl-5-bromo-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (15)
To a solution of thiophene derivative (480 mg, 1.1 mmol)
in benzene (10 mL) was added benzoyl peroxide (24 mg, 0.1
mmol), and the mixture was heated under reflux. At reflux-
ing conditions, a mixture of NBS (196 mg, 1.1 mmol) and
benzoyl peroxide (24 mg, 0.1 mmol) was added, and the
reflux was continued for 6 h. The solvent was removed under
reduced pressure, and the residue, dissolved in EtOAc (15
mL), was washed successively with a saturated solution of
NaHCO3 (5 mL), water (5 mL), and brine (5 mL). The or-
ganic phase was dried (Na2SO4) and concentrated under vac-
uum to give a yellow powder. This residue, was suspended
with petroleum ether (20 mL), the mixture was stirred for 30
min, and filtered. The resulting residue 15 was used for the
next reaction without purification. Yellow solid, 78%, mp
petroleum ether 1-1), furnished 5g as a yellow solid. Yield:
1
68%, mp 125-126 °C. H NMR (CDCl ): ꢀ 3.73 (s, 6H),
3
5.59 (bs, 2H), 6.24 (s, 1H), 6.32 (s, 1H), 6.71 (s, 2H), 7.03
(m, 5H). MS (ESI): [M]+ 356.4.
Synthesis of (2-amino-4-methylthiophen-3-yl)(3,4,5-tri-
methoxyphenyl)methanone (11)
To an ice cooled (+ 4 °C) suspension of 3-oxo-3-(3,4,5-
trimethoxyphenyl)-propionitrile (940 mg, 4 mmol) and 2,5-
dimethyl-2,5-dihydroxy-1,4-dithiane (360 mg, 2 mmol) in
EtOH (10 mL) was added TEA (0.7 mL, 5 mmol). After stir-
ring for 2 h at 70 °C, the solvent was evaporated and the
residue diluted with DCM (20 mL). Mixture was washed
successively with an aqueous HCl solution (0.1 N, 5 mL),
water (2x5 mL), and brine (5 mL). The organic phase was
dried and evaporated. The crude product was purified by
silica gel column chromatography (petroleum ether-EtOAc
1
183-184 °C. H NMR (CDCl3) (400 MHZ): ꢀ 2.27 (s, 3H),
3.62 (s, 3H), 3.80 (s, 6H), 6.91 (s, 2H), 7.70 (m, 2H), 7.77
(m, 2H).
Synthesis of 2-[4-methyl-5-phenyl-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (16)
2:8) afforded 11 as a yellow powder (50% yield); mp 148-
A mixture of thiophene derivative (517 mg, 1 mmol), po-
tassium carbonate (207 mg, 1.5 mmol, 1.5 equiv.), phenylbo-
ronic acid (244 mg, 2 mmol, 2 equiv.) and tetrakis (triphen-
ylphosphine)palladium (85 mg, 0.06 mmol) in dry toluene
(20 mL) was stirred at 120 °C under nitrogen for 18 h,
cooled to ambient temperature and evaporated in vacuo. The
residue was dissolved with DCM (30 mL), and the resultant
solution was washed sequentially with an aqueous 5% Na-
HCO3 solution (10 mL), water (10 mL) and brine (10 mL).
The organic layer was dried, filtered and evaporated, and the
residue was purified by flash chromatography on silica gel
(EtOAc-petroleum ether 4:6). The product 16 was obtained
1
150 °C. H NMR (CDCl ): ꢀ 1.82 (s, 3H), 3.89 (m, 9H),
3
5.86 (s, 1H), 6.43 (bs, 2H), 6.77 (s, 2H). Anal.
(C15H17NO4S): C, H, N.
Synthesis of 2-[4-methyl-3-(3,4,5-trimethoxybenzoyl)-2-
thienyl]-1H-isoindole-1,3(2H)-dione (12)
To a suspension of thiophene (6 g., 19.5 mmol) in acetic
acid (200 mL) was added phthalic anhydride (3.5 g., 23.4
mmol). After stirring for 18 h at reflux, the solvent was
evaporated and the residue dissolved in EtOAc (100 mL).
The organic solution was washed with a saturated solution of
NaHCO3 (2x50 mL), water (50 mL), and brine (40 mL),
dried and concentrated. The crude product was purified by
column chromatography on silica gel (EtOAc-petroleum
ether 4-6 as eluent) to afford 12 as a yellow solid. Yield
1
as a yellow cream solid, yield: 87%, mp 162-164 °C. H
NMR (CDCl3): ꢀ 2.33 (s, 3H), 3.62 (s, 3H), 3.82 (s, 6H),
6.98 (s, 2H), 7.48 (m, 5H), 7.75 (m, 4H).
Synthesis of 2-[4-bromomethyl-5-phenyl-3-(3,4,5-trimeth-
oxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (17)
1
87%, mp 108-110 °C. H NMR (CDCl3) (400 MHZ): ꢀ 2.34
(s, 3H), 3.61 (s, 3H), 3.79 (s, 6H), 6.91 (s, 2H), 7.08 (s, 1H),
7.70 (m, 2H), 7.77 (m, 2H).
A solution of thiophene derivative (431 mg, 0.84 mmol)
in carbon tetrachloride (20 mL) was heated under reflux. At
refluxing conditions a mixture of NBS (178 mg, 1 mmol)
and benzoyl peroxide (20 mg, 0.08 mmol) was added, and
the reflux was continued for 5 h. The mixture was diluted
with DCM (20 mL), and the solution was washed with water
(10 mL) and brine (10 mL), dried (Na2SO4) and concentrated
to give a residue, which was purified by flash chromatogra-
phy on silica gel (EtOAc-petroleum ether 4:6). The product
17 was obtained as a yellow solid, yield: 96%, mp 194-196
Synthesis of 2-[4-bromomethyl-3-(3,4,5-trimethoxyben-
zoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (13)
To a suspension of thiophene derivative (656 mg, 1.5
mmol) in carbon tetrachloride (30 mL) was added benzoyl
peroxide (36 mg, 0.15 mmol), and the mixture was heated
under reflux: At refluxing conditions, a mixture of NBS (320
mg, 1.8 mmol) and benzoyl peroxide (36 mg, 0.15 mmol)
was added, and the reflux was continued for 5 h. The mixture
was filtered on a pad of celite, and DCM (30 mL) was added
1
°C. H NMR (CDCl3) (400 MHZ): ꢀ 3.81 (s, 3H), 3.82 (s,

336 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
6H), 4.81 (s, 2H), 6.98 (s, 2H), 7.52 (m, 3H), 7.62 (m, 2H),
7.72 (m, 4H).
Synthesis of 2-[5-phenyl-4-(4-fluorobenzyl)-3-(3,4,5-trime-
thoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (18f)
Following general procedure (C), the product 18f was ob-
tained as a yellow solid. Yield: 62%, mp 96-98 °C. 1H NMR
(CDCl3): ꢀ 3.58 (s, 3H), 3.73 (s, 6H), 4.21 (s, 2H), 6.65 (s,
2H), 6.75 (d, J=8.8 Hz, 1H), 6.79 (d, J=8.8 Hz, 1H), 7.02 (m,
2H), 7.45 (m, 3H), 7.55 (m, 2H), 7.71 (m, 4H).
General Procedure C (Suzuki Coupling) for the Synthesis
of Compounds 14a-d and 18e-h
A mixture of thiophene derivative 13 (206 mg, 0.4 mmol)
or 17 (237 mg, 0.4 mmol), potassium carbonate (85 mg, 0.6
mmol, 1.5 equiv.), the appropriate aryl boronic acid (0.8
mmol, 2 equiv.) and tetrakis (triphenylphosphine)palladium
(85 mg, 0.06 mmol) in dry toluene (10 mL) was stirred at
120 °C under nitrogen for 18 h, cooled to ambient tempera-
ture and evaporated in vacuo. The residue was dissolved
with EtOAc (30 mL), and the resulting solution was washed
sequentially with an aqueous 5% NaHCO3 solution (10 mL),
water (10 mL) and brine (10 mL). The organic layer was
dried, filtered and evaporated, and the residue was purified
by flash chromatography on silica gel (EtOAc-petroleum
ether 3:7).
Synthesis of 2-[5-phenyl-4-(4-chlorobenzyl)-3-(3,4,5-trime-
thoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (18g)
Following general procedure (C), the product 18g was
1
obtained as a yellow solid. Yield: 64%, mp 91-93 °C. H
NMR (CDCl3): ꢀ 3.59 (s, 3H), 3.73 (s, 6H), 4.20 (s, 2H),
6.65 (s, 2H), 6.91 (d, J=8.4 Hz, 2H), 7.01 (d, J=8.4 Hz, 2H),
7.43 (m, 3H), 7.53 (m, 2H), 7.72 (m, 4H).
Synthesis of 2-[5-phenyl-4-(4-methoxybenzyl)-3-(3,4,5-
trimethoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-
dione (18h)
Synthesis of 2-[4-benzyl-3-(3,4,5-trimethoxybenzoyl)-2-
thienyl]-1H-isoindole-1,3(2H)-dione (14a)
Following general procedure (C), the product 14a was
obtained as a yellow solid. Yield: 72%, mp 181-182 °C. H
Following the general procedure (C) the product 18h was
obtained as a white solid. Yield: 61%, mp 72-73 °C. H
1
1
NMR (CDCl3): ꢀ 3.70 (s, 6H), 3.72 (s, 3H), 4.10 (s, 2H),
6.76 (s, 2H), 6.97 (s, 1H), 7.17 (m, 3H), 7.21 (d, J=3,6 Hz,
2H), 7.67 (m, 2H), 7.74 (m, 2H).
NMR (CDCl3): ꢀ 3.57 (s, 3H), 3.60 (s, 3H), 3.64 (s, 3H),
3.73 (s, 3H), 4.18 (s, 2H), 6.57 (d, J=8.6 Hz, 2H), 6.64 (s,
2H), 6.88 (d, J=8.6 Hz, 2H), 7.44 (m, 3H), 7.57 (m, 2H),
7.74 (m, 4H).
Synthesis of 2-[4-(4-chlorobenzyl)-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (14b)
General Procedure (D) for the Synthesis of Compounds
19a-d
Following general procedure (C), the product 14b was
obtained as a yellow solid. Yield: 55%, mp 184-186 °C. H
1
To a stirred solution of compound 13 (258 mg, 0.5
mmol) in dry N,N-dimethylformamide (10 mL) was added
K2CO3 (104 mg, 0.75 mmol, 1.1 equiv.). The mixture was
cooled with a bath of ice/water, and then the appropriate
phenol (0.75 mmol, 1.5 equiv.) was added. The mixture was
then stirred at room temperature for 18 h, the solvent evapo-
rated in vacuo, and the residue diluted with DCM (10 mL)
and washed with water (5 mL) and then brine (5 mL). The
organic layer was dried and concentrated in vacuo to give a
residue which was purified by column chromatography on
silica gel (EtOAc-petroleum ether 3-7).
NMR (CDCl3): ꢀ 3.52 (s, 2H), 3.78 (s, 6H), 3.87 (s, 3H),
6.57 (d, J=8.4 Hz, 2H), 6.69 (s, 2H), 7.08 (d, J=8.4 Hz, 2H),
7.28 (s, 1H), 7.66 (m, 4H).
Synthesis of 2-[4-(4-methylbenzyl)-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (14c)
Following general procedure (C), the product 14c was
1
obtained as a yellow solid. Yield: 68%, mp 163-165 °C. H
NMR (CDCl3): ꢀ 2.25 (s, 3H), 3.49 (s, 2H), 3.78 (s, 6H),
3.88 (s, 3H), 6.58 (d, J=8.2 Hz, 2H), 6.78 (s, 2H), 6.96 (d,
J=8.2 Hz, 2H), 7.30 (s, 1H), 7.70 (m, 4H).
Synthesis of 2-[4-(phenoxymethyl)-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (19a)
Synthesis of 2-[4-(3-methoxybenzyl)-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (14d)
Following general procedure (C), the product 14d was
obtained as a yellow solid. Yield: 65%, mp 172-174 °C. H
Following general procedure (D), the product 19a was
obtained as a yellow solid. Yield: 70%, mp 183-185 °C. H
1
1
NMR (CDCl3): ꢀ 3.48 (s, 2H), 3.67 (s, 3H), 3.78 (s, 6H),
3.85 (s, 3H), 6.30 (d, J= 8.4 Hz, 1H), 6.60 (s, 2H), 6.96 (m,
2H), 7.10 (m, 1H), 7.28 (s, 1H), 7,85 (m, 4H).
NMR (CDCl3): ꢀ 3.75 (s, 3H), 3.77 (s, 6H), 5.18 (s, 2H),
6.85 (s, 2H), 7.05 (d, J=8.4 Hz, 2H), 7.32 (m, 3H), 7.52 (s,
1H), 7.73 (m, 4H).
Synthesis of 2-[4-benzyl-5-phenyl-3-(3,4,5-trimethoxy-
benzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (18e)
Synthesis of 2-[4-[(4-chlorophenoxy)methyl]-3-(3,4,5-trime-
thoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (19b)
Following general procedure (C), the product 18e was
obtained as a white solid. Yield: 64%, mp 112-114 °C. H
NMR (CDCl3): ꢀ 3.54 (s, 3H), 3.70 (s, 6H), 4.25 (s, 2H),
6.63 (s, 2H), 6.96 (m, 3H), 7.02 (m, 2H), 7.42 (m, 3H), 7.56
(m, 2H), 7.72 (m, 4H).
1
Following general procedure (D), the product 19b was
obtained as a yellow solid. Yield: 55%, mp 187-189 °C. H
NMR (CDCl3): ꢀ 3.73 (s, 3H), 3.76 (s, 6H), 5.19 (s, 2H),
6,08 (d, J=8.4 Hz, 2H), 6.85 (s, 2H), 7.22 (d, J=8.4 Hz, 2H),
7.53 (s, 1H), 7.73 (m, 4H).
1

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 337
Synthesis of 2-[4-[(4-methylphenoxy)methyl]-3-(3,4,5-trime-
thoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (19c)
NMR (CDCl3): ꢀ 3.57 (s, 3H), 3.69 (s, 6H), 3.71 (s, 3H),
4.44 (s, 2H), 6.22 (s, 1H), 6.30 (m, 2H), 6.80 (s, 2H), 7.02 (t,
J=8.6 Hz, 1H), 7.40 (s, 1H), 7.71 (m, 2H), 7.74 (m, 2H).
Following general procedure (D), the product 19c was
1
obtained as a yellow solid. Yield: 60%, mp 172-174 °C. H
General Procedure (F) for the Synthesis of Compounds
20a-d
NMR (DMSO-d6): ꢀ 2.19 (s, 3H), 3.35 (s, 3H), 3.37 (s, 6H),
5.11 (bs, 2H), 6.65 (s, 2H), 6.76 (d, J=8.6 Hz, 2H), 7.05 (d,
J=8.6 Hz, 2H), 7.85 (m, 4H), 8.51 (s, 1H).
To a stirred solution of compound 13 (258 mg, 0.5
mmol) in dry DCM (5 mL) was added K2CO3 (83 mg, 0.6
mol, 1.2 equiv.). The mixture was cooled with a bath of
ice/water, and then the appropriate N-substituted piperazine
(1.5 mmol, 3 equiv.) dissolved in DCM (1 mL) was slowly
added. The mixture was then stirred at room temperature for
one hour, diluted with DCM (10 mL), and washed with wa-
ter (5 mL) and brine (5 mL). The organic layer was dried and
concentrated in vacuo to give a residue that was purified by
column chromatography on silica gel using (EtOAc-
petroleum ether 3-7 v/v) as eluent.
Synthesis of 2-[4-[(3,5-dimethoxyphenoxy)methyl]-3-(3,4,
5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione
(19d)
Following general procedure (D), the product 19d was
1
obtained as a yellow solid. Yield: 64%, mp 178-180 °C. H
NMR (CDCl3): ꢀ 3.58 (s, 3H), 3.73 (s, 6H), 3.76 (s, 6H), 5.18
(s, 2H), 6.67 (s, 3H), 6.88 (s, 2H), 7.54 (s, 1H), 7.76 (m, 4H).
General Procedure (E) for the Synthesis of Compounds
19e-h
Synthesis of 2-[4-[(4-phenylpiperazin-1-yl)methyl]-3-(3,4,
5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione
(20a)
To a stirred solution of compound 13 (258 mg, 0.5
mmol) in dry DCM (10 mL) was added TEA (84 μL, 0.6
mmol, 1.2 equiv.). The mixture was cooled with a bath of
ice/water, and then the appropriate aniline derivative (0.6
mmol, 1.2 equiv.) was added. The mixture was then stirred at
room temperature for 5 h, diluted with DCM (10 mL) and
washed with water (10 mL) and then brine (10 mL). The
organic layer was dried and concentrated in vacuo to give a
residue that was purified by column chromatography on sil-
ica gel (EtOAc-petroleum ether 2-8).
Following general procedure (F), the product 20a was
1
obtained as a yellow solid. Yield: 96%, mp 188-190 °C. H
NMR (CDCl3): ꢀ 2.10 (t, J=4.8 Hz, 4H), 2.90 (m, J=4.8 Hz,
4H), 3.12 (s, 2H), 3.84 (s, 6H), 3.86 (s, 3H), 6.65 (s, 2H),
6.96 (m 2H), 7.12 (m, 3H), 7.25 (s, 1H), 7.60 (m, 4H).
Synthesis of 2-[4-{[4-(3-fluorophenyl)piperazin-1-yl]me-
thyl}-3-(3,4,5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-
1,3(2H)-dione (20b)
Synthesis of 2-[4-(anilinomethyl)-3-(3,4,5-trimethoxyben-
zoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione (19e)
Following general procedure (F), the product 20b was
obtained as a yellow solid. Yield: 84%, mp 176-179 °C. H
NMR (CDCl3): ꢀ 2.12 (t, J=4.8 Hz, 4H), 2.91 (t, J=4.8 Hz,
4H), 3.15 (s, 2H), 3.85 (s, 6H), 3.87 (s, 3H), 6.70 (s, 2H),
7.15 (m, 4H), 7.28 (s, 1H), 7.49 (m, 4H).
1
Following general procedure (E), the product 19e was
obtained as a yellow solid. Yield: 78%, mp 160-163 °C. H
NMR (CDCl3): ꢀ 3.48 (s, 3H), 3.62 (s, 6H), 4.40 (s, 2H),
6.68 (s, 2H), 6.72 (d, J=8.4 Hz, 2H), 7.00 (m, 3H), 7.28 (s,
1H), 7.64 (m, 4H).
1
Synthesis of 2-[4-{[4-(4-chlorophenyl)piperazin-1-yl]me-
thyl}-3-(3,4,5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-
1,3(2H)-dione (20c)
Synthesis of 2-[4-{[(3-fluorophenyl)amino]methyl}-3-(3,4,
5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione
(19f)
Following general procedure (F), the product 20c was ob-
1
tained as a yellow solid. Yield: 88%, mp 192-194 °C. H
Following general procedure (E), the product 19f was ob-
tained as a yellow solid. Yield: 62%, mp 165-167 °C. H
NMR (CDCl3): ꢀ 3.51 (s, 3H), 3.64 (s, 6H), 4.36 (s, 2H),
6.65 (s, 2H), 6.85 (m, 4H), 7.27 (s, 1H), 7.63 (m 4H).
1
NMR (CDCl3): ꢀ 2.11 (t, J=4.8 Hz, 4H), 2.93 (t, J=4.8 Hz,
4H), 3.13 (s, 2H), 3.85 (s, 6H), 3.88 (s, 3H), 6.42 (d, J=8.6
Hz, 2H), 6.66 (s, 2H), 7.01 (d, J=8.6 Hz, 2H), 7.23 (s, 1H),
7.44 (m, 4H).
Synthesis of 2-[4-{[(4-chlorophenyl)amino]methyl}-3-(3,4,
5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-dione
(19g)
Synthesis of 2-[4-{[4-(3-methoxyphenyl)piperazin-1-yl]
methyl}-3-(3,4,5-trimethoxybenzoyl)-2-thienyl]-1H-isoin-
dole-1,3(2H)-dione (20d)
Following general procedure (E), the product 19g was
Following general procedure (F), the product 20d was
1
obtained as a yellow solid. Yield: 60%, mp 168-170 °C. H
1
obtained as a yellow solid. Yield: 90%, mp 182-184 °C. H
NMR (CDCl3): ꢀ 3.64 (m, 3H), 3.66 (s, 6H), 4.36 (s, 2H),
6.40 (d, J=8.2 Hz, 2H), 6.69 (s, 2H), 6.96 (d, J=8.2 Hz, 2H),
7.29 (s, 1H), 7.65 (m 4H).
NMR (CDCl3): ꢀ 2.15 (t, J=4.8 Hz, 4H), 2.99 (t, J=4.8 Hz,
4H), 3.17 (s, 2H), 3.84 (s, 3H), 3.88 (s, 6H), 3.90 (s, 3H),
6.67 (s, 2H), 6.79 (d, J=8.4 Hz, 1H), 6.93 (m, 2H), 7.05 (t,
J=8.4 Hz, 1H), 7.25 (s, 1H), 7.87 (m, 4H).
Synthesis of 2-[4-{[(3-fluorophenyl)amino]methyl}-3-(3,4,
5-trimethoxybenzoyl)-2-thienyl]-1H-isoindole-1,3(2H)-
dione (19h)
General Procedure (G) for the Synthesis of Compounds
6a-h, 7a-h and 8a-d
Following general procedure (E), the product 19h was
obtained as a yellow solid. Yield: 66%, mp 167-168 °C. H
A stirred suspension of thiophene derivatives (0.5 mmol)
and 100% hydrazine monohydrate (1.2 eq, 0.6 mmol, 29 μL)
1

338 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
in abs. ethanol (10 ml) was refluxed for 2 h. After this time,
the solvent was evaporated, and the residue was suspended
in DCM(15 mL) and water (5 mL). The separated organic
phase, washed with brine (2 mL) and dried, was then con-
centrated under vacuum to yield a residue that was purified
by column chromatography on silica gel (EtOAc-petroleum
ether 3-7 as eluent).
Synthesis of (4-(4-chlorobenzyl)-2-amino-5-phenylthio-
phen-3-yl)(3,4,5-trimethoxyphenyl)methanone (6g)
Following general procedure (G), the product 6g was ob-
tained as a yellow solid. Yield: 67%, mp 78-80 °C. 1H NMR
(CDCl3): ꢀ 3.76 (s, 2H), 3.78 (s, 6H), 3.90 (s, 3H), 6.41 (bs,
2H), 6.50 (s, 2H), 6.52 (d, J=8.2 Hz, 2H), 7.02 (d, J=8.2 Hz,
2H), 7.34 (m, 5H). MS (ESI): [M]+ 493.3.
Synthesis of (2-amino-4-benzylthiophen-3-yl)(3,4,5-trime-
thoxyphenyl)methanone (6a)
Synthesis of (4-(4-methoxybenzyl)-2-amino-5-phenylthio-
phen-3-yl)(3,4,5-trimethoxyphenyl)methanone (6h)
Following general procedure (G), the product 6a was ob-
tained as a yellow solid. Yield: 83%, mp 115-116 °C. H
NMR (CDCl3): ꢀ 3.56 (s, 2H), 3.78 (s, 6H), 3.89 (s, 3H),
5.84 (s, 1H), 6.61 (bs, 2H), 6.77 (d, J=8.0 Hz, 2H), 7.12 (m,
3H). MS (ESI): [M]+ 383.1.
Following general procedure (G), the product 6h was ob-
tained as a yellow solid. Yield: 72%, mp 82-84 °C. 1H NMR
(CDCl3): ꢀ 3.72 (s, 6H), 3.74 (s, 3H), 3.78 (s, 2H), 3.91 (s,
3H), 6.33 (bs, 2H), 6.48 (d, J=8.2 Hz, 2H), 6.52 (s, 2H), 6.58
(d, J=8.2 Hz, 2H), 7.35 (m, 5H). MS (ESI): [M]+ 489.6.
1
Synthesis of (4-(4-chlorobenzyl)-2-aminothiophen-3-yl)(3,
4,5-trimethoxyphenyl)methanone (6b)
Synthesis of (2-amino-4-(phenoxymethyl)thiophen-3-yl)
(3,4,5-trimethoxyphenyl)methanone (7a)
Following general procedure (G), the product 6b was ob-
tained as a yellow solid. Yield: 77%, mp 179-181 °C. H
NMR (CDCl3): ꢀ 3.56 (s, 2H), 3.79 (s, 6H), 3.89 (s, 3H),
5.90 (bs, 2H), 6.57 (s, 2H), 6.66 (d, J=8.4 Hz, 2H), 7.08 (d,
J=8.4 Hz, 2H). MS (ESI): [M]+ 417.4.
Following general procedure (G), the product 7a was ob-
tained as a yellow solid. Yield: 80%, mp 88-89 °C. 1H NMR
(CDCl3): ꢀ 1.60 (bs, 2H), 3.77 (s, 3H), 3.80 (s, 6H), 4.48 (s,
2H), 6,36 (s, 1H), 6.61 (dd, J=8.8 and 1.0 Hz, 2H), 6.76 (s,
2H), 6.88 (t, J=7.6 Hz, 1H), 7.15 (t, J=7.6 Hz, 2H). MS
(ESI): [M]+ 399.5.
1
Synthesis of (4-(4-methylbenzyl)-2-aminothiophen-3-yl)
(3,4,5-trimethoxyphenyl)methanone (6c)
Synthesis
of
(4-((4-chlorophenoxy)methyl)-2-amino-
thiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (7b)
Following general procedure (G), the product 6c was ob-
1
Following general procedure (G), the product 7b was ob-
tained as a yellow solid. Yield: 87%, mp 118-120 °C. H
NMR (CDCl3): ꢀ 3.72 (m, 6H), 3.74 (s, 3H), 4.40 (s, 2H),
6.26 (s, 1H), 6.32 (bs, 2H), 6.47 (d, J=8.8 Hz, 2H), 6.69 (s,
2H), 7.04 (d, J=8.8 Hz, 2H). MS (ESI): [M]+ 433.5.
tained as a yellow solid. Yield: 70%, mp 168-170 °C. H
1
NMR (CDCl3): ꢀ 2.26 (s, 3H), 3.51 (s, 2H), 3.79 (s, 6H),
3.89 (s, 3H), 5.84 (bs, 2H), 6.62 (s, 2H), 6.66 (d, J=7.6 Hz,
2H), 6.96 (d, J=7.6 Hz, 2H). MS: (ESI): [M]+ 397.2.
Synthesis of (4-(3-methoxybenzyl)-2-aminothiophen-3-yl)
(3,4,5-trimethoxyphenyl)methanone (6d)
Synthesis of (4-((4-methylphenoxy)methyl)-2-amino-
thiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (7c)
Following general procedure (G), the product 6d was ob-
tained as a yellow solid. Yield: 77%, mp 92-94 °C. 1H NMR
(CDCl3): ꢀ 3.54 (s, 2H), 3.69 (s, 3H), 3.79 (s, 6H), 3.89 (s,
3H), 5.84 (bs, 2H), 6.31 (s, 1H), 6.40 (d, J=8.4 Hz, 1H), 6.63
(s, 2H), 6.64 (d, J=8.4 and 2.8 Hz, 2H), 7.06 (t, J=8.4 Hz,
1H). MS (ESI): [M]+ 413.2.
Following general procedure (G), the product 7c was ob-
tained as a yellow solid. Yield: 81%, mp 117-119 °C. H
NMR (CDCl3): ꢀ 2.23 (s, 3H), 3.80 (s, 6H), 3.82 (s, 3H),
3.96 (bs, 2H), 4.46 (s, 2H), 6.35 (s, 1H), 6.52 (d, J=8.6 Hz,
2H), 6.76 (s, 2H), 6.95 (d, J=8.6 Hz, 2H). MS (ESI): [M]+
413.6.
1
Synthesis of (2-amino-5-phenyl-4-benzylthiophen-3-yl)(3,
4,5-trimethoxyphenyl)methanone (6e)
Synthesis of (4-((3,5-dimethoxyphenoxy)methyl)-2-amino-
thiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (7d)
Following general procedure (G), the product 6e was ob-
tained as a yellow solid. Yield: 78%, mp 102-104 °C. H
NMR (CDCl3): ꢀ 3.75 (s, 6H), 3.78 (s, 2H), 3.89 (s, 3H),
6.37 (bs, 2H), 6.49 (s, 2H), 6.59 (t, J=8.2 Hz, 2H), 7.04 (m,
3H), 7.29 (m, 5H). MS (ESI): [M]+ 459.6.
1
Following general procedure (G), the product 7d was ob-
tained as a yellow solid. Yield: 75%, mp 75-77 °C. 1H NMR
(CDCl3): ꢀ 3.70 (s, 6H), 3.80 (s, 6H), 3.82 (s, 3H), 4.44 (s,
2H), 5.81 (d, J=2.2 Hz, 2H), 6.01 (t, J=2.2 Hz, 1H), 6.35 (s,
1H), 6.46 (bs, 2H), 6.76 (s, 2H). MS (ESI): [M]+ 459.3.
Synthesis of (4-(4-fluorobenzyl)-2-amino-5-phenylthio-
phen-3-yl)(3,4,5-trimethoxyphenyl)methanone (6f)
Synthesis of [2-amino-4-(anilinomethyl)-3-thienyl](3,4,5-
trimethoxyphenyl)methanone (7e)
Following general procedure (G), the product 6f was ob-
tained as a yellow solid. Yield: 71%, mp 89-90 °C. 1H NMR
(CDCl3): ꢀ 3.75 (s, 2H), 3.78 (s, 6H), 3.90 (s, 3H), 6.32 (bs,
2H), 6.52 (s, 2H), 6.54 (m, 2H), 6.76 (t, J=8.2 Hz, 2H), 7.34
(m, 5H). MS (ESI): [M]+ 477.6.
Following general procedure (G), the product 7e was ob-
tained as a yellow solid. Yield: 90%, mp 143-144 °C. H
NMR (CDCl3): ꢀ 3.80 (s, 6H), 3.82 (s, 3H), 3.84 (bs, H),
4.05 (bs, 2H), 6.00 (bs, 2H), 6.20 (s, 1H), 6.75 (s, 2H), 7.03
(m, 5H). MS (ESI): [M]+ 398.4.
1

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 339
Synthesis of (4-((3-fluorophenylamino)methyl)-2-amino-
thiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (7f)
(CDCl3): ꢀ 2.11 (t, J=4.8 Hz, 4H), 2.96 (t, J=4.8 Hz, 4H),
3.10 (s, 2H), 3.76 (s, 3H), 3.86 (s, 6H), 3.89 (s, 3H), 5.94
(bs, 2H), 6.16 (s, 1H), 6.37 (m, 2H), 6.40 (m, 1H), 6.86 (s,
2H), 7.19 (t, J=7.2 Hz, 1H). MS (ESI): [M]+ 497.6.
Following general procedure (G), the product 7f was ob-
1
tained as a yellow solid. Yield: 84%, mp 122-124 °C. H
NMR (CDCl3): ꢀ 3.80 (s, 6H), 3.83 (s, 3H), 3.86 (bs, 1H),
4.00 (bs, 2H), 6.08 (bs, 2H), 6.23 (s, 1H), 6.79 (s, 2H), 6.98
(m, 4H). MS (ESI): [M]+ 416.5.
2.3. Biological Assays
2.3.1. Cell Growth Inhibitory Activity
Synthesis of (4-((4-chlorophenylamino)methyl)-2-amino-
thiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (7g)
Murine leukemia L1210 and mammary carcinoma FM3A
and human T-lymphocyte Molt 4 and CEM cells were ob-
tained from the American Type Culture Collection (ATCC).
All the tested compounds were dissolved in DMSO at 1
mg/mL and stored as stock solutions at -20 °C. Further dilu-
tions were made in colture medium before addition to the
cells. In all experiments, the final concentration of DMSO
did not exceed 0.3% (v/v), a concentration non-toxic to the
cells. Cell lines were cultured in RPMI 1640 medium
(GIBCO) supplemented with 10% fetal calf serum (FCS)
(Flow, Irvine, U.K.), 2 mM L-glutamine (GIBCO), 10 mM
ꢀ-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL
streptomycin in a humidified atmosphere of 95% air and 5%
CO2 at 37 °C. Cells were maintained at a density of 1x106
cells/mL. To determine the effects of the studied compounds
on in vitro cell growth, the cells were suspended at 300,000-
500,000 cells/mL of culture medium, and 100 μL of a cell
suspension was added to 100 μL of an appropriate dilution of
the test compounds in wells of 96-well microtiter plates. Af-
ter incubation at 37 °C for 48 h, cell number was determined
using a ZF-Coulter Counter (Beckman Coulter Electronics,
Following general procedure (G), the product 7g was ob-
tained as a yellow solid. Yield: 77%, mp 162-164 °C. 1H NMR
(CDCl3): ꢀ 3.81 (m, 6H), 3.85 (s, 3H), 3.86 (bs, 1H), 4.14 (bs,
2H), 6.11 (bs, 2H), 6.22 (s, 1H), 6.32 (d, J=8.2 Hz, 2H), 6.78 (s,
2H), 7.00 (d, J=8.2 Hz, 2H). MS (ESI): [M]+ 432.6.
Synthesis of (4-((3-methoxyphenylamino)methyl)-2-amino-
thiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (7h)
Following general procedure (G), the product 7h was ob-
1
tained as a yellow solid. Yield: 85%, mp 134-136 °C. H
NMR (CDCl3): ꢀ 3.70 (s, 3H), 3.81 (s, 9H), 3.85 (s, 2H),
3.84 (bs, 1H), 5.93 (s, 1H), 6.01 (d, J=8.6 Hz, 1H), 6.20 (bs,
2H), 6.21 (m, 1H), 6.22 (s, 1H), 6.78 (s, 2H), 6.98 (t, J=8.6
Hz, 1H). MS (ESI): [M]+ 428.6.
Synthesis of {2-amino-4-[(4-phenylpiperazin-1-yl)me-
thyl]-3-thienyl}(3,4,5-trimethoxyphenyl)methanone (8a)
Following general procedure (G), the product 8a was ob-
tained as a yellow solid. Yield: 79%, mp 64-66 °C. 1H NMR
(CDCl3): ꢀ 2.12 (t, J=4.8 Hz, 4H), 2.97 (t, J=4.8 Hz, 4H),
3.11 (s, 2H), 3.85 (s, 6H), 3.89 (s, 3H), 5.97 (bs, 2H), 6.17
(s, 1H), 682 (d, J=7.6 Hz, 2H), 6.86 (s, 2H), 7.24 (m, 3H).
MS (ESI): [M]+ 467.3.
Hialeah, Fla., USA). Results were expressed as IC (dose
50
causing 50% inhibition of cell growth in treated cultures
relative to untreated controls). All experiments were repeated
at least twice. For each drug concentration, duplicate cultures
were used.
Synthesis of (2-amino-4-{[4-(3-fluorophenyl)piperazin-1-
yl]methyl}-3-thienyl)(3,4,5-trim ethoxyphenyl)methanone
(8b)
2.3.2. Effects on Tubulin Polymerization and Colchicine
Binding
To evaluate the effect of the compounds on tubulin as-
sembly in vitro, varying concentrations were preincubated
with 10 μM tubulin in 0.8 M monosodium glutamate buffer
at 30 °C and then cooled to 0 °C. After addition of 0.4 mM
GTP, the mixtures were transferred to 0 °C cuvettes in a re-
cording spectrophotometer and warmed to 30 °C, and the
assembly of tubulin was observed turbidimetrically. The IC50
was defined as the compound concentration that inhibited the
extent of assembly by 50% after 20 min at 30 °C. In the col-
chicine binding assay, tubulin at 1.0 μM was incubated with
5.0 μM [3H]colchicine and tested compounds at 1.0 μM for
10 min at 37 °C.
Following general procedure (G), the product 8b was ob-
tained as a yellow solid. Yield: 71%, mp 56-58 °C. 1H NMR
(CDCl3): ꢀ 2.09 (t, J=4.8 Hz, 4H), 2.09 (t, J=4.8 Hz, 4H),
3.10 (s, 2H), 3.86 (s, 6H), 3.89 (s, 3H), 5.94 (s, 2H), 6.16 (s,
1H), 6.46 (s, 1H), 6.50 (m, 1H), 6,59 (d, 1H), 6.86 (s, 2H),
7.19 (q, J=7.2 Hz, 1H). MS (ESI): [M]+ 485.3.
Synthesis of (2-amino-4-{[4-(4-chlorophenyl)piperazin-1-
yl]methyl}-3-thienyl)(3,4,5-trim ethoxyphenyl)methanone
(8c)
Following general procedure (G), the product 8c was ob-
1
tained as a yellow solid. Yield: 75%, mp 194-195 °C. H
NMR (CDCl3): ꢀ 2.10 (t, J=4.8 Hz, 4H), 2.96 (t, J=4.8 Hz,
4H), 3.15 (s, 2H), 3.84 (s, 6H), 3.88 (s, 3H), 5.90 (bs, 2H),
6.05 (s, 1H), 6.38 (d, J=8.6 Hz, 2H), 6.86 (s, 2H), 6.90 (d,
J=8.6 Hz, 2H). MS (ESI): [M]+ 502.4.
2.3.3. Cell Cycle Analysis
The effects of the most active compounds of the series on
cell cycle distribution were studied on K562 cells (a chronic
myeloblastic leukemia cell line) by flow cytometric analysis
after staining with propidium iodide [19]. K562 cells were
cultured in RPMI 1640 containing 10% FCS, 100 U/ml peni-
cillin, 100 μg/ml streptomycin (Gibco), and 2 mM L-
glutamine in a 5% CO2 atmosphere at 37 °C. Cells were
exposed 48 h to each compound used at a concentration cor-
responding to the IC50 determined after a 24 h incubation.
Synthesis of (2-amino-4-{[4-(3-methoxyphenyl)piperazin-
1-yl]methyl}-3-thienyl)(3,4,5-trim ethoxyphenyl)metha-
none (8d)
Following general procedure (G), the product 8d was ob-
tained as a yellow solid. Yield: 78%, mp 62-64 °C. 1H NMR

340 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
After treatment, the cells were washed once in ice-cold
phosphate-buffered saline (PBS) and resuspended at 1ꢀ106
per mL in a hypotonic fluorochrome solution containing
propidium iodide at 50 μg/mL in 0.1% sodium citrate plus
0.03% (v/v) nonidet P-40. After a 30 min incubation, the
fluorescence of each sample was analyzed as a single-
parameter frequency histogram by using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). The distribu-
tion of cells in the cell cycle was analyzed using MultiCycle
for Windows (Phoenix Flow Systems, San Diego, CA).
2.3.4. Statistical Analysis
The statistical significance of difference in between dif-
ferent treatments, was analyzed, when appropriate, using
one-way analysis of variance (ANOVA) and the Student-
Newman Keuls test. p values lower than 0.05 were consid-
ered statistically significant.
3. RESULTS AND DISCUSSION
Table 1 summarizes the growth inhibitory effects of de-
rivatives 5a-g, 6a-h, 7a-h and 8a-d against murine leukemia
Table 1. In Vitro Inhibitory Effects of Compounds 4a, 5a-f, 6a-h, 7a-h and 8a-d on Proliferation of L1210, FM3A Molt/4 and CEM
Cells
IC50 (μM)
Compound
L1210
FM3A/0
Molt4/C8
CEM/0
4a
5a
5b
5c
5d
5e
5f
0.012±0.001
10±0
0.096±0.039
14±3
0.030±0.001
7.5±0.8
61±12
0.029± 0.001
6.8±3.7
55±5
>100
>100
>100
>100
>100
>100
>100
>100
69±12
56±1
2.8±0.2
4.5±0.8
42 ± 19
42±3
5.0±1.1
9.1±0.7
85 ± 19
40±5
1.8±0.3
1.7±0.2
24 ± 14
22±3
1.6±0.4
2.2±0.1
42 ± 1
22±4
5g
6a
6b
6c
6d
6e
6f
10±0.1
2.1±0.1
45±0
12±1
7.0±1.9
1.4±0.6
7.9±0
7.9±1.0
1.3±0.4
12±3
2.0±0.6
38±7
1.6±0.2
8.7±0.3
1.9±0.7
8.9±0.9
2.4±0.1
39±1
1.7±0.3
8.6±0.1
1.5±0.3
8.3± 0.3
15±1
0.61±0.22
4.9±0.8
1.5±0.1
7.6±0.1
1.7±0.0
20±12
1.4±0.4
7.3±0.6
1.9±0.1
10±1
6g
6h
7a
7b
7c
7d
7e
7f
1.7±0.1
25±6
52±5
32±2
34±1
11±0
11±1
45±2
42±1
24±2
31±9
13±3
17±6
10±1
10±0
10±0
9.6±1.9
44±6
8.5±0.7
46±1
8.9±0.4
43±0
7g
7h
8a
8b
8c
8d
62±9
8.9±0
8.7±0.7
39±6
8.1±0.8
24±6
7.4±0.6
20±7
32±12
12±0
16±2
8.7±0.5
8.5±0.2
7.2±0.9
8.6±0.9
7.5±0.6
6.4±1.1
8.8±0.3
8.4±0.1
9.6±0.9
8.6±0.2
aIC50= compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SE from the dose-response curves of at least three independent
experiments.

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 341
(L1210), murine mammary carcinoma (FM3A) and human
T-lymphoblastoid (Molt/4 and CEM) cells, using 4a as refer-
ence compound. In general, the antiproliferative activities of
the compounds were greater against the Molt/4 and CEM
cells as compared with the two murine cell lines. In detail, it
is noteworthy that while 6g inhibits cell growth of all the
employed cell line to similar extent (differences of the IC50
values were not statistically significant), and the effect of 6e
was particularly interesting on Molt/4, the antiproliferative
activities of compounds 5e and 6c were more pronounced
against human Molt/4 and CEM as compared with murine
L1210 and FM3A cells (p<0.05). Therefore, different
mechanisms of action of these molecules are expected to
lead to cell growth inhibition. Of all tested compounds, the
4-benzyl-5-phenyl thiophene analogue 6e possessed the
highest potency and inhibited the growth of L1210, FM3A,
Molt/4 and CEM cancer cell lines with IC50’s of 1.6, 1.7,
0.61 and 1.4 μM, respectively. The two most potent com-
pounds of the series of 5-unsubstituted derivatives bear the
p-methyl benzyl (6c) and phenoxymethyl (7a) substitution at
the 4-position of 2-amino-3-(3’,4’,5’-trimethoxybenzoyl)
thiophene skeleton.
human lines and had no effect on activity with the murine
lines. The compounds with either a para-fluoro (6f) or para-
methoxy (6h) group were less active than both 6e and 6g.
In the series of 4-aryloxymethyl derivatives 7a-d, both
electron-withdrawing (para-chloro, 7b) or electron-releasing
(para-methyl and meta, meta’-dimethoxy for 7c and 7d)
groups were detrimental to antiproliferative activity com-
pared with the unsubstituted phenoxy derivative 7a.
Among the arylaminomethyl derivatives 7e-h, only mi-
nor differences in antiproliferative activity were observed
between meta-methoxy and meta-fluoro (7f and 7h, respec-
tively) and the unsubstituted 7e, while there was a 2-6-fold
reduction in potency with the meta-fluoro (7f) relative to the
para-chloro (7g) phenyl substituted and unsubstituted (7e)
compounds.
In the series of arylpiperazine derivatives 8a-d, while the
unsubstituted phenyl derivative 8a displayed modest antipro-
liferative activity, the presence of a substituent on the phenyl
ring was beneficial for the activity. The introduction of a
para-chloro group (8c) led from 3- to 4-fold increased activ-
ity with respect to the unsubstituted derivative 8a. Derivative
8c showed activities comparable to those observed with ana-
logues with ither a meta-fluoro (8b) or a meta-methoxy (8d)
substituent.
In the series of 2-amino-3-(3’,4’,5’-trimethoxybenzoyl)-
4-aryl thiophene derivatives 5a-d, the unsubstituted 4-phenyl
analogue 5a was the most active compound (p<0.01). The
aryl substituted derivatives 5b-d showed little inhibition of
cell proliferation, with IC50 values > 50 μM. The presence
either of electron-withdrawing (F and Cl for 5b and 5d, re-
spectively) or electron-releasing (OMe for 5d) groups caused
substantial loss of activity. Comparing 5e-f with 5a demon-
strated that the 3’,4’,5’-trimethoxybenzoyl substitution pre-
sent in 5a was not crucial for potent cell growth inhibition.
Its replacement with either a 3’,4’-dimethoxybenzoyl or a 4’-
methoxybenzoyl moiety (compounds 5e and 5f, respectively)
led to increased antiproliferative inhibition activity (p<0.01).
Comparing the biological activity of compound 5a with that
of 5g, in which the 4’-methoxy was replaced with a 4’-
hydroxyl, resulted in a 4- to 6-fold reduction un antiprolif-
erative activity. The increased lipophilicity of 5a vs. 5g may
facilitate the penetration of the compound into the tumor
cells, explaining the greater activity of 5a.
To investigate whether the antiproliferative activities of
these compounds were related to an interaction with tubulin,
we examined the effects of compounds 5e, 6c, 6e and 6g on
the polymerization of purified tubulin [18], in comparison
with the potent antimitotic compound CA-4 (Table 2).
Compound 6e did not greatly alter tubulin assembly at
concentrations as high as 40 μM. As noted above, this com-
pound had the greatest antiproliferative of all those whose
synthesis is presented here. Thus, 6e may exert its antiprolif-
erative effect by a mechanism other than inhibition of tubulin
polymerization. The order of inhibitory action on tubulin
assembly was CA4>6c>5e>6g>>6e, which was not consis-
tent with the results of the antiproliferative assays. The re-
maining three compounds did have relatively weak inhibi-
tory effects on tublin assembly, with the order of activity
CA4>>6c>5e>>6g, which was not consistent with the results
of the antiproliferative assays (of the three compounds with
antitubulin activity, 6g had the greatest antiproliferative ac-
tivity, although the differences were not great).
In the series of 4-benzyl derivatives 6a-d, the presence of
electron-withdrawing (chlorine) or electron-releasing
(methyl) substituents at the para-position of the phenyl ring,
derivatives 6b and 6c, respectively, significantly enhanced
antiproliferative activity relative to the unsubstituted 6a
(p<0.01), with the greatest activity being observed with 6c.
The introduction of a 3-methoxy group (compound 6d) led to
no change in activity, relative to 6a, against the murine tu-
mor cells, while activity against the human lines was in-
creased.
Table 2. Inhibition of Tubulin Polymerization by Com-
pounds CA-4, 5e, 6c, 6e and 6g
Tubulin assemblya
Compound
IC50±SD (μM)
In compounds 6e-6h, a phenyl substituent was added at
the 5-position of the thiophene ring. This change resulted in
increased antiproliferative activity, comparing 6e with 6a
and 6g with 6b (p<0.01). In fact, 6e had the greatest antipro-
liferative activity of any compound whose synthesis is de-
scribed in this paper, and 6g was nearly as active. In contrast
to the situation with 6b vs. 6a, where the para-chloro group
enhanced activity 3-4-fold, with the 5-phenyl compounds the
para-chloro group caused a small loss in activity against the
5e
6c
6e
6g
7.9±0.1
7.0±0.2
>40
25±2
CA-4
1.0±0.1
aInhibition of tubulin polymerization. Tubulin was at 10 μM.

342 Medicinal Chemistry, 2010, Vol. 6, No. 6
Romagnoli et al.
However, the tumor cell growth assay and the tubulin po-
lymerization assay differ in a number of further important
ways, such as the tubulin concentration present within the
cells and during microtubule formation outside a cell, the
presence of different types of microtubule-associated pro-
teins and the possible effects of regulatory proteins ex-
pressed in the cells but being absent in the polymerization
assay.
IC50 values obtained with K562 cells were determined after
48 h and are shown in Table 3.
The relative antiproliferative activity of the three com-
pounds with the K562 cells was similar to that obtained with
the other cell lines examined (Table 1). After this analysis,
the distribution of cells in different phases of the cell cycle
was determined after 48 h of growth in each compound at its
IC50. Fig. 1 (A and C) shows representative analyses ob-
tained on K562 cells cultured in the absence (Fig. 1A) or in
the presence of compound 5e (Fig. 1C). Treatment with 5e
caused an increase in the proportion of G2-M cells, and this
is quantified in Fig. 1C.
Because molecules exhibiting effects on tubulin assem-
bly should cause alteration of cell cycle parameters, with
preferential G2-M blockade, we examined cell cycle effects
of derivatives 5e, 6c and 6g using flow cytometric analysis
on K562 cells. The K562 cell line was used because of its
rapid proliferation and high sensitivity to standard anticancer
agents and in order to determine whether these compounds
had activity against human transformed cells [19, 20]. The
The data from the cell cycle analyses (averages from
three independent experiments) are shown in Table 3. These
data were used to generate the G2-M/(G1+S) ratios shown in
Fig. (1B), while in the Fig. (1D) data demonstrate the
Table 3. Effects of 5e, 6c and 6g on Cell Proliferation and Cell Cycle of K562 Cells
Cell cycleb
Cell proliferation IC50 ± SD
% ± SD (μM
Treatment
(μM)a
G1
S
G2-M
-
n.a.c
40.2 ± 3.3c
38.5 ± 4.2
38.1 ± 0.5
38.1 ± 0.5
40.9 ± 0.5
33.8 ± 1.0
38.0 ± 0.6
36.4 ± 1.5
18.8 ± 3.8
27.7 ± 3.2
23.9 ± 0.9
27.8 ± 3.6
5e
6c
6g
0.80 ± 0.12c
0.70 ± 0.06
0.31 ± 0.04
aData represent the average ± S.D. of three independent experiments.
bCell cycle distribution was analyzed on K562 cells treated for 48 h with IC50 concentrations of 5e, 6c or 6g.
cn.a. = not applicable.
Fig. (1). A,C. Flow cytometric analysis of the cell cycle distribution of untreated K562 cells (A) and K562 cells treated for 48 h with a con-
centration of compound 5e causing 50% inhibition of cells growth (C). B. Effects of compounds 5e, 6c and 6g on the ratios G2-M/(G1+S)
obtained after flow cytometric analysis and experimental conditions described in the text. Effects of compounds 5e, 6c and 6g on the propor-
tion of sub-G1 cells. Data of panels B and D represent the average ± S.D. of three independent experiments.

Synthesis and Cellular Pharmacology Studies
Medicinal Chemistry, 2010, Vol. 6, No. 6 343
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marked increase in sub-G1 cells (indicating activation of
apoptotic pathways) following treatment of the cells with 5e,
6c or 6g. The increase both in G2-M cells and in apoptotic
cells is characteristic of cellular response to antitubulin
agents [15].
4. CONCLUSIONS
The synthesis and biological evaluation of a new class of
synthetic compounds based on the 2-(3’,4’,5’-trimethoxy-
benzoyl)-3-aminothiophene skeleton were described here.
The biological evaluation included extensive antiprolifera-
tive studies with four cancer cell lines and examination of
some of the most active compounds on tubulin assembly and
disruption of the cell cycle. In the latter studies, we observed
accumulation of G2-M and apoptotic cells, effects expected
for compounds that interact with tubulin. The most active of
the new compounds (6e) against cell growth, however, did
not affect tubulin assembly and probably has a different in-
tracellular target.
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SAR aspects of our study of antiproliferative effects of
the substituents at the 4-.position of the thiophene ring in-
cluded the introduction of different substituents (aryl, benzyl,
aryloxymethyl, arylaminomethyl and arylpiperazino methyl).
Generally, the human Molt4 and CEM cells proved to be
more sensitive than the murine cells to the antiproliferative
of the tested compounds. The structures of the 4-position
substituents on the thiophene ring had highly variable and
generally unpredictable effects on the antiproliferative activ-
ity of the compounds. Notably, a phenyl group at the 5-
position of the thiophene ring (compounds 6e-h) had a posi-
tive effect on the activity of these compounds when com-
pared with the unsubstituted derivatives 6a-d.
[10]
[11]
[12]
[13]
[14]
The strong antiproliferative activity of 4-methylbenzyl
thiophene 6c, along with the 4-benzyl and 4-p-chlorobenzyl-
5-phenyl thiophene derivatives 6e and 6g justify further ex-
ploration of their potential antitumor activity and the prepa-
ration of additional analogues with the goals of further eluci-
dating important SAR features and identifying other active
congeners. Further studies are underway to clarify additional
details of the molecular mechanism of action of these com-
pounds and their ability to inhibit the growth of additional
cancer cells.
[15]
[16]
[17]
ACKNOWLEDGMENTS
RG is supported by grants from the AIRC and the “Fon-
dazione Cassa di Risparmio di Padova e Rovigo”. The
authors would like to thank Dr. Alberto Casolari for the
technical assistance
[18]
[19]
[20]
Lampronti, I.; Bianchi, N.; Borgatti, M.; Fibach, E.; Prus, E.; Gam-
bari, R. Accumulation of gamma-globin mRNA in human erythroid
cells treated with angelicin. Eur. J. Haematol., 2003; 71, 189-195.
Khan, M. T., Lampronti, I.; Martello, D.; Bianchi, N.; Jabbar, S.;
Choudhuri, M.S.; Datta, B. K.; Gambari, R. Identification of pyro-
gallol as an antiproliferative compound present in extracts from the
medicinal plant Emblica officinalis: effects on in vitro cell growth
of human tumor cell lines. Int. J. Oncol., 2002; 21, 187-192.
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Received: August 19, 2010
Revised: September 29, 2010
Accepted: October 03, 2010