Synthesis and evaluation of new β-carboline-3-(4-benzylidene)- 4H-oxazol-5-one derivatives as antitumor agents

Article abstract of DOI:10.3390/molecules17056100

In the present work, we report the synthesis and in vitro anticancer and antimicrobial activity evaluation of a new series of 1-substituted-β- carboline derivatives bearing a 4-benzylidene-4H-oxazol-5-one unity at C-3. The compound 2-[1-(4- methoxyphenyl)-9H-β-carbolin-3-yl]-4-(benzylidene)-4H- oxazol-5-one (11) was the most active derivative, exhibiting a potent cytotoxic activity against glioma (U251), prostate (PC-3) and ovarian (OVCAR-03) cancer cell lines with IC₅₀ values of 0.48, 1.50 and 1.07 μM, respectively. An in silico study of the ADME properties of the novel synthesized β-carboline derivatives was also performed.

Full text of DOI:10.3390/molecules17056100

Molecules 2012, 17, 6100-6113; doi:10.3390/molecules17056100
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of New β-Carboline-3-(4-benzylidene)-
4H-oxazol-5-one Derivatives as Antitumor Agents
Franciele Cristina Savariz 1, Mary Ann Foglio 2, João Ernesto de Carvalho 2, Ana Lúcia T. G. Ruiz 2,
Marta C. T. Duarte 2, Mauricio Ferreira da Rosa 3, Emerson Meyer 1 and
Maria Helena Sarragiotto 1,*
1
Departamento de Química, Centro de Ciências Exatas, Universidade Estadual de Maringá,
Av. Colombo, 5790, Maringá, 87020-900 PR, Brazil
2
Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA),
Universidade Estadual de Campinas, 6171, Campinas, 13083-970 SP, Brazil
3
Centro de Engenharias e Ciências Exatas, Universidade Estadual do Oeste do Paraná,
Rua da Faculdade, 645, Toledo, 85903-000 PR, Brazil
* Author to whom correspondence should be addressed; E-Mail: mhsarragiotto@uem.br;
Tel.: +55-44-3261-3657; Fax: +55-44-3011-4125.
Received: 5 April 2012; in revised form: 5 May 2012 / Accepted: 7 May 2012 /
Published: 21 May 2012
Abstract: In the present work, we report the synthesis and in vitro anticancer and
antimicrobial activity evaluation of a new series of 1-substituted-β-carboline derivatives
bearing a 4-benzylidene-4H-oxazol-5-one unity at C-3. The compound 2-[1-(4-
methoxyphenyl)-9H-β-carbolin-3-yl]-4-(benzylidene)-4H-oxazol-5-one (11) was the most
active derivative, exhibiting a potent cytotoxic activity against glioma (U251), prostate
(PC-3) and ovarian (OVCAR-03) cancer cell lines with IC50 values of 0.48, 1.50 and 1.07 µM,
respectively. An in silico study of the ADME properties of the novel synthesized
β-carboline derivatives was also performed.
Keywords: -carboline; 4H-oxazol-5-one; cytotoxic activity; antimicrobial activity
1. Introduction
Natural and synthetic tetrahydro--carbolines and -carbolines are a class of alkaloids with a large
spectrum of important pharmacological properties [1–7]. Among the activities presented, the antitumor

Molecules 2012, 17
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activity has received special attention, and several studies on structure-activity relationship of
-carbolines have focused their anticancer activities [816]. The SAR studies have demonstrated that
the introduction of appropriate substituents into the positions −1, −2, −3 and −9 of the -carboline
nucleus resulted in more potent antitumor -carboline derivatives, with reduced toxicity.
The potential of -carbolines and the importance of the search for new antitumor agents led our
group to a continuing study of this class of compounds. In previous works, we reported the synthesis
and in vitro antitumor activities, against a panel of human cancer cell lines, of a series of 1-substituted
-carboline derivatives bearing different substituents at C-3, such as 2-substituted-1,3,4-oxadiazole
and 5-substituted-1,2,4-triazole rings [17], 3-alkylamino(methyl)-2-thioxo-1,3,4-oxadiazole groups [18]
and N-(substituted-benzylidene)-carbohydrazide groups [19]. The anticancer assay results indicated
several compounds with potent anticancer activity, with IC50 values lower than 1.0 M for some of the
human cancer cell lines tested [17–19]. In addition to the antitumor activity, -carbolines containing
the 2-thioxo-1,3,4-oxadiazole group at C-3 exhibited antimicrobial activity towards the fungus
Candida albicans and the bacterium Bacillus subtilis [18]. Antimicrobial activity of -carboline
derivatives, mainly against Bacillus subtilis, Escherichia coli and Staphylococcus aureus bacteria and
against Candida albicans fungi, was also reported in literature [20,21].
With the aim of evaluating the influence of different substituents at position-3 of 1-substituted--
carbolines, in this work we propose the incorporation of a benzylidene-4H-oxazol-5-one unity at C-3,
expecting an improvement of the antitumor and antimicrobial activities in relation to the most active
-carboline derivatives reported in our previous work [17–19]. The 4H-oxazol-5-one ring is found in
natural and synthetic compounds possessing important biological activities, such as antimicrobial [22–24],
antiviral [25], antiangiogenic [26], inhibitory of tyrosinase [27], cytotoxic and immunomodulatory
properties [28]. Due their biological importance, several methods were reported for the synthesis of
oxazolones [29–31].
Thus, in the present work, we report the synthesis and in vitro cytotoxic and antimicrobial activity
evaluation of some novel 1-substituted -carboline derivatives bearing a 4-(benzylidene)-4H-oxazol-5-
one moiety at C-3. Additionally, an in silico study of the ADME properties of the novel synthesized
β-carboline derivatives was carried out by investigating their Lipinski’s parameters, topological polar
surface area (TPSA) and percentage of absorption (% ABS).
2. Results and Discussion
2.1. Chemistry
The synthetic route for the -carboline-3-(4-benzylidene)-4H-oxazol-5-ones 8–11 is outlined in
Scheme 1. The 1-substituded -carboline-3-carboxylic acids 5a–c were prepared from commercial
L-tryptophan (1), according to the synthetic protocol described by our group [17–19]. The Pictet-
Spengler condensation of L-tryptophan methyl ester (2) with benzaldehyde, 4-hydroxybenzaldehyde
and 4-methoxybenzaldehyde afforded the 1,2,3,4-tetrahydro--carbolines 3a–c. Oxidation of 3a–c
with sulfur in refluxing xylene, led to the methyl -carboline-3-carboxylates 4a–c, which were
hydrolyzed under basic conditions to give the -carboline-3-carboxylic acids 5a–c.

Molecules 2012, 17
6102
In order to synthesize the -carboline-3-(4-benzylidene)-4H-oxazol-5-ones 8–11, the Erlenmeyer-
Plöchl reaction, the most common route to oxazolones, was employed [31]. The derivatives 5a–c were
converted to the respective N-(1-benzylidene-β-carboline-3-carbonyl)-glycine ethyl esters 6a–c by
activation of the -carboline-3-carboxylic acids 5a–c with N,N'-dicyclohexylcarbodiimide (DCC) and
dimethylaminopyridine (DMAP), in pyridine, followed by treatment with glycine ethyl ester
hydrochloride [32]. To prepare the -carboline-3-carbonyl-amino acids 7a–c, the corresponding esters
6a–c were submitted to hydrolysis with sodium carbonate in refluxing methanol/water. Erlenmeyer-
Plöchl reaction of 7a and 7c with 3-nitrobenzaldehyde or benzaldehyde, afforded the corresponding
-carboline-3-oxazolones 8–11. The reaction of the derivative 7b, bearing the 4-hydroxyphenyl
substituent at C-1, led to the oxazolone 9 with the hydroxyl group acetylated.
To evaluate the effects of electron-donating groups at the R2 position of the benzylidene-4H-oxazol-
5-one moiety on activity, compounds 7a and 7c were subjected to Erlenmeyer-Plöchl reaction with
4-N,N-dimethylaminobenzaldehyde and 4-methoxybenzaldehyde. However, these reactions failed to
furnish the corresponding -carboline-3-oxazolones, probably due to the relative low electrophilicity
of the aldehydes employed.
Scheme 1. The synthetic route to compounds 8–11.
COOCH3
COOCH3
COOR
a, b
c
N
NH
NH2
N
N
N
H
H
H
1: R=H
2: R=CH3
R1
R1
4 a-c
3 a-c
O
O
COOH
f
e
N
d
H
N
O
N
N
H
N
H
R2
R1
R1
6 a-c
5 a-c
1"
O
O
N
4
R1
R2
5
8
4
OH
4b
4a
2
6
7
5
N
O
3
g
H
8
9
H
m-NO2
N
N
O
9a
8a
N
N
1
1'
H
H
p-OCOCH3 m-NO2
10 p-OCH3
11 p-OCH3
m-NO2
R1
R1
H
7 a-c
8-11
R1 =
a) H
b) 4-OH
c) 4-OCH3
Reagents and conditions: (a) CH3OH, H2SO4, reflux, 48 h, 95%; (b) Aldehyde (R1CHO), TFA,
CH2Cl2, reflux, 48 h, 75–95%; (c) Sulfur, xylene, reflux, 48 h, 75–85%; (d) NaOH, H2O:CH3OH
2:1, reflux, 24 h and HCl 5 M, 0 °C, 3 h, 85–90%; (e) HCl.NH2CH2COOCH2CH3, DMAP, DCC,
pyridine, CH2Cl2, RT, 48 h, 45–56%; (f) Na2CO3, H2O:CH3OH 2:1, reflux, 24 h and HCl 5 M,
0 °C, 3h, 68–77%; (g) Aldehyde (R2CHO), AcONa, (CH3CO)2O, 80 °C, 24 h, 30–42%.

Molecules 2012, 17
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1
All novel compounds were characterized by their spectroscopic data (IR, HR-ESIMS, H and
13C-NMR), which are described in the Experimental section. The (4-benzylidene)-4H-oxazol-5-one
1
group at C-3 of 8–11 was evidenced by the presence of a singlet at H 7.54–7.56 in the H-NMR
spectra, corresponding to the benzylidene hydrogen, which showed a correlation with the carbon
signals at C 126.4–131.5, in the HSQC spectra. The signals at C 164.0–164.5 and 168.0–166.9, in the
13C-NMR spectra, were assigned to the C-2 and C-5 carbons, respectively, of the 4H-oxazol-5-one ring.
The IR spectra showed absorption bands characteristic for C=O stretching in the 1,780–1,816 cm−1 region.
The structures of 8–11 were also confirmed by their HR-ESI and EI mass spectra. The compounds
showed the presence of the molecular ions [M] consistent with the expected structures, and a base
peak at m/z [Msubstituted-benzylidene oxazolone] corresponding to the cleavage between C-3 of
-carboline and C-2 of the 4H-oxazol-5-one ring.
2.2. Cytotoxic Activity
The IC50 values obtained for the synthesized compounds are shown in Table 1. Analysis of the IC50
values (Figure 1, Table 1) showed that compound 8 was active only against the melanoma (UACC-62)
cell line, with IC50 of 7.52 μM. The same result was observed for the derivative 9, where the phenyl
group at C-1 was changed for a 4-acetoxyphenyl group.
On the other hand, the substitution of the phenyl group at C-1, in compound 8, for a 4-methoxy-
phenyl group resulted in the more active compound 10, showing that a good electron-donating
substituent at the phenyl ring is important for the cytotoxicity. A potent activity against glioma (U251)
and ovarian (OVCAR-03) cell lines was observed for compound 10, which presented IC50 values of
0.35 and 2.18 M, respectively. Keeping the 4-methoxyphenyl group at C-1 and in order to evaluate
the effect on activity of other groups at the oxazolone moiety, the 3-nitrophenyl group was substituted
for a phenyl group, resulting in compound 11, which displayed the better activity in comparison to all
other synthesized compounds. Derivative 11 showed IC50 values ≤ 10.00 μM for four of the cells lines
tested (Table 1). A potent activity was observed mainly against glioma (U251), prostate (PC-3) and
ovarian (OVCAR-03) cell lines with IC50 values of 0.48, 1.50 and 1.07 μM, respectively.
Table 1. IC50 values (in μM) for compounds 8–11.
Glioma Melanoma Breast Prostate
Ovarian
Colon VERO
U251
0.03
62.42
80.20
0.35
UACC-62 MCF7
PC-3
0.05
>100
>100
5.28
1.50
OVCAR-03 HT-29
Doxorubicin a
0.04
7.52
0.06
>100
>100
47.63
23.44
0.42
63.19
>100
2.18
0.18
>100
>100
>100
67.88
0.50
>100
>100
>100
63.17
8
9
10
11
8.76
15.93
10.00
0.48
1.07
a Doxorubicin was the positive control.
In addition to the effective growth inhibition (IC50 values), compound 11 showed also cytostatic
activity, with IC100 values of 15.50, 56.83, 64.17 and 112.42 μM for glioma (U251), ovarian (OVCAR-03),
prostate (PC-3) and melanoma (UACC-62) human cell lines, respectively (Figure 1).

Molecules 2012, 17
Figure 1. Concentration (μM) versus cell growth (%) for compound 11.
6104
100
50
0
U251
UACC-62
MCF7
PC-3
OVCAR-3
HT29
Vero
-50
-100
10-3
10-2
10-1
100
101
102
103
Concentration (M)
The comparison of IC50 data of 11 (Table 1) with those of the most active β-carboline derivatives
reported in our previous work [17–19] demonstrated, in general, a lower cytotoxic activity for this
compound. However, specifically for the prostate (PC-3) and ovarian (OVCAR-03) cancer cell lines,
the cytotoxic activity of 11 was similar to that of some active reported compounds, such as 1-(4-N,N-
dimethylaminophenyl)-3-(5-thioxo-1,2,4-triazol-3-yl)--carboline (prostate: IC50 = 1.37 μM; ovarian:
IC50 = 1.09 μM) [17] and N'-(2-chlorobenzylidene)-1-(4-hydroxyphenyl)-β-carboline-3-carbohydrazide
(prostate: IC50 = 1.83 μM, ovarian: 1.65 μM) [19].
The IC50 data shown in Table 1 were also compared with those of previously reported β-carboline
derivatives bearing the 4-methoxyphenyl group at position-1 and the 2-thioxo-1,3,4-oxadiazole,
2-methylthio-1,3,4-oxadiazole and 5-thioxo-1,2,4-triazole groups [17], and (substituted benzylidene)-
carbohydrazide groups [19], at position-3. This comparison pointed of that, except for 1-(4-
methoxyphenyl)-3-(2-methylthio-1,3,4-oxadiazol-5-yl) β-carboline (ovarian: IC50 = 2.13 μM) [17], the
synthesized oxazoles 10 and 11 showed higher cytotoxic activity, particularly for prostate (PC-3) and
ovarian (OVCAR-03) cancer cell lines, than all other 1-(4-methoxyphenyl)-3-substituted-β-carboline
derivatives previously tested [17,19]. These data demonstrate the importance of the (substituted
benzylidene)-4H-oxazol-5-one moiety on the cytotoxic activity of compounds 10 and 11.
2.3. Antimicrobial Activity
Compounds 8–11 were assayed against the bacteria Bacillus subtilis ATCC 2576, Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 13388, Staphylococus aureus ATCC 6538, and against
the fungi Candida albicans ATCC 10231, Candida parapsilosis ATCC-22019 and Candida tropicalis
ATCC-28707. Contrarily to the expectation, the assay results showed no activity for compounds 8–11
(IC50 > 100 µM), indicating that the (substituted-benzylidene)-4H-oxazol-5-one group at C-3 did not
contribute to the antimicrobial activity.

Molecules 2012, 17
2.4. In Silico Study
6105
An in silico computational study of the synthesized -carboline-3-(4-benzylidene)-4H-oxazol-5-
ones 8–11 was performed by determining the Lipinski’s parameters, topological polar surface area
(TPSA) and percentage of absorption (% ABS) [33–35]. Calculations were performed using the
Molinspiration Online Property Calculation Toolkit (www.molinspiration.com) [34] and OSIRIS
Property Explorer (www.organic-chemistry.org/prog/peo) [35]. The percentage of absorption was
estimated using the equation: % ABS = 109 − 0.345 × TPSA [33]. These data are shown in Table 2.
In vivo absorption of the new synthesized derivatives was tentatively assessed by means of
theoretical calculations following Lipinski’s rule of five, which establishes that the absorption or
permeation of an orally administered compound is more likely to be good if the drug satisfies the
following criteria: (a) hydrogen bond donors ≤ 5 (OH and NH groups); (b) hydrogen bond acceptors
≤10 (N and O atoms); (c) molecular weight < 500; (d) calculated logP < 5 [33–35]. Compounds
violating more than one of these rules may present bioavailability problems.
Table 2. Lipinsk’s parameters and % ABS, TPSA, LogS for compounds 8–11.
Lipinsk’s parameters
TPSA a
(A2)
nHBA
(NO)
nHBD
(OHNH)
n
Comp.
%ABS
logP a
MW
logS a
violations
8
9
10
11
68.42
59.35
65.24
81.05
117.61
143.91
126.85
81.02
8
10
9
1
1
1
1
5.45
5.00
5.50
5.57
460.45
518.48
490.47
445.48
1
1
1
1
−7.41
−7.71
−7.43
−6.97
6
a www.molinspiration.com; b www.organic-chemistry.org/prog/peo; %ABS = 109 − 0.345 × TPSA;
Number hydrogen bond acceptor (NO)= nHBA ≤ 10; Number hydrogen bond donors (OHNH) =
nHBD ≤ 5; MW ≤ 500; Octanol-water partition coefficient = LogP < 5; Solubility = LogS > −4.
Our results (Table 2) revealed that the -carboline-3-(4-benzylidene)-4H-oxazol-5-ones 8–11
violated only one of the Lipinski’s rules. Compound 9 has a molecular weight larger than 500 g/moL
and the derivatives 8, 10 and 11 showed octanol-water partition coefficients (LogP) larger than 5.0.
All derivatives have number of hydrogen bond acceptors (n-ON = 6–10) and donors (n-OHNH = 1)
in agreement with Lipinski’s rule. The calculated percent absorption (% ABS) of all derivatives ranged
between 59.35 and 81.05%, indicating that these compounds should have good cellular plasmatic
membrane permeability.
3. Experimental
3.1. Chemistry
Melting points were determined in a Micro-Química MQAPF-301 apparatus and are uncorrected.
1H- and 13C-NMR spectra were recorded in a Varian model Mercury plus 300 spectrometer (Palo Alto,
CA, USA) at 300 MHz and 75.5 MHz, respectively, with DMSO-d6 as solvent and TMS as the internal
standard. EI-MS spectra were recorded in a Thermoelectron Corporation Focus-DSQ II spectrometer
(Austin, TX, USA). HR-ESI mass spectra were recorded on a Q-TOF (Micromass) spectrometer in

Molecules 2012, 17
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positive ion mode (Manchester, UK). IR spectra were recorded on a BOMEM model MB-100
spectrometer (Quebec, CA, USA). For TLC, Merck precoated plates (silica gel 60 G254) were used.
Silica gel 60 Merck (230–400 mesh) was used in the column chromatography purification of some
compounds. All reagents were purchased from commercial suppliers.
3.2. General Method for Synthesis of N-(1-benzylidene-β-carboline-3-carbonyl)-glycine Ethyl Esters
6a–c
To a solution of -carboline-3-carboxylic acids 5a–c (0.7 mmol) in pyridine (5 mL) at 0 °C was
added glycine ethyl ester hydrochloride (0.7 mmol), DMAP (0.07 mmol) in CH2Cl2 (5 mL). The
mixture was stirred for 5 min and a solution of DCC (0.7 mmol) in CH2Cl2 (5 mL) was added,
followed by stirring for 2 h, at 0 °C, and for 24 h at room temperature. This step was repeated with a
new amount of DCC (0.7 mmol) in CH2Cl2 (5 mL), followed by evaporation of the solvent, addition of
CH2Cl2 and filtration of the DCU precipitate formed. The solvent was removed from the filtrate and
the residual DCU separated by precipitation with acetone and filtration. The solvent was removed and
the residue obtained purified on a chromatographic column (silica gel, CHCl3/CH3OH 5%) to give the
pure -carbolines 6a–c in yields ranging from 45–56%.
N-(1-Phenyl-β-carboline-3-carbonyl)-glycine ethyl ester (6a). Yield: 55%, m.p. 194–195 °C. IR (KBr)
1
max cm−1: 1737 (C=O); 1657 (C=O), 1622 (C=N); 1591–1494 (C=C). H-NMR (CDCl3):  8.80 (s,
H-4), 8.18 (d, J = 7.8 Hz, H-5), 7.35 (td, J = 7.3 Hz and 1.5 Hz, H-6), 7.49–7.63 (m, H-7), 7.49–7.63
(m, H-8), 8.89 (s, NH-9), 7.99 (dd, J = 6.9 Hz and 1.5 Hz, H-2'), 7.51–7.64 (m, H-3'/5'), 7.49–7.64 (m,
H-4'), 7.99 (dd, J = 6.9 Hz and 1.5 Hz, H-6'), 8.65 (t, J = 5.7 Hz, NH-2''), 4.31 (d, J = 5.7 Hz,
NHCH2), 4.27 (q, J = 7.2 Hz, OCH2CH3), 1.33 (t, J = 7.2 Hz, CH3CH2O). 13C-NMR (CDCl3):  141.2
(C-1), 139.8 (C-3), 113.7 (C-4), 130.6 (C-4a), 122.4 (C-4b), 122.3 (C-5), 121.1 (C-6), 129.1 (C-7), 112.1
(C-8), 140.9 (C-8a), 137.9 (C-9a), 135.0 (C-1'), 128.4 (C-2'), 129.3 (C-3'/5'), 129.3 (C-4'), 128.4 (C-6'),
166.0 (C=O), 41.7 (NHCH2), 170.5 (C=O), 61.7 (OCH2CH3), 14.4 (CH3CH2O). EIMS, 70 eV, m/z
(rel. int. %): 373 (40, M+·), 243 (100), 271 (40).
N-[1-(4-Hydroxyphenyl)-β-carboline-3-carbonyl]-glycine ethyl ester (6b). Yield: 45%, m.p. 168–170 °C.
IR (KBr) max cm−1: 1729 (C=O), 1626 (C=N), 1538–1464 (C=C). 1H-NMR (CDCl3):  8.72 (s, H-4),
8.27 (d, J = 7.8 Hz, H-5), 7.28 (t, J = 7.8 Hz, H-6), 7.54 (t, J = 7.8 Hz, H-7), 7.68 (d, J = 7.8 Hz, H-8),
11.69 (s, NH-9), 8.01 (d, J = 8.7 Hz, H-2'/6'), 7.04 (d, J = 8.7 Hz, H-3'/5'), 9.71 (brs, OH), 8.97 (t,
J = 6.0 Hz, NH-2''), 4.17 (d, J = 6.0 Hz, NHCH2), 4.18 (q, J = 7.2 Hz, OCH2CH3), 1.27 (t, J = 7.2 Hz,
13
CH3CH2O). C-NMR (CDCl3):  141.3 (C-1), 138.6 (C-3), 111.9 (C-4), 127.9 (C-4a), 121.1 (C-4b),
121.3 (C-5), 119.7 (C-6), 128.2 (C-7), 112.4 (C-8), 141.0 (C-8a), 133.9 (C-9a), 129.3 (C-1'), 129.8
(C-2'/6'), 115.3 (C-3'/5'), 158.2 (C-4'), 165.2 (C=O), 40.9 (NHCH2), 169.8 (C=O), 60.3 (OCH2CH3),
13.9 (CH3CH2O). EIMS, 70 eV, m/z (rel. int. %): 389 (25, M+), 259 (100), 343 (10).
N-[1-(4-Methoxyphenyl-β-carboline-3-carbonyl]-glycine ethyl ester (6c). Yield: 56%, m.p. 184–186 °C.
1
IR (KBr) max cm−1: 1742 (C=O); 1656 (C=O), 1609 (C=N), 1561–1464 (C=C). H-NMR (CDCl3):
 8.79 (s, H-4), 8.19 (d, J = 7.8 Hz, H-5), 7.35 (td, J = 7.0 Hz; J = 1.5 Hz, H-6), 7.53–7.61 (m, H-7),
7.53–7.61 (m, H-8), 8.81 (s, NH-9), 7.95 (dd, J = 8.7 Hz and 1.8 Hz, H-2'/6'), 7.13 (dd, J = 8.7 Hz and

Molecules 2012, 17
6107
1.8 Hz, H-3'/5'), 3.92 (s, OCH3), 8.67 (t, J = 5.5 Hz, NH-2''), 4.32 (d, J = 5.5 Hz, NHCH2), 4.27 (q,
J = 7.0 Hz, OCH2CH3), 1.33 (t, J = 7.0 Hz, CH3CH2O). 13C-NMR (CDCl3):  141.2 (C-1), 139.8
(C-3), 113.3 (C-4), 122.5 (C-4b), 122.3 (C-5), 121.1 (C-6), 128.9 (C-7), 112.0 (C-8), 140.8 (C-8a),
134.9 (C-9a), 130.5 (C-1'), 129.7 (C-2'/6'), 114.8 (C-3'/5'), 160.6 (C-4'), 55.6 (OCH3), 166.1 (C=O),
170.5 (C=O), 41.7 (NHCH2), 61.7 (OCH2CH3), 14.4 (CH3CH2O). EIMS, 70 eV, m/z (rel. int. %):
403 (60, M+), 301 (45), 273 (100), 258 (30).
3.3. General Method for the Synthesis of 1 N-(1-substituted-β-carboline-3-carbonyl)-glycine 7a–c
A suspension of N-(1-substituted-β-carboline-3-carbonyl)-glycine ethyl esters 6a–c (0.5 mmol) and
Na2CO3 (1.5 mmol) in H2O:MeOH 2:1 (5 mL) was stirred for 24 h, at 80 °C. The mixture was cooled
at 0 °C, and after stirring for 2 h, the solution was neutralized with a solution of 5 M HCl solution. The
product was collected by filtration, dried and crystallized from ethanol, furnishing the compounds 7a–c
in 68–77% yield.
N-(1-Phenyl-β-carboline-3-carbonyl)-glycine (7a). Yield: 77%, m.p. 236–238 °C. IR (KBr) max cm−1:
1731 (C=O); 1636 (C=N), 1593–1464 (C=C). 1H-NMR (DMSO-d6):  8.83 (s, H-4), 8.43 (d, J = 7.8 Hz,
H-5), 7.32 (t, J = 7.8 Hz; H-6), 7.55–7.71 (m, H-7), 7.55–7.71 (m, H-8), 11.87 (s, NH-9), 8.14 (dd,
J = 7.8 Hz and 1.2 Hz, H-2'/6'), 7.55–7.71 (m, H-3'/5'), 7.55–7.71 (m, H-4'), 8.87 (t, J = 5.1 Hz, NH-2''),
13
3.89 (d, J = 5.1 Hz, NHCH2). C-NMR (DMSO-d6):  141.6 (C-1), 139.5 (C-3), 112.9 (C-4), 129.9
(C-4a), 122.0 (C-4b), 121.2 (C-5), 120.2 (C-6), 128.9 (C-7), 112.7 (C-8), 140.6 (C-8a), 137.5 (C-9a),
134.2 (C-1'), 128.6 (C-2'/6'), 128.8 (C-3'/5'), 128.8 (C-4'), 164.7 (C=O), 41.9 (NHCH2), 171.4 (C=O).
EIMS, 70 eV, m/z (rel. int. %): 345 (20, M+), 301 (20), 243 (95), 60 (100).
N-[1-(4-Hydroxyphenyl)-β-carboline-3-carbonyl]-glycine (7b). Yield: 74%, mp decomp. IR (KBr)
1
max cm−1: 1729 (C=O), 1609 (C=N), 1540–1498 (C=C). H-NMR (DMSO-d6):  8.76 (s, H-4), 8.40
(d, J = 7.6 Hz, H-5), 7.31 (t, J = 7.6 Hz, H-6), 7.59 (t, J = 7.6 Hz, H-7), 7.70 (d, J = 7.6 Hz, H-8),
12.64 (s, NH-9), 8.04 (d, J = 8.7 Hz, H-2'/6'), 7.04 (d, J = 8.7 Hz, H-3'/5'), 9.86 (brs, OH), 8.95 (t,
J = 6.0 Hz, NH-2''), 4.08 (d, J = 6.0 Hz, NHCH2), 11.76 (s, OH). 13C-NMR (DMSO-d6):  141.5 (C0-1),
139.2 (C-3), 112.3 (C-4), 128.4 (C-4a), 121.3 (C-4b), 121.9 (C-5), 120.1 (C-6), 128.3 (C-7), 112.7
(C-8), 141.0 (C-8a), 133.9 (C-9a), 129.6 (C-1’), 130.1 (C-2'/6'), 115.6 (C-3'/5'), 158.4 (C-4'), 165.2
(C=O), 41.2 (NHCH2), 171.6 (C=O). C20H15N3O4 EIMS, 70 eV, m/z (rel. int. %): 361 (20, M+), 260
(100), 229 (20), 60 (10).
N-[1-(4-Methoxyphenyl-β-carboline-3-carbonyl]-glycine (7c). Yield: 68%, m.p. 247–249 °C. IR (KBr)
1
max cm−1: 1741 (C=O), 1638 (C=N), 1545–1464 (C=C). H-NMR (DMSO-d6):  8.76 (s, H-4), 8.28
(d, J = 7.5 Hz, H-5), 7.30 (t, J = 7.5 Hz, H-6), 7.56 (t, J = 7.5 Hz, H-7), 7.69 (d, J = 7.5 Hz, H-8),
11.74 (s, NH-9), 8.11 (d, J = 8.7 Hz, H-2'/6'), 7.91 (d, J = 8.7 Hz, H-3'/5'), 3.91 (s, OCH3), 8.90 (t,
J = 5.7 Hz, NH-2''), 4.13 (d, J = 5.7 Hz, NHCH2). 13C-NMR (DMSO-d6):  141.5 (C-1), 138.6 (C-3),
112.3 (C-4), 121.1 (C-4b), 121.4 (C-5), 119.9 (C-6), 128.1 (C-7), 112.5 (C-8), 140.4 (C-8a), 134.0
(C-9a), 129.5 (C-1'), 129.9 (C-2'/6'), 113.8 (C-3'/5'), 159.8 (C-4'), 55.0 (OCH3), 164.8 (C=O), 40.9
(NHCH2), 171.2 (C=O). EIMS, 70 eV, m/z (rel. int. %): 375 (25, M+·), 274 (100), 229 (25), 60 (40).

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3.4. General Method for Synthesis of 1-Substituted β-carboline-3-(4-benzylidene)-4H-oxazol-5-ones 8–11
To a solution of derivatives 7a–c (0.3 mmol), in acetic anhydride (5 mL), was added 4-nitro-
benzaldehyde or benzaldehyde (0.75 mmol), followed by addition of sodium acetate (1.5 mmol). The
solution was stirred for 24 h at 80 °C and then, for 1h at 0 °C. The solid formed was filtered under
vacuum and washed with cold acetone to afford the compounds 8–11.
2-(1-Phenyl-9H-β-carbolin-3-yl)-4-(3-nitrobenzylidene)-4H-oxazol-5-one (8). Yield: 42%, m.p.
271–273 °C. IR (KBr) max cm−1: 3395 (N–H), 1810 (C=O); 1625 (C=N), 1547–1497 (C=C). 1H-NMR
(DMSO-d6):  9.17 (s, H-4), 8.49 (d, J = 7.8 Hz, H-5), 7.41 (t, J = 7.8 Hz, H-6), 7.59–7.66 (m, H-7),
7.75 (d, J = 7.8 Hz, H-8), 12.17 (s, NH-9), 8.12 (d, J = 8.0 Hz, H-2'/6'), 7.68 (d, J = 8.0 Hz, H-3'/5'),
7.59–7.66 (m, H-4'), 7.54 (s, C=CH), 9.30 (s, H-2''), 8.34 (d, J = 7.5 Hz, H-4''), 7.85 (t, J = 7.5 Hz,
H-5''), 8.80 (d, J = 7.5 Hz, H-6''). 13C-NMR (DMSO-d6):  143.1 (C-1), 137.2 (C-3), 116.9 (C-4), 129.1
(C-4a), 120.9 (C-4b), 122 (C-5), 120.8 (C-6), 129.3 (C-7), 113.0 (C-8), 141.5 (C-8a), 134.8 (C-9a), 132.3
(C-1'), 128.6 (C-2'/6'), 128.9 (C-3'/5'), 129.0 (C-4'), 167.0 (C=O), 164.4 (C=N), 135.7 (C=CH), 126.8
(C=CH), 135.1 (C-1''), 126.2 (C-2''), 148.2 (C-3''), 124.8 (C-4''), 130.4 (C-5''), 137.8 (C-6''). EIMS, 70 eV,
m/z (rel. int. %): 460 (30, M+), 243 (100), 271 (30), 432 (5). HR-ESIMS: calcd for C27H17N4O4:
461.1250 [M+H]+; found: 461.1296.
2-[1-(4-Acetoxyphenyl)-9H--carbolin-3-yl]-4-(3-nitrobenzylidene)-4H-oxazol-5-one (9). Yield: 30%,
1
m.p. 255–257 °C. IR (KBr) max cm−1: 1816 cm−1 (C=O), 1655 (C=N), 1553–1528 (C=C). H-NMR
(DMSO-d6):  9.20 (s, H-4), 8.51 (d, J = 8.1 Hz, H-5), 7.41 (t, J = 8.1 Hz, H-6), 7.67 (d, J = 8.1 Hz,
H-7), 7.75 (d, J = 8.1 Hz, H-8), 12.26 (s, NH-9), 8.20 (d, J = 8.4 Hz, H-2'/6'), 7.45 (d, J = 8.4 Hz,
H-3'/5'), 2.37 (s, CH3), 7.56 (s, C=CH), 9.32 (s, H-2''), 8.34 (dd, J = 7.8 Hz and 2.1 Hz, H-4'), 7.87 (t,
J = 7.8 Hz, H-5''), 8.83 (d, J = 7.8 Hz, H-6''). 13C-NMR (DMSO-d6):  142.3 (C-1), 141.5 (C-3), 117.0
(C-4), 129.1 (C-4a), 120.9 (C-4b), 122.1 (C-5), 121.0 (C-6), 129.4 (C-7), 113.0 (C-8), 144.1 (C-8a),
134.7 (C-9a), 132.3 (C-1'), 129.8 (C-2'/6'), 122.3 (C-3'/5'), 151.3 (C-4'), 20.9 (CH3), 169.2 (OCOCH3),
166.9 (C=O), 164.4 (C=N), 135.7 (C=CH), 126.4 (C=CH), 135.1 (C-1''), 126.2 (C-2''), 148.2 (C-3''),
124.8 (C-4''), 130.4 (C-5''), 137.8 (C-6''). HR-ESIMS: calcd for C29H19N4O6 [M+H]+: 519.1305; found:
519.1400.
2-[1-(4-Methoxyphenyl)-9H--carbolin-3-yl]-4-(3-nitrobenzylidene)-4H-oxazol-5-one (10). Yield:
32%, m.p. 201–203 °C. IR (KBr) max cm−1: 3330 (N–H), 1781 cm−1 (C=O), 1609 (C=N), 1547–1494
(C=C). 1H-NMR (DMSO-d6):  9.15 (s, H-4), 8.48 (d, J = 7.6 Hz, H-5), 7.40 (t, J = 7.6 Hz, H-6), 7.65
(t, J = 7.6 Hz, H-7), 7.71 (d, J = 7.6 Hz, H-8), 12.14 (s, NH-9), 8.12 (d, J = 8.7 Hz, H-2'/6'), 7.24 (d,
J = 8.7 Hz, H-3'/5'), 3.91 (s, OCH3), 7.55 (s, C=CH), 9.32 (s, H-2''), 8.35 (d, J = 8.0 Hz, H-4''), 7.87 (t,
J = 8.0 Hz, H-5”), 8.51 (d, J = 8.0 Hz, H-6''). 13C-NMR (DMSO-d6):  143.1 (C-1), 138.0 (C-3), 116.6
(C-4), 129.2 (C-4a), 120.0 (C-4b), 122.0 (C-5), 121.0 (C-6), 128.9 (C-7), 109.9 (C-8), 142.0 (C-8a),
134.7 (C-9a), 129.6 (C-1'), 130.1 (C-2'/6'), 114.3(C-3'/5'), 160.2 (C-4'), 55.4 (OCH3), 167.1 (C=O),
164.5 (C=N), 135.8 (C=CH), 126.7 (C=CH), 135.2(C-1''), 126.2 (C-2''), 148.2 (C-3''), 124.8 (C-4''),
131.0 (C-5''), 137.8 (C-6''). EIMS, 70 eV, m/z (rel. int. %): 490 (60, M+·), 301 (30), 273 (100), 258
(35). HR-ESIMS: calcd for C28H19N4O5 [M+H]+: 491.1355; found: 491.1309.

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2-[1-(4-Methoxyphenyl)-9H--carbolin-3-yl]-4-benzylidene-4H-oxazol-5-one (11). Yield: 40%, m.p.
245–247 °C. IR (KBr) max cm−1: 3330 (N–H), 1781 cm−1 (C=O), 1648 (C=N), 1573–1494 (C=C).
1H-NMR (DMSO-d6):  9.18 (s, H-4), 8.55 (d, J = 7.5 Hz, H-5), 7.41 (m, H-6), 7.64 (t, J = 7.5 Hz,
H-7), 7.74 (d, J = 7.5 Hz, H-8), 12.00 (s, NH-9), 8.06 (d, J = 8.4 Hz, H- 2'/6'), 7.24 (d, J = 8.4 Hz,
H-3'/5'), 3.90 (s, OCH3), 7.56 (s, C=CH), 8.40 (d, J = 7.2 Hz, H-2''/6''), 7.58 (d, J = 7.2 Hz, H-3''/H-5''),
7.37 (m, H-4''). 13C-NMR (DMSO-d6):  143.4 (C-1), 138.2 (C-3), 116.7 (C-4), 129.6 (C-4a) 121.5 (C-4b),
122.7 (C-5), 121.1 (C-6), 129.4 (C-7), 113.4 (C-8), 141.9 (C-8a), 135 (C-9a), 133.1 (C-1'), 130.5
(C-2'/6'), 114.7 (C-3'/5'), 160.6 (C0-4'), 55.5 (OCH3), 168.0 (C=O), 164.0 (C=N), 134.1 (C=CH), 131.5
(C=CH), 134 (C-1''), 132.7 (C-2''/6), 129.4 (C-3''/5''), 130.0 (C-4''). EIMS, 70 eV, m/z (rel. int. %): 445
(35, M+), 301 (30), 273 (100), 258 (35), 242 (15). HR-ESIMS: calcd for C28H20N3O3 [M+H]+:
446.1505; found: 446.1529.
3.2. Biological Assays
3.2.1. Cytotoxic Assay
The synthesized compounds were evaluated in vitro against six human cancer cell lines consisting
of glioma (U251), melanoma (UACC-62), breast (MCF-7), prostate (PC-3), ovarian (OVCAR-03) and
colon (HT-29). Cell lines were obtained from National Cancer Institute (Frederick, MD, USA).
Normal cell line (VERO, renal, green monkey), from the Rio de Janeiro Cell Bank, was also used.
Stock cultures were grown in medium containing 5 mL RPMI 1640 (GIBCO BRL) supplemented
with 5% fetal bovine serum (FBS, GIBCO), at 37 °C with 5% CO2. Penicillin:streptomycin
(1000 μg/L:1000 U/L, 1 mL/L) was added to the experimental cultures. Cells in 96-well plates
(100 μL cells well−1) were exposed to compounds 8–11 in DMSO (concentrations of 0.25, 2.5, 25, and
250 μg·mL−1) at 37 °C, 5% of CO2 in air for 48 h. Final DMSO concentration did not affect the cell
viability. Afterwards cells were fixed with 50% trichloroacetic acid and cell proliferation determined
by spectrophotometric quantification (540 nm) of cellular protein content by using sulforhodamine B
assay and doxorubicin as the positive control [36]. Three measurements were obtained: at time zero
(To, at the beginning of incubation) and 48 h post-incubation for compound-free (C) and tested (T) cells.
Cell proliferation was determined according to the equation 100 × [(T − T0)/C − T0], for T0 < T ≤ C,
and 100 × [(T − T0)/T0], for T ≤ T0 and a concentration-response curve for each cell line was plotted
using software ORIGIN 8.0® (OriginLab Corporation). Using the concentration–response curve for
each cell line, the IC50 values (concentration that produces a 50% reduction in cellular growth when
compared to untreated control cells) and IC100 values (concentration that promotes total growth
inhibition) were determined through non-linear regression analysis using software ORIGIN 8.0®
(OriginLab Corporation) [37]. Compounds with IC50 values < 100 μM were considered actives.
3.2.2. Antimicrobial Activity Assay
Antibacterial and antifungal assays for compounds 8–11 were carried out according to previously
reported experimental protocols [38,39]. The bacterial strains were grown overnight at 36 °C in
Nutrient Agar (Merck, Darmstadt, Germany), and the strains were grown in Saboraud Dextrose Agar.
Inoculum for the assays was prepared by diluting scraped cell mass in 0.85% NaCl solution, adjusted

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to McFarland scale 0.5 and confirmed by spectrophotometrical reading at 580 nm. Cell suspensions
were finally diluted to 104 UFC·mL1 for use in the activity assays. Minimal Inhibitory Concentration
(MIC) tests were carried out using Müller-Hinton broth (bacteria) or RPMI-1640 (yeasts) on a tissue
culture test plate (96 wells). Each compound was tested in duplicate. The stock solutions of
compounds firstly in DMSO and subsequently in Tween 80 water solution (0.1%) were diluted and
transferred into the first well, and serial dilutions were performed so that concentrations in the range of
250–1.6 μg·mL1 were obtained. Chloramphenicol and nystatin (Merck) were used as the reference
antibiotic control. The inoculum was added to all wells and the plates were incubated at 36 °C for 24 h.
Antibacterial activity was detected by adding 0.5% aqueous solution of ntriphenyltetrazolium chloride
(TTC, 20 μL, Merck). MIC was defined as the lowest concentration of the compounds that inhibited
visible growth, as indicated by TTC staining (dead cells are not stained by TTC). For antifungal
activity evaluation, after the incubation period changes in the RPMI-1640 medium color were verified
from pink (original color) to yellow. The change indicates an acidification from medium by the
microorganisms’ growth.
4. Conclusions
In conclusion, four 1-substituted -carboline-3-(4-benzylidene)-4H-oxazol-5-ones 8–11 were
prepared and assayed for their antitumor and antimicrobial activities. The compound 2-[1-(4-
methoxyphenyl)-9H-β-carbolin-3-yl]-4-(benzylidene)-4H-oxazol-5-one (11) was the most active
derivative, exhibiting potent cytotoxic activity against glioma (U251), prostate (PC-3) and ovarian
(OVCAR-03) cancer cell lines with IC50 values of 0.48, 1.50 and 1.07 μM, respectively. In silico
studies indicate that compounds of this class are potential new anticancer drug candidates.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/3/6099/s1.
Acknowledgments
This work was supported by Fundação Araucária (Brazil, PR), Fundação de Amparo a Pesquisa de
São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
Brazil). We thank Fundação Araucária, CAPES and CNPq for fellowships (FCS, MAF, JEC, MCTD,
MFR, MHS).
Conflict of Interest
The authors declare no conflict of interest.
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