Synthesis and antitumor activity of β-carboline 3-(substituted- carbohydrazide) derivatives

Article abstract of DOI:10.1016/j.bmc.2011.08.059

A series of β-carboline derivatives bearing a substituted- carbohydrazide moiety at C-3 were synthesized and evaluated for their antitumor activity against eight human cancer cell lines. The b-carboline N-(substituted-benzylidene)carbohydrazides showed, i

Full text of DOI:10.1016/j.bmc.2011.08.059

Bioorganic & Medicinal Chemistry 19 (2011) 6400–6408
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumor activity of b-carboline 3-(substituted-carbohydrazide)
derivatives
Valéria Aquilino Barbosa a, Anelise S. Nazari Formagio a, Franciele Cristina Savariz a, Mary Ann Foglio b,
Humberto Moreira Spindola b, João Ernesto de Carvalho b, Emerson Meyer a, Maria Helena Sarragiotto a,
⇑
a Departamento de Química, UEM, Universidade Estadual de Maringá, 87020-900 Maringá, PR, Brazil
b Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, UNICAMP, Universidade Estadual de Campinas, 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:
Received 4 June 2011
Revised 19 August 2011
Accepted 28 August 2011
Available online 1 September 2011
A series of b-carboline derivatives bearing a substituted-carbohydrazide moiety at C-3 were synthesized
and evaluated for their antitumor activity against eight human cancer cell lines. The b-carboline N-
(substituted-benzylidene)carbohydrazides showed, in general, a greater antitumor activity than their
N-(alkylidene)carbohydrazide analogues. The N9-methylation of b-carboline N-(substituted-benzylidene)
carbohydrazides resulted in a decrease of antitumor activity. Among compounds tested, the benzylidene-
carbohydrazides 3, 4, 11, 13, 16, 21 and 22 were the most active, possessing IC50 less than 10
the eight tumor cell lines assayed. The derivative 4 displayed the most significant activity toward all
tested cell lines, with a remarkable cytotoxicity against renal (786-0) cell lines (IC50 = 0.04 M). Com-
pound 4 was assayed for its in vivo antineoplastic activity in the Ehrlich solid carcinoma assay.
Ó 2011 Elsevier Ltd. All rights reserved.
lM for six of
Keywords:
b-Carboline derivatives
Carbohydrazides
Antitumor activity
Ehrlich tumor
l
1. Introduction
relationship studies of b-carbolines, we designed a series of new
1-phenyl-substituted b-carboline derivatives bearing a substi-
Synthetic and naturally occurring compounds containing the
b-carboline nucleus possess a large spectrum of important phar-
macological properties, including potent antitumor activity. The
potential of b-carboline compounds as anticancer agents have
stimulated studies into their synthesis and structure–activity-rela-
tionship (SAR) with an aim to the improvement of their antitumor
potential.
tuted-carbohydrazide moiety at C-3. Recent investigations of our
research group showed that b-carboline-3-carbohydrazide deriva-
tives were effective against poliovirus and herpes simplex virus
(HSV-1).28
In continuation of our studies to develop novel potent antitumor
agents, in this work we synthesized and evaluated the in vitro anti-
tumor activity of a series of 1-(substituted-phenyl)-b-carbolines
SAR studies on a variety of synthetic b-carboline derivatives
have demonstrated that the introduction of appropriated substitu-
ents into position-1, -2, -3 and -9 of the b-carboline skeleton re-
sulted in more potent antitumor derivatives, with reduced
toxicity.1–17 The anticancer mode of action of these alkaloids has
been also widely investigated. Multiple mechanisms, such as DNA
intercalation4–17 and inhibition of Topoisomerases I and II,18,19 IkB
kinase (IKK),20 cyclin-dependent kinases (CDKs),21,22 mitogen acti-
vated protein kinase-activated protein kinase 2 (MK-2),23 polo-like
kinase (PLK1)24 and kinesin-like protein Eg525 were pointed out
from these investigations.
Our previous studies showed that b-carboline derivatives con-
taining a phenyl-substituted group at C-1, and the 1,3,4-oxadiazole
and 1,2,4-triazole units at C-3, presented significant antitumoral
activities.26,27 In order to evaluate the influence of different groups
at 3-position and to provide additional data for structure–activity
and 1-(substituted-phenyl)-9-methyl-b-carbolines, bearing
a
substituted-carbohydrazide moiety at C-3, against several human
cancer cell lines. Also, the most active derivative was tested for its
in vivo antineoplastic activity in the Ehrlich solid carcinoma assay.
2. Results and discussion
2.1. Chemistry
The synthetic strategies followed for the preparation of the b-
carboline derivatives are depicted in Scheme 1. The methyl esters
1a–h were prepared through a Pictet–Spengler condensation of
the L-tryptophan with appropriate aromatic aldehydes, in acidic
media, and subsequent esterification of the resulting carboxylic
acids with methanol and sulfuric acid, as previously reported.26,27
Reaction of 1a–h with hydrazine hydrate afforded the respective
carbohydrazides 2a–h.26,27 The N-(substituted benzylidene)-b-
carboline-3-carbohydrazides (3–26) were synthesized according
to a previously described methodology.28 The compounds of the
⇑
Corresponding author. Tel.: +55 44 3261 3657; fax: +55 44 3011 4125.
E-mail address: mhsarragiotto@uem.br (M.H. Sarragiotto).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.059

V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
6401
O
2
aldehyde
N
R
CONHNH
CO CH
2
2
3
or
ketone
b
N
H
3
R
a
N
N
N
N
H
N
N
H
H
1
1
1
R
R
R
2a-h
1a-h
3-50
2
R
4"
c
O
CHO
3"
N
1"
5
CO CH
2
CONHNH
2
4
3
N
3
4a
H
7
2
N
N
1
N
R
9
N
d
a
N
N
2'
CH
3
CH
CH
3
3
1
1
1
R
R
R
4'
53-56
52a-c
51a-c
Scheme 1. Reagents and conditions: (a) NH2NH2ꢀH2O, EtOH, reflux, 48 h; (b) aldehyde or ketone, EtOH, reflux 30 h; (c) CH3I, NaOH, DMF, rt, 48 h; (d) EtOH, H2SO4 (cat), reflux
48 h.
N-alkylidene-carbohydrazide series 27–50 were prepared by the
condensation of the 2a–h with cyclohexanone, cyclopentanone
and propanone, in ethanol under reflux (Scheme 1).
In order to evaluate the influence of a methyl group at the 9-po-
sition, some of the active carbohydrazides of the N-substituted-
benzylidene series were N-methylated, using CH3I and NaOH, in
DMF at room temperature, to generate the corresponding deriva-
tives 53–56 (Scheme 1).
All novel compounds were characterized by their spectral data
(IR, EIMS, 1H and 13C NMR). Compounds 3–26 were characterized
by comparison with NMR data previously reported.23 The N-meth-
ylated derivatives 53–56 were characterized by the presence of an
additional signal at dH/dC 3.50/33.5, corresponding to the N9-
methyl group attached to the b-carboline nucleus. The presence
of an alkylidene-carbohydrazide moiety for the novel compounds
27–50 was confirmed by the signals at dC 154.8–162.8 (C@N) and
dC 159.7–176.5 (C@O) in the 13C NMR spectra, together with the
signals for the respective alkyl groups.
activity. An analysis of the relation between the IC50 data and effect
of the substituents at carboydrazide moiety showed that the phe-
nyl and 2-chlorophenyl groups are present in four (compounds 11,
13, 16 and 21) of the seven more active compounds, demonstrating
the importance of these substituents for the antitumor activity of
the b-carboline N-benzylidene-carbohydrazide series.
In order to improve the antitumor activity, a series of b-carbo-
line 3-(substituted-alkylidene)-carboydrazides (27–50) were syn-
thesized and tested. Comparing the activity of the benzylidene
and alkylidene groups in the carbohydrazide moiety indicated
the decreased antitumor activity of derivatives possessing the later
group. Among the compounds tested, only four derivatives pre-
sented significant cytotoxicity, with IC50 less than 20 lM against
six of the eight human tumor cell lines, two of the cyclohexylidene
(compounds 27 and 31) and two of isopropylidene (compounds 42
and 44) series. Concerning the activity against individual cell lines,
the best activities were observed for compounds 31, 34 and 42 to-
ward breast cell lines (MCF-7), with IC50 values ranging of 6.17–
8.33 lM.
2.2. Anticancer activity
With the aim to evaluate the effect of a methyl group at the 9-
position, some compounds of the alkylidene-carbohydrazide series
(compounds 4, 8, 11 and 21) were N9-methylated, to afford the cor-
responding N-methyl derivatives 53, 54, 55 and 56. The results of
antitumor assays showed that the incorporation of the methyl
group had a detrimental effect on the cytotoxicity. However, the
N-methylation resulted in a remarkable increase in selectivity to-
ward 786-0 renal cancer cells line for 53 and 54, in addition to a
small increase in their cytotoxic potential.
2.2.1. In vitro assays
The in vitro anticancer activities of all synthesized compounds
were evaluated against eight human cancer cell lines and the re-
sults are summarized in Table 1. Regarding the N-(substituted-
benzylidene)-3-carbohydrazide series, the derivatives 3, 4, 11, 13,
16, 21 and 22 possesses potent antitumor activity with IC50 less
than 10 lM against six of the eight human tumor cell lines assayed.
Four compounds (4, 11, 16 and 21) were found to be the most po-
tent derivatives of the N-benzylidene-carbohydrazide series, with
2.2.2. In vivo assays
IC50 values ranging of 0.04–10.38
l
M against all the human tumor
In order to validate the in vitro antineoplastic activity of com-
pounds, in vivo assays were used to assess the activity of the most
active derivative 4. The most active compound 4 was submitted to
the Ehrlich solid carcinoma experimental model, which consists of
a rapidly growing carcinoma inoculated into the mice paw, used to
evaluate if the tested derivative will be able to reduce tumor devel-
opment compared to a control group (vehicle treatment). This tu-
mor model possesses a very aggressive behavior and is able to
grow in almost all mice strains, which suggests that the recogni-
tion and immune responses to this tumor are independent of major
histocompatibility complex. This characteristic suggests that the
control of the Ehrlich tumor is related more with innate immunity,
specially the inflammatory response, than with T cell responses.29
cell lines tested. Among these compounds, the derivative 4, bearing
a 30-nitrophenyl group at C-1 and a 4-dimethylaminophenyl group
attached to the carbohydrazide moiety, displayed the most signif-
icant activity toward all tested cell lines, with remarkable cytotox-
icity against the renal (786-0) cell line, with an IC50 value of
0.04
lM. The derivatives 12 and 14 showed high selectivity toward
ovarian cancer cell line (OVCAR) and were active against the her-
pes virus HSV-1 (EC50 = 22.9 lM) and poliovirus (EC50 = 2.67 lM),
respectively, as demonstrated in our previous work.28 On the other
hand, the derivatives 9, 10, 24 and 25 were shown to be inactive
against all cell lines, suggesting the importance of aromatic func-
tionality with m-substituted electron withdrawal group for the

6402
V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
Table 1
In vitro antitumor activity (IC50 in
l
M) of 1-(substituted-phenyl) b-carboline 3-(substituted-carbohydrazide) derivatives 3–50 and 53–56
Compd
R1
R2
R3
Melanoma
UACC-62
Breast
MCF7
Lung
NCI-
460
Leukemia
K-562
Ovarian
OVCAR
Prostate
PCO-3
Colon
HT29
Renal
786-0
3
4
5
6
7
8
9
3-NO2
3-NO2
3-NO2
3-NO2
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OH
4-OH
4-OH
4-OH
4-OH
H
C6H5
4-N(CH3)2C6H5
4-NO2C6H5
2-ClC6H5
4-OCH3C6H5
C6H5
4-N(CH3)2C6H5
4-NO2C6H5
2-ClC6H5
4-OCH3C6H5
C6H5
4-N(CH3)2C6H5
4-NO2C6H5
2-ClC6H5
4-OCH3C6H5
C6H5
4-N(CH3)2C6H5
4-NO2C6H5
2-ClC6H5
4-OCH3C6H5
C6H5
4-N(CH3)2C6H5
4-NO2C6H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.19
1.34
3.27
35.71
91.44
3.45
>100
>100
5.57
3.85
3.18
88.18
4.46
1.56
65.59
25.87
87.14
4.78
2.16
9.69
11.78
>100
>100
2.53
11.73
31.17
>100
7.86
>100
>100
21.64
76.37
>100
>100
2.61
0.63
1.93
2.11
6.76
28.89
35.14
3.45
>100
>100
1.43
1.24
0.24
4.26
68.08
>100
3.22
>100
>100
2.52
87.38
4.31
>100
3.73
1.65
14.55
79.54
>100
4.29
1.45
7.54
14.42
>100
>100
6.35
10.35
55.90
87.14
10.27
NT
6.14
0.19
NT
4.18
1.93
9.80
7.34
3.76
0.04
12.84
14.12
71.41
35.99
>100
>100
10.38
>100
8.94
>100
12.27
7.01
11.04
1568
75.91
21.26
6.13
46.73
>100
>100
32.28
>100
>100
7.43
0.37
1.26
1.26
51.04
1.83
24.84
16.58
>100
26.53
>100
>100
4.52
NT
2.25
NT
12.79
2.03
59.38
11.63
87.14
8.60
2.52
7.54
15.93
>100
>100
18.03
10.35
27.51
>100
7.68
>100
>100
18.28
>100
>100
>100
>100
17.03
>100
93.37
>100
>100
2.52
61.98
4.32
>100
9.58
15.35
>100
35.99
>100
>100
1.26
95.59
4.32
>100
3.87
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
31
32
33
34
35
37
38
9.82
19.83
69.54
>100
2.18
59.38
8.49
>100
5.16
4.57
1.71
3.22
10.0
38.50
>100
>100
15.08
1.25
>100
>100
>100
29.69
2.16
14.09
29.88
>100
>100
2.75
18.55
>100
>100
14.96
>100
72.80
24.51
>100
>100
97.04
H
H
H
H
10.56
75.91
14.42
2.84
21.40
2.05
>100
>100
4.55
12.75
78.05
>100
13.20
>100
>100
20.75
>100
>100
>100
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-OCH3
H
3-NO2
4-OH
4-N(CH3)2
4-NO2
2-Cl
4-OCH3
3-NO2
3-OH. 4-
OCH3
4-OH
4-N(CH3)2
4-NO2
2-Cl
8.19
4.58
8.92
25.04
>100
>100
18.02
9.14
32.50
>100
8.33
>100
25.64
6.19
>100
>100
>100
13.78
>100
>100
38.08
20.17
68.87
>100
13.20
>100
>100
54.14
>100
>100
>100
18.82
>100
>100
3.85
13.96
47.30
>100
10.27
NT
>100
40.42
>100
>100
NT
2-ClC6H5
–CH2(CH2)3CH2–
–CH2(CH2)3CH2–
–CH2(CH2)3CH2–
–CH2(CH2)3CH2–
–CH2(CH2)3CH2–
–CH2(CH2)3CH2–
–CH2(CH2)3CH2–
–CH2(CH2)2CH2–
–CH2(CH2)2CH2–
–CH2(CH2)2CH2–
NT
NT
>100
>100
NT
39
40
41
42
43
44
45
46
–CH2(CH2)2CH2–
–CH2(CH2)2CH2–
–CH2(CH2)2CH2–
–CH2(CH2)2CH2–
CH3
CH3
CH3
CH3
>100
18.78
>100
11.67
40.71
10.13
>100
>100
78.14
22.60
>100
7.37
>100
14.17
>100
>100
>100
80.90
>100
13.78
>100
19.77
>100
>100
>100
13.44
>100
NT
>100
6.69
>100
>100
>100
>100
>100
11.19
>100
26.37
>100
>100
>100
20.41
>100
14.99
>100
15.39
>100
>100
>100
55.58
>100
20.08
>100
19.77
>100
>100
>100
>100
>100
11.67
>100
16.06
>100
70.00
4-OCH3
H
CH3
CH3
CH3
CH3
3-NO2
3-OH. 4-
OCH3
4-OH
4-N(CH3)2
4-NO2
2-Cl
3-NO2
4-OCH3
4-OCH3
H
47
48
49
50
53
54
55
56
CH3
CH3
CH3
CH3
4-N(CH3)2C6H5
C6H5
2-ClC6H5
2-ClC6H5
CH3
CH3
CH3
CH3
—
—
—
—
35.35
10.14
>100
19.13
>100
>100
>100
>100
24.28
16.76
>100
26.72
>100
>100
14.04
>100
38.14
25.42
>100
>100
>100
>100
>100
>100
18.14
23.37
>100
21.69
NT
>100
NT
NT
>100
40.45
>100
64.17
>100
>100
>100
>100
19.70
18.99
>100
25.63
>100
>100
>100
>100
NT
24.28
26.51
>100
>100
1.24
1.39
>100
>100
35.58
>100
38.89
>100
NT
>100
>100
NT = not tested; Compounds with IC50 >100 lM were considered not active.
Inflammation, an environment rich in inflammatory cells, growth
factors, activated stroma, and DNA damage, is a physiologic pro-
cess directly involved in the Ehrlich tumor development. Several
studies have established the link between cancer and chronic
inflammatory processes. Epidemiological evidence supports such
relationships and suggests that more than 25% of cancer diseases
are related to chronic infections or other types of inflammation.
Some reports have demonstrated that the neutrophilic inflamma-
tory response is essential for controlling Ehrlich tumor growth,
and the high influx of these cells promotes tumor develop-
ment.30,31 This effect is probably related with the angiogenesis
and growth factors induced by inflammation that are necessary
for the tumor growth. The Ehrlich solid tumor implantation in-
duces per se a local inflammatory reaction, with increasing vascu-
lar permeability, which results in an intense edema formation,
cellular migration and a progressive ascitic fluid formation, which
is essential for tumor growth, since this fluid constitutes the direct
nutritional source for tumor cells.32,33
The preliminary in vivo study with compound 4, concerning the
acute toxicological effects, determined the doses of 30 mg/kg as
the higher one to be used on the following in vivo experiments,
based on the fact that this dose did not produced toxicological ef-
fects (not shown). Therefore, doses of 10 and 30 mg/kg of com-
pound 4 were determined to be evaluated on the Ehrlich solid
carcinoma.
The results of the Ehrlich carcinoma assay for compound 4 dem-
onstrate its systemic effectiveness as a tumor growth inhibitor.
This assertion is supported by the fact that the compound given
by intraperitoneal route (ip) every 3 days during the 15-days per-
iod of experiment, was capable of reducing tumor growth (such
as observed for the positive control 5-Fluoruracil) when compared
to the negative control group (vehicle). It was observed that in the

V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
6403
initial phase of the tumor growth (first 3 days after inoculation),
3. Conclusion
representing the inflammation process involving several media-
tors, such as prostaglandins, leukotrienes, histamine and bradyki-
nin,34 the treatment with the 30 mg/kg doses of compound 4
reduced the paw edema compared to control group (⁄P <0.05).
The result discussed above suggests that compound 4 may act
decreasing pro-inflammatory factors. The inflammatory factors
promote tumor growth due to their role in the relation between tu-
mor and stroma cells thus establishing a tumor microenvironment
which facilitates angiogenesis and evades the immune system at-
tack. Also, leukotrienes modulate proliferation, tumor differentia-
tion, apoptosis and interfere with migration, and invasion of the
tumor cells. In the tumor microenvironment, cells of the immune
system and endothelium are recruited to produce more pro-
inflammatory mediators, including eicosanoids, growth, and angio-
genic factors.34
Starting from the ninth day trial of the experiment, the results
of the present study also showed that, during the exponential pro-
liferative phase of the tumor growth with cell proliferation, pro-
duction of tumor and inflammation factors, compound 4 was
capable of decrease the tumor growth. It derives from the fact that
the positive control 5-FU (10 mg/kg) and both doses (10 and
30 mg/kg) of the compound reduced the tumor volume (⁄P <0.05)
of the mice paw compared to the control group (vehicle). More-
over, compound 4 (30 mg/kg) and 5-FU maintained the activity un-
til the end of the experiment, demonstrating that in the 12-day
(⁄P <0.05; ⁄⁄P <0.01) and 15-day (⁄P <0.05; ⁄⁄⁄P <0.001) evaluation
of tumor growth, both groups significantly reduced the mice paw
edema (Fig. 1).
In summary, in this work we have synthesized and evaluated
the antitumor activity of a number of b-carboline 3-N-substi-
tuted-carbohydrazides against
lines. In general, we have demonstrated that the presence of
carbohydrazide moiety at C-3 of 1-substituted-phenyl
a panel of human cancer cell
a
a
b-carboline nucleus can enhance the antitumor activity. The
b-carboline 3-N-benzylidene-carbohydrazides presented greater
activity than the 3-N-alkylidene-carbohydrazide analogues. The
N9-methylation of the b-carboline 3-benzylidene-carbohydra-
zides resulted in a decrease of antitumor activity.
The derivative 4 displayed significant activity towards all cell
lines tested, being highly active against renal (786-0) cell lines
with an IC50 of 0.04 lM. The results of the Ehrlich carcinoma assay
for compound 4 demonstrate its systemic effectiveness, as a tumor
growth inhibitor. The treatment with the 30 mg/kg doses of com-
pound 4 reduced the Ehrlich tumor volume of the mice paw, sug-
gesting that its may act decreasing pro-inflammatory factors.
4. Experimental
4.1. General
All reagents were purchased from commercial suppliers. The
reactions were monitored by thin layer chromatography con-
ducted on Merck TLC plates (Silica Gel 60 F254). 1H and 13C spectra
were recorded on a Varian spectrometer model Mercury Plus BB at
300 MHz and 75 MHz, respectively. Mass spectra (MS) were re-
corded on a Thermoelectron Corporation Focus-DSQ II spectrome-
ter. IR spectra were obtained on a BOMEM spectrometer model
MB-100.
The complexity of the tumor biology related to its interaction
with the associated stroma, often leads to failure when drugs with
a good in vitro profile enter to in vivo experimentation.35 However,
it is not the case of compound 4, which displayed a promising
in vivo activity. Considering the complex processes involved in
Ehrlich carcinoma growth, the effectiveness of compound 4 may
be related to several mechanisms, such as the cell proliferation
or in the inflammatory responses that facilitate tumor growth.
Thus, further studies are necessary to clarify the specific mecha-
nism of action of compound 4.
4.2. General procedure for the synthesis of N0-benzylidene-1-
(substituted-phenyl)-b-carboline-3-carbohydrazides (3–26)
The synthesis and spectral characterization of the N-(substi-
tuted-benzylidene)-b-carboline-3-carbohydrazides (3–26) were
described in a previous work.28
4.3. General procedure for the synthesis of N0-alkylidene-1-
(substituted-phenyl)-b-carboline-3-carbohydrazides (27–50)
0.4
Vehicle
5-Fluorouracil 10 mg/kg
Compound 4 30 mg/kg
To a solution of the carbohydrazides 2a–h (4.0 mmol) in EtOH
(20 mL) was added cyclohexanone, cyclopentanone and propanone
(3 equiv), and the resulting solution was refluxed for 30 h. The
reaction mixture was then cooled and kept at 0 °C for 3 h. The
resulting solids were filtered off and washed with cold EtOH to fur-
nish the products in a pure form.
0.3
Compound 4 10 mg/kg
0.2
*
*
4.3.1. N0-Cyclohexylidene-1-(4-methoxyphenyl)-b-carboline-3-
carbohydrazide (27)
*
**
0.1
0.0
***
Yield: 82%; mp 267–269 °C; IR (KBr)
m
max: 3227 (N-H), 1609
;
(C@N), 1684 (C@O), 1558–1347 (C@C) cmꢁ1
1H NMR (300 MHz,
**
9
CDCl3/CD3OD 3:1): d 1.68–1.78 (m, 6H, H2-300, H2-400 and H2-500),
2.47 (t, J = 6.3 Hz, 2H, H2-600), 2.53 (t, J = 6.3 Hz, 2H, H2-200), 3.93
(s, 3H), 7.17 (d, J = 8.7 Hz, 2H, H-30 and H-50), 7.37 (t, J = 7.8 Hz,
1H, H-6), 7.54–7.62 (m, 2H, H-7 and H-8), 7.93 (d, J = 8.7 Hz, 2H,
H-20 and H-60), 8.23 (d, J = 7.8 Hz, 1H, H-5), 8.71 (s, 1H, NH), 8.93
(s, 1H, H-4), 11.11 (s, 1H, 9-NH); 13C NMR (75.5 MHz): d 25.5
(CH2, C-300), 26.0 (CH2, C-500), 26.9 (CH2, C-600), 27.0 (CH2, C-400),
35.5 (CH2, C-200), 55.4 (O–CH3), 112.2 (CH, C-4), 113.5 (CH,C-8),
114.4 (CH, C-30 and C-50), 120.6 (CH, C-6), 121.7 (CH, C-5), 128.7
(CH, C-7), 129.5 (CH, C-20 and C-60), 130.1 (C, C-4b), 130.3 (C, C-
3), 134.8 (C, C-10), 138.2 (C, C-9a), 140.9 (C, C-8a), 141.4 (C, C-1),
*
0
15
3
Experimental time (days)
Figure 1. Effect of compound 4 on Ehrlich solid tumor expressed as tumor volume
(mL) per day of treatment. Treatment with 4 at 30 mg/kg doses reduced tumor
volume starting at the third day (acute inflammatory response, ⁄P <0.05), and from
the ninth until the end of experiment (15° day, ⁄P <0.05), which represents the
tumor growth itself. A dose of 10 mg/kg only reduced tumor volume on the ninth
day (⁄P <0.05). 5-Fluorouracil was the positive control and vehicle was saline 0.9%.

6404
V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
160.4 (C, C-40), 161.4 (C@N), 162.6 (C@O); MS m/z (%): 412.05 (M+,
85), 369.00 (53), 273.00 (98), 272.97 (100), 257.95 (50).
H-60), 8.21 (d, J = 8.1 Hz, 1H, H-5), 8.86 (s, 1H, H-4); 13C NMR
(75.5 MHz): d 25.5 (CH2, C-300), 26.1 (CH2, C-500), 27.0 (CH2, C-600),
27.1 (CH2, C-400), 35.3 (CH2, C-200), 112.2 (CH, C-4), 113.4 (CH, C-
8), 115.9 (2 CH, C-30and C-50), 120.6 (CH, C-6), 121.8 (C, C-4a),
121.9 (CH, C-5), 128.6 (CH, C-7), 129.4 (C, C-4b), 129.7 (2 CH, C-
20 and C-60), 130.2 (C, C-3), 135.1 (C, C-10), 138.4 (C, C-9a), 141.4
(C, C-8a), 141.7 (C, C-1), 158.1 (C, C-40), 162.2 (C@N), 162.8
(C@O); MS m/z (%): 398.07 (M+, 50), 355.02 (28), 258.99 (100).
4.3.2. N0-Cyclohexylidene-1-phenyl-b-carboline-3-
carbohydrazide (28)
Yield: 85%; mp 284–286 °C; IR (KBr)
m
max: 3154 (N-H), 1623
;
(C@N), 1676 (C@O), 1556-1345 (C@C) cmꢁ1
1H NMR (300 MHz,
CDCl3/CD3OD 3:1): d 1.65–1.82 (m, 6H, H2-300, H2-400 and H2-500),
2.46 (t, J = 6,3 Hz, 2H, H2-600), 2.52 (t, J = 6.3 Hz, 2H, H2-200), 7.38
(dd, J = 6.3 Hz, J = 1.5 Hz, 1H, H-6), 7.54–7.60 (m, H-30, H-40, H-500,
H-7 and H-8), 7.99 (dd, J = 7.6 Hz, J = 1,4 Hz, 2H, H-20 and H-60),
8.23 (d, J = 7.8 Hz, 1H, H-5), 8.79 (s, 1H, N H), 9.02 (s, 1H, H-4),
4.3.6. N0-Cyclohexylidene-1-[4-(dimethylamino)phenyl]-b-
carboline-3-carbohydrazide (32)
Yield: 90%; mp >300 °C; IR (KBr)
mmax: 1623 (C@N), 1676 (C@O),
13
11.10 (s, 1H, 9-NH); C NMR (75.5 MHz,): d 25.2 (CH2, C-300),
1554–1347 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3/CD3OD 3:1): d
;
26.0 (CH2, C-500), 26.9 (CH2, C-600), 27.0 (CH2, C-400), 35.3 (CH2, C-
200), 112.2 (CH, C-4), 113.9 (CH, C-8), 120.6 (CH, C-6), 121.8 (CH,
C-5), 128.2 (2CH, C-20 and C-60), 128.7 (CH, C-7), 129.0 (2CH, C-30
and C-50), 129.1 (C, C-40), 130.5 (C, C-4a), 135.1 (C, C-10), 137.9 (C,
C-9a), 138.6 (C, C-3), 141.1 (C, C-8a), 141.3 (C, C-1), 161.9 (C@N),
162.4 (C@O); MS m/z (%): 382.05 (M+, 48), 243.99 (63), 242.98
(100).
1.68–1.89 (m, 6H, H2-300, H2-400 and H2-500), 2.49 (t, J = 6.3 Hz, 2H,
H2-600), 2.53 (t, J = 6.3 Hz, 2H, H2-200), 3.10 (s, 6H, N(CH3)2), 6.94
(d, J = 8.8 Hz, 2H, H-30 and H-50), 7.36 (t, J = 7.8 Hz, 1H, H-6),
7.52–7.61 (m, 1H, H-7), 7.59 (d, J = 7.8 Hz, 1H, H-8), 7.89 (d,
J = 8.8 Hz, 2H, H-20 and H-60), 8.21 (d, J = 7.8 Hz, 1H, H-5), 8.72 (s,
1H, NH), 8.93 (s, 1H, H-4), 11.10 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d 25.5 (CH2, C-300), 26.0 (CH2, C-500), 26.9 (CH2, C-600),
27.0, (CH2, C-400) 35.3 (CH2, C-200), 40.3 (N–(CH3)2), 112.1 (CH, C-
4), 112.5 (2CH, C-30 and C-50), 112.9 (CH, C-8), 120.5 (CH, C-6),
121.8 (CH, C-5), 122.0 (C, C-4b), 125.6 (C, C-10), 128.4 (CH, C-7),
129.0 (2 CH,C-20 and C-60), 129.9 (C, C-4a), 134.7 (C, C-9a), 138.5
(C, C-8a), 141.1 (C, C-3), 141.8 (C, C-1), 151.0 (C, C-40), 162.1
(C@N), 162.1 (C@O); MS m/z (%): 425.11 (M+, 92), 382.05 (63),
286.02 (100), 241.97 (82).
4.3.3. N0-Cyclohexylidene-1-(3-nitrophenyl)-b-carboline-3-
carbohydrazide (29)
Yield: 88%; mp 279–281 °C; IR (KBr)
m
max: 3250 (N-H), 1624
;
(C@N), 1667 (C@O), 1557–1345 (C@C) cmꢁ1
1H NMR (300 MHz,
CDCl3/CD3OD 3:1): d 1.70–1.79 (m, 6H, H2-300, H2-400 and H2-500),
2.48–2.51 (m, 2H, H2-600), 2.51–2.55 (m, 2H, H2-200), 7.42 (dd,
J = 8.1 Hz, J = 1.6 Hz, 1H, H-6), 7.60–7.68 (m, 2H, H-7 and H-8),
7.84 (t, J = 7.9 Hz, 1H, H-50), 8.25 (d, J = 8.1 HZ, 1H, H-5), 8.36–
8.41 (m, 2H, H-40 and H-60), 8.84 (s, 1H, NH), 8.93 (t, J = 1.8 Hz,
1H, H-20), 9.08 (s, 1H, H-4), 11.04 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d 25.4 (CH2, C-300), 25.9 (CH2, C-500), 26.8 (CH2, C-600),
27.0 (CH2, C-400), 35.2 (CH2, C-200), 112.4 (CH, C-8), 114.6 (CH, C-
4), 120.9 (CH, C-6), 121.5 (C, C-4b), 121.7 (CH, C-5), 123.4 (CH, C-
20), 129.9 (CH, C-40), 129.9 (CH, C-60), 131.5 (C, C-4a), 133.9 (CH,
C-7), 133.9 (C, C-10), 133.9 (CH, C-50), 134.9 (C, C-9a), 138.5 (C, C-
3), 139.5 (C, C-8a), 141.7 (C, C-1), 148.7 (C, C-30), 162.8 (C@N),
162.8 (C@O); MS m/z (%): 427.07 M+, 58), 384.01 (38), 289.00
(60), 241.98 (100).
4.3.7. N0-Cyclohexylidene-1-(4-nitrophenyl)-b-carboline-3-
carbohydrazide (33)
Yield: 90%; mp 292–295 °C; IR (KBr)
m
max: 3308 (N-H), 1628
1H NMR (300 MHz,
(C@N), 1669 (C@O), 1557–1341 (C@C) cmꢁ1
;
DMSO-d6): d 1.65–1.68 (m, 6H, H2-300, H2-400 and H2-500), 2.36–
2.50 (m, 4H, H2-200 and H2-600), 7.36 (t, J = 8.1 Hz, 1H, H-6), 7.64
(d, J = 8.1 Hz, 1H, H-8), 7.69 (t, J = 8.1 Hz, 1H, H-7), 8.43–8.49 (m,
3H, H-30, H-50 and H-5), 8.49–8.51 (m, 2H, H-20and H-60), 8.79 (s,
1H, NH), 9.00 (s, 1H, H-4), 12.12 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d25.0 (CH2, C-300), 25.5 (CH2, C-500), 26.6 (CH2, C-600),
27.0 (CH2, C-400), 35.0 (CH2, C-200), 112.6 (CH, C-8), 114.6 (CH, C-
4), 120.6 (CH, C-6), 121.0 (C, C-4b), 122.4 (CH, C-5), 123.9 (2CH,
C-30 and C-50), 129.1 (CH, C-7), 130.0 (2CH, C-20 and C-60), 130.7
(C, C-4a), 134.6 (C, C-10), 138.0 (C, C-9a), 139.5 (C, C-3), 141.7 (C,
C-8a), 143.5 (C, C-1), 147.5 (C, C-40), 160.0 (C@N), 163.2 (C@O);
MS m/z (%): 427.07 (M+, 60), 289.00 (65), 241.98 (100).
4.3.4. N0-Cyclohexylidene-1-(3-hydroxy-4-methoxyphenyl)-b-
carboline-3-carbohydrazide (30)
Yield: 90%; mp 288–290 °C; IR (KBr)
m
max: 3321 (N-H), 1623
1H NMR (300 MHz,
(C@N), 1680 (C@O), 1562–1349 (C@C) cmꢁ1
;
DMSO-d6/CDCl3 3:1): d 1.73–1.79 (m, 6H, H2-300, H2-400 and H2-
500), 2.51 (t, J = 6.0 Hz, 4H, H2-200 and H2-600), 4.01 (s, 3H, OMe),
7.35 (t, J = 6.6 Hz, 1H, H-6), 7.53–7.61 (m, 4H, H-20, H-50, H-60
and H-70), 7.63 (d, J = 7.8 Hz, 1H, H-8), 8.23 (d, J = 8.1 Hz, 1H, H-
4.3.8. N0-Cyclohexylidene-1-(2-chlorophenyl)-b-carboline-3-
carbohydrazide (34)
Yield: 78%; mp 276–279 °C; IR (KBr)
m
max: 3312 (N-H), 1623
;
13
5), 8.88 (s, 1H, H-4); C NMR (75.5 MHz): d 25.4 (CH2, C-400),
(C@N), 1673 (C@O), 1596–1347 (C@C) cmꢁ1
1H NMR (300 MHz,
26.0 (CH2, C-500), 26.8 (CH2, C-300), 26.9 (CH2, C-600), 35.2 (CH2, C-
200), 55.7 (C, C-OMe), 111.4 (CH, C-20), 112.1 (CH, C-8), 113.4
(CH, C-4), 115.2 (CH, C-50), 120.5 (CH, C-6), 121.1 (CH, C-60),
121.7 (CH, C-5), 121.8 (C, C-4b), 128.6 (CH, C-7), 129.7 (C, C-10),
130.3 (C, C-4a), 134.9 (C, C-9a), 138.3 (C, C-3), 141.3 (C, C-8a),
141.4 (C, C-1), 147.2 (C, C-40), 147.9 (C, C-30), 161.9 (C@N),
162.1 (C@O); MS m/z (%): 428.08 (M+, 100), 385.03 (72), 290.02
(98), 256.98 (90).
CDCl3): d 1.62–1.91 (m, 6H, H2-300, H2-400 and H2-500), 2.42 (t,
J = 6.3 Hz, 2H, H2-600), 2.49 (t, J = 6.3 Hz, 2H, H2-200), 7.37 (dd,
J = 8.1 Hz, J = 1.2 Hz, 1H, H-6), 7.47–7.52 (m, 2H, H-30 and H-600),
7.55–7.71 (m, 4H, H-40, H-50, H-7 and H-8), 8.23 (d, J = 8.1 Hz, 1H,
H-5), 8.58 (s, 1H, NH), 9.06 (s, 1H, H-4), 10.97 (s, 1H, 9-NH); 13C
NMR (75.5 MHz): d 25.8 (CH2, C-300), 26.2 (CH2, C-500), 27.1 (CH2,
C-400), 27.1 (CH2, C-600), 35.8 (CH2, C-200), 112.0 (CH, C-8), 115.0
(CH, C-4), 121.3 (CH, C-6), 122.2 (C, C-4b), 122.5 (CH, C-5), 127.6
(CH, C-7), 129.3 (CH, C-50), 130.6 (CH, C-30), 130.8 (CH, C-60),
131.8 (CH, C-40), 133.2 (C, C-20), 135.6 (C, C-10), 136.2 (C, C-9a),
139.2 (C, C-3), 139.8 (C, C-8a), 140.8 (C, C-1), 161.3 (C@N), 161.6
(C@O); MS m/z (%): 416.05 (M+, 50), 277.98 (70), 241.98 (100).
4.3.5. N0-Cyclohexylidene-1-(4-hydroxyphenyl)-b-carboline-3-
carbohydrazide (31)
Yield: 87%; mp >300 °C; IR (KBr)
m
max: 3446 (N-H), 1611 (C@N),
;
1659 (C@O), 1557–1347 (C@C) cmꢁ1
1H NMR (300 MHz, DMSO-
d6/CDCl3 3:1): d 1.71–1.80 (m, 6H, H2-300, H2400 and H2-500), 2.47–
2.52 (m, 4H, H2-200 and H2-600), 7.08 (d, J = 8.4 Hz, 2H, H-30 and H-
50), 7.33 (td, J = 8.1 Hz, J = 1.4 Hz, 1H, H-6), 7.56 (t, J = 8.1 Hz, 1H,
H-7), 7.60 (d, J = 8.1 Hz, 1H, H-8), 7.88 (d, J = 8.4 Hz, 2H, H-20 and
4.3.9. N0-Cyclopentylidene-1-(4-methoxyphenyl)-b-carboline-3-
carbohydrazide (35)
Yield: 78%; mp 265–267 °C; IR (KBr)
m
max: 3238 (NH), 1623
;
(C@N), 1678 (C@O), 1559–1345 (C@C) cmꢁ1
1H NMR (300 MHz,

V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
6405
CDCl3/CD3OD 3:1): d 1.84 (quint, J = 6.8 Hz, 2H, H2-400), 1.96 (quint,
4.3.13. N0-Cyclopentylidene-1-(4-hydroxyphenyl)-b-carboline-
J = 6.8 Hz, 2H, H2-300), 2.47 (t, J = 6.8 Hz, 2H, H2-500), 2.59 (t;
J = 6.8 Hz, 2H, H2-200), 3.98 (s, 3H, OCH3), 7.16 (d, J = 8.1 Hz, 2H,
H-30and H-500), 7.33 (dd, J = 7.5 Hz, J = 1,0 Hz, 1H, H-6), 7.57 (dd,
J = 7.5 Hz, J = 1,0 Hz, 1H, H-7), 7.62 (d, J = 7.5 Hz, 1H, H-8), 7.97
(d, J = 8.1 Hz, 2H, H-20 and H-60), 8.19 (d, J = 7.5 Hz, 1H, H-5), 8.84
3-carbohydrazide (39)
Yield: 80%; mp 300 °C, decomp; IR (KBr)
m
max: 1609 (C@N), 1656
(C@O), 1446–1346 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6/
;
CDCl3 3:1): d 1.87 (quint., J = 6.9 Hz, 2H, H2-400), 1.96 (quint.,
J = 6.9 Hz, 2H, H2-300), 2.51 (t, J = 6.9 Hz, 2H, H2-500), 2.61 (t,
J = 6.9 Hz, 2H, H2-200), 7.08 (d, J = 8.4 Hz, 2H, H-30 and H-50), 7.30
(t, J = 7.5 Hz, 1H, H-6), 7.55 (t, J = 7.5 Hz, 1H, H-7), 7.69 (d,
J = 7.5 Hz, 1H, H-8), 7.88 (d, J = 8.4 Hz, 2H, H-20 and H-60), 8.24 (d,
J = 7.5 Hz, 1H, H-5), 8.89 (s, 1H, H-4), 10.81 (s, 1H, NH), 11.76 (s,
13
(s, 1H, H-4); C NMR (75.5 MHz): d 24.7 (CH2, C-400), 24.8 (CH2,
C-300), 27.5 (CH2, C-500), 33.2 (CH2, C-200), 55.4, 112.2 (CH, C-8),
113.5 (CH, C-4), 114.4 (2CH, C-30 and C50), 120.6 (CH, C-6), 121.8
(CH, C-5), 121.9 (C, C-4b), 128.6 (CH, C-7), 129.5 (2CH, C-20 and
60), 130.3 (C, C-10), 130.4 (C, C-4a), 134.9 (C, C-9a), 138.3 (C, C-3),
141.0 (C, C-8a), 141.3 (C, C-1), 160.3 (C, C-40), 161.6 (C@N), 168.1
(C@O); MS m/z (%): 398.09 (M+, 83), 369.05 (75), 273.02 (100).
13
1H, 9-NH); C NMR (75.5 MHz): d 24.2 (CH2, C-400), 24.3 (CH2, C-
300), 26.8 (CH2, C-500), 32.8 (CH2, C-200), 112.4 (CH, C-4), 112.5 (CH,
C-8), 115.5 (2CH, C-30 and 50), 119.9 (CH, C-6), 121.1 (C, C-4b),
125.4 (CH, C-5), 128.1 (CH, C-7), 128.2 (C, C-4a), 129.5 (C, C-9a),
129.5 (C-20 and C-60), 134.0 (C, C-10), 138.1 (C, C-3), 140.9 (C, C-
8a), 141.3 (C, C-1), 158.3 (C, C-40), 160.0 (C@N), 166.3 (C@O); MS
m/z (%): 384.08 (M+, 47), 355.02 ((40), 259.00 (100).
4.3.10. N0-Cyclopentylidene-1-phenyl-b-carboline-3-
carbohydrazide (36)
Yield: 80%; mp 268–271 °C; IR (KBr)
mmax: 1624 (C@N), 1690
(C@O), 1557–1344 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6/
;
CDCl3 3:1): d 1.80 (quint., J = 6.7 Hz, 4H, H2-300 and H2-400), 2.48–
2.54 (m, 4H, H2-200 and H2-500), 7.31 (t, J = 7.8 Hz, 1H, H-6), 7.53–
7.71 (m, 5H, H-7, H-8, H-30, H-40 and H-50), 8.14–8.10 (m, 2H, H-
20 and H-60), 8.34 (d, J = 7.8 Hz, 1H, H-5), 8.90 (s, 1H, H-4), 10.79
(s, 1H, NH), 11.80 (s, 1H, 9-NH); 13C NMR (75.5 MHz): d 24.2
(CH2, C-400), 24.3 (CH2, C-300), 26.8 (CH2, C-500), 32.9 (CH2, C-200),
112.5 (CH, C-8), 113.2 (CH, C-4), 120.0(CH, C-6), 121.0 (C, C-4b),
121.6 (CH, C-5), 128.2 (CH, C-40), 128.3 (2CH, C-20 and C-60),
128.6 (2CH, C-30and C-50), 128.7 (CH, C-7), 129.9 (C, C-4a), 134.4
(C, C-10), 137.4 (C, C-9a), 138.3 (C, C-3), 140.4 (C, C-8a), 141.5 (C,
C-1), 159.9 (C@N), 166.4 (C@O); MS m/z (%): 368.07 (M+, 38),
339.04 (32), 243.00 (100).
4.3.14. N0-Cyclopentylidene-1-[4-(dimethylamino)phenyl]-b-
carboline-3-carbohydrazide (40)
Yield: 85%; mp 269–270 °C; IR (KBr)
mmax: 1622 (C@N), 1675
(C@O), 1555–1347 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3/CD3OD
;
3:1): d 1.84 (quint., J = 6.9 Hz, 2H, H2-400), 1.95 (quint., J = 6.9 Hz,
2H, H2-300), 2.48 (t, J = 6.9 Hz, 2H, H2-500), 2.59 (t, J = 6.9 Hz, 2H,
H2-200), 3.09 (s, 6H, (CH3)2), 6.95 (d, J = 9.0 Hz, 2H, H-30 and H-50),
7.33–7.36 (m), 7.54–7,61 (m, 2H, H-7 and H-8), 7.92 (d,
J = 9.0 Hz, 2H, H-20 and H-60), 8.20 (d, J = 7.8 Hz, 1H, H-5), 8.82 (s,
13
1H, H-4); C NMR (75.5 MHz): d 24.6 (CH2, C-400), 24.7 (CH2, C-
300), 27.4 (CH2, C-500), 33.1 (CH2, C-200), 40.1 (CH3)2, 112.1 (CH, C-
4), 112.4 (2CH, C-30 and C-50), 112.7 (CH, C-8), 120.3 (CH, C-6),
121.5 (C, C-4b), 121.8 (CH, C-5), 125.4 (C, C-4a), 128.3 (CH, C-7),
128.9 (2CH, C-20 and C-60), 129.9 (C, C-10), 134.7 (C, C-9a), 137.9
(C, C-3), 141.2 (C, C-8a), 141.9 (C, C-1), 151.0 (C, C-40), 161.9
(C@N), 168.2 (C@O); MS m/z (%): 411.12 (M+, 92), 382.07 (87),
286.07 (100), 242 (85).
4.3.11. N0-Cyclopentylidene-1-(3-nitrophenyl)-b-carboline-3-
carbohydrazide (37)
Yield: 77%; mp 300 °C, decomp.; IR (KBr)
m
max: 3214 (N-H), 1624
1H NMR (300 MHz,
(C@N), 1683 (C@O), 1561–1349 (C@C) cmꢁ1
;
CDCl3/CD3OD 3:1): d 1.82–1.89 (m, 2H, H2-400), 191–1.98 (m, 2H,
H2-300), 2.45 (t, J = 6.0 Hz, 2H, H2-500), 2.61 (t, J = 6.0 Hz, 2H, H2-
200), 7.58–7.62 (m, 2H, H-7 and H-8), 7.83 (t, J = 7.8 Hz, 1H, H-40),
8.37–8.43 (m, 2H, H-50 and H-60), 8.86–8.87 (m, 1H, H-20), 8.18
(d, J = 7.8 Hz, 1H, H-5), 8.91 (s, 1H, H-4); 13C NMR (75.5 MHz): d
24.6 (CH2, C-300), 24.6 (CH2, C-400), 27.4 (CH2, C-500), 33.1 (CH2, C-
200), 112.3 (CH, C-8), 114.5 (CH, C-4), 120.8 (CH, C-6), 121.4 (C, C-
4b), 121.6 (CH, C-5), 123.3 (CH, C-20), 123.4 (CH, C-60), 129.0 (CH,
C-40), 129.9 (CH, C-7), 131.3 (C, C-4a), 133.8 (CH, C-50), 135.0 (C,
C-10), 137.9 (C, C-9a), 138.1 (C, C-3), 139.4 (C, C-8a), 141.7 (C, C-
1), 148.6 (C, C-30), 161.1 (C@N), 168.7 (C@O); MS m/z (%): 413.06
(M+, 38), 384.03 (42), 288.00 (50), 242.00 (100) .
4.3.15. N0-Cyclopentylidene-1-(4-nitrophenyl)-b-carboline-3-
carbohydrazide (41)
Yield: 88%; mp 291–293 °C; IR (KBr)
mmax: 1624 (C@N), 1682
(C@O), 1555–1341 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d
;
1.75 (quint, J = 6.3 Hz, 2H, H2-400), 1.85 (quint, J = 6.3 Hz, 2H, H2-
300), 2.44–2.51 (m, 2H, H2-200), 2.44–2.51 (m, 2H, H2-500), 7.36 (t,
J = 7.8 Hz, 1H, H-6), 7.65 (t, J = 7.8 Hz, 1H, H-7), 7.72 (d,
J = 7.8 Hz, 1H, H-8), 8.44 (d, J = 9.0 Hz, 2H, H-30 and H-50), 8.46
(d, J = 7.8 Hz, 1H, H-5), 8.50 (d, J = 9.0 Hz, 2H, H-20 and H-60),
9.03 (s, 1H, H-4), 10.7 (s, 1H, NH), 12.1 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d 24.3 (CH2, C-400), 24.4 (CH2, C-300), 27.1 (CH2, C-
500), 32.9 (CH2, C-200), 112.6 (CH, C-8), 114.6 (CH, C-4), 120.6
(CH, C-6), 121.0 (CH, C-4b), 122.4 (CH, C-5), 124.0 (2CH, C-30
and C-50), 129.2 (CH, C-7), 129.9 (2CH, C-20 and C-60), 130.8 (C,
C-4a), 134.7 (C, C-10), 138.0 (C, C-9a), 139.1 (C, C-3), 141.7 (C,
C-8a), 143.5 (C, C-1), 147.5 (C, C-40), 159.5 (C@N), 167.7 (C@O);
MS m/z (%): 413.06 (M+, 53), 384.03 (45), 288.00 (58), 258.02
(58), 242.00 (100).
4.3.12. N0-Cyclopentylidene-1-(3-hydroxy-4-methoxyphenyl)-b-
carboline-3-carbohydrazide (38)
Yield: 77%; mp 296–297 °C; IR (KBr)
mmax: 1623 (C@N), 1682
(C@O), 1598–1346 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6/
;
CDCl3 3:1): d 1.82 (quint., J = 6.9 Hz, 2H, H2-400), 1.91 (quint.,
J = 6.9 Hz, 2H, H2-300), 2.49–2.57 (m, 4H, H2-200 and H2-500), 3.98
(s, 3H, OCH3), 7.06 (d, J = 8.1 Hz, 1H, H-50), 7.30 (t, J = 7.8 Hz,
1H, H-6), 7.53–7.59 (m, 2H, H-60 and H-7), 7.68–7.71 (m, 2H, H-
20 and H-8), 8.33 (d, J = 7.8 Hz, 1H, H-5), 8.82 (s, 1H, H-4), 9.37
(s, 1H, OH), 10.87 (s, 1H, NH), 11.82 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d 24.1 (CH2, C-400), 24.2 (CH2, C-300), 26.8 (CH2, C-
500), 32.7 (CH2, C-200), 55.3 (OCH3), 111.8 (CH, C-20), 112.4 (CH,
C-8), 112.5 (CH, C-50), 115.3 (CH, C-4), 119.9 (CH, C-6), 121.0 (C,
C-4b), 121.1 (CH, C-5), 121.5 (CH, C-60), 128.1 (C, C-4a), 128.5
(C, C-10), 129.5 (C, C-7), 134.0 (C, C-9a), 138.1 (C, C-3), 140.8 (C,
C-8a), 141.3, (C, C-1) 147.7 (C, C-40), 147.8 (C, C-30), 159.9
(C@N), 166.2 (C@O); MS m/z (%): 414.06 (M+, 100), 385.02 (95),
290.00 (95), 256.97 (90).
4.3.16. N0-Cyclopentylidene-1-(2-chlorophenyl)-b-carboline-3-
carbohydrazide (42)
Yield: 77%; mp 280–282 °C; IR (KBr)
mmax: 1623 (C@N), 1669
(C@O), 1563–1345 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d
;
1.79 (quint., J = 7.2 Hz, 2H, H2-400), 1.89 (quint., J = 7.2 Hz, 2H, H2-
300), 2.41 (t, J = 7.2 Hz, 2H, H2-500), 2.56 (t, J = 7.2 Hz, 2H, H2-200),
7.39 (ddd; J = 7.8 Hz, J = 6.7 Hz, J = 1.5 Hz, 1H, H-6), 7.46–7.51 (m,
2H, H-7 and H-8), 7.55–7.71 (m, 4H, H-30, H-40 H-50and H-60),
8.22 (d, J = 7.8 Hz, 1H, H-5), 8.67 (s, 1H, NH), 9.05 (s, 1H, H-4),
13
10.67 (s, 1H, 9-NH); C NMR (75.5 MHz): d 24.9 (CH2, C-400), 25.0
(CH2, C-300), 27.6 (CH2, C-500), 33.6 (CH2, C-200), 112.0 (CH, C-8),

6406
V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
115.0 (CH, C-4), 121.3 (CH, C-6), 122.2 (C, C-4b), 122.4 (CH, C-5),
127.6 (CH, C-7), 129.3 (CH, C-50), 130.5 (CH, C-30), 130.6 (C, C-4a),
130.8 (CH, C-60), 131.7 (CH, C-40), 133.2 (C, C-20), 135.6 (C, C-10),
136.2 (C, C-9a), 139.2 (C, C-3), 139.5 (C, C-8a), 140.8 (C, C-1),
161.1 (C@N), 167.4 (C@O); MS m/z (%): 401.99 (M+, 50), 276.94
(67), 241.97 (100).
(CH3, C-300), 25.1 (CH3, C-200), 55.5 (OCH3), 112.2 (CH, C-8),
112.6 (CH, C-4), 112.7 (CH, C-20), 115.5 (CH, C-50), 120.2 (CH,
C-6), 121.2 (C, C-4a), 121.2 (CH, C-60), 122.0 (CH, C-5), 128.5
(CH, C-7), 129.7 (C, C-10), 134.0 (C, C-9a), 138.6 (C, C-3), 140.9
(C, C-8a), 141.4 (C, C-1), 147.9 (C, C-40), 148.1 (C, C-30), 154.9
(C@N), 159.9 (C@O); MS m/z (%): 388.03 (M+, 90), 373.00 (95),
28.99 (100), 256.95 (87).
4.3.17. N0-2-Propylidene-1-(4-methoxyphenyl)-b-carboline-3-
carbohydrazide (43)
4.3.21. N0-2-Propylidene-1-(4-hydroxyphenyl)-b-carboline-3-
Yield: 80%; mp 285–288 °C; IR (KBr)
m
max: 1624 (C@N), 1686
carbohydrazide (47)
(C@O), 1561–1346 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d
;
Yield: 76%; mp >300 °C; IR (KBr)
mmax: 3315 (N-H), 1666
2.07 (s, 3H, H3-300), 2.20 (s, 3H, H3-200), 3.94 (s, 3H, OCH3), 7.16 (d,
J = 8.7 Hz, 2H, H-30 and H-50), 7.37 (dd, J = 8.1 Hz, J = 1.5 Hz, 1H,
H-6), 7.54–7.63 (m, 2H, H-7 and H-8), 7.93 (d, J = 8.7 Hz, 2H, H-20
and H-60), 8.22 (d, J = 8.1 Hz, 1H, H-5), 8.73 (s, 1H, NH), 8.98 (s,
1H, H-4), 11.03 (s, 1H, 9-NH); 13C NMR (75.5 MHz): d 16.8 (CH3,
C-300), 25.8 (CH3, C-200), 55.7 (OCH3), 111.9 (CH, C-8), 114.1 (CH,
C-4), 115.0 (2CH, C-30 and C-50), 121.3 (CH, C-6), 122.3 (C, C-4b),
122.5 (CH, C-5), 129.1 (CH, C-7), 129.4 (2CH,C-20 and C-60), 129.6
(C, C-4a), 130.6 (C, C-3), 130.7 (C, C-10), 134.9 (C, C-9a), 140.0 (C,
C-8a), 140.7 (C, C-1), 154.8 (C@N), 160.6 (C-40), 161.3 (C@O); MS
m/z (%): 372.04 (M+, 55), 357.02 (57), 272.99 (100).
(C@O), 1561–1381 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d
;
2.03 (s, 3H, H3-300), 2.06 (s, 3H, H3-200), 7.03 (d, J = 8.4 Hz, 2H, H-
30 and H-50), 7.32 (t, J = 7.5 Hz, 1H, H-6), 7.60 (t, J = 7.5 Hz, 1H,
H-7), 7.70 (d, J = 7.5 Hz, 1H, H-8), 8.00 (d, J = 8.4 Hz, 2H, H-20
and H-60), 8.43 (d, J = 7.5 Hz, 1H, H-5), 8.84 (s, 1H, H-4), 9.90 (s,
1H, NH), 10.98 (s, 1H, OH), 11.85 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d 16.4 (CH3, C-300), 25.0 (CH3, C-200), 112.6 (CH, C-4),
112.7 (CH, C-8), 115.7 (2CH, C-30 and C-50), 120.2 (CH, C-6),
121.2 (C, C-4a), 122.0 (C, C-5), 128.2 (C, C-4b), 128.5 (CH, C-7),
129.7 (C, C-3), 129.9 (2CH, C-20 and C-60), 134.9 (C, C-10), 138.6
(C, C-9a), 140.9 (C, C-8a), 141.4 (C, C-1), 155.2 (C, C-40), 158.5
(C@N), 159.9 (C@O); MS m/z (%): 358.04 (M+, 32), 343.03 (30),
258.99 (100).
4.3.18. N0-2-Propylidene-1-phenyl-b-carboline-3-
carbohydrazide (44)
Yield: 82%; mp 284–286 °C; IR (KBr)
m
max: 1625 (C@N), 1678
4.3.22. N0-2-Propylidene-1-[4-(dimethylamino)phenyl]-b-
carboline-3-carbohydrazide (48)
(C@O), 1560–1344 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d
;
2.05 (s, 3H, H3-300), 2.18 (s, 3H, H3-200), 7.37 (dd, J = 7.8 Hz,
J = 1.8 Hz, 1H, H-6), 7.52–7.57 (m, 2H, H-7 and H-8), 7.59–7.66
(m, 2H, H-30 and H-50), 7.97–8.00 (m, 2H, H-20 and H-60), 8.21 (d,
J = 7,8 Hz, 1H, H-5), 8.87 (s, 1H, NH), 9.00 (s, 1H, H-4), 11.01 (s,
Yield: 82%; mp 272–274 °C; IR (KBr)
mmax: 1671 (C@O), 1555–
1347 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.05 (s, 3H, H3-
;
300), 2.16 (s, 3H, H3-200), 3.05 (s, 6H, N(Me)2), 6.90 (d, J = 8.7 Hz,
2H, H-30 and H-50), 7.29–7.38 (m, 1H, H-6), 7.56–7.58 (m, 2H, H-
7 and H-8), 7.89 (d, J = 8.7 Hz, 2H, H-20 and H-60), 8.16 (d,
J = 7.8 Hz, 1H, H-5), 8.87 (s, 1H, H-4), 8.95 (s, 1H, NH), 11.08 (s,
13
1H, 9-NH); C NMR (75.5 MHz): d 16.7 (CH3, C-300), 25.7 (CH3, C-
200), 112.2 (CH, C-8), 114.3 (CH, C-4), 121.2 (CH, C-6), 122.2 (C, C-
4b), 122.3 (CH, C-5), 128.2 (2CH, C-20 and C-60), 129.1 (CH, C-7),
129.3 (CH, C-40), 129.4 (2CH, C-30 and C-50), 130.8 (C, C-4a), 135.1
(C, C-10), 138.0 (C, C-9a), 139.6 (C, C-3), 140.9 (C, C-8a), 141.0 (C,
C-1), 154.9 (C@N), 161.3 (C@O); MS m/z (%): 342.02 (M+, 28),
327.00 (28), 242.98 (100).
13
1H, 9-NH); C NMR (75.5 MHz): d 16.7 (CH3, C-300), 25.7 (CH3, C-
200), 40.5 (CH3)2, 111.9 (CH, C-8), 112.7 (2CH, C-30 and C-50), 113.3
(CH, C-4), 121.0 (CH, C-6), 122.3 (CH, C-5), 122.5 (C, C-4b), 125.6
(C-4a), 128.7 (CH, C-7), 129.0 (2CH, C-20 and C-60), 130.3(C, C-10),
134.8 (C-9a), 139.7 (C, C-3), 140.7 (C, C-8a), 141.6 (C, C-1), 151.1
(C, C-40), 154.5 (C@N), 161.4 (C@O); MS m/z (%): 385.07 (M+, 85),
370.05 (77), 286.03 (100), 241.98 (72).
4.3.19. N0-2-Propylidene-1-(3-nitrophenyl)-b-carboline-3-
carbohydrazide (45)
Yield: 85%; mp >300 °C; IR (KBr)
m
max: 1623 (C@N), 1674 (C@O),
4.3.23. N0-2-Propylidene-1-(4-nitrophenyl)-b-carboline-3-
carbohydrazide (49)
1561–1345 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 2.05 (s,
;
3H, H3-300), 2.07 (s, 3H, H3-200), 7.36 (t, J = 7.5 Hz, 1H, H-6), 7.63
(d, J = 7.5 Hz, 1H, H-8), 7.70 (t, J = 7.5 Hz, 1H, H-7), 7.95 (t,
J = 8.0 Hz, 1H, H-50), 8.41 (dd, J = 8.0 Hz, J = 2.3 Hz, 1H, H-40), 8.50
(d, J = 8.0 Hz, 1H, H-60), 8.62 (d, J = 7.5 Hz, 1H, H-5), 8.95 (t,
J = 1.8 Hz, 1H, H-20), 9.00 (s, 1H, H-4), 10.97 (s, 1H, NH), 12.16 (s,
Yield: 85%; mp >300 °C; IR (KBr)
m
max: 3249 (N-H), 1626
1H NMR (300 MHz,
(C@N), 1672 (C@O), 1515–1341 (C@C) cmꢁ1
;
DMSO-d6/CDCl3 3:1): d 2.03 (s, 3H, H3-300), 2.07 (s, 3H, H3-200),
7.36 (t, J = 7.5 Hz, 1H, H-6), 7.65 (t, J = 7.5 Hz, 1H, H-7), 7.71 (d,
J = 7.5 Hz, 1H, H-8), 8.43–8.52 (m, 5H, H-20, H-30, H-50, H-60 and
H-5), 9.02 (s, 1H, H-4), 10.92 (s, 1H, NH), 12.48 (s, 1H, 9-NH);
13C NMR (75.5 MHz): d 16.4 (CH3, C-300), 25.0 (CH3, C-200), 112.4
(CH, C-8), 114.3 (CH, C-4), 120.3 (CH, C-6), 120.9 (C, C-4a),
121.9 (CH, C-5), 123.6 (2CH, C-30 and C-50), 128.8 (CH, C-7),
129.6 (2CH, C-20 and C-60), 130.7 (C, C-10), 134.6 (C, C-3), 138.9
(C, C-9a), 141.6 (C, C-8a), 143.5 (C, C-1), 147.3 (C, C-40), 155.5
(C@N), 159.7 (C@O); MS m/z (%): 387.02 (M+, 55), 371.99 (48),
287.96 (75), 241.97 (100).
13
1H, 9-NH); C NMR (75.5 MHz): d 16.5 (CH3, C-300), 25.1 (CH3, C-
200), 112.6 (CH, C-8), 114.3 (CH, C-4), 120.1 (CH, C-6), 121.1 (C, C-
4b), 122.4 (CH, C-60), 123.4 (CH, C-40), 123.6 (CH, C-20), 129.1 (CH,
C-7), 130.7 (CH, C-50), 134.4 (C, C-4a), 134.9 (CH, C-5), 137.8 (C,
C-10), 138.7 (C, C-9a), 139.1 (C, C-3), 141.6 (C, C-8a), 148.3 (C, C-
1), 156.2 (C, C-30), 159.6 (C@N), 176.5 (C@O); MS m/z (%): 387.01
(M+, 52), 372.00 (50), 287.98 (70), 241.97 (100).
4.3.20. N0-2-Propylidene-1-(3-hydroxy-4-methoxyphenyl)-b-
carboline-3-carbohydrazide (46)
4.3.24. N0-2-Propylidene-1-(2-chlorophenyl)-b-carboline-3-
Yield: 81%; mp >300 °C; IR (KBr)
m
max: 3327 (N-H), 1624
1H NMR
carbohydrazide (50)
(C@N), 1675 (C@O), 1524–1347 (C@C) cmꢁ1
;
Yield: 75%; mp 233–236 °C; IR (KBr)
mmax: 1624 (C@N), 1682
(300 MHz, DMSO-d6): d 2.06 (s, 3H, H3-200), 2.06 (s, 3H, H3-300),
3.92 (s, 3H, OCH3), 7.03 (d, J = 7.8 Hz, 1-H, H-50), 7.32 (t,
J = 7.8 Hz, 1H, H-6), 7.57–7.62 (m, 2H, H-7 and H-8), 7.70 (d,
J = 7.8 Hz, 1H, H-60), 7.74 (d, J = 1.8 Hz, 1 H, H-20), 8.43 (d,
J = 7.8 Hz, 1H, H-5), 8.85 (s, 1H, H-4), 9.49 (s, 1H, NH), 11.03
(s, 1H, OH), 11.88 (s, 1H, 9-NH); 13C NMR (75.5 MHz): d 16.3
(C@O), 1595–1345 (C@C) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d
;
1.93 (s, 3H, H3-300), 2.03 (s, 3H, H3-200), 7.30–7.36 (m, 1H, H-6),
7.57–7.65 (m, 4H, H-50, H-60, H-7 and H-8), 7.73–7.76 (m, 1H, H-
30), 7.79–7.82 (m, 1H, H-40), 8.47 (d, J = 7.8 Hz, 1H, H-5), 8.99 (s,
1H, H-4), 10.79 (s, 1H, NH), 11.81 (s, 1H, 9-NH); 13C NMR
(75.5 MHz): d 16.4 (CH3, C-300), 25.0 (CH3, C-200), 112.4 (CH, C-8),

V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
6407
114.1 (CH, C-4), 120.3 (CH, C-6), 121.0 (C, C-4b), 122.3 (CH, C-5),
4.4.3. N0-(2-Chlorobenzylidene)-1-(4-methoxyphenyl)-9-
methyl-b-carboline-3-carbohydrazide (55)
127.6 (CH, C-7), 128.9 (CH, C-50), 129.4 (C, C-20), 129.4 (C, C-4a),
130.2 (C, C-30), 130.7 (CH, C-60), 132.4 (CH, C-40), 135.2 (C, C-10),
135.8 (C,C-9a), 138.4 (C, C-3), 139.3 (C, C-8a), 141.4 (C, C-1),
155.3 (C@N), 159.8 (C@O); MS m/z (%): 376.01 (M+, 45), 361.00
(48), 276.97 (85), 241.99 (100).
Yield: 78%; mp 217.0–220.0 °C; IR (KBr)
mmax: 1673 (C@O),
1510–1357 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.55 (s, 3H,
;
9-N-CH3), 3.95 (s, 3H, OCH3), 7.13 (d, J = 8.7 Hz, 2H, H-30 and H-
50), 7.30–7.36 (m, 3H, H-6, H-500 and H-600), 7.40 (t, J = 7.5 Hz, 1H,
H-400), 7.47 (d, J = 7.5 Hz, 1H, H-8), 7.62 (d, J = 8.7 Hz, 2H, H-20
and H-60), 7.67 (d, J = 7.5 Hz, 1H, H-300), 8.28 (d, J = 7.5 Hz, 1H, H-
5), 8.31–8.33 (m, 1H, H-7), 8.65 (s, 1H, N@C), 9.04 (s, 1H, H-4),
11.26 (s, 1H, NH); 13C NMR (75.5 MHz): d 33.2 (N–CH3), 55.6
(OCH3), 110.2 (CH, C-8), 114.0 (2CH, C-30 and C-50), 114.4 (CH, C-
4), 121.1 (CH, C-6), 121.7 (C, C-4b), 122.2 (CH, C-5), 127.2 (CH, C-
600), 128.3 (CH, C-7), 129.2 (CH, C-300), 129.8 (CH, C-500), 130.9 (C,
C-4a), 131.1 (2CH, C-20 and C-60), 131.3 (C, C-400), 131.6 (CH, C-
200), 131.7 (C, C-100), 134.3 (C, C-10), 136.8 (C, C-9a), 138.2 (C, C-3),
142.5 (C, C-1), 143.4 (C, C-8a), 144.2 (N@CH), 160.3 (C, C-40),
161.6 (C@O); MS m/z (%): 468.01 (M+, 0.5), 331.02 (100), 314.03
(50), 288.03 (70).
4.4. General procedure for the synthesis of N0-(substituted-
benzylidene)-1-(substituted-phenyl)-9-methyl-b-carboline-3-
carbohydrazides (53–56)
To a solution of the methyl esters 1a–c (1.9 mmol) in anhydrous
DMF (15 mL) were added NaOH (0.304 mg,7.6 mmol) and methyl
iodide (0.118 mL, 19 mmol) at room temperature. The mixture
was stirred for 48 h, diluted with H2O (250 mL) and extracted with
EtOAc. The organic layer was dried over N2SO4, filtered and evapo-
rated. The solid residues obtained were recrystallized from metha-
nol to afford the products 51a–c. The N9-methylated derivatives
51a–c (4.0 mmol) in EtOH (50 mL) were treated with hydrazine
hydrate (53 mmol) and the mixture was refluxed for 72 h and then
cooled to 0 °C. The formed solids were collected by filtration and
washed with EtOH, to furnish the corresponding carbohydrazides
52a–c.
4.4.4. N0-(2-Chlorobenzylidene)-9-methyl-1-phenyl-b-
carboline-3-carbohydrazide (56)
Yield: 82%; mp 260.0–263.0 °C; IR (KBr)
mmax: 1692 (C@O),
1557–1351 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.52 (s, 3H,
;
A solution of the carbohydrazides 52a–c (1.0 mmol) in H2O
(10 mL) and two drops of concentrated H2SO4 was heated to
65 °C, and the respective aldehydes, dissolved in EtOH (10 mL),
were added. The resulting solution was refluxed for 48 h. After
cooling, the mixture was poured onto ice-water, neutralized with
10% aqueous Na2CO3 and the formed solids were collected by fil-
tration and washed with water.
N-CH3), 7.31–7.49 (m, 5H, H-6, 7, 8, H-500 and H-600), 7.60–7.71
(m, 7H, H-20, H-30, H-40, H-50, H-60, H-300 and H-400), 8.28–8.33 (m,
1H, H-5), 8.66 (s, 1H, N@CH), 9.08 (s, 1H, H-4), 11.26 (s, 1H, NH);
13C NMR (75.5 MHz): d 33.2 9 (N–CH3), 110.2 (CH, C-8), 114.7
(CH, C-4), 121.2 (CH, C-6), 121.7 (C-4b), 122.3 (CH, C-5), 127.2
(CH, C-500), 128.3 (CH, C-300), 128.6 (2CH, C-30 and C-50), 129.2
(CH, C-400), 129.3 (C, C-40), 129.8 (CH, C-7), 129.9 (2CH, C-20 and
C-60), 131.0 (C, C-4a), 131.3 (CH, C-600), 131.6 (C, C-10), 134.4 (C,
C-100), 136.7 (C, C-200), 138.3 (C, C-3), 139.4 (C, C-9a), 142.6 (C, C-
8a), 143.4 (C, C-1), 144.3 (C@N), 161.6 (C@O); MS m/z (%): 438.02
(M+, 0.5), 301.03 (90), 284.02 (40), 258.02 (100), 257.02 (90).
4.4.1. N0-(4-Dimethylaminobenzylidene)-9-methyl-1-(3-
nitrophenyl)-b-carboline-3-carbohydrazide (53)
Yield: 75%; mp 232.0ꢁ234.0 °C; IR (KBr)
m
max: 1671 (C@O),
1525–1348 (C@C) cmꢁ1
;
1H NMR (300 MHz, CDCl3): d 3.03 (s, 6H,
N(CH3)2), 3.55 (s, 3H, N-CH3), 6.71 (d, J = 8.7 Hz, 2H, H-300 and H-
500), 7.43 (t, J = 8.1 Hz, 1H, H-7), 7.50 (d, J = 8.1 Hz, 1H, H-8), 7.71
(d, J = 8.7 Hz, 2H, H-200 and H-600), 7.80 (t, J = 8.1 Hz, 1H, H-6),
8.15 (s, 1H, N@CH), 8.31 (d, J = 8.1 Hz, 1H, H-5), 8.42-8.45 (m, 2H,
H-50 and H-60), 8.58 (t, J = 1.8 Hz, 1H, H-20), 9.11 (s, 1H, H-4), 10.8
(s, 1H, NH); 13C NMR (75.5 MHz): d 33.7 (N–CH3), 40.4 (CH3)2,
110.3 (CH, C-8), 111.8 (2CH, C-300 and C-500), 115.0 (CH, C-4),
121.5 (C, C-4b), 121.5 (CH, C-6), 121.5 (CH, C-7), 122.4 (CH,C-5),
123.8 (CH, C-50), 124.9 (CH, C-20), 129.5 (2CH, C-200 and C-600),
129.6 (C, C-100), 129.7 (C, C-10), 131.9 (CH, C-60), 135.9 (CH,C-40),
136.5 (C, C-3), 139.4 (C, C-9a), 141.2 (C, C-8a), 143.6 (C, C-1),
148.3 (C, C-30), 149.3 (N@CH), 152.0 (C, C-400), 160.6 (C@O); MS
m/z (%): 492.09 (M+, 40), 346.01 (40), 303.00 (75), 256.1 (57),
145.99 (100).
4.5. Biological assays
4.5.1. Animals
Balb/C mice (20–35 g) obtained from CEMIB-Unicamp were
maintained in a room with controlled temperature 25 2 °C for
12 h light/dark cycle, with free access to food and water. Animal
care, research and animal sacrifice protocols were in accordance
with the principles and guidelines adopted by the Brazilian College
of Animal Experimentation (COBEA) and approved by the Biology
Institute/UNICAMP—Ethical Committee for Animal Research.
4.5.2. In vitro anticancer assay
The anticancer activity was assessed by sulforodamine B (SRB)
colorimetric assay developed by the National Cancer Institute,
using doxorubicin as a positive control.36 Assays were performed
in a 96-well plate using four concentrations at 10-fold dilutions
(0.25–250 mg mLꢁ1) for each tested compound. The anticancer
activity was deduced from concentration–response curves. The
IC50 value refers to the drug concentration that produces a 50%
reduction in cellular growth when compared to untreated control
cells.37
4.4.2. N0-Phenylbenzylidene-1-(4-methoxyphenyl)-9-methyl-b-
carboline-3-carbohydrazide (54)
Yield: 83%; mp 216.0–219.0 °C; IR (KBr)
mmax: 1686 (C@O), 1610
(C@N), 1511–1358 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.54
;
(s, 3H, N-CH3), 3.95 (s, 3H, O-CH3), 7.12 (d, J = 8.4 Hz, 2H, H-30 and
H-50), 7.35–7.47 (m, 4H, H-6, 7, 8 and H-400), 7.59–7.66 (m, 4H, H-20,
60, H-200 and H-600), 7.79–7.82 (m, 2H, H-300 and H-500), 8.25 (s, 1H,
N@CH), 8.27 (s, 1H, H-5), 9.01 (s, 1H, H-4), 11.14 (s, 1H NH); 13C
NMR (75.5 MHz): d 33.2 (N–CH3), 55.6 (O-CH3), 110.2 (CH, C-8),
113.9 (2CH,C-30 and C-50), 114.2 (CH, C-4), 121.0 (CH, C-6), 121.7
(C, C-4b), 122.2 (CH,C-5), 127.9 (2CH, C-300 and C-500), 128.8 (2CH,
C-200 and C-600), 129.1 (CH, C-7), 130.4 (CH, C-400), 130.9 (C, C-4a),
131.1 (2CH,C-20 and C-60), 131.8 (C, C-10), 134.1 (C, C-100), 136.8
(C, C-9a), 138.4 (C, C-3), 142.4 (C, C-8a), 143.4 (C, C-1), 148.0
(N@C), 160.3 (C, C-40), 161.5 (C@O); MS m/z (%): 434.07 (M+, 12),
331.04 (100), 314.04 (53), 288.04 (70).
4.5.3. In vivo antineoplastic assay: Ehrlich solid carcinoma in
mice paw
The most active compound 4 was tested for its in vivo in the
Ehrlich solid carcinoma assay model. The procedures were devel-
oped according to Vendramini-Costa et al.,35 with few modifica-
tions on data analysis. The Ehrlich ascitic tumor (EAT), derived
from a spontaneous murine mammary adenocarcinoma, was
maintained in the ascitic form by sequential passages in Swiss
mice, by means of weekly ip transplantations of 5 ꢂ 105 tumor

6408
V. A. Barbosa et al. / Bioorg. Med. Chem. 19 (2011) 6400–6408
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cells, in order to prepare cells for the following test. The ascitic
fluid was removed by opening the belly and carefully collecting
all the fluid using a sterile 3 mL syringe. Ascitic tumor cell counts
were performed in a Neubauer hemocitometer, and the total num-
ber was determined by the Trypan blue dye exclusion method,
with tumor cell viability always higher than 90%. The cells were
then diluted in 0.9% phosphate buffer saline (PBS) for final inocula-
tion density (2.5 ꢂ 106 cells/mL). For the solid form implantation,
2.5 ꢂ 106 viable tumor cells in a volume of 60
lL were injected
in sub-plantar site of the right hind paw of Balb-C mice.38 After tu-
mor cell inoculation, the foot volume was measured every three
days using a plethysmometer apparatus (Panlab, Spain) till the
15th day when the animals were sacrificed. The tumor growth
was measured considering the following formula: Volume mea-
sured ꢁ Basal volume = Tumor volume.
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This work was supported by Fundação Araucária (Brazil, PR)
and Fundação de Amparo a Pesquisa de São Paulo (FAPESP). We
thank Fundação Araucária, CAPES and CNPq for fellowships
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