New 3-tetrazolyl-β-carbolines and β-carboline-3-carboxylates with anti-cancer activity

Article abstract of DOI:10.1016/j.ejmech.2019.05.085

The synthesis and in vitro anticancer activity of novel β-carbolines is reported. New tryptamines have been prepared via hetero-Diels-Alder reaction of nitrosoalkenes with indoles and used to prepare functionalized β-carbolines by the Pictet-Spengler appr

Full text of DOI:10.1016/j.ejmech.2019.05.085

Accepted Manuscript
New 3-tetrazolyl-β-carbolines and β-carboline-3-carboxylates with anti-cancer activity
Manuela Ribeiro Panice, Susana M.M. Lopes, Mariana Cecchetto Figueiredo, Ana
Lucia T. Goes Ruiz, Mary Ann Foglio, Anelise S. Nazari Formagio, Maria Helena
Sarragiotto, Teresa M.V.D. Pinho e Melo
PII:
S0223-5234(19)30503-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.085
EJMECH 11395
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 May 2019
Revised Date: 24 May 2019
Accepted Date: 29 May 2019
Please cite this article as: M.R. Panice, S.M.M. Lopes, M.C. Figueiredo, A.L.T. Goes Ruiz, M.A. Foglio,
A.S. Nazari Formagio, M.H. Sarragiotto, T.M.V.D. Pinho e Melo, New 3-tetrazolyl-β-carbolines and β-
carboline-3-carboxylates with anti-cancer activity, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.05.085.
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ACCEPTED MANUSCRIPT
New 3-Tetrazolyl-β-Carbolines and β-Carboline-3-Carboxylates
with Anti-cancer Activity
Manuela Ribeiro Panice,a,b Susana M. M. Lopes,b Mariana Cecchetto Figueiredo,c Ana
Lucia T. Goes Ruiz,c Mary Ann Foglio,c Anelise S. Nazari Formagio,a Maria Helena
Sarragiotto,a Teresa M. V. D. Pinho e Melob,*
aDepartamento de Química, Universidade Estadual de Maringá, 87020-900, Maringá,
PR, Brazil
bCQC and Department of Chemistry, University of Coimbra, 3004-535 Coimbra,
Portugal
cCentro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA),
Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil
Email: tmelo@ci.uc.pt
Abstract - The synthesis and in vitro anticancer activity of novel β-carbolines is
reported. New tryptamines have been prepared via hetero-Diels-Alder reaction of
nitrosoalkenes with indoles and used to prepare functionalized β-carbolines by the
Pictet-Spengler approach. These included 6-substituted-β-carboline-3-carboxylates and
3-(1H-tetrazol-5-yl)-β-carbolines, whose synthesis is reported for the first time.
Carboline-3-carboxylates derived from L-tryptophan methyl ester were also prepared.
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ACCEPTED MANUSCRIPT
The structural diversity that was achieved allowed the discovery of impressive activities
against a range cancer cell lines with the selectivity depending on the type of
substitution pattern of the β-carboline core. We have identified at least one β-carboline
derivative with GI50 ≤ 1µM for each of the following human tumor cell lines:
glioblastoma (U251), melanona (UACC-61), breast (MCF-7), ovarian expressing
multiple-drug-resistance phenotype 4 (NCI-ADR/RES), renal (786-0), lung (NCI-
H460), ovarian cancer (OVCAR-3), leukemia (K-562) and colon (HT29). These results
demonstrated that the new β-carboline derivatives are very promising anticancer agents.
Keywords: β-Carbolines; Anti-cancer Agents; Tetrazoles; Cycloaddition;
Nitrosoalkenes
Introduction
Tetrahydro-β-carbolines and β-carboline are a large family of indole-based alkaloids
displaying important biological properties.1 Over the years, naturally occurring as well
as synthetic β-carboline derivatives have demonstrated excellent antitumoral,2
antifungical,3 anti-HSV-1,4 anti-HIV,5 anti-Malarial,6 antitrypanosomal (Chagas’
disease)7 and antileismanial8 activities. Among these diverse biological activities,
antitumoral activity of β-carbolines isolated from natural sources stands out (Figure
1).1a, 9
Figure 1. β-Carboline alkaloids with anticancer activity.
Due to their relevance in medicinal chemistry, numerous synthetic methods have been
developed in order to obtained β-carboline derivatives.10 The most common approach
involves the Pictet-Spengler reaction of a tryptamine with an aldehyde followed by
oxidation of the so formed tetrahydro-β-carboline.11 We envisioned that one way to
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widen the β-carbolines’ structural diversity, which would potentially enable the
modulation of their biological activity, would be to further explore this chemistry by
developing novel tryptophan derivatives to be used as synthetic building blocks.
One way to achieve this goal is to explore the chemistry of nitrosoalkenes.12 In fact, we
have previously reported the generation of novel 3-tetrazolyl-nitrosoalkenes and their
reactivity towards electron-rich olefins and heterocycles.13 The study included the
synthesis of tryptophan analogues where the carboxylic group was replaced by the
bioisosteric tetrazolyl group.14 The reaction of in situ generated 3-tetrazolyl-
nitrosoalkenes 2 with indole gave the corresponding open-chain oximes 4, resulting
from the ring-opening and concomitant rearomatization of the indole unit of the initially
formed hetero-Diels-Alder cycloadducts 3. The reduction of the oxime moiety of
functionalized indoles 4 afforded the target tryptophan analogues 5 and 6. The
deprotection of the tetrazolyl moiety of compound 6 was also carried out affording
tetrazolyl-tryptamine 7 (Scheme 1).13 In drug discovery, this type of building blocks is a
particularly interesting synthetic tool to explore carboxylic acid/tetrazole bioisosterism.
H
N
R
R
R
N
N
N
N
N
N
N
- HX
N
N
N
N
N
Na2CO3
Diels-Alder
reaction
Br
N
N
N
H
NOH
O
O
N
2
3
1a R = Bn
1b R = Ph
1,2-oxazine
ring-opening
R
Ph
N
N
Al/Hg
N
N
THF/10% H2O, rt
R = Ph
N
N
N
N
NH2
HN
HN
HO
5
4
AcOH/Zn
THF, reflux
R = Bn
Bn
N
N
HN
NH
N
N
HCO2NH4, Pd/C 10%
MeOH, reflux
N
N
N
HN
NH
HN
Ac
7
6
Ac
Scheme 1. Tryptophan analogues derived from nitrosovinyltetrazoles.13
A similar strategy can be used to prepare tryptophan derivatives functionalized in the
indole moiety by using functionalized indoles as the starting material. In fact, one
3

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example has been reported on the synthesis of a tryptophanyl ester by this route, which
was used to prepare a β-carboline derivative.2j
In this context, we set out to further explore the synthesis of tryptophan analogues based
on the chemistry of nitrosoalkenes and the use of these building blocks in the
preparation of functionalized β-carbolines (Scheme 2). Our focus was on obtaining a
diversity of β-carbolines and carrying out their anticancer activity evaluation in order to
identify structure-activity relationships to enable the finding of potent anticancer agents.
N
N
Bn
N
Bn
Bn
N
N
N
N
N
N
N
N
N
Br
N
NOH
NH2
HN
HN
Ar
N
H
R1
via Hetero-Diels-Alder reaction of
nitrosoalkenes with indoles
via Pictet-Spengler approach
CO2Et
CO2Et
NH2
CO2Et
Br
R1
N
NOH
Ar
N
New building blocks
H
Scheme 2. Synthetic strategy towards new β-carbolines.
Results and Discussion
Chemistry
Initially, the hetero-Diels-Alder reaction of 3-tetrazolyl-nitrosoalkene 2a (R = Bn),
generated in situ by treatment of α-bromooxime 1a with base, with indole was carried
out following our previously reported procedure,13a affording the expected open-chain
oxime 4 in 64% yield (Scheme 3). We have previously demonstrated that the reduction
of oxime 4 could be carried out by using Zn/AcOH at reflux but the corresponding N-
acetyl derivative was obtained (see Scheme 1). In order to obtain directly the
unprotected amine 8, oxime 4 was treated with an excess of metallic zinc/aqueous
formic acid in tetrahydrofuran at room temperature for 24 hours, giving the target
tryptophan analogue in 76% yield.15
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H
N
Bn
Bn
Bn
N
N
N
N
N
N
Zn, HCO2H (aq)
N
N
N
N
N
THF, rt, 24 h
N
Br
Na2CO3
N
NH2
8 76%
CH2Cl2, rt, 48 h
NOH
HN
HN
HO
1a
4 64%
CHO
R
TFA
CH2Cl2, rt, 48 h
Bn
N
N
N
N
N
N
N
N
Bn
HN
N
N
N
N
S, xylene
t-BuOK, DMSO
N
NH
reflux, 24 h
O2, rt, 7 h
R = F
N
H
N
N
H
H
R
F
R
cis/trans
70% (39:61)
9b R = OMe 63% (42:58)
9c R = F 70% (37:63)
11 80%
10a R = H
10b R = OMe 78%
10c R = F
71%
9a R = H
95%
Scheme 3. Synthetic sequence to obtain 3-tetrazolyl-β-carbolines 10a-c and 11.
The next step was the synthesis of tetrahydro-β-carboline derivatives 9 through the
Pictet-Spengler reaction, starting from amine 8 and aromatic aldehydes (benzaldehyde,
p-methoxybenzaldehyde and p-fluorobenzaldehyde) in the presence of TFA (Scheme 3).
Tetrahydro-β-carbolines 9a-c were obtained in good yields (63-70%) and isolated as
mixtures of cis/trans isomers, being the major component the trans isomer (NMR
analysis). Heterocycles 9a-c were subjected to oxidation with sulfur in xylene at reflux
for 24 hours8a affording β-carbolines 10a-c in very good yields (71-95%) with simple
isolation procedures.
The N-debenzylation of the tetrazolyl group of β-carboline 10c was also studied
(Scheme 3). Attempts to carry out the deprotection reaction using ammonium formate
as catalytic hydrogen transfer agent, reaction conditions used to obtain tryptophan
analogue 11 (see Scheme 1), were unsuccessful. Thus, an alternative method reported
in the literature for the deprotection of aromatic heterocycles was explored.16 Treatment
of the N-benzylated-3-tetrazolyl-β-carboline 10c with potassium tert-butoxide/DMSO
and oxygen at room temperature afforded the 3-tetrazolyl-β-carboline 11 in excellent
yield (80%).
There are few reports on the synthesis and biological activity of 6-substituited-β-
carbolines.1 Thus, we decided to prepare 6-substituited-tryptophan derivatives via
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ACCEPTED MANUSCRIPT
hetero-Diels-Alder reaction of ethyl 2-nitrosoacrylate with 5-substituited-indoles which
were used to prepare the target compounds by the Pictet-Spengler approach (Scheme 4).
Ethyl 2-nitrosoacrylate, generated in situ from α-bromooxime 12, reacted with 5-
methylindole, 5-methoxyindole and 5-nitroindole to give the corresponding open-chain
oximes 13a-c in good yields (53-66%). Reduction of the oximes 13a and 13b group
with metallic zinc in acetic acid afforded the corresponding amines 14a and 14b in 89%
and 92% yield, respectively (Scheme 4). It should be noticed that compound 13c, which
contains a nitro group in the indole ring, underwent reduction of the oxime group along
with the reduction of the nitro group, giving the diamine derivative 14c in moderate
yield (49%).
R1
R1
R1
N
NOH
Zn, AcOH
rt, 25-48 h
H
CO2Et
Br
CO2Et
Na2CO3
EtO2C
CH2Cl2, rt
21-47 h
12
N
NH2
14a R1 = OMe 89%
HN
HN
HO
13a R1 = OMe 65%
14b R1 = Me
14c R1 = NH2
92%
52%
13b R1 = Me
13c R1 = NO2
53%
66%
CHO
R2
CH2Cl2, TFA
rt, 48 h
CO2Et
CO2Et
3
R1
R1
NH
N
S, xylene
1
reflux, 48 h
R2
R2
N
H
N
H
16a R1 = OMe, R2 = H
70%
75%
81%
80%
74%
80%
86%
92%
15a R1 = OMe, R2 = H
94%
82%
61%
56%
67%
50%
50%
58%
16b R1 = OMe, R2 = p-OMe
16c R1 = OMe, R2 = o-Cl
16d R1 = OMe, R2 = p-OH
16e R1 = OMe, R2 = m-NO2
16f R1 = OMe, R2 = p-NMe2
16g R1 = Me, R2 = H
15b R1 = OMe, R2 = p-OMe
15c R1 = OMe, R2 = o-Cl
15d R1 = OMe, R2 = p-OH
15e R1 = OMe, R2 = m-NO2
15f R1 = OMe, R2 = p-NMe2
15g R1 = Me, R2 = H
16h R1 = Me, R2 = p-OMe
15h R1 = Me, R2 = p-OMe
Scheme 4. Synthesis of 6-substituted-β-carboline-3-carboxylates 16.
Only 6-substituited-tryptophan derivatives 14a and 14b were used to prepare 6-
substituted-β-carbolines since the condensation of diamine 14c with benzaldehyde led
to a complex mixture. Thus, 6-substituted-tetrahydro-β-carbolines 15a-h were isolated
in high yields (50-94%) through the Pictet-Spengler condensation of amines 14a and
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14b with different aromatic aldehydes in the presence of trifluoroacetic acid. Although
in all cases a mixture of cis:trans isomers was obtained, it was possible to separate them
by column chromatography. The structural assignment of the cis and trans isomers in
each case was achieved based on NMR analysis. This can be illustrated for the case of
15a. In the two-dimensional NOESY spectrum of the cis isomer cross peaks could be
observed between proton H-1 (δ = 3.94 ppm) and proton H-3 (δ = 5.22 ppm). On the
other hand, this connectivity was not observed in the NOESY spectrum of the trans
1
isomer. In the H NMR spectrum the value of the chemical shift observed for protons
H-3 of the cis isomers is slightly lower than the one observed for trans isomers. The
intensity of these signals was used to estimate the cis:trans ratio. It was possible to
conclude that the trans isomer was the major component in most mixtures, with the
exception of mixtures 15c and 15g in which the major component was the cis isomer
(see experimental section).
Finally, the 6-substituted-tetrahydro-β-carbolines 15a-h, used as cis:trans mixtures,
were converted into the corresponding 6-substituted-β-carbolines 16a-h in excellent
yields (70-92%) by oxidation with sulfur in xylene at reflux (Scheme 4).
Anticancer Activity
The synthetic work described above gave access to 1-aryl-3-tetrazolyl-β-carbolines and
6-substituted-1-aryl-β-carboline-3-carboxylates which could not be obtained directly
from L-tryptophan esters. However, having β-carboline-3-carboxylates with only an
additional substituent at C-1, it would be particularly interesting to evaluate the effect
on the anticancer activity of the carboxylic acid/tetrazole bioisosterism or the addition
of a substituent in the indole moiety. This could enable a more complete overview of
the structural-activity relationships. Thus, β-carboline methyl ester derivatives 17a-f,
with different aryl groups at C-1 were also synthetized from L-tryptophan methyl ester
following known general procedures17,2f,4a (Figure 2).
CO2Me
N
17a R = H
17b R = p-OMe
17c R = o-Cl
C
A
17d R = p-OH
17e R = m-NO2
17f R = p-NMe2
R
N
H
D
7

ACCEPTED MANUSCRIPT
Figure 2. β-Carbolines derived from L-tryptophan methyl ester.
The in vitro anticancer activities of the synthetized β-carboline derivatives (17a-f, 10a-
c, 11 and 16a-h) were evaluated against the human tumor cell lines melanona (UACC-
61), breast (MCF-7), ovarian expressing multiple-drug-resistance phenotype 4 (NCI-
ADR/RES), renal (786-0), lung (NCI-H460), prostate (PC-3), ovarian (OVCAR-3),
leukemia (K-562) and colon (HT29). The biological activity of β-carboline derivatives
(10a-c, 11 and 16a-h) against glioblastoma cell lines (U251) was also carried out. The
response GI50 was calculated for each compound and cell line tested, and the results are
summarized in Table 1-3. The GI50 values refer to the drug concentration that inhibits in
50% the cellular growth when compared to untreated control cells. Compounds with
GI50 ≥ 100 µM were considered inactive.
Among the β-carboline-3-carboxylates 17 studied, compound 17b (R = OMe) proved to
be the most active against five cancer cell lines (Table 1). It was particularly active
against leukemia (K-562, GI50 = 0.04 µM), lung carcinoma (NCI-H460, GI50 = 0.38
µM), ovarian resistant cancer (NCI-ADR-RES, GI50 = 1.11 µM) and melanoma
(UACC-61, GI50 = 2.61 µM). β-Carboline 17a (R = H) was not active except against
melanoma cancer cell line UACC-61, showing a GI50 value of 5.45 µM. These results
indicate that the replacement of the phenyl group by p-methoxyphenyl leads to a
significant enhancement of the anticancer activity. The other compounds 17c, 17d, 17e
and 17f were also selective against melanoma with GI50 ranging from 3.61 µM to 11.99
µM. The activity observed for β-carboline 17e (R = m-NO2) against the leukemia cell
line (GI50 = 3.05 µM) should also be highlighted.
Table 1. In vitro anticancer activity (GI50 in µM) of β-carbolines 17.
DOXOa
Cell lines
UACC-61
(Melanoma)
MCF-7 (Breast
Adenocarcinoma)
NCI-ADR-RES
(Ovarian Resistant)
786-0 (Renal
17a
17b
17c
17d
17e
17f
0.047
0.073
1.44
5.45
2.61
5.76
11.99
7.02
3.61
>100
>100
52.7
1.11
36.41
27.57
27.47
25.69
>100
>100
36.8
11.60
0.15
0.37
>100
>100
32.69
0.38
42.35
26.75
29.53
28.48
31.67
>100
20.36
15.32
Adenovarcinoma)
NCI-H460 (Lung
8

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Carcinoma)
PC-3 (Prostate
0.74
>100
>100
10.24
39.44
22.31
21.64
20.67
21.05
>100
>100
9.56
Adenocarcinoma)
OVCAR-3 (Ovarian
Carcinoma)
0.0087
28.39
HT29 (Colorectal
Adenocarcinoma)
2.58
1.11
>100
>100
24.76
0.04
25.30
13.54
25.08
>100
>100
3.05
34.04
21.76
K-562 (Leukemia)
a Doxorubicin used as positive control.
The evaluation of the biological activity of 1-aryl-3-tetrazolyl-β-carbolines 10a-c and
11 against several cancer cell lines was also carried out (Table 2). Compound 10a,
containing a phenyl group at C-1 showed poor activity, although it was selective against
breast cell line (MCF-7), presenting a GI50 value of 4.40 µM. It was again observed that
the replacement of the phenyl group at C-1 by a p-methoxyphenyl group has a
significant impact on the anticancer activity, going from β-carboline 10a with low/no
activity to β-carboline 10b with very interesting features as anticancer agent. The latter
was very active against the human tumor cell lines tested, with the exception of
melanoma and colorectal adenocarcinoma. In fact, β-carboline 10b proved to be a very
promising anticancer agent against breast adenocarcinoma (MCF-7), lung carcinoma
(NCI-H460) and ovarian carcinoma (OVCAR-3) with GI50 between 1.32 and 1.62 µM.
3-Tetrazolyl-β-carboline 10c, bearing 4-fluorophenyl at the C-1, was also very active
against several cell lines. In some cases, the activity was even higher than the one
observed for β-carboline 10b, namely against ovarian carcinoma (OVCAR-3, GI50
=
0.57 µM), but less active against other cell lines (e.g. NCI-H460). It should be
highlighted that β-carboline 10c was also the most active among the 3-tetrazolyl-β-
carbolines studied against leukemia (K-562, GI50 = 0.69 µM) and glioblastoma (U251,
GI50 = 1.38 µM).
To investigate the effect of the deprotection of the tetrazole ring, the anticancer activity
of 3-(1H-tetrazol-5-yl)-β-carboline 11 was evaluated (Table 2). In general, this
structural modulation contributed to the decrease in activity against the majority of the
cancer cell lines studied, compared to the corresponding protected β-carboline 10c.
However, β-carboline 11 proved to be selective and very active against ovarian
carcinoma (OVCAR-3, GI50 = 0.27 µM).
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A trend can be identified with regard to the activity of the 3-(1H-tetrazol-5-yl)-β-
carboline derivatives 10b, 10c and 11 against ovarian cancer cell lines. These three
compounds showed high activity in OVCAR-3 and β-carbolines 10b, 10c were also
active against NCI-ADR-RES (Table 2).
Table 2. In vitro anticancer activity (GI50 in µM) of 3-tetrazolyl-β-carbolines 10a-c and
11.
DOXOa
Cell lines
10a
10b
10c
11
U251 (Glioblastoma)
0.21
63.53
3.82
1.38
8.17
UACC-61
(Melanoma)
0.047
36.95
4.40
88.11
1.32
2.70
2.17
1.59
5.73
1.62
>100
1.04
2.07
0.69
20.16
6.11
0.57
63.33
10.11
36.63
64.45
14.83
71.81
0.27
MCF-7 (Breast
Adenocarcinoma)
NCI-ADR-RES
(Ovarian Resistant)
786-0 (Renal
Adenovarcinoma)
NCI-H460 (Lung
Carcinoma)
PC-3 (Prostate
Adenocarcinoma)
OVCAR-3 (Ovarian
Carcinoma)
0.073
1.44
70.99
70.56
31.95
>100
>100
0.15
0.37
0.74
0.0087
HT29 (Colorectal
Adenocarcinoma)
2.58
1.11
>100
27.43
18.33
7.14
6.51
0.69
25.64
10.69
K-562 (Leukemia)
a Doxorubicin used as positive control.
In order to evaluate the effect of the β-carbolines’ ring A functionalization on the
anticancer activity, 6-substituted-β-carbolines 16a-h were also studied (Table 3).
Adding the 6-methoxy substituent led, in some cases, to decrease of the anticancer
activity ability of the β-carboline derivatives. In fact, compounds 16a, 16b, 16c and 16e
showed overall lower activity than the corresponding 6-unsubstituted-β-carboline-3-
carboxylates (17a, 17b, 17c and 17e, respectively). In contrast with this observation, the
presence of the 6-methoxy substituent in 1-(4-hydroxyphenyl)-β-carboline 16d resulted
in a very impressive impact since this compound showed potent activity against nine of
the cell lines studied, with GI50 values in the range 0.66-7.74 µM (17d is the
corresponding 6-unsubstituted derivative). Particularly interesting was the low GI50
value observed for β-carboline 16d against ovarian carcinoma (OVCAR-3, GI50 = 0.66
µM). The functionalization of β-carboline 17f leading to 1-(4-(dimethylamino)phenyl)-
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β-carboline 16f resulted in an increase in activity which was particularly relevant in
three cell lines, melanoma (UACC-61, GI50 = 0.28 µM), ovarian resistant cancer (NCI-
ADR-RES, GI50 = 4.29 µM) and renal adenovarcinoma (786-0, GI50 = 1.40 µM). This
compound 16f was also very active against glioblastoma (U251, GI50 = 0.95 µM).
Regarding β-carboline-3-carboxylates 16g and 16h, the most striking effect of the
presence of the 6-methyl substituent was the high activity observed against ovarian
carcinoma (OVCAR-3, GI50 = 0.60-61 µM), when compared to the activity of the
corresponding 6-unsubstituted derivatives 17a and 17 b, respectively (Table 3). In the
case of β-carboline 16g, an improvement of activity for most of cancer cell lines tested
was observed. namely against leukaemia (K-562, GI50 = 1.24 µM) and ovarian resistant
(NCI-ADR-RES, GI50 = 2.66 µM).
Table 3. In vitro anticancer activity (GI50 in µM) 6-substituted-β-carbolines 16a-h.
16a
16b
16c
16d
16e
16f
16g
16h
DOXOa
0.21
Cell lines
R1 = OMe
R1 = Me
U251
(Glioblastoma)
UACC-61
(Melanoma)
MCF-7 (Breast
Adenocarcinoma)
NCI-ADR-RES
(Ovarian
>100 70.79 22.05 7.26 >100 0.95
7.44
7.07
0.047
0.073
>100 >100 7.71 >100 19.65 0.28 28.17 23.27
79.59 >100 >100 6.57 >100 >100 5.08 32.21
1.44
>100 >100 >100 6.54 >100 4.29
2.66 41.64
Resistant)
786-0 (Renal
Adenovarcinoma)
NCI-H460 (Lung
Carcinoma)
PC-3 (Prostate
Adenocarcinoma)
OVCAR-3
0.15
0.37
0.74
>100 20.87 12.18 7.51 >100 1.40 10.83 4.27
>100 74.81 65.59 6.95 >100 12.73 6.96 11.57
>100 >100 >100 3.28 >100 7.74 20.61 >100
(Ovarian
Carcinoma)
HT29 (Colorectal
Adenocarcinoma)
K-562
0.0087 >100 >100 >100 0.66 >100 >100 0.60
0.61
>100 >100 >100 7.74 >100 >100 7.14 80.10
>100 >100 >100 4.36 >100 >100 1.24 25.11
2.58
1.11
(Leukemia)
a Doxorubicin used as positive control.
Conclusion
Herein, the synthesis and anticancer activity of novel β-carbolines is reported. Aiming
to widen the β-carboline’s structural diversity, which could enable the finding of potent
11

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anticancer agents, novel tryptamines have been prepared via hetero-Diels-Alder reaction
of nitrosoalkenes with indoles. These building blocks were used to prepare
functionalized β-carbolines by the Pictet-Spengler approach, including 6-substituted-β-
carboline-3-carboxylates and 3-(1H-tetrazol-5-yl)-β-carbolines whose synthesis is
reported for the first time. β-Carboline-3-carboxylates derived from L-tryptophan
methyl ester were also prepared.
The results allowed to identify the new β-carboline derivatives as very promising
anticancer agents. Among the L-tryptophan derived β-carboline-3-carboxylates, 1-
methoxyphenyl-β-carboline-3-carboxylate proved to be the most active against five
cancer cell lines. It was particularly active against leukemia, lung carcinoma and
ovarian resistant cancer with GI50 values in the range 0.04-1.11 µM. It was observed that
the replacement of the 1-phenyl group by the p-methoxyphenyl group led to a
significant enhancement of the anticancer activity.
3-(1-Benzyl-tetrazolyl)-β-carboline, bearing 4-fluorophenyl at the C-1, was also very
active against several cell lines, namely against ovarian carcinoma, leukemia, renal
adenovarcinoma and breast adenocarcinoma with GI50 ≤ 1µM. The corresponding β-
carboline bearing the unprotected tetrazole ring showed significantly lower activity
against the majority of the cancer cell lines studied, being nevertheless selective and
very active against ovarian carcinoma (OVCAR-3, GI50 = 0.27 µM).
The study of the 6-substituted-β-carboline-3-carboxylates allowed to conclude that the
addition of a 6-methoxy substituent can lead to lower anticancer activity. In contrast
with this observation, the 6-methoxy-1-(4-hydroxyphenyl)-β-carboline derivative
showed high activity against nine of the cell lines studied, with GI50 = 0.66 against
ovarian carcinoma (OVCAR-3). The potent activity against melanoma (UACC-61, GI50
= 0.28 µM) and glioblastoma (U251, GI50 = 0.95 µM) observed for 1-(4-
(dimethylamino)phenyl)-β-carboline derivative should also be highlighted.
Regarding 6-methyl-β-carboline-3-carboxylates, the most striking effect of the presence
of the 6-methyl substituent was the enhanced activity observed against ovarian
carcinoma (OVCAR-3, GI50 = 0.60-61 µM).
12

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Experimental section
Chemistry
General information
1H NMR spectra were recorded on an instrument operating at 400 MHz. 13C NMR
spectra were recorded on an instrument operating at 100 MHz. The solvent was
deuterochloroform or hexadeuterodimethyl sulfoxide unless otherwise indicated.
Chemical shifts are expressed in parts per million relative to internal TMS, and coupling
constants (J) are in Hz. Infrared spectra (IR) were recorded in a Fourier Transform
spectrometer coupled with a diamond Attenuated Total Reflectance (ATR) sampling
accessory. High-resolution mass spectra (HRMS) were obtained on a TOF VG
Autospect M spectrometer with electrospray ionization (ESI) or electronic impact (EI).
Melting points were determined in open glass capillaries and are uncorrected. Thin-
layer chromatography (TLC) analyses were performed using precoated silica gel plates.
Flash column chromatography was performed with silica gel 60 as the stationary phase.
E
thyl bromopyruvate oxime (12)18 and 1-(1-benzyl-1H-tetrazol-5-yl)-2-(1H-indol-3-
yl)ethanone oxime (4)13a were prepared according the literature procedures.
General procedure for the hetero-Diels-Alder reaction
Sodium carbonate (23.8 mmol) was added to a solution of α-bromooxime 12 (4.76
mmol) and the appropriate indole (1.1 or 1.2 equiv.) in dry dichloromethane (65 mL).
The reaction mixture was stirred at room temperature for the time indicated in each
case, monitored by TLC. Upon completion, the mixture was filtered through a Celite
pad, which was washed with ethyl acetate (2 x 30 mL). The solvent was evaporated, and
the product was purified by flash chromatography.
Ethyl 2-(hydroxyimino)-3-(5-methoxy-1H-indol-3-yl)propanoate (13a):19 Obtained
from oxime 12 and 5-methoxylindole (1.1 equiv.; 5.24 mmol), reaction time: 47 h.
Purification of the crude product by flash chromatography [ethyl acetate/hexane (1:2)
and (1:1)] gave compound 13a as a beige solid (0. 85 g, 65%). 1H NMR (DMSO-d6) δ:
1.24 (t, J = 6.8 Hz, 3H), 3.79 (s, 3H), 3.92 (s, 2H), 4.19 (q, J = 6.8 Hz, 2H), 6.76 (dd, J
= 8.8 and 2.4 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 7.26 (d, J =
8.8 Hz, 1H), 10.7 (br s, 1H), 12.4 (s, 1H).
13

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Ethyl 2-(hydroxyimino)-3-(5-methyl-1H-indol-3-yl)propanoate (13b): Obtained
from oxime 12 and 5-methylindole (1.2 equiv.; 5.71 mmol), reaction time: 21 h.
Purification of the crude product by flash chromatography [ethyl acetate/hexane (1:2)
and (1:1)] gave compound 13b as a beige solid (0.66 g, 53%). mp 145.8-146.0 °C (from
1
ethyl acetate/hexane). IR (ATR) ν 998, 1028, 1210, 1324, 1717 and 3369 cm-1. H
NMR (DMSO-d6) δ: 1.19 (t, J = 7.2 Hz, 3H), 2.36 (s, 3H), 3.87 (s, 2H), 4.14 (q, J = 7.2
Hz, 2H), 6.88 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H),
7.35 (s, 1H), 10.7 (br s, 1H), 12.3 (s, 1H). 13C NMR (DMSO-d6) δ: 13.9, 20.0, 21.3,
60.7, 107.8, 111.0, 118.1, 122.5, 123.7, 126.6, 127.1, 134.3, 150.3, 163.8. HRMS (EI-
TOF) m/z for C14H16N2O3 [M]+ calcd 260.1161, found 260.1166.
Ethyl 2-(hydroxyimino)-3-(5-nitro-1H-indol-3-yl)propanoate (13c): Obtained from
oxime 12 and 5-nitroindole (1.1 equiv, 5.24 mmol), reaction time: 22 h. Purification of
the crude product by flash chromatography [ethyl acetate/hexane (1:2) and (1:1)] gave
compound 13c as an yellow solid (0.91 g, 66%). mp 181.1-182.0 ºC (from ethyl
1
acetate/hexane). IR (ATR) ν 999, 1018, 1097, 1131, 1316, 1718 and 3327 cm-1. H
NMR (DMSO-d6) δ: 1.19 (t, J = 7.2 Hz, 3H), 3.98 (s, 2H), 4.16 (q, J = 7.2 Hz, 2H), 7.39
(s, 1H), 7.51 (d, J = 9.2 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 8.65 (s, 1H), 11.7 (br s, 1H),
13
12.6 (br s, 1H). C NMR (DMSO-d6) δ: 13.9, 19.8, 60.9, 111.6, 111.9, 116.0, 116.4,
126.1, 127.9, 139.0, 140.3, 149.5, 163.5. HRMS (EI-TOF) m/z for C13H13N3O5 [M]+
calcd 291.0855, found 291.0849.
1-(1-Benzyl-1H-tetrazol-5-yl)-2-(1H-indol-3-yl)ethanamine (8): Oxime 4 (2.96
mmol) was dissolved in a smallest amount of tetrahydrofuran (12 mL) and 70% aqueous
formic acid (65 mL) was added. The resulting solution was cooled with an ice bath and
zinc powder (88.8 mmol) was added in small portions over 30 min. The reaction
mixture was stirred for 24 hours at room temperature, filtered on Celite pad and washed
with ethyl acetate (2 x 30 mL). The filtrate was neutralized with a concentrated
ammonia solution to pH 8 and then extracted with ethyl acetate (3 x 30 mL). The
combined organic extracts were washed with water (2 x 20 mL), dried over anhydrous
Na2SO4 and concentrated in vacuo to give the crude product. Purification of the crude
product by flash chromatography [ethyl acetate and ethyl acetate/MeOH (97:3)] gave
14

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compound 8 as a beige solid (0.71 g, 76%). mp 124.8-124.9 °C. IR (ATR) 721, 953,
1026, 1111, 1359, 1437, 1451, 2919 and 3253 cm-1. 1H NMR (CDCl3) δ: 3.22-3.33 (m,
2H), 4.39 (t, J = 7.6 Hz, 1H), 5.07 (d, J = 15.2 Hz, 1H), 5.41 (d, J = 15.2 Hz, 1H), 6.87
(d, J = 2.4 Hz, 1H), 7.01-7.04 (m, 2H), 7.10-7.14 (m, 1H), 7.20-7.24 (m, 1H), 7.27-7.30
(m, 3H), 7.36-7.39 (m, 2H), 8.09 (br s, 1H). 13C NMR (CDCl3) δ: 33.5, 47.7, 50.7,
110.8,111.5, 118.3, 119.9, 122.5, 123.2, 127.1, 127.5, 128.7, 129.1, 133.8, 136.2, 157.9.
HRMS (EI-TOF) m/z for C18H18N6 [M]+ calcd 318.1593, found 318.1590.
General procedure for the reduction of oximes 13
Zinc powder (6-12 equiv) was added portion wise to a solution of the oxime 13a-c (2.48
mmol) in glacial acetic acid (16.6 mL) at 0 ºC. The reaction mixture was stirred at room
temperature for the time indicated in each case, monitored by TLC. Upon completion,
the mixture was filtered through a Celite pad, which was washed with ethyl acetate (2 x
30 mL). The filtrate was neutralized with aqueous solution NaOH 2M and extracted
with ethyl acetate (3 x 20 mL). The organic extracts were dried over Na2SO4 and the
solvent evaporated off. The amine was pure enough to be used in the next step.
Ethyl 2-amino-3-(5-methoxy-1H-indol-3-yl)propanoate (14a): Obtained from oxime
13a using zinc powder (6 equiv, 14.9 mmol) added in 3 equiv portions at each 8 hours,
reaction time: 25 h. Amine 14a was obtained as an orange solid (0.58 g, 89%). IR %).
1
IR (ATR) 1056, 1204, 1481, 1723 and 3291 cm-1. H NMR (CDCl3) δ: 1.24 (t, J = 8.0
Hz, 3H), 3.02 (dd, J = 14.4 and 7.6 Hz, 1H), 3.24 (dd, J = 14.4 and 5.2 Hz, 1H), 3.81
(dd, J = 7.6 and 5.2 Hz, 1H), 3.86 (s, 3H), 4.09-4.22 (m, 2H), 6.85 (dd, J = 8.8 and 2.4
Hz, 1H), 7.04 (dd, J = 16.0 and 2.4 Hz, 2H), 7.23 (d, J = 8.8 Hz, 1H), 8.14 (br s, 1H).
13C NMR (CDCl3) δ: 14.2, 30.8, 55.1, 56.0, 61.0, 100.7, 111.0, 111.9, 112.3, 123.7,
128.0, 131.5, 154.1, 175.4. HRMS (EI-TOF) m/z for C14H18N2O3 [M]+ calcd 262.1317,
found 262.1313.
Ethyl 2-amino-3-(5-methyl-1H-indol-3-yl)propanoate (14b): Obtained from oxime
13b using zinc powder (9 equiv, 22.3 mmol) added in 3 equiv portions at each 8 hours,
reaction time: 48 h. Amine 14b was obtained as a colorless oil (0.56 g, 92%). IR (ATR)
1
791, 1180, 1720, 2916 and 3360 cm-1. H NMR (CDCl3) δ: 1.26 (t, J = 6.8 Hz, 3H),
2.45 (s, 3H), 3.02 (dd, J = 14.4 and 7.6 Hz, 1H), 3.25 (dd, J = 14.4 and 4.8 Hz, 1H),
15

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3.81 (dd, J = 7.6 and 4.8 Hz, 1H), 4.14-4.20 (m, 2H), 7.01-7.03 (m, 2H), 7.24 (d, J = 8.4
Hz, 1H), 7.40 (s, 1H), 8.05 (br s, 1H). 13C NMR (CDCl3) δ: 14.2, 21.5, 30.8, 55.0, 60.9,
110.7, 110.9, 118.4, 123.1, 123.8, 127.8, 128.7, 134.6, 175.4. HRMS (EI-TOF) m/z for
C14H18N2O2 [M]+ calcd 246.1368, found 246.1367.
Ethyl 2-amino-3-(5-amino-1H-indol-3-yl)propanoate (14c): Obtained from oxime
13c using zinc powder (12 equiv, 29.8 mmol) added in 3 equiv portions at each 8 hours,
reaction time: 48 h. Amine 14c was obtained as a beige solid (0.32 g, 52%). mp > 120
ºC (with decomposition) (from ethyl acetate/hexane). IR (ATR) 1186, 1464, 1578,
1718, 2922 and 3174 cm-1. 1H NMR (DMSO-d6) δ: 1.12 (t, J = 7.2 Hz, 3H), 2.78 (dd, J
= 14.0 and 6.8 Hz, 1H), 2.90 (dd, J = 14.0 and 6.0 Hz, 1H), 3.55 (t, J = 6.8 Hz, 1H),
4.02 (q, J = 7.2 Hz, 2H), 6.46 (dd, J = 8.4 and 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H),
6.91 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 8.4 Hz, 1H), 10.34 (br s, 1H). 13C NMR (DMSO-
d6) δ: 14.1, 31.1, 55.0, 59.8, 101.5, 108.4, 111.4, 111.7, 123.4, 128.2, 130.0, 140.7,
175.3. HRMS (EI-TOF) m/z for C13H17N3O2 [M]+ calcd 247.1321, found 247.1330.
General procedure for the synthesis of tetrahydo-β-carbolines 9 and 15.
Trifluoroacetic acid (2 mmol) was added to a solution of amine 8 or 14 (1 mmol) and
the appropriate aromatic aldehyde (1 mmol) in dichloromethane (8 mL). The reaction
mixture was stirred for 48 h at room temperature and then was concentrated under
reduce pressure. The residue was dissolved in ethyl acetate, neutralized with aqueous
solution of Na2CO3 10% and then extracted with ethyl acetate (2 x 30 mL). The organic
phase was dried over Na2SO4 and the solvent evaporated off to give the product. The
product was purified by recrystallized in methanol an was obtained as a mixture of
cis/trans isomers.
3-(1-Benzyl-1H-tetrazol-5-yl)-1-(phenyl)-2,3,4,9-tetrahydo-β-carboline
(9a):
Obtained from amine 8 with benzaldehyde, as a mixture of cis/trans isomers [0.29 g,
70%, (39:61)].
Major component (trans-9a): IR (ATR) 1072, 1248, 1324, 1454 and 3281 cm-1. 1H
NMR (CDCl3) δ: 3.23 (dd, J = 16.0 and 4.4 Hz, 1H), 3.58 (ddd, J = 16.0, 10.4 and 1.2
Hz, 1H), 4.17 (dd, J = 10.4 and 4.4 Hz, 1H), 5.28 (d, J = 15.2 Hz, 1H), 5.33 (s, 1H),
16

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5.50 (dd, J = 15.2 Hz, 1H), 6.58 (d, J = 7.6 Hz, 1H), 7.71-7.25 (m, 9H), 7.40-7.43 (m,
3H), 7.51-7.59 (m, 1H), 7.78 (s, 1H). HRMS (EI-TOF) m/z for C25H22N6 [M]+ calcd
406.1906, found 406.1919.
3-(1-Benzyl-1H-tetrazol-5-yl)-1-(p-methoxyphenyl)-2,3,4,9-tetrahydo-β-carboline
(9b): Obtained from amine 8 with p-methoxybenzaldehyde, as a mixture of cis/trans
isomers [0.28 g, 63%, (42:58)].
Major component (trans-9b): IR (ATR) 1031, 1172, 1244, 1450, 1508, 1610 and 3289
1
cm-1. H NMR (CDCl3) δ: 3.23 (dd, J = 16.0 and 4.4 Hz, 1H), 3.55 (ddd, J = 16.0, 10.8
and 1.2 Hz, 1H), 3.83 (s, 3H), 4.18 (dd, J = 10.8 and 4.4 Hz, 1H), 4.33 (d, J = 14.8 Hz,
1H), 5.28 (s, 1H), 5.51 (d, J = 14.8 Hz, 1H), 6.63 (d, J = 7.2 Hz, 1H), 6.90-6.92 (m,
2H), 7.12-7.35 (m, 10H), 7.50-7.58 (m, 1H), 7.80 (s, 1H). HRMS (EI-TOF) m/z for
C26H24N6O [M]+ calcd 436.2012, found 436.2017.
3-(1-Benzyl-1H-tetrazol-5-yl)-1-(p-fluorophenyl)-2,3,4,9-tetrahydo-β-carboline
(9c): Obtained from amine 8 with p-fluorobenzaldehyde, as a mixture of cis/trans
isomers [0.30 g, 70%, (37:63)].
Major component (trans-9c): IR (ATR) 1093, 1155, 1224, 1455, 1507 and 3290 cm-1.
1H NMR (CDCl3) δ: 3.23 (dd, J = 16.0 and 4.4 Hz, 1H), 3.56 (ddd, J = 16.0, 10.4 and
1.2 Hz, 1H), 4.14 (dd, J = 10.4 and 4.0 Hz, 1H), 5.30 (d, J = 15.2 Hz, 1H), 5.31 (s, 1H),
5.52 (d, J = 15.2 Hz, 1H), 6.63 (d, J = 7.2 Hz, 1H), 7.05-7.35 (m, 12H), 7.50-7.58 (m,
1H), 7.78 (s, 1H). HRMS (EI-TOF) m/z for C25H21N6F [M]+ calcd 424.1812, found
424.1815.
Ethyl 6-methoxy-1-(phenyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate (15a).
Obtained from amine 14a with benzaldehyde. Purification of the crude product by flash
chromatography gave, in order or elution: compound cis-15a as a white solid (0.24 g,
68%) and compound trans-15a as a white solid (0.091 g, 26%).
Compound cis-15a: mp 100.1-102.0 ºC (from ethyl acetate/hexane) °C. IR (ATR)
1023, 1132, 1208, 1439, 1731, 2827 and 3138 cm-1. 1H NMR (CDCl3) δ: 1.34 (t, J = 7.2
Hz, 3H), 2.99 (ddd, J = 13.6, 11.2 and 2.4 Hz, 1H), 3.19 (ddd, J = 15.2, 4.4 and 2.2 Hz,
1H), 3.86 (s, 3H), 3.95 (dd, J = 11.2 and 4.4 Hz, 1H, H-3), 4.23-4.32 (m, 2H), 5.22 (s,
1H, H-1), 6.79 (dd, J = 8.8 and 2.4 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 7.09 (d, J = 8.8
Hz, 1H), 7.35-7.39 (m, 6H). 13C NMR (CDCl3) δ: 14.3, 25.8, 56.0, 57.0, 58.8, 61.3,
17

ACCEPTED MANUSCRIPT
100.4, 108.8, 111.6, 111.8, 127.6, 128.5, 128.6, 129.0, 131.3, 135.7, 140.8, 154.2,
172.8. HRMS (EI-TOF) m/z for C21H22N2O3 [M]+ calcd 350.1630, found 350.1640.
Compound trans-15a: mp 168.9-169.8 ºC (from ethyl acetate/hexane). IR (ATR) 1021,
1
1122, 1204, 1433, 1724 and 2825 cm-1. H NMR (CDCl3) δ: 1.26 (t, J = 6.8 Hz, 3H),
3.06 (dd, J = 15.2 and 7.2 Hz, 1H), 3.23 (dd, J = 15.6 and 5.2 Hz, 1H), 3.86 (s, 3H),
3.92 (dd, J = 6.8 and 5.6 Hz, 1H, H-3), 4.12-4.21 (m, 2H), 5.35 (s, 1H, H-1), 6.80 (dd, J
= 8.8 and 2.4 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 7.22-7.24 (m,
2H), 7.29-7.32 (m, 3H), 7.59 (br s, 1H). 13C NMR (CDCl3) δ: 14.3, 24.9, 52.5, 55.2,
56.1, 61.2, 100.7, 108.5, 111.7, 111.8, 127.5, 128.2, 128.5, 128.9, 131.4, 134.2, 142.1,
154.2, 173.7. HRMS (EI-TOF) m/z for C21H22N2O3 [M]+ calcd 350.1630, found
350.1646.
Ethyl
6-methoxy-1-(p-methoxyphenyl)-2,3,4,9-tetrahydro-β-carboline-3-
carboxylate (15b): Obtained from amine 14a with p-methoxybenzaldehyde, as a
mixture of cis/trans isomers [0.31 g, 82%, (7:93)].
Major component (trans-15b): IR (ATR) 1023, 1172, 1199, 1237, 1509, 1721 and
3368 cm-1. 1H NMR (CDCl3) δ: 1.27 (t, J = 7.2 Hz, 3H), 3.07 (ddd, J = 15.2, 6.8 and 1.2
Hz, 1H), 3.22 (dd, J = 15.2 and 4.4 Hz, 1H), 3.79 (s, 3H), 3.87 (s, 3H), 3.93 (dd, J = 6.8
and 5.6 Hz, 1H), 4.13-4.23 (m, 2H), 5.34 (s, 1H), 6.81 (dd, J = 8.8 and 2.4 Hz, 1H),
6.85 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 2.4 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 7.17 (d, J =
8.8 Hz, 2H), 7.48 (br s, 1H). HRMS (EI-TOF) m/z for C22H24N2O4 [M]+ calcd 380.1736,
found 380.1748.
Ethyl 6-methoxy-1-(o-chlorophenyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate
(15c): Obtained from amine 14a with o-chlorobenzaldehyde, as a mixture of cis/trans
isomers [0.23 g, 61%, (45:55)].
1
Major component (trans-15c): IR (ATR) 1033, 1214, 1431, 1711 and 3349 cm-1. H
NMR (CDCl3) δ: 1.28 (t, J = 7.2 Hz, 3H), 2.95-3.09 (m, 1H), 3.17-3.25 (m, 1H), 3.88 (s,
3H), 3.97 (dd, J = 10.8 and 4.0 Hz, 1H), 4.14-4.23 (m, 2H), 5.88 (s, 1H), 6.78-6.84 (m,
1H), 6.95 (dd, J = 7.6 and 1.6 Hz, 1H), 7.01 (dd, J = 10.0 and 2.4 Hz, 1H), 7.11-7.16
(m, 2H), 7.21-7.27 (m, 1H), 7.43-7.46 (m, 1H), 7.56 (s, 1H). HRMS (EI-TOF) m/z for
C21H21N2O3Cl [M]+ calcd 384.1241, found 384.1239.
18

ACCEPTED MANUSCRIPT
Ethyl
6-methoxy-1-(p-hydroxyphenyl)-2,3,4,9-tetrahydro-β-carboline-3-
carboxylate (15d): Obtained from amine 14a with p-hydroxybenzaldehyde, as a
mixture of cis/trans isomers [0.21 g, 56%, (61:39)].
Major component (cis-15d): IR (ATR) 1028, 1213, 1449, 1729, 3254 and 3349 cm-1.
1H NMR (DMSO-d6) δ: 1.28 (t, J = 7.2 Hz, 3H), 2.74-2.84 (m, 1H), 2.99-3.04 (m, 1H),
3.75 (s, 3H), 3.80-3.83 (m, 1H), 4.14-4.20 (m, 2H), 5.09 (br s, 1H), 6.62-6.71 (m, 2H),
6.75 (d, J = 8.4 Hz, 2H), 6.95 (br s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 9.37 (s, 1H), 10.1 (s,
1H). HRMS (EI-TOF) m/z for C21H22N2O4 [M]+ calcd 366.1580, found 366.1592.
Ethyl 6-methoxy-1-(m-nitrophenyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate
(15e): Obtained from amine 14a with m-nitrobenzaldehyde, as a mixture of cis/trans
isomers [0.26 g, 67%, (48:52)].
Major component (trans-15e): IR (ATR) 1031, 1212, 1347, 1524, 1719 and 3316 cm-1.
1H NMR (CDCl3) δ: 1.27 (t, J = 6.8 Hz, 3H), 3.11 (ddd, J = 15.6, 6.4 and 1.2 Hz, 1H),
3.19-3.24 (m, 1H), 3.87 (s, 3H), 3.90 (dd, J = 12.0 and 6.0 Hz, 1H), 4.13-4.23 (m, 2H),
5.51 (s, 1H), 6.80-6.85 (m, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.10-7.16 (m, 1H), 7.46-7.57
(m, 2H), 7.64 (d, J = 7.6 Hz, 1H), 8.14-8.22 (m, 1H), 8.28-8.29 (m, 1H). HRMS (EI-
TOF) m/z for C21H21N3O5 [M]+ calcd 395.1481, found 395.1491.
Ethyl
6-methoxy-1-(p-dimethylaminephenyl)-2,3,4,9-tetrahydro-β-carboline-3-
carboxylate (15f): Obtained from amine 14a with m-dimethylaminebenzaldehyde, as a
mixture of cis/trans isomers [0.20 g, 50%, (45:55)].
Major component (trans-15f): IR (ATR) 1031, 1113, 1216, 1433, 1521, 1719 and
1
2903 cm-1. H NMR (CDCl3) δ: 1.27 (t, J = 7.2 Hz, 3H), 2.93 (s, 6H), 3.06 (ddd, J =
15.2, 8.8 and 1.6 Hz, 1H), 3.22 (ddd, J = 15.2, 6.4 and 0.8 Hz, 1H), 3.87 (s, 3H), 3.92-
3.97 (m, 1H), 4.13-4.22 (m, 2H), 5.31 (s, 1H), 6.67 (d, J = 8.8, Hz, 2H), 6.77-6.81 (m,
1H), 6.99-7.01 (m, 1H), 7.08 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.8 Hz, 2H), 7.45 (s, 1H).
HRMS (EI-TOF) m/z for C23H27N3O3 [M]+ calcd 393.2052, found 393.2058.
Ethyl 6-methyl-1-(phenyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate (15g):
Obtained from amine 14b with benzaldehyde, as a mixture of cis/trans isomers [0.17 g,
50%, (73:27)].
Major component (cis-15g): IR (ATR) 1031, 1124, 1219, 1304, 1723 and 3238 cm-1.
1H NMR (CDCl3) δ: 1.34 (t, J = 7.2 Hz, 3H), 2.45 (s, 3H), 2.98 (ddd, J = 15.2, 11.2 and
19

ACCEPTED MANUSCRIPT
2.4 Hz, 1H), 3.20 (ddd, J = 15.2, 4.0, 1.6 Hz, 1H), 3.95 (dd, J = 11.2 and 4.4 Hz, 1H),
4.23-4.33 (m, 2H), 5.25 (t, J = 2.0 Hz, 1H), 6.96-7.00 (m, 1H), 7.10 (d, J = 8.0 Hz, 1H),
7.32-7.39 (m, 7H). HRMS (EI-TOF) m/z for C21H22N2O2 [M]+ calcd 334.1681, found
334.1687.
Ethyl 6-methyl-1-(p-methoxyphenyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylate
(15h): Obtained from amine 14b with p-methoxybenzaldehyde, as a mixture of cis/trans
isomers [0.20 g, 58%, (26:74)].
Major component (trans-15h): IR (ATR) 1029, 1175, 1217, 1508, 1726 and 2905 cm-
1. 1H NMR (CDCl3) δ: 1.26 (t, J = 7.2 Hz, 3H), 2.46 (s, 3H), 3.07 (ddd, J = 15.2, 7.2 and
1.2 Hz, 1H), 3.22 (ddd, J = 15.2, 5.2 and 0.8 Hz, 1H), 3.78 (s, 3H), 3.89-3.94 (m, 1H),
4.12-4.21 (m, 2H), 5.35 (s, 1H), 6.82-6.84 (m, 2H), 6.95-6.99 (m, 1H), 7.08-7.12 (m,
1H), 7.15-7.17 (m, 2H), 7.34 (s, 1H), 7.48 (s, 1H). HRMS (EI-TOF) m/z for C22H24N2O3
[M]+ calcd 364.1787, found 364.1800.
General procedure for a synthesis of β-carbolines 10 and 16
Sulphur powder (2.11 mmol) was added to a solution of tetrahydro-β-carboline 9 or 15
(0.70 mmol) in xylene (6.0 mL). The reaction mixture was stirred for 24 hours
(tetrahydro-β-carbolines 9) or 48 hours (tetrahydro-β-carbolines 15) under reflux and
then the solvent evaporated off. The precipitated was triturated with diethyl ether,
filtered and dried under vacuum to give the products in a pure form.
3-(1-Benzyl-1H-tetrazol-5-yl)-1-(phenyl)-β-carboline
(10a):
Obtained
from
o
tetrahydro-β-carboline 9a, as a beige solid (0.20 g, 71%). mp 279.9-280.7 C (from
diethyl ether). IR (ATR) 1105, 1251, 1430 and 1493 cm-1. 1H NMR (DMSO-d6) δ: 6.36
(s, 2H), 7.19-7.22 (m, 2H), 7.27-7.30 (m, 3H), 7.34-7.38 (m, 1H), 7.55-7.66 (m, 4H),
7.72 (d, J = 8.4 Hz, 1H), 7.94-7.96 (m, 2H), 8.49 (d, J = 8.0 Hz, 1H), 9.12 (s, 1H), 12.0
13
(s, 1H). C NMR (DMSO-d6) δ: 51.8, 112.8, 115.4, 120.4, 120.9, 122.3, 127.2, 127.9,
128.4, 128.6, 128.9, 129.0, 129.2, 130.0, 132.8, 133.6, 135.8, 137.2, 141.7, 142.2,
152.7. HRMS (EI-TOF) m/z for C25H18N6 [M]+ calcd 402.1593, found 402.1590.
3-(1-Benzyl-1H-tetrazol-5-yl)-1-(p-methoxyphenyl)-β-carboline (10b): Obtained
o
from tetrahydro-β-carboline 9b, as a beige solid (0.24 g, 78%). mp 235.8-237.0 C
20

ACCEPTED MANUSCRIPT
1
(from diethyl ether). IR (ATR) 1246, 1431, 1492, 1511 and 1608 cm-1. H NMR
(DMSO-d6) δ: 3.88 (s, 3H), 6.37 (s, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.19-7.21 (m, 2H),
7.27-7.35 (m, 4H), 7.60-7.64 (m, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H),
8.46 (d, J = 7.6 Hz, 1H), 9.06 (s, 1H), 11.9 (s, 1H). 13C NMR (DMSO-d6) δ: 51.8, 55.4,
112.8, 114.3, 114.9, 120.4, 120.9, 122.2, 127.3, 127.9, 128.7, 128.9, 129.6, 129.8,
132.7, 133.3, 135.8, 141.6, 142.2, 152.8, 160.1. HRMS (EI-TOF) m/z for C26H20N6O
[M]+ calcd 432.1699, found 432.1700.
3-(1-Benzyl-1H-tetrazol-5-yl)-1-(p-fluorophenyl)-β-carboline (10c): Obtained from
o
tetrahydro-β-carboline 9c, as a beige solid (0.28 g, 95%). mp 272.0-273.6 C (from
1
diethyl ether). IR (ATR) 1156, 1224, 1433, 1494 and 1510 cm-1. H NMR (DMSO-d6)
δ: 6.34 (s, 2H), 7.18-7.20 (m, 2H), 7.27-7.38 (m, 4H), 7.43 (t, J = 8.8 Hz, 2H), 7.64 (t, J
= 7.6 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.97 (dd, J = 8.8 and 5.6 Hz, 2H), 8.48 (d, J =
8.0 Hz, 1H), 9.11 (s, 1H), 12.0 (s, 1H). 13C NMR (DMSO-d6) δ: 51.8, 112.7, 115.4,
115.8 (d, J = 21.4 Hz, 2C), 120.5, 120.8, 122.3, 127.2, 127.9, 128.7, 129.1, 130.1, 130.7
(d, J = 8.4 Hz, 2C), 132.7, 133.5, 133.6 (d, J = 2.9 Hz, 1C), 141.2, 141.7, 152.7, 162.6
(d, J = 245.3 Hz, 1C). HRMS (EI-TOF) m/z for C25H17N6F [M]+ calcd 420.1499, found
420.1490.
Ethyl 6-methoxy-1-(phenyl)-β-carboline-3-carboxylate (16a): Obtained from
o
tetrahydro-β-carboline 15a, as a beige solid (0.17 g, 70%). mp 259.3-259.5 C (from
1
diethyl ether). IR (ATR) 1145, 1211, 1308, 1437 and 1701 cm-1. H NMR (DMSO-d6)
δ: 1.40 (t, J = 7.2 Hz, 3H), 3.90 (s, 3H), 4.42 (q, J = 7.2 Hz, 2H), 7.25 (dd, J = 8.8 and
2.8 Hz, 1H), 7.55-7.67 (m, 4H), 8.02-8.04 (m, 3H), 8.94 (s, 1H), 11.8 (s, 1H). 13C NMR
(DMSO-d6) δ: 14.4, 55.6, 60.5, 103.5, 113.6, 116.8, 118.9, 121.6, 128.5, 128.8, 128.9,
128.9, 134.9, 136.2, 136.3, 137.6, 142.1, 154.1, 165.5. HRMS (EI-TOF) m/z for
C21H18N2O3 [M]+ calcd 346.1317, found 346.1329.
Ethyl 6-methoxy-1-(p-methoxyphenyl)-β-carboline-3-carboxylate (16b): Obtained
o
from tetrahydro-β-carboline 15b, as a beige solid (0.20 g, 75%). mp: 253.9-254.1 C
1
(from diethyl ether). IR (ATR) 1172, 1208, 1307, 1511 and 1701 cm-1. H NMR
(DMSO-d6) δ: 1.40 (t, J = 7.2 Hz, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 4.41 (q, J = 7.2 Hz,
2H), 7.20 (d, J = 8.8 Hz, 2H), 7.24 (dd, J = 8.8 and 2.4 Hz, 1H), 7.60 (d, J = 9.2 Hz,
1H), 7.99 (d, J = 8.8 Hz, 2H), 8.00 (s, 1H), 8.88 (s, 1H), 11.7 (s, 1H). 13C NMR
21

ACCEPTED MANUSCRIPT
(DMSO-d6) δ: 14.4, 55.3, 55.6, 60.5, 103.5, 113.6, 114.1, 116.3, 118.8, 121.6, 128.7,
129.9, 130.1, 134.7, 136.1, 136.2, 141.9, 154.1, 159.9, 165.6. HRMS (EI-TOF) m/z for
C22H20N2O4 [M]+ calcd 376.1423, found 376.1419.
Ethyl 6-methoxy-1-(o-chlorophenyl)-β-carboline-3-carboxylate (16c): Obtained
o
from tetrahydro-β-carboline 15c, as a yellow solid (0.22 g, 81%). mp: 283.5-283.8 C
1
(from diethyl ether). IR (ATR) 1148, 1211, 1305, 1436 and 1701 cm-1. H NMR
(DMSO-d6) δ: 1.38 (t, J = 7.2 Hz, 3H), 3.89 (s, 3H), 4.39 (q, J = 7.2 Hz, 2H), 7.23 (dd, J
= 8.8 and 2.4 Hz, 1H), 7.50 (d, J = 9.2 Hz, 1H), 7.55-7.64 (m, 3H), 7.70-7.72 (m, 1H),
8.03 (d, J = 2.4 Hz, 1H), 9.00 (s, 1H), 11.6 (s, 1H). 13C NMR (DMSO-d6) δ: 14.4, 55.6,
60.6, 103.8, 113.3, 117.5, 119.0, 121.5, 127.4, 128.0, 129.6, 130.6, 131.9, 132.5, 135.8,
135.8, 136.1, 136.4, 141.2, 154.1, 165.5. HRMS (EI-TOF) m/z for C21H17N2O3Cl [M]+
calcd 380.0928, found 380.0936.
Ethyl 6-methoxy-1-(p-hydroxyphenyl)-β-carboline-3-carboxylate (16d): Obtained
from tetrahydro-β-carboline 15d, as a beige solid (0.20 g, 80%). mp > 223.4-224.9 oC
1
(from diethyl ether). IR (ATR) 1107, 1209, 1306, 1494, 1681 and 3372 cm-1. H NMR
(DMSO-d6) δ: 1.40 (t, J = 7.2 Hz, 3H), 3.89 (s, 3H), 4.41 (q, J = 7.2 Hz, 2H), 7.02 (d, J
= 8.4 Hz, 2H), 7.23 (dd, J = 8.8 and 2.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.88 (d, J =
13
8.4 Hz, 2H), 7.99 (d, J = 2.4 Hz, 1H), 8.85 (s, 1H), 9.85 (br s, 1H), 11.6 (s, 1H). C
NMR (DMSO-d6) δ: 14.4, 55.6, 60.5, 103.4, 113.6, 115.5, 116.1, 118.7, 121.6, 128.4,
128.6, 129.9, 134.6, 136.1, 136.1, 142.4, 154.0, 158.3, 165.6. HRMS (EI-TOF) m/z for
C21H18N2O4 [M]+ calcd 362.1267, found 362.1269.
Ethyl 6-methoxy-1-(m-nitrophenyl)-β-carboline-3-carboxylate (16e): Obtained from
tetrahydro-β-carboline 15e, as a yellow solid (0.20 g, 74%). mp 282.6-283.8 oC (from
1
diethyl ether). IR (ATR) 1106, 1218, 1308, 1522 and 1704 cm-1. H NMR (DMSO-d6)
δ: 1.41 (t, J = 7.2 Hz, 3H), 3.91 (s, 3H), 4.44 (q, J = 7.2 Hz, 2H), 7.28 (dd, J = 8.8 and
2.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.94 (t, J = 8.0 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H),
8.42 (dd, J = 8.4 and 2.0 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.79 (s, 1H), 9.02 (s, 1H),
11.9 (s, 1H). 13C NMR (DMSO-d6) δ: 14.4, 55.6, 60.7, 103.7, 113.5, 117.6, 119.2,
121.6, 123.3, 123.5, 129.6, 130.4, 134.9, 135.0, 136.3, 136.5, 139.0, 139.5, 148.1,
154.6, 165.6. HRMS (EI-TOF) m/z for C21H17N3O5 [M]+ calcd 391.1168, found
391.1176.
22

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Ethyl
6-methoxy-1-(p-dimethylaminephenyl)-β-carboline-3-carboxylate
(16f):
Obtained from tetrahydro-β-carboline 15f, as a beige solid (0.22 g, 80%). mp 240.9-
241.2 oC (from diethyl ether). IR (ATR) 1095, 1207 1301, 1522, 1609 and 1701 cm-1. 1H
NMR (DMSO-d6) δ: 1.40 (t, J = 7.2 Hz, 3H), 3.04 (s, 6H), 3.89 (s, 3H), 4.41 (q, J = 7.2
Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 7.22 (dd, J = 9.2 and 2.8 Hz, 1H), 7.60 (d, J = 8.8 Hz,
13
1H), 7.93 (d, J = 9.0 Hz, 2H), 7.97 (d, J = 2.4 Hz, 1H), 8.81 (s, 1H), 11.6 (s, 1H). C
NMR (DMSO-d6) δ: 14.4, 19.3, 55.6, 60.4, 103.4, 112.0, 113.6, 115.6, 118.5, 121.7,
125.2, 125.7, 128.4, 129.3, 134.5, 136.1, 136.2, 142.7, 150.7, 154.0, 165.7. HRMS (EI-
TOF) m/z for C23H23N3O3 [M]+ calcd 389.1739, found 389.1748.
Ethyl 6-methyl-1-(phenyl)-β-carboline-3-carboxylate (16g): Obtained from
o
tetrahydro-β-carboline 15g, as a beige solid (0.22 g, 86%). mp 199.2-200.6 C (from
1
diethyl ether). IR (ATR) 1099, 1198, 1258, 1301 and 1710 cm-1. H NMR (DMSO-d6)
δ: 1.40 (t, J = 7.2 Hz, 3H), 2.51 (s, 3H), 4.41 (q, J = 7.2 Hz, 2H), 7.44 (dd, J = 8.4 and
0.8 Hz, 1H), 7.57-7.60 (m, 2H), 7.63-7.67 (m, 2H), 8.01-8.03 (m, 2H), 8.21 (s, 1H),
8.86 (s, 1H), 11.8 (s, 1H). 13C NMR (DMSO-d6) δ: 14.4, 21.0, 60.6, 112.5, 116.5, 121.2,
121.4, 128.6, 128.7, 128.8, 128.9, 129.3, 130.1, 134.7, 136.7, 137.6, 139.7, 142.0,
165.5. HRMS (EI-TOF) m/z for C21H18N2O2 [M]+ calcd 330.1368, found 330.1372.
Ethyl 6-methyl-1-(p-methoxyphenyl)-β-carboline-3-carboxylate (16h): Obtained
o
from tetrahydro-β-carboline 15h, as an orange solid (0.23 g, 92%). mp 238.4-239.8 C
1
(from diethyl ether). IR (ATR) 1198, 1250, 1299, 1512 and 1708 cm-1. H NMR
(DMSO-d6) δ: 1.39 (t, J = 7.2 Hz, 3H), 2.51 (s, 3H), 3.89 (s, 3H), 4.41 (q, J = 7.2 Hz,
2H), 7.20 (d, J = 8.4 Hz, 2H), 7.43 (dd, J = 8.4 and 1.2 Hz, 1H), 7.59 (d, J = 8.0 Hz,
1H), 7.99 (d, J = 8.4 Hz, 2H), 8.19 (s, 1H), 8.81 (s, 1H), 11.8 (s, 1H). 13C NMR
(DMSO-d6) δ: 14.4, 21.0, 55.3, 60.5, 112.5, 114.1, 116.0, 121.2, 121.3, 125.7, 128.6,
129.2, 129.9, 130.0, 134.5, 136.6, 139.7, 141.8, 159.9, 165.5. HRMS (ESI-TOF) m/z for
C22H21N2O3 [M+H]+ calcd 361.1547, found 361.1545.
3-(1H-Tetrazol-5-yl)-1-(p-fluorophenyl)-β-carboline (11):
Potassium tert-butoxide (0.84 mmol) was added to a solution of β-carboline 10c (0.12
mmol) in dimethylsulfoxide (5 mL). The reaction mixture was stirred at room
temperature under O2 atmosphere for 5 hours. After this time, saturated solution of
23

ACCEPTED MANUSCRIPT
NH4Cl was added and the mixture extracted with ethyl acetate (3 x 10 mL). The organic
extracts were dried over Na2SO4 and the solvent evaporated off. The product was
purified by a flash chromatography [ethyl acetate/hexane (2:1)] and obtained as a
yellow solid (31.7 mg, 80%). mp 282.1-283.4 ºC (from ethyl acetate/hexane). IR (ATR)
1
1156, 1218, 1453, 1509, 1626 and 3271 cm-1. H NMR (DMSO-d6) δ: 7.35 (t, J = 7.6
Hz, 1H), 7.51 (t, J = 8.8 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 8.27
13
(dd, J = 8.8 and 6.0 Hz, 2H), 8.47 (d, J = 8.0 Hz, 1H), 9.01 (s, 1H), 11.9 (s, 1H). C
NMR (DMSO-d6) δ: 112.7, 113.5, 115.6 (d, J = 21.4 Hz, 2C), 120.4, 120.9, 122.2,
128.9, 130.1, 131.0 (d, J = 8.4 Hz, 2C), 132.8, 133.6, 133.7 (d, J = 3.4 Hz, 1C), 141.5,
141.6, 155.6, 162.7 (d, J = 245.0 Hz, 1C). HRMS (EI-TOF) m/z for C18H11N6F [M]+
calcd 330.1029, found 330.1040.
Anticancer Activity
The compounds 10a-c, 11, 16a-h and 17a-f were evaluated in vitro against the tumor
cell lines glioma (U251), melanona (UACC-61), breast adenocarcinoma (MCF-7),
ovarian expressing multiple-drug-resistance phenotype 4 (NCI-ADR/RES), renal (786-
0), lung (NCI-H460), prostate (PC-3), ovarian (OVCAR-3), leukemia (K-562) and
colon (HT29). kindly provided by Frederick MA (National Cancer Institute, Bethesda,
MD, USA). Stock and experimental cultures were grown in medium containing 5 mL
RPMI 1640 (GIBCO BRL) supplemented with 5% fetal bovine serum (GIBCO BRL).
Penicillin/Streptomicin mixture (1000 U/mL:1000 µg/mL, 1 mL/L RPMI) was added to
the experimental cultures. Cells in 96-well plates (100 µL cells well-1) were exposed to
sample concentrations in DMSO/RPMI (0.25, 2.5, 25, 250 µg mL-1) in triplicate at 37
oC, 5% of CO2 in air for 48 h. The final DMSO concentration did not affect cell
viability. Doxorubicin (0.025–25 µg/mL) was used as positive control. Before (T0 plate)
and after the sample addition (T1 plates), cells were fixed with 50% trichloroacetic acid,
and cell proliferation was determined by spectrophotometric quantification (540 nm) of
cellular protein using the sulforhodamine B assay. All compounds were tested in
triplicate each concentration. Using the dose–response curve for each cell line, the
concentration that inhibits cell growth in 50% (GI50) was determined through non-linear
24

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regression analysis using ORIGIN software version 9.0 (OriginLab Corporation).20
Compounds with GI50 values >100 µM were considered inactive.
Acknowledgements
We would like to thank CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico) for the fellowships Science without Borders (Ciência sem Fronteiras),
Process number 201583/2014-3. Coimbra Chemistry Centre (CQC) is supported by the
Portuguese Agency for Scientific Research, Fundação para a Ciência e a Tecnologia
(FCT) through project UID/QUI/00313/2019. We acknowledge the UC-NMR facility
for the NMR spectroscopic data (www.nmrccc.uc.pt).
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Highlights
•
Novel tryptamines were prepared via hetero-Diels-Alder reaction of nitrosoalkenes
with indoles
•
•
•
•
These building blocks afforded novel β-carbolines via the Pictet-Spengler approach
The synthesis of 3-(1H-tetrazol-5-yl)-β-carbolines is reported for the first time
β-Carbolines with impressive anti-cancer activity were discovered
β-Carbolines with GI50 ≤ 1µM 8 different cancer cell lines studied were identified