Antileishmanial activity of new thiophene–indole hybrids: Design, synthesis, biological and cytotoxic evaluation, and chemometric studies

Article abstract of DOI:10.1016/j.bmc.2016.04.057

In the present work, thirty-two hybrid compounds containing cycloalka[b]thiophene and indole moieties (TN5, TN5 1–7, TN6, TN6 1–7, TN7, TN7 1–7, TN8, TN8 1–7) were designed, synthesized and evaluated for their cytotoxic and antileishmanial activity against Leishmania amazonensis promastigotes. More than half of the compounds (18 compounds) exhibited significant antileishmanial activity (IC₅₀lower than 10.0 μg/L), showing better performance than the reference drugs (tri- and penta-valent antimonials). The most active compounds were TN8-7, TN6-1 and TN7 with respective IC₅₀values of 2.1, 2.3 and 3.2 μg/mL. Demonstrating that all of the compounds were less toxic than the reference drugs, even at the highest evaluated concentration (400 μg/mL), no compound tested presented human erythrocyte cytotoxicity. Compound TN8-7's effectiveness against a trivalent antimony-resistant culture was demonstrated. It was observed that TN8-7's antileishmanial activity is associated with DNA fragmentation of L. amazonensis promastigotes. Chemometric studies (CPCA, PCA, and PLS) highlight intrinsic solubility/lipophilicity, and compound size and shape as closely related to activity. Our results suggest that hybrid cycloalka[b]thiophene–indole derivatives may be considered as lead compounds for further development of new drugs for the treatment of leishmaniasis.

Full text of DOI:10.1016/j.bmc.2016.04.057

Accepted Manuscript
Antileishmanial Activity of new Thiophene-indole Hybrids: Design, Synthesis,
Biological and Cytotoxic Evaluation, and Chemometric Studies
Mayara B. Félix, Edson R. de Souza, Maria do C.A. de Lima, Daiana Karla G.
Frade, Vanessa de L. Serafim, Klinger Antonio da F. Rodrigues, Patrícia Lima
do N. Néris, Frederico F. Ribeiro, Luciana Scotti, Marcus T. Scotti, Thiago M.
de Aquino, Francisco Jaime B. Mendonça-Junior, Márcia R. de Oliveira
PII:
S0968-0896(16)30312-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.057
BMC 12972
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 February 2016
19 April 2016
26 April 2016
Please cite this article as: Félix, M.B., de Souza, E.R., de Lima, M.d.C.A., Frade, D.K.G., Serafim, V.d.L., Rodrigues,
A.d.F., Néris, L.d.N., Ribeiro, F.F., Scotti, L., Scotti, M.T., de Aquino, T.M., Mendonça-Junior, F.J.B., de Oliveira,
M.R., Antileishmanial Activity of new Thiophene-indole Hybrids: Design, Synthesis, Biological and Cytotoxic
Evaluation, and Chemometric Studies, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.04.057
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Antileishmanial Activity of new Thiophene-indole Hybrids: Design, Synthesis,
Biological and Cytotoxic Evaluation, and Chemometric Studies
Mayara B. Félix a, Edson R. de Souza b, Maria do C. A. de Lima b, Daiana Karla G. Frade a, Vanessa
de L. Serafim a, Klinger Antonio da F. Rodrigues c, Patrícia Lima do N. Néris c, Frederico F. Ribeirod,
Luciana Scotti e, Marcus T. Scottic, Thiago M. de Aquinof, Francisco Jaime B. Mendonça-Juniorg,
Márcia R. de Oliveiraa*
aDepartamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza,Universidade
Federal da Paraíba, João Pessoa, Paraíba 58059-900, Brazil;
bDepartamento de Antibióticos, Universidade Federal de Pernambuco, Recife-PE 50670-910,Brazil;
cPrograma de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos. Universidade Federal da
Paraíba, João Pessoa, Paraíba 58059-900, Brazil;
dPrograma de Pós-Graduação em Inovação Terapêutica, Universidade Federal de Pernambuco,
Campus I, Recife-PE, 50670-901, Brazil;
eFederal University of Paraíba, Campus I, João Pessoa, PB, 58051-970, Brazil;
fInstituto de Quimica e Biotecnologia, Universidade Federal de Alagoas, Maceió, AL, 57072-970,
Brazil;
gDepartamento de Ciências Biológicas, Universidade Estadual da Paraíba, CCBSA, João Pessoa-PB
58070-540, Brazil;
* Corresponding author
Márcia Rosa de Oliveira
Departamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza, Universidade
Federal da Paraíba, Campus I, Castelo Branco, João Pessoa, Paraíba, CEP 58059-900, Brazil
Tel: +55 83 3216 7436
E-mail: mrosajp@gmail.com

Abstract
In the present work, thirty-two hybrid compounds containing cycloalka[b]thiophene
and indole moieties (TN5, TN5 1-7, TN6, TN6 1-7, TN7, TN7 1-7, TN8, TN8 1-7) were
designed, synthesized and evaluated for their cytotoxic and antileishmanial activity against
Leishmania amazonensis promastigotes. More than half of the compounds (18 compounds)
exhibited significant antileishmanial activity (IC50 lower than 10.0 µg/L), showing better
performance than the reference drugs (tri- and penta-valent antimonials). The most active
compounds were TN8-7, TN6-1 and TN7 with respective IC50 values of 2.1, 2.3 and 3.2
µg/mL. Demonstrating that all of the compounds were less toxic than the reference drugs,
even at the highest evaluated concentration (400 µg/mL), no compound tested presented
human erythrocyte cytotoxicity. Compound TN8-7’s effectiveness against a trivalent
antimony-resistant culture was demonstrated. It was observed that TN8-7’s antileishmanial
activity is associated with DNA fragmentation of L. amazonensis promastigotes.
Chemometric studies (CPCA, PCA, and PLS) highlight intrinsic solubility/lipophylicity, and
compound size and shape as closely related to activity. Our results suggest that hybrid
cycloalka[b]thiophene-indole derivatives may be considered as lead compounds for further
development of new drugs for the treatment of leishmaniasis.
Keywords: Leishmaniasis; Leishmania amazonensis; cytotoxicity; 2-aminothiophene; indole,
Chemometric studies.
Leishmaniasis can be defined as the array of diseases caused by protozoans of the
genus Leishmania (Order Kinetoplastida, Family Trypanosomatidae)[1,2]. It is considered a
public health concern and afflicts over two million people each year. About 350 million
people live in endemic areas under risk of infection [3].
The life cycle of Leishmania sp. starts when the insect invertebrate host feeds on
infected mammalian blood, thus imbibing amastigotes present within macrophages. In the
vector-insect intestine, the amastigote form of the parasite becomes a procyclical, and later a
metacyclic promastigote. When the insect bites a mammalian host again, inoculated virulent
promastigotes enter the blood stream and are internalized by macrophages, where they
differentiate again into amastigotes, completing the cycle [4].

In spite of its epidemiological importance, the recommended treatment for
leishmaniasis is far from satisfactory. The conventional therapy (via parenteral) uses
pentavalent antimonials [5,6]. The cure rate is high, but the treatment involves difficulties
such as high toxicity and possible therapeutic failure [7]. There is an urgent need to find new
antileishmanial drugs that are efficient against the pathogen yet present low toxicity to the
patient [8].
The thiophenes belong to a group of aromatic heterocyclic compounds [9,10], among
which 2-aminothiophene compounds are well known for their therapeutic applications
[11,12,13]. There are reports of thiophene derived compounds with antipromastigote activity
(IC50 at 7.7 µg/mL) against Leishmania amazonensis as reported by Takahashi et al, [14]. In
vitro activity has also been found against the intracellular amastigote form in murine
peritoneal macrophages, with IC50 values appearing from 1.56 to >80 µM [15].
2-Aminothiophene compounds can be condensed to an indole nucleus, as described
by Alves et al. [16]. Taha et al. [17], have reported the antileishmanial potential of indol-2-
hydrazone compounds with IC50 values varying between 8.18 and 16.14 µM. Sangshetti et al.
[18] also reported the antileishmanial activity of hybrid indole-coumarin compounds against
Leishmania donovani promastigote forms, with IC50 values between 95.0 and 287.5 µg/mL.
The most active compound (C26H17Cl2NO – IC50 95.0 µg/mL) did not show cytotoxicity
against HeLa cells.
Previous studies from our group identified promising in vitro antileishmanial activity
in 2-aminothiophene compounds, demonstrating their potential for leishmaniasis treatment.
The compounds displayed activity against promastigote forms of L. amazonesis with IC50
values reaching 3.37 µM, without cytotoxicity against blood cells or macrophages. An
apoptosis associated action mechanism involving externalization of phosphatidylserine and
DNA fragmentation, with cellular death by secondary necrosis was observed. The
compounds were effective against infection in macrophages, and their anti-amastigote
activity was associated with host immune response modulation [19]. The most effective
compounds in the study presented a 2-aminothiophene hybridized indole ring,
which might be involved in the increase of antileishmanial activity.
The present work reports the design and synthesis of a series of new hybrid
compounds, all containing a combination of 2-aminothiophene and indole cores in a single
chemical structure. These compounds were evaluated for antileishmanial activity against L.
amazonensis promastigote forms, cytotoxicity against blood cells, and their mechanism of

action thru death by DNA fragmentation. Finally, the compounds were subjected to
computer-aided drug design (CADD) studies using Consensus PCA (CPCA), Principal
Component Analysis (PCA), and Partial Least Squares (PLS) regression in order to extract
structural information relating to antileishmanial activity, and to attempt a predictive model
able to direct the synthesis of new derivatives with even greater antileishmanial activity.
The synthesis of the hybrid 2-[(indolyl)methyleneamino]- cycloalka[b]thiophene-3-
carbonitrile derivatives (TN5, TN5 1-7, TN6, TN6 1-7, TN7, TN7 1-7, TN8, TN8 1-7) was
performed according to procedures previously described by our group [20,21] and is depicted
in Scheme 1.
Scheme 1. Synthesis of thiophene-indole derivatives; compounds TN5, TN5 1-7, TN6, TN6 1-7,
TN7, TN7 1-7, TN8, TN8 1-7; Reagents and conditions: (a) Morpholine, EtOH, 5-10°C, 3h; (b)
AcOH, EtOH, rt, 24 h.
CN
R
X
CN
N
S
+
+
S
O
NH
NH
NH
NH
H2C
TN5, TN5 1-7
CN
(CH2)n
CN
(a)
n = 1, 2, 3, 4
R
R
R
X
N
S
CN
OHC
R
n(H2C)
TN6, TN6 1-7
+
(b)
NH2
X
S
N
CN
H
X
X
.
COMPOUNDS
R
H
X
CH
CH
N
S
TN5, TN6, TN7, TN8
TN5 1, TN6 1, TN7 1, TN8 1 5-Me
TN5 2, TN6 2, TN7 2, TN8 2 5-OMe CH
TN5 3, TN6 3, TN7 3, TN8 3 7-Me CH
TN5 4, TN6 4, TN7 4, TN8 4 4-NO2 CH
TN5 5, TN6 5, TN7 5, TN8 5 [g]benzo CH
TN7, TN7 1-7
CN
N
TN5 6, TN6 6, TN7 6, TN8 6
TN5 7, TN6 7, TN7 7, TN8 7
H
5-CN
N
S
CH
TN8, TN 1-8
All compounds were obtained in satisfactory yields (except TN7-6 and TN7-7). All
hybrid derivatives, with the exception of the nitro derivatives (TN5-4, TN6-4, TN7-4 and
TN8-4), presented yellow colors, the nitro compounds presented orange colors.

The chemical structures of all new compounds were then characterized on the basis of
their physicochemical, and spectral data (available in Supporting data), and were in full
agreement with the proposed structures
Table 1. Chemical structures and yields of the new hybrid 2-[(indolyl)-methyleneamino]-
cycloalka[b]thiophene-3-carbonitrile derivatives (TN5, TN5 1-7, TN6, TN6 1-7, TN7, TN7 1-7,
TN8, TN8 1-7).
CN
(CH2)n
R
N
S
X
N
H
Yield
(%)
Yield
(%)
Compound
R
X
n
Compound
R
X
n
H
CH
CH
CH
CH
CH
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
57
77
70
74
81
59
52
58
58
65
59
75
96
83
56
74
H
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
90
63
65
77
69
64
32
49
39
60
72
57
61
73
77
51
TN5
TN7
5-CH3
5-OCH3
7-CH3
4-NO2
5-CH3
5-OCH3
7-CH3
TN5-1
TN5-2
TN5-3
TN5-4
TN5-5
TN5-6
TN5-7
TN6
TN7-1
TN7-2
TN7-3
TN7-4
TN7-5
TN7-6
TN7-7
TN8
4-NO2
[g]Benzo CH
[g]Benzo CH
H
N
H
N
5-CN
H
CH
CH
CH
CH
CH
CH
5-CN
H
CH
CH
CH
CH
CH
CH
5-CH3
5-OCH3
7-CH3
4-NO2
5-CH3
5-OCH3
7-CH3
4-NO2
TN6-1
TN6-2
TN6-3
TN6-4
TN6-5
TN6-6
TN6-7
TN8-1
TN8-2
TN8-3
TN8-4
TN8-5
TN8-6
TN8-7
[g]Benzo CH
[g]Benzo CH
H
N
H
N
5-CN
CH
5-CN
CH

1
The H NMR and 13C NMR data for all obtained compounds are given in the
Supplementary Material and all proposed structures were confirmed. As observed in Souza et
al. [21], the 1H NMR spectra did not display signs of the precursor amino groups, but rather
CH=N protons, which demonstrates successful pharmacophore (2-aminothiophene and
indole) coupling.
The CH=N proton signals were observed as a singlet in the 8.14–9.02 ppm region,
almost always as the second and more de-shielded signal. The most de-shielded signal was
the broad singlet associated with the N-H proton of the indole ring, which appears between
11.93 and 12.95 ppm. The most shielded NMR signals were attributed to cycloalkane protons
1
13
and carbons. These appeared at 1.37–2.89 ppm in H NMR, and at 16.61–31.33 ppm in C
NMR.
The HRMS of all compounds exhibited (M+H)+ molecular ion peaks, which is in
agreement with their molecular formulas. For TN6-3, the molecular ion peak found was [M-
CH3]+ which is likely due to the high incidence of α fragmentation which occurs in C-
heteroatomic bonds.
The majority of the hybrid derivatives evaluated displayed antipromastigote activity,
some of the compounds tested revealed higher activity than both penta- and trivalent
antimony (reference drugs), as can be seen in the IC50 values (Table 2) (Supporting data,
Gra. S1).
Table 2. Data on 50% growth inhibition of the parasite population (IC50), cellular viability (HC50) and
selectivity index (SI).
Series
HC50
(µg/mL)
>400
>400
>400
n.d.
Group IC50 (µg/mL)
SI
TN5
-
48.9
7.1
124.7
91.6
171.2
7.9
289.0
7.4
4.5
>8.2
>56.6
>3.2
n.e.
n.e.
TN5-1
TN5-2
TN5-3
TN5-4
TN5-5
TN5-6
TN5-7
TN6
TN6-1
TN6-2
TN6-3
TN6-4
TN6-5
TN6-6
1
2
3
4
5
6
7
-
n.d.
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>50.7
>1.4
>53.7
>88.8
>172.4
>66.8
>1.2
>1.3
>13.5
>1.7
1
2
3
4
5
6
2.3
6.0
322.0
299.0
29.7
228.0

TN6-7
TN7
TN7-1
TN7-2
TN7-3
TN7-4
TN7-5
TN7-6
TN7-7
TN8
TN8-1
TN8-2
TN8-3
TN8-4
TN8-5
TN8-6
7
-
4.7
3.2
6.1
10.2
786.0
1067.0
18.7
8.7
3.4
5.4
6.5
6.4
4.1
9.4
9.4
651.0
2.1
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
681.2
22.4
>84.7
>124.6
>65.6
>39.1
>0.5
1
2
3
4
5
6
7
-
1
2
3
4
5
6
7
>0.3
>21.4
>45.8
>115.6
>73.2
>61.3
>62.7
>96.6
>42.5
>42.6
>0.6
TN8-7
>193.2
0.02
124.5
38.2
32100.6
0.2
Glucantime
Amphotericin B
Trivalent
Antimony
347.2
9.0
Analyzing the cyclopenta[b]thiophene (TN5) series, we observe that the eight tested
compounds present antileishmanial activity with IC50 values between 7.07 and 171.2 µg/mL.
Among the eight tested compounds, those presenting stronger antipromastigote activity were
the compounds presenting 5-methyl (TN5-1), benzo[g]indol (TN5-5), and 5-cyano (TN5-7)
groups. We also observed that the incorporation of such groups promoted significant
increases in antileishmanial activity as compared to the non-substituted base nucleus
(compound TN5). The incorporation of the pyrrolo[2,3-b]pyridin (TN5-6) resulted in
compounds with low activity (Supporting data, Gra. S2).
Five compounds from the cyclohexa[b]thiophene (TN6) series showed
antipromastigote activity with IC50 values ranging from 2.3 to 29.7 µg/mL. Three compounds
displayed higher activity: TN6-1 (5-methyl), TN6-2 (5-methoxy) e TN6-7 (5-cyano),
presenting more pronounced antileishmanial activity when compared to the non-substituted
derivative (TN6). Substituting the indole ring with 7-methyl (TN6-3), 4-nitro (TN6-4) and
pyrrolo[2,3-b]pyridin (TN6-6) groups resulted in negative contributions to the
antipromastigote activity (Supporting data, Gra. S3).
The cyclohepta[b]thiophene (TN7) series revealed six compounds with
antipromastigote activity; IC50 values ranging from 3.2 to 18.7 µg/mL. However, we stress
that besides the 5-cyano derivative (TN7-7), incorporation of substitutes to the base nucleus
of this series resulted in decreased anti-parasitic activity as compared to the non-substituted

derivative (TN7). The greatest difference was observed with the insertion of 7-methyl (TN7-
3) and 4-nitro (TN7-4) groups; resulting in compounds with non-significant antipromastigote
activity (Supporting data, Gra. S4).
The cycloocta[b]thiophene (TN8) series showed the best antileishmanial profile;
seven hybrid compounds were active against the parasite (IC50 values ranging from 2.1 to 9.4
µg/mL). Analyzing the results of the non-substituted (8CN) compound, and comparing with
the rest of the compounds in this series, one may note that incorporation of 7-methly (TN8-3)
and 5-cyano (TN8-7) groups promoted significant increases in anti-Leishmania activity
(Supporting data, Gra. S5).
Analyzing the influence of indole group bound substitutes; there is an emerging
pattern of increasing antipromastigote activity when the methyl group is changed from
position 7 to 5 on the indole ring in series TN5, TN6 and TN7. It is important to highlight
that TN6-3 and TN7-3 showed respectively low antileishmanial activity (IC50 = 322.0 and
786.0 µg/mL). With this alteration in the placement of the methyl group in TN6-1 and TN7-
1, a noticeable increment in the antipromastigote activity was detected (IC50 = 2.3 µg/mL e
6.1 µg/mL, respectively).
Concerning position C-5 substituent influence; within the same series a methyl group
resulted in compounds with higher activity than substitutions with a methoxyl group, as was
observed in compounds TN5-1 e TN5-2 (IC50 = 124.7 and 7.1 µg/mL, respectively), and
TN7-1 e TN7-2 (IC50 = 10.2 and 6.1 µg/mL, respectively).
Substitutions resulting in indole ring size increases (compounds with benzo[g]indole
moiety) were well tolerated, resulting in compounds with low IC50 values, respectively (7.9,
9.4, 18.7, and 29.7 µg/mL) for compounds TN5-5, TN8-5, TN7-5 and TN6-5,.
We observed a variation in biological activity for compounds with the indole portion
substituted by a 4-nitro group, with respective values for IC50 at TN5-4 171.2 µg/mL, TN8-4
9.4 µg/mL, TN6-4 299.0 µg/mL, and TN7-4 and 1067.0 µg/mL.
The introduction of a 5-cyano group to the indole ring resulted in the most active
compounds among the series studied; TN8-7 and TN7-7 with respective IC50 values of 2.1
and 3.4 µg/mL; indicating that substitutions in position 5 by electron-withdrawing groups are
well tolerated, and result in compounds with more expressive biological activity.
We may infer that the quantity of methylene groups (CH2) bound to the thiophene
ring hybrid derivative exerts a progressively positive influence on antipromastigote activity.
Accordingly observed in our study, the greater the size of the cycloalkane ring (more
methyene groups) the more pronounced the antipromastigote activity, i.e. it was observed that

the cycloocta[b]thiophene derivatives (TN8 series) showed the best activity profile. We
believe that the drug’s binding site to the drug target in the parasite might be large and
hydrophobic, allowing a better interaction with larger rings.
Some thiophene derivatives extracted from plants have been referred to as effective
against promastigote forms of certain Leishmania species [22], hinting at the potential of such
molecules as antileishmanial agents. Synthetic compounds derived from indole, as reported
by Gupta et al. [23] demonstrated the potential of indole derivatives using [1,2,4] triazine
[5,6-b]indol-3-ylthio-1,3,5-triazine; the antileishmanial activity of such compounds were
verified against Leishmania donovani.
Studies with other synthetic thiophene derivatives have also reported antileishmanial
potential against promastigote forms. Rodrigues et al. [19] reported utilizing 2-
aminothiophene derivatives; demonstrating activity against promastigote forms with IC50
values ranging from 3.37 to 189.3 μM. The highest value was related to the compound
containing the unsubstituted thiophene ring. When compared with our indole derivatives, one
notes that an indole nucleus increases antipromastigote activity, evidenced by the low IC50
values registered. It is important to stress that certain drugs evaluated in the present study
displayed IC50 values similar to those registered in our previous works [19]. For example, our
two most promising compounds TN6-1 and TN8-7 (IC50: 2.3 µg/mL (7.3 μM), and 2.1
µg/mL (5.1 μM), respectively) as compared to the most promising drugs in the cited work;
SB-200, SB-44, and SB-83 (9.2 µg/mL (3.6 μM), 2.3 µg/mL (7.3 μM), and 1.3 µg/mL (3.3
μM)), made evident the positive antileishmanial effect of indoles to the hybrid derivatives
tested.
None of the hybrid derivatives tested showed cytotoxicity against human erythrocytes
up to the highest evaluated concentration (400 µg/mL). This demonstrated that all of the
evaluated compounds are less toxic to blood cells than the three reference drugs (penta- and
trivalent antimonials, and Amphotericin B). It is important to highlight that these drugs, (with
hemolytic activity), are the currently applied antileishmanial therapeutics, and present high
cytotoxicity in humans [24]. We report that incorporation of certain groups in non-substituted
compounds did not alter cytotoxicity for the new indole derivatives (Table 2). The in vitro
results suggest that these compounds might not present significant in vivo toxicity.
Nevertheless, in vivo toxicity assays are necessary to confirm this hypothesis.
We also report that all of the derivatives tested were more toxic to the promastigote
forms of L. amazonensis than to human erythrocytes, displaying selective activity for the
parasite (Table 2).

In comparison with the reference drugs, we note that based on the selectivity index
most of the new thiophene-indole derivatives are significantly more selective than the penta-
and trivalent antimonials. Also, compounds TN6-1 (SI >172.4), TN7 (SI >124.6) and TN8-7
(SI >193.2) were more selective than Amphotericin B (SI: 124.5).
From the 32 studied molecules, 2-[(1H-indol-3-ilmethylene)-amino]-4,5,6,7,8,9-
hexahydro-cycloocta[b]thiophene-3-carbonitrile (TN8-7) was selected for subsequent studies.
This choice was made based on its lower IC50 value when compared to its own (and other)
series. The results obtained confirm better selectivity (by index) than all three of the selected
reference drugs.
We therefore performed a parasite growth inhibition test using trivalent antimony (Sb
(III)) resistant strains and obtained similar values as compared to those obtained for sensitive
strains, previously exposed to the same substance (Graph 1). For the resistant strains and the
sensitive ones we registered respective IC50 values of 2.8 µg/mL, and 2.1 µg/mL. These
results demonstrate the efficiency of the new drug when applied against strains with
resistance to the most often applied current therapeutic.
Graph 1. Antileishmanial activity of the TN8-7 drug on promastigote forms of Leishmania
amazonensis whether sensitive or resistant to trivalent antimony.
The promastigotes treated with TN8-7 exhibited DNA fragmentation, which was
previously reported by Rodrigues et al. [19]. As represented in Figure 1, the samples exposed
to 1x, 2x, and 4x the IC50 showed DNA bands fragmented in three portions, as compared to
the control (first sample). We also verified an increase of last band intensity with increasing
drug concentration, suggesting higher fragmentation at four times the IC50. Reports indicate

that treatment with some drugs induces endonuclease liberation, which is responsible for
DNA fragmentation in Leishmania parasites [25]. TN8-7 might well induce parasitic cells to
enter apoptosis. Yet, more detailed studies are necessary to clarify the mechanism of action,
not only for the current substance, but for the other derivatives synthesized.
Figure 1. The genomic DNA of Leishmania amazonensis promastigotes treated with different
concentrations of TN8-7. Agarose gel (1%) was stained with GelRed (1:500), and analysis was
performed under UV light. (1) control; (2) 1x IC50; (3), 2x IC50 (4), and 4x IC50;
2
3
4
1
The CPCA (Consensus PCA) study was developed using Volsurf+ software
considering 13 blocks of descriptors (independent variables), with 128 independent
variables/descriptors. After auto-scaling of the data and considering PC1 and PC2, we
observed that blocks H2O and DRY had greater weights in explaining the variance of the
model. (Figure available in Supporting data) The two main principal components (PC1 and
PC2) were found using a cross-validation technique, explaining 61.266% of the total variance
in antileishmanial activity.
Explaining 61.266% of the total variance in antileishmanial activity, the two main
principal components (PC1 and PC2) were found using a cross-validation technique, (a table
of the explained variance on CPCA is available in Supporting data).
The PCA analysis was generated considering the 40 higher weight descriptors in the
highlighted CPCA. More than 60% of the total variance (60.544%) was explained by PC1
and PC2.

As can be observed in Figure 2, the plot score from the PCA (using the DRY and
OH2 VolSurf+ descriptors) was able to discriminate the most active compounds (large
spheres), from the less active compounds (smaller spheres).
Figure 2. Object Positions in relation to PC1 and PC2 components in the PCA. Large spheres
represent more active compounds and smaller spheres represent less active compounds.
To carry out the Partial Least Squares (PLS) 33 compounds were selected to compose
the training set (TN5, TN5-1, TN5-5, TN6, TN6-1, TN6-5, TN6-7, TN7, TN7-2, TN7-5,
TN7-7, TN8, TN8-1, TN8-2, TN8-3, TN8-5, TN8-7, 6CN10, 6CN09, SB-80, SB-63, SB-81,
SB-44 e SB-83), and 9 compounds were used for the test set (TN5-2, TN5-7, TN6-2, TN7-1,
TN7-6, TN8-4, 6CN01, SB-68 e SB-200) (Compounds 6CN10, 6CN09, 6CN01, SB-80, SB-
68, SB-63, SB-81, SB-200, SB-44 and SB-83 may be found in Rodrigues et al. [19].).
We applied leave-one-out, and the program used was Pentacle software. The best
model was able to explain 78.17% of the data variance with five latent variables and showed
respective r2, q2cv and R2 values of 0.99, 0.89 and 0.44. The descriptors found thru PLS were
generated in interaction with the probes lipophilicity (DRY-DRY), and the molecular shape
(TIP-TIP), highlighting hydrophobicity and molecular shape as important for activity to occur
[21, 26, 27].
In conclusion, thirty-two hybrid compounds containing cycloalka[b]thiophene and
indole moieties (TN5, TN5 1-7, TN6, TN6 1-7, TN7, TN7 1-7, TN8, TN8 1-7) were
designed, synthesized in good yields, and well characterized by physicochemical and spectral
data (1H NMR, 13C NMR and HRMS). These compounds were investigated for their
cytotoxic and antileishmanial properties against promastigote forms of Leishmania

amazonensis. It was observed that many of the substances evaluated, whose IC50 values were
less than 10 µg/mL, demonstrated higher activity than the reference drugs (penta- and
trivalent antimonials). The most active compounds were TN8-7, TN6-1 and TN7 with
respective IC50 values of 2.1, 2.3, and 3.2 µg/mL.
To the highest concentration tested, none of the thiophene-indole derivatives
evaluated revealed cytotoxicity to human erythrocytes, demonstrating that all of the evaluated
compounds are less toxic to blood cells than the three reference drugs. The 2-aminothiophene
derivatives were more selective for the parasite than for blood cells, presenting a high
selectivity index, sometimes higher than the reference drugs. Compound TN8-7 showed the
greatest antileishmanial activity, its action associated with DNA fragmentation. The molecule
demonstrated its effectiveness against antimony-resistant parasites.
For the designed compounds, the chemometric studies generated robust models with
good statistical indices; especially after internal validation; highlighting that the descriptors
related to hydrophobicity and molecular shape are closely related to antileishmanial activity.
Based on our data, we suggest that hybrid cycloalka[b]thiophene-indole compounds
may be taken as lead compounds for further development as new drugs for the treatment of
leishmaniasis.
Acknowledgements
Thanks to all our contributors; UFPB, UFPE, UEPB, and CAPES for the financial and
structural support demanded to perform this work
Supplementary data
Supplementary data associated with this article can be found, in the online version at;
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Graphical abstract

HIGHLIGHTS




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32 hybrid thiophene-indolyl derivatives were synthesized and characterized;
Various compounds showed good antileishmanial activity (IC50 < 10µg/mL);
Most of the compounds presenting Selectivity Indexes greater than reference drugs;
Compound TN8-7 showed activity associated with DNA fragmentation;
Chemometric tools found Lipophilicity and Size/Shape as important for the activity;