Synthesis and preliminary evaluation of 3-thiocyanato-1H-indoles as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.04.039

A novel series of twenty 3-thiocyanato-1H-indoles, carrying diversification at positions N-1, C-2 and C-5 of the heterocyclic core, were synthesized; their antiproliferative activity against four human cancer cell lines (HL60, HEP-2, NCI-H292 and MCF-7) was evaluated, employing doxorubicin as positive control. Indole, N-methylindole and 2-(4-chlorophenyl)-N-methylindole demonstrated to be essentially inactive, whereas several of their congener 3-thiocyanato-1H-indoles displayed good to excellent levels of potency (IC₅₀ ≤ 6 μM), while being non-hemolytic. N-Phenyl-3-thiocyanato-1H-indole and 1-methyl-2-(4-chlorophenyl)-3-thiocyanato-1H-indole showed good to high potency against all the cell lines. On the other side, the N-(4-chlorophenyl)-, 2-(4-chlorophenyl)- and 2-phenyl- 3-thiocyanato-1H-indole derivatives were slightly less active against the test cell lines. Overall, these results suggest that the indole-3-thiocyanate motif can be suitably decorated to afford highly cytotoxic compounds and that the substituted indole can be employed as a useful scaffold toward more potent compounds.

Full text of DOI:10.1016/j.ejmech.2016.04.039

European Journal of Medicinal Chemistry 118 (2016) 21e26
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and preliminary evaluation of 3-thiocyanato-1H-indoles as
potential anticancer agents
,
*
Margiani P. Fortes a, Paulo B.N. da Silva b, Teresinha G. da Silva c, Teodoro S. Kaufman d
,
b
a, **
~
Gardenia C.G. Militao , Claudio C. Silveira
a Departamento de Química, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil
b
ꢀ
Universidade Federal de Pernambuco, Departamento de Fisiologia e Farmacologia, CCB R. Prof. Moraes Rego, Cidade Universitaria, 50670-901 Recife, PE,
Brazil
c
ꢀ
ꢀ
Universidade Federal de Pernambuco, Departamento de Antibioticos, CCB R. Prof. Moraes Rego, Cidade Universitaria, 50670-901 Recife, PE, Brazil
d Instituto de Química Rosario (IQUIR, CONICET-UNR), Suipacha 531, 2000 Rosario, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of twenty 3-thiocyanato-1H-indoles, carrying diversification at positions N-1, C-2 and C-5
of the heterocyclic core, were synthesized; their antiproliferative activity against four human cancer cell
lines (HL60, HEP-2, NCI-H292 and MCF-7) was evaluated, employing doxorubicin as positive control.
Indole, N-methylindole and 2-(4-chlorophenyl)-N-methylindole demonstrated to be essentially inactive,
whereas several of their congener 3-thiocyanato-1H-indoles displayed good to excellent levels of po-
Received 20 February 2016
Received in revised form
14 April 2016
Accepted 15 April 2016
Available online 19 April 2016
tency (IC50 ꢀ 6
m
M), while being non-hemolytic.
and
N-Phenyl-3-thiocyanato-1H-indole
1-methyl-2-(4-chlorophenyl)-3-thiocyanato-1H-indole
Keywords:
showed good to high potency against all the cell lines. On the other side, the N-(4-chlorophenyl)-, 2-(4-
chlorophenyl)- and 2-phenyl- 3-thiocyanato-1H-indole derivatives were slightly less active against the
test cell lines.
Overall, these results suggest that the indole-3-thiocyanate motif can be suitably decorated to afford
highly cytotoxic compounds and that the substituted indole can be employed as a useful scaffold toward
more potent compounds.
3-Thiocyanato-1H-indoles
Bioactive heterocycles
Cytotoxic compounds
HL60 and HEP-2
NCI-H292 and MCF-7 cells
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
indolyl benzimidazoles (2) [5], tricyclic and tetracyclic cyclo-
alkanoindoles (3) [6], and the 2-acyl-1H-indole-4,7-diones (4) [7]
One of the most fruitful paradigms for the discovery of new
bioactive chemical entities is to start with established structural
cores, known to be part of other bioactive molecules [1]. The indole
skeleton is a privileged scaffold [2]; hence, there is an enhanced
likelihood that indole derivatives will have biological activity.
Therefore, this heterocyclic motif is one of the most important
morphological sub-units for the discovery of new drug candidates.
Indoles are the most abundant heterocycles among biologically
active natural products, pharmaceuticals and agrochemicals. Many
of them possess interesting biological and pharmacological prop-
erties [3]; thus, analogs of the aplicyanines A, B and E (1aec) [4], 3-
are cytotoxic and antiproliferative.
Historically, natural products have inspired the synthesis of new
pharmacological agents. However, naturally-occurring thiocya-
nates are rather scarce [8]. Hence, not surprisingly, there have been
few reports on bioactive natural thiocyanates.
Further, most of the natural thiocyanates are terpenoids [9], but
linear alkanes as the thiocyanatins [10] and aminoacid derivatives
as psammaplin B [11], have also been disclosed. In addition, the
thiocyanato group occurs in certain anticancer natural products
formed by deglycosylation of cruciferous glucosinolates [12].
The naturally-occurring fasicularin (5), is bioactive in a DNA
repair-deficient strain of yeasts lacking the RAD 52 gene, and
cytotoxic against Vero cells, acting as a guanidine alkylating agent
[13], while the related cylindricines were toxic in the brine shrimp
assay [14].
* Corresponding author.
** Corresponding author.
E-mail addresses: kaufman@iquir-conicet.gov.ar (T.S. Kaufman), silveira@
quimica.ufsm.br (C.C. Silveira).
The inclusion of the thiocyanato functionality in potentially
bioactive compounds is infrequent and only scattered reports are
http://dx.doi.org/10.1016/j.ejmech.2016.04.039
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

22
M.P. Fortes et al. / European Journal of Medicinal Chemistry 118 (2016) 21e26
available [15]. Simple aromatic thiocyanates (6) are active against
Trypanosoma cruzi, the causative agent of Chagas' disease [16],
whereas others (7) hold promise for the treatment of leishmanial
infections [17].
2. Results and discussion
2.1. Chemistry
In addition, benzyl thiocyanate was isolated from Lepidium
sativum; its chlorinated derivatives (8) were shown to cause mitotic
arrest, acting as a sulfhydryl alkylating reagent [18], and 2,5,6-
substituted imidazo[2,1-b][1,3,4] thiadiazole derivatives (9) have
been found to be potent antiproliferative agents [19].
On the other hand, isatin thiocyanates (10) have shown signif-
icant in vitro anticancer activity, whilst thiocyanate-substituted
derivatives exhibited growth inhibition against melanoma
UACC903 cells [20]. Further, indoles bearing benzimidazole carba-
mate (11) and glyoxylamide moieties have been successfully
explored as antiproliferative agents [21], and different thiocyanates
have been tested as cytotoxics [22] Fig. 1.
The complete set of compounds submitted to the cytotoxicity
tests is detailed in Table 1. All of them were obtained in good overall
yields and in few reaction steps. Compounds 12aee and 13aee
were prepared and fully characterized as previously reported [25].
On the other hand, the 2-arylindoles 20aee, which were
employed as precursors of the 2-aryl-3-thiocyanato-1H-indole
derivatives 14aee and 15aee, were synthesized by means of two
different strategies, disclosed by the groups of Rossi and Samsoniya,
respectively, as shown in Scheme 1.
In the first method [26], the anion of the starting acetophenones
16aec (17aec) was prepared by reaction of the corresponding ke-
tones with potassium tert-butoxide in anhydrous DMSO, and
Finally, in spite that to date the bioactivity of 3-thiocyanato-1H-
indole derivatives has not been explored, these compounds have
been employed as synthetic intermediates toward complex bioac-
tive compounds [21a]; some of the latter have been shown to act as
estrogen receptor ligands, non-nucleoside reverse transcriptase
inhibitors and agents for treatment of respiratory syncytial virus
infection [23].
Considering our previous work with indole [24] and thiocyanato
[25] derivatives, and from a combined literature analysis on
bioactive compounds carrying these structural features, we con-
jectured that the combination of a suitable decorated indole nu-
cleus with the thiocyanato functionalization could afford novel
cytotoxic compounds. Therefore, herein we report the synthesis
and biological activity results of the first generation of 3-
thiocyanato-1H-indole derivatives, as antiproliferative agents.
coupled to 2-iodoaniline (18) through
a
photostimulated
(l
¼ 254 nm) SRN1 reaction at room temperature. The resulting
ketones 19aec spontaneously cyclized in situ, to afford the target 2-
arylindoles 20aec in 45e60% yield, under acid-free conditions.
The second procedure [27], which entailed the acetic acid-
promoted condensation of acetophenones 16d,e with phenyl-
hydrazine (21) and the subsequent Fischer synthesis-type poly-
phosphoric acid (PPA)-mediated [3,3]-sigmatropic rearrangement
of the resulting phenylhydrazone intermediates 22d,e, followed by
ammonia elimination and aromatization, afforded the expected 2-
arylindoles 20d and 20e in 28% and 20% yield, respectively.
Next, the indoles 20aee were N-methylated by treatment with
KOH in DMSO, followed by MeI quench, furnishing the corre-
sponding derivatives 23aee in 80e94% yield. The final stage
comprised the desired thiocyanation, which was accomplished by
exposure of the indoles to the NH4SCN-oxone reagent system and
resulted in 80e98% yields of the 3-thiocyanato-1H-indoles 15aee.
Analogously, submission of the indole derivatives 20aee to thio-
cyanation under similar conditions furnished 82e98% yield of the
expected 3-thiocyanato-1H-indoles 14aee.
H
N
R
R4
R1
N
R1
N
N
H
The structures of the synthesized compounds were established
on the basis of exhaustive infrared, 1H and 13C NMR and mass
spectral analyses. They were also characterized by their melting
point. The infrared and 13C NMR spectra provided the most useful
confirmatory evidences. For instance, the IR spectra of compounds
in series 14 and 15 evidenced the stretching absorption of the C^N
N
R3
H
N
Br
R3
1, Aplicyanins
R2
3, Cycloalkanoindoles
R1= H, NH2
N
R2
N
H
R2= H, OMe
a, A R1=R2=R3= H
R3= H, OMe
2, R= H, Cl, NO2, Br
b, B R1= Ac, R2=R3= H
R4= H, OH, OMe
triple bond of the thiocyanates as sharp signals at 2156 10 cmꢁ1
.
c, E R1= H, R2= OMe, R3= Br
On the other side, in their 13C NMR spectra, it was observed that
the resonance of C-3, which supported the SCN moiety, appeared as
O
H
N
R
a signal at
rivatives (14aee); however, this resonance experienced a slight
upfield shift ( 110.4e110.6 ppm) when the nitrogen atom was
d 111.7e112.2 ppm in the nitrogen unsubstituted de-
C6H13
O
O
N
H
O
H
d
H
R
SCN
O
SCN
5, Fasicularin
alkylated (15aee).
4, R= Me, Ar
6, R= Cl, CF3
In addition, the diagnostic signal of the carbon atom of the
thiocyanato moiety also appeared in a very narrow region (
d
O
SCN
Cl
N
86.5e90.8 ppm). With the exception of 14d, these resonances were
shifted slightly upfield in the nitrogen-unsubstituted compounds
compared with their N-alkyl congeners (15aee).
SCN
N
N
N
SCN
R
R1
R3
S
N
R2
Cl
8
2.2. Biological activity
7
9, R= H, Cl, OMe
The antiproliferative activity of the 3-thiocyanato-1H-indole
derivatives was assessed by investigating their effects on a panel of
four different cell lines, including HL60 (human promyelocytic
leukemia), MCF-7 (human breast adenocarcinoma), NCI-H292
(human lung cancer), and HEP-2 (human cervical carcinoma).
The MTT colorimetric assay was employed to assess the potency
of the compounds. In this assay, the formazan product resulting
from the reduction of MTT is dissolved and quantitated
R1
O
Br
S
H
N
N
O
N
N
SCN
CO2Me
R2
N
H
R
Br
10, R= CH2, (CH2)2, C6H4
Fig. 1. Selected bioactive simple indole and thiocyanato derivatives.
H
11 R1= H, CN; R2= H, Me

M.P. Fortes et al. / European Journal of Medicinal Chemistry 118 (2016) 21e26
23
Table 1
Chemical structures of the 3-thiocyanato-1H-indole derivatives synthesized for testing.
SCN
R3
R2
Cl
N
N
N
N
R1
H
Me
Me
23e
12-15
24
25
Compound no
R1
R2
R3
Compound no
R1
R2
R3
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
24
H
H
H
H
H
Ph
Me
H
H
H
H
H
H
H
H
H
H
H
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
23e
H
H
H
H
C6H5
H
H
H
H
H
H
H
H
H
H
4-MeeC6H4
OMe
Br
CN
4-MeeC6H4
4-MeOeC6H4
3-CF3eC6H4
4-CleC6H4
C6H5
4-MeeC6H4
4-MeOeC6H4
3-CF3eC6H4
4-CleC6H4
H
H
Me
Me
Me
Me
Me
4-MeeC6H4
4-MeOeC6H4
Me
4-CleC6H4
Indole
H
H
H
2-(4-Chlorophenyl)-N-methylindole
25
N-Methylindole
I
NH2
18
Compounds with IC50 ꢀ 3
m
M were judged as highly active,
O-
O
O
whereas thiocyanates bearing IC50 values in the range 3e6
regarded as having good activity. On the other hand, compounds
with IC50 between 6 M and 12 M were considered of moderate
activity, while indole derivatives displaying IC50 values in the range
12e24 M were regarded as bearing low activity. Finally, the
thiocyanates exhibiting IC50 > 24 M were deemed inactive. Under
mM were
a
Me
R
R
R
b
NH2
m
m
19a-c
17a-c
16
m
H
N
m
a R= H
b R= 4-Me
c R= 4-MeO
d R= 3-CF3
e R= 4-Cl
NH2
these conditions, it was concluded that the HL60 cell line was the
most sensitive one, since all the tested compounds exhibited good
d
c
21
to high levels of potency (IC50 ¼ 0.63e5.63
However, since the HL60 cells were also highly sensitive to
M), thiocyanato derivatives with high
mM).
R
doxorubicin (IC50 ¼ 0.02
m
Me
R
e
potency towards HL60 were observed as being more than 50 times
less potent than doxorubicin. Conversely, the MCF-7 cell line was
the least sensitive. Not a single compound was highly potent, a few
exhibited good activity (13a, 13e, 14e and 15e) and many indole
derivatives displayed low activity (12a, 12d, 14a, 15a and 15d) or
were inactive (13d, 14b and 14c).
N
N
H
H
N
H
22d,e
20a-e
g
f
SCN
1
R
R
4
The HEP-2 cells were slightly more sensitive. Few thiocyanates
were highly potent (13a, 15e) while many compounds exhibited
low activity (12a, 12b, 12d and 15c) or were inactive (12c, 13b, 13d
and 15d). Finally, the remaining cell line (NCI-H292) exhibited a
quite similar profile, with more compounds with high (13a, 14e and
15e) and good (13c, 13e, 14a and 15a) potency, and a smaller
number of inactive substituted thiocyanate-1H-indoles (12c and
15d).
g
N
N
2
3
Me
R1
14a-e R1= H; 15a-e R1= Me
23a-e
Scheme 1. Reagents and conditions: a) KtBuO, DMSO, r.t., 15 min; b) 2-iodoaniline; c) h
(254 nm, 400 W), r.t., 3 h (R ¼ 4-H, 4-Me, 4-MeO); d) PhNHNH2, AcOH (cat.), EtOH,
reflux, 6 h; e) 1. PPA, 80 ꢂC, 30 min; 2. 1 M NaOH (R ¼ 3-CF3, 4-Cl); f) 1. KOH, DMSO, r.t.;
2. MeI, r.t.; g) NH4SCN, oxone, MeOH, r.t. 0.3e1 h.
n
Analysis of the series of compounds 12e15 brought about
additional conclusions. The series of compounds 12aee (entries
4e8) comprises the 3-thiocyanato-1H-indole derivatives carrying
only C-5 substituents. The profile of potency of the unsubstituted 3-
thiocyanato-1H-indole 12a improved very slightly upon introduc-
tion of an electron withdrawing group (CN) at C-5 (12e). However,
the presence of substituents able to release electrons resulted in
low general performances, as those observed with the 5-tolyl and
5-bromo derivatives 12b and 12d; furthermore, compound 12c
which carries an electron donating group (MeO) exhibited the
worst profile.
spectrophotometrically. The results are summarized in Table 2,
where Doxorubicin was used as a positive control (entry 24).
Owing to the wide range of molecular weights of the tested
compounds (174.2e332.3) and also taking into account that doxo-
rubicin is a complex, high molecular weight small organic molecule
(MW ¼ 543.5), the potencies of these thiocyanates were compared
taking into account their micromolar concentrations. The 50%
inhibitory concentrations (IC50) and their 95% confidence intervals
were calculated by non-linear regression.
On the other hand, the series of compounds 13aee (entries
9e13) includes the 3-thiocyanato-1H-indole derivatives displaying
substitution of the nitrogen atom. In this case, the introduction of
an unsubstituted phenyl moiety considerably improved the activity
(13a vs 12a), resulting in the N-phenyl-3-thiocyanato derivative
13a, that exhibited good potency against MCF-7, being highly active
At the beginning of the study, it was demonstrated that indole
(24), N-methylindole (25) and 2-(4-chlorophenyl)-N-methylindole
(23e) were essentially inactive under the test conditions (entries
1e3). Next, a general analysis of the performance of the cell lines
and the tested compounds was carried out, according to the
sensitivity of the former and the substitution pattern of the latter.

24
M.P. Fortes et al. / European Journal of Medicinal Chemistry 118 (2016) 21e26
Table 2
Cytotoxic activity of the 3-thiocyanato-1H-indole derivatives against four different cell lines, and their hemolytic activity against human erythrocytes.a
Entry no
Compound nob
IC50 and confidence intervals, in mM
HL60
HEP-2
NCI-H292
MCF-7
1
2
3
24
25
23e
>50
>50
>50
>50
>50
>50
>50
>50
NT
NT
NT
20.7 (14.8e30.5)
3.21 (2.58e3.96)
2.53 (1.74e3.75)
5.63 (3.36e9.40)
3.00 (2.64e3.39)
5.62 (5.11e6.17)
1.43 (1.19e1.71)
4.05 (2.83e5.81)
1.42 (1.35e1.53)
1.54 (1.06e2.18)
3.58 (3.33e4.10)
0.63 (0.20e2.19)
2.27 (2.04e2.49)
4.45 (3.95e4.99)
0.69 (0.37e1.31)
1.08 (0.80e1.40)
1.39 (0.90e1.74)
1.50 (1.01e2.23)
1.35 (1.29e1.39)
2.56 (2.38e2.79)
1.10 (0.79e1.53)
0.02 (0.02e0.04)
4
5
6
7
8
9
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
15a
18.9 (16.1e22.3)
15.7 (13.2e18.7)
40.7 (36.2e45.9)
19.9 (12.1e32.9)
11.89 (9.78e14.5)
2.79 (1.56e4.95)
24.2 (13.2e44.2)
10.8 (7.5e26.7)
35.6 (18.1e70.1)
8.49 (5.51e13.2)
5.03 (4.31e5.83)
10.21 (7.90e12.9)
9.1 (7.9e8.16)
4.1 (3.45e5.34)
8.74 (4.53e6.04)
8.09 (6.16e10.7)
3.62 (2.67e4.92)
22.01 (13.9e35.0)
37.3 (24.8e56.5)
2.80 (2.0e3.75)
1.29 (0.55e2.58)
14.9 (12.2e18.3)
21.5 (6.73e9.65)
27.8 (25.0e31.0)
13.7 (10.7e17.6)
7.22 (6.67e7.83)
2.07 (1.95e2.19)
20.9 (14.1e30.9)
5.99 (5.1e7.02)
21.9 (15.7e30.8)
3.47 (2.94e4.03)
5.43 (4.79e6.11)
17.3 (11.2e26.7)
10.16 (7.09e14.5)
2.07 (1.66e2.54)
7.28 (6.49e8.18)
4.23 (3.44e5.18)
9.90 (8.52e11.5)
11.58 (8.28e16.1)
44.23 (16.4e119.7)
2.07 (1.87e2.28)
0.37 (0.18e0.55)
14.7 (10.3e20.9)
6.85 (5.45e8.63)
6.2 (1.17e33.1)
22.3 (10.7e46.2)
8.73 (6.97e10.9)
3.95 (3.07e5.07)
NT
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
NT
>50
6.81 (3.72e12.4)
13.46 (9.50e19.2)
47.43 (21.4e104.7)
25.68 (17.9e35.0)
4.96 (3.74e6.60)
3.96 (2.63e6.04)
17.81 (11.7e27.2)
11.8 (7.03e19.7)
NT
15b
15c
15d
15e
21.0 (9.08e48.7)
4.05 (1.32e12.4)
0.55 (0.37e0.92)
Doxorubicin (positive control)
a
Data are presented as IC50 values obtained by non-linear regression. NT ¼ Not tested.
b
The series of compounds 12e15 were not hemolytic, with EC50 > 500 mM.
against the other cell lines.
non-methylated congener (14a). In addition, no clear trend could
be found when the N-methylated compounds 15b (which carries a
2-tolyl moiety) and 15c were compared with their respective non-
methylated precursors 14b and 14c.
However, para-substitution of the phenyl moiety either with
chlorine (13e) or methoxy (13c) groups resulted in compounds
with lower potency with regards of the N-phenyl derivative (13a).
Changing to an N-alkyl derivative was of no help, as the N-methyl
substituted compound 13d exhibited a poor profile, being inactive
against HEP-2 and MCF-7 cells. In addition, as expected, the per-
formance of the indoles could not be improved by introduction of a
5-tolyl substitution. Comparison of the potency against HL60, HEP-
2 and NCI-H292 and their confidence intervals suggested that the
resulting N-methylindole 13b exhibited an activity pattern with
slightly lower quality than that of its non-methylated 5-tolyl
congener 12b.
Compounds 14aee (entries 14e18) helped to establish the effect
of the presence of a C-2 substituent. The introduction of a phenyl
group resulted in a general increase of potency with regards to the
unsubstituted congener (14a vs 12a), which was more evident
against HL60, HEP-2 and NCI-H292. However, lower activities were
observed when the phenyl moiety was substituted with a 4-methyl
motif, such as in the 2-tolyl derivative 14b, and the performance of
the tested compound worsened by addition of a 4-methoxy group
as example of an electron releasing substituent (14c), which
became the worst compound of this series. Both, 14b and 14c were
considered inactive against the MCF-7 cells.
Interestingly, N-methylation deeply affected the general per-
formance of the 3-trifluoromethylated compound (15d vs 14d),
which became inactive against both, HEP-2 and NCI-H292 cells, and
exhibited low activity against the MCF-7 cell line. Conversely, N-
methylation notably improved the potency profile of 14e. The
resulting chloro-derivative 15e displayed good potency against
MCF-7, while being highly active against the other cells. The profile
of activity of 15e was very similar to that of the N-phenyl derivative
13a, carrying no substituent on C-2, being a strong improvement
over that of 15a, unsubstituted on the heterocyclic nitrogen atom.
In order to demonstrate that the cytotoxic effects presented by
the substituted indoles do not relate to unspecific cellular mem-
brane disruption, and as a measure of their toxicity to mammalian
cells, the hemolytic activity of the 3-thiocyanato-1H-indole de-
rivatives was assessed, against the highly sensitive human eryth-
rocytes. The release of hemoglobin was monitored by measuring
the absorbance at 540 nm, against negative (erythrocytes in saline
solution, without test compound) and positive (0.1% w/v Triton X-
100 in saline solution) controls.
No hemolytic activity was observed among the compounds,
Slight improvements, compared with the unsubstituted 14a,
could be achieved by adding 4-Cl and 3-CF3 moieties as electron
withdrawing groups (14d and 14e, respectively), concluding that
the 2-(3-trifluoromethyl-phenyl) substitution pattern (14d) despite
leading to a moderately active compound against the MCF-7 cell
line, was the best overall choice within this series.
Finally, the lot of compounds 15aee (entries 19e23), which
embraces the N-methylated 2-substituted 3-thiocyanato-1H-indole
derivatives, was analyzed. It was observed that introduction of a C-
2 phenyl ring increased the potency of the resulting N-methylated
indole derivative (15a vs 13d); however, the overall performance of
the N-methyl,2-phenyl indole 15a was slightly lower than that of its
under test concentrations up to 500 mM, suggesting that the
mechanism of cytotoxicity is not a result of membrane damage. As a
result of this screen, it may be concluded that the whole cell panel
was more sensitive only to the N-phenyl indole 13a and the N-
methylated 2(4-chlorophenyl) derivative 15e, whereas compounds
13e [3(4-chlorophenyl)], 14a (2-phenyl) and 14e [2(4-
chlorophenyl)] can also be considered promising compounds, dis-
playing suitable potency against at least three of the cell lines.
3. Conclusions
In summary, a series of 3-thiocyanato-1H-indole derivatives,

M.P. Fortes et al. / European Journal of Medicinal Chemistry 118 (2016) 21e26
25
carrying three different points of diversification, has been designed
and synthesized, following simple and straightforward protocols.
All the synthesized compounds were spectroscopically character-
ized and screened for their growth inhibitory activity against a
panel of four different human cancer cell lines, including HL60, NCI-
H292, Hep-2 and MCF-7.
from peripheral blood of healthy volunteers. The protocol was
approved by the Ethics Committee of the Federal University of
Pernambuco. The erythrocytes were separated from plasma by
centrifugation at 1500 rpm for 5 min. For the experiment 100 mL of
a 2% erythrocytes suspension in 0.85% NaCl containing 10 mM CaCl2
was added at each well.
Indole (24), N-methylindole (25) and 2-(4-chlorophenyl)-N-
methylindole (23e) were essentially inactive, while all of the 3-
thiocyanato-1H-indole derivatives displayed good to high potency
toward at least one of the test cells, suggesting that the thiocyanate
moiety plays a key role in the observed bioactivity. Further, many of
the tested compounds exhibited interesting levels of growth
inhibitory activity against the whole panel and all of them
A solution of Triton X-100 (1% w/v) was used as positive control
(100%); saline (0.85% NaCl containing 10 mM CaCl2) was employed
as a negative control (blank) and the samples were tested at a
concentration of 500 mM. After incubation at room temperature for
1 h the supernatant was removed and the liberated hemoglobin
was measured spectrophotometrically as the absorbance at
540 nm. Each sample was tested in triplicate. Compounds with
demonstrated to be non-lytic at doses >500
mM in the hemolytic
EC50 > 500 mM were considered non-hemolytic. The percentage of
activity test. The 3-thiocyanato-1H-indoles 13a (N-phenyl) and 15e
[N-methylated 2(4-chlorophenyl) derivative] showed the best
profile of potency.
Against HL60, they were 55e71.5 times less potent than doxo-
rubicin; however, confronted with the HEP-2, NCI-H292 and MCF-
7 cells, the most active compounds were only 2.2e6.8 times less
potent than this reference compound.
hemolysis [Hemolysis (%)] was calculated using Eq. (1)
Hemolysis (%) ¼ [(Asample e Ablank)/(A100% e Ablank)] ꢃ 100
(1)
where Asample, Ablank and A100% are the absorbances of the sample,
and the negative and positive controls, respectively [29].
These results, which suggest that the 3-thiocyanato-1H-indole
motif can be used as a versatile and valuable scaffold to afford
potent cytotoxic compounds, represent an important advance in
this field. Therefore, with the hypothesis that suitably decorated 3-
thiocyanato-1H-indole can afford more potent compounds, further
studies involving redesign of the active moieties are in progress;
their results will be disclosed in due time.
Acknowledgments
The authors are grateful to FAPERGS, CNPq (P. 473139/2013-8)
and CAPES for financial support and scholarships. TSK also thanks
ANPCyT (PICT 2014-0445), CONICET and SECTeI.
Appendix A. Supplementary data
4. Experimental section
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.039.
4.1. In vitro cytotoxicity
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