Synthesis and biological evaluation of 2-substituted-4-(3′,4′, 5′-trimethoxyphenyl)-5-aryl thiazoles as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2012.10.001

Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of potency being NHCH₃ > Me ? N(CH₃)₂. The N-methylamino substituent significantly improved antiproliferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the C2-position from N-methylamino to N,N-dimethylamino caused a 1-2 log decrease in activity. The 2-N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative agents, with IC₅₀ values from low micromolar to single digit nanomolar, and, in addition, they are also active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was probably caused by the compounds binding to the colchicines site of tubulin polymerization and disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.

Full text of DOI:10.1016/j.bmc.2012.10.001

Bioorganic & Medicinal Chemistry 20 (2012) 7083–7094
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 2-substituted-4-(30,40,50-trimethoxy-
phenyl)-5-aryl thiazoles as anticancer agents
Romeo Romagnoli a, , Pier Giovanni Baraldi a, , Maria Kimatrai Salvador a, M. Encarnacion Camacho a,
⇑
⇑
Delia Preti a, Mojgan Aghazadeh Tabrizi a, Marcella Bassetto b, Andrea Brancale b, Ernest Hamel c,
Roberta Bortolozzi d, Giuseppe Basso d, Giampietro Viola d,
⇑
a Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19, Università di Ferrara, 44121 Ferrara, Italy
b School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
c Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory
for Cancer Research, National Institutes of Health, Frederick, MD 21702, USA
d Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2012
Revised 28 September 2012
Accepted 2 October 2012
Available online 12 October 2012
Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable
attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for
the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of
the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on
antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the
thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of
potency being NHCH3 > Me ꢀ N(CH3)2. The N-methylamino substituent significantly improved antipro-
liferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the
C2-position from N-methylamino to N,N-dimethylamino caused a 1–2 log decrease in activity. The 2-
N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative
agents, with IC50 values from low micromolar to single digit nanomolar, and, in addition, they are also
active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was
probably caused by the compounds binding to the colchicines site of tubulin polymerization and
disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through
the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.
Keywords:
Tubulin
Thiazole
Combretastatin-A4
Colchicine site
Apoptosis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
inhibits cell growth at nanomolar concentrations, exhibiting inhib-
itory effects even on multidrug resistant cancer cell lines.6 The
Microtubules are key components of the cytoskeleton and are
involved in a wide range of critical cellular functions, such as cell
division, where they are responsible for mitotic spindle formation
and proper chromosomal separation.1 Antimitotic agents are one
of the main class of cytotoxic drugs for cancer treatment, and tubu-
lin represents a known target for numerous small natural and
synthetic molecules that inhibit the formation of the mitotic spin-
dle.2,3 Combretastatin A-4 (CA-4, 1a; Chart 1) isolated from the
bark of the South African tree Combretum caffrum,4 is one of the
well-known natural molecules that inhibits the spindle formation
through its interaction with tubulin at the colchicine site.5 CA-4
disodium phosphate prodrug of CA-4, named CA-4P (1b),7 has been
prepared as a water-soluble derivative, and there have been prom-
ising results with 1b as a tumor vascular disrupting agent in phase
II clinical trials.8
Structure–activity relationship (SAR) studies of CA-4 have
underlined that the presence of the 3,4,5-trimethoxy substituted
A-ring and the 4-methoxy substituted B-ring separated by a dou-
ble-bond with cis-configuration are fundamental for optimal anti-
proliferative activity.9 It has also been reported that 3-hydroxy
group on the B-ring is not necessary for potent activity.10 The activ-
ity of CA-4 is hampered by isomerization of the active cis-stibene
configuration into the corresponding inactive trans analogue.
Replacement of the olefinic bond with a five-membered heterocy-
clic ring permitted retention of the correct geometric orientation of
the two phenyl rings of CA-4, placing them at an appropriate
distance for efficient interaction with the colchicines site of
tubulin.11
⇑
Corresponding authors. Tel.: +39 0 532455303; fax: +39 0 532455953 (R.R.);
tel.: +39 0 532455293; fax: +39 0 532455953 (P.G.B.); tel.: +39 0 498211451;
fax: +39 0 498211462 (G.V.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi),
giampietro.viola1@unipd.it (G. Viola).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.001

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R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
MeO
MeO
OMe
MeO
MeO
N
S
A
B
NH2
R
OMe
OR
2a-e
OMe
2a, R=naphth-2-yl
2b, R=4'-Me-C6H4
2c, R=4'-CF3-C6H4
2d, R=4'-OMe-3'-F-C6H3
2e, R=4'-OEt-C6H4
R=H, Combretastatin A-4 (CA-4), 1a
R=PO3Na2, CA-4P, 1b
3a, R1=NHCH3, R2=naphth-2-yl
3b, R1=NHCH3, R2=4'-Me-C6H4
3c, R1=NHCH3, R2=4'-CF3-C6H4
3d, R1=NHCH3, R2=4'-OMe-3'-F-C6H3
3e, R1=NHCH3, R2=4'-OEt-C6H4
3f, R1=N(CH3)2, R2=naphth-2-yl
3g, R1=N(CH3)2, R2=4'-Me-C6H4
3h, R1=N(CH3)2, R2=4'-CF3-C6H4
3i, R1=N(CH3)2, R2=4'-OMe-3'-F-C6H3
3j, R1=N(CH3)2, R2=4'-OEt-C6H4
3k, R1=CH3, R2=naphth-2-yl
MeO
MeO
OMe
N
S
R1
R2
3l, R1=CH3, R2=4'-Me-C6H4
3m, R1=CH3, R2=4'-CF3-C6H4
3n, R1=CH3, R2=4'-OMe-3'-F-C6H3
3o, R1=CH3, R2=4'-OEt-C6H4
Chart 1. Lead structures of tubulin polymerization inhibitors
In our previous study, we reported the biological evaluation of a
ate 5 with piperidine.13 Position 5 of C2-substituted thiazoles
6b–d was then brominated using N-bromosuccinimide in CHCl3,
to yield the 5-bromothiazole derivatives 7a–c. These molecules
were subjected to Suzuki–Miyaura coupling conditions in the
presence of various commercially available arylboronic acids,
giving rise to the corresponding 5-aryl thiazole derivatives 3f–
o and 8a–e. These latter compounds were transformed by ethan-
olysis into the final products 3a–e.
series of 2-amino-4,5-diarylthiazoles with general structure 2,
with the 2-aminothiazole moiety retaining the cis-olefin configura-
tion of CA-4, with the 30,40,50-trimethoxyphenyl ring of CA-4 at the
C4-position of the thiazole ring.12 On the basis of in vitro antipro-
liferative activity data, the best substituents for the C5-position of
the thiazole ring, corresponding to the B-ring of CA-4, were a naphth-
2-yl (2a) or a phenyl substituted at its para-position with a Me
(2b), CF3 (2c) MeO (2d) and EtO (2e) moiety.
Taking into consideration these results, in the present study we
have synthesized a new series of analogues with general structure
3, all of which retain at the C4- and C5-positions of the thiazole
ring the same substituents as derivatives 2a–e, examining the ef-
fect on biological activity derived by replacement of the amino
group at the C2-position of the thiazole nucleus with different moi-
eties, such as N-methylamine (3a–e), N,N-dimethylamine (3f–j)
and methyl (3k–o).
3. Biological results and discussion
3.1. In vitro antiproliferative activities
The series of 2-substituted-4-(30,40,50-trimethoxyphenyl)-
5-arylthiazoles 3a–o were evaluated for their inhibition of the
growth of a panel of six different human cancer cell lines in com-
parison with the 2-aminothiazole analogues 2a–e and the refer-
ence compound CA-4 (1a) as positive controls. From the
antiproliferative data reported in Table 1, the potency of the new
synthesized molecules 3a–o was found to be highly dependent
on the substituent at the 2-position of thiazole ring, and, in addi-
tion, some interesting trends were noted. The substituent at the
2-position of the thiazole ring clearly affected inhibitory effects
on cell growth, with decreasing activity generally observed as fol-
lows: NH2 > NHCH3 > CH3 ꢀ N(CH3)2.
2. Chemistry
2-Substituted-4-(30,40,50-trimethoxyphenyl)-5-aryl thiazole
derivatives with general structure 3 were prepared following
the reaction sequence shown in Scheme 1. 4-Substituted thia-
zoles 6a and 6c were formed by condensation of 2-bromo-
1-(30,40,50-trimethoxyphenyl)ethanone 4 with N-methylthiourea
or thioacetamide, respectively, in refluxing ethanol. The former
compound 6a was transformed into the corresponding N-acetyl
analogue 6d by treatment with a refluxing mixture of acetic
anhydride and sodium acetate. The 2-N,N-dimethylamino
thiazole 6b were prepared by ‘one-pot’ cyclization in refluxing
With the exception of 40-trifluoromethylphenyl derivative 3c,
all N-methylamino derivatives 3a–e had significant antiprolifera-
tive activity against all tested human cancer cell lines, some of
0
them even had IC50 s in the low to mid nanomolar range. Com-
pound 3e, bearing 40-ethoxyphenyl and N-methylamino moieties
at the 2- and 5-positions, respectively, of thiazole ring displayed
the greatest antiproliferative activity among the new synthesized
compounds 3a–o, with IC50 values ranging from 1.7 to 38 nM
ethanol of N,N-dimethylcyanamide with the anion of
a-mercapto
3,4,5-trimethoxyacetophenone, generated in situ by treating
O-ethyl-S-[2-oxo-2-(3,4,5-trimethoxyphenyl)-ethyl] dithiocarbon-

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
7085
MeO
MeO
OMe
MeO
MeO
MeO
MeO
MeO
MeO
O
O
a
b
Br
S
S
N
S
5
4
OEt
6a-c
R1
a, R1=NHCH3
b, R1=N(CH3)2
c, R1=Me
c
d. R1=N(CH3)COCH3
d
MeO
MeO
OMe
a. R1=N(CH3)COCH3
b, R1=N(CH3)2
c, R1=Me
N
R1
Br
S
7a-c
e
MeO
OMe
MeO
N
S
8a-e and 3f-o
R1
R2
3f, R1=N(CH3)2, R2=naphth-2-yl
3g, R1=N(CH3)2, R2=4'-Me-C6H4
3h, R1=N(CH3)2, R2=4'-CF3-C6H4
3i, R1=N(CH3)2, R2=4'-OMe-3'-F-C6H3
3j, R1=N(CH3)2, R2=4'-OEt-C6H4
3k, R1=CH3, R2=naphth-2-yl
8a, R1=N(CH3)COCH3, R2=naphth-2-yl
3a, R1=NHCH3, R2=naphth-2-yl
8b, R1=N(CH3)COCH3, R2=4'-Me-C6H4
3b, R1=NHCH3, R2=4'-Me-C6H4
8c, R1=N(CH3)COCH3, R2=4'-CF3-C6H4
3c, R1=NHCH3, R2=4'-CF3-C6H4
8d, R1=N(CH3)COCH3, R2=4'-OMe-3'-F-C6H3
3d, R1=NHCH3, R2=4'-OMe-3'-F-C6H3
8e, R1=N(CH3)COCH3, R2=4'-OEt-C6H4
3e, R1=NHCH3, R2=4'-OEt-C6H4
f
f
f
f
3l, R1=CH3, R2=4'-Me-C6H4
3m, R1=CH3, R2=4'-CF3-C6H4
3n, R1=CH3, R2=4'-OMe-3'-F-C6H3
3o, R1=CH3, R2=4'-OEt-C6H4
f
Scheme 1. Reagents and conditions: (a) N-Methylthiourea or thioacetamide for 6a and 6c, respectively, EtOH, reflux; (b) (CH3)2NCN, piperidine, EtOH, reflux; (c) AcONa,
Ac2O, reflux; (d) NBS, CHCl3, rt for 6b–d; (e) PdCl2(DPPF), ArB(OH)2, CsF, 1,4-dioxane and 65 °C for 7a and 7c or toluene and 75 °C for 7b; (f) 1 N NaOH, EtOH, reflux.
against the six cell lines. Derivative 3e was 5-, 15- and 1000-fold
more potent than CA-4 against A549, MCF-7 and HT-29 cells, but
it was 2- to 4-fold less potent than CA-4 against the other three
lines. However, compound 3e was one to two orders of magnitude
less active than its 2-aminothiazole counterpart 2e in four of the
six cancer cell lines, the exceptions being the MCF-7 and HT-29
cells, in which 3e was 2- and 10-fold more potent than 2e, respec-
tively. In human colon adenocarcinoma HT-29 cells, compound 3e
was 4- to 70-fold more potent than derivatives 2a–e.
Comparing compounds with the same substituent at the C5-
position of the thiazole ring, replacement of the 2-amino group
of thiazole derivatives 2a–e with a N-methylamino group (com-
pounds 3a–e) decreased antiproliferative activity against four of
the six cell lines, while with MCF-7 and HT-29 cells there were
variable effects. With the exception of 40-trifluoromethylphenyl
derivative 3c, the MCF-7 breast cancer cell line was more sensitive
toward N-methylamino analogues 3a–b and 3d–e than the
corresponding amino counterparts 2a–b and 2d–e, with 3b being
the most potent of all the newly synthesized derivatives 3a–o in
this cell line. Moreover, 3b was almost 28-fold more potent than
2b against the MCF-7 cells. In HT-29 cells, the 40-methoxyphenyl
(3d) and 40-ethoxyphenyl (3e) derivatives were 2- and 10-fold
more potent than their 2-amino counterparts 2d and 2e.
Comparing compounds 3k–o, which shared a common methyl
moiety at the thiazole C2-position, the napht-2-yl derivative 3k
had the highest antiproliferative activities in five of the six human
cancer cell lines, the exception being the HT-29 cells, in which 3k
was less active than 3m and 3o. All the N,N-dimethylamino deriv-
atives 3f–j showed lower antiproliferative activities than did their
N-methylamino analogues 3a–e, and 3f–j had relatively little activ-
ity against all the cell lines examined.
3.2. Effect of compounds 3e and 3k on normal human cells
To obtain more insights into the cytotoxic potential of test com-
pounds for normal human cells, the most active compounds were
assayed in vitro against peripheral blood lymphocytes (PBL) from
healthy donors as previously reported.14 Compounds 3e and 3k,
proved moderately cytotoxic having
a GI50 of 443 29 and
1272 307 nM in resting PBL, respectively, whereas in PHA-
stimulated PBL we found a GI50 of 120 24 (3e) and 285 57 nM
(3k) suggesting that this derivatives acts preferentially on prolifer-

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R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
Table 1
In vitro cell growth inhibitory effects of compounds 2a–e, 3a–o and CA-4 (1)
Compd
IC50 a(nM)
HeLa
A549
HL-60
Jurkat
MCF-7
HT-29
2ab
2bb
2cb
2db
2eb
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
1.6 0.6
0.73 0.2
3.2 0.4
2.3 0.2
0.03 0.005
128 49
3.8 0.6
42.8 5.7
357 65
20.2 1.7
0.09 0.01
151 36
125 22
4565 536
138 26
38.6 7.3
2900 320
3960 465
>10,000
>10,000
>10,000
119 22
1950 156
4287 258
2563 421
3321 352
180 50
2.7 0.3
3.3 0.9
3.9 0.6
8.5 1.2
2.1 0.4
51.2 9.2
279.5 69.8
0.4 0.1
23.9 3.9
12.4 5.1
221 50.2
11.7 3.9
38.2 11.4
38.2 6.1
423 107
1235 149
6.3 1.0
3.9 1.1
5.1 1.1
3.2 1.2
0.9 0.3
143 67
18.1 5.2
3166 188
27.3 7.2
1.8 0.8
2568 834
3479 359
>10,000
1893 424
303 26
205 37
1512 45
>10,000
60.9 8.4
44.0 6.3
21.1 4.0
10.1 4.1
7285 851
37.6 14.1
24.5 14.2
2385 574
>10,000
>10,000
8362 1291
>10,000
33.4 8.2
1743 374
>10,000
0.14 0.05
51.1 13
19.0 8.2
3014 769
34.3 7.2
1.7 0.8
836 135
6656 1762
>10,000
7599 1262
4150 1242
48.6 11.1
517 192
7109 1677
1755 838
1744 656
69.1 13
3401 292
70.1 12.3
13.2 6.3
1404 222
4141 165
>10,000
3371 366
3264 937
82.1 4.0
1100 58
3826 311
500 25.2
1866 145
3.2 0.4
729 108
3916 1470
>10,000
497 72
6027 190
702 142
722 235
396 164
1417 250
487 67
3k
3l
3m
3n
3o
CA-4
3427 80
253 19
606 12
42.3 8.2
370 100
4
1
1
0.2
5
0.6
3100 100
a
IC50 = 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.
b
Data taken from Ref. 12.
ating cells. Nevertheless these results pointed out also that com-
pounds 3e and 3k are less cytotoxic respect to the lymphoblastic
cell line Jurkat (see Table 1).
ity in all three of the drug resistant cell lines. The activity in the
two P-glycoprotein overexpressing cell lines demonstrated that
these derivatives are not substrates for this important drug pump.
3.3. Effect of compounds 3k, 3d and 3e on multidrug resistant
cells
3.4. Inhibition of tubulin polymerization and colchicine binding
To investigate whether the antiproliferative activities of com-
pounds 3a–b, 3d–k and 3o derived from an interaction with tubu-
lin, these agents were evaluated for their inhibition of tubulin
polymerization and for effects on the binding of [3H]colchicine to
tubulin.18,19 For comparison, 2-amino thiazoles 2a–b and 2d–e,
as well as CA-4, were examined in contemporaneous experiments.
To investigate whether these derivatives are substrates of drug
efflux pumps, some of the most active compounds (3d–e, 3k) were
tested against a panel of drug resistant cell lines that either over-
express P-glycoprotein (LovoDoxo and CemVbl100 15,16
or are associ-
)
ated with tubulin gene mutations (A549-T12)17 that result in
modified tubulin with impaired polymerization properties. As
shown in Table 2, the tested compounds exhibited cytotoxic activ-
Data for inactive compounds in the assembly assay (IC50 >20 lM)
are not shown in Table 3. All the N,N-dimethylamino derivatives
3f–j did not inhibit tubulin assembly at a concentration as high
as 20
l
M. Compound 3e was found to be the most active derivative
Table 2
in the in vitro tubulin polymerization assay (IC50, 0.89
in agreement with 3e being the compound with the greatest
l
M). This is
In vitro cell growth inhibitory effects of compound 3d–e, 3k on drug resistant cell
lines
antiproliferative activity. Compounds 3a–b, 3d, 3k and 3o had
a
Compd
IC50 (nM)
Resistance ratiob
LoVo
LoVoDoxo
3d
3e
3k
190 70
31 4.5
72 16
152 14
14.5 1.2
150 25
0.8
0.5
2.1
109.6
Table 3
Inhibition of tubulin polymerization and colchicine binding by compounds 2a–b,
2d–e, 3a–b, 3d–e, 3k, 3o and CA-4
Doxorubicinc
120 30
13150 210
b
Compd
Tubulin assemblya IC50
(
l
M)
Colchicine binding (%)
CEM
CEMVbl100
15 4.6
1.2 0.2
88 12
Resistance ratiob
1.5
2.9
1.4
256.2
Resistance ratiob
0.9
0.5
3d
3e
3k
10 3.5
0.8 0.1
61 18
0.8 0.1
2a
2b
2d
2e
3a
3b
3d
3e
0.74 0.01
0.44 0.01
1.1 0.07
0.61 0.05
0.96 0.01
1.2 0.07
1.3 0.07
0.89 0.06
1.1 0.07
1.2 0.07
1.2 0.07
94 0.49
88 0.71
79 0.21
95 0.71
78 2.1
Vinblastine
205 46
A549
A549-T12
125 23
21.5 9.0
121 36
3d
3e
138 26
38.6 7.3
119 22
7.2 0.1
68 1.4
61 0.28
83 0.28
66 0.21
51 2.8
3k
1.0
10.4
Taxolc
75.2 12.5
3k
3o
CA-4 (1)
a
IC50 = 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.
98 0.42
a
Inhibition of tubulin polymerization. Tubulin was at 10
l
M.
Inhibition of [3H]colchicine binding. Tubulin, colchicine and tested compound
M, respectively. Data are expressed as the mean SE of two
independent experiments.
b
b
The values express the ratio between the IC50’s determined in resistant and
non-resistant cell lines.
were at 1, 5 and 5 l
c
Data from Ref. 8.

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
7087
IC50 values of 0.96–1.3
lM, essentially equivalent to that of CA-4
(IC50, 1.2 M), although 3o was less effective as an inhibitor of cell
l
growth than CA-4.
When comparing inhibition of tubulin polymerization with
antiproliferative effect, we found a good correlation for most of
the active compounds, but not for all of them. Thus, compounds
3b, 3d and 3o were similar as inhibitors of tubulin assembly, but
3b and 3d were much more active than 3o as antiproliferative
agents.
The free amino group on compounds 2a and 2e is not critical for
inhibition of tubulin assembly. Substituting the amino group with
a methyl, as in compounds 3k and 3o, results in retention of
activity.
In the colchicine binding studies, derivative 3e was almost (83%
inhibition) as potent as CA-4, which in these experiments inhibited
colchicine binding by 98%. Inhibition of colchicine binding by com-
pounds 3b, 3d, 3k and 3o was lower, varying within the 51–68%
range, and they were also less potent than 3a, which inhibited
colchicine binding by 78%. Although, many agents in the present
series have activities comparable (3b, 3d, 3k and 3o) or superior
(3a and 3e) to that of CA-4 as inhibitors of tubulin assembly, none
was as active as CA-4 as an inhibitor of colchicine binding to
tubulin.
Figure 1. Proposed binding pose of 2e to tubulin. Before the molecular dynamics:
carbon atoms of 2e in purple, carbon atoms of tubulin residues in orange; after the
molecular dynamics: carbon atoms of 2e in cyan, carbon atoms of tubulin residues
in turquoise. Other atoms indicated as follows: red, oxygen; blue, nitrogen; yellow,
sulphur; Hydrogen atoms are not shown.
The results are consistent with the conclusion that inhibition of
cell growth of these new compounds derives from an interaction
with the colchicine site of tubulin and interference with microtu-
bule assembly.
Comparing the antiproliferative activity of 2-N-methylamino-
thiazole derivatives 3d–e with those of 2-amino counterparts
2d–e, the data indicated that a free amino group at the thiazole
C2-position was an essential requisite for potent activity. In the
inhibition of tubulin assembly, compounds 2d and 3d, as well as
2e and 3e, exhibited similar antitubulin activity, although both
2-amino derivatives had somewhat greater activity than their
methylated analogues. The discrepancy between antiproliferative
activity and antitubulin potency has been reported previously.
The lower antiproliferative activities of 3d–e as compared with
2d–e may result from poor permeability into cells, poor solubility
in the tissue culture medium or any other mechanism limiting the
accessibility of molecules 3d–e to cellular tubulin. Nevertheless,
we cannot exclude the possibility that 2d–e may affect other
molecular targets in addition to microtubules and thus cause their
enhanced antiproliferative activity.
3.5. Molecular modeling
In an attempt to rationalize the biological results observed, a
series of molecular docking simulations were carried out following
the procedure used previously.12 Indeed, the binding mode ob-
served for the compounds reported here is very similar to that ob-
served for 2a–e, with the trimethoxyphenyl ring in close contact
with Cys241 and the second aromatic moiety deep in the binding
pocket (Fig. 1). Interestingly, the N,N-dimethylamino analogues
3f–j also presented the same binding mode, providing little struc-
tural rationale for the loss of biological activity observed with
these compounds. More closely examining the docking results
(Fig. 2), however, we observed a minor steric clash between the
dimethylamino group and tubulin residue Thr179. To assess the
significance of this observation, we performed a series of short
molecular dynamics (MD) simulations on compounds 2e, 3e and
3j in complex with tubulin. The results obtained clearly show that
the presence of the two methyl groups induce a significant confor-
mational change of the binding site residues in close contact with
the N,N-dimethylamino moiety (Fig. 2). Furthermore, there is a sig-
nificant shift of the compound itself within the pocket, suggesting
an overall binding instability for this compound.
Figure 2. Proposed binding pose of 3j to tubulin. Before the molecular dynamics:
carbon atoms of 3j in brown, carbon atoms of tubulin residues in orange; after the
molecular dynamics: carbon atoms of 3j in green, carbon atoms of tubulin residues
in turquoise. Other atoms indicated as follows: red, oxygen; blue, nitrogen; yellow,
sulphur; Hydrogen atoms are not shown.
Moreover, in the case of compound 2e, the binding complex re-
mained stable during the simulation time, and 2e also established
two hydrogen bonds between the aminothiazole ring and Asp251
(Fig. 1). The simulation on compound 3e presented a very similar
profile to the one observed for 2e (Fig. 3), confirming the overall
binding stability of this compound and providing a possible
justification for the different inhibition of tubulin polymerization
profiles of 2e, 3e and 3j.

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R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
3.6. Analysis of cell cycle effects
The effect of 3e and 3k on cell cycle progression was examined
by flow cytometry in Hela and Jurkat cells. 3e treatment resulted in
the rapid accumulation of cells in the G2/M phase, with a concom-
itant reduction in cells in both the S and G1 phases, in both cell
lines. These changes occurred in a concentration-dependent man-
ner (Fig. 4, panels A and B), but changes were observed even at
the lowest concentrations (31 nM in HeLa cells, 62 nM in Jurkat
cells) used. In contrast, compound 3k induced an increase in G2/
M arrested cells only at higher concentrations (a small rise was
first observed with 125 nM compound in both cell lines).
Next, we investigated the association between 3e and 3k-
induced G2/M arrest and alterations in G2/M regulatory protein
expression in HeLa cells. As shown in Figure 4 (panel C), both
compounds caused phosphorylation of the phosphatase cdc25c.
The phosphorylation of cdc25c directly stimulates its phospha-
tase activity, and this is necessary to activate cdc2/cyclin B on en-
try into mitosis.20 In good agreement, we also observed at both 24
and 48 h of incubation, a dephosphorylation at Tyr15 of cdc2 ki-
nase. The expression of cyclin B remained practically constant after
24 h of treatment with both compounds, followed at 48 h with
compound 3e by a slight decrease in cyclin B expression.
Figure 3. Proposed binding pose of 3e to tubulin. Before the molecular dynamics:
carbon atoms of 3e in dark green, carbon atoms of tubulin residues in orange; after
the molecular dynamics: carbon atoms of 3e in purple, carbon atoms of tubulin
residues in turquoise. Other atoms indicated as follows: red, oxygen; blue, nitrogen;
yellow, sulphur; Hydrogen atoms are not shown.
100
100
A
G1
G2/M
S
3k
3e
80
60
40
20
0
80
60
40
20
0
0
125
250
375
500
0
125
250
375
500
Concentration (nM)
Concentration (nM)
B
C
100
100
80
60
40
20
0
3e
3k
80
60
40
20
0
0
125
250
375
500
0
125
250
375
500
Concentration (nM)
Concentration (nM)
Figure 4. Effect of compounds 3e and 3k cell on cycle distribution of HeLa (panel A) and Jurkat cells (panel B). Cells were treated with different compound concentrations
ranging from 31 to 500 nM for 24 h. Then the cells were fixed and stained with PI to analyze DNA content by flow cytometry. Data are presented as mean SEM of three
independent experiments. Panel C: Effect of 3e and 3k on G2/M regulatory proteins. HeLa cells were treated for 24 or 48 h with the indicated concentration of the compound.
The cells were harvested and lysed for the detection of cyclin B, p-cdc2Y15 and cdc25c expression by western blot analysis. To confirm equal protein loading, each membrane
was stripped and reprobed with anti-b-actin antibody.

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
7089
(Fig. 6, panel A). Of note, treatment with 3e and 3k did not induce
activation of caspase-9, (data not shown), one of the major initiator
caspases in the intrinsic (mitochondrial) apoptosis pathway. In
particular, mitochondrial functions such as mitochondrial polariza-
tion and ROS production were only slightly affected by treatment
with the two compounds (data not shown). Interestingly, we ob-
served a clear caspase-8 and caspase-2 activation following treat-
ment with compounds 3e and 3k (Fig. 6, panel A). The activation
of caspase-2 is indicated by disappearance of the uncleaved protein
in the western blot, while the activation of caspase-8 is indicated
by appearance of the cleavage product. In this context, caspase-2
is a unique caspase with characteristics of both initiator and effec-
tor caspases.21 Recently, its key role in several apoptosis signaling
cascades has emerged. In particular, caspase-2 has been implicated
in the cell death induced by different antimitotic agents.22,23
A
A-/PI-
A+/PI-
A+/PI+
A-/PI+
100
80
60
40
20
0
24 h
48 h
3.9. Effect of 3e and 3k on BH3 protein and IAP expression
A-/PI-
A+/PI-
A+/PI+
A-/PI+
100
80
60
40
20
0
B
There is increasing evidence that regulation of the Bcl-2 family
of proteins shares the signaling pathways induced by antimicrotu-
bule compounds.20a,24 Several pro-apoptotic family proteins (e.g.,
Bax, Bid, Bim and Bak) promote the release of cytochrome c,
whereas anti-apoptotic members (Bcl-2, Mcl-1) are capable of
antagonizing the pro-apoptotic proteins and preventing the loss
of mitochondrial membrane potential. Our results showed that
Bcl-2 expression is strongly reduced after treatment with the two
compounds (Fig. 6, panel B), while, in contrast, expression of Bax,
a proapoptotic protein of the Bcl-2 family, was unchanged. Mcl-1
is an anti-apoptotic member of the Bcl-2 family, and recently it
has been reported that sensitivity to antimitotic drugs is regulated
by Mcl-1 levels.25,26 As shown in Figure 6 (panel B) we observed
disappearance of the Mcl-1 band after 24 h treatments with both
compounds.
Xiap and survivin are members of IAP family (Inhibitors of
apoptosis protein), and, in general the IAP proteins function
through direct interactions to inhibit the activity of several caspas-
es, including caspase-3, caspase-7 and caspase-9, thereby inhibit-
ing the processing and activation of the caspases.27 Our results (
Fig. 6) showed that expression of Xiap was almost eliminated after
a 24 h treatment with either compound. Of note, survivin was
phosphorylated on Thr32 upon treatment with the two com-
pounds at both 24 and 48 h. This effect is consistent with cell cycle
arrest in mitosis.28 In addition, since survivin phosphorylation on
Thr32 may regulate apoptosis at cell division via an interaction
with caspase-9,29 our results suggest that activation of caspase-2
may be a compensatory mechanism that leads to cell death.
24 h
48 h
Figure 5. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells
with 3k (panel A) and 3e (panel B) at the indicated concentrations after incubation
for 24 or 48 h. The cells were harvested and labeled with annexin-V-FITC and PI and
analyzed by flow cytometry. Data are represented as mean SEM of three
independent experiments.
3.7. Compounds 3e and 3k induced apoptosis
To evaluate the mode of cell death induced by compounds 3e
and 3k, we performed a biparametric cytofluorimetric analysis
using propidium iodide (PI) and annexin-V-FITC, which stain
DNA and phosphatidylserine (PS) residues, respectively.
After treatment with the two compounds at either 250 or
500 nM for 24 or 48 h, HeLa cells were labeled with the two dyes,
and the resulting red (PI) and green (FITC) fluorescence was
monitored by flow cytometry. As shown in Figure 5 (panel A),
3k caused a significant induction of apoptotic cells (A+/PIꢁ and
A+/PIꢁ) after 24 h, especially with the compound at 500 nM. The
percentage of annexin-V positive cells then further increased at
48 h. Analogous behaviour but with a stronger effect was observed
for 3e (Fig. 5, panel B). These data were supported by findings using
the MTT assay. These findings prompted us to further investigate
the apoptotic process after treatment of cells with 3e and 3k.
4. Conclusions
In this manuscript we have reported a series of thiazole deriva-
tives modified at their C2-position, which shared with previously
published compounds 2a–e, the 30,40,50-trimethoxyphenyl and aryl
substituents at their C4- and C5-positions, respectively. The results
showed that small changes at the 2-position of thiazole ring had a
major impact on antiproliferative activity, with the new synthe-
sized compounds 3a–o generally less active than the correspond-
ing 2-aminothiazole analogues 2a–e. However, the most potent
compound of this series, derivative 3e, displayed similar or more
potent antiproliferative activity than CA-4, with IC50 values ranging
from 1.7 to 38 nM against the tested cancer cell lines. Compound
3e was also the most active inhibitor of tubulin polymerization
3.8. Effect of 3e and 3k on caspases activation
The activation of caspases plays a central role in apoptotic cell
death. To determine which caspases were involved in cell death in-
duced by compounds 3e and 3k, the expression of different caspas-
es was measured by immunoblot analysis. We observed a clear
activation of caspase-3, as well as cleavage of the caspase-3 sub-
strate PARP, after 24 and 48 h exposures to the two compounds
(IC50 = 0.89 l
M) and strongly inhibited the binding of [3H]colchicine
to tubulin (83% inhibition), with activities not greatly different
from those of CA-4. For this latter derivative, a good correlation
was observed between antiproliferative activity, inhibition of

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R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
Figure 6. Effect of 3e and 3k on the expression of caspases (panel A) and Bcl-2 family members and IAP proteins (panel B). HeLa cells were treated with the indicated
concentrations of compounds for 24 or 48 h. Then the cells were harvested and lysed for the detection of protein expression with specific antibodies by western blot analysis.
tubulin polymerization and inhibition of colchicine binding. Com-
paring compounds characterized by the same substituent at the
C5-position of the thiazole ring, with the exception of MCF-7 and
enhanced the apoptotic pathway induced by this class of
compounds.
HT-29 cells, replacement of the 2-amino group with
a N-
5. Experimental section
5.1. Chemistry
methylamino, N,N-dimethylamino and methyl moieties produced
a decrease of activity, with the potency decreasing in the following
order: NH2 > NHCH3 > CH3 ꢀ N(CH3)2. With the exception of 40-
trifluorophenyl derivative 2c, for derivatives 2a–b and 2d–e
replacement of the amino group with a N-methylamino at the
2-position of the thiazole skeleton (compounds 3a–e) led to an
increase in activity against MCF-7 cells. This effect was more
evident for the 40-tolyl analogue 3b, which was 4- to 30-fold more
potent than derivatives 2a–b and 2d–e. The reduced antiprolifera-
tive activities of compounds 3f–j indicated that the presence of an
additional methyl group on the N-methylamino moiety of
analogues 3a–e was detrimental to activity. The 2-N-methylamino
thiazole derivatives 3d–e retain the tubulin affinity of the parent
2-amino compounds 2d–e and CA-4. The greater antiproliferative
activity of 2d–e as compared with 3d–e despite similar antitubulin
effects suggests either differences in intracellular transport or,
possibly, an additional cellular target for the former compounds.
The most active compounds strongly induced apoptosis, and
this effect was followed by caspase activation. An interesting find-
ing of this study was that, at least in HeLa cells, we did not observe
a mitochondrial apoptotic pathway but instead the activation of
caspase-8 and caspase-2 following treatment of cells with the most
active compounds. In addition, the strong decrease of the anti-
apoptotic proteins of Bcl-2 and Mcl-1 along with reduction of Xiap
5.1.1. Materials and methods
1H NMR spectra were recorded on a Varian VXR 200 spectrom-
eter. Chemical shifts (d) are given in ppm upfield, and the spectra
were recorded in appropriate deuterated solvents, as indicated.
Positive-ion electrospray ionization (ESI) mass spectra were re-
corded on a double-focusing Finnigan MAT 95 instrument with
BE geometry. Melting points (mp) were determined on a Buchi–
Tottoli apparatus and are uncorrected. All products reported
showed 1H NMR spectra in agreement with the assigned struc-
tures. The purity of tested compounds was determined by combus-
tion elemental analyses conducted by the Microanalytical
Laboratory of the Chemistry Department of the University of
Ferrara with a Yanagimoto MT-5 CHN recorder elemental analyzer.
All tested compounds yielded data consistent with a purity of at
least 95% as compared with the theoretical values. All reactions
were carried out under an inert atmosphere of dry nitrogen, unless
otherwise indicated. Standard syringe techniques were used for
transferring dry solvents. Reaction courses and product mixtures
were routinely monitored by TLC on silica gel (precoated F254
Merck plates), and compounds were visualized with aqueous
KMnO4. Flash chromatography was performed using 230–400

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
7091
mesh silica gel and the indicated solvent system. Organic solutions
were dried over anhydrous Na2SO4. Arylboronic acids are commer-
cially available and used as received. All chemicals and reagents
were purchased from Aldrich (Sigma–Aldrich) or Lancaster (Alfa
Aesar, Johnson Matthey Company).
The crude product suspended in petroleum ether (15 mL) and stir-
red for 1 h, furnished the title compound 6d as a brownish solid.
Yield: 89%, mp 197–198 °C. 1H NMR (CDCl3) d: 2.44 (s, 3H), 3.82
(s, 3H), 3.88 (s, 3H), 3.94 (s, 6H), 7.10 (s, 1H), 7.12 (s, 2H). MS
(ESI): [M]+ = 322.5.
5.2. General procedure (A) for the synthesis of compounds (6a)
and (6c)
5.5. General procedure (B) for the synthesis of compounds (7a–c)
A solution of the appropriate thiazole 6b–d (2 mmol) in anhy-
drous chloroform (10 mL) was cooled to 0 °C and treated with
N-bromosuccinimide (392 mg, 2.2 mmol) under nitrogen. The
reaction was allowed to warm to room temperature and then stir-
red for 4 h. After quenching with saturated Na2S2O3 (5 mL), the
resulting mixture was diluted with dichloromethane (10 mL). The
organic phase was washed with water (5 mL) and brine (5 mL),
dried (MgSO4) and evaporated.
A mixture of 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone 4
(1.45 g, 5 mmol) and thioacetamide or N-methylthiourea
(5.5 mmol) in anhydrous EtOH (20 mL) was heated to reflux for
1.5 h. After that, the solvent was removed in vacuo, and saturated
aqueous NaHCO3 (5 mL) was added to make the mixture basic (pH
8–9). Then the mixture was extracted with CH2Cl2 (3 ꢂ 15 mL). The
combined organic phases were washed with water (10 mL) and
brine (10 mL), dried with anhydrous Na2SO4 and concentrated un-
der vacuum.
5.5.1. Synthesis of N-(5-bromo-4-(3,4,5-
trimethoxyphenyl)thiazol-2-yl)-N-methylacetamide (7a)
Following general procedure (B), after work-up the solid residue
was stirred in ethyl ether (10 mL) for 30 min. After filtration, the
title compound 7a was isolated as a cream coloured solid. Yield
86%, mp 177–178 °C. 1H NMR (CDCl3) d: 2.43 (s, 3H), 3.74 (s, 3H),
3.86 (s, 3H), 3.92 (s, 6H), 7.22 (s, 2H). MS (ESI): [M]+ = 400.3,
[M+2]+ = 402.5.
5.2.1. Synthesis of 4-(3,4,5-trimethoxyphenyl)-N-methylthiazol-
2-amine (6a)
Following general procedure (A), after work-up the orange oil
residue was suspended in ethyl ether (20 mL) and stirred for 1 h
at +4 °C. The solid was collected by filtration to give the title com-
pound 6a as a white powder. Yield 56%, mp 123–125 °C. 1H NMR
(CDCl3) d: 2.99 (d, J = 5.2 Hz, 3H), 3.88 (s, 3H), 3.92 (s, 6H), 5.34
(bs, 1H), 6.64 (s, 1H), 7.03 (s, 2H). MS (ESI): [M]+ = 280.4.
5.5.2. Synthesis of 5-bromo-4-(3,4,5-trimethoxyphenyl)-N,N-
dimethylthiazol-2-amine (7b)
5.2.2. Synthesis of 4-(3,4,5-trimethoxyphenyl)-2-methylthiazole
(6c)
Following general procedure (B), after work-up the residue was
purified by column chromatography, using ethyl acetate/petroleum
ether 6–4 v/v as eluent, to afford 7b as a white solid. Yield 68%, mp
96–97 °C. 1H NMR (CDCl3) d: 3.10 (s, 3H), 3.11 (s, 3H), 3.87 (s, 3H),
3.91 (s, 6H), 7.16 (s, 2H). MS (ESI): [M]+ = 372.2, [M+2]+ = 374.3.
Following general procedure (A), after work-up the title com-
pound 6c was obtained as an oil, which became a white solid at
+4 °C. Yield 96%, mp 82–83 °C. 1H NMR (CDCl3) d: 2.77 (s, 3H),
3.87 (s, 3H), 3.92 (s, 6H), 7.11 (s, 2H), 7.23 (s, 1H). MS (ESI):
[M]+ = 265.5.
5.5.3. Synthesis of 5-bromo-4-(3,4,5-trimethoxyphenyl)-2-
methylthiazole (7c)
5.3. Synthesis of 4-(3,4,5-trimethoxyphenyl)-N,N-
dimethylthiazol-2-amine (6b)
Following general procedure (B), after work-up the residue was
purified by column chromatography, using ethyl acetate/petro-
leum ether 3–7 v/v as eluent, to afford a colorless oil, which, when
triturated with hexane, furnished 7c as a white solid. Yield 74%, mp
98–99 °C. 1H NMR (CDCl3) d: 2.70 (s, 3H), 3.89 (s, 3H), 3.93 (s, 6H),
7.15 (s, 2H). MS (ESI): [M]+ = 343.1, [M+2]+ = 345.1.
Piperidine (0.88 mL, 8.8 mmol) was added to a stirred solution
of dithiocarbonic acid O-ethyl ester S-[2-oxo-2-(3,4,5-trimethoxy-
phenyl)-ethyl] ester 5 (1.32 g., 4 mmol) dissolved in ethanol
(20 mL). The reaction mixture was stirred for 30 min at room tem-
perature, and N, N0-dimethylcyanamide (0.32 mL, 4 mmol) was
added. The solution was stirred at reflux for 4 h, after which ethanol
was removed under reduced pressure and the residue portioned in
a mixture of dichloromethane (15 mL) and a 3 N aqueous solution
of HCl (30 mL). The aqueous acid phase was basified (pH 9)
with sodium carbonate and washed with dichloromethane
(3 ꢂ 15 mL). The combined organic phase was washed with brine
(10 mL) and dried over sodium sulfate. After concentration under
reduced pressure, the residue was triturated with petroleum ether
(10 mL), and this furnished the final compound as a brown solid.
Yield: 62%, mp 80–81 °C. 1H NMR (CDCl3) d: 3.15 (s, 6H), 3.86 (s,
3H), 3.92 (s, 6H), 6.62 (s, 1H), 7.07 (s, 2H). MS (ESI): [M]+ = 294.4.
5.6. General procedure (C) for the synthesis of compounds 3k–o
and 8a–e
A stirred suspension of 5-bromo-4-(3,4,5-trimethoxyphenyl)-
thiazole derivative 7a or 7c (0.5 mmol) and the appropriate
phenylboronic acid (1 mmol) in dioxane (6 mL containing 3 drops
of water) was degassed under a stream of nitrogen for 10 min, then
treated with [1,10-bis(diphenylphosphino)ferrocene] dichloropalla-
dium (II) methylene chloride complex (41 mg, 0.05 mmol) and cesium
fluoride (190 mg, 1.25 mmol). The reaction mixture was heated un-
der nitrogen at 45 °C for 30 min, then at 65 °C for 5 h. The reaction
mixture was cooled to ambient temperature, diluted with CH2Cl2
(10 mL), filtered through a pad of celite and evaporated in vacuo.
The residue was dissolved with CH2Cl2 (15 mL), and the resultant
solution was washed sequentially with water (5 mL) and brine
(5 mL). The organic layer was dried and evaporated, and the residue
was purified by flash chromatography on silica gel.
5.4. Synthesis of N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-N-
methylacetamide (6d)
Sodium acetate (250 mg, 3 mmol) was added to a stirred solu-
tion of 6a (840 mg, 3 mmol) in acetic anhydride (30 mL). The mix-
ture was refluxed for 18 h, the solvent evaporated in vacuo and the
residue portioned in a mixture of dichloromethane (25 mL) and
water (10 mL). The organic extract was washed with brine
(10 mL), dried over Na2SO4 and concentrated at reduced pressure.
5.6.1. 4-(3,4,5-Trimethoxyphenyl)-2-methyl-5-(naphthalen-2-
yl)thiazole (3k)
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 6:4

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R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
(v:v) as eluent furnished 3k as a yellow oil. Yield 71%. 1H NMR
(CDCl3) d: 2.78 (s, 3H), 3.58 (s, 6H), 3.82 (s, 3H), 6.77 (s, 2H), 7.34
(d, J = 8.8 Hz, 1H), 7.44 (m, 2H), 7.79 (m, 3H), 7.88 (s, 1H). MS
(ESI): [M]+ = 391.3. Anal. (C23H21NO3S): C, H, N.
(CDCl3) d: 2.46 (s, 3H), 3.70 (s, 6H), 3.75 (s, 3H), 3.84 (s, 3H), 6.71 (s,
2H), 7.49 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H). MS (ESI):
[M]+ = 466.5.
5.6.9. N-(4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)
thiazol-2-yl)-N-methylacetamide (8d)
5.6.2. 4-(3,4,5-Trimethoxyphenyl)-2-methyl-5-p-tolylthiazole
(3l)
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 8d as a yellow oil. Yield: 69% yield. 1H
NMR (CDCl3) d: 2.44 (s, 3H), 3.68 (s, 6H), 3.78 (s, 3H), 3.81 (s,
3H); 3.84 (s, 3H), 6.80 (s, 2H), 6.87 (d, J = 8.8 Hz, 2H), 7.31 (d,
J = 8.8 Hz, 2H). MS (ESI): [M]+ = 428.6.
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 3l as a yellow oil. Yield 71%. 1H NMR
(CDCl3) d: 2.34 (s, 3H), 2.77 (s, 3H), 3.66 (s, 6H), 3.72 (s, 3H), 6.70
(s, 2H), 7.10 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H). MS (ESI):
[M]+ = 355.5. Anal. (C20H21NO3S): C, H, N.
5.6.10. N-(5-(4-Ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)
thiazol-2-yl)-N-methylacetamide (8e)
5.6.3. 5-(4-(Trifluoromethyl)phenyl)-4-(3,4,5-trimethoxy-
phenyl)-2-methylthiazole (3m)
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 8e as a colorless oil. Yield 72%. 1H NMR
(CDCl3) d: 1.41 (t, J = 6.8 Hz, 3H), 2.44 (s, 3H), 3.68 (s, 6H), 3.78
(s, 3H), 3.83 (s, 3H); 4.05 (q, J = 6.8 Hz, 2H), 6.81 (s, 2H), 6.86 (d,
J = 8.6 Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H). MS (ESI): [M]+ = 442.5.
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 3m as a colorless oil. Yield 66%. 1H
NMR (CDCl3) d: 2.77 (s, 3H), 3.66 (s, 6H), 3.84 (s, 3H), 6.67 (s,
2H), 7.49 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H). MS (ESI):
[M]+ = 409.2. Anal. (C20H18F3NO3S): C, H, N.
5.7. General procedure (D) for the synthesis of compounds (3a–e)
5.6.4. 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)-2-
methylthiazole (3n)
A mixture of N-acetyl thiazole derivative 8a–e (0.5 mmol), 1 N
aqueous NaOH (0.55 mL, 0.55 mmol) and EtOH (8 mL) was refluxed
for 1 h, and the solvent was removed by evaporation. The residue
was dissolved in a mixture of water (5 mL) and dichloromethane
(10 mL). The organic phase was washed with brine (5 mL), dried
(Na2SO4) and concentrated in vacuo. Ethyl ether (5 mL) was added
to the residue, and the resulting precipitate was collected by filtra-
tion to furnish the title compound as a solid.
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 6:4
(v:v) as eluent, furnished 3n as a colorless oil. Yield 68%. 1H NMR
(CDCl3) d: 2.73 (s, 3H), 3.68 (s, 6H), 3.81 (s, 3H), 3.83(s, 3H), 6.74
(s, 2H), 6.86 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H). MS (ESI):
[M]+ = 371.3. Anal. (C20H21NO4S): C, H, N.
5.6.5. 5-(4-Ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2-
methylthiazole (3o)
5.7.1. 4-(3,4,5-Trimethoxyphenyl)-N-methyl-5-(naphthalen-2-
yl)thiazol-2-amine (3a)
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 3o as a yellow oil. Yield 79%. 1H NMR
(CDCl3) d: 1.41 (t, J = 6.8 Hz, 3H), 2.73 (s, 3H), 3.68 (s, 6H), 3.83
(s, 3H), 4.04 (q, J = 6.8 Hz, 2H), 6.75 (s, 2H), 6.84 (d, J = 8.8 Hz,
2H), 7.26 (d, J = 8.8 Hz, 2H). MS (ESI): [M]+ = 385.5. Anal.
(C21H23NO4S): C, H, N.
Following general procedure (D), after work-up 3a was isolated
as a white solid. Yield 81%, mp 194–195 °C. 1H NMR (CDCl3) d: 3.03
(d, J = 4.8 Hz, 3H), 3.59 (s, 6H), 3.83 (s, 3H), 5.25 (bs, 1H), 6.76 (s,
2H), 7.34 (d, J = 6.8 Hz, 1H), 7.47 (m, 2H), 7.72 (m, 3H), 7.84 (s,
1H). MS (ESI): [M]+ = 406.7. Anal. (C23H22N2O3S): C, H, N.
5.7.2. 4-(3,4,5-Trimethoxyphenyl)-N-methyl-5-p-tolylthiazol-2-
amine (3b)
5.6.6. N-(4-(3,4,5-Trimethoxyphenyl)-5-(naphthalen-2-
yl)thiazol-2-yl)-N-methylacetamide (8a)
Following general procedure (D), after work-up 3b was isolated
as a yellow solid. Yield 82%, mp 189–190 °C. 1H NMR (CDCl3) d:
2.32 (s, 3H), 2.98 (d, J = 5.2 Hz, 3H), 3.67 (s, 6H), 3.82 (s, 3H), 5.22
(bs, 1H), 6.72 (s, 2H), 7.09 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz,
2H). MS (ESI): [M]+ = 370.5. Anal. (C20H22N2O3S): C, H, N.
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished the 8a precursor as a colorless oil. Yield
82%. 1H NMR (CDCl3) d: 2.46 (s, 3H), 3.58 (s, 6H), 3.70 (s, 3H),
3.81 (s, 3H), 6.82 (s, 2H), 7.46 (m, 3H), 7.78 (m, 3H), 7.93 (s, 1H).
MS (ESI): [M]+ = 448.5.
5.7.3. 5-(4-(Trifluoromethyl)phenyl)-4-(3,4,5-trimethoxy-
phenyl)-N-methylthiazol-2-amine (3c)
5.6.7. N-(4-(3,4,5-Trimethoxyphenyl)-5-p-tolylthiazol-2-yl)-N-
methylacetamide (8b)
Following general procedure (D), after work-up 3c was isolated
as a white solid. Yield 78%, mp 183–184 °C. 1H NMR (CDCl3) d: 4.03
(d, J = 5.2 Hz, 3H), 3.67 (s, 6H), 3.84 (s, 3H), 5.23 (bs, 1H), 6.69 (s,
2H), 7.37 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H). MS (ESI):
[M]+ = 424.9. Anal. (C20H19F3N2O3S): C, H, N.
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 8b as a yellow oil. Yield 71%. 1H NMR
(CDCl3) d: 2.34 (s, 3H), 2.44 (s, 3H), 3.67 (s, 6H), 3.78 (s, 3H), 3.84
(s, 3H), 6.78 (s, 2H), 7.16 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz,
2H). MS (ESI): [M]+ = 412.5.
5.7.4. 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)-N-
methylthiazol-2-amine (3d)
Following general procedure (D), after work-up 3d was isolated
as a brown solid. Yield 84%, mp 174–176 °C. 1H NMR (CDCl3) d:
2.99 (d, J = 4.4 Hz, 3H), 3.67 (s, 6H), 3.79 (s, 3H), 3.82 (s, 3H), 5.20
(bs, 1H), 6.73 (s, 2H), 6.83 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.8 Hz,
2H). MS (ESI): [M]+ = 386.6. Anal. (C20H22N2O4S): C, H, N.
5.6.8. N-(5-(4-(Trifluoromethyl)phenyl)-4-(3,4,5-trimethoxy-
phenyl)thiazol-2-yl)-N-methylacetamide (8c)
Following general procedure (C), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 8c as a colorless oil. Yield 67%. 1H NMR

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
7093
5.7.5. 5-(4-Ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-N-
methylthiazol-2-amine (3e)
d: 1.38 (t, J = 7.0 Hz, 3H), 3.14 (s, 6H), 3.67 (s, 6H), 3.81 (s, 3H), 4.02
(q, J = 7.0 Hz, 2H), 6.77 (s, 2H), 6.81 (d, J = 8.8 Hz, 2H), 7.22 (d,
J = 8.8 Hz, 2H). MS (ESI): [M]+ = 414.6. Anal. (C22H26N2O4S): C, H, N.
Following general procedure (D), after work-up 3e was isolated
as a white solid. Yield 82%, mp 136–137 °C. 1H NMR (CDCl3) d: 1.40
(t, J = 7.0 Hz, 3H), 2.99 (d, J = 5.2 Hz, 3H), 3.67 (s, 6H), 3.82 (s, 3H),
4.03 (q, J = 7.0 Hz, 2H), 5.20 (bs, 1H), 6.73 (s, 2H), 6.82 (d, J = 8.8 Hz,
2H), 7.21 (d, J = 8.8 Hz, 2H). MS (ESI): [M]+ = 400.7. Anal.
(C21H24N2O4S): C, H, N.
5.9. Biology experiments
5.9.1. Antiproliferative assays
Human T-leukemia (Jurkat) and human promyelocytic leuke-
mia (HL-60) cells were grown in RPMI-1640 medium, (Gibco,
Milano, Italy). Breast adenocarcinoma (MCF-7), human non-small
cell lung carcinoma (A549), human cervix carcinoma (HeLa) and
human colon adenocarcinoma (HT-29) cells were grown in DMEM
medium (Gibco, Milano, Italy). Both media were supplemented
5.8. General procedure (E) for the synthesis of compounds 3f–j
To a stirred suspension of 7b (0.5 mmol) and the appro-
priate phenylboronic acid (1 mmol) in toluene (7 mL) was
added [1,10-bis(diphenylphosphino)ferrocene]dichloropalladium
(II) methylene chloride complex (41 mg, 0.05 mmol) and cesium
fluoride (190 mg, 1.25 mmol). The reaction mixture was heated
under nitrogen at 55 °C for 45 min, then at 75 °C for 20 h. The reac-
tion mixture was cooled to ambient temperature, diluted with
CH2Cl2 (10 mL), filtered through a pad of celite and evaporated in
vacuo. The residue was dissolved with CH2Cl2 (15 mL), and the
resultant solution was washed sequentially with water (5 mL)
and brine (5 mL). The organic layer was dried and evaporated,
and the residue was purified by flash chromatography on silica gel.
with 115 units/mL of penicillin G (Gibco, Milano, Italy), 115 lg/
mL of streptomycin (Invitrogen, Milano, Italy) and 10% fetal bovine
serum (Invitrogen, Milano, Italy). All these cell lines were pur-
chased by ATCC. LoVoDox°Cells are a doxorubicin resistant subclone
of LoVo cells15 and were grown in complete Ham’s F12 medium
supplemented with doxorubicin (0.1 l
g/mL). CEMVbl-100 cells are
a multidrug-resistant line selected against vinblastine.16 LoVoDoxo
and CEMVbl-100 were a kind gift of Dr. G. Arancia (Istituto Superiore
di Sanità, Rome, Italy) A549-T12 cells are a non-small cell lung car-
cinoma line exhibiting resistance to taxol17 were kindly donated by
Professor I. Castaglioulo (University of Padova). They were grown
in complete DMEM medium supplemented with taxol (12 nM).
Stock solutions (10 mM) of the different compounds were obtained
by dissolving them in DMSO. Individual wells of a 96-well tissue
5.8.1. 4-(3,4,5-Trimethoxyphenyl)-N,N-dimethyl-5-(naphthalen-
3-yl)thiazol-2-amine (3f)
Following general procedure E, the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 2.5:7.5
(v:v) as eluent, furnished 3f as a yellow oil. Yield 63%. 1H NMR
(CDCl3) d: 3.18 (s, 6H), 3.58 (s, 6H), 3.82 (s, 3H), 6.80 (s, 2H), 7.34
(d, J = 8.8 Hz, 1H), 7.42 (m, 2H), 7.76 (m, 3H), 7.88 (s, 1H). MS
(ESI): [M]+ = 420.6. Anal. (C24H24N2O3S): C, H, N.
culture microtiter plate were inoculated with 100 lL of complete
medium containing 8 ꢂ 103 cells. The plates were incubated at
37 °C in a humidified 5% CO2 incubator for 18 h prior to the exper-
iments. After medium removal, 100 lL of fresh medium containing
the test compound at different concentrations was added to each
well and incubated at 37 °C for 72 h. The percentage of DMSO in
the medium never exceeded 0.25%. This was also the maximum
DMSO concentration in all cell-based assays described below. Cell
viability was assayed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide test as previously described.30 The
IC50 was defined as the compound concentration required to inhi-
bit cell proliferation by 50%, in comparison with cells treated with
the maximum amount of DMSO (0.25%) and considered as 100%
viability.
5.8.2. 4-(3,4,5-Trimethoxyphenyl)-N,N-dimethyl-5-p-
tolylthiazol-2-amine (3g)
Following general procedure (E), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 3:7
(v:v) as eluent, furnished 3g as a brown solid. Yield 67%, mp
77–78 °C. 1H NMR (CDCl3) d: 2.32 (s, 3H), 3.14 (s, 3H), 3.15
(s, 3H), 3.66 (s, 6H), 3.82 (s, 3H), 6.76 (s, 2H), 7.10 (d, J = 8.0 Hz,
2H), 7.18 (d, J = 8.0 Hz, 2H). MS (ESI): [M]+ = 384.5. Anal.
(C21H24N2O3S): C, H, N.
5.9.2. Effects on tubulin polymerization and on colchicine
binding to tubulin
5.8.3. 5-(4-(Trifluoromethyl)phenyl)-4-(3,4,5-trimethoxy-
phenyl)-N,N-dimethylthiazol-2-amine (3h)
To evaluate the effect of the compounds on tubulin assembly in
Following general procedure (E), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 4:6
(v:v) as eluent, furnished 3h as an orange oil. Yield 68%. 1H NMR
(CDCl3) d: 3.17 (s, 6H), 3.68 (s, 6H), 3.83 (s, 3H), 6.69 (s, 2H), 7.37
(d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.2 Hz, 2H). MS (ESI): [M]+ = 438.6.
Anal. (C21H21F3N2O3S): C, H, N.
vitro,18 varying concentrations of compounds were preincubated
with 10 lM bovine brain tubulin in glutamate buffer at 30 °C for
15 min and then cooled to 0 °C. After addition of 0.4 mM GTP,
the mixtures were transferred to 0 °C cuvettes in a recording
spectrophotometer and warmed to 30 °C. Tubulin assembly was
followed turbidimetrically at 350 nm. The IC50 was defined as the
compound concentration that inhibited the extent of assembly
by 50% after a 20 min incubation. The capacity of the test com-
pounds to inhibit colchicine binding to tubulin was measured as
5.8.4. 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)-N,N-
dimethylthiazol-2-amine (3i)
Following general procedure (E), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 3:7
(v:v) as eluent, furnished 3i as a cream colored solid. Yield 72%,
mp 140–142 °C. 1H NMR (CDCl3) d: 3.14 (s, 6H), 3.67 (s, 6H), 3.79
(s, 3H), 3.81 (s, 3H), 6.76 (s, 2H), 6.82 (d, J = 8.8 Hz, 2H), 7.22 (d,
J = 8.8 Hz, 2H). MS (ESI): [M]+ = 400.6. Anal. (C21H24N2O4S): C, H, N.
described,19 except that the reaction mixtures contained 1
tubulin, 5 M test compound.
M [3H]colchicine and 5
lM
l
l
5.9.3. Molecular modeling
All molecular modeling studies were performed on a MacPro
dual 2.66 GHz Xeon running Ubuntu 10. The tubulin structure
was downloaded from the PDB data bank (http://www.rcsb.org/—
PDB code:1SA0).31 Hydrogen atoms were added to the protein,
using the Protonate3D function of molecular operating environ-
ment (MOE).32 Ligand structures were built with MOE and
minimized using the MMFF94x forcefield until a RMSD gradient
of 0.05 kcal molꢁ1 Åꢁ1 was reached. The docking simulations were
5.8.5. 5-(4-Ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-N,N-
dimethylthiazol-2-amine (3j)
Following general procedure (E), the crude residue, purified by
flash chromatography using ethyl acetate/petroleum ether 2.5:7.5
(v:v) as eluent, furnished 3j as a red oil. Yield 73%. 1H NMR (CDCl3)

7094
R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 7083–7094
performed using plants.33 Molecular dynamics was performed
with the Gromacs 4.534 with the Amber99 force field. The structure
was solvated using TIP3P water molecules, providing a minimum
of 9.0 Å of water between the protein surface and any periodic
box edge. The system was neutralized, minimized and then a posi-
tion restrain dynamics simulation was carried out for 150 ps. The
production simulation was conducted for 2 ns at 298 K using a
NPT environment. Inhibitors were parameterized by Antechamber
of AmberTool 1.5.35 Trajectories analysis were carried out by
VMD.35
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.001.
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Acknowledgment
The authors would like to thank Dr. Alberto Casolari for excel-
lent technical assistance.
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