Synthesis and anticancer activity of analogues of phenstatin, with a phenothiazine A-ring, as a new class of microtubule-targeting agents

Article abstract of DOI:10.1016/j.bmcl.2012.10.135

A new family of microtubule-targeting agents with a phenothiazine A-ring was synthesized and evaluated for anti-proliferative activity and interaction with tubulin. These new derivatives showed significant activities against cellular proliferation and tub

Full text of DOI:10.1016/j.bmcl.2012.10.135

Bioorganic & Medicinal Chemistry Letters 23 (2013) 147–152
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer activity of analogues of phenstatin,
with a phenothiazine A-ring, as a new class of microtubule-targeting agents
Cristina-Maria Abuhaie a, Elena Bîcu a, Benoît Rigo b,c, Philippe Gautret b,c, Dalila Belei a, Amaury Farce b,d
,
Joëlle Dubois e, Alina Ghinet a,b,c,
⇑
a Department of Organic Chemistry, ‘Al. I. Cuza’ University of Iasi, Faculty of Chemistry, Bd. Carol I nr. 11, 700506 Iasi, Romania
b Univ Lille Nord de France, F-59000 Lille, France
c UCLille, EA 4481 (GRIIOT), Laboratoire de Pharmacochimie, HEI, 13 rue de Toul, F-59046 Lille, France
d Institut de Chimie Pharmaceutique Albert Lespagnol, EA GRIIOT (4481), IFR114, 3 Rue du Pr Laguesse, B.P. 83, F-59006 Lille, France
e Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new family of microtubule-targeting agents with a phenothiazine A-ring was synthesized and evalu-
ated for anti-proliferative activity and interaction with tubulin. These new derivatives showed significant
activities against cellular proliferation and tubulin polymerization, rather similar to those of phenstatin.
Phenothiazine derivative 21 proved to be the most potent compound synthesized with GI50 values rang-
ing from 29 to 93 nM on different cell lines. The same compound showed a better inhibition of COLO 205,
A498, and MCF7 cell lines than the parent phenstatin.
Received 19 September 2012
Revised 26 October 2012
Accepted 30 October 2012
Available online 8 November 2012
Keywords:
Tubulin
Ó 2012 Elsevier Ltd. All rights reserved.
Anticancer agent
Phenothiazine derivative
Microtubule-targeting agent
Tubulin is a heterodimer of closely related and tightly linked
globular - and b-tubulin proteins, capable to polymerize in hol-
group.14 It was also reported that if the benzene ring was changed
by simple heteroaryl or other benzenic systems, the activity dimin-
ishes in several orders of magnitude or even disappears.15 In this
context, we were puzzled by a recent report on the strong inhibi-
tion of tubulin polymerization and anti-proliferative activities of
phenoxazine and phenothiazine derivatives such as compounds 4
and 5 (Fig. 1).16 In these products, the heterocycle system seems
to correspond to the A-ring of the standard tubulin polymerization
agents. Because of our expertise in phenothiazine chemistry,17 we
decided to synthesize phenstatin analogs utilizing phenothiazine
as an A-ring and different B-rings in order to explore some struc-
ture–activity relationships in this new family of compounds.
Nucleophilic reactions of phenothiazine with appropriately
substituted benzoyl (13–16) and indolecarbonyl (17–19) chlorides,
carried out in toluene, provided new phenstatin analogs 20–26
with a phenothiazine A-ring (Scheme 1). The syntheses of the acid
chlorides (13–19) were realized starting from the commercially
available carboxylic acids (6–12) and thionyl chloride.19 The reac-
tion of the 1H-indole-3-carbonyl chloride 17 with the phenothia-
zine did not lead to the N-acylation product, but gave the
Friedel–Crafts acylation product 24 (Scheme 1). The high stability
of the intermediate carbocation could explain the ring-acylation
in the absence of a Lewis acid.
a
low tubes called microtubules. The microtubule system is involved
in many essential cell functions,1 and their importance in cell divi-
sion makes microtubules an important target for anticancer drugs.2
To date there are at least 28 compounds targeting tubulin in clin-
ical development.3 Most of the antimitotic drugs in clinical use or
in development, bind at three major sites on tubulin: the vinca,
taxane, and colchicine sites.4 Combretastatin A-4 (CA-4) (1,
Fig. 1), extracted from a South African tree Combretum caffrum by
Pettit et al,5 depicts high affinity for the colchicine binding site,
and is one of the most potent antimitotic agents.6 Research on
combretastatin A-4 in order to improve its in vivo activity led to
the discovery of phenstatin (2, Fig. 1)7 then later of isocombretast-
atin A-4 (3, Fig. 1).8 In these compounds, the cisoid disposition of
the two aromatic rings and the 3,4,5-trimethoxyphenyl ring A
are considered both essential for the biological activity.9 However,
it has been demonstrated that some modifications of the A-ring
such as 2,3,4-trimethoxyphenyl,10 halogeno-methoxyphenyl,11
2,3-methylenedioxy-4-phenyl,12 3,4,5-trimethylphenyl13 moieties
led to equal or better activity, and pharmacophore model indicates
that other hydrogen bond acceptors could replace the trimethoxy
The recent identification of phenstatin analogues bearing 5,6-
fused bicyclic azaheterocyclic scaffolds as B-rings with potent
⇑
Corresponding author. Tel.: +33 328 384 858; fax: +33 328 384 604.
E-mail address: alina.ghinet@hei.fr (A. Ghinet).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.135

148
C.-M. Abuhaie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 147–152
O
OH
OH
MeO
MeO
MeO
MeO
MeO
MeO
A
OH
OMe
B
OMe
OMe
OMe
OMe
OMe
Phenstatin
Isocombretastatin A-4
3
ITP IC50 = 1.5 µM18
Combretastatin A-4
1
2
ITP IC50 = 3.43 µM (Lit. 1.0µM7)
CN
ITP IC50 = 3.0 µM10
O
O
OH
OH
N
N
O
S
OMe
OMe
5
4
ITP IC50 = 0.52 µM16
ITP IC50 = 1.40 µM16
Figure 1. Structure and inhibitory activity on tubulin polymerization (ITP) of combretastatin A-4 (1),10 phenstatin (2),7 isocombretastatin A-4 (3)18 and of compounds 4 and
5.16
in vitro anticancer activity20 led us to synthesize compounds with
similar B-rings. In this light, the construction of the indolizine unit
of compounds 27, 28 (Scheme 2) was realized by [3+2] cycloaddi-
tion reaction of the corresponding ylide 34, 35 with ethyl propio-
late, followed by spontaneous aromatization of intermediates 36,
37. Ylides 34, 35 were obtained in situ by base treatment of pyrid-
inium salts 32, 33 resulting from the reaction of substituted pyri-
dines 29, 30 with 10-(chloroacetyl)-10H-phenothiazine 31
(Scheme 2).
The synthesis of imidazo[1,2-a]pyridine 38 was realized by the
alkylation of the amidine 39 by the compound 31, then cyclization
of the resulting cycloimonium salt 41 upon dimethylamine hydro-
chloride elimination. Intermediate 39 was obtained via the con-
densation reaction of 2-aminopyridine 40 with DMF/DMA
(Scheme 3).
In order to complete the preliminary study, and to establish
structure–activity relationships, we become interested in the syn-
thesis of compounds with urea scaffold. Target compounds 42, 43
(Scheme 4) were prepared by reacting commercially available 10H-
phenothiazine-10-carbonyl chloride 44 with corresponding indole
derivatives 45, 46, following a similar synthetic procedure de-
scribed in the literature.22
The activity of synthesized compounds was first evaluated on
the tubulin polymerization inhibition.23 The results are summa-
rized in Table 1. The replacement of the trimethoxyphenyl unit
by a 10H-phenothiazine moiety, and the modification of the B-ring
by a 4-methoxy-3-fluorophenyl in compound 20 (Scheme 1) lead
to the best antitubulin activity in this study, with an IC50 value
in the micromolar range. Compound 21 (Scheme 1), with a 3-ami-
no-4-methoxyphenyl B-ring, showed slightly decreased antitubu-
lin potential compared to 3-fluoro derivative 20 (21:
IC50 = 11.8 lM; 20: 9.5 lM, Table 1). Interestingly, the replacement
of the A-ring by a 10H-phenothiazin-3-yl moiety and the introduc-
tion of 1H-indol-3-yl as the B-ring in compound 24 (Scheme 1)
conserved an antitubulin activity but less potent than derivatives
20 and 21.
Molecules 22, 25–28, and 38 showed very modest tubulin poly-
merization inhibition at a 10À4 M concentration (values raging
from 31 to 48%) (Table 1). The two other tested compounds 23
(Scheme 1) and 43 (Scheme 4) were inactive.
All synthesized compounds were selected by the National Can-
cer Institute (NCI) for screening against 60 human tumor cell
lines.24 They were tested initially at a single high dose (10À5 M)
in the full NCI 60 cell panel. Only products which satisfied pre-
determined threshold inhibition criteria have progressed to the
5-dose screen in order to determine their GI50 values. Results are
summarized in Table 2) and Table 3 (supplementary data).
The phenothiazine derivative 21 exhibited the most potent
in vitro cytotoxic activity: inhibition of K-562, SR, NCI-H522,
HCT-15, KM12, SW-620, M14, MDA-MB-435, SK-MEL-5, NCI/
ADR-RES, SF-295, MCF-7, and MDA-MB-468 cell lines with GI50
values ranging from 29 to 93 nM (Table 2). The closely related
compound 20, which posseses a fluoro substituent instead of an
amino group at the meta position of the B-ring, showed slightly de-
creased cytotoxicity compared to compound 21. However, both
phenothiazine derivatives 20 and 21 exhibited more potent anti-
proliferative activity than phenstatin (2) against several types of
cancer cells, such as COLO 205, A498, and MCF7 cell lines (Table 2).
O
O
S
O
O
(i)
(ii)
N
Ar
Ar
or
S
Ar
OH
Ar
Cl
N
H
6: Ar=3-fluoro-4-methoxyphenyl
7: Ar=3-amino-4-methoxyphenyl
8: Ar=3-chloro-4-ethoxyphenyl
9: Ar=2,4-difluoro-3-methoxyphenyl
10: Ar=1H-indol-3-yl
11: Ar=5,6-dimethoxy-1H-indol-2-yl
12: Ar=5-fluoro-1H-indol-2-yl
20: Ar=3-fluoro-4-methoxyphenyl, 66%
21: Ar=3-amino-4-methoxyphenyl, 42%
22: Ar=3-chloro-4-ethoxyphenyl, 73%
23: Ar=2,4-difluoro-3-methoxyphenyl, 59%
25: Ar=5,6-dimethoxy-1H-indol-2-yl, 46%
26: Ar=5-fluoro-1H-indol-2-yl, 53%
24: Ar=1H-indol-3-yl, 51%
13-19
Scheme 1. Reagents and conditions: (i) 1.5 equiv SOCl2, DMF (cat. amount), CH2Cl2, rt, inert atmosphere, 1-6 h, quantitative yield;19 (ii) 0.91 equiv 10H-phenothiazine,
toluene, 90 °C, inert atmosphere, 12–24 h.16

C.-M. Abuhaie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 147–152
149
2
2
R
R
1
R
2
+
+
1
R
O
O
H
1
R
N
H
R
N
_
N
Cl-
..
(ii)
(iii)
N
O
H
CO2Et
S
N
N
S
S
36-37
-H2
32: R1=Me, R2=H, 81%
34-35
33: R1=H, R2=NMe2, 93%
Cl
1
R
2
O
2
O
(i)
R
N
R
N
N
N
+
S
1
R
S
CO2Et
29: R1=Me, R2=H
27: R1=Me, R2=H, 53%
31
30: R1=H, R2=NMe2
28: R1=H, R2=NMe2, 45%
Scheme 2. Reagents and conditions: (i) EtOAc, reflux; (ii) 1.2 equiv NEt3, CH3CN, rt; (iii) 1.5 equiv ethyl propiolate, rt, 24 h.
O
+
N
N
N
N
(i)
(ii)
-
N
Cl
O
N
+
S
N
NH2
N
N
N
-Me2NH2]Cl-
N
S
39
40
38 65%
41
Scheme 3. Reagents and conditions: (i) 1.1 equiv DMF/DMA, toluene, reflux, 12 h; (ii) 1 equiv 10-(chloroacetyl)-10H-phenothiazine 31, MeOH, reflux, 16 h.21
derivative 21 (Table 2). A loss in potency was observed for com-
pound 23, which presents a 2,4-difluoro-3-methoxy substitution
on the B-ring. However, the replacement of the B-ring by a 5,6-
dimethoxy-1H-indol-2-yl (compound 25, Scheme 1) resulted in
an increase of the cell growth inhibition compared to compound
23. Also, the activity of the 5-fluoroindolyl analogue 26 (Scheme 1)
was diminished compared to the dimethoxy derivative 25, indicat-
ing that the fluoro group at the 5-position and elimination of the
second methoxy unit at the 6-position did not offer any advantage
in terms of biological potential. Interestingly, the results obtained
from NCI revealed that the unexpected Friedel–Crafts acylation
product 24 had also a cytotoxic effect (e.g., GI50 (MDA-MB-
435) = 112 nM, Table 2). The replacement of the B-ring by a ethyl
5-methyl-indolizine-1-carboxylate (compound 27, Scheme 2) in-
duced moderate anti-proliferative potency (GI50 values, generally
in the micromolar range) (Table 2). The urea derivative 43
(Scheme 4) showed similar activity compared to indolizine 27
(Table 2).
Phenothiazine derivatives 26, 28, 38 and 42 did not satisfy pre-
determined threshold inhibition criteria in the initial single-dose
assay and did not progress to the 5-dose screen. Results from these
studies are listed in Table 3 (Supplementary data).
The docking studies were realized in the colchicine binding site
of tubulin,26 on compounds that presented a biological potential
(20, 21, 22, and 24) (Fig. 2).
The fluoro derivative 20 adopts a single conformation which
occupies the pocket in the same global position as phenstatin
(Fig. 2(a)). Compound 21, which has an amino substitution at the
meta position of the B-ring instead a fluorine as in compound 20,
displays a preferential conformation with 23 solutions. It behaves
differently, the carbonyl function forms hydrogen bonds with Val
181 and with the side chain of Asn 258 (Fig. 2(b)).
O
O
R
(i)
N
Cl
N
R
N
+
S
S
N
H
44
45: R=COMe
46: R=CH2NMe2
42: R=COMe, 76%
43: R=CH2NMe2, 11%
Scheme 4. Reagents and conditions: (i) 1.3 equiv Et3N, 0.1 equiv DMAP, CH2Cl2, rt,
72 h.22
Table 1
Inhibitory activities on tubulin polymerization23
Compd. no.
% ITPa
IC50
(
lM
SD)a
20
21
22
23
24
25
26
27
95
99
48
10
66
36
32
31
42
38
9
9.48 0.60
11.82 1.96
N.D.b
N.D.
38.91 3.63
N.D.
N.D.
N.D.
N.D.
N.D.
28
38
43
N.D.
3.43 0.70
1.76 0.44
Phenstatin 2
99
100
Desoxypodophyllotoxin
a
Inhibition of tubulin polymerization at a 100
Not determined.
lM concentration.
b
Phenothiazine derivative 22 bearing a 3-chloro-4-ethoxy phe-
nyl as B-ring (Scheme 1) showed comparable activities to fluoro-

150
C.-M. Abuhaie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 147–152
Table 2
Results of the in vitro human cancer cell growth inhibitiona,b for compounds 20-25, 27, and 43
Cell type
Leukemia
Compound
Cell line
Phenstatin 2
20
21
22
23
GI50 (
24
25
27
43
l
M)a,b
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
—
—
—
0.300
0.267
0.143
0.442
0.259
0.271
0.382
0.964
0.539
0.209
0.393
0.372
0.102
0.268
0.245
0.040
0.380
0.328
0.030
0.187
N.D.e
0.226
0.277
0.437
0.289
0.035
0.361
0.315
0.277
0.408
0.424
0.142
0.620
N.D.
0.512
0.272
0.999
0.368
0.221
5.000
3.990
3.240
4.290
3.220
2.330
4.070
N.D.
8.370
N.D.
4.890
3.670
2.700
0.380
0.382
0.395
3.750
0.761
0.382
0.634
11.600
0.670
0.970
0.943
0.429
0.248
0.589
0.387
0.437
1.540
0.902
0.303
0.656
N.D.
0.838
0.054
7.140
0.396
0.335
2.390
3.080
3.780
3.720
2.160
2.420
3.810
5.840
6.140
2.690
3.550
3.750
1.820
2.000
2.220
1.950
2.130
1.840
1.760
1.680
N.D.
1.680
N.D.
4.850
1.810
1.900
Non-small
Cell lung
Cancer
A549/ATCC
EKVX
—
—
HOP-62
HOP-92
NCI-H23
NCI-H460
NCI-H522
0.006c
Colon
Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
4.860d
—
—
—
0.244
0.314
0.375
0.405
0.251
0.239
0.358
0.193
0.683
0.166
0.073
0.151
0.097
0.090
0.354
2.700
0.457
0.373
0.320
0.426
0.349
6.470
11.700
3.600
3.070
3.240
4.010
3.680
0.435
0.976
0.404
0.489
0.368
0.365
0.406
0.595
23.400
0.537
0.442
0.395
0.620
0.418
3.130
6.710
2.890
4.520
2.950
2.940
3.140
1.850
1.850
1.740
1.700
1.790
1.890
1.840
KM12
SW-620
—
Melanoma
M14
—
—
—
—
—
0.216
0.035
0.355
0.543
0.518
0.590
0.991
0.671
0.335
0.312
6.560
0.522
0.384
0.112
0.322
0.572
0.090
0.029
0.134
0.407
0.127
0.881
N.D.
1.940
0.053
0.101
1.210
N.D.
0.373
0.095
0.395
0.817
0.451
0.750
0.446
0.564
0.333
0.231
3.750
0.527
0.596
0.376
0.623
1.020
3.860
1.800
3.450
4.930
2.680
4.720
3.090
6.510
2.950
4.200
10.300
24.100
10.000
2.970
12.200
4.870
0.372
0.112
0.397
0.647
0.532
0.446
0.997
0.581
0.408
0.404
16.500
1.750
1.040
0.247
0.533
0.620
0.599
0.184
0.873
0.829
0.529
3.310
0.503
N.D.
0.300
0.366
1.750
2.590
1.630
0.571
0.723
1.880
2.600
0.672
N.D.
1.970
1.740
2.230
1.720
1.720
1.800
1.990
1.600
1.600
1.950
2.040
1.500
1.780
2.530
2.020
1.730
MDA-MB-435
SK-MEL-2
SK-MEL-28
UACC-62
LOX IMVI
MALME-3M
UACC-257
SK-MEL-5
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
3.500
2.550
4.570
6.130
3.240
2.550
4.240
7.930
65.800
3.760
2.170
4.040
5.780
Ovarian
Cancer
0.002d
—
—
—
—
—
0.366
0.080
0.244
0.721
IGROV1
Renal
Cancer
A498
0.380d
0.211
0.561
0.272
0.760
2.220
0.590
1.890
0.586
0.303
0.419
0.129
0.290
0.130
0.798
0.650
0.524
0.472
0.575
0.291
0.310
0.210
0.457
0.236
0.777
0.520
0.481
0.447
0.857
0.476
0.390
2.220
4.010
2.290
6.070
6.520
7.570
7.960
10.200
14.000
2.840
0.251
0.462
1.270
0.888
1.030
1.900
1.780
2.140
1.420
0.654
0.215
0.573
0.363
3.860
N.D.
0.941
0.611
8.310
0.679
0.435
1.910
6.230
2.770
5.530
3.850
4.170
4.850
6.730
8.220
2.200
1.240
1.530
1.210
1.980
1.610
1.750
1.500
1.730
1.980
1.620
CAKI-1
RXF 393
SN12C
UO-31
786-0
ACHN
TK-10
DU-145
PC-3
—
—
—
—
Prostate
Cancer
0.034d
Central
Nervous
System
Cancer
SF-268
SF-295
SF-539
SNB-75
—
0.819
0.229
0.290
0.222
0.958
0.071
0.164
0.257
1.760
0.254
0.239
0.283
6.500
6.100
5.030
2.320
1.500
0.323
0.576
0.326
10.200
0.329
0.517
0.392
7.110
4.890
4.130
1.880
2.920
1.750
1.640
1.650
0.052c
—
Breast Cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
0.437d/0.034f
—
—
0.316
0.570
0.285
0.604
0.468
0.249
0.093
0.310
0.424
0.877
N.D.
0.419
0.531
0.573
0.613
0.466
0.257
5.500
3.660
6.920
5.210
5.240
1.700
0.293
1.110
0.702
0.398
0.502
0.314
0.368
0.184
1.960
6.060
0.804
0.204
3.030
6.040
1.500
2.930
4.160
N.D.
1.790
1.170
2.080
2.960
2.040
1.160
MDA-MB-468
—
0.050
a
b
c
d
e
f
Data obtained from NCI’s in vitro 60 cell 5-dose screen.24
GI50 is the molar concentration of synthetic compound causing 50% growth inhibition of tumor cells.
Data obtained from Ref. 9g.
Data obtained from Ref. 7.
Not determined.
Data obtained from Ref. 25.
Compound 22 has a different substitution pattern of the B-ring
Compound 24, bearing a 10H-phenothiazin-3-yl unit, displays a
single conformation, which establishes hydrogen bonds between
the nitrogen atom of the phenothiazinic ring and the carbonyl of
Val 238. Only minor rotations are permitted round the carbonyl
linker and therefore, there are a rather low number of conforma-
tions differentiated by their indole position.
compared to compounds 20 and 21, and adopts a unique confor-
mation, similar to product 21 (Fig. 2(c)), in which the carbonyl
function is in a favorable position to form hydrogen bonds with
Val 181 and Asn 258.

C.-M. Abuhaie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 147–152
151
O
O
F
NH2
N
N
S
S
Ser 178
Ser 178
OMe
OMe
20
21
Thr 179
Thr 179
Asn 258
Asn 258
Val181
Val181
Val 238
Val 238
(a)
(b)
O
O
N
S
Cl
N
Ser 178
Ser 178
S
N
H
OEt
H
24
22
Thr 179
Thr 179
Asn 258
Asn 258
Val181
Val181
Val 238
Val 238
(c)
(d)
MeO
MeO
OH
Ser 178
OMe
OMe
1
Thr 179
Asn 258
Val181
Val 238
(e)
Figure 2. Structure and docking of phenothiazine derivatives in the tubulin binding site: (a) compound 20, (b) compound 21, (c) compound 22, (d) compound 24, and (e)
combretastatin A-4 (1).

152
C.-M. Abuhaie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 147–152
K.; Hamel, E.; Pettit, R. K.; Chapuis, J. C.; Schmidt, T. M. J. Med. Chem. 2002, 45,
In conclusion, a new class of microtubule-targeting agents bear-
2534; (h) Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C.
C.; Yang, Y. N.; Chang, J. Y.; Lee, S. J.; Hsieh, H. P. J. Med. Chem. 2004, 47, 4247; (i)
Ghinet, A.; Rigo, B.; Hénichart, J.-P.; Le Broc-Ryckewaert, D.; Pommery, J.;
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ing a phenothiazine core was synthesized. In these series, the
replacement of the classical B-ring by various substituents (e.g.,
compounds 22, 23, 25–28, 42 or 43) was also realized. The biolog-
ical results obtained are important for further structure-activity
relationships in these series since there are no reports disclosing
similar modifications to the B-ring in the phenstatin series.
These new derivatives showed significant activities against cel-
lular proliferation and tubulin polymerization, rather similar to
those of phenstatin (2).
Phenothiazine derivative 21 proved to be the most potent com-
pound synthesized with GI50 values ranging from 29 to 93 nM on
different cell lines. The same compound showed a better inhibition
of COLO 205, A498, and MCF7 cell lines than the parent phenstatin
(2). Moreover, compound 21 showed increased inhibitory potential
on K-562 (leukemia cell line) compared to the best phenothiazine
derivative issued from a precedent study (e.g., GI50 = 40.0 nM (Ta-
ble 2) versus 210 nM16). Further investigation on the metabolic
profile of the best candidates issued from this study will be real-
ized in due course.
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Acknowledgments
The authors gratefully acknowledge the CommScie (project
POSDRU/89/1.5/S/63663) for financial support, the ‘Programul
Operational Sectorial Dezvoltarea Resurselor Umane 2007–2013’
(project POSDRU/88/1.5/S/47646) for C.-M. A.’s scholarship, and
the NCI (National Cancer Institute) for the biological evaluation
of synthesized compounds on their 60-cell panel.
18. Hamze, A.; Rasolofonjatovo, E.; Provot, O.; Mousset, C.; Veau, D.; Rodrigo, J.;
Bignon, J.; Liu, J.-M.; Wdzieczak-Bakala, J.; Thoret, S.; Dubois, J.; Brion, J.-D.;
Alami, M. ChemMedChem 2011, 6, 1.
19. Clayton, J.; Clayden, J. Tetrahedron Lett. 2011, 52, 2436.
20. Tung, Y.-S.; Coumar, M. S.; Wu, Y.-S.; Shiao, H.-Y.; Chang, J.-Y.; Liou, J.-P.;
Shukla, P.; Chang, C.-W.; Chang, C.-Y.; Kuo, C.-C.; Yeh, T.-K.; Lin, C.-Y.; Wu, J.-S.;
Wu, S.-Y.; Liao, C.-C.; Hsieh, H.-P. J. Med. Chem. 2011, 54, 3076.
Supplementary data
21. Georgescu, F.; Georgescu, E.; Draghici, C.; Iuhas, P. C.; Filip, P. I. Rev. Roum.
Chim. 2005, 50, 349.
Supplementary data (Synthesis details, physico–chemical char-
acterization for all new compounds and data obtained from the
NCI’s in vitro disease-oriented human tumor cell screen at 10 lM
concentration for compounds 26, 28, 38, and 42 (Table 3) are avail-
able in the ‘supplementary data’ section) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2012.10.135.
22. Beemelmanns, C.; Lentz, D.; Reissig, H.-U. Chem. Eur. J. 2011, 17, 9720.
23. Tubulin studies: sheep brain tubulin was purified according to the method of
Shelanski (Shelanski, M. L.; Gaskin, F.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A.,
1970, 70, 765) by two cycles of assembly buffer containing 0.1 M MES, 0.5 mM
MgCl2, 1 mM EGTA, and 1 mM of GTP, pH 6.6 (the concentration of tubulin was
about 2-3 mg/mL). Tubulin assembly was monitored by fluorescence according
to reported procedure (Barron, D. M.; Chatterjee, S. K.; Ravindra, R.; Roof, R.;
Baloglu, E.; Kingston, D. G. I.; Bane, S. Anal. Biochem. 2003, 315, 49) using DAPI
as fluorescent molecule. Assays were realized on 96-well plates prepared with
Biomek NKMC and Biomek 3000 from Beckman coulter and read at 37 °C on
Wallac Victor fluorimeter from Perkin-Elmer. The IC50 value of each compound
was determined as tubulin by 50% compared to the rate in the absence of
compound. The IC50 values for all compounds were compared to the IC50 of
phenstatin and desoxypodophyllotoxin and measured the same day under the
same conditions.
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a and b-
tubulin was taken from the 1sa043 entry of the RCSB Protein Data Bank
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site was thought to be that of the co-crystallised thiocolchicine. Flexible
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2002, 1, 11) and
a visual assessment of the consistency of the docking
solutions, expressed as the closeness of the thirty generated conformations.