Synthesis and in vitro antitumour evaluation of benzothiazole-2- carbonitrile derivatives

Article abstract of DOI:10.1016/S0223-5234(99)00130-0

Novel benzothiazole derivatives have been synthesised via the corresponding imino-1,2,3-dithiazoles. The cytotoxicity of some of these polyheterocyclic compounds was studied. Our results show that 2-cyano derivatives exhibit a medium in vitro antitumour a

Full text of DOI:10.1016/S0223-5234(99)00130-0

Eur. J. Med. Chem. 34 (1999) 1053−1060
1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
1053
©
Original article
Synthesis and in vitro antitumour evaluation
of benzothiazole-2-carbonitrile derivatives
a
a
a
b
c
Valérie Bénéteau , Thierry Besson *, Jérôme Guillard , Stéphane Léonce , Bruno Pfeiffer
a
Laboratoire de Génie Protéique et Cellulaire, UPRES 2001, Pôle Sciences et Technologie, Université de La Rochelle, avenue Marillac,
F-17042 La Rochelle cedex 1, France
b
Institut de Recherche Servier, 11, rue des Moulineaux, 92150 Surennes, France
c
A.D.I.R., 1, rue Carle Hébert, 92415 Courbevoie cedex, France
(Received 20 May 1999; revised 7 July 1999; accepted 15 July 1999)
Abstract – Novel benzothiazole derivatives have been synthesised via the corresponding imino-1,2,3-dithiazoles. The cytotoxicity of some
of these polyheterocyclic compounds was studied. Our results show that 2-cyano derivatives exhibit a medium in vitro antitumour activity.
©
1999 Éditions scientifiques et médicales Elsevier SAS
imino-1,2,3-dithiazoles / antitumour activity / benzothiazoles / benzodioxines
1
. Introduction
completely stable in a dry inert atmosphere but reacts
slowly with moisture to form 4-chloro-1,2,3-dithiazol-5-
one. This compound is readily prepared from chloro-
acetonitrile and disulfur dichloride [9], reacts rapidly
with 2,5-dimethoxy-aniline, in dichloromethane at room
temperature, to give the stable N-arylimino-1,2,3-
dithiazoles 2 in high yield (84%). Pyrolysis of these
imines gave 2-cyanobenzothiazoles 3 by cyclisation of
the ortho carbon onto sulfur with liberation of the other
sulfur atom and hydrogen chloride [10] (figure 1).
The benzothiazole ring is present in various marine or
terrestrial natural compounds which have useful biologi-
cal activities [1–4]. Because we are interested in hetero-
cyclic systems with potential pharmacological value, we
decided to synthesise new benzothiazole derivatives
which are related to synthetic thiazoles which have
shown antitumour activity [5]. Novel dioxinobenzothiaz-
oles were also prepared with the aim to enhance the
antiproliferative activity.
Removal (hydrolysis and decarboxylation) of the cy-
ano group in the thiazole ring was performed by vigorous
heating of compound 3a in concentrated hydrochloric
acid. The decyanated benzothiazole 4 was isolated in
good yield (70%) (figure 2). Using standard conditions
for the transformation of cyano groups into carboxylic
acid or amido groups [11], the starting thiazole 3a was
treated with aqueous sodium hydroxide to give the acid 5,
or with sulfuric acid to provide the amide 6, in quite good
yields. The substituted amide 7 was prepared by treat-
ment of the acid 5 with N,N-dimethylethylenediamine in
the presence of 1-(3-dimethylaminopropyl)-3-ethyl car-
bodiimide hydrochloride (EDCI) and 1-hydroxy-
benzotriazole (HOBT), a method commonly used for the
coupling of amino acids (figure 2).
In this paper, we describe the biological evaluation of
,7-dimethoxybenzothiazoles and dioxinobenzothiazoles
prepared via N-arylimino-1,2,3-dithiazoles which have
proved to be highly versatile intermediates in hetero-
cyclic chemistry [6–8].
4
2
. Chemistry
2
.1. 4,7-Dimethoxybenzothiazoles
4,5-Dichloro-1,2,3-dithiazolium chloride 1 is a pale
greenish yellow solid, insoluble in organic solvents. It is
*
Correspondence and reprints

1
054
Figure 1. Reactions and conditions: a) pyridine, dichloromethane, –15 °C, 3 h (2a, 40%) or room temperature, 3 h (2b, 84%);
b) toluene, sealed tube, reflux, 4 h (3a, 70%); c) PyHBr , pyridine, reflux, 1.5 h (3b, 72%).
3
Decyanation of compound 3b, using the conditions
described above, afforded the amino decyanated ben-
zothiazole 8, in which the amino group was easily
reprotected, to give 9, by treatment with acetic anhydride
in the presence of pyridine. The quinone 10 was obtained
by oxidation of 3b with cerium ammonium nitrate (fig-
ure 3).
Starting from the commercially available isothiocyan-
ate 11, the 2-alkoxyderivative 13 was synthesised by a
Jacobson process via the intermediate thiocarbamate 12
the starting 6-amino-2,3-dihydro-1,4-benzodioxin (fig-
ure 5). The bromination (NBS, CCl , AIBN)–debromina-
4
tion (NaI, acetone) sequence previously described in
several syntheses of benzodioxins [13], lead to the ex-
pected products 19 and 20 (figure 5), whilst heating of the
brominated intermediate 21 in pyridine, in the presence
of one equivalent of copper iodide (CuI), allowed an
alternative access to the linear compound 15 (the use of
the standard method afforded the angular brominated
derivative 22) (figure 6).
(
figure 4). In this case, an electron releasing group is
present in the 2-position of the benzothiazole ring.
3. Pharmacology
2.2. Dioxinobenzothiazoles [12]
Fifteen compounds were evaluated in vitro for their
The chemistry of the salt 1 described above also allows
a rapid access to the dioxinobenzothiazoles 15–18, from
antiproliferative activity using the murine L1210 leu-
kaemia cell line [14]. The results expressed as IC50
Figure 2. Reactions and conditions: a) conc. HCl, reflux, 8 h, 70%; b) NaOH 10%, 80 °C, 3.5 h, 64%; c) conc. H SO , room
2
4
temperature, 2 h, 72%; d) N,N-dimethylethylenediamine, EDCI, HOBT, DMF, 0 °C, 4 days, 43%.

1
055
Figure 3. Reactions and conditions: a) conc. HCl, reflux, 1.5 h, 83%; b) CAN, CH CN, H O, room temperature, 15 min, 56%;
3
2
c) Ac O, pyridine, room temperature, 12 h, 56%.
2
(concentration reducing the cell proliferation by 50%) are
4. Results and discussion
reported in table I. Cell cycle perturbations induced by
the most active compounds (IC50 < 20 µM) were also
investigated.
The 2-cyano-4,7-dimethoxybenzothiazole derivatives
3a and 3b were first evaluated and found practically
Figure 4. Reactions and conditions: a) PrOH, NaH, 83%; b) K FeCN , NaOH, 28%.
3
6
Figure 5. Reagents and conditions: a) PyHBr (1 eq.), DMF/pyridine, reflux, 90 min, 58% (29% for 15 and 29% for 16); b): HCl,
3
reflux, 2.5 h, 80% (17) and 65% (18); c) i) NBS/AIBN, CCl , hν, reflux, 10 h; ii) NaI, acetone, reflux, 1.5 h, 69% (19) and 27% (20);
4
d) Br (1 eq.), CH COOH, room temperature, 2 h, 98%.
2
3

1
056
results already published on dioxinocoumarins [15, 16].
The dioxinobenzothiazole 19 was found able to block
L1210 cells in the G + M phase of the cell cycle.
2
5. Conclusion
Figure 6. Reagents and conditions: a) PyHBr (1 eq.), DMF/
pyridine, reflux, 2 h, 25%; b) CuI, pyridine, reflux, 90 min,
3
In conclusion, we have described the synthesis of
novel benzothiazoles and dioxinobenzothiazoles, among
which the 2-cyano derivatives exhibit interesting in vitro
antitumour activity. Presence of unsaturation in the dioxin
moiety, in combination with the cyano group in the
5
5%.
equipotent on cell proliferation with IC ’s of, respec-
50
2
-position of the thiazole ring, did not really involve any
tively, 20.6 µM and 25.2 µM. Also, both were found to be
specific effect on the cell cycle. Our results suggest that
introduction of the thiazolo-2-carbonitrile ring into more
extended and more complexe heterocyclic moieties could
open the door to promising applications.
able to almost totally block the cells in the G + M phase
2
of the cell cycle. Suppression of the 2-cyano substituent
(compounds 4 and 9) or its replacement by a 2-carboxy
(compound 5), a 2-aminocarbonyl (compound 6), a 2-[2-
(N,N-dimethylamino)ethylaminocarbonyl] (compound 7)
or a 2-propoxy (compound 13) substituent led to inactive
derivatives. Compound 10 which is a quinone derivative
was found significantly more active that its 4,7-
dimethoxy counterpart 3b with an IC50 of 5µM versus
6
. Experimental protocols
6.1. Chemistry
25.2 µM. This compound was unfortunately devoid of
Melting points were determined using a Kofler banc
any specific effect on the cell cycle.
and are uncorrected. IR spectra were recorded on a
1
13
All the 2-cyano-dioxinobenzothiazoles have also
shown a relatively interesting antiproliferative activity.
As for the 4,7-dimethoxybenzothiazoles, removal of the
cyano substituent present in the 2-position of the dioxino-
benzothiazole ring (compound 17) involved the lost of
any activity (IC50 > 100 µM). The unsaturated com-
pounds 19 and 20 were found significantly more active
than their saturated conterparts 15 and 18, with IC ’s of,
Perkin-Elmer Paragon 1000PC instrument. H- and C-
NMR were recorded on a JEOL JNM LA400 (400 Mhz)
spectrometer (Centre Commun d’Analyse, Université de
La Rochelle); chemical shifts (δ) are reported in parts per
million (ppm) downfield from tetramethylsilane (TMS),
which was used as internal standard. Mass spectra were
recorded on a Varian MAT311 in the Centre de Mesure
Physiques de L’Ouest (C.R.M.P.O.), Université de
Rennes. Chromatography was carried out on silica gel 60
50
respectively, 18.2 µM and 31.7 µM, confirming the
Table I. Characteristics and pharmacological activity of synthesised compounds.
a
Compound
M.p. (°C)
Formula
IC50 (µΜ)
% of L1210 cells in the G + M phase (µM)
2
3
3
4
5
6
7
9
1
1
1
1
1
1
2
2
a
b
174
173
108
> 230
240
144
154
242
73
178
156
142
176
150
250
C H N O S
20.6
25.2
64% (50)
10 8 2 2
C H N O S
77% (100)
12 11 3 3
b
C H NO S
> 100
> 100
> 100
> 100
> 100
5
> 100
58.4
42.3
n.e.
9
9
2
C H NO S
n.e.
n.e.
n.e.
n.e.
10
9
4
C H N O S
10 10 2 3
C H N O S
14 19 3 3
C H N O S
11 12 2 3
0
3
5
6
7
9
0
2
C H N O S
non specific
n.e.
10 5 3 3
C H NO S
12
15
3
b
C H N O S
n.e.
10 6 2 2
C H N O S
n.e.
n.e.
41% (50)
n.e.
n. e.
10 6 2 2
C H NO S
> 100
18.2
31.7
9
7
2
C H N O S
10 4 2 2
C H N O S
10 4 2 2
C H BrN O S
80.8
10
5
2 2
a
b
2
4% of untreated control cells were in the G + M phase of the cell cycle. n.e.: not evaluated (for IC > 30 µM).
2
50

1
057
at medium pressure and the sample mixtures were applied
to the column preadsorbed onto silica. Light petroleum
refers to the fraction b.p. 40–60 °C. Further solvents were
used without purification. Thin-layer chromatography
was performed on Merck Kieselgel 60 F254 aluminium
backed plates.
6.1.3. 4,7-Dimethoxybenzothiazole-2-carbonitrile 3a
In a closed system, a solution of 2a (871 mg,
3.02 mmol) in toluene (5 mL) was heated in an oil bath at
200–210 °C for 8 h. After cooling, the toluene was
removed in vacuo. The crude residue was purified by
column chromatography (eluent: dichloromethane/light
petroleum: 6/4) and recrystallised from ethanol to afford
Spectral data for compounds 14–22 are consistent with
assigned structures as previously described in ref. [12].
3
a (471 mg, 70%) as yellow needles; m.p. 174 °C; IR
(KBr): ν 2 229 (nitrile), 1 595 (C=N), 1 501, 1 454,
–1 1
1
280, 1 140, 1 094, 1 046, 969 cm ; H-NMR (CDCl ):
3
6
.1.1.
2,5-Dimethoxy-N-(4-chloro-5H-dithiazol-5-yli-
δ 3.98 (s, 3H, OCH ), 4.05 (s, 3H, OCH ), 6.93 (s, 2H,
5-H and 6-H); C-NMR (CDCl ): δ 56.36, 56.49,
3
3 3
13
dene)aniline 2a
1
1
08.10, 108.44, 112.97, 126.32, 135.55, 143.75, 147.59,
Under an inert atmosphere, 4,5-dichloro-1,2,3-
dithiazolium chloride (15.42 g, 73.7 mmol) and pyridine
+
+
48.91; MS (EI): m/z 220 (M ), 205 (M –CH ), 191;
3
HRMS: calc. for C H N O S: 220.0306, found:
2
(
10.8 mL, 134 mmol) were added to a stirred solution of
,5-dimethoxyaniline (10.3 g, 67 mmol) in dichloro-
10 8 2 2
20.0303.
2
methane (150 mL) cooled at –15 °C. After 2 h, the
mixture was filtered and the solvent removed in vacuo.
The crude residue was purified by column chromatogra-
phy (eluent: light petroleum/ethyl acetate: 9/1) to afford
compound 2a as an orange oil (7.8 g, 40%); IR (film): ν
6
.1.4. 6-Acetamido-4,7-dimethoxybenzothiazole-2-carbo-
nitrile 3b
Under an inert atmosphere, a mixture of 2b (1.60 g,
.63 mmol) and pyridinium perbromide (1.63 g,
4
5
2
.10 mmol) in pyridine (20 mL) was heated at reflux for
.5 h. After cooling, pyridine was removed in vacuo and
2
1
938 and 2 833 (CH ), 1 586 (C=N), 1 498, 1 464,
278, 1 223, 1 195, 1 165, 1 044 cm ; H-NMR
3
–1
1
the crude residue purified by column chromatography
eluent: dichloromethane/ethyl acetate: 95/5). Recrystal-
lisation from ethanol afforded compound 3b (927 mg,
2%) as yellow needles; m.p. 173 °C; IR (KBr): ν 3 390
NH), 2 226 (nitrile), 1 683 (C=O), 1 600 (C=N), 1 523,
(
CDCl ): δ 3.78 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 6.69
3 3 3
(
(
d, 1H, J = 2.9 Hz, 6-H), 6.75 (dd, 1H, J = 2.9 Hz and
J≠ = 8.8 Hz, 4-H), 6.93 (d, 1H, J≠ = 8.8 Hz, 3-H);
1
3
7
(
C-NMR (CDCl ): δ 56.65, 57.21, 106.11, 112.91,
3
1
14.43, 141.71, 144.68, 148.48, 154.97, 161.02; MS (EI):
–1
1
+
1
448, 1 420, 1 240, 1 132, 1 050 cm ; H-NMR
m/z 288 (M ), 188, 148; HRMS: calc. for
(CDCl ): δ 2.30 (s, 3H, NHCOCH ), 3.96 (s, 3H, OCH ),
C H ClN O S : 287.9794, found: 287.9801.
3 3 3
1
0
9
2 2 2
4
.08 (s, 3H, OCH ), 7.86 (bs, 1H, NH), 8.34 (s, 1H, 5-H);
3
1
3
C-NMR (CDCl ): δ 25.20, 56.68, 59.88, 101.65,
3
6
.1.2. 4-Acetamido-2,5-dimethoxy-N-(4-chloro-5H-1,2,3-
12.78, 128.05, 132.37, 132.79, 134.56, 139.33, 151.25,
+
+
dithiazol-5-ylidene) aniline 2b
3
Under an inert atmosphere, 4,5-dichloro-1,2,3-
dithiazolium chloride (220 mg, 1.04 mmol) was added to
a stirred solution of 4-acetamido-2,5-dimethoxyaniline
HRMS: calc. for C12
277.0524.
H N O S: 277.0521, found:
11 3 3
(200 mg, 0.95 mmol) in dichloromethane (10 mL). After
6.1.5. 4,7-Dimethoxybenzothiazole 4
1
h, pyridine (110 mg, 1.39 mmol) was added and the
A suspension of compound 3a in concentrated hydro-
chloric acid (20 mL) was heated at reflux for 8 h. The
mixture was cooled at 0 °C, neutralised to pH 8 using
saturated aqueous sodium hydrogen carbonate and ex-
tracted with dichloromethane. The combined extracts
mixture stirred for 15 min. The solvent was removed in
vacuo and the crude residue purified by column chroma-
tography (eluent: dichloromethane/ethyl acetate: 4/1) to
afford compound 2b (275 mg, 84%) as an orange powder;
m.p. 155 °C; IR (KBr): ν 3 506 (NH), 1 671 (C=O),
were dried over MgSO and the solvent removed in
4
–1 1
1
483, 1 402, 1 214, 1 041, 862 cm ; H-NMR (CDCl ):
vacuo. After recrystallisation from hexane, compound 4
3
δ 2.22 (s, 3H, NHCOCH ), 3.85 (s, 3H, OCH ), 3.87 (s,
(177 mg, 70%) was obtained as colourless needles; m.p.
3
3
3
1
H, OCH ), 6.72 (s, 1H, 6-H), 7.80 (s, 1H, NH), 8.28 (s,
108 °C; IR (KBr): ν 3 074 (CHunsat.), 2 959 (CH ), 1 594
3
3
13
H, 3-H); C-NMR (CDCl ): δ 25.01, 56.20, 56.24,
(C=N), 1 500, 1 456, 1 439, 1 345, 1 263, 1 184,
3
–1 1
1
02.98, 104.85, 126.62, 133.91, 141.64, 143.81, 147.80,
1 150 cm ; H-NMR (CDCl ): δ 3.97 (s, 3H, OCH ),
3
3
+
158.88, 168.24; MS (EI): m/z 345 (M ), 252 (M–CNSCl),
4.03 (s, 3H, OCH ), 6.78 (d, 1H, J = 8.6 Hz, H
(d, 1H, J = 8.6 Hz, Harom.), 8.91 (s, 1H, 2-H); C-NMR
), 6.86
3
arom.
1
3
1
95; HRMS: calc. for C H ClN O S : 345.0009,
12
12
3 3 2
found: 345.0016.
(CDCl ): δ 55.29, 55.63, 104.81, 106.39, 123.95, 144.11,
3

1
058
47.59, 152.49, 152.53; MS (EI): m/z 195 (M ), 180
+
1
slowly. Water was then added with cooling and the crude
material extracted with ethyl acetate. The combined
extracts were washed with water (three times) and dried
+
(
M –CH ), 166; HRMS: calc. for C H NO S: 195.0354,
3 9 9 2
found: 195.0361.
over MgSO . After removal of the solvent in vacuo, the
4
6
.1.6. 4,7-Dimethoxybenzothiaole-2-carboxylic acid 5
A suspension of compound 3a (204 mg, 0.93 mmol) in
0% aqueous sodium hydroxide (10 mL) was heated at
0 °C for 3.5 h. After cooling to room temperature, the
product was purified by column chromatography (eluent:
dichloromethane/methanol: 95/5) and recrystallised from
hexane/ethanol to afford compound 7 (393 mg, 43%) as
pale yellow needles; m.p. 144 °C; IR (KBr): ν 3 168
1
8
mixture was poured onto iced water and acidified to pH 1
using 10% aqueous hydrochloric acid. The yellow pre-
cipitate that separated was filtered under vacuum and
dried. The product was purified by column chromatogra-
phy (eluent: hexane/ethyl acetate/methanol: 1/1/0.5 then
methanol). Recrystallisation from water afforded com-
pound 5 (141 mg, 64%) as yellow needles; m.p. 230 °C
(
NH), 2 950 (CH ), 2 824 (CH ), 1 662 (C=O), 1 540,
276, 1 188, 1 143, 1 090, 1 045 cm ; H-NMR
3 2
–1
1
1
(CDCl ): δ 2.24 (s, 6H, N(CH ) ), 2.50 (t, 2H, J = 6.25
3 3 2
Hz, CH –N), 3.54 (q, 2H, J = 6.25 Hz, NH–CH ), 3.91 (s,
2
2
3
H, OCH ), 3.99 (s, 3H OCH ), 6.77 (d, 1H, J = 8.7 Hz,
3 3
Harom.), 6.82 (d, 1H, J = 8.7 Hz, Harom.), 7.73 (bs, 1H,
13
NH); C-NMR (CDCl ): δ 37.47, 45.29 (×2), 56.05,
56.34, 57.86, 106.40, 107.07, 128.11, 144.29, 148.29,
1
((CH ) N=CH ); HRMS: calc. for C H N O S:
309.1147, found: 309.1155.
3
(
dec); IR (KBr): ν 3 367 (broad: OH), 2 838 (CH ), 1 632
3
(C=O), 1 499, 1 383, 1 263, 1 189, 1 097, 1 044,
+
48.39, 159.89, 163.77; MS (EI): m/z 309 (M ), 58
–
1 1
9
3
74 cm ; H-NMR (DMSO-d ): δ 3.87 (s, 3H, OCH ),
+
3 2 2 14 19 3 3
6
3
13
.88 (s, 3H, OCH ), 6.90 (s, 2H, Harom.); C-NMR
3
(
DMSO-d ): δ 55.92, 56.11, 106.04, 107.53, 126.56,
6
1
2
44.48, 147.54, 148.19, 162.07, 169.85; MS (EI): m/z
6
.1.9. 6-Amino-4,7-dimethoxybenzothiazole 8
A solution of compound 3b (93 mg, 0.34 mmol) in
09 (M–CH O), 195 (M–CO ), 180 (M–CO , CH ), 166;
2
2
2
3
HRMS could not be measured due to the absence of the
molecular pic.
concentrated hydrochloric acid (7 mL) was heated at
reflux for 1.5 h. After cooling at room temperature, the
mixture was basified to pH 8 using saturated aqueous
sodium hydrogen carbonate and extracted with dichloro-
methane. The combined extracts were washed with water
6.1.7. 4,7-Dimethoxybenzothiazole-2-carboxamide 6
A solution of compound 3a (703 mg, 3.19 mmol) in
concentrated sulfuric acid (5 mL) was stirred at room
temperature for 2 h. After cooling at 0 °C, the mixture
was basified using 10% aqueous sodium hydroxide and
then extracted with ethyl acetate. The combined extracts
and brine, and dried over MgSO . The solvent was
4
removed in vacuo and the crude residue was purified by
column chromatography (eluent: light petroleum/ethyl
acetate: 1/1) to afford compound 8 (59 mg, 83%) as a
brown powder; m.p. 153 °C; IR (KBr): ν 3 440/3 310
were dried over MgSO . Removal of the solvent in vacuo
4
followed by a recrystallisation from ethanol gives com-
pound 6 (548 mg, 72%) as amber needles; m.p. 240 °C;
IR (KBr): ν 3 406 (NH), 1 682 (C=O), 1 600 (C=N),
(NH ), 1 614 (C=N), 1 500, 1 463, 1 398, 1 237, 1 043,
2
–1 1
9
87 cm ; H-NMR (CDCl ): δ 3.86 (s, 3H, OCH ), 3.99
3 3
–1 1
(s, 3H, OCH ), 6.40 (s, 1H, 5-H), 8.60 (s, 1H, 2-H);
3
1
558, 1 505, 1 118, 799 cm ; H-NMR (CDCl ): δ 3.97
3
13
C-NMR (CDCl ): δ 56.09, 58.75, 98.02, 128.61,
3
(s, 3H, OCH ), 4.04 (s, 3H, OCH ), 5.73 (bs, 1H, NH),
3 3
1
33.24, 137.35, 137.48, 148.11, 150.38; MS (EI): m/z 210
6
.84 (d, 1H, J = 8.6 Hz, Harom.), 6.89 (d, 1H, J = 8.6 Hz,
+
+
+
3 3 4
13
(M ), 195 (M –CH ), 154 (M –C H O).
Harom.), 7.41 (bs, 1H, NH); C-NMR (DMSO-d ): δ
6
5
1
5.98, 56.15, 107.50, 108.33, 126.58, 143.89, 147.44,
+
6.1.10. 6-Acetamido-4,7-dimethoxybenzothiazole 9
Acetic anhydride (2.2 mL, 23.33 mmol) was added to a
stirred solution of 8 (265 mg, 1.26 mmol) in pyridine
48.33, 161.17, 163.60; MS (EI): m/z 238 (M ), 223
+
(
M –CH ), 209, 192, 180; HRMS: calc. for
C H N O S: 238.0412, found: 238.0415.
3
1
0 10 2 3
(15 mL). The mixture was strirred overnight at room
6
.1.8. 4,7-Dimethoxybenzothiazole-2-[2-(N,N-dimethyl-
temperature. Then, water was added, the product was
extracted with ethyl acetate, purified by column chroma-
tography (eluent: ethyl acetate/light petroleum: 7/3) and
recrystallised from dichloromethane/light petroleum to
afford compound 9 (179 mg, 56%) as pale yellow
needles; m.p. 154 °C; IR (KBr): ν 3 296 (NH), 2 963,
1 668 (C=O), 1 606 (C=N), 1 532, 1 463, 1 386, 1 254,
amino)ethyl]carboxamide 7
Under an inert atmosphere, 1-(3-dimethylamino-
propyl)-3-ethyl-carbodiimide hydrochloride (1.22 g, 6.47
mmol), 1-hydroxybenzotriazole (840 mg, 6.47 mmol)
and N,N-dimethylethylenediamine (0.7 mL, 6.47 mmol)
were added to a stirred solution of compound 5 (703 mg,
–1
1
2.94 mmol) in DMF (20 mL) at 0 °C. The mixture was
1 222, 1 042 cm ; H-NMR (CDCl ): δ 2.25 (s, 3H,
3
stirred for 4 days, allowing the temperature to increase
NHCOCH ), 3.93 (s, 3H, OCH ), 4.04 (s, 3H, OCH ),
3
3
3

1
059
7
.78 (bs, 1H, NH), 8.18 (s, 1H, 5-H), 8.79 (s, 1H, 2-H);
(Gibco) supplemented with 10% foetal calf serum, 2 mM
L-glutamine, 100 units/mL penicillin, 100 µg/mL strep-
tomycin, and 10 mM HEPES buffer (pH = 7.4).
Cytotoxicity was measured by the microculture tetra-
zolium assay as described in ref. [14]. Cells were exposed
to graded concentrations of the compounds for 48 h and
results expressed as IC50 (concentration which reduced
by 50% the optical density of treated cells with respect to
untreated controls).
13
C-NMR (CDCl ): δ 25.09, 56.35, 59.74, 100.80,
3
1
26.26, 128.94, 135.58, 140.60, 149.89, 150.81, 168.42;
+
+
MS (EI): m/z 252 (M ), 237 (M –CH ), 195; HRMS:
3
calc. for C H N O S: 252.0569, found: 252.0567.
11 12 2 3
6
.1.11. 6-Acetamido-2-cyano-4,7-dioxobenzothiazole 10
A solution of cerium ammonium nitrate (2.59 g,
.72 mmol) in water (10 mL) was added to a stirred
4
solution of 3b (524 mg, 1.89 mmol) in acetonitrile
20 mL) over 10 min. After 15 min the mixture was
5
For the cell cycle analysis, L1210 cells (2.5 × 10
(
cells/mL) were incubated for 21 h with various concen-
trations of the compounds, then fixed by 70% ethanol
(v/v), washed and incubated in PBS containing 100 µg/
extracted with ethyl acetate, the combined extracts were
dried over MgSO and the solvents removed in vacuo.
4
The crude residue was purified by column chromatogra-
phy (eluent: dichloromethane/ethyl acetate: 95/5) to af-
ford compound 10 (261 mg, 56%) as orange needles;
m.p. 242 °C; IR (KBr): ν 3 266 (NH), 3 108 (CHunsat.),
mL RNAse and 25 µg/mL propidium iodide for 30 min at
4
2
0 °C. For each sample, 1 × 10 cells were analysed on an
ATC3000 flow cytometer (Brucker, France) using an
argon laser (Spectra-Physics) emitting 400 mW at
2
1
238 (nitrile), 1 708 (C=O), 1 522, 1 427, 1 331, 1 194,
4
88 nm. The fluorescence of propidium iodide was col-
–1
1
141, 1 014 cm ; H-NMR (CDCl ): δ 2.32 (s, 3H,
3
lected through a 615 nm long-pass filter.
NHCOCH ), 7.94 (s, 1H, 5-H), 8.13 (bs, 1H, NH);
3
Data are displayed as linear histograms and results are
13
C-NMR (CDCl ): δ 25.04, 111.15, 115.47, 138.29,
3
expressed as the percentage of cells found is the G + M
2
1
40.03, 142.88, 153.75, 169.02, 175.34, 178.42; MS (EI):
phase of the cell cycle.
+
m/z 247 (M ), 205; HRMS: calc. for C H N O S:
10 5 3 3
247.0052, found: 247.0046.
Acknowledgements
6.1.12. 4,7-Dimethoxy-2-propyloxybenzothiazole 13
A mixture of 12 (698 mg, 2.73 mmol) and 30% aque-
ous sodium hydroxide (2.9 mL, 21.84 mmol) in ethanol
We thank the Communauté de Villes de
l’Agglomération de La Rochelle, the society ADIR
(
(
3 mL) was added dropwise to a solution of K FeCN6
3.6 g, 10.62 mmol) in water (5 mL) heated at 85 °C.
3
(Groupe SERVIER) and the Comité de Charente-
Maritime de la Ligue Nationale Contre le Cancer for
financial support.
After 1.5 h, the mixture was allowed to cool to room
temperature and extracted with dichloromethane. The
combined extracts were dried over MgSO4 and the
solvents removed in vacuo. The crude residue was
purified by column chromatography (eluent: light
petroleum/ethyl acetate: 7/1). Recrystallisation from hex-
ane afforded compound 13 (191 mg, 28%) as colourless
needles; m.p. 73 °C; IR (KBr): ν 2 964 and 2 833
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–1
1
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3
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1
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3
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12
15
3
7
2
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.2. Antiproliferative activity
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