Synthesis and antitumoral activity of novel 3-(2-substituted-1,3,4-oxadiazol-5-yl) and 3-(5-substituted-1,2,4-triazol-3-yl) β-carboline derivatives

Article abstract of DOI:10.1016/j.bmc.2008.10.008

Several novel 1-substituted-phenyl β-carbolines bearing the 2-substituted-1,3,4-oxadiazol-5-yl and 5-substituted-1,2,4-triazol-3-yl groups at C-3 were synthesized and evaluated for their in vitro anticancer activity. The assay results pointed thirteen compounds with growth inhibition effect (GI₅₀ < 100 μM) for all eight different types of human cancer cell lines tested. The β-carbolines 7a and 7h, bearing the 3-(2-metylthio-1,3,4-oxadiazol-5-yl) group, displayed high selectivity and potent anticancer activity against ovarian cell line with GI₅₀ values lying in the nanomolar concentration range (GI₅₀ = 10 nM for both compounds). The 1-(N,N-dimethylaminophenyl)-3-(5-thioxo-1,2,4-triazol-3-yl) β-carboline (8g) was the most active compound, showing particular effectiveness on lung (GI₅₀ = 0.06 μM), ovarian and renal cell lines. The potent anticancer activity presented for synthesized compounds 7a, 7h, and 8g, together with their easiness of synthesis, makes these compounds promising anticancer agents.

Full text of DOI:10.1016/j.bmc.2008.10.008

Bioorganic & Medicinal Chemistry 16 (2008) 9660–9667
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumoral activity of novel 3-(2-substituted-1,3,4-oxadiazol-
5-yl) and 3-(5-substituted-1,2,4-triazol-3-yl) b-carboline derivatives
Anelise S. Nazari Formagio a, Lilian T. Düsman Tonin a, Mary Ann Foglio b, Christiana Madjarof b,
João Ernesto de Carvalho b, Willian Ferreira da Costa a, Flávia P. Cardoso a, Maria Helena Sarragiotto a,
*
a Departamento de Química, Universidade Estadual de Maringá, Av. Colombo, 5790 CEP: 87020-900, Maringá, PR, Brazil
b Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, CP: 6171, 13083-970 Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel 1-substituted-phenyl b-carbolines bearing the 2-substituted-1,3,4-oxadiazol-5-yl and 5-
Received 29 June 2008
Revised 2 October 2008
Accepted 3 October 2008
Available online 7 October 2008
substituted-1,2,4-triazol-3-yl groups at C-3 were synthesized and evaluated for their in vitro anticancer
activity. The assay results pointed thirteen compounds with growth inhibition effect (GI50 < 100 lM) for
all eight different types of human cancer cell lines tested. The b-carbolines 7a and 7h, bearing the 3-(2-
metylthio-1,3,4-oxadiazol-5-yl) group, displayed high selectivity and potent anticancer activity against
ovarian cell line with GI50 values lying in the nanomolar concentration range (GI50 = 10 nM for both com-
pounds). The 1-(N,N-dimethylaminophenyl)-3-(5-thioxo-1,2,4-triazol-3-yl) b-carboline (8g) was the
Keywords:
Antitumor activity
b-Carboline derivatives
1,3,4-Oxadiazoly
1,2,4-Triazolyl
most active compound, showing particular effectiveness on lung (GI50 = 0.06 lM), ovarian and renal cell
lines. The potent anticancer activity presented for synthesized compounds 7a, 7h, and 8g, together with
their easiness of synthesis, makes these compounds promising anticancer agents.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
series of novel 1-substituted-phenyl b-carbolines bearing the 2-
substituted-1,3,4-oxadiazol-5-yl and 5-substituted-1,2,4-triazol-
The b-carbolines alkaloids are a class of synthetic and naturally
occurring compounds that possess a large spectrum of important
pharmacological properties.1–6 Recent reports7–16 have pointed b-
carboline alkaloids as potential antitumoral agents, which can act
through multiple mechanisms, such as intercalating into DNA,17
3-yl at C-3, expecting that the incorporation of these substituents
may improve the antitumoral activities of b-carbolines.
2. Results and discussion
2.1. Chemistry
inhibiting Topoisomerase
I
and II,18 CDK (cyclin-dependent
kinases)19,20 and I B kinase complex).21 Studies on a variety
jK (Ij
of synthetic b-carboline derivatives have demonstrated the influ-
ence of both molecule planarity and nature of the substituents in
positions-1, -3 and -9 of the b-carboline skeleton on the antitumor
activities.7–16 Appropriate substituents on these positions could
lead to more potent compounds with reduced toxicity. Conse-
quently, the synthesis of novel b-carboline derivatives with differ-
ent substituents are needed in order to provide more data for
structure–activity relationship studies of this class of compounds.
Some studies demonstrated that b-carboline derivatives
containing a phenyl-substituted group in 1-position present anti-
tumoral activities.4 Furthermore, several compounds having
1,3,4-oxadiazole and 1,2,4-triazole units were reported to possess
antitumoral activities.22–25 Therefore, in the present work we pro-
posed the synthesis and in vitro anticancer activity evaluation of a
The synthetic routes for the preparation of 1,3-disubstituted
b-carbolines are presented in Schemes 1 and 2. The methyl tet-
rahydro-b-carboline-3-carboxylates 3a–h were prepared through
a Pictet-Spengler condensation of the L-tryptophan 1 with appro-
priate aromatic aldehydes, in acid media, and subsequent ester-
ification of the carboxylic acids 2a–h with methanol and sulfuric
acid. Conversion of the derivatives 3a–h to the corresponding b-
carboline-3-carbohydrazides 5a–h was carried out by oxidation
with sulfur in refluxing xylene of methyl 1,2,3,4-tetrahydro-9H-
b-carboline-3-carboxilates 3a–h, followed by reaction of methyl
b-carboline-3-carboxylates 4a–h with hydrazine hydrate, in eth-
anol under reflux, according to the procedures described in the
literature.12 For preparation of 3-(2-thioxo-1,3,4-oxadiazol-5-yl)
b-carbolines 6a–h, the key intermediates 5 were subjected to
reaction with carbon disulfide in presence of KOH and ethanol,
under reflux. The 1,3,4-oxadiazolyl b-carbolines 6a–h were sub-
sequently S-methylated with methyl iodide in the presence of
K2CO3 at room temperature to afford the 3-(2-methylthio-1,3,
* Corresponding author. Tel.: +55 44 3261 3657.
E-mail
addresses:
mhsarragiotto@uem.br,
mhelena@wnet.com.br
(M.H. Sarragiotto).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.008

Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
9661
COOCH3
COOH
COOH
a
b
NH
NH
NH2
N
H
N
H
N
H
R
R
1
3a-h
2a-h
CONHNH2
3
COOCH3
3
c
N
d
N
N
H
N
H
1
1
R
R
5a-h
4a-h
a: R= H
e: R= 4-NO2
b: R= 4-OCH3
c: R= 4-OH
d: R= 3-NO2
f: R= 3-OCH3, 4-OH
g: R= 4-N(CH3)2
h: R= 2-Cl
Scheme 1. Reagents and conditions: (a) acetic acid, RCHO, reflux, 2 h, and adjusted pH 5 with NH4OH; (b) CH3OH, H2SO4, reflux, 24 h; (c) S, xylene, reflux 24 h, at 4 °C, 3 h; (d)
NH2NH2ꢀH2O, EtOH, reflux, 48 h.
N
N
N
N
NH
O
N
SCH3
SH
S
O
O
N
a
N
N
N
H
b
N
H
N
H
7a-h
R
6a-h
R
R
N
N
NH
5
8
4
N
CONHNH2
3
6
7
SH
S
N
N
H
N
N
N
H
H
N
1
N
H
c
N
H
'
'
'
2
6
'
3
5
R
'
4
R
R
8a, b, c, g
5a-h
N
NH
N
N
N
N
SCH3
S
SH
N
N
N
N
N
N
NH2
NH2
NH2
d
N
H
e
N
H
N
H
R
R
R
10a, h
9a, c, g, h
a: R = H
e: R = 4-NO2
b: R = 4-OCH3
c: R = 4-OH
d: R = 3-NO2
f: R = 3-OCH3, 4-OH
g: R = 4-N(CH3)2
h: R = 2-Cl
Scheme 2. Reagents and conditions: (a) CS2/KOH, EtOH, reflux, 48 h, acidified dilute HCl; (b) CH3I, K2CO3, THF, rt, 48 h; (c) KSCN, HCl, EtOH, reflux, 48 h, NaOH 2 M, reflux,
48 h; (d) CS2/KOH, EtOH, reflux, 48 h, NH2NH2.H2O, reflux, 4 days; (e) CH3I, KOH, EtOH, rt, 48 h.
4-oxadiazol-5-yl) b-carbolines 7a–h.26 The 3-(5-thioxo-1,2,4-tria-
zol-3-yl) b-carbolines 8a–c and 8g were prepared by reflux of
corresponding compounds 5 with KSCN in ethanol and HCl and
further alkaline cyclization of the thiosemicarbazides intermedi-
ates.27 The synthesis of compounds 9a, 9c, and 9g–h were accom-
plished by refluxing compounds 5 with carbon disulfide in
presence of KOH, in ethanol, followed by reaction of the potas-
sium dithiocarbazate salt with hydrazine hydrate, in ethanol, un-
der reflux.28 Reaction of 9a and 9h with methyl iodide and KOH
produces the S-methyl derivatives 10a and 10h.29

9662
Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
The chemical structures of all novel compounds were confirmed
2.2. Anticancer activity
by spectral data (1H NMR, 13C NMR, IR, and HR-ESIMS). The spec-
troscopic data are given in Section 4. The 3-(2-thioxo-1,3,4-oxa-
diazol-5-yl) b-carbolines 6 and the 3-(5-thioxo-1,2,4-triazol-3-yl)
b-carbolines 8 and 9 may exist in thione-thiol tautomeric struc-
tures, as indicated by the –NH and –SH protons signals at 13.7–
14.1 ppm in the 1H NMR, as broad singlet integrating for one
hydrogen each. In the state solid, these compounds are present
in thione (–C@S) form, as indicated in their IR spectrum by absence
of the absorption band at 2500 cmꢁ1 (–SH stretching) and the pres-
ence of two absorption bands at 1230–1339 cmꢁ1, characteristic of
the –C@S group for this class of compounds.
The anticancer activity of all synthesized 1,3-disubstituted b-
carboline derivatives was evaluated in vitro against eight human
tumor cell lines: melanoma (UACC-62), breast (MCF7), lung (NCI-
460), leukemia (K-562), ovarian (OVCAR), prostate (PCO-3), colon
(HT-29), and renal (786-0). Three response parameters (GI50, TGI,
and LC50) were calculated for each compound and cell line tested
and the results were summarized in Tables 1 and 2. The GI50 values
(growth inhibitory activity) (Table 1) refer to the drug concentra-
tion that produce a 50% reduction of cellular growth when com-
pared to untreated control cells. The TGI (cytostatic activity) and
Table 1
GI50 values (in
lM) and GI50 MG-MID (in lM) for compounds 6a–h, 7a–h, 8a–c, 8g, 9a, 9c, 9g–h, 10a, and 10h
Compound
Melanoma UACC-62
Breast MCF-7
Lung NCI-460
Leukemia K-562
Ovarian OVCAR
Prostate PCO-3
Colon HT-29
Renal 786-0
MG-MIDa
Cancer cell lines
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8g
9a
9c
9g
9h
10a
10h
>100
25.54
24.88
88.70
27.93
27.56
32.17
18.84
26.93
12.19
31.21
7.42
79.75
>100
12.66
>100
16.78
>100
>100
19.8
20.60
9.20
15.33
26.44
21.47
10.98
46.66
31.66
9.55
20.30
0.43
0.18
10.80
21.32
>100
22.57
>100
0.17
31.77
>100
>100
22.31
>100
5.87
14.72
45.87
0.01
25.54
17.31
81.07
12.20
26.01
25.94
14.72
21.43
23.62
>100
10.46
44.18
>100
7.53
92.60
1.03
4.11
>100
0.50
1.37
25.54
20.26
>100
27.93
31.34
17.31
40.46
17.43
16.22
>100
12.85
68.14
>100
7.31
87.40
8.02
35.5
>100
46.2
8.63
27.95
31.55
94.88
32.38
73.13
22.45
14.72
27.89
32.04
>100
11.78
>100
>100
15.35
>100
48.55
7.56
7.48
0.17
0.04
10.9
0.26
0.97
39.5
>100
16.8
28.75
24.06
78.65
24.59
48.20
23.51
17.90
23.65
4.65
12.05
12.47
38.03
100
11.65
97.39
2.03
13.21
29.00
6.58
16.76
>100
24.10
37.99
17.33
23.82
18.08
99.48
29.08
19.07
16.38
>100
17.80
>100
15.19
6.36
66.8
10.6
1.74
24.3
22.5
50.4
20.2
>100
18.6
>100
73.41
>100
89.29
19.09
20.75
3.42
1.28
2.13
17.82
24.47
>100
4.82
>100
1.64
6.36
0.10
6.69
0.06
1.59
0.15
0.20
15.4
>100
13.9
13.52
21.32
>100
16.37
>100
0.01
64.9
>100
6.41
1.09
12.0
27.5
>100
6.80
3.20
>100
>100
11.1
9.93
29.9
>100
62.4
2.04
8.63
15.0
61.2
>100
70.1
1.23
10.26
8.87
23.65
27.18
61.48
13.07
10.9
25.5
>100
24.7
>100
22.2
15.0
34.0
>100
33.0
>100
20.4
>100
20.1
>100
11.2
1.92
a
GI50 (in lM) mean-graph midpoint (MG-MID)=average sensitivity of all cell lines toward the test compounds.
Table 2
TGI ( M) and LC50 (lM; values in parentheses) and TGI and LC50 MG-MID (lM) for compounds 6a–h, 7a–h, 8a–c, 8g, 9a, 9c, 9g–h, 10a, and 10h
l
Melanoma UACC-62
Cancer cell lines
Breast MCF-7
Lung NCI-460
Leukemia K-562
Ovarian OVCAR
Prostate PCO-3
Colon HT-29
Renal 786-0
MG-MIDa
6a
6b
6c
6d
6e
6f
6h
7a
7b
7c
7d
7f
7h
8c
8g
9a
9c
9g
9h
10h
>100
47.2 (>100)
>100
>100
>100
47.9 (>100)
80.1 (>100)
>100
>100
>100
69.4 (>100)
48.6 (>100)
>100
>100
>100
>100
>100
>100
86.1 (>100)
59.0 (>100)
>100
97.0 (>100)
>100
>100
>100
>100
>100
31.3 (>100)
>100
23.4 (80.3)
>100
28.1 (61.2)
51.9 (>100)
>100
37.0 (>100)
32.5 (>100)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
74.7 (>100)
56.6 (>100)
73.4 (>100)
99.2 (>100)
>100
54.4 (>100)
>100
87.0 (>100)
86.7 (>100)
>100
>100
>100
>100
>100
>100
>100
>100
0.1 (1.6)
>100
35.0 (>100)
>100
59.8 (>100)
0.1(10.1)
>100
7.28 (47.9)
25.3 (52.5)
98.4 (>100)
>100
49.9 (>100)
38.4 (>100)
>100
41.1 (>100)
>100
35.7 (>100)
99.2 (>100)
97.3 (>100)
>100
86.6 (>100)
>100
>100
55.3 (>100)
>100
>100
>100
>100
>100
>100
>100
35.0 (>100)
>100
39.4 (>100)
99.6 (>100)
>100
51.5
86.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.8 (51.9)
>100
36.3 (>100)
>100
23.8 (>100)
1.24 (37.3)
49.4 (>100)
94.1 (>100)
80.0 (>100)
>100
92.67 (>100)
75.36 (>100)
98.27 (>100)
86.36 (>100)
99.90 (>100)
90.90 (>100)
93.67 (>100)
26.03 (57.50)
69.84 (>100)
37.28 (83.20)
93.03 (>100)
68.06 (94.05)
16.56 (72.25)
74.69 (>100)
17.85 (80.63)
52.65 (92.26)
76.14 (89.26)
93.30 (>100)
79.73 (>100)
73.86 (>100)
>100
74.0 (>100)
10.4 (74.7)
>100
28.8(93.1)
81.5 (>100)
>100
68.4 (>100)
>100
>100
>100
>100
>100
>100
24.8 (59.2)
>100
39.4 (>100)
3.8 (73.5)
40.7 (>100)
6.68 (>100)
30.6 (>100)
>100
>100
>100
>100
4.2 (>100)
>100
8.96 (>100)
54.7 (>100)
12.2 (40.3)
71.8 (>100)
38.0 (>100)
41.8 (>100)
>100
>100
>100
93.5 (>100)
a
TGI (lM) and LC50 (lM; values in parentheses) mean-graph midpoint (MG-MID) = average sensitivity of all cell lines toward the test compounds.

Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
9663
LC50 (cytotoxic activity) values parameters (Table 2) refer to the
drug concentration for total growth inhibition and for killing 50%
of the cells, respectively. Compounds with GI50 values < 100 lM
were considered active. A mean-graph midpoint values (MG-
MID) and selectivity of the compounds toward tumor cell lines
derivatives 7a–h by reaction of compounds 6 with methyl iodide
and evaluated the effect of the S-methylation of 6a–h on the anti-
tumoral activity. The analysis of GI50 and TGI (MG-MID) values (Ta-
bles 1 and 2) showed an increase of the activities for compounds
7a, 7b, 7c, 7f, and 7h compared to the 2-thioxo-1,3,4-oxadiazol-
5-yl analogues. The b-carbolines 7a and 7h, with the phenyl and
o-chlorophenyl substituents in position-1, respectively, were the
most active derivatives for this series, displaying high selectivity
and potent anticancer activity against OVCAR ovarian cell line both
were calculated for each of the parameters GI50, TGI, and LC50
.
MG-MID values furnished an averaged activity parameter for all
cell lines. For the measurement of the selectivity, the MG-MID
(lM) value for each compound was divided by their GI50, TGI or
LC50 values for a particular cell line. Selectivity (d) was considered
low if <10, moderate >10, and <100, and high if >100.
The anticancer assay results pointed thirteen compounds (6d,
6f, 6g, 6h, 7a, 7c, 7f, 7h, 8g, 9a, 9c, 9h, and 10h) with growth
presenting GI50 of 0.01
both compounds displayed significant activity against leukemia
(K-562) cell line with GI50 values of 0.43 and 0.17 M, for 7a and
7h, respectively. A potent activity against this cell line was also
observed for compound 7b (GI50 = 0.18 M), containing the p-
lM and TGI of 0.1 lM (Tables 1 and 2). Also,
l
inhibition effect (GI50 < 100
lines tested, showing GI50 (MG-MID) values in the range of
1.23–27.18 M (Table 1). Compounds 7a, 7h, 8c, 8g, 9a, and 9c
lM) for all eight human cancer cell
l
methoxyphenyl group at position-1. The influence of the S-methyl
group on activity was particularly significant for 6c, with GI50
l
exhibited broad spectrum antitumor and potent activity at GI50
and TGI levels, showing effective growth inhibition GI50 (MG-
values increasing in the range of 21.47 to >100
MID = 78.65 M) in 6c to 7.42–19.07 (GI50 MG-MID =
12.47 M) in the S-methylated analog 7c. On the other hand, the
S-methylation of the moderately active derivative 6e (GI50 MG-
MID = 48.20 M) and of active 6g (GI50 MG-MID = 17.90 M)
M,
lM (GI50 MG-
l
lM
MID) values of 4.65, 2.03, 6.58, 1.23, 10.26, and 8.87
1), respectively, and cytostatic activity TGI (MG-MID) values of
26.03, 16.56, 74.69, 17.85, 52.65, and 76.14 M (Table 2), respec-
l
M (Table
l
l
l
l
tively. In addition, compounds 7a, 7h, 8g, 9a, and 9c displayed
resulted in 7e and 7g (GI50 MG-MID values of 100 and 97.39 l
cytotoxic efficacy with LC50 (MG-MID) values of 57.50, 72.25,
respectively) with complete loss of the antitumoral activity.
80.63, 92.26, and 89.26
7a, 7h, and 8g proved to be the most active derivatives in this
study.
In relation to the sensitivity against some particular cell line,
compounds 7a and 7h presented the highest growth inhibitory
l
M (Table 2), respectively. Compounds
The substitution of 2-thioxo-1,3,4-oxadiazol-5-yl group (com-
pounds 6) at C-3 of the b-carboline ring system for a 5-thioxo-
1,2,4-triazol-3-yl group (compounds 8) resulted in a increase of
the antitumoral activity, particularly for 8c and 8g derivatives.
The 1-(4-N,N-dimethylaminophenyl)-3-(5-thioxo-1,2,4-triazol-3-
yl) b-carboline 8g was the most active among all compounds
tested, with particular effectiveness against NCI-470 lung, OVCAR
ovarian, and 786-0 renal cell lines.
activity towards OVCAR ovarian cancer cell lines (GI50 = 0.01
Table 1). Compound 8g was particular effectiveness on NCI-460
lung (GI50 = 0.06 M, TGI = 8.96 M), OVCAR ovarian
M, TGI = 7.28 and LC50 47.9 M) and 786-0 renal
M, TGI = 1.24 and LC50 37.3 M) cell lines. Analysis
lM;
l
l
(GI50 = 1.09
(GI50 = 0.04
l
l
l
l
The 4-amination of the 1,2,4-triazol-3-yl heterocyclic ring of 8g
led to the compound 9g with reduced activity, which showed only
selective growth inhibition of the NCI-460 lung cell line with GI50
of the selectivity data (Table 3) showed that compounds 7a, 7h,
8b, and 9g demonstrated high relative selectivity towards a partic-
ular tumor cell line (dGI50 in the range of 118.20–465.08; Table 3).
Additionally, compounds 7a and 7h presented high selectivity at
TGI level, with dTGI values 260.27 and 165.55, respectively.
From the series containing the 2-thioxo-1,3,4-oxadiazol-5-yl
substituent at C-3 of the b-carboline system, compounds 6g and
6h with p-N,N-dimethylaminophenyl and o-chlorophenyl substitu-
ents at C-1, respectively, were the most actives with GI50 values
values of 0.20
pound of this series, presenting GI50 values minor than 25
all cell lines. S-methylation of 9a and 9h resulted in the reduction
of the activity for the derivative 10a (GI50 MG-MID = 61.48 M;
Table 1) and increase of the activity for 10h (GI50 MG-
MID = 13.07 M; Table 1).
lM. The 4-amino analog 9a was de most active com-
lM for
l
l
Analyzing the bioassays results we can observe an interestingly
anticancer effect for 5-substituted-1,2,4-triazol-3-yl derivatives.
Except for 10a, the derivatives of this series showed a specific anti-
tumoral activity towards human lung (NCI-460) and renal (786-0)
minor of 50 lM against all human cell lines. In order to improve
the activity we prepared the 2-methylthio-1,3,4-oxadiazol-5-yl
cell lines with GI50 values in the range of 0.06–15.4
39.5 M, respectively.
lM and 0.04–
l
Table 3
3. Conclusions
Selectivity (d) toward most sensitive tumor cell lines for compounds 7a–b, 7h, 8b–c,
8g, 9c, 9g, and 10a
In conclusion, several novel 3-(2-substituted-1,3,4-oxadiazol-5-
yl) and 3-(5-substituted-1,2,4-triazol-3-yl) b-carboline derivatives
displaying potential antitumoral activity were synthesized from
Compound
Cell lines
Selectivitya
(d) for GI50
(d) for TGI
7a
K-562
OVCAR
K-562
10.9
465.08
66.24
11.94
202.7
289.96
13.17
38.77
20.46
30.83
59.13
34.5
the commercially available L-tryptophan in few steps and high
260.27
yields. Particularly, compounds 7a, 7h, 8c, 8g, 9a, and 9c exhibited
broad spectrum antitumor and potent activity at GI50, TGI, and LC50
levels. Compounds 7a and 7h inhibits high and selectively OVCAR
ovarian cell line with GI50 values lying in the nanomolar concentra-
tion range (GI50 = 10 nM). Compound 8g was the most active
7b
7h
K-562
OVCAR
NCI-460
PCO-3
786-0
NCI-460
786-0
NCI-460
786-0
NCI-460
786-0
165.55
14.39
8b
8c
among all compounds tested with GI50 values less than 11 lM
8g
9c
9g
against all tumor cell lines. The potent anticancer activity
presented for the synthesized compounds, especially 7a, 7h, and
8g, together with their easiness of synthesis makes these com-
pounds promising anticancer agents. Our current investigation
corroborated the previous reports that the antitumor activity of
b-carboline derivatives is dependent of the substituents at the
positions-1 and -3 of the b-carboline ring.
118.2
24.2
30.11
10a
K-562
a
Selectivity was considered low if <10, moderate >10, and <100, high if >100.

9664
Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
123.0, 124.6, 124.8, 130.5, 131.2, 132.1, 133.1, 135.9, 140.4,
141.2, 142.2, 143.4, 150.0, 162.9, 180.7; HR-ESIMS: calcd for
19H11N5O3S [M+H]+ 390.0549; found 390.0661.
4. Experimental
4.1. Chemistry
C
4.1.2.5. 1-(4-Nitrophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl) b-
carboline (6e). Yield: 72%; mp 260.0–262.0 °C; IR(KBr) mmax
3408 (N–H), 1623 (C@N), 1523–1448 (C@C), 1343, 1242 (C@S),
1175 (C–O–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.35 (t,
4.1.1. General
:
Melting points were determined in a Micro-Química apparatus
MQAPF-301 model and are uncorrected. 1H and 13C spectra were
;
recorded in
a Varian spectrometer model Mercury plus BB
J = 7.8 Hz, 1H, H-6), 7.61–7.71 (m, 2H, H-7, H-8), 8.34 (d,
J = 8.7 Hz, 2H, H-20, H-60), 8.46 (d, J = 7.8 Hz, 1H, H-5), 8.48 (d,
J = 8.7 Hz, 2H, H-30, H-50), 8.93 (s, 1H, H-4), 12.14 (s, 1H, 9-NH);
13C NMR (75.45 MHz, DMSO-d6): d 112.8, 114.6, 120.0, 131.4,
122.5, 123.7, 124.0, 129.4, 129.9, 130.2, 134.1, 139.9, 141.7,
143.4, 147.5, 161.0, 178.1; HR-ESIMS: calcd for C19H11N5O3S
[M+H]+ 390.0661; found 390.0549.
300 MHz with DMSO as solvent and TMS as internal standard.
HR-ESI mass spectra were recorded on a Q-Tof (Micromass) spec-
trometer in a positive ion mode. IR spectra were recorded as potas-
sium bromide pellets on a BOMEM spectrometer model MB-100.
All reagents were purchased from commercial suppliers.
4.1.2. General procedure for preparation of 1-(substituted
phenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl) b-carbolines (6a–h)
To a solution of derivatives 5a–h (1 mmol) in ethanol (10 mL)
was added carbon disulfide (5 mmol) and potassium hydroxide
(1 mmol) at 0 °C. The resulting solution was refluxed for 48 h.
The solvent was evaporated and the residue dissolved in water
and acidified with a diluted solution of HCl. The solid obtained
was filtered and crystallized from methanol.
4.1.2.6. 1-(3-Methoxy-4-hydroxyphenyl)-3-(2-thioxo-1,3,4-oxa-
diazol-5-yl) b-carboline (6f). Yield: 68%; mp 188.0–190.0 °C;
IR(KBr)
m
max: 3391 (N–H), 1645 (C@N), 1516–1435 (C@C), 1370,
1272 (C@S), 1173 (C–O–C) cmꢁ1
;
1H NMR (300 MHz, CDCl3/
CD3OD): d 4.01 (s, 3H, OMe), 7.05 (d, J = 8.1 Hz, 1H, H-50), 7.36 (t,
J = 7.8 Hz, 1H, H-6), 7.50 (dd, J = 8.1 Hz and 2.0 Hz, 1H, H-60), 7.56
(d, J = 2.0 Hz, 1H, H-20), 7.60–7.64 (m, 2H, H-7, H-8), 8.21 (d,
J = 7.8 Hz, 1 H, H-5), 8.61 (s, 1H, H-4); 13C NMR (75.45 MHz,
CDCl3/CD3OD): d 55.4; 112.8, 113.0, 113.5, 115.1, 120.7, 121.3,
121.5, 121.7, 128.9, 129.6, 130.5, 134.5, 141.5, 144.4, 147.4,
147.9, 161.4, 178.4; HR-ESIMS: calcd for C20H14N4O3S [M+H]+
391.0865; found 391.0767.
4.1.2.1. 1-Phenyl-3-(2-thioxo-1,3,4-oxadiazol-5-yl) b-carboline
(6a). Yield: 72%; mp 230.0–232.0 °C; IR (KBr)
1623 (C@N), 1556–1441 (C@C), 1368, 1237 (C@S), 1163 (C–O–C)
cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.32 (t, J = 7.0 Hz, 1H, H-
mmax: 3056 (N–H),
;
6), 7.55 (m, 1H, H-7), 7.57–7.69 (m, 4H, H-20, H-30, H-50 and H-
60), 8.03 (d, J = 7.0 Hz, 2H, H-8 and H-40), 8.44 (d, J = 7.2 Hz, 1H,
H-5), 8.83 (s, 1H, H-4), 12.03 (s, 1H, 9-NH); 13C NMR (75.45 MHz,
DMSO-d6): d 112, 113.9, 122.3, 120.5, 120.9, 128.6, 128.9, 129.0,
129.2, 129.5, 131.0, 134.0, 137.2, 141.6, 142.8, 161.2, 177.6; HR-
ESIMS calcd for C19H12N4OS [M+H]+: 345.0810; found 345.0681.
4.1.2.7. 1-(4-N,N-Dimethylaminophenyl)-3-(2-thioxo-1,3,4-oxa-
diazol-5-yl) b-carboline (6g). Yield: 70%. mp: 186.0–188.0 °C;
IR(KBr)
m
max: 3053 (N–H), 1607 (C@N), 1590–1454 (C@C), 1365,
1237 (C@S), 1167 (C–O–C) cmꢁ1
;
1H NMR (300 MHz, DMSO-d6):
d 3.05 (s, 6H, -N(Me)2), 7.08 (d, J = 8.7 Hz, 2H, H-30, H-50), 7.31 (t,
J = 8.1 Hz, 1H, H-6), 7.58 (t, J = 8.1 Hz, 1H, H-7), 7.70 (d, J = 8.1 Hz,
1H, H-8), 7.97 (d, J = 8.7 Hz, 2H, H-20, H-60), 8.43 (d, J = 8.1 Hz, 1H,
H-5), 8.75 (s, 1H, H-4); 13C NMR (75.45 MHz, DMSO-d6): d 40.7,
112.0, 112.8, 113.0, 120.4, 120.9, 122.1, 128.7, 129.1, 129.5,
130.7, 133.4, 134.3, 141.3, 141.5, 143.2, 161.3, 177.5; HR-ESIMS:
calcd for C21H17N5OS [M+H]+ 388.1232; found 388.1078.
4.1.2.2. 1-(4-Methoxyphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)
b-carboline (6b). Yield: 75%; mp 180.0–182.0 °C; IR(KBr) mmax
3300 (N–H), 1610 (C@N), 1514–1432 (C@C), 1372, 1242 (C@S),
1178 (C–O–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 3.88 (s, 3H,
:
;
OMe), 7.20 (d, J = 8.7 Hz, 2H, H-30, H-50), 7.32 (t, J = 8.1 Hz, 1H, H-
6), 7.60 (t, J = 8.1 Hz, 1H, H-7), 7.69 (d, J = 8.1 Hz, 1H, H-8), 8.02
(d, J = 8.7 Hz, 2H, H-20, H-60), 8.44 (d, J = 8.1 Hz, 1H, H-5), 8.80 (s,
1H, H-4), 11.9 (s, 1H, 9-NH); 13C NMR (75.45 MHz, DMSO-d6): d
55.2 (OMe), 112.4, 113.4, 114.4, 120.7, 121.3, 121.5, 128.9, 129.5,
129.6, 129.9, 130.7, 134.4, 141.4, 143.9, 160.4, 161.6, 178.3; HR-
ESIMS: calcd for C20H14N4O2S [M+H]+ 375.0916; found 375.0750.
4.1.2.8. 1-(2-Chlorophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl) b-
carboline (6h). Yield: 65%; mp 258.0–260.0 °C; IR(KBr)
(N–H), 1626 (C@N), 1595–1438 (C@C), 1315, 1247 (C@S), 1170 (C–
O–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.33 (m, 1H, H-6),
mmax: 3185
;
7.55–7.66 (m, 5H, H-7, H-8, H-40, H-50, H-60), 7.72 (dd, J = 7.2 and
2.0 Hz, 1H, H-30), 8.48 (d, J = 7.2 Hz, 1H, H-5), 8.94 (s, 1H, H-4),
11,8 (s, 1H, 9-NH); 13C NMR (75.45 MHz, DMSO-d6): d 112.6,
114.7, 120.5, 120.8, 122.5, 127.6, 128.7, 129.2, 129.8, 130.5,
130.9, 131.9, 132.5, 135.0, 136.0, 141.5, 142.0, 161.0, 177.6;
HR-ESIMS: calcd for C19H11ClN4OS [M+H]+ 379.0420; found
379.0175.
4.1.2.3. 1-(4-Hydroxyphenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl)
b-carboline (6c). Yield: 73%; mp 160.0–162.0 °C; IR(KBr) mmax
3349 (N–H), 1604 (C@N), 1557–1461 (C@C), 1366, 1266 (C@S),
1167 (C–O–C) cmꢁ1 1H NMR (300 MHz, CDCl3/CD3OD): d 7.00 (d,
:
;
J = 8.7 Hz, 2H, H-30, H-50), 7.34 (t, J = 8.1 Hz, 1H, H-6); 7.53 (t,
J = 8.1 Hz, 1H, H-7), 7.66 (d, J = 8.1 Hz, 1H, H-8), 7.90 (d,
J = 8.7 Hz, 2H, H-20, H-60), 8.24 (d, J = 8.1 Hz, 1H, H-5), 8.61 (s, 1H,
H-4). 13C NMR (75.45 MHz, CDCl3/CD3OD): d 13.5 (C-4), 114.1(C-
8), 116.7, 121.7, 122.6, 122.7, 129.8, 129.9, 130.9, 131.3, 132.4,
135.6, 143.1, 145.6, 160.0, 163.0, 180.2; HR-ESIMS: calcd for
C19H12N4O2S [M+H]+ 361.0759; found 361.0707.
4.1.3. General procedure for the preparation of 1-(substituted-
phenyl)-3-(2-methylthio-1,3,4-oxadiazol-5-yl) b-carbolines
(7a–h)
To a solution of derivatives 6a–h (0.6 mmol) in anhydrous THF
(10 mL) was added potassium cabonate (0.6 mmol). The resulting
solution was stirred at room temperature for 1 h. Methyl iodide
(0.8 mmol) was then added and the mixture was stirred for an
additional 48 h. The solvent was removed and the residue obtained
was crystallized from methanol.
4.1.2.4. 1-(3-Nitrophenyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl) b-
carboline (6d). Yield: 70%; mp 226.0–228.0 °C; IR(KBr) mmax
3079 (N–H), 1623 (C@N), 1525–1440 (C@C), 1349, 1235 (C@S),
1163 (C–O–C) cmꢁ1 1H NMR (300 MHz, CDCl3/CD3OD): d 7.34 (t,
:
.
J = 7.8 Hz, 1H, H-6), 7.63 (d, J = 7.8 Hz, 2H, H-8, H-60), 7.84 (t,
J = 7.8 Hz, 1H, H-7), 8.27 (d, J = 7.8 Hz, 1H, H-40), 8.36–8.42 (m,
2H, H-5, H-50), 8.69 (s, 1H, H-4), 8.84 (d, J = 2,0 Hz, 1H, H-20); 13C
NMR (75.45 MHz, CDCl3/CD3OD): d 113.6, 115.2, 119.3, 122.0,
4.1.3.1. 1-Phenyl-3-(2-methylthio-1,3,4-oxadiazol-5-yl) b-carb-
oline (7a). Yield: 53%; mp 216.0–218.0 °C; IR(KBr)
mmax: 3461 (N–
H), 1625 (C@N), 1557–1447 (C@C), 1195 (C–O–C) 725 (C–S–C)

Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
9665
cmꢁ1
;
1H NMR (300 MHz, DMSO-d6): d 2.80 (s, 3H, SMe), 7.34 (t,
DMSO-d6): d 14.4, 55.6, 112.5, 112.8, 113.3, 115.8, 120.5, 121.0,
121.6, 121.7, 128.7, 129.2, 131.5, 132.9, 133.8, 141.6, 143.4,
147.9, 148.3, 164.5, 165.9; HR-ESIMS: calcd for C21H16N4O3S
[M+H]+ 405.1021; found 405.1204.
J = 7.2 Hz, 1H, H-6), 7.55–7.70 (m, 5H, H-7, H-8, H-30, H-40, H-50),
8.04 (d, J = 8.1 Hz, 2H, H-20, H-60), 8.47 (d, J = 7.8 Hz, 1H, H-5),
8.97 (s, 1H, H-4), 11.85 (s, 1H, 9-NH); 13C NMR (75.45 MHz,
DMSO-d6): d 14.4, 112.8, 114.1, 120.3, 120.9, 122.2, 128.5, 128.8,
129.1, 129.7, 131.5, 134.2, 137.3, 137.3, 142.7, 164.5, 165.9; HR-
ESIMS: calcd for C20H14N4OS [M+H]+ 359.0966; found 359.0862.
4.1.3.7. 1-(4-N,N-Dimethylaminophenyl)-3-(2-methylthio-
1,3,4-oxadiazol-5-yl) b-carboline (7g). Yield: 48%; mp 168.0–
170.0 °C; IR(KBr)
m
max: 3376 (N–H), 1624 (C@N), 1565–1435
4.1.3.2. 1-(4-Methoxyphenyl)-3-(2-methylthio-1,3,4-oxadiazol-
(C@C), 1175 (C–O–C), 725 (C–S–C) cmꢁ1
;
1H NMR (300 MHz,
5-yl) b-carboline (7b). Yield: 50%; mp 235.0–237.0 °C; IR(KBr)
DMSO-d6): d 2.80 (s, 3H, SMe), 3.70 (s, 6H, N(Me)2), 7.26 (t,
J = 7.3 Hz, 1H, H-6), 7.56 (t, J = 7.3 Hz, 1H, H-7), 7.70 (d, J = 7.3 Hz,
1H, H-8), 8.19 (d, J = 8.7 Hz, 2H, H-30, H-50), 8.43 (d, J = 7.3 Hz, 1H,
H-5), 8.52 (d, J = 8.7 Hz, 2H, H-20, H-60), 8.95 (s, 1H, H-4). 13C
NMR (75.45 MHz, DMSO-d6): d 14.4, 114.2, 114.5, 119.4, 121.9,
122.5, 128.1, 129.7, 130.3, 131.2, 133.5, 139.7, 140.1, 146.9,
164.0, 166.2; HR-ESIMS: calcd for C22H19N5OS [M+H]+ 402.1389;
found 402.1459.
m
max: 3415 (N–H), 1624 (C@N), 1513–1447 (C@C), 1177 (C–O–C),
730 (C–S–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 2.79 (s, 3H,
;
SMe), 3.87 (s, 3H, OMe), 7.20 (d, J = 8.5 Hz, 2H, H-30, H-50), 7.32
(t, J = 7.5 Hz, 1H, H-6), 7.60 (t, J = 7.5 Hz, 1H, H-7), 7.69 (d,
J = 7.8 Hz, 1H, H-8), 8.01 (d, J = 8.5 Hz, 2H, H-20, H-60), 8.43 (d,
J = 7.8 Hz, 1H, H-5), 8.89 (s, 1H, H-4), 11.89 (s, 1H, 9-NH); 13C
NMR (75.45 MHz, DMSO-d6): d 14.4, 55.4, 112.8, 113.5, 114.3,
120.3, 120.9, 122.2, 128.7, 129.4, 129.7, 130.1, 131.5, 133.8,
138.4, 141.7, 142.6, 160.1, 161.7; HR-ESIMS: calcd for C21H16N4O2S
[M+H]+ 389.1072; found 389.1039.
4.1.3.8. 1-(2-Chlorophenyl)-3-(2-methylthio-1,3,4-oxadiazol-5-
yl) b-carboline (7h). Yield: 56%; mp 240.0–242.0 °C; IR(KBr) mmax
3453 (N–H), 1625 (C@N), 1562–1453 (C@C), 1198 (C–O–C), 730
(C–S–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 2.78 (s, 3H, SMe),
:
4.1.3.3. 1-(4-Hydroxyphenyl)-3-(2-methylthio-1,3,4-oxadiazol-
;
5-yl) b-carboline (7c). Yield: 45%; mp 252.0–254.0 °C; IR(KBr)
7.33 (t, J = 7.0 Hz, 1H, H-6), 7.54–7.67 (m, 4H, H-7, H-40, H-50, H-
60), 7.59 (d, J = 8.0 Hz, 1H, H-8), 7.72 (dd, J = 7.2 and 1.5 Hz, 1H,
H-30), 8.46 (d, J = 7.8 Hz, 1H, H-5), 9.03 (s, 1H, H-4), 11.81 (s, 1H,
9-NH); 13C NMR (75.45 MHz, DMSO-d6): d 14.4, 112.5, 114.7,
120.3, 120.8, 122.5, 127.5, 128.8, 129.1, 129.8, 130.9, 131.2,
131.9, 132.5, 134.9, 136.1, 141.5, 164.6, 165.8; HR-ESIMS: calcd
for C20H13ClN4OS [M+H]+ 375.0916; found 375.0940.
m
max: 3399 (N–H), 1622 (C@N), 1561–1447 (C@C), 1172 (C–O–C),
734 (C–S–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 2.80 (s, 3H,
;
SMe), 7.00 (d, J = 8.7 Hz, 2H, H-30, H-50), 7.30 (t, J = 7.8 Hz, 1H, H-
6), 7.56 (t, J = 7.8 Hz, 1H, H-7), 7.67 (d, J = 7.8 Hz, 1H, H-8), 7.95
(d, J = 8.7 Hz, 2H, H-20, H-60), 8.40 (d, J = 7.8 Hz, 1H, H-5), 8.84 (s,
1H, H-4); 13C NMR (75.45 MHz, DMSO-d6): d 14.7, 113.4, 113.6,
118.9, 121.4, 122.3, 122.5, 129.5, 130.0, 130.5, 130.9, 132.4,
135.5, 142.9, 159.8, 167.8, 166.9; HR-ESIMS: calcd for C20H14N4O2S
[M+H]+ 379.0420; found 379.0414.
4.1.4. General procedure for preparation of 1-(substituted-
phenyl)-3-(5-thioxo-1,2,4-triazol-3-yl) b-carboline (8a–c, 8g)
A mixture of carbohydrazide (5a–c, 5g) (1.0 mmol) and potas-
sium thiocyanate (4.0 mmol) was refluxed for 48 h in ethanol
(50 mL) containing 1.0 mL of concentrated hydrochloric acid. The
precipitate formed was collected by filtration and dried. A solution
of intermediate, which was used without further purification, in
2 N sodium hydroxide aqueous (25 mL) was refluxed for 48 h. After
cooling to room temperature, concentrated hydrochloric acid was
added. The precipitate was filtered and washed several times with
distilled water and crystallized from methanol.
4.1.3.4. 1-(3-Nitrophenyl)-3-(2-methylthio-1,3,4-oxadiazol-5-
yl) b-carboline (7d). Yield: 47%; mp 220.0–223.0 °C; IR(KBr) mmax
3388 (N–H), 1623 (C@N), 1524–1437 (C@C), 1174 (C–O–C), 727
(C–S–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 2.81 (s, 3H, SMe),
:
;
7.36 (t, J = 7.8 Hz, 1H, H-6), 7.63 (t, J = 7.8 Hz, 1H, H-7), 7.70 (d,
J = 7.8 Hz, 1H, H-8), 7.95 (t, J = 8.1 Hz, 1H, H-50), 8.42 (dd, J = 8.1
and 1.5 Hz, 1H, H-40), 8.49 (d, J = 7.8 Hz, 1H, H-5), 8.54 (dd, J = 7.8
and 1.5 Hz, 1H, H-60), 8.85 (brs, 1H, H-20), 9.04 (s, 1H, H-4), 12.14
(s, 1H, 9-NH); 13C NMR (75.45 MHz, DMSO-d6): d 14.5, 112.9,
114.9, 120.5, 120.9, 122.4, 123.4, 123.6, 129.2, 130.3, 130.5,
131.6, 134.6, 135.1, 138.8, 140.1, 142.2, 148.2, 164.7, 165.6; HR-
ESIMS: calcd for C20H13N5O3S [M+H]+ 404.0817; found 404.0804.
4.1.4.1. 1-Phenyl-3-(5-thioxo-1,2,4-triazol-3-yl)
(8a). Yield: 80%; mp 326.0–328.2 °C; IR(KBr)
b-carboline
m
max: 3419 (N–H),
1625 (C@N), 1576–1457 (C@C), 1321, 1237 (C@S) cmꢁ1 1H NMR
;
(300 MHz, DMSO-d6): d 7.30 (t, J = 7.5 Hz, 1H, H-6), 7.55–7.60 (m,
2H, H-7, H-40), 7.62 (d, J = 7.5 Hz, 2H, H-30, H-50), 7.69 (d,
J = 8.4 Hz, 1H, H-8), 8.32 (d, J = 7.5 Hz, 2H, H-20, H-60), 8.39 (d,
J = 7.8 Hz, 1H, H-5), 8.76 (s, 1H, H-4),11.9 (s, 1H, 9-NH), 13.7 (s,
1H), 13.9 (s, 1H); 13C NMR (75.45 MHz, DMSO-d6): d 111.7, 112.8,
120.2, 120.9, 122.1, 128.7, 128.9, 129.0, 129.1, 130.2, 133.4,
133.9, 137.3, 141.6, 141.7, 151.6, 167.0; HR-ESIMS: calcd for
C19H13N5S [M+H]+ 344.0970; found 344.1064.
4.1.3.5. 1-(4-Nitrophenyl)-3-(2-methylthio-1,3,4-oxadiazol-5-
yl) b-carboline (7e). Yield: 43%; mp 218.0–220.0 °C; IR(KBr) mmax
3337 (N–H), 1625 (C@N), 1574–1451 (C@C), 1173 (C–O–C), 728
(C–S–C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 2.81 (s, 3H, SMe),
:
;
7.41 (t, J = 7.5 Hz, 1H, H-6), 7.68 (t, J = 7.5 Hz, 1H, H-7), 7.70 (d,
J = 7.5 Hz, 1H, H-8), 8.28 (d, J = 7.5 Hz,1H, H-5), 8.42 (d, J = 8.7 Hz,
2H, H-20, H-60), 8.82 (s, 1H, H-4), 9.34 (d, J = 8.7 Hz, 2H, H-30, H-
50); 13C NMR (75.45 MHz, DMSO-d6): d 14.4, 112.0, 114.6, 116.8,
121.3, 122.4, 123.2, 126.6, 129.5, 130.0, 130.5, 131.4, 138.5,
139.9, 146.1, 156.3, 163.5, 166.2; HR-ESIMS: calcd for C20H13N5O3S
[M+H]+ 404.0817; found 404.0804.
4.1.4.2. 1-(4-Methoxyphenyl)-3-(5-thioxo-1,2,4-triazol-3-yl) b-
carboline (8b). Yield: 77%; mp 324.0–326.0 °C; IR(KBr) mmax
3418 (NH), 1625 (C@N), 1510–1425 (C@C), 1327, 1238 (C@S)
cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 3.88 (s, 3H, OMe), 7.19 (d,
:
;
4.1.3.6. 1-(3-Methoxy-4-hydroxyphenyl)-3-(2-methylthio-1,3,4-
oxadiazol-5-yl) b-carboline (7f). Yield: 40%; mp 210.0–212.0 °C;
J = 8.7 Hz, 2H, H-30, H-50), 7.31 (t, J = 7.8 Hz, 1H, H-6), 7.59 (t,
J = 7.8 Hz, 1H, H-7), 7.69 (d, J = 7.8 Hz, 1H, H-8), 8.04 (d,
J = 8.7 Hz, 2H, H-20, H-60), 8.40 (d, J = 7.8 Hz, 1H, H-5), 8.85 (s, 1H,
H-4), 11.0 (s, 1H, 9-NH), 13.7 (s, 1H), 13.8 (s, 1H). 13C NMR
(75.45 MHz, DMSO-d6): d 55.4, 112.8, 114.2, 115.8, 120.4, 121.2,
122.0, 128.6, 129.3, 129.9, 130.1, 134.3, 137.2, 141.5, 141.6,
151.1, 160.0, 166.9; HR-ESIMS: calcd for C20H15N5OS [M+H]+
374.1075; found 374.1089.
IR(KBr)
m
max: 3353 (N-H), 1625 (C@N), 1556–1448 (C@C), 1120
(C–O–C), 730 (C–S–C) cmꢁ1
;
1H NMR (300 MHz, DMSO-d6): d
2,80 (s, 3H, SMe), 3.89 (s, 3H, OMe), 7.00 (d, J = 8.1 Hz, 1H, H-50),
7.36 (t, J = 7.8 Hz, 1H, H-6), 7.40 (dd, J = 8.1 Hz, J = 1.8 Hz, 1H, H-
60), 7.50 (d, J = 1.8 Hz, 1H, H-20), 7.56–7.69 (m, 2H, H-7, H-8), 8.21
(d, J = 7.8 Hz, 1H, H-5), 8.84 (s, 1H, H-4); 13C NMR (75.45 MHz,

9666
Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
4.1.4.3. 1-(4-Hydroxyphenyl)-3-(5-thioxo-1,2,4-triazol-3-yl) b-
carboline (8c). Yield: 75%; mp 336.5–338.0 °C; IR(KBr) mmax
3303 (NH), 1625 (C@N), 1513–1434 (C@C), 1358, 1239 (C@S)
cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.03 (d, J = 8.7 Hz, 2H, H-
4.1.5.3. 1-(4-N,N-Dimethylaminophenyl)-3-(4-amino-5-thioxo-
1,2,4-triazol-3-yl) b-carboline (9g). Yield: 82%; mp. 360.0–
:
362.0 °C; IR(KBr) mmax
:
3411 (NH), 1624 (C@N), 1530–1426
;
(C@C), 1354, 1208 (C@S) cmꢁ1
;
1H NMR (300 MHz, DMSO-d6): d
30, H-50), 7.29 (t, J = 7.8 Hz, 1H, H-6), 7.58 (t, J = 7.8 Hz, 1H, H-7),
7.71 (d, J = 7.8 Hz, 1H, H-8), 8.18 (d, J = 8.7 Hz, 2H, H-20, H-60),
8.35 (d, J = 7.8 Hz, 1H, H-5), 8.70 (s, 1H, H-4), 11.9 (s, 1H, 9-NH),
13.7 (s, 1H), 13.8 (s, 1H). 13C NMR (75.45 MHz, DMSO-d6): d
111.0, 112.9, 115.5, 120.2, 121.0, 122.0, 127.7, 128.8, 130.0,
130.6, 133.0, 133.2, 141.8, 142.0, 151.4, 158.7, 166.9; HR-ESIMS:
calcd for C19H13N5OS [M+H]+ 360.0919; found 360.0906.
3.07 (s, 6H, N(Me)2), 7.13 (d, J = 8.4 Hz, 2H, H-30, H-50), 7.34 (t,
J = 7.5 Hz, 1H, H-6), 7.63 (t, J = 7.5 Hz, 1H, H-7), 7.74 (d, J = 7.5 Hz,
1H, H-8), 8.01 (d, J = 8.4 Hz, 2H, H-20, H-60), 8.40 (d, J = 7.8 Hz, 1H,
H-5), 8.82 (s, 1H, H-4), 12.1 (s, 1H, 9-NH), 14.1 (s, 1H). 13C NMR
(75.45 MHz, DMSO-d6): d 40.8, 113.0, 113.4, 113.7, 120.7, 120.8,
122.3, 129.4, 129.9, 130.1, 132.3, 132.7, 141.2, 142.2, 146.5,
149.3, 150.2, 164.6; HR-ESIMS: calcd for C21H19N7S [M+H]+
402.1501; found 402.1694.
4.1.4.4. 1-(4-N,N-Dimethylaminophenyl)-3-(5-thioxo-1,2,4-tria-
zol-3-yl) b-carboline (8g). Yield: 78%; mp 345.0–347.0 °C; IR(KBr)
4.1.5.4. 1-(2-Chlorophenyl)-3-(4-amino-5-thioxo-1,2,4-triazol-
3-yl) b-carboline (9h). Yield: 84%; mp 295.5–297.4 °C; IR(KBr)
mmax: 3449 (NH), 1625 (C@N), 1557–1411 (C@C), 1324, 1269 (C@S)
cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d 3.10 (s, 6H, N(Me)2), 7.28 (d,
J = 8.4 Hz, 2H, H-30, H-50), 7.30 (d, J = 7.7 Hz, 1H, H-6), 7.58 (t,
J = 7.7 Hz, 1H, H-7), 7.69 (d, J = 7.7 Hz, 1H, H-8), 8.30 (d,
J = 8.4 Hz, 2H, H-20, H-60), 8.36 (d, J = 7.8 Hz, 1H, H-5), 8.70 (s, 1H,
H-4), 11.9 (s, 1H, 9-NH), 13.7 (s, 1H), 13.9 (s, 1H). 13C NMR
(75.45 MHz, DMSO-d6): d 41.9, 111.1, 112.8, 114.7, 120.2, 120.9,
122.0, 128.8, 130.0, 130.2, 133.0, 133.4, 141.5, 141.6, 141.8,
148.8, 151.4, 166.9; HR-ESIMS: calcd for C21H18N6O [M+H]+
387.1392; found 387.1368.
m
max: 3245, 3137 (NH), 1626 (C@N), 1579–1434 (C@C), 1339,
1243 (C@S) cmꢁ1 1H NMR (300 MHz, DMSO-d6): 6.40 (s, 2H,
;
NH2), 7.33–7.80 (m, 7H, H-6, H-7, H-8, H-30, H-40, H-50, H-60),
8.39 (d, J = 7.8, 1H, H-5), 8.92 (s, 1H, H-4), 12.2 (s, 1H, 9-NH),
14.1 (s, 1H). 13C NMR (75.45 MHz, DMSO-d6): d 112.5, 114.8,
120.3, 120.7, 122.2, 127.6, 129.0, 129.1, 130.0, 130.8, 132.0,
132.4, 133.9, 134.2, 136.1, 140.5, 141.6, 147.3, 163.9; HR-ESIMS:
calcd for C19H13ClN6S [M+H]+ 393.0689; found 393.0782.
4.1.6. Procedure for preparation of 1-(phenyl)-3-(4-amino-5-
methylthio-1,2,4-triazol-3-yl) b-carboline (10a,h)
4.1.5. General procedure for preparation of 1-(substituted-
phenyl)-3-(4-amino-5-thioxo-1,2,4-triazol-3-yl) b-carboline (9a,
9c, 9g–h)
To a suspension of 9a,h (0.5 mmol) in ethanol (10 mL) was
added dropwise, at 0oC an aqueous solution of potassium hydrox-
ide (2 M, 1 mmol). To this mixture, methyl iodide (1 mmol) was
added dropwise with vigorous stirring and the solution kept at
room temperature for 48 h. Then, water (10 mL) was added, and
the precipitate formed was separated by filtration, washed with
water, and recrystallized from ethanol.
To a cold solution (0–5 oC) of potassium hydroxide (1.5 mmol)
and carbohydrazide 5 (1.0 mmol) in ethanol (25 mL) was added
dropwise carbon disulfide (1.5 mmol) with stirring. The mixture
was refluxed for 48 h and after cooled, diluted with 30 mL of ethyl
ether. The precipitate formed was collected by filtration, washed
with ethyl ether and dried. A suspension of the intermediate and
hydrazine hydrate (20 mL) was refluxed for four days. The color
of the reaction mixture changed to green, hydrogen sulfide was
evolved and a homogeneous solution resulted. The reaction mix-
ture was diluted with 20 mL of cold water and acidified with con-
centrated HCl. The product was collected by filtration, washed
several times with cold water, dried and crystallized from ethanol.
4.1.6.1. 1-Phenyl-3-(4-amino-5-methylthio-1,2,4-triazol-3-yl)
b-carboline (10a). Yield: 50%; mp 228.0–230.0 °C; IR(KBr) mmax
:
;
3295 (NH), 1625 (C = N), 1594–1434 (C = C), 714 (C–S–C) cmꢁ1
1H NMR (300 MHz, DMSO-d6): d 2.64 (s, 3H, SMe), 6.73 (s, 2H,
NH2), 7.31 (t, J = 7.8 Hz, 1H, H-6), 7.55–7.70 (m, 5H, H-7, H-8, H-
30, H-40, H-50), 8.10 (d, J = 7.5 Hz, 2H, H-20, H-60), 8.44 (d, 7.8 Hz,
1H, H-5), 8.88 (s, 1H, H-4), 11.8 (s, 1H, 9-NH). 13C NMR
(75.45 MHz, DMSO-d6): d 13.2, 112.7, 112.8, 120.2, 120.9, 122.3,
128.5, 128.9, 129.2, 129.1, 130.3, 132.9, 136.5, 137.6, 141.2,
141.7, 151.8, 152.9; HR-ESIMS: calcd for C20H16N6S [M+H]+
373.1235; found 373.1394.
4.1.5.1. 1-Phenyl-3-(4-amino-5-thioxo-1,2,4-triazol-3-yl)
carboline (9a). Yield: 85%; mp. 358.0–360.0 °C; IR(KBr) mmax
3242, 3138 (NH), 1625 (C@N), 1557–1494 (C@C), 1348, 1239
(C@S) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 6.20 (brs, 2H, NH2),
b-
:
;
7.32 (t, J = 7.5 Hz, 1H, H-6), 7.54–7.61 (m, 2H, H-7, H-40), 7.63 (d,
J = 7.8 Hz, 2H, H-30, H-50), 7.72 (d, J = 8.1 Hz, 1H, H-8), 8.10 (d,
J = 7.8 Hz, 2H, H-20, H-60), 8.38 (d, J = 7.8 Hz, 1H, H-5), 8.85 (s, 1H,
H-4), 12.0 (s, 1H, 9-NH), 14,0 (s, 1H). 13C NMR (75.45 MHz,
DMSO-d6): d 112.9, 114.1, 120.4, 120.9, 122.1, 128.7, 129.0,
129.1, 129.2, 129.2, 130.0, 133.3, 134.2, 137.2, 141.5, 141.8,
147.4, 163.9; HR-ESIMS: calcd for C19H14N6S [M+H]+ 359.1079;
found 359.1102.
4.1.6.2. 1-(2-Chlorophenyl)-3-(4-amino-5-methylthio-1,2,4-
triazol-3-yl) b-carboline (10h). Yield: 52%; mp 215.0–217.4 °C;
IR(KBr)
m
max: 3200 (NH), 1625 (C@N), 1595–11436 (C@C), 716
(C–S–C) cmꢁ1
;
1H NMR (300 MHz, DMSO-d6): d 2.63 (s, 3H, SMe),
6.68 (s, 2H, NH2), 7.30 -7.80 (m, 7H, H-6, H-7, H-8, H-30, H-40, H-
50, H-6), 8.45 (d, J = 7.8, 1H, H-5), 8.93 (s, 1H, H-4), 11.7 (s, 1H, 9-
NH). 13C NMR (75.45 MHz, DMSO-d6): d 13.3, 112.4, 113.5, 120.1,
120.8, 122.4, 127.7, 129.1, 129.6, 130.1, 130.7, 132.0, 132.3,
133.8, 135.8, 136.1, 139.9, 141.6, 151.6, 152.6; HR-ESIMS: calcd
for C20H15ClN6S [M+H]+ 407.0846; found 407.0858.
4.1.5.2. 1-(4-Hydroxyphenyl)-3-(4-amino-5-thioxo-1,2,4-tria-
zol-3-yl) b-carboline (9c). Yield: 80%; mp 355.0–356.8 °C; IR(KBr)
mmax: 3423 (NH), 1625 (C@N), 1557–1426 (C@C), 1352, 1281 (C@S)
4.2. Anticancer assays
cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d 7.04 (d, J = 8.7 Hz, 2H, H-30,
H-50), 7.32 (t, J = 7.5 Hz, 1H, H-6), 7.61 (t, J = 7.5 Hz, 1H, H-7), 7.71
(d, J = 7.5 Hz, 1H, H-8), 7.95 (d, J = 8.7 Hz, 2H, H-20, H-60), 8.37 (d,
J = 7.5 Hz, 1H, H-5), 8.78 (s, 1H, H-4), 11.9 (s, 1H, 9-NH), 14.0 (s,
1H, NH). 13C NMR (75.45 MHz, DMSO-d6): d 112.9, 113.3, 115.9,
120.4, 120.9, 122.1, 127.5, 129.0, 129.8, 130.1, 132.9, 133.6,
141.8, 141.9, 147.1, 158.8, 164.0; HR-ESIMS: calcd for C19H14N6OS
[M+H]+ 375.1028; found 375.1168.
The synthesized compounds were evaluated in vitro against a
eight-cell panel lines consisting of melanoma UACC-62, breast
MCF-7, lung NCI-460, leukemia K-562, ovarian OVCAR, prostate
PCO-3, colon HT29, and renal 786-0. The tests were performed
by the colorimetric method with sulforrodamina B, according to
NCI standard protocol and doxorubicin was used as positive
control.30 Assays were performed in a 96-well plate using four

Anelise S. Nazari Formagio et al. / Bioorg. Med. Chem. 16 (2008) 9660–9667
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