Isobrassinin and its analogues: Novel types of antiproliferative agents

Article abstract of DOI:10.1016/j.bmcl.2006.09.016

Isobrassinin (2-(S-methyldithiocarbamoylaminomethyl)indole (7a), a regioisomer of the cruciferous phytoalexin brassinin (1), exerted marked antiproliferative effects on the HeLa, A431 and MCF7 cell lines (>78.6% inhibition at 30 μM). For structure-activity relationships, further analogues were synthesized. The highest cytotoxic effect was displayed by 2-phenylimino-1,3-thiazino[5,6-b]indole (10) (10 μM, 76.8%-HeLa and 46.3%-MCF7). The effect of the natural phytoalexin brassinin was also determined.

Full text of DOI:10.1016/j.bmcl.2006.09.016

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6273–6276
Isobrassinin and its analogues: Novel types of
antiproliferative agents
a,b
c
c
a,b,
*
P e´ ter Csom o´ s, Istv a´ n Zupk o´ , Borb a´ la R e´ thy, Lajos Fodor,
c
a
George Falkay and G a´ bor Bern a´ th
a
Institute of Pharmaceutical Chemistry, University of Szeged and Research Group for Heterocyclic Chemistry,
Hungarian Academy of Sciences, H-6701, PO Box 427, Hungary
b
Central Laboratory, County Hospital, H-5701 Gyula, PO Box 46, Hungary
Department of Pharmacodynamics and Biopharmacy, University of Szeged, H-6701, PO Box 427, Hungary
c
Received 2 August 2006; revised 5 September 2006; accepted 7 September 2006
Available online 25 September 2006
Abstract—Isobrassinin (2-(S-methyldithiocarbamoylaminomethyl)indole (7a), a regioisomer of the cruciferous phytoalexin brassi-
nin (1), exerted marked antiproliferative effects on the HeLa, A431 and MCF7 cell lines (>78.6% inhibition at 30 lM). For
structure–activity relationships, further analogues were synthesized. The highest cytotoxic effect was displayed by 2-phenylimino-
1
,3-thiazino[5,6-b]indole (10) (10 lM, 76.8%—HeLa and 46.3%—MCF7). The effect of the natural phytoalexin brassinin was also
determined.
2006 Elsevier Ltd. All rights reserved.
ꢀ
Substituted dithiocarbamic acid esters are a common
class of organic molecules. The different derivatives in
this group exhibit a wide range of biological effects (anti-
bacterial, antifungal, antioxidant activity, inhibition
4
of cardiac hypertrophy, etc.). A steadily increasing
number of studies have been published on dithiocarba-
mates and their anticancer activity. 4-Metha-
nesulfinylbutyl dithiocarbamic acid methyl ester
3-cyano-3,3-diphenylpropyl esters have been found to
1
be effective against the HL-60 and Bel-7402 cell lines.
1
Different metal [Pt(II), Pd(II), Au(III), Cu(II)]
complexes of dithiocarbamate derivatives (methyl- and
ethylsarcosinedithiocarbamate, N,N-dimethyldithiocar-
bamate, S-methyl-N,N-dimethyldithiocarbamate and
diethyldithiocarbamate) have been prepared and their
1
2
3
1
2–14
cytotoxicities were studied.
The Pt(II) complexes
(
Sulforamate) has proved to be a potential cancer che-
of these sulfur-containing molecules can act as chemo-
protectants in platinum-based chemotherapy, modulat-
ing cisplatin nephrotoxicity.
5
mopreventive compound as a phase II enzyme inducer.
A series of alkyl/arylsulfonyl-N,N-diethyldithiocarba-
mates display moderate to powerful tumour growth-in-
hibitory properties against several cancer cell lines
in vitro. 4(3H)-Quinazolinone derivatives with a
dithiocarbamate side chain exhibit antitumour activity
against human myelogenous leukaemia K562 cells.
Pyrrolidine dithiocarbamate stimulates apoptosis by
suppressing the activation of nuclear factor jB
1
5
Besides the compounds mentioned above, probably the
most interesting group of dithiocarbamates exhibiting
antitumour activity are the phytoalexins from crucifer-
ous plants. The phytoalexins are a group of structurally
diverse, low molecular weight, generally lipophilic anti-
microbial substances formed in plants. They are not
present in healthy plant tissue, but are synthetized in
response to pathogen attack or physical or chemical
stress, probably as a result of the de novo synthesis of
6
7
,8
(
leukaemia and pancreatic adenocarcinoma ).
NF-jB) in various cancer cells (e.g., acute myelogenous
9 10
A
variety of 4-substituted-piperazine-1-carbodithioic acid
1
6
enzymes. Some of the cruciferae species that have been
examined accumulate a series of specific indole-sulfur
compounds. The basic structures are characterized by
an indole ring variably substituted at positions 2
and/or 3 with nitrogen- and sulfur-containing substitu-
Keywords: 2-Aminomethylindole; Dithiocarbamate; Thiazino[5,6-b]in-
dole; Antitumour activity.
*
Corresponding author. Tel.: +36 66 463763; fax: +36 66 526539;
e-mail: fodor@pandy.hu
1
7
ents. Typical representatives of dithiocarbamate and
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.016

6274
P. Csom o´ s et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6273–6276
thiazino[6,5-b]indole-type phytoalexins from cruciferous
plants are brassinin (1), 1-methoxybrassinin (2), 4-meth-
oxybrassinin (3), cyclobrassinin (4) and sinalbin B (5)
performed with lithium aluminium hydride in
tetrahydrofuran.
(
Fig. 1).
Starting from amine 6, 2-(S-methyldithiocarbamoylam-
2
8
inomethyl)indole (7a, isobrassinin ) was prepared with
carbon disulfide, using chloroform as solvent and trieth-
Among these compounds, brassinin (1) and cyclobrass-
inin (4) proved active in inhibiting the formation of pre-
neoplastic mammary lesions in culture. The former
also exerts an antiproliferative effect in human acute
T-lymphoblastic leukaemia cells. Brassinin and its
derivatives are inhibitors of indolamine 2,3-dioxygenase,
a new cancer immunosuppression target. These com-
pounds can serve as lead compounds for the generation
ylamine and catalytic 4-dimethylaminopyridine as base,
29,30
1
8
followed by treatment with methyl iodide. When
benzyl bromide was used instead of the latter alkylating
agent, 2-(S-benzyldithiocarbamoylaminomethyl)indole
1
9
3
0
(7b) was obtained in good yield. The selective Hugersc-
hoff ring-closure of 7a,b with phenyltrimethylammoni-
um tribromide gave 2-methylthio-1,3-thiazino[5,
2
0
2
1
31
of more efficient analogues.
6-b]indole (8a) and its benzyl analogue 8b. Thiourea
derivatives 9a,b were synthesized from 2-aminomethy-
In our work on the chemistry of sulfur- and nitrogen-con-
2–25
lindole with methyl and phenyl isothiocyanates in reflux-
32
2
taining condensed-skeleton heterocycles,
we earlier
prepared brassinin (1) and cyclobrassinin (4) and some
ing chloroform. The oxidative ring-closure of 9b with
phenyltrimethylammonium tribromide and basic treat-
2
6
of their derivatives. As a continuation, our present
aim was the preparation of regioisomers of cruciferous
phytoalexins (1, 4), 2-(S-methyldithiocarbamoylami-
nomethyl)indole (7a, isobrassinin), 2-methylthio-1,3-
thiazino[5,6-b]indole (8a, isocyclobrassinin) and their
analogues, and investigation of their antiproliferative
effects against human cell lines.
ment provided 2-phenylimino-1,3-thiazino[5,6-b]indole
3
derivative 10 (Scheme 1).
1
The cytotoxic activities of the synthetized compounds
were determined by the MTT [3-(4,5-dimethylthiazol-
3
3
2-yl)-2,5-diphenyltetrazolium bromide] assay, using
3
4
the HeLa, MCF7 and A431 cell lines. The results are
summarized in Table 1.
The key intermediate, 2-aminomethylindole (6) (Scheme
1), was prepared from commercial indole-2-carboxylic
acid. In the first step, indole-2-carboxamide was ob-
tained by the method of Larock et al. The reduction
of indole-2-carboxamide to 2-aminomethylindole was
Table 1. In vitro tumour cell growth inhibition data (HeLa, MCF7
and A431 cell lines) for the compounds synthesized
2
7
a
Compound Concentration
lM)
Inhibition (%) ± SEM
(
HeLa
MCF7
A431
Cisplatin
1
10
30
30
30
30
30
30
30
30
10
42.6 ± 2.9 53.0 ± 2.3 88.6 ± 0.5
b
R1
S
25.2 ± 5.5 21.5 ± 2.5 NT
83.6 ± 1.9 86.2 ± 1.8 78.6 ± 7.0
76.1 ± 2.2 89.0 ± 0.9 70.7 ± 5.2
7
7
a
b
N
H
S
Me
N
N
R
N
S
SMe
8a
8b
44.5 ± 1.5 <10.0
45.8 ± 2.1 37.3 ± 3.2 NT
<10.0 <10.0 <10.0
<10.0
2
b
R
9
9
1
a
b
0
1
2
1
2
3
R = R = H
4 R = H
50.0 ± 1.8 57.4 ± 1.9 45.7 ± 2.2
b
1
2
R = OMe, R = H
5 R = OMe
99.8 ± 0.3 83.8 ± 1.9 NT
76.8 ± 1.2 46.3 ± 4.4 NT
1
b
R = H, R = OMe
10
a
SEM—standard error of the mean.
Not tested.
Figure 1. Dithiocarbamate and 1,3-thiazino[6,5-b]indole phytoalexins
from cruciferous plants.
b
a
c
H
H
H
R1
N
N R
2
N
S
NH2
N
H
N
H
N
H
S
N
S
6
7
a,b
9a,b
b
2
b
R = Ph
1
7
a, 8a: R = Me
SR1
1
S
NPh
S
7b, 8b: R = benzyl
2
9
a: R = Me
NH
2
N
H
N
H
8
9b: R = Ph
10
a,b
Scheme 1. Reagents and conditions: (a) CHCl
1% (7b); (b) CH Cl , PhMe NBr , rt 5 min, Et
reflux, 2 h, 92% (9a), 95% (9b).
3
, Et
3
N, cat. DMAP, CS
N, rt 10 min, 76% (8a), 82% (8b), 72% (10); (c) CHCl
2
, 0 ꢁC then rt 2 h, MeI (for 7a) or benzyl bromide (for 7b), rt 3 h, 85% (7a),
, dioxane MeNCS (for 9a) or PhNCS (for 9b),
9
2
2
3
3
3
3

P. Csom o´ s et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6273–6276
6275
2
-(S-Methyldithiocarbamoylaminomethyl)indole (7a)
6. Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2000, 10, 1887.
displayed marked antiproliferative effects on the HeLa,
A431 and MCF7 cell lines: >78.6% inhibition was ob-
served at 30 lM. Replacement of the methyl group of
isobrassinin by a benzyl group resulted in similarly high
activity: 7b induced 70.7–89.0% inhibition. A compari-
son of the literature data on the in vitro antiproliferative
activities of brassinin (1) and cyclobrassinin (2) reveals
that the latter (containing a thiazine ring) has a lower
cytotoxic effect. Isocyclobrassinin (8a) also exerted less
pronounced activity (<10.0–44.5%) than that of 7a. The
isosteric transformation of the dithiocarbamate moiety
of isobrassinin to thiourea led to a loss of inhibitory
activity. For the methyl thiourea derivative 9a, no sub-
stantial inhibition was observed, but the phenyl ana-
logue still exhibited >45.7% activity for all three cell
lines. The most interesting results emerged from the oxi-
dative ring-closure of phenyl thiourea 9b. The highest
cytotoxic effect was induced by 10, which at 10 lM re-
duced the cell growth of HeLa and MCF7 cells by
7
8
9
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Lai, C.; Li, R. Bioorg. Med. Chem. Lett. 2006, 16, 4214.
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4, 1867.
14. Viola-Rhenals, M.; Rieber, M. S.; Rieber, M. Biochem.
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7
6.8% and 46.3%, respectively. This is comparable to
the activity of cisplatin (10 lM, 42.6%—HeLa,
3.0%—MCF7). The effects of natural phytoalexin
15. Giovagnini, L.; Ronconi, L.; Aldinucci, D.; Lorenzon, D.;
Sitran, S.; Fregona, D. J. Med. Chem. 2005, 48, 1588.
1
6. Purkayastha, R. P. In Handbook of phytoalexin metabo-
lism and action. In Progress in Phytoalexin Research
During the Past 50 Years; Daniel, M., Purkayasta, R. P.,
Eds.; Marcell Dekker: New York, 1995; pp 1–39.
7. Pedras, M. S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A.
Q. Phytochemistry 2000, 66, 391.
5
2
6
brassinin (1) were also determined (30 lM, 25.2%—
HeLa, 21.5%—MCF7).
1
1
In summary, indolylmethyldithiocarbamates and some
analogues were prepared, and were found to have note-
worthy in vitro antiproliferative effects. Isobrassinin (7a)
and its benzyl analogue (7b) are a new type of dithiocar-
bamate antiproliferative agent. The 2-phenylimino-1,3-
thiazino[5,6-b]indole derivative 10, a sulfur analogue of
b-carboline proved to be a novel type of antitumour
compound. Work on the preparation of further deriva-
tives and the screening of their antiproliferative effects
is in progress.
8. Metha, R. G.; Liu, J.; Constantinou, A.; Thomas, C. F.;
Hawthorne, M.; You, M.; Gerh a¨ user, C.; Pezutto, J. M.;
Moon, R. C.; Moriarty, M. R. Carcinogenesis 1995, 16,
399.
ˇ
1
9. Pil a´ tov a´ , M.; Sari sˇ sk y´ , M.; Kutschy, P.; Miro sˇ sˇ ay; Mez-
ˇ
encev, R.; Curillov a´ , Z.; Such y´ , M.; Monde, K.; Mirossay,
L.; Moj zˇ i sˇ , J. Leuk. Res. 2005, 29, 415.
2
0. Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A.
J.; Prendergast, G. C.; DuHadaway, J.; Bennett, S.;
Donovan, A. M. J. Med. Chem. 2006, 49, 684.
2
2
1. Sporn, M. B. Cancer Res. 1999, 59, 4743.
2. Fodor, L.; Szab o´ , J.; Sz u} cs, E.; Bern a´ th, G.; Soh a´ r, P.;
Tam a´ s, J. Tetrahedron 1984, 40, 4089.
Acknowledgments
2
2
3. Fodor, L.; MacLean, D. B. Can. J. Chem. 1987, 65, 18.
4. Szab o´ , J.; Fodor, L.; Bani-Akoto, E.; Talpas, G. S.;
Bern a´ th, G.; Soh a´ r, P. Tetrahedron 1992, 48, 4957.
5. Csom o´ s, P.; Fodor, L.; Sinkonen, J.; Pihlaja, K.; Bern a´ th,
G. Tetrahedron Lett. 2006, 47, 5665.
The authors express their thanks to the Hungarian
Scientific Research Foundation (OTKA) for financial
support. We are indebted to Dr. Tam a´ s A. Martinek
and Istv a´ n M. M a´ ndity for helpful discussions and
Mrs. E. Juh a´ sz Dinya for technical assistance.
2
2
2
6. Csom o´ s, P.; Fodor, L.; Soh a´ r, P.; Bern a´ th, G. Tetrahedron
2
005, 61, 9257.
7. Pletnev, A. A.; Tian, Q.; Larock, R. C. J. Org. Chem.
002, 67, 9276.
2
References and notes
28. Pedras, M. S. C.; Suchy, M.; Ahiahonu, P. W. K. Org.
Biomol. Chem. 2006, 4, 691.
1
. Imamura, H.; Ohtake, N.; Jona, H.; Shimizu, A.; Moriya,
M.; Sato, H.; Sugimoto, Y.; Ikeura, C.; Kiyonaga, H.;
Nakano, M.; Hagano, R.; Abe, S.; Yamade, K.; Hashiz-
ume, T.; Morishima, H. Bioorg. Med. Chem. Lett. 2000,
29. Melting points were determined on a Kofler apparatus and
are uncorrected. Elemental analyses were performed with
a Perkin-Elmer 2400 CHNS elemental analyser. Merck
Kieselgel 60F254 plates were used for TLC and Merck
Silica gel 60 (0.063–0.100) for column chromatography.
1
0, 109.
2
3
4
. Len, C.; Boulognemerlo, A. S.; Posted, D. J. J. Agric.
Food Chem. 1996, 44, 2856.
NMR spectra were acquired with a Bruker Spectrospin
spectrometer operating at 400.13 MHz for
1
H and
1
3
. Schreck, R.; Meier, B.; Mannel, D. N.; Droge, W.;
Baeuerle, P. A. J. Exp. Med. 1992, 175, 1181.
. Ha, T.; Li, Y.; Gao, X.; McMullen, J. R.; Shioi, T.; Izumo,
S.; Kelley, J. L.; Zhao, A.; Haddad, G. E.; Williams, D.
L.; Browder, I. W.; Kao, R. L.; Li, C. Free Radic. Biol.
Med. 2005, 39, 1570.
100.61 MHz for C. Spectra were recorded at 25 ꢁC in
CDCl or DMF-d as solvent in 5 mm NMR tubes.
3
7
Proton and carbon spectra were referenced internally to
the TMS signal at 0.00 ppm. All spectra were measured by
using the standard pulse programs installed by Bruker.
30. General procedure for dithiocarbamates 7a,b: To a stirred
solution of 2-aminomethylindole (6) (0.5 g, 3.5 mmol) in
chloroform (20 mL), triethylamine (0.48 ml, 3.5 mmol)
5
. Gerhauser, C.; You, M.; Pezzuto, J. M. Cancer Res. 1997,
5
7, 272.

6276
P. Csom o´ s et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6273–6276
0 0
2
–NH), 9.04 (1H, bs, NH-5), 7.78 (2H, d, H-2 ,6 ), 7.47
and 4-dimethylaminopyridine (0.06 g, 0.5 mmol) were
added. Carbon disulfide (0.23 mL, 3.8 mmol) was next
added dropwise under ice cooling and the mixture was
stirred at the same temperature for 2 h. Methyl iodide or
benzyl bromide (3.5 mmol) in chloroform (5 mL) was then
added dropwise to the solution and it was stirred for 3 h at
RT. The organic phase was extracted in turn with 3%
hydrochloric acid (10 mL) and with water (10 mL), dried
CH
(1H, d, J = 7.8 Hz, H-9), 7.39 (1H, d, J = 7.8 Hz, H-6),
0
0
0
7.28 (2H, t, J = 7.7, H-3 ,5 ), 7.16 (1H, t, J = 7.7, H-4 ),
7.07 (1H, t, J = 7.8, H-7), 7.00 (1H, t, J = 7.8, H-8), 4.83
1
3
2 7
(2H, s, CH ); C NMR d (DMF-d ): 163.1, 137.2, 129.1
(2· C), 128.4, 126.2, 122.3, 122.2, 122.1, 120.0, 119,4
(2· C), 117,4, 112,5, 98.4, 45,4. Anal. Calcd for
16 13 3
C H N S (279.36): C, 68.79; H, 4.69; N, 15.04; S,
(
Na
2
SO
4
) and evaporated. The residue was subjected to
11.48. Found: C, 68.62; H, 4.91; N, 15.10; S, 11.62.
32. General procedure for thioureas 9a,b: To a stirred solution
of 2-aminomethylindole (6) (0.29 g, 2 mmol) in chloro-
form (10 mL) and dioxane (10 mL), the corresponding
isothiocyanate (2 mmol) was added in one portion. The
reaction mixture was refluxed for 2 h. After evaporation
the residue was triturated with n-hexane, decanted and
purified by column chromatography using n-hexane/ethyl
column chromatography using n-hexane/ethyl acetate 4:1.
Compound 7a: light pink crystalline powder; mp 95–97 ꢁC
(
NH-1), 7.56 (1H, d, J = 7.8 Hz, H-4), 7.33 (1H, d,
J = 7.8 Hz, H-7) 7.31 (1H, bs, NH–CH ), 7,18 (1H, t,
2
J = 7.8, H-6), 7.08 (1H, t, J = 7.8, H-5), 6.41 (1H, s, H-3),
5.08 (2H, d, J = 5.5 Hz, CH
28
1
Lit. mp 82–84 ꢁC); H NMR d (CDCl
3
): 8.90 (1H, bs,
1
3
2
), 2.67 (3H, s, CH
3
);
C
): 201.7, 136.9, 135.0, 128.2, 123.2, 121,1,
NMR d (CDCl
3
acetate 4:1 as eluent. Compound 9a: white crystalline
1
1
C
2
20.7, 111,9, 102.7, 44.5, 19.1. Anal. Calcd for
(236.36): C, 55.90; H, 5.12; N, 11.85; S,
7.13. Found: C, 55.75; H, 4.88; N, 11.83; S, 27.38.
Compound 7b: light pink crystalline powder; mp 124–
powder; mp 99–100 ꢁC; H NMR d (CDCl ): 9.51 (1H, bs,
3
11
H
12
N
2
S
2
NH-1), 7.55 (1H, d, J = 7.8 Hz, H-4), 7.35 (1H, d,
J = 8 Hz, H-7), 7,17 (1H, t, J = 7.8 Hz, H-6), 7,09 (1H, t,
J = 7.8, H-5), 6.36 (1H, s, H-3), 6.05 (2H, bs, 2· NH–CS),
1
13
1
26 ꢁC; H NMR d (CDCl ): 8.83 (1H, bs, NH-1), 7.55
4.89 (1H, s, CH ), 2.87 (3H, d, J = 4.4, CH ); C NMR d
3
2
3
(1H, d, J = 7.8 Hz, H-4), 7.48–7.22 (7H, m, H-7, NH–CH
and C
7
2
(CDCl
112.0, 101.6, 42.4, 31.1. Anal. Calcd for C11H N S
3
): 182.9, 136.9, 136.7, 128.1, 123.0, 121.0, 120.6,
6
H
5
, overlapping signals), 7,18 (1H, t, J = 7.8, H-6),
.12 (1H, t, J = 7.8, H-5), 6.40 (1H, s, H-3), 5.06 (2H, d,
13 3
(219.31): C, 60.24; H, 5.97; N, 19.16; S, 14.62. Found: C,
60.10; H, 6.04; N, 19.01; S, 14.81. Compound 9b: white
1
3
J = 5.5 Hz, NH–CH
CDCl
): 200.0, 136.9, 136.6, 134.7, 129.7 (2· C), 129.4 (2·
C), 128.3, 128.2, 123.2, 121.1, 120.7, 111.9, 102.8, 44.5,
2
), 4.56 (2H, s, S–CH
2
); C NMR d
1
(
3
crystalline powder; mp 168–169 ꢁC; H NMR d (CDCl
9.51 (1H, bs, NH–CS), 8.10 (1H, bs, NH-1), 7.52 (1H, d,
3
):
4
5
1.0. Anal. Calcd for C H N S (312.45): C, 65.35; H,
1
.16; N, 8.97; S, 20.53. Found: C, 65.11; H, 5.27; N, 8.92;
J = 7.8 Hz, H-4), 7.48–7.18 (7H, m, H-7, H-6 and C H ,
6
7
16
2
2
5
overlapping signals), 7,12 (1H, t, J = 7.8 Hz, H-5), 6.45
(1H, bs, NH–CS), 6.29 (1H, s, H-3), 4.96 (2H, d,
S, 20.66.
1. General procedure for 1,3-thiazino[5,6-b]indoles 8a,b and
0: To an intensively stirred solution of 8a,b or 10
0.85 mmol) in dichloromethane (10 mL) at RT, phenyl-
1
3
3
J = 6.0 Hz, CH2); C NMR d (CDCl ): 182.0, 136.9,
3
1
(
136.5, 136.2, 131.0 (2· C), 128.6 (2· C), 128.1, 126.3,
122.9, 121.0, 120.4, 112.0, 101.9, 42.9. Anal. Calcd for
trimethylammonium tribromide (0.32 g, 0.85 mmol) was
added in one portion. After stirring for 5 min, triethyl-
amine (0.36 mL, 2.6 mmol) was added in one portion.
After 10 min the mixture was evaporated (water bath
<50 ꢁC) and the residue was purified by column chroma-
tography, using n-hexane/ethyl acetate 3:2 as eluent.
16 15 3
C H N S (281.38): C, 68.30; H, 5.37; N, 14.93; S, 11.40.
Found: C, 68.11; H, 5.53; N, 14.91; S, 11.67.
33. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
34. Cervix adenocarcinoma (HeLa), breast adenocarcinoma
(MCF7) and squamous skin carcinoma (A431) cells were
maintained in minimal essential medium supplemented
with 10% foetal bovine serum, antibiotic–antimycotic
agents and non-essential amino acids. Briefly, human
cancer cells were seeded onto a 96-well microplate. On the
second day of the procedure, the original medium was
removed and 200 lL new medium containing the test
substances was added. The tested compounds were
dissolved in DMSO, in a final concentration never
exceeding 0.1%, which has no substantial effect on cell
growth. After an incubation period of 72 h, living cells
were assayed by the addition of 20 lL of 5 mg/mL MTT
solution. MTT was converted by intact mitochondrial
reductase and precipitated as blue crystals during a 4 h
contact period. The medium was then removed and the
precipitated crystals were dissolved in 100 mL DMSO
during a 60 min period of shaking. Finally, the reduced
MTT was assayed at 545 nm by using a microplate reader.
All in vitro experiments were carried out on two micro-
plates with at least 5 parallel wells.
Compound 8a: grey crystalline powder; mp 121–124 ꢁC
1
(
decomp.); H NMR d (CDCl
3
): 8.07 (1H, bs, NH-5), 7.42
(
(
1H, d, J = 7.8 Hz, H-9), 7.33 (2H, d, J = 7.8 Hz H-6), 7,20
1H, t, J = 7.8, H-7), 7.14 (1H, t, J = 7.8, H-8), 4.98 (2H, s,
1
3
2 3 3
CH ), 2.52 (3H, s, CH ); C NMR d (CDCl ): 158.7,
1
1
4
1
36.7, 126.3, 125.6, 123.3, 121.1, 118.4, 112.1, 99.7, 49.4,
6.1. Anal. Calcd for C H N S (234.34): C, 56.38; H,
1
1
10
2 2
.30; N, 11.95; S, 27.37. Found: C, 56.22; H, 4.51; N,
1.83; S, 27.45. Compound 8b: light grey crystalline
1
powder; mp 173–175 ꢁC (dec); H NMR d (CDCl
3
): 8.08
(
1H, bs, NH-5), 7.41 (1H, d, J = 7.8 Hz, H-9), 7.40–7.20
6H, m, H-6 and C , overlapping signals), 7,26 (1H, t,
(
J = 7.8, H-7), 7.18 (1H, t, J = 7.8, H-8), 5.00 (2H, s, NH–
6 5
H
1
3
2 2 3
CH ), 4.35 (2H, s, S–CH ); C NMR d (CDCl ): 157.5,
1
1
37.7, 136.7, 131.6, 129.7 (2· C), 129.2 (2· C), 128.0,
26.2, 123.4, 121.1, 118.5, 112.0, 100.0, 49.5, 37.6. Anal.
14 2 2
Calcd for C17H N S (310.44): C, 65.77; H, 4.55; N, 9.02;
S 20.66,. Found: C, 65.52; H, 4.71; N, 9.01; S, 20.91.
Compound 10: light brown crystalline powder; mp 200–
35. Mezencev, R.; Moj zˇ i sˇ , J.; Pil a´ tov a´ , M.; Kutschy, P.
Neoplasma 2003, 50, 239.
1
2
04 ꢁC (dec); H NMR d (DMF-d
7
): 11.43 (1H, bs,