C.S. Francisco et al. / European Journal of Medicinal Chemistry 87 (2014) 298e305
303
1
0), 120.91 (C-3), 118.86 (C-4a), 112.03 (C-7), 108.25 (C-11), 106.56
1), 8.17 (d, 1H, J ¼ 8.4 Hz, H-5), 8.08 (d, 1H, J ¼ 7.2 Hz, H-8), 7.75 (d,
(
C-5), 16.66 (CH
3
) ppm. HRMS (ESI-TOF) calcd for C16
84.02402; found 285.0250 (MH ).
H
9
ClO
3
1H, J ¼ 2.4 Hz, H-4), 7.51 (t,1H, J ¼ 7.6 Hz, H-6), 7.42 (t,1H, J ¼ 7.6 Hz,
þ
13
2
4
2
H-7), 7.11 (dd, 1H, J ¼ 8.8 e 2.8 Hz, H-2), 3.87 (s, 3H, OCH
3
) ppm.
C
6
NMR (100.6 MHz, DMSO-d ): d 159.31 (CHO), 156.70 (C-3), 138.15
.1.5. 3-Chloro-4-methylpyrano[2,3-b]carbazole-2(10H)-one (5)
To a mixture of 2-hydroxycarbazole (1.0 g, 5.43 mmol) and ethyl
-chloroacetoacetate (1.14 mL, 8.19 mmol), 80% H SO (2.50 mL)
(C-8a), 130.74 (C-9a), 127.24 (C-7), 125.07 (C-4b), 123.91 (C-6),
120.87 (C-5), 116.75 (C-1), 115.16 (C-2), 112.23 (C-4a), 111.41 (C-8),
104.07 (C-4), 55.64 (OCH ) ppm.
3
2
4
was added and the mixture was stirred at room temperature for
8 h. The mixture was poured onto ice, the brown precipitate was
separated by filtration, washed with cold water and recrystallized
from EtOAc/light petroleum. The yellow solid formed was sepa-
rated by filtration and dried. Yield: 0.254 g (0.90 mmol, 16%). mp
4
4
.1.9. 3-Hydroxy-9H-carbazole-9-carbaldehyde (9)
To a cold solution of compound 8 (0.069 g, 0.31 mmol) in dry
CH
CH
2
Cl
Cl
2
(5 mL) under nitrogen atmosphere a 1 M solution of BBr
(0.60 mL, 0.60 mmol) was slowly added. After reaching
3
in
2
2
ꢀ
1
2
(
1
(
(
1
1
1
47e250 C. H NMR (300 MHz, DMSO-d
s, 1H, H-5), 8.23 (d, 1H, J ¼ 7.8 Hz, H-6), 7.50 (dd, 1H, J ¼ 8.1 and
.2 Hz, H-9), 7.43 (dt,1H, J ¼ 1.2 e 7.7 Hz, H-8), 7.38 (s,1H, H-11), 7.23
6
): d 11.66 (s, 1H, NH), 8.59
room temperature the mixture was kept under stirring for 24 h and
then it was poured onto ice and extracted with CH Cl
(3 ꢁ 10 mL).
The organic phase was dried (MgSO ), filtered and evaporated to
dryness. The residue obtained was purified by preparative layer
chromatography (eluent: diethyl ether/light petroleum, 1:1). The
compound 9 was obtained as a solid. Yield: 0.031 g (0.14 mmol,
2
2
4
13
dt, 1H, J ¼ 1.2 and 7.5 Hz, H-7), 2.67 (s, 3H, CH
75.4 MHz, DMSO-d ): 156.66 (C]O), 150.00 (C-10a or C-11a),
49.81 (C-10a or C-11a), 141.81 (C-5a), 140.95 (C-9a), 122.08 (C-5b),
26.46 (C-8), 120.75 (C-4), 120.70 (C-6), 119.64 (C-7), 117.68 (C-5),
15.19 (C-3), 112.07 (C-4a), 111.26 (C-9), 97.10 (C-11), 16.48 (CH
ppm. HRMS (ESI-TOF) calcd for C16 10ClNO 283.0400; found
3
) ppm. C NMR
6
d
ꢀ
1
4
8%). mp 149e150 C. H NMR (300 MHz, DMSO-d ): d 9.79 (s, 1H,
6
3
)
CHO), 9.60 (sl, 1H, OH), 8.25 (d, 1H, J ¼ 8.4 Hz, H-1), 8.06 (d, 2H,
J ¼ 8.4 Hz, H-5 and H-8), 7.49 (t, 1H, J ¼ 7.6 Hz, H-7), 7.46 (d, 1H,
J ¼ 2.4 Hz, H-4), 7.39 (t, 1H, J ¼ 7.6 Hz, H-6), 6.94 (dd, 1H, J ¼ 8.8 and
H
2
2
83.0405.
13
2
6
.0 Hz, H-2) ppm. C NMR (100.6 MHz, DMSO-d ): d 159.08 (CHO),
4
.1.6. 6-Methoxy-2,3,4,9-tetrahydro-1H-carbazole (6)
To a solution of cyclohexanone (0.30 mL, 2.86 mmol) in glacial
acetic acid (1 mL), methyl 4-methoxyphenyl hydrazine (0.501 g,
.87 mmol) was added over 1 h. The mixture was refluxed for 3.5 h
154.70 (C-3), 138.15 (C-8a), 129.84 (C-9a), 127.09 (C-7), 125.06 (C-
4
4
b), 123.85 (C-6), 120.69 (C-5), 116.76 (C-1), 115.64 (C-2), 112.05 (C-
a), 111.33 (C-8), 105.73 (C-4) ppm.
2
and left stirring until reaching room temperature. It was then
cooled in ice for 20 min and then 75% (v/v) aqueous MeOH (4 mL)
was added. The solid formed was filtered and washed with 12 mL of
the same aqueous methanol to give a beige solid. Yield: 0.409 g
4
.1.10. (E)-Ethyl 3-(3-hydroxy-9H-carbazol-9-yl)acrylate (10)
To a solution of compound 9 (0.070 g, 0.33 mmol) in N,N -
0
diethylaniline (2.0 mL) carbethoxymethylenetriphenylphosphor-
ane (0.160 g, 0.46 mmol) was added and the mixture was refluxed
for 15 h. After cooling, water was added (10 mL) and extracted with
ꢀ
1
(
2.03 mmol, 71%). mp 93e95 C. H NMR (300 MHz, CDCl
br, 1H, NH), 7.18 (d, 1H, J ¼ 9.0 Hz, H-1), 6.93 (d, 1H, J ¼ 2.4 Hz, H-4),
), 2.72e2.68
3
): d 7.56 (s
CH
2
Cl
2
(4 ꢁ 5 mL). The organic phase was washed with 5% HCl
6
.77 (dd, 1H, J ¼ 8.7 and 2.4 Hz, H-2), 3.86 (s, 3H, OCH
m, 4H, H-6 and H-7),1.90e1.89 (m, 4H, H-5 and H-8) ppm. C NMR
75.4 MHz, CDCl ): 153.84 (C-3), 153.06 (C-8a), 130.68 (C-9a),
28.17 (C-4a), 110.90 (C-1), 110.50 (C-2), 110.02 (C-4b), 100.23 (C-4),
5.95 (OCH ), 23.33 (C-6 or C-7), 23.18 (C-5 and C-8), 20.94 (C-6 or
C-7) ppm. Anal. calcd for C13 15NO: C, 77.91; H, 7.223; N, 6.983%;
found: C, 77.58; H, 7.51; N, 6.96%.
3
13
(5 ꢁ 5 mL), dried (MgSO
4
), filtered and evaporated to dryness. The
(
(
1
residue obtained was purified by preparative thin layer chroma-
3
d
tography (eluent: diethyl ether/light petroleum, 7:3) and a yellow
solid was obtained. Yield: 0.021 g (0.09 mmol, 28%). m.p. > 200 C
ꢀ
5
3
1
(dec.). H NMR (400 MHz, acetone-d
6
):
d
8.54 (d, 1H, J ¼ 14.4 Hz, H-
H
0
0
3
), 8.57 (s,1H, OH), 8.10 (dd,1H, J ¼ 8.0 and 0.8 Hz, H-5 or H-8 ), 7.90
0
0
0
(
1
d, 1H, J ¼ 8.4 Hz, H-5 or H-8 ), 7.81 (d, 1H, J ¼ 8.8 Hz, H-1 ), 7.61 (d,
0
0
H, J ¼ 2.4 Hz, H-4 ), 7.58 (td, 1H, J ¼ 8.0 and 1.2 Hz, H-7 ), 7.39 (td,
4.1.7. 3-Methoxy-9H-carbazole (7)
0
0
1
6
H, J ¼ 8.0 and 0.8 Hz, H-6 ), 7.15 (dd, 1H, J ¼ 2.4 and 8.8 Hz, H-2 ),
To a solution of compound 6 (0.409 g, 2.03 mmol) in p-cymene
and H O (5 mL, 4:1) 10% Pd/C (0.206 g) was added and the mixture
2
heated under reflux for 48 h. After cooling it was filtered over Celite
and washed with boiling EtOAc. The filtrate was evaporated to
.32 (d, 1H, J ¼ 14.0 Hz, H-2), 4.28 (q, 2H, J ¼ 7.2 Hz, CH
2
), 1.35 (t, 3H,
): 168.18 (C]
13
J ¼ 7.2 Hz, CH
3
) ppm. C NMR (100.6 MHz, acetone-d
6
0
0
0
O), 154.66 (C-3 ), 140.64 (C-8 a), 137.65 (C-3), 133.61 (C-9 a), 127.98
0 0 0 0 0
(C-7 ), 127.64 (C-4 a), 126.14 (C-4 b), 123.31 (C-6 ), 121.27 (C-5 or C-
dryness, and the product was obtained as an oil. Yield: 0.210 g
0
0
0
0
0
0
1
8 ), 116.49 (C-2 ), 113.97 (C-1 ), 112.50 (C-5 or C-8 ), 106.72 (C-4 ),
00.90 (C-2), 60.50 (CH ), 14.74 (CH ) ppm.
(
1.07 mmol, 53%). H NMR (300 MHz, CDCl
3
):
d
8.04 (d, 1H,
1
2
3
J ¼ 8.1 Hz, H-5), 7.57 (d, 1H, J ¼ 2.4 Hz, H-4), 7.41e7.40 (m, 2H, H-7
and H-8), 7.34 (d, 1H, J ¼ 8.7 Hz, H-1), 7.24e7.20 (m, 1H, H-6), 7.07
(
dd,1H, J ¼ 9.0 and 2.4 Hz, H-2), 3.94 (s, 3H, OCH
3
) ppm. NH was not
4.2. Tumour cell growth assay
observed.
The compounds were evaluated for their anti-proliferative effect
on human cancer cell lines MDA MB231 (breast adenocarcinoma)
and TCC-SUP (bladder transitional cell carcinoma). The MDA
MB231 epithelial cell line was established from a pleural effusion
obtained from a 51-year-old female patient with breast cancer. The
TCC-SUP cell line was established from a tumour specimen resected
from the urinary bladder transitional cell carcinoma (undifferen-
tiated, grade IV) of a 67-year-old woman. All the cell lines were
4
.1.8. 3-Methoxy-9H-carbazole-9-carbaldehyde (8)
To a solution of dry DMF (0.10 mL, 1.30 mmol) in dry CH
2
Cl
2
ꢀ
(
5 mL) cooled to 0 C, POCl
3
(0.07 mL, 0.75 mmol) was slowly added
and the mixture was kept under stirring for 30 min. Then a solution
of compound 7 (0.100 g, 0.51 mmol) in dry CH Cl (10 mL) was
added and the reaction mixture was heated under reflux for 3 h.
After cooling, water (10 mL) was added and the phases were
separated. The organic phase was sequentially washed with 10%
2
2
kindly provided by IPATIMUP (Portugal). The cells were maintained
ꢀ
HCl (5 mL) and saturated NaHCO
filtered and evaporated to dryness. The product 8 was obtained as a
3
(2 ꢁ 5 mL), dried (MgSO
4
),
in an incubator with a 5% CO
2
atmosphere and at 37 C. The culture
medium used was the Dulbecco's modified Eagle medium (DMEM)
ꢀ
1
®
solid, m.p. 67e70 C. Yield: 0.087 g (0.39 mmol, 76%). H NMR
300 MHz, DMSO-d ):
9.81 (s, 1H, CHO), 8.34 (d, 1H, J ¼ 8.4 Hz, H-
(GIBCO , Invitrogen, Barcelona, Spain) supplemented with Foetal
®
(
6
d
Bovine Serum (FBS) (GIBCO , Invitrogen, Barcelona, Spain) (10% for