New trimethoxybenzamides and trimethoxyphenylureas derived from dimethylcarbazole as cytotoxic agents. Part i

Article abstract of DOI:10.1002/jhet.1951

A convenient synthesis of novel functionalized 1,4-dimethylcarbazole derivatives containing 3,4,5-trimethoxybenzamido-ureido or N-(3,4,5- trimethoxyphenyl)ureido group starting from their corresponding indole derivatives is reported. Three derivatives pre

Full text of DOI:10.1002/jhet.1951

E294
New Trimethoxybenzamides and Trimethoxyphenylureas Derived from
Dimethylcarbazole as Cytotoxic Agents. Part I
Vol 51
Antonella Panno,a,b† Maria Stefania Sinicropi,b† Anna Caruso,a,b Hussein El-Kashef, Jean-Charles Lancelot,a
a,c
*
Geneviève Aubert,d Aurélien Lesnard,a Thierry Cresteil,d and Sylvain Raulta
aUniversité de Caen Basse-Normandie, Centre d’Etudes et de Recherche sur le Médicament de Normandie UPRES EA
4258–FR CNRS 3038 INC3M, Bd Becquerel, 14032 Caen Cedex, France
bDipartimento di Scienze Farmaceutiche, Università della Calabria, 87036 Arcavacata di Rende Cosenza, Italy
cChemistry Department, Faculty of Science, Assiut University, 71516, Assiut, Egypt
dInstitut de Chimie des Substances Naturelles, UPR 2301, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
*
E-mail: anna.caruso@unical.it
†These authors equally contributed to the manuscript.
Received October 18, 2012
DOI 10.1002/jhet.1951
Published online 1 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
O
O
R
H
N
O
O
NH2
R
N
R
H
N
H
N
H
H
O
R
H
N
N
O
O
N
O
H
A convenient synthesis of novel functionalized 1,4-dimethylcarbazole derivatives containing 3,4,
5-trimethoxybenzamido-ureido or N-(3,4,5-trimethoxyphenyl)ureido group starting from their corresponding
indole derivatives is reported. Three derivatives prepared (5g, 6c, and 6g) were active against leukemia cell lines
HL60. Both 5g and 6g showed potent antiproliferative activity against KB cell lines, likely associated with the
inhibition of tubulin polymerization.
J. Heterocyclic Chem., 51, 294 (2014).
INTRODUCTION
RESULTS AND DISCUSSION
Many of carbazole derivatives show antioxidative and
biological activities, such as antitumor, psychotropic, anti-
inflammatory, antihistaminic, and antibiotic activities [1].
The carbazole-based naturally occurring heterocycles,
Mahanine (I) [2] and Ellipticine (II) [3] (Fig. 1), are among
the anti-cancer drugs acting selectively on tumor cells by
inducing apoptosis or pseudo-apoptosis. Mahanine has
been shown to exhibit a wide range of pharmacological
effects: antimutagenicity against heterocyclic amines;
antimicrobial activity against Gram positive bacteria;
anti-inflammatory effect; and acts as a potent apoptosis-
inducing agent in HL-60 cells [4–7]. Ellipticine, a DNA
intercalator, is used for the treatment of osteolytic metasta-
ses of breast cancer [8].
Numerous derivatives of DMC are potent cytostatic
agents [9], and probably their action is due to intercalat-
ing mechanism into DNA. They may interact directly or
indirectly with nuclear DNA leading to severe chromo-
some aberrations. However, experimental evidences
showed that these compounds may bind to DNA not
only through intercalation but also by covalent binding
or generation of strand breaks [10] and exert their cyto-
toxic effects by enhancing enzyme-mediated DNA
breaking within the genome [11–14]. Moreover, they
can target topoisomerases, the enzymes that modulate
DNA topology in vivo, and involved in virtually every
aspect of DNA metabolism [15].
Rescifina et al. showed that in order to achieve an effec-
tive intercalation in the DNA, it is necessary that, in addition
to the presence of fused polyaromatic systems, these systems
may carry amino and/or amide groups. These groups favor a
good interaction through the formation of hydrogen bonding
with DNA bases [16].
On the other hand, the important naturally occurring
antimitotic tubulin-binding agents Combretastatin A-4 (III)
and Colchicine (IV) involve a common trimethoxybenzene
nucleus in their structures, which plays a crucial role in their
biological activity (Fig. 2).
Thus, it deemed of interest to incorporate this tri-
methoxybenzene moiety in a carbazole nucleus with the
aim that such a combination may result in an enhanced
anticancer activity.
Currently, anticancer therapy, on the basis of microtu-
bule-targeting agents, is receiving growing attention [17].
Tubulin heterodimers, the major component of microtu-
bules, are the molecular target of these agents. Inhibition
© 2014 HeteroCorporation

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New Trimethoxybenzamides and Trimethoxyphenylureas Derived from
Dimethylcarbazole as Cytotoxic Agents. Part I
E295
IC50 values between 0.012 and 0.298 mmol/L. Also, some
carbazole-3-sulfonamides (VI) (Fig. 3) related to Combre-
tastatin A-4 (III) (Fig. 2) have been prepared and evaluated
for their antiproliferative activity [22]. The biological
activities of urea-containing compounds have been also
reported [23].
Figure 1. Naturally occurring heterocycles Mahanine (I) and Ellipticine (II).
In this context, we wish to report herein the synthesis
novel functionalized DMC derivatives containing 3,4,5-
trimethoxybenzamido- and N(3,4,5-trimethoxyphenyl)
ureido group 5a–h and 6a–h, respectively as potential anti-
cancer agents (Scheme 1). These compounds were prepared
starting from known 3-aminocarbazole derivatives 4a–h that
were synthesized previously by us [24] starting from the
indole derivatives 1a–h (Scheme 1).
Figure 2. Combretastatin A-4 (III) and Colchicine (IV).
Thus, the reaction of 3-aminocarbazoles 4a–h with
stoichiometric amounts of 3,4,5-trimethoxybenzoic acid
(7) in chloroform at room temperature in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and
4-dimethylaminopyridine gave the corresponding 3-
(3,4,5-trimethoxybenzamido)carbazoles 5a–h in 41–70
% yield (Scheme 2, Table 1).
On the other hand, when 4a–h were reacted with 3,4,
5-trimethoxybenzoyl azide 8 in refluxing anhydrous aceto-
nitrile, the N-(3,4,5-trimethoxyphenyl)ureas 6a–h were
obtained in good yield (58–74 %) (Scheme 3, Table 2).
The acid azide 8 has been prepared earlier by converting
the 3,4,5-trimethoxybenzoic acid into the corresponding
acid chloride followed by treatment with sodium azide
under phase-transfer catalysis [25]. However, we have
prepared this azide following a more convenient one-pot
reaction through the formation of its mixed acid anhydride,
as shown in the Experimental part.
of tubulin polymerization or interfering with microtubule
disassembly disrupts several cellular functions, including
cell motility and mitosis [18–20].
De Martino et al. [17] have shown that 3-(3,4,5-
trimethoxyphenylthio)indoles have potencies comparable
with those of the reference compounds Colchicine and Com-
bretastatin A-4 in both tubulin assembly and cell growth
inhibition assays.
Yue-Ming Wang [21] has synthesized a new tubulin
ligand N-(2,6-dimethoxypyridine-3-yl)-9-methylcarbazole-
3-sulfonamide (IG-105) (V) (Fig. 3), which showed a potent
activity against human leukemia and solid tumors in
breast, liver, prostate, lung, skin, colon, and pancreas with
BIOLOGICAL EVALUATION
All the carbazole derivatives prepared 5a–h and 6a–h were
Figure 3. Carbazole-3-sulfonamides. IG-105 (V).
evaluated for their anticancer activity against KB cell lines
Scheme 1. Novel functionalized 1,4-dimethylcarbazole derivatives. Reaction conditions: (i) acetonylacetone, p-TSA, reflux EtOH; (ii) HNO3,
(CH3CO)2O, rt; (iii) SnCl2, HCL, reflux or Pd/C, H2, EtOH; (iv) 3,4,5-trimethoxybenzoic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide (EDCI), 4-dimethylaminopyridine (DMAP), CHCl3 rt; and (v) 3,4,5-trimethoxybenzoyl azide, reflux CH3CN.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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A. Panno, M. S. Sinicropi, A. Caruso, H. El-Kashef,
Vol 51
J.-C. Lancelot, G. Aubert, A. Lesnard, T. Cresteil, and S. Rault
Scheme 2
Table 1
Mini-library of synthesized amidocarbazoles 5a-h.
Starting compound
R
R0
Product
R
R0
Yield (%)
4a
4b
4c
Cl
F
Br
H
H
H
5a
5b
5c
Cl
F
Br
H
H
H
H
70
60
43
56
4d
4e
NH2
H
H
5d
5e
CH2CH3
H
CH2CH3
H
43
41
4f
OH
H
5f
4g
4h
CH3
OCO2C2H5
H
H
5g
5h
CH3
OCO2C2H5
H
H
42
51
at 10–6 and 10–5 M concentrations (Table 3). All the
compounds tested showed no inhibition at a concentration
of 10–6 M with the exception of the two compounds 6a and
substituent. Similarly, in the trimethoxyphenylcarbamido
series 5a–h, compound 5g having also a methyl group at
position 6 showed the highest activity. The substitution of
this methyl group by bromine atom led to a decrease of
activity, whereas its substitution with fluorine atom strongly
decreased the activity. Complete abolishing of activity
was observed when the methyl group was substituted with
chlorine, trimethoxybenzamido, trimethoxybenzoyloxy, or
ethoxycarbonyloxy substituent.
6c that showed very low inhibition. However, at 10ꢀ5
M
concentration, some of the tested compounds showed
inhibitory activity. In the trimethoxyureido series 6a–h,
compound 6g that possesses a methyl group at position
6 displayed the highest inhibition. The substitution of this
methyl group by halogens led to a decrease of activity in
the order Br › F › Cl 6c, 6b, 6a [24,26]. However, the ac-
tivity is abolished when the methyl group was substituted
by trimethoxybenzoylamino or trimethoxybenzoyloxy
Within the two series compounds, 5g, 6h, and 6g
showed the highest inhibition values of 81, 91, and 100,
respectively at a concentration of 10ꢀ5 M.
Scheme 3
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Table 2
Mini-library of synthesized ureidocarbazoles 6a-h.
Starting compd.
R
R0
Product
R
R0
Yield (%)
4a
4b
4c
Cl
F
Br
H
H
H
6a
6b
6c
Cl
F
Br
H
H
H
H
70
60
74
60
4d
NH2
H
6d
4e
4f
4g
4h
H
OH
CH3
CH2CH3
6e
6f
6g
6h
H
OH
CH3
CH2CH3
62
58
68
67
H
H
H
H
H
H
OCO2C2H5
OCO2C2H5
spectrometer at ionizing potential of 70eV (EI) or by using a
spectrometer LC-MS Waters alliance 2695 (ESI+). 1H NMR
(400 MHz) and 13C NMR (100MHz) spectra were recorded on a
JEOL Lambda 400 spectrometer. Chemical shifts are expressed in
parts per million downfield from tetramethylsilane as an internal
standard. Thin layer chromatography was performed on silica gel
60F-264 (Merck).
These three latter derivatives were also screened against
HL60 cell lines (Table 4) and for their inhibition activity of
tubulin polymerization (Table 5).
As shown in Table 3, compounds 5g and 6g proved to
be potent inhibitors of cell proliferation, whereas the
replacement of the methyl group of 6g by a bromine atom
6c resulted in a reduction of its antiproliferative activity
(62% at 10ꢀ4 M). These three molecules showed compara-
ble activities when tested against the leukemia cell lines
HL60 (Table 4).
Preparation of 1,4-dimethyl-8-ethyl-9H-carbazole derivatives
(2e, 3e, 4e).
Compound 2e has been prepared by the reaction
of the indole derivative 1e (10 g, 0.069 mol) with hexane-2,5-dione
(11.77 g, 0.103 mol) in the presence of p-toluenesulfonic acid
(13.09 g, 0.069 mol) in boiling ethanol (100 mL) as reported by
Cranwell and Saxton reaction [27].
The 1,4-dimethylcarbazole 2e (2 g, 0.009 mol) obtained has
been treated with concentrated HNO3 (0.56g, 0.009mol) in acetic
anhydride (30 mL) at room temperature to give the 3-nitro carbazole
3e (2 g, 0.007 mol), which was in turn transformed into the amino de-
rivative 4e by the reduction of corresponding nitro group of 3e with
stannous chloride (10 g, 0.045 mol), in hydrochloric acid 20 mL.
In an attempt to elucidate the mode of action of
carbazole derivatives, the inhibition of tubulin polymerization
was investigated. As expected, compounds are weak inhibi-
tors of tubulin assembly (Table 5).
CONCLUSION
1,4-Dimethyl-8-ethyl-9H-carbazole (2e).
Gray powder
ꢀ1
(yield 85%). Mp 118 C. IR (KBr): n = 3470 (NH) cm
.
1H
Several novel functionalized DMC derivatives containing
3,4,5-trimethoxybenzamido- or N-(3,4,5-trimethoxyphenyl)
ureido group have been synthesized starting from their
corresponding indole derivatives. Out of all the carbazoles
prepared, three derivatives (5g, 6c, and 6g) were active
against leukemia cell lines HL60. Both 5g and 6g showed
potent antiproliferative activity against KB cell lines, likely
associated with the inhibition of tubulin polymerization;
however, the presence of a bromine atom in the structure
of 6c diminishes the antiproliferative activity.
ꢁ
ꢀ
NMR (DMSO-d6): d 1.36 (t, J = 7.3 Hz, 3H, CH2CH3); 2.61 (s,
3H, CH3); 2.78 (s, 3H, CH3); 3.05–3.11 (q, J = 7.3 Hz, 2H,
CH2CH3); 6.87 (d, J = 6.8 Hz, 1H, Ar); 7.10 (d, J = 6.8 Hz, 1H,
Ar); 7.15 (t, J = 7.8 Hz, 1H, Ar); 7.25 (d, J = 6.8 Hz, 1H, Ar); 7.98
(d, J = 7.8 Hz, 1H, Ar); 10.72 (s, 1H, NH). 13C NMR (DMSO-d6):
14.62; 17.28; 20.35; 23.88; 117.84; 118.96; 119.59; 120.11;
121.08; 123.43; 123.61; 125.80; 126.55; 129.43; 138.35; 139.09.
MS (ESI+): 224 (M + H)+. Anal. Calcd for C16H17N: C, 86.05; H,
7.67; N, 6.27. Found: C, 86.09; H, 7.70; N, 6.31.
1,4-Dimethyl-8-ethyl-3-nitro-9H-carbazole (3e).
powder (yield 75%). Mp 232ꢁC. IR (KBr): ꢀn = 3354 (NH); 1304
(NO2) cmꢀ1 1H NMR (DMSO-d6): d 1.30 (t, J = 7.3 Hz, 3H,
Yellow
.
CH2CH3); 2.59 (s, 3H, CH3); 2.90 (s, 3H, CH3); 2.99–3.03 (q,
J = 7.3 Hz, 2H, CH2CH3); 7.17–7.21 (m, 1H, Ar); 7.30 (d,
J = 6.8 Hz, 1H, Ar); 7.80 (s, 1H, Ar); 8.03 (d, J = 7.8 Hz, 1H, Ar);
11.28 (s, 1H, NH). 13C NMR (DMSO-d6): 13.58; 17.38; 20.32;
24.00; 114.20; 117.17; 118.11; 120.09;122.93; 123.98; 124.53;
127.00; 128.80; 130.43; 145.70; 146.28. MS (ESI+): 269
(M + H)+. Anal. Calcd for C16H16N2O2: C, 71.62; H, 6.01; N,
10.44. Found: C, 71.58; H, 6.04; N, 10.48.
EXPERIMENTAL
Chemistry. Commercial reagents were purchased from Aldrich
(Saint-Quentin Fallavier, France), Acros Organics (Geel, Belgium),
and Alfa Aesar (Schiltigheim, France) and used without additional
purification. Melting points were determined on a Kofler melting
point apparatus. IR spectra were recorded on a Perkin Elmer BX FT-
IR. Mass spectra were obtained by using a JEOL JMS GCMate
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Table 3
Antiproliferative activity of carbazoles 5a–h and 6a–h against human cell lines KB.
Compound number
Structure
10ꢀ5 M (%)
10ꢀ6 M (%)
5a
0
0
5b
5c
5d
2
35
0
0
0
0
5e
5f
7
0
0
0
5g
5h
81
0
0
0
(Continued)
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Dimethylcarbazole as Cytotoxic Agents. Part I
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Table 3
(Continued)
Compound number
Structure
10ꢀ5 M (%)
10ꢀ6 M (%)
6a
51
3
6b
33
0
6c
25
5
0
6d
0
6e
6f
50
0
0
0
0
6g
100
6h
91
0
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Table 4
C24H23ClN2O4: C, 65.68; H, 5.28; N, 6.38. Found: C, 65.64; H,
5.31; N, 6.42.
Antiproliferative activity of selected carbazoles against KB and HL60
cell lines.
N-(1,4-Dimethyl-6-fluoro-9H-carbazol-3-yl)-3,4,5-trimethoxy-
benzamide (5b). Yellow powder (yield 30%). Mp > 260ꢁC. IR
1
(KBr): ꢀn = 3240 (NH); 1622 (C═O)cmꢀ1. H NMR (DMSO-d6):
Compound number
KB
HL60
d 2.46 (s, 3H, CH3); 2.56 (s, 3H, CH3); 3.70 (s, 3H, OCH3); 3.83
(s, 6H, OCH3); 7.08 (s, 1H, Ar); 7.22 (t, J = 8.7 Hz, 1H, Ar); 7.35
(s, 2H, Ar); 7.47-7.51 (m, 1H, Ar); 7.86 (d, J = 7.8Hz, 1H, Ar);
9.95 (s, 1H, NHC═O); 11.28 (s, 1H, NH). 13C NMR (DMSO-d6):
15.08; 16.57; 56.06; 60.15; 105.12; 107.52; 111.81; 112.54;
117.66; 120.55; 123.59; 126.51; 126.62; 127.11; 129.81; 136.76;
138.69; 140.10; 152.70; 156.32; 165.14. MS (ESI+): 423
(M+ H)+. Anal. Calcd for C24H23FN2O4: C, 68.23; H, 5.49; N,
6.63. Found: C, 68.27; H, 5.46; N, 6.59.
ꢀ6
IC50 (mM) 10ꢀ5
>10
M
IC50 (mM)
9.1 ꢂ 0.3
6c
6g
5g
52 ꢂM5//170ꢂ 11
6.7 ꢂ 3.0
9.6 ꢂ 0.1
97 ꢂ 1/5 ꢂ 8
5.3 ꢂ 0.6
>10
26 ꢂ 3/7 ꢂ 3
Table 5
Tubulin polymerization inhibitory activity of selected carbazoles.
N-(6-Bromo-1,4-dimethyl-9H-carbazol-3-yl)-3,4,5-trimethoxy-
benzamide (5c). White powder (yield 43%). Mp> 260ꢁC. IR
Compound number
Inhibition of tubulin polymerization
ꢀ
(KBr): n = 3242 (NH); 2939 (OCH3); 1644 (C═O amide); 1130
(CN); 1580; 1491; 1005; 737 cmꢀ1. 1H NMR (DMSO-d6): d 2.52
(s, 3H, CH3); 2.60 (s, 3H, CH3); 3.73 (s, 3H, OCH3); 3.86 (s, 6H,
OCH3); 7.13 (s, 1H, Ar); 7.38 (s, 2H, Ar); 7.51 (s, 2H, Ar); 8.25
(s, 1H, Ar); 9.98 (s, 1H, NHC═O); 11.46 (s, 1H, NH). 13C NMR
(DMSO-d6): 15.20; 16.57; 56.07; 60.15; 105.17; 110.70;
113.01; 117.73; 119.89; 124.20; 125.29; 126.69; 126.77;
127.32; 127.57; 129.76; 138.06; 138.96; 140.14; 152.71;
165.18. MS (ESI+): 483 and 485 (M + H)+. Anal. Calcd for.
C24H23BrN2O4: C, 59.64; H, 4.80; N, 5.80. Found: C, 59.67;
H, 4.78; N, 5.77.
6c
6g
8% at 6.7 ꢃ 10ꢀ5
50% at 6.7 ꢃ 10ꢀ5
3% at 3.3 ꢃ 10ꢀ5
20% at 6.7 ꢃ 10ꢀ5
M
M
M
M
a
5g
aThe concentration was reduced to 3.3 ꢃ 10ꢀ5 M because of the optical
interference at 350 nm.
3-Amino-1,4-dimethyl-8-ethyl-9H-carbazole (4e).
Yellow
N-(1,4-Dimethyl-6-(3,4,5-trimethoxybenzamido)-9H-carbazol-3-
ꢁ
ꢀ
yl)-3,4,5-trimethoxy benzamide (5d).
White powder (yield
powder (yield 45%). Mp 182 C. IR (KBr): n = 3410, 3344 (NH2);
3254 (NH) cmꢀ1. 1H NMR (DMSO-d6): d 1.29 (t, J = 7.8 Hz, 3H,
CH2CH3); 2.45 (s, 3H, CH3); 2.48 (s, 3H, CH3); 2.93–3.00 (q,
J = 7.8 Hz, 2H, CH2CH3); 4.35 (bs, 2H, NH2); 6.63(s, 1H, Ar);
6.98 (t, J = 7.8 Hz, 1H, Ar); 7.09 (d, J = 6.8 Hz, 1H, Ar); 7.92 (d,
J = 7.7 Hz, 1H, Ar); 10.16 (s, 1H, NH). 13C NMR (DMSO-d6):
13.75; 14.65; 17.42; 23.95; 111.89; 116.74; 117.72; 118.16;
119.82; 122.11; 123.04; 123.64; 126.37; 132.83; 138.68; 138.99.
MS (EI) m/z (%): 238 (M+. 100); 208 (74). Anal. Calcd for
C16H18N2: C, 80.63; H, 7.61; N, 11.75. Found: C, 80.59; H, 7.65;
N, 11.79.
36%). Mp > 260 C. IR (KBr):
ꢁ
ꢀn = 3367–3215 (NH); 1631 (C═O
amide); cmꢀ1. 1H NMR (DMSO-d6): d 2.53 (s, 3H, CH3); 2.64 (s,
3H, CH3); 3.73 (s, 6H, OCH3); 3.87 (s, 12H, OCH3); 7.08 (s, 1H,
Ar); 7.34 (s, 2H, Ar); 7.39 (s, 2H, Ar); 7.52 (d, J = 8.7Hz, 1H,
Ar); 7.71 (d, J = 8.7Hz, 1H, Ar); 8.58 (s, 1H, Ar); 9.96 (s, 1H,
NHC═O); 10.18 (s, 1H, NHC═O); 11.22 (s, 1H, NH). 13C
NMR (DMSO-d6): 15.10; 16.64; 56.09; 56.14; 60.18;
105.17; 105.22; 110.73; 114.98; 117.55; 119.90; 120.84;
123.24; 126.06; 126.32; 127.13; 129.86; 130.45; 130.66;
137.28; 138.20; 140.10; 140,14; 152.67; 152.73; 164.69;
165.19. MS (ESI+): 614 (M + H)+. Anal. Calcd for
C34H35N3O8: C, 66.55; H, 5.75; N, 6.85. Found: C, 66.52;
H, 5.72; N, 6.88.
General procedure for the preparation of 3,4,5-trimethoxyben-
zamides (5a–h). To a stirred solution of aminocarbazole 4a–h (2g,
0.009mol) in CHCl3 (80 mL), 3,4,5-trimethoxybenzoic acid (2,4g
0.012mol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.2g
0.012mol), and 4-dimethylaminopyridine have been added
successively. Stirring was continued at rt for 16h. The reaction
mixture was then diluted with chloroform, and the organic layer
was separated, washed with saturated aqueous solution of
NaHCO3 and then with water. It was then dried over anhydrous
magnesium sulfate, filtered, and concentrated under reduced pressure.
The solid precipitate obtained was filtered and recrystallized from
acetonitrile.
N-(1,4-Dimethyl-8-ethyl-9H-carbazol-3-yl)-3,4,5-trimethoxy-
benzamide (5e).
(KBr):
Yellow powder (yield 23%). Mp 172ꢁC. IR
ꢀn = 3283 (NH); 1645 (C═O amide) cmꢀ1
.
1H NMR
(DMSO-d6): d 1.33 (t, J = 7.3Hz, 3H, CH3); 2.58 (s, 3H, CH3);
2.60 (s, 3H, CH3); 3.04 (q, J = 6.8 Hz, 2H, CH2CH3); 3.73 (s, 3H,
OCH3); 3.86 (s, 6H, OCH3); 7.07 (s, 1H, Ar); 7.12 (t, 1H, Ar);
7.22 (d, 1H, Ar); 7.38 (s, 2H, Ar); 7.99 (d, 1H, Ar); 9.96 (s, 1H,
NHC═O); 10.73 (s, 1H, NH). 13C NMR (DMSO-d6): 14.58;
15.29; 17.09; 23.79; 56.07; 60.16; 105.13; 117.75; 119.09;
119.71; 121.36; 123.52; 123.73; 125.91; 126.22; 126.75; 127.24;
129.89; 137.57; 138.76; 140.09; 152.72; 165.13. MS (ESI+): 433
(M+ H)+. Anal. Calcd for C26H28N2O4: C, 72.20; H, 6.53; N,
6.48. Found: C, 72.23; H, 6.50; N, 6.51.
N-(6-Chloro-1,4-dimethyl-9H-carbazol-3-yl)-3,4,5-trimethoxy-
benzamide (5a). White powder (yield 40%). Mp> 260ꢁC. IR
(KBr): ꢀn = 3243 (NH); 2939 (OCH3); 1700–1623 (C═O amide);
1
1131 (C–N); 1580; 1493; 814; 766 cmꢀ1. H NMR (DMSO-d6):
d 2.52 (s, 3H, CH3); 2.60 (s, 3H, CH3); 3.73 (s, 3H, OCH3); 3.86
(s, 6H, OCH3); 7.13 (s, 1H, Ar); 7.37–743 (m, 3H, Ar); 7.55 (d,
J = 8.7 Hz, 1H, Ar); 8.12 (s, 1H, Ar); 9.98 (s, 1H, NHC═O);
11.44 (s, 1H, NH). 13C NMR (DMSO-d6): 15.19; 16.57; 56.07;
60.16; 105.16; 112.51; 117.73; 120.01; 121.33; 122.88; 124.60;
124.71; 126.69; 126.74; 127.53; 129.78; 138.25; 138.71; 140.14;
152.71; 165.18. MS (ESI+): 438 (M+ H)+. Anal. Calcd for
5,8-Dimethyl-6-(3,4,5-trimethoxybenzamide)-9H-carbazol-3-
yl-3ꢁ,4,5-trimethoxybenzoate (5f). White powder (yield 41%). Mp
amide) cmꢀ1. H NMR (DMSO-d6): d 2.55 (s, 3H, CH3); 2.59 (s,
3H, CH3); 3.73 (s, 3H, OCH3); 3.78 (s, 3H, OCH3); 3.86 (s, 6H,
OCH3); 3.88 (s, 6H, OCH3); 7.12 (s, 1H, Ar); 7.28 (d, J=8.7Hz,
1H, Ar); 7.38 (s, 2H, Ar); 7.46 (s, 2H, Ar); 7.59 (d, J= 11.1 Hz,
150 C. IR (KBr):
ꢀn = 3361 (NH); 1715 (C═O ester); 1646 (C═O
1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

2014
New Trimethoxybenzamides and Trimethoxyphenylureas Derived from
Dimethylcarbazole as Cytotoxic Agents. Part I
E301
1H, Ar); 7.99 (s, 1H, Ar) 9.97 (s, 1H, NHC═O); 11.38 (s, 1H, NH).
13C NMR (DMSO-d6): 15.14; 16.64; 56.08; 56.17; 60.16; 60.32;
105.17; 107.16; 111.38; 114.83; 117.69; 119.19; 120.66; 123.56;
124.42; 126.46; 126.56; 127.30; 129.82; 138.10; 138.45; 140,14;
142.27; 143.34; 152.72; 152.94; 165.13; 165.18. MS (ESI+): 615
(M + H)+. Anal. Calcd for C34H34N2O9: C, 66.44; H, 5.58; N, 4.56.
Found: C, 66.41; H, 5.61; N, 4.59.
124.19; 125.45; 127.57; 132.09; 136.58; 136.76; 137.86; 152.87;
154.05; 156.23. MS (ESI+): 438 (M + H)+. Anal. Calcd for
C24H24FN3O4: C, 65.89; H, 5.53; N, 9.61. Found: C, 65.91; H,
5.50; N, 9.58.
N-(6-Bromo-1,4-dimethyl-9H-carbazol-3-yl)-N0-(3,4,5-trimetho-
xyphenyl)urea (6c). Silver powder (yield 74%). Mp 212ꢁC. IR
1
(KBr): ꢀn = 3345–3251 (NH); 1644 (C═O amide) cmꢀ1. H NMR
3,4,5-Trimethoxy-N-(1,4,6-trimethyl-9H-carbazol-3-yl)
(DMSO-d6): d 2.49 (s, 3H, CH3); 2.63 (s, 3H, CH3); 3.59 (s, 3H,
OCH3); 3.72 (s, 6H, OCH3); 6.81 (s, 2H, Ar); 7.33 (s, 1H, Ar);
7.48 (s, 2H, Ar); 7.94 (s, 1H, Ar); 8.23 (s, 1H, NHC═O); 8.68 (s,
1H, NHC═O); 11.34 (s, 1H, NH). 13C NMR (DMSO-d6): 14.62;
16.69; 55.65; 60.16; 95.69; 110.49; 112.93; 117.44; 119.78;
124.05; 124.20; 125.29; 125.52; 127.16; 128.04; 132.11; 136.54;
137.16; 138.94; 152.88; 153.99. MS (ESI+): 498, 500 (M + H)+.
Anal. Calcd for C24H24BrN3O4: C, 57.84; H, 4.85; N, 8.43. Found:
C, 57.81; H, 4.83; N, 8.47.
benzamide (5g).
IR (KBr): ꢀn = 3249 (NH); 1644 (C═O amide)cmꢀ1
White powder (yield 22%). Mp> 260ꢁC.
.
1H NMR
(DMSO-d6): d 2.49 (s, 6H, CH3); 2.61 (s, 3H, CH3); 3.73 (s, 3H,
OCH3); 3.86 (s, 6H, OCH3); 7.04 (s, 1H, Ar); 7.21 (d, J = 7.8 Hz,
1H, Ar); 7.38 (s, 2H, Ar); 7.42 (d, J = 7.8 Hz, 1H, Ar); 7.95 (s, 1H,
Ar) 9.94 (s, 1H, NHC═O); 11.08 (s, 1H, NH). 13C NMR
(DMSO-d6): 15.27; 16.62; 21.39; 56.06; 60.14; 105.12; 110.78;
117.21; 120.58; 122.00; 123.72; 125.72; 126.17; 126.37; 126.92;
127.12; 129.87; 137.82; 138.51; 140.06; 152.68; 165.09. MS
(ESI+): 419 (M + H)+. Anal. Calcd for C25H26N2O4: C, 71.75; H,
6.26; N, 6.69. Found: C, 71.78; H, 6.23; N, 6.72.
N-(1,4-Dimethyl-6-(3,4,5-trimethoxyureido)-9H-carbazol-3-yl)-
N0-(3,4,5-trimethoxyphenyl)urea (6d).
Pink powder (yield
60%). Mp> 260ꢁC. IR (KBr): n = 3289 (NH); 1640 (C═O
amide)cmꢀ1. 1H NMR (DMSO-d6): d 2.49 (s, 3H, CH3); 2.65 (s,
3H, CH3); 3.60 (s, 3H, OCH3); 3.62 (s, 3H, OCH3); 3.73 (s, 6H,
OCH3); 3.75 (s, 6H, OCH3); 6.81 (s, 2H, Ar); 6.83 (s, 2H, Ar);
7.24 (s, 1H, Ar); 7.38–7.46 (m, 2H, Ar); 7.91 (s, 1H, NHC═O);
8.24 (s, 1H, NHC═O); 8.52 (s, 1H, NHC═O); 8.54 (s, 1H,
NHC═O); 8.67 (s, 1H, Ar); 11.00 (s, 1H, NH). 13C NMR
(DMSO-d6): 14.39; 16.43; 55.66; 55.76; 60.00; 96.12; 96.50;
110.64; 112.89; 116.96; 118.25; 120.59; 123.40; 123.94; 124.68;
127.29; 130.91; 132.43; 132.68; 136.02; 136.36; 136.46; 137.29;
152.76; 152.80; 153.16; 153.94. MS (ESI+): 644 (M+ H)+. Anal.
Calcd for C34H37N5O8: C, 63.44; H, 5.79; N, 10.88. Found: C,
63.41; H, 5.81; N, 10.84.
ꢀ
Ethyl (1,4-dimethyl-3-(3,4,5-trimethoxybenzamido)-9H-carbazol-
6-yl)carbonate (5h). White powder (yield 31%). Mp 206ꢁC. IR
ꢀ
(KBr): n = 3366–3265 (NH); 2935 (OCH3); 1733 (C═O ester);
1645 (C═O amide); 1129 (CN); 1583; 1492; 1266; 1003;
762 cmꢀ1. 1H NMR (DMSO-d6): d 1.30 (t, J = 6.83 Hz, 3H, CH3);
2.53 (s, 3H, CH3); 2.58 (s, 3H, CH3); 3.73 (s, 3H, OCH3); 3.86 (s,
6H, OCH3); 4.26 (q, J = 6.8Hz, 2H, CH2CH3); 7.11 (s, 1H, Ar);
7.25 (d, J = 8.7 Hz, 1H, Ar); 7.38 (s, 2H, Ar); 7.54 (d, J = 8.7Hz,
1H, Ar); 7.95 (s, 1H, Ar) 9.97 (s, 1H, NHC═O); 11.36 (s, 1H,
NH). 13C NMR (DMSO-d6): 14.13; 15.12; 16.59; 56.07; 60.15;
64.43; 105.14; 111.29; 114.32; 117.65; 118.61; 120.58; 123.37;
126.44; 126.54; 127.29; 129.80; 138.04; 138.46; 140.10; 143.60;
152.70; 154.03; 165.10. MS (ESI+): 493 (M + H)+. Anal. Calcd
for C27H28N2O7: C, 65.84; H, 5.73; N, 5.69. Found: C, 65.87; H,
5.70; N, 5.72.
N-(1,4-Dimethyl-8-ethyl-9H-carbazol-3-yl)-N’-(3,4,5-trimetho-
xyphenyl)urea (6e). Yellow powder (yield 62%). Mp 240ꢁC. IR
(KBr): ꢀn = 3307 (NH); 1640 (C═O amide)cmꢀ1 1H NMR
.
General procedure for the preparation of N-(carbazol-3-yl)-
N0-(trimethoxyphenyl) ureas (6a–h). To a stirred solution of
the aminocarbazole 4a–h (2 g, 0.009 mol) in anhydrous
acetonitrile (50 mL) was added 3,4,5-trimethoxybenzoyl azide,
and the reaction mixture was heated at reflux for 3 h. The solid
precipitate obtained was filtered and purified by washing with
diethyl ether.
(DMSO-d6): d 1.32 (t, J =7.3 Hz, 3H, CH3); 2.56 (s, 3H, CH3);
2.64 (s, 3H, CH3); 3.03 (q, J = 6.8 Hz, 2H, CH2CH3); 3.60 (s, 3H,
OCH3); 3.74 (s, 6H, OCH3); 6.82 (s, 2H, Ar); 7.06–7.11 (m, 1H,
Ar); 7.18–7.22 (m, 1H, Ar); 7.25 (s, 1H, Ar); 7.92 (s, 1H,
NHC═O); 7.98 (d, J = 7.8Hz, 1H, Ar); 8.67 (s, 1H, NHC═O);
10.62 (s, 1H, NH). 13C NMR (DMSO-d6): 14.20; 14.45; 16.89;
23.49; 56.66; 60.00; 96.15; 117.33; 118.72; 119.45; 121.22;
123.34; 123.44; 123.79; 124.68; 126.47; 127.58; 132.45; 136.38;
136.68; 138.67; 152.77; 153.96. MS (ESI+): 448 (M + H)+. Anal.
Calcd for C26H29N3O4: C, 69.78; H, 6.53; N, 9.39. Found: C,
69.81; H, 6.55; N, 9.41.
N-(6-Chloro-1,4-dimethyl-9H-carbazol-3-yl)-N’-(3,4,5-trimetho-
xyphenyl)urea (6a). Brown powder (yield 70%). Mp 210ꢁC. IR
(KBr):ꢀn= 3254 (NH); 1642 (C═O amide) cmꢀ1. 1H NMR (DMSO-
d6): d 2.49 (s, 3H, CH3); 2.63 (s, 3H, CH3); 3.59 (s, 3H, OCH3);
3.72 (s, 6H, OCH3); 6.81 (s, 2H, Ar); 7.32 (s, 1H, Ar); 7.35–7.38
(m, 1H, Ar); 7.51 (d, J = 8.79 Hz, 1H, Ar); 7.96 (s, 1H, Ar); 8.10
N-(1,4-Dimethyl-6-hydroxy-9H-carbazol-3-yl)-N0-(3,4,5-
trimethoxyphenyl)urea (6f).
Silver powder (yield 58%). Mp
(s, 1H, NHC═O); 8.70 (s, 1H, NHC═O); 11.33 (s, 1H, NH). 13
C
210ꢁC. IR (KBr): ꢀn = 3479–3291 (OH, NH); 1631 (C═O
amide)cmꢀ1. 1H NMR (DMSO-d6): d 2.46 (s, 3H, CH3); 2.60 (s,
3H, CH3); 3.59 (s, 3H, OCH3); 3.73 (s, 6H, OCH3); 6.81 (s, 2H,
Ar); 6.88 (d, J = 7.8 Hz, 1H, Ar); 7.17 (s, 1H, Ar); 7.30 (d,
J = 7.8Hz, 1H, Ar); 7.50 (s, 1H, Ar); 7.86 (s, 1H, NHC═O); 8.63
(s, 1H, OH); 8.84 (s, 1H, NHC═O); 10.76 (s, 1H, NH). 13C NMR
(DMSO-d6): 14.53; 16.69; 55.60; 60.12; 95.59; 107.05; 111.26;
114.07; 116.90; 120.50; 124.06; 124.10; 124.68; 126.80; 131.98;
134.32; 136.61; 137.53; 150.15; 152.81; 154.08. MS (ESI+): 436
(M+ H)+. Anal. Calcd for C24H25N3O5: C, 66.19; H, 5.79;
N, 9.65. Found: C, 66.22; H, 5.76; N, 9.62.
NMR (DMSO-d6): 14.61; 16.69; 55.63; 60.15; 95.66; 112.40;
117.42; 119.98; 121.31; 122.66; 124.07; 124.54; 124.59; 125.51;
128.00; 132.08; 136.57; 137.33; 138.67; 152.86; 154.00. MS
(ESI+): 454 (M+ H)+. Anal. Calcd for C24H24ClN3O4: C, 63.50;
H, 5.33; N, 9.26. Found: C, 63.53; H, 5.37; N, 9.23.
N-(1,4-Dimethyl-6-fluoro-9H-carbazol-3-yl)-N0-(3,4,5-trimetho-
xyphenyl)urea (6b). Brown powder (yield 60%). ꢀM1p 208ꢁC. IR
ꢀ
(KBr): n = 3331 (NH); 1644 (C═O amide) cm
.
1H NMR
(DMSO-d6): d 2.54 (s, 3H, CH3); 2.63 (s, 3H, CH3); 3.59 (s, 3H,
OCH3); 3.73 (s, 6H, OCH3); 6.82 (s, 2H, Ar); 7.21 (t, J=8.7Hz,
1H, Ar); 7.29 (s, 1H, Ar); 7.47–7.51 (m, 1H, Ar); 7.87 (d,
J= 7.7 Hz, 1H, Ar); 7.95 (s, 1H, NHC═O); 8.68 (s, 1H, NHC═O);
11.19 (s, 1H, NH). 13C NMR (DMSO-d6): 14.51; 16.69; 55.64;
60.15; 95.67; 107.48; 111.70; 112.37; 117.40; 120.46; 123.58;
N-(1,4,6-Trimethyl-9H-carbazol-3-yl)-N0-(3,4,5-trimethoxyphenyl)
urea (6g). Brown powder (yield 68%). Mp 244ꢁC. IR (KBr):
ꢀ
n = 3298 (NH); 2934 (OCH3); 1641 (C═O amide); 1131 (CN);
1506; 1228; 999; 797cmꢀ1
.
1H NMR (DMSO-d6): d 2.49 (s,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E302
A. Panno, M. S. Sinicropi, A. Caruso, H. El-Kashef,
Vol 51
J.-C. Lancelot, G. Aubert, A. Lesnard, T. Cresteil, and S. Rault
REFERENCES AND NOTES
6H, CH3); 2.67 (s, 3H, CH3); 3.60 (s, 3H, OCH3); 3.73 (s, 6H,
OCH3); 6.83 (s, 2H, Ar); 7.19 (d, J = 7.8 Hz, 1H, Ar); 7.24 (s, 1H,
Ar); 7.40 (d, J = 7.8 Hz, 1H, Ar); 7.90 (s, 1H, NHC═O); 7.95 (s,
1H, Ar); 8.66 (s, 1H, NHC═O); 10.60 (s, 1H, NH). 13C NMR
(DMSO-d6): 14.75; 16.76; 21.39; 55.64; 60.16; 95.67; 110.73;
116.98; 120.52; 122.03; 123.74; 124.03; 124.67; 126.05; 126.94;
127.36; 132.08; 136.64; 137.03; 138.54; 152.88; 145.10. MS
(ESI+): 434 (M + H)+. Anal. Calcd for C25H27N3O4: C, 69.27; H,
6.28; N, 9.69. Found: C, 69.30; H, 6.26; N, 9.66.
[1] (a) Chakraborty, D. P. The Alkaloids 1993, 44, 257; (b)
Chakraborty, D. P.; Roy, R. S. Prog Chem Org Nat Prod 1991, 57, 71;
(c) Tachibana, Y.; Kikuzaki, H.; Lajis, N. H.; Nakatani, N. J Agric Food
Chem 2001, 49, 5589; (d) Sÿtolc, S. Life Sci 1999, 65, 1943; (e) Caruso,
A.; Voisin-Chiret, A. S.; Lancelot, J. C.; Sinicropi, M. S.; Garofalo, A.;
Rault, S. Molecules 2008, 13, 1312.
[2] Roy, M. K.; Thalang, V. N.; Trakoontivakorn, G.; Nakahara,
K. Brit J Pharmacol 2005, 145, 145.
[3] Goodwin, S.; Smith, A. F.; Horning, E. C. J Am Chem Soc
1959, 81, 1903.
Ethyl (1,4-dimethyl-3-(3,4,5-thrimethoxyphenylureido)-9H-
carbazol-6-yl)carbonate (6h).
Yellow powder (yield 67%).
[4] Ramsewak, R. S.; Nair, M. G.; Strasburg, G. M.; Dewitt, D. L.;
Nitiss, J. L. J Agric Food Chem 1999, 47, 444.
[5] Tachibana, Y.; Kikuzaki, H.; Lajis, N. H.; Nakatani, N. J Agric
Food Chem 2001, 49, 5589.
ꢁ
ꢀ
Mp 218 C IR (KBr): n = 3428–3247 (NH); 2932 (OCH3); 1740
(C═O ester); 1646 (C═O amide); 1129 (CN); 1507; 1006;
1
782 cmꢀ1. H NMR (DMSO-d6): d 1.30 (t, J = 6.8 Hz, 3H, CH3);
2.50 (s, 3H, CH3); 2.62 (s, 3H, CH3); 3.59 (s, 3H, OCH3); 3.73
(s, 6H, OCH3); 4.26 (q, J = 6.8 Hz, 2H, CH2CH3); 6.82 (s, 2H,
Ar); 7.22 (d, J = 10.7Hz, 1H, Ar); 7.31 (s, 1H, Ar); 7.50
(d, J = 8.7 Hz, 1H, Ar); 7.94 (s, 1H, Ar); 7.96 (s, 1H, NHC═O);
8.71 (s, 1H, NHC═O); 11.26 (s, 1H, NH). 13C NMR (DMSO-d6):
14.13; 14.58; 16.71; 55.64; 60.16; 64.43; 95.70; 111.20; 114.34;
117.40; 118.43; 120.52; 123.40; 124.00; 125.28; 127.78; 132.12;
136.60; 137.60; 138.06; 143.50; 152.89; 154.04; 154.08. MS
(ESI+): 508 (M + H)+. Anal. Calcd for C27H29N3O7: C, 63.89; H,
5.76; N, 8.28. Found: C, 63.90; H, 5.79; N, 8.31.
[6] Nakahara, K.; Trakoontivakorn, G.; Alzoreky, N. S.; Ono, H.;
Onishi-Kameyama, M.; Yoshida, M. J Agric Food Chem 2002, 50, 4796.
[7] Roy, M. K.; Vipaporn, N. T.; Trakoontivakorn, G.; Nakahara,
K. Biochem Pharma 2004, 67, 41.
[8] (a) Clarysse, A.; Brugarolas, A.; Siegenthaler, P.; Abele, R.;
Cavalli, F.; de Jager, R.; Renard, G.; Roseneweig, M.; Hansen, H. H. Eur J
Cancer Clin Oncol 1984, 20, 243; (b) Sopkovà-de Oliveira Santos, J.; Car-
uso, A.; Lohier, J. F.; Lancelot, J. C.; Rault, S. Acta Cryst 2008, C64, o453.
[9] (a) Stiborova, M.; Rupertova, M.; Schmeiser, H. H.; Frei,
E. Biomed Pap Med 2006, 150, 13; (b) Caruso, A.; Voisin-Chiret, A.
S.; Lancelot, J. C.; Sinicropi, M. S.; Garofalo, A.; Rault, S. Heterocycles
2007, 71, (10), 2203; (c) Caruso, A.; Lancelot, J. C.; El-Kashef, H.;
Sinicropi M. S.; Legay, R.; Lesnard, A.; Rault, S. Tetrahedron 2009, 65,
10400; (d) Caruso, A.; Chimento, A.; El-Kashef, H.; Lancelot, J. C.;
Panno, A.; Pezzi, V.; Saturnino, C.; Sinicropi, M. S.; Sirianni, R.; Rault,
S. J Enzym Inhib Med Ch 2012, 27 (4), 609.
Preparation of 3,4,5-trimethoxybenzoyl azide (8).
A
mixture of triethylamine (2.47 g, 0.235 mol) in acetone (10 mL)
was added to a solution of the acid 7 (5g, 0.235mol) in acetone–
water mixture (50/5 mL) at 0ꢁC. After stirring for 30 min, ethyl
chloroformate (2.55g, 0.235mol) was added with stirring for 1 h.
Finally, a solution sodium azide (1.68g, 0.258 mol) in cold water
(10 mL) was added and stirring was further continued for 1.5 h,
keeping the temperature at 0ꢁC. The reaction mixture was
then diluted with water (70 mL), and the solid formed was
collected by filtration. The azide formed was used without
any further purification. White powder (yield 75%). Mp
[10] Braga, S. F.; de Melo, L. C.; Barone, P. M. V. B. J Mol Struc
(Theochem) 2004, 710, 51.
[11] Le Pecq, J. B.; Dat-Xuong, N.; Gosse, C.; Paoletti, C. Proc Nat
Acad Sci 1974, 71, 5078.
[12] Corbett, A. H.; Osheroff, N. Chem Res Toxicol 1993, 6, 585.
[13] Zhang, H.; D’Arpa, P.; Liu, L. F. Cancer Cell 1990, 2, 23.
[14] Giaccone, G. Anticancer Res 1994, 14, 269.
[15] Froelich-Ammon, S. J.; Patchan, M. W.; Osheroff, N.;
Thompson, R.B. J Biol Chem 1995, 270, 14998.
ꢀ1
ꢁ
ꢀ
96 C. IR (KBr): n = 2145 (CON3); 1674 (C═O) cm
.
Biology. Cell culture and cell proliferation assay.
The
[16] Rescifina, A.; Corsaro, A.; Piperno, A.; Iannazzo, D. XIII
Convegno Nazionale sulle Reazioni Pericicliche, Pavia Chem Abst 2009.
[17] De Martino, G.; Edler, M. C.; La Regina, G.; Coluccia, A.;
Barbera, M. C.; Barrow, D.; Nicholson, R. I.; Chiosis, G.; Brancale, A.;
Hamel, E.; Artico, M.; Silvestri, R. J Med Chem 2006, 49, 947.
[18] Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry
1989, 28, 6984.
[19] Beckers, T.; Mahboobi, S. Drugs Future 2003, 28, 767.
[20] Medina, J. C.; Houze, J.; Clark, D. L.; Schwendner, S.;
Beckmann, H.; Shan, B. Abstracts of Papers, 222nd American Chemical Soci-
ety National Meeting, Chicago, August 26-30, 2001; ACS: Washington, DC.
[21] Wang, Y. M.; Hu, L. X.; Liu, Z. M.; You, X. F.; Zhang, S. H.; Qu,
J. R.; Li, Z. R.; Li, Y.; Kong, W. J.; He, H. W.; Shao, R. G.; Zhang, L. R.;
Peng, Z. G.; Boykin, D. W.; Jiang, J. D. Clin Cancer Res 2008, 14, 6218.
[22] Hu, L.; Li, Z. R.; Li, Y.; Qu, J.; Ling, Y. H.; Jiang, J. D.;
Boykin, D. W. J Med Chem 2006, 49, 6273.
[23] Takeuchi, T.; Oishi, S.; Watanabe, T.; Ohno, H.; Sawada, J. I.;
Matsuno, K.; Asai, A.; Asada, N.; Kitaura, K.; Fujii, N. J Med Chem
2011, 54, 4839.
[24] Tabka, T.; Héron, J. F.; Gauduchon, P.; Le Talaer, J. Y.;
Lancelot, J. C.; Rault, S.; Robba, M. J Med Chem 1988, 23, 119.
[25] Pfister, J.; Wymann, W. Synthesis 1983, 38.
[26] (a) Tabka, T.; Letois, B.; Lancelot, J. C.; Gauduchon, P.;
Héron, J. F.; Le Talaer, J. Y.; Rault, S.; Robba, M. J Med Chem 1989, 24,
605; (b) Letois, B.; Lancelot, J. C.; Rault, S.; Robba, M.; Tabka, T.; Gaudu-
chon, P.; Bertreux E.; Le Talaer, J. Y. J Med Chem 1990, 25, 775.
[27] Cranwell, A.; Saxton, J. E. J Chem Soc 1962, 3482.
[28] Zavala, F.; Guenard D.; Robin J. P.; Brown E. J Med Chem
1980, 23, 546.
human cell lines KB (month epidermoid carcinoma) were
obtained from ECACC (Salisbury, UK) and grown in DMEM
medium supplemented with 10% fetal calf serum (Invitrogen), in
the presence of penicillin, streptomycin, and Fungizone in 75 cm2
flask under 5% CO2. HL60 cells (acute promyelocytic leukemia)
were similarly grown in supplemented RPMI medium.
Cells were plated in 96-well tissue culture microplates at a den-
sity of 650 or 2000 cells/well, respectively, for KB and HL60 in
200 mL medium and treated 24 h later with compounds dissolved
in DMSO with concentrations that ranged from 0.5 nM to 10 mM
with the use of a Biomek 3000 automate (Beckman Coulter).
Controls received the same volume of DMSO (1% final volume).
After 72 h exposure, MTS reagent (Promega) was added and incu-
bated for 3 h at 37ꢁC: the absorbance was monitored at 490nm and
the results were expressed as the inhibition of cell proliferation cal-
culated as the ratio [(1-(OD490 treated/OD490 control))ꢃ 100].
For IC50 determinations (50% inhibition of cell proliferation),
experiments were performed in separate duplicate.
Tubulin polymerization inhibition. Sheep brain microtubule
proteins were incubated at 37ꢁC for 10min and at 0ꢁC for 5 min
with the tested compounds at a concentration of 6.7ꢄ10ꢀ5 M. The
tubulin polymerization rate was measured by turbidimetry at
350 nm using deoxypodophyllotoxin as positive control [28].
Because of the optical interference of 6g at 350nm, the
concentration was reduced to 3.3ꢄ10ꢀ5 M.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet