New thiophene–acridine compounds: Synthesis, antileishmanial activity, DNA binding, chemometric, and molecular docking studies

Article abstract of DOI:10.1111/cbdd.13176

In this study, we synthesized eight new compounds containing the 2-amino-cycloalkyl[b]thiophene and acridine moieties (ACT₀₁ and ACS₀₁-ACS₀₇). None tested compounds presented human erythrocyte cytotoxicity. The new compounds presented antipromastigote activity, where ACS₀₁ and ACS₀₂ derivatives presented significant antileishmanial activity, with better performance than the reference drugs (tri and pentavalent antimonials), with respective IC₅₀ values of 9.60?±?3.19 and 10.95?±?3.96?μm. Additionally, these two derivatives were effective against antimony-resistant Leishmania (Leishmania) amazonensis strains. In addition, binding and fragmentation DNA assays were performed. It was observed that the antileishmanial activity of ACS₀₁ is not associated with DNA fragmentation of the promastigote forms. However, it interacted with DNA with a binding constant of 10⁴?m^?1. In partial least-squares studies, it was observed that the most active compounds (ACS₀₁ and ACS₀₂) showed lower values of amphiphilic moment descriptor, but there was a correlation between the lipophilicity of the molecules and antileishmanial activity. Furthermore, the docking molecular studies showed interactions between thiophene–acridine derivatives and the active site of pyruvate kinase enzyme with the major contribution of asparagine 152 residue for the interaction with thiophene moiety. Thus, the results suggested that the new thiophene–acridine derivatives are promising molecules as potential drug candidates.

Full text of DOI:10.1111/cbdd.13176

PROFESSOR MARIA DE LIMA (Orcid ID : 0000-0002-8277-3458)
Article type : Research Article
New Thiophene-Acridine Compounds: Synthesis, Antileishmanial Activity,
DNA Binding, Chemometric and Molecular Docking Studies
Vanessa de Lima Serafima,f; Mayara Barbalho Félixa,f; Daiana Karla Frade Silvaa,f; Klinger
Antônio da Franca Rodriguesa,f, Patrícia Néris Andradea,f; Sinara Mônica Vitalino de
Almeidac; Sanderssonilo de Albuquerque dos Santosd; Jamerson Ferreira de Oliveirad; Maria
do Carmo Alves de Limad; Francisco Jaime Bezerra Mendonça-Juniorb,f; Marcus Tullius
Scottif; Márcia Rosa de Oliveiraa; Ricardo Olímpio de Mourab,e*
.
aLaboratório de Leishmanioses, Departamento de Biologia Molecular, CEP 58051-970,
Universidade Federal da Paraíba, João Pessoa, PB, Brazil;
bLaboratório de Síntese e Vetorização de Moléculas, Universidade Estadual da Paraíba,
Departamento de Ciências Biológicas, CEP 58070-450, João Pessoa, PB, Brazil;
cLaboratório de Imunopatologia Keizo Asami (LIKA) and Departamento de Bioquímica,
Universidade Federal de Pernambuco, Recife 50670-901, PE, Brazil;
dLaboratório de Química e Inovação Terapêutica (LQIT) - Departamento de Antibióticos,
Universidade Federal de Pernambuco, Recife 50670-901, PE, Brazil;
eDepartamento de Ciências Farmacêuticas, Centro de Ciências Biológicas e da Saúde,
Universidade Estadual da Paraíba - Bodocongó, CEP: 58429-600, Campina Grande, PB,
Brazil;
fPrograma de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos. Universidade
Federal da Paraíba, João Pessoa, Paraíba 58059-900, Brazil;
To whom correspondence should be addressed: Maria do Carmo Alves de Lima
*Email: nenalima.mariadocarmo@gmail.com
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13176
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Fax: +55 8121268346
Abstract
In the present study, we synthesized eight new compounds containing the 2-amino-
cycloalkyl[b]thiophene and acridine moieties (ACT01 and ACS01-ACS07). None tested
compounds presented human erythrocyte cytotoxicity. The new compounds presented
antipromastigote activity, where ACS01 and ACS02 derivatives presented significant
antileishmanial activity, with better performance than the reference drugs (tri and pentavalent
antimonials), with respective IC50 values of 9.60±3.19 and 10.95±3.96 µM. Additionally,
these two derivatives were effective against antimony-resistant L. (L.) amazonensis strains. In
addition, binding and fragmentation DNA assays were performed. It was observed that the
antileishmanial activity of ACS01 is not associated to DNA fragmentation of the promastigote
forms. However, it interacted with DNA with a binding constant of 104 M-1. In partial least-
squares (PLS) studies, it was observed that the most active compounds (ACS01 and ACS02)
showed lower values of amphiphilic moment descriptor, but there was a correlation between
the lipophilicity of the molecules and antileishmanial activity. Furthermore, the docking
molecular studies showed interactions between thiophene-acridine derivatives and the active
site of pyruvate kinase enzyme with the major contribution of asparagine 152 residue for the
interaction with thiophene moiety. Thus, the results suggested that the new thiophene-
acridine derivatives are promising molecules as potential drug candidates.
Keywords: Leishmaniasis; 2-amino-thiophene; acridine; DNA binding; molecular docking.
1. Introduction
Neglected Tropical Diseases (NTDs) are a group of diverse contagious diseases that
occur under tropical and subtropical climates in countries with precarious conditions,
constituting a severe public health concern. Among the NTDs, leishmaniasis stands out for
having the second highest incidence parasitism in the world, surpassed only by malaria, with
a high level of mortality and morbidity [1,2,3]
.
Currently, the treatment applied to leishmaniasis is based on chemotherapy, although
the available drugs are limited and most cause severe toxic side effects. Another concern is
the increasing resistance developed by the parasite, diminishing the efficacy of the drugs,
being necessary the discovery of new promising molecules to antileishmanial drugs [4]
.
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The strategy of molecular hybridization, among the medicinal chemistry techniques,
presents potential to the planning and developing of new drugs. This technique is based on
the combination of specific structural characteristics of different bioactive moieties in order
to produce new compounds, which displays better target affinity and biological efficacy when
compared to its precursors. The new hybrid derivatives could also present a modified
selectivity profile, as well as diminishing the undesirable side effects [5]
.
A lead chemical structure that have been explored for the synthesis of new active
[6]
compounds is the acridine ring, by the condensation with different lateral chemical chain
.
The resulting acridine derivatives are particularly interesting because the wide array of
biological activities described in the literature such as antitumor, antimicrobial, anti-
inflammatory, anti-malarial, anti-alzheimer and antitrypanosomal [6-12]. One of the main
mechanisms that characterize biological activity of these compounds is attributed to the
planarity of its aromatic structures, which can intercalate on the double helix of DNA and
thus interfere in cellular and enzymatic functions [13-17]
.
Studies have reported the antileishmanial activity of acridine derivatives. Di Giorgio
[18]
et al.
synthetized 6-mono-substituted and 3,6-di-substituted acridine derivatives and
verified the anti-amastigote activity (1.1 µM) on L. (L.) infantum. Wong et al. [19] synthesized
a series of compounds from quinacrine, an acridine analogous, and verified that these
compounds restored the sensibility of L. (L.) donovani and L. (L.) enrietti to pentamidine, and
that this action mechanism can be mediated by multiple targets.
Another class of compounds with antileishmanial potential are the 2-amino-thiophene
[20]
derivatives. Rodrigues et al.
evaluated the effect of 2-amino-cycloalkyl[b]thiophene-
carbonitrile derivatives on L. (L.) amazonensis. All tested compounds were active against the
two forms of the parasite, presented low toxicity to murine macrophages and erythrocytes
with inhibition of trypanothione reductase activity as suggested mechanism of action. Félix et
al. [21] studied the importance of the molecular hybridization of 2-amino-thiophene and indole
nuclei, evaluating the potential of the new hybrid compounds and verified that the majority of
the compounds exhibited significant antileishmanial activity (IC50 < 10 µg/mL), and that the
compounds were less toxic that the reference drugs (tri and pentavalent antimonials). Based
on the previous works, here it was proposed the synthesis of new compounds by linkage of
acridine and thiophene rings and evaluation of the antileishmanial activity.
Additionally, the enzyme pyruvate kinase has been extensively studied as a biological
[22]
target for drug candidates, as it is important for the survival of the parasite
.
Trypanosomatids, in the case of the parasites of the genus Leishmania, depend only on the
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glycolytic pathway for ATP generation [23]. Thus, inhibition of this enzyme would cause cell
[24]
death
.
The present study described the synthesis of eight new thiophene-acridine hybrid
compounds and the antileishmanial activity on promastigote forms of L. (L.) amazonensis.
We also evaluated their cytotoxicity on human erythrocytes, and the action mechanism
profile through DNA interaction and fragmentation assays, chemometric studies and
molecular docking.
2. Experimental
2.1. Material and Methods
All reagents were commercial and used without further purification. Chromatography
was performed on silica gel (60-120 mesh and 100-200). All reactions were monitored by
thin layer chromatography (TLC) on Fluka Analytical silica gel thickness 0.2 mm plates with
fluorescent indicator 254 nm and the disclosures were made by UV light (254 or 365nm).
Fusion bands of the synthesized molecules were determined in open capillaries on the unit
Quimis® Model Q-340M apparatus and were not corrected. The Nuclear Magnetic
Resonance spectra of Hydrogen and Carbon were obtained on Agilent-NMR spectrometer,
mod. Mercury Plus multinuclear 300MHz, magneto-NMR OXFORD 300 (operating at 400
1
or 300 MHz for H and 100 or 75 MHz for 13C) using DMSO-d6 as solvent and
tetramethylsilane (TMS) as internal standard. The processing of the spectra was done in
MestReC 4.8.6.0 software. Chemical shifts are reported in parts per million. The
multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublet (dd), triplet
(t) and multiplet (m). Infrared spectra were obtained with KBr discs on IRPrestige-21
spectrometer. The data were processed with Origin 8.0 software. The measures the exact
masses of the molecular ions were obtained on a Shimadzu electrospray LC / MS-IT-TOF in
a positive way. UV–Vis spectra were measured on an Ultraspec 3000 PRO UV–Visible
spectrophotometer (Biochrom Ltd., Cambridge, UK) and fluorescence spectra on a JASCO
FP-6300 (Jasco Corporation, Tokyo, Japan) spectrofluorometer.
The 6,9-Dichloro-2-methoxy-acridine (CAS 8638-4) was obtained commercially
(Aesar® Alpha). The nucleus thiophen were obtained by Gewald classical reaction, and the
condensed ring acridine were: 5CN: 2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
carbonitrile; 6CN: 2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbonitrile; 7CN: 2-
Amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbonitrile;
8CN:
2-Amino-
4,5,6,7,8,9-hexahydro-cycloocta[b]thiophene-3-carbonitrile;
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6EST: 2-Amino-4,5,6,7-

tetrahydro-benzo[b]thiophene-3-carboxylic acid ethyl ester; 7EST: 2-Amino-5,6,7,8-
tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid ethyl ester; 6AMD: 2-Amino-
4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid amide. All these compounds were
obtained and characterized by our group, as previously reported [25, 26]. The acridine-9-
[7]
carboxaldehyde was obtained with the methodology described by Barros et al.
obtained satisfactorily.
and
2.2. General procedure for synthesis of new thiophene-acridine hybrids (ACT01)
The mixture of acridine-9-carboxaldehyde (AC02) (1 equivalent) and the thiophene
derivative 5CN in anhydrous ethanol (10 mL) was stirred at room temperature for 4-6 h. The
product formation was accompanied by TLC with eluting system n-hexane/AcOEt 7:3. Since
the reaction was complete, the material was filtered and washed with cold ethanol (20 mL) to
give pure crystals.
2.2.1.
2-((acridin-9-ylmethylene)amino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
carbonitrile (ACT01)
Brown solid. Formula: C22H15N3S; Yield: 58.6%; Melting point: 192 ºC; Rf 0.57 (n-
hexane/AcOEt 7:3). IR (KBr. cm-1): 3446,79; 2846.93(=C-H); 2218.13 (C≡N); 1830.44;
1406.10; 1159.21; 945.11; 752.23; NMR 1H (DMSO-d6, 400 MHz, δ ppm): 9.73 (1H, s); 8.92
(2H, d, J= 8.7 Hz); 8.36 (2H, d, J=8.7 Hz); 7.85 (2H, dt, J1= 8.7Hz, J2=1.2 Hz); 7.72 (2H, dt,
J1= 8.7 Hz, J2= 1.2Hz); 3.03 (2H, t, J1= 14.4Hz, J2= 7.2Hz); 2.95 (2H, t, J1= 14.7 Hz, J2=7.5
Hz); 2.60 (2H, q, J1= 14.7 Hz, J2= 7.2 Hz).
2.3 General procedure for synthesis of new thiophene-acridine hybrids (ACS)
The compounds 6,9-Dichloro-2-methoxy-acridine (1 equivalent) and thiophene
derivatives in anhydrous ethanol (10 mL) were stirred at 60 ° C for 4-6 h. The product
formation was accompanied by TLC with eluting system n-hexane/AcOEt 7:3. Since the
reaction was complete, the material was filtered and washed with cold ethanol (20 mL) to
give pure crystals.
2.3.1. 2-(6-chloro-2-methoxy-acridin-9-ylamino)-5,6-dihydro-4H-cyclopenta[b]- thiophene-
3-carbonitrile (ACS01)
Beige solid. Formula: C22H16ClN3OS; Yield: 87%; Melting point: 196 ºC; Rf 0.56 (n-
hexane/AcOEt 7:3). IR (KBr. cm-1): 3392.78 (NH); 2208.49 (C≡N); 1631.77 (C=C); 1255.65
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1
and 1032.57 (Ar-O-C); 936.97; NMR H (DMSO-d6, 400 MHz, δ ppm): 12.04 (1H, s, NH);
8.19 (1H, d, CH aromatic, J= 9.6Hz); 8.15 (1H, d, CH aromatic; J= 9.2 Hz); 7.84 (1H, d, CH
aromatic, J= 9.2Hz); 7.58 – 7.52 (1H, m, CH aromatic); 7.40 (1H, dd, CH aromatic; J1=
8.8Hz, J2= 2.4Hz); 7.22 (1H, dd, CH aromatic, J1= 8.8 Hz, J2= 2.0 Hz); 3.84 (3H, s, OCH3);
2.89 (2H, t, cycloalkyl, J1= 13.6Hz, J2= 6.8Hz); 2.74 (2H, t, cycloalkyl, J1= 14Hz, J2=
13
6.8Hz); 2.36 (2H, q, cycloalkyl, J1= 14.4Hz, J2= 7.4Hz; NMR C (DMSO-d6. 100 MHz, δ
ppm): 26.88; 27.93; 29.64; 55.43; 104.93; 113.96; 116.20; 116.91; 117.42; 119.27; 120.46;
123.51; 124.41; 127.73; 128.24; 135.40; 137.52; 141.07; 142.54; 153.17; 154.27; 155.25.
HRMS m/z [M + H]+: calculated for C22H16ClN3OS: 406.070; found: 406.078.
2.3.2. 2-(6-Chloro-2-methoxy-acridin-9-ylamino)-4,5,6,7-tetrahydro-benzo[b]- thiophene-3-
carbonitrile (ACS02)
Mustard solid. Formula: C23H18ClN3OS; Yield: 94.9%; Melting point: 237 ºC; Rf 0.69
(chloroform/methanol 9:1). IR (KBr, cm-1): 3329.13 (NH); 2941.44 (CH); 2221.99 (C≡N);
1
1631.77 (C=C); 1438.89; 1234.44 and 1085.92 (Ar-O-C); 933.54 (aromatic); NMR H
(DMSO-d6, 300 MHz, δ ppm): 12.04 (1H, NH); 8.13 (1H, d, CH aromatic, J= 9.6Hz); 7.92-
7.84 (2H, m, CH aromatic); 7.60-7.51 (2H, m, CH aromatic); 7.40 (1H, d, CH aromatic, J=
7.8Hz); 3.83 (3H, s, OCH3); 2.65 (2H, s, cycloalkyl); 2.34 (2H, d, cycloalkyl, J= 22.5Hz);
1.73 ppm (4H, q, cycloalkyl, J1= 22.8Hz, J2= 4.2Hz); NMR 13C (DMSO-d6, 75 MHz, δ
ppm): 21.67; 22.73; 24.04; 24.11; 55.20; 104.96; 114.47; 116.21; 117.13; 119.29; 120.29;
121.03; 122.95; 124.45; 127.98; 128.27; 133.00; 135.26; 135.62; 137.74; 139.81; 141.07;
154.30. HRMS m/z [M + H]+: calculated for C23H18ClN3OS: 420.085; found: 420.085.
2.3.3.
2-(6-Chloro-2-methoxy-acridin-9-ylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]-
thiophene-3-carbonitrile (ACS03)
Orange solid. Formula: C24H20ClN3OS; Yield: 94.3%; Melting point: 194 ºC; Rf 0.72
(chloroform/methanol 9:1). IR (KBr, cm-1): 3649.31 (NH); 3275.12; 2922.15 (CH); 2638.62;
2204.63 (C≡N); 1627.92 (C=C); 1552.68; 1271.08 and 1083.99 (Ar-O-C); 929.68 (aromatic);
NMR 1H (DMSO-d6, 300 MHz, δ ppm): 11.93 (1H, s, NH); 8.07 (1H, d, CH aromatic, J = 8.7
Hz); 7.59 (1H, d, CH aromatic, J= 2.4 Hz); 7.54 (1H, d, CH aromatic, J= 4.8); 7.50 (1H, d,
CH aromatic, J= 4.2 Hz); 7.43 (1H, m, CH aromatic, J= 4.2 Hz); 7.23 – 7.18 (1H, m, CH
aromatic); 3.65 (3H, OCH3, s,); 2.70 (2H, t, cycloalkyl, J1=9.9 Hz, J2=4.5Hz); 2.60 (2H, t,
13
cycloalkyl, J1=9.9 Hz, J2=4.5Hz); 1.82- 1.54 (6H, m, cycloalkyl); NMR C (DMSO-d6, 75
MHz, δ ppm): 26.94; 27.03; 28.74 (2C); 31.34; 54.93; 105.66; 115.59; 116.21; 116.54;
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116.88; 119.27; 119.82; 121.06; 121.79; 124. 48; 128.41; 129.89; 134.87; 135.59; 137.07;
137.65; 139.96; 154.06. HRMS m/z [M + H]+: calculated for C24H20ClN3OS: 434.101; found:
434.103.
2.3.4.
2-(6-Chloro-2-methoxy-acridin-9-ylamino)-4,5,6,7,8,9-hexahydro-cycloocta[b]-
thiophene-3-carbonitrile (ACS04)
Brown solid. Formula: C25H22ClN3OS; Yield: 89.3%; Melting point: 253 ºC; Rf 0.67
(chloroform/methanol 9:1). IR (KBr, cm-1): 3446.79 (NH); 2926.01(CH); 2218.13 (C≡N);
1
1625.99 (C=C); 1492.90; 1234.44 and 1080.13 (Ar-O-C); 931.61; NMR H (DMSO-d6, 400
MHz, δ ppm): 11.87 (1H, s, NH); 8.21 (1H, d, CH aromatic, J=8.8 Hz); 7.61 (1H, d, CH
aromatic, J=3.2Hz); 7.55 (1H, d, CH aromatic, J= 1.6Hz); 7.52 (1H, d, CH aromatic,
J=9.2Hz); 7.42 (1H, dd, CH aromatic, J=9.2Hz, J=3.2 Hz); 7.22 (1H, dd, CH aromatic, J=
8.8 Hz, J=2.0 Hz); 3.85 (3H, s, OCH3); 2.78 (2H, t, cycloalkyl, J= 6.0 Hz); 2.64 (2H, t,
13
cycloalkyl, J=12Hz, J=6.0Hz); 1.60 (4H, s, cycloalkyl); 1.43 (4H, s, cycloalkyl); NMR C
(DMSO-d6, 100 MHz, δ ppm): δ=22.10; 23.14; 24.31; 25.30 (3C); 55.37; 104.96; 114.84;
116.21; 119.29; 121. 05; 124.48; 127.15; 127.34; 128.28; 128.39; 129.47; 129.51; 133.81;
139.84; 141.07; 154.31; 175.54; HRMS m/z [M + H]+: calculated for C25H22ClN3OS:
448.117; found: 448.114.
2.3.5. 2-(6-Chloro-2-methoxy-acridin-9-ylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-
carboxylic acid ethyl ester (ACS05)
Orange solid. Formula: C23H20ClN3O2S; Yield: 81.5%; Melting point: 245 ºC; Rf 0.68
(chloroform/methanol 9:1). IR (KBr, cm-1): 3404.36 (NH); 2937.58 (CH); 2636.69; 2360.87;
1707.00 (C=O ester); 1489.04; 1274.94 and 1029.98 (Ar-O-C); 937.40; NMR 1H (DMSO-d6,
300 MHz, δ ppm): 11.91 (1H, s, NH); 8.20 (1H, d, CH aromatic, J= 8.1Hz); 7.72 (1H, d, CH
aromatic, J= 3.0Hz); 7.55-7.54 (1H, m, CH aromatic); 7.51-7.47 (1H, m, CH aromatic); 7.42
(1H, dd, CH aromatic, J= 8.7Hz, J= 3.0Hz); 7.22 (1H, dd, CH aromatic, J= 8.7Hz,
J=1.8Hz); 4.13 (2H, q, CH2 ester, J= 14.1Hz, J= 6.9Hz); 3.85 (3H, s, OCH3); 2.58 (2H, d,
cycloalkyl, J= 6.0Hz); 2.39 (2H, d, cycloalkyl, J= 5.1Hz); 1.77-1.64 (4H, m, cycloalkyl);
0.67 (3H, t, CH3 ester, J= 14.1Hz, J=6.9Hz); NMR 13C (DMSO-d6, 75 MHz, δ ppm): 11. 37;
22.39; 23.91; 24.49; 26.50; 55.40; 59.60; 104.97; 112.30; 115.47; 116.22; 119.30; 121.08;
122.00; 122.80; 124.51; 127.55; 128.31; 132.10; 134.22; 135.63; 137.62; 138.40; 141.08;
154.34. HRMS m/z [M + H]+: calculated for C23H20ClN3O2S: 467.111; found: 467.110.
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2.3.6.
2-(6-Chloro-2-methoxy-acridin-9-ylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxylic acid ethyl ester (ACS06)
Orange solid. Formula: C25H23ClN2O3S; Yield: 44.8%; Melting point: 211 ºC; Rf 0.71
(chloroform/methanol 9:1). IR (KBr, cm-1): 3404.36 (NH); 2912.51 (CH); 2692.62; 1712.78
(C=O ester); 1514.12(C=C); 1271.08 and 1029.98 (Ar-O-C); 937.40; NMR1H (DMSO-d6,
300 MHz, δ ppm): 11.90 (1H, s, NH); 8.20 (1H, d, CH aromatic, J=8.1 Hz); 7,60 (1H, d, CH
aromatic, J= 3.0 Hz); 7.55 (1H, d, CH aromatic, J= 1.8Hz); 7.52 (1H, d, CH aromatic, J= 9.3
Hz); 7.42 (1H, dd, CH aromatic, J1= 9.3 Hz; J2= 3.0 Hz); 7.22 (1H, dd, CH aromatic, J= 9.0
Hz; J= 1.8 Hz); 4.16 (2H, q, CH2 ester, J1=14.1 Hz, J2= 6.9 Hz); 3.85 (3H, s,OCH3); 3.42
(2H, s, cycloalkyl); 2.90 - 2.83 (2H, m, cycloalkyl); 1.90 - 1.50 (6H, m, cycloalkyl); 0.62
13
(3H, t, CH3 ester, J1= 14.1 Hz, J2= 7.2 Hz); NMR C (DMSO-d6, 75 MHz, δ ppm): 14.37;
22.39; 23.91; 24.49; 26.50; 55.40; 59.60; 104.97; 112.30; 115.47; 116.22; 119.30; 121.08;
122.00; 122.80; 124.51; 127.55; 128.31; 132.10; 134.22; 135.63; 137.62; 138.40; 141.08;
154.34. HRMS m/z [M + H]+: calculated for C25H23ClN2O3S: 481.127; found: 481.002.
2.3.7. 2-(6-Chloro-2-methoxy-acridin-9-ylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-
carboxylic acid amide (ACS07)
Yellow solid. Formula: C26H25ClN2O3S; Yield: 86.2%; Melting point: 212 ºC; Rf 0.61 (n-
hexane/AcOEt 7:3). IR (KBr, cm-1): 3429.43 (NH); 3138.18; 2939.65; 2362.79; 1635.63
1
(C=O amide); 1492.90; 1244.08; 860.25; 835.17 (aromatic); NMR H (DMSO-d6, 300 MHz,
δ ppm): δ= 11.86 (1H, s, NH); 8.21 (1H, d, CH aromatic, J= 8.1Hz); 7.94-7.86 (1H, m CH
aromatic); 7.65-7.51 (2H, m, CH aromatic); 7.41 (1H, d, CH aromatic, J= 6.6Hz); 7.21 (1H,
d, CH aromatic, J= 6.9Hz); 4.02 (1H, s, NH amide); 3.85 (3H, s, OCH3); 3.71 (1H, s, NH
13
amide); 2.68 (4H, s, cycloalkyl); 1.77 (4H, s, cycloalkyl); NMR C (DMSO-d6, 75 MHz, δ
ppm): 22.62 (2C); 24.04; 24.11; 55.34; 105.03; 114.47; 116.14; 117.13; 119.14; 120.97;
121.03; 122.95; 124.36; 127.98; 128.22; 135.56; 133.02; 137.74; 139.9; 141.05; 154.28;
166.01. HRMS m/z [M + H]+: calculated for C26H25ClN2O3S: 438.096; found: 438.100.
2.4. Antileishmanial activity evaluation
2.4.1. Parasites and in vitro maintenance
Promastigote forms of L. (L.) amazonensis (IFLA/BR/67/PH8) used in the assays
coming from cultures of Bank Institute of Biological Sciences at University Federal de Minas
Gerais, Brazil. They were cultivated in test tubes containing NNN (Novy-McNeal-Nicolle)
blood agar culture medium associated to Schneider liquid medium (Sigma, USA),
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supplemented by 20% Fetal Bovine Serum (FBS) and 1% streptomycin (100 µg/mL) and
penicillin (100 U.I./mL). Cells were incubated at 25±1 °C and the medium was changed
weekly to maintain cellular viability.
2.4.2. Antipromastigote activity evaluation and determination of 50% growth inhibition
concentration (IC50)
For the in vitro tests, compounds were diluted in DMSO (Sigma - Aldrich ™, St
Louis, USA) to prepare a stock solution, and posteriorly diluted in Schneider liquid medium
to final work solutions (5-200 µg/mL). The final DMSO concentration in culture did not
overpassed 1.0%, concentration that does not present cytotoxicity for promastigote forms of
Leishmania sp. and human erythrocytes.
The determination of growth inhibition of promastigote forms of L. (L.) amazonensis
by the studies substances was performed in screw capped test tubes containing 1.0 mL of
Schneider medium supplemented with 20% FBS and streptomycin (100 µg/mL) and
penicillin (100 UI/mL). The density of 1 x 106 of L. (L.) amazonensis promastigotes/mL in
logarithmic growth phase were added to the test tubes in absence (control) and presence of
different concentrations of the evaluated substances.
After 72 hours of incubation at 25 ºC±1, samples of each culture were obtained,
diluted in isotonic solution (10.5 g citric acid, 7 g NaCl, 15 mL formaldehyde in 1 L distilled
water), analysed and quantified directly under optic microscope in a Neubauer chamber. The
L. (L.) amazonensis cultures were evaluated for their sensitivity to the pentavalent antimony
Glucantime® (Aventis Pharma ™, SP, Brazil), Potassium antimonyl tartrate trihydrate
(trivalent antimony source) (Sigma-Aldrich – code: 383376) and Amphotericin B ®
(Cristália, SP, Brazil), used as reference drugs. The assays were performed in duplicated and
repeated at least three times. The control cultures, whose growth was considered 100%, were
maintained in drug absence and the calculation of 50% growth inhibition concentration (IC50)
for each compound was obtained by comparison to the control cultures. The 50% inhibitory
concentration (IC50) was calculated by Probit analysis (SPSS for Windows 13.0).
2.5. Hemolytic activity evaluation
The hemolytic activity was evaluated by incubation of the thiophene-acridine
derivatives diluted in PBS solution with 80 µL of a 5% suspension of human erythrocytes
(O+) during one hour at 37 °C. The reaction was stopped by adding 200 µL of PBS solution,
followed by centrifugation at 1,000 g for 10 minutes. The supernatant was collected and
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cellular lysis was measured via spectrophotometer (540 nm). The absence of hemolysis and
total hemolysis (positive control) were determined by substituting the solution containing the
tested substances (25-800 µg/mL) by an equal volume of PBS or Milli-Q sterile water,
respectively. The results were determined by comparing the percentage of hemolysis to the
positive control (100% hemolysis). All assays were performed in triplicates at least twice [20]
.
This study was approved by the Ethics Committee of the Human Health Sciences Center
Research (CCS), UFPB (Protocol number 0511/15, CAAE: 49005615.7.0000.5188), Brazil.
2.6. Determination of selectivity index
The antipromastigote and human erythrocytes hemolysis data were used for the IC50
(concentration capable of inhibit 50% of parasitic growth) and HC50 (concentration needed to
induce lysis in 50% of the erythrocytes) estimation, respectively. These data allowed to
calculate the Selectivity Index (SI) by the ratio between HC50 and IC50 (SI: HC50/ IC50). The
SI indicates if the tested compound is more selective for the parasite (Leishmania) than to the
mammal cells for in vitro assays performed. The compounds which presented higher SI
values were selected for posterior assays.
2.7. Evaluation of antipromastigote activity against Sb (III) resistant strains
The compounds ACS01 and ACS02 (with highest SI) were selected for evaluation the
activity against L. (L.) amazonensis strains resistant to trivalent antimonial (22.5 times more
resistant than wild). For this analysis, it was used the same method described in the item
2.4.2.
2.8. DNA fragmentation analysis by agarose gel electrophoresis
The ACS01 and ACS02 were selected to evaluate its capacity of causing fragmentation
of the DNA parasite as an indication of apoptosis. Promastigote forms of L. amazonensis in
logarithmic phase of growth were incubated in absence (negative control) and presence of
ACS01 at concentration of 1, 2 and 4 higher than IC50. The cultures were incubated overnight
at 25 ± 1°C, followed by DNA extraction. The Leishmania DNA extraction was performed
using the chloroform/phenol method [20]. The concentration and degree of purity of the DNA
extracted from the L. amazonensis cultures were estimated by spectrophotometer (Thermo
Scientific NanoDrop 2000). The DNA with GelRed Nucleic Acid Gel Stain 1:500 (10.000x
Biotium®) colorant was added to 1% agarose gel (Gibco, USA) and the electrophoresis was
performed in TBE (Tris/Borato/EDTA) solution at 60 V, 50 mA. After the electrophoresis,
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the gels were analysed under UV transilluminator (ImageMaster®). The assays were
performed in duplicates and repeated once.
2.9. DNA binding assay
2.9.1. UV-Vis Absorption Measurements
All experiments involving interactions of derivatives and ctDNA were carried out in 10
mM Tris-HCl buffer (pH 7.6). The solution of ctDNA in Tris buffer was sonicated for 5 min
and the DNA concentration was determined using the molar extinction coefficient 6.600 M−1
cm−1 at 260 nm [27]. The purity of DNA was determined by monitoring the value of the
A260/A280 ratio. DNA concentration was expressed as micromolar equivalents of the base
pairs. Compounds ACS01 and ACS02 were dissolved in DMSO at a concentration of 1 mM
(stock solution) from which working solutions of concentrations ranging from 10 to 50 μM
were prepared by dilution using Tris buffer. After compound concentration optimization, the
absorption spectral titration experiment was performed by keeping the compound
concentration (30 µM) while varying the DNA concentration (0-100 μM bp). All
measurements were performed at 25 °C in a rectangular quartz cuvette with a 1-cm path
length. The intrinsic binding constant (Kb) with ctDNA was obtained by fitting the data to
Equation (1) [28]
:
[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb (εb − εf)
(1)
where εa, εb and εf are the apparent, bound, and free extinction coefficients for each
compound, respectively. Plot fitting of exponential equation [DNA]/(εa − εf) vs. [DNA] was
used for obtaining the Kb values. The binding data were fitted using the Origin 8.0 software.
2.9.2. Fluorescence Measurements
All fluorescence measurements of non-bound and ctDNA-bound derivatives were
performed with solution concentration of 10 μM in 10 mM Tris buffer, pH 7.6. Emission
spectra were recorded in the region 440–600 nm using an excitation wavelength of 417 nm for
both compounds. All measurements were performed at 25 °C in a rectangular quartz cuvette
with a 1-cm path length. Fluorescence intensities were expressed in arbitrary units.
Fluorescence titrations were conducted by the addition of increasing amounts of ctDNA (0–
100 μM bp) directly into the cell containing solutions of the derivatives. Fluorescence
intensities of compound solutions exposed to different ctDNA concentrations were used to
calculate the relative fluorescence decreasing by I/I0 that are fluorescence intensities in the
presence and absence of ctDNA.
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2.10. Chemometric Studies
2.10.1. VolSurf Descriptors
Three-dimensional structures (3D) were used as input data in the VolSurf+ program v.
1.0.7, and were subjected to molecular interaction fields (MIF) to generate descriptors using
the following probes: N1 (amide nitrogen-hydrogen bond donor probe), O (carbonyl oxygen-
hydrogen bond acceptor probe), H2O (water probe), and DRY (hydrophobic probe).
Additional non-MIF-derived descriptors were generated to create a total of 128 descriptors
[29]. VolSurf descriptors have been previously used to predict the activity of against neglected
protozoan diseases [30, 31]
.
2.10.2. Partial Least-Squares (PLS) model
PLS regression were applied to construct models considering a training set of 16
compounds [32]. The number of latent variables (LV), as well as original variables, were
selected by using a Variable Influence on Projection (VIP) plot, together with the plot that
reports, for each PLS component, the explained variance in fitting (R2) and the explained
2
variance in predicting (Qcv ; cross-validation by leave-one-out). The VIP (Variable
Importance in Projection) plot condenses the importance of the independent variables, either
for the explained variances among themselves, or for the dependent variable of the PLS
2
model. The model with the highest value of cross-validation correlation coefficient (Qcv ) was
selected.
2.10.3. Molecular Docking
L. amazonensis and L. mexicana share several genotypic features [33], therefore, the
structures of pyruvate kinase and arginase in complex, respectively, with 2(s)-amino-6-
[34]
boronohexanoic acid (ABH, PDB ID 4IU0) [33], and suramin in (PDB ID 3PP7)
from L.
mexicana
were
downloaded
from
the
protein
data
bank
(http://www.rcsb.org/pdb/home/home.do). The structures of thiophene derivatives were
submitted to molecular docking using the Molegro Virtual Docker, v. 6.0.1 (MVD) [35]. All
the water molecules were deleted from the enzyme structure, and the enzyme and compound
structures were prepared using the same default parameter settings in the same software
package (Score function: MolDock Score; Ligand evaluation: Internal ES, Internal HBond,
Sp2-Sp2 Torsions, all checked; Number of runs: 10 runs; Algorithm: MolDock SE;
Maximum Interactions: 1500; Max. population size: 50; Max. steps: 300; Neighbor distance
factor: 1.00; Max. number of poses returned: 5). The docking procedure was performed using
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a GRID of 15 Å in radius and 0.30 in resolution to cover the ligand-biding site of both pdb
files. Templates with features expected to be relevant for ligand binding extracted for both
ligands were generated to perform docking. The Moldock score [GRID] algorithm was used
as the score function, and the Moldock search algorithm was used [35]. Docking was validated
redocking the original ligand rosiglitazone in active site of enzymes as observed in
crystallography pdb file (PDB ID 4IU0 and 3PP7) [34, 36]
.
2.11. Statistical analysis
The data obtained were presented as mean±standard error of the mean (SEM).
Student’st-test was used to evaluate the individual data significance, and one-way analysis of
variance (ANOVA) for comparison between groups. For statistical analysis GraphPad Prism
software version 5.0 for Windows (GraphPad Software, San Diego, CA, USA) was
used. Only values of p≤0.05 were considered as significant.
3. Results and discussion
3.1 Synthesis of thiophene-acridine compounds
The synthesis of 2-amino-thiophene the thiophene-acridine compounds is presented in
Scheme 1. The 2-amino-thiophene derivatives (5CN, 6CN, 7CN, 8CN, 6EST, 7EST,
6AMD) were obtained using the classic Gewald reaction, as previously described [25, 26]. The
thiophene-acridine derivatives were obtained through linkage reactions between different 2-
amino-thiophene compounds and acridine moieties, via nucleophilic aromatic substitution,
resulting in the final compounds ACS01 to ACS07 and ACT01, with good efficiency.
All new thiophene-acridine derivatives presented over 44% yields and their
physicochemical characteristics and structures were determined by spectrometric data
1
13
according to the structures proposed. The H and C NMR and HRMS data are available in
supporting material which demonstrated that all structures were confirmed.
1
The H NMR of the ACS series shows signals (δ) between 1.43 and 3.42 ppm that
corresponded to cycloalkyl to all the compounds. The linkage between the amine function
and the 6,9-dichloro-2-methoxy-acridine core conducted to a substituted amine (NH-Ar) with
chemical shifts signals between 11.86 - 12.4 ppm, observed to all compounds which prove
the linkage between the moieties. It was also possible to observe singlet signals between 3.65
and 3.85 ppm, corresponding to the methoxy chemical shifts [18], and acridine peaks between
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7.22 and 8.21 [17]. In addition, for the ACS05 and ACS06, it was observed the chemical shifts
of the ester function between 0.62 - 0.67 (CH3) and 4.13 - 4.16 (CH2) ppm [37]
.
The IR spectre results demonstrated the carbonitrile from the amide function
(HNC=O) in ACS07 by absorption at 1635 cm-1, according to the absorption values (1630 -
1690 cm-1). The NH2 function from the amide was observed at 3138 cm-1. To all compounds,
the absorption band for the aromatic ether group (Ar-O-C) was between 1029 - 1085 and
1234 - 1271 cm-1 [37]. On regard to the nitriles (CN), the absorption on the region was
associated to axial deformation vibrations on the triple bonds occurring between 2204 - 2221
cm-1 [32, 33]. Regarding ACS05 and ACS06 derivatives, the ester function presented absorption
of 1707 and 1712 cm-1, respectively [37]. Moreover, HRMS confirmed the identity of all the
synthesized compounds (see supporting material).
3.2 Biological activity
3.2.1 Cytotoxicity and antileishmanial activities
None of the new thiophene-acridine derivatives tested presented cytotoxicity against
human erythrocytes up to the highest concentration evaluated (1000 µM), which
characterized that the compounds obtained are no toxic to blood cells. In addition, it is
important to note that Amphotericin B, a drug used in the current therapy of leishmaniasis,
showed haemolytic activity (HC50 24.25±2.52 µM), being more toxic than all the derivatives
evaluated in this study [38]. It was observed that the addition of chemical groups to the
compounds (nitrile, ester and amide), as well as the increase of the carbon numbers in the
cycloalkyl group, did not alter the cytotoxicity of the new derivatives (Table 1).
Regarding antiparasitic activity, the 2-amino-thiophene, acridine-9-carboxyaldehyde
and 6,9-dichloro-2-methoxy-acridine moieties were tested separately, as all are considered
privileged structures, to check the success of the new compounds synthesised. All the
moieties presented low activity on promastigote forms of L. (L.) amazonensis, presenting
antileishmanial activity with IC50 values < 80 µM, with the exception of AC02 and 8CN
(Table 1).
The linkage of acridine-9-carboxyaldehyde with 2-amino-thiophene moieties
conducted to a loss of activity (IC50 > 500 µM). However, the new compounds obtained by
the 6,9-dichloro-2-methoxy-acridine and the 2-amino-thiophene moieties favoured the
antipromastigote activity, once they presented antileishmanial activity with IC50 between 9.62
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and 69.11 µM. Also, some of the tested compounds presented higher activity than the tri and
pentavalent antimonials (reference drugs), as seen by the IC50 values (Table 1).
It was observed that the size of the cycloakyl ring interferes in the antileishmanial
activity, as the cyclopentane [ACS01 (IC50 9.60±3.19 µM)] and cyclohexane [ACS02 (IC50
10.95±3.96 µM)] presented higher activity than the cycloheptane [ACS03 (IC50 32.26±5.50
µM)] and cyclooctane [ACS04 (IC50 68.97±4.85 µM)]. Thus, these results suggest that the
increase in the number of methylene carbon groups can cause increased lipophilic and steric
effects, affecting the pharmacological response. In addition, the presence of the nitrile radical
improved the biological activity of the compounds [ACS02 (IC50 10.95±3.96 µM)], when
compared to the compounds ACS05 and ACS07 [ACS05 (IC50 26.55±3.98 µM) and ACS07
(IC50 51.37±13.14 µM)], with ester and amide radicals, respectively. The importance of the
[39]
nitrile group bound to heterocyclic nuclei was studied by McGeary et al.
where it was
shown that this functionality would be related to a greater interaction with an enzymatic
catalytic site and consequently a better biological response.
In attempt to evaluate the selective activity for the parasite by the new compounds, the
cytotoxic against human erythrocytes was performed. The results demonstrated that all tested
compounds were more toxic to the promastigote forms of L. (L.) amazonensis than to human
erythrocytes. From the results, the selectivity indexes (SI) both to the reference drugs as well
as for the new derivatives, were obtained. The SI values of the ACS series of thiophene-
acridine compounds demonstrated better selectivity than the antimonials, with ACS01 and
ACS02 presenting higher SI values than all reference drugs used, as shown in Table 1.
Based on the selectivity index, ACS01 and ACS02 derivatives were selected for further
tests. The choice was based on the IC50 values of the compounds, as well as the absence of
toxicity against erythrocytes and higher selectivity for the parasite. It was performed growth
inhibition tests using parasite strains resistant to trivalent antimony (SbIII) and the values
were similar to the ones obtained in sensitive strains, previously exposed to the same
compounds (Table 2). The compound ACS01 presented IC50 of 14.83±0.44 µM for the
resistant strains, compared to 14.04±1.46 µM for the sensitive strains. ACS02 presented IC50
of 16.36±1.72 µM for the resistant strains, with 16.05±1.38 µM for the sensitive ones. The
results prove the efficacy of ACS01 and ACS02 derivatives on the chemotherapy when applied
on strains resistant to the current therapy.
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3.2.2 DNA assays
DNA interaction and fragmentation tests were performed to verify the possible action
mechanism of the compounds, whereas 9-amino-6-chloro-2-methoxyacridine derivatives are
well known as DNA intercalator [40]. It was performed the analysis of DNA binding in vitro
with ctDNA by UV-vis absorption and fluorescence emission of ACS01 and ACS02
derivatives. Acridine derivatives present absorption and emissive properties due the chemical
structure of fused aromatic rings [13]. Besides this, the planarity of acridine ring allows the
intercalation between pair bases of DNA [15]. These characteristics have been widely used in
several studies in order to establish the biological action mechanism of acridine derivatives
after chemical modifications [14,17]. Based on this information, it was supposed that the effects
of ACS01 and ACS02 on DNA could constitute one of the mechanism against Leishmania.
The interaction of ACS01 and ACS02 with ctDNA was monitored by
spectrophotometric titrations in aqueous buffer [17]. The absorption spectra of ACS01 and
ACS02 displayed absorption bands in the region of 350-600 nm (See supplementary material).
The changes observed in the complexes absorption spectra in the presence of increasing
concentration of ctDNA were used for determining the interaction of derivatives with duplex
DNA. A red shift of the absorption maximum was observed upon addition of DNA for both
compounds (9 and 24 nm for ACS01 and ACS02, respectively), besides an increase of the
absorption intensity. The absorption spectra of derivatives in the absence and presence of
ctDNA are depicted in Figure 1.
The data of the spectrophotometric titrations were used to estimate the binding
constants (Kb) according to the model of McGhee and von Hippel [27]. ACS01 and ACS02
derivatives bind to ctDNA with values of Kb = 104 M-1. Typical Kb for intercalation
complexes between organic dyes and DNA range from 1 × 104 to 1 × 106 M−1 [41]. Similar
[14]
DNA binding constants were found by Lafayette et al.
for thiazacridine and
imidazacridine derivatives. The 6-chloro-2-methoxy-acridine moiety found in our compounds
(ACS01 and ACS02 derivatives), are of great biological importance since it is reported in the
[42]
literature the possibility of interaction with DNA Zhang et al.
probably by the
establishment of hydrogen bonding with this biomacromolecule. These results indicate that
the substitution of acridine ring with chloro and methoxy moieties did not impair DNA
interaction. Furthermore, it was not observed significant differences in DNA binding
properties between ACS01 and ACS02 through spectrophotometric titrations.
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The interaction of ACS01 and ACS02 with ctDNA it also was investigated by
fluorescence spectroscopy. Both derivatives showed emissive properties with excitation and
emission wavelengths at 417 and 463 nm, respectively (Figure 2). Fluorescence spectra were
monitored at a fixed concentration of 10 µM of each derivative and different ctDNA
concentrations. The fluorescence of both compounds was quenched upon binding to ctDNA
bases, but the decrease in fluorescence emission of ACS02 was lower than those observed for
ACS01. This emission-quenching phenomenon confirms the interaction between the
compounds and ctDNA [13,14], and indicates that the methoxy moiety influences the DNA
interaction.
After to identify that ACS01 and ACS02 binding to ctDNA, a DNA fragmentation
assay was carried to verify if the compounds would cause fragmentation besides DNA
intercalation. The results demonstrated that the promastigote forms of the parasite treated
with ACS01 did not presented DNA fragmentation (data not shown), with no DNA bands
formation in any tested concentrations one, two and four times the IC50 compared to the
positive control (Amphotericin B), with results like the negative control (untreated
promastigotes). This result was similar to those described for 2-(acridin-9-ylmethylene)-N-
phenylhydrazinecarbothioamide against human breast adenocarcinoma cells where the
derivative did not promote DNA fragmentation of the treated cells [43]
.
Acridine derivatives are considered as DNA intercalators and inhibitor of essential
cell replicative enzymes. In addition, these derivatives also have the capacity to be dual
inhibitors, being potent in reducing resistance to multiple drugs through other mechanisms of
action [44]. These evidences lead us to believe that the mechanism of death of the parasite is
not associated with DNA fragmentation, but may be associated with the inhibition of
enzymes critical for its survival. However, these mechanisms of action should be investigated
in future tests.
3.2.3 PLS Studies
The dataset that was evaluated is composed by 16 thiophene derivatives - synthetic
intermediate and final compounds - (See supplementary material). Auto scaling preprocess
was applied to the PLS analysis using VolSurf descriptors as independent variables and pIC50
(-log IC50), value concentration necessary to inhibition growth of 50% promastigote cells of
L. amazonensis, as dependent variables. After the variable selection using the Variable
Important Projection the PLS generated significant statistical measures (leave-one-out cross-
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2
validation correlation coefficient, Qcv = 0.80; and the coefficient of determination, R2 =
0.89), using only two latent variables (LV). The first LV explains 78.6 % and LV 28.5%,
summarizing 87.1 % of the total variance of independent variables. Figure 3A shows that the
2
model containing three LVs presented an increment in R2 value, but the Qcv value began to
decrease.
Visualizing the plot of experimental pIC50 values versus recomputed (Figure 3B) and
predicted (Figure 3C) can be noticed that both depict the adjustment regarding a straight line
of the points used for the calibration of the model and validation.
The coefficients plot (Figure 4A) shows the 12 variables selected and together with
the loadings plot (Figure 4B), it is possible to notice that Amphiphilic moment (A) and
Hydrophobic Integy moment, ID1 and ID3, are the main descriptors with a negative
contribution to pIC50. The 3D Dry-Acceptor-Acceptor (DRDRAC) shows low positive
contribution to pIC50 values [29]. Analyzing the coefficients and the loadings plot of the
selected descriptors, one realizes that the model extract the physical-chemical features that
are undesirable for antileishmanial activity for this set.
The scores plot (Figure 5) differentiate the more active compounds AC02, ACS01 and
ACS02 (in the first quadrant) from most inactive compound 8 (6AMD) localized in the third
quadrant.
The Amphiphilic moment descriptor is defined as a vector pointing from the center of
the hydrophobic domain to the center of the hydrophilic domain. The vector length is
proportional to the strength of the amphiphilic moment. It is possible to check the association
among higher values of this descriptor for the inactive compounds such ACS, 6CN and
6AMD. The most active compounds AC02, ACS01 and ACS02 show lower values of
Amphiphilic moment descriptor, therefore the symmetric distribution of hydrophobic and
hydrophilic regions causes the near positioning of both center domain. Compound 6AMD
show two distinct hydrophilic and lipophilic regions on two different sides of the structure,
on the other hand compound ACS01 these regions are distributed in such way that there is not
formation of poles (Figure 6A) [29]
.
The Hydrophobic Integy moment ID1 and ID3 describe the unbalance between the
center of mass of a molecule and the barycenter of its hydrophobic region generated at -0.2
and at -0.6 kcal/mol respectively. The symmetry of the hydrophobic region of the molecule is
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significantly noticeable in the active compounds as it is possible to visualize in Figure 6B
comparing compound 6AMD and AC02. The 6AMD shows antisymmetric hydrophobic
[29]
region, unlike compound AC02
.
Descriptor DRDRAC is a 3D (three-dimensional) pharmacophore descriptor. This
kind of parameter is based on the triplets of pharmacophoric points descriptors. At first the
atoms (points) of a structure are classified as Dry, H-bond donor and H-bond acceptor, then
all possible triplet of distances between these atoms are generated. The DRDRAC descriptor
is the maximum area (over all possible conformers) of the triangles derived from triplet Dry-
Dry-Acceptor (H-bond acceptor).
3.2.4 Molecular Docking
For better understanding the chemical group influence on antileishmanial activity,
molecular docking studies were performed [45]. It is worthwhile to highlight that for a
neglected pathogen such as Leishmania, there are just a few representative structures of
enzymes on the PDB, and only some of them have multiple structures (e.g., pyruvate kinase
of L. mexicana) as demonstrated recently [46]. In our case, the analysis was focused on
pyruvate kinase enzyme. Docking results of pyruvate kinase show a clear correlation among
pIC50 values against L. amazonensis promastigote and MolDock energies of the set of
compounds excluding compound 9 (AC02) (r =-0.76, table 2 in supporting data). Analysing
the compounds with pIC50 values above of 4.29 show energies lower than -107 kJ/mol, being
minor than the ligand ABH (-99.18 kJ/mol). The results of the arginase show not only very
low correlation, but positive (r =0.57, table 2 in supporting data), in other words, more active
compounds have higher MolDock energies, therefore more instable interactions. Figure 7A
shows interactions between compound ACS01 and the active site of pyruvate kinase. Residue
asparagine 152 is responsible for the interaction with thiophene moiety. The compounds
ACS01 to ACS04 almost show the same interactions in the pocket (Figure 7B). Even the dock
energies of these compounds are similar, increasing the number of carbons of the fused ring
with thiophene hamper the hydrogen bond between the carbonitrile fragment with asparagine
152 (Figure 7C). Even the conformations of ACS05 and ACS06 aren’t the same, but show the
same hydrogen bond interactions of ether oxygen with residues asparagine 143, threonine
148, valine 149 and, carbonyl oxygen with serine 150 and asparagine 152. These results
indicated that pyruvate kinase enzyme can be a putative target of the thiophene-acridine
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derivatives synthesized in this work. Future analysis will be performed to better elucidate the
mechanism of action against leishmaniasis.
4. Conclusion
Eight new thiophene-acridine compounds were designed, synthesized with good
efficiency and well characterized by physicochemical and spectral data (1H and 13C NMR, IR
and HRMS). These compounds were investigated for their cytotoxic and antileishmanial
properties against promastigote forms of L. (L.) amazonensis. It was observed that the
evaluated substances, whose IC50 values were lower than 10 µM, presented better activity
than the reference drugs (tri and pentavalent antimony). The most active compounds were
ACS01 and ACS02, with IC50 of 9.60±3.19 and 10.95±3.96 µM, respectively. On the highest
concentration tested, none of the compounds tested presented cytotoxicity for human
erythrocytes, demonstrating that all compounds evaluated are less toxic for blood cells than
the reference drugs. The thiophene-acridine derivatives were more selective for the parasite
than for blood cells, with selectivity index better than those for reference drugs. The
derivatives ACS01 and ACS02 were efficient against parasites resistant to the trivalent
antimony. The activity antileishmanial of ACS01 was not associated to DNA fragmentation.
However, the compounds ACS01 and ACS02 presented in vitro DNA Kb of 104 M-1,
characteristic of DNA intercalating agents. There was a correlation between the lipophilicity
of the molecules and antileishmanial activity highlighted by the PLS data. Molecular docking
findings demonstrated correlation between the values of pIC50 and inhibition of pyruvate
kinase enzyme, where the presence of ring thiophene, especially with ester and nitrile
functions were important for interaction with the target. In addition, the methoxy-acridine
nucleus was able to intercalate into the DNA and also interacted with the enzyme pyruvate
kinase through its methoxyl, which indicates the duality of mechanism of action of the new
derivatives. Based on the results, it can be suggested that the thiophene-acridine compounds
can be considered promising for the development of new drugs for the treatment of
leishmaniasis.
Acknowledgements
We would like to extend our gratitude to our supporters and partners; UFPB, UFPE,
UEPB and CAPES for the financial and structural support for this study.
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Supplementary data
Supplementary data associated with this article can be found in the online version.
Figure Legends
Scheme 1. Synthesis of 2-amino-thiophene and the new hybrid thiophene-acridine
compounds. Reagents and conditions: (a) morpholine, sulfur, EtOH, 5 - 10°C, 3h; (b) 9-
acrinaldehyde (AC02), EtOH, 60ºC, 5h. (c) 6,9-dichloro-2-methoxy-acridine (ACS), EtOH,
AcOH, rt, 4h.
Figure 1. (A) Absorption spectra of ACS01 and ACS02 (30 µM) with increasing
concentrations of ctDNA. [DNA] = 0 (black), 10 (red), 20 (green), 40 (yellow), 60 (blue), 80
(pink) and 100 (light blue) µM. Arrow () refers to hyperchromic effect. (B) The plot of
[DNA]/(εa − εf) as function of DNA concentration as determined from the absorption spectral
data.
Figure 2. Fluorescence spectra of ACS01 (A) and ACS02 (B) (10 µM) with increasing
concentrations of ctDNA. [DNA] = 0 (black), 10 (red), 20 (green), 40 (yellow), 60 (blue), 80
(pink) and 100 (light blue) µM. Arrow () refers to hypochromic effect.
Figure 3. (A) Plot of r2 and q2 versus the number of latent variables (LV) used in PLS
models. (B) Plot of experimental versus recomputed values of pIC50 generated with the PLS
model by using 2 latent variables and 15 descriptors for 16 structures. (C) Plot of
experimental versus predicted values of pIC50 values by leave-one-out cross-validation
method.
Figure 4. (A) Coefficients plot generated from the selected PLS model. (B) Loadings plot
generated from the selected PLS model.
Figure 5. Scores plot of the selected PLS mode (see table 2 in supporting data).
Figure 6. (A) Hydrophobic (green) and hydrophilic (blue) regions of compounds 6AMD
(pIC50 = 3.37) and ACS01 (pIC50 = 5.01) generated at -1.0 kcal/mol and -3.0kcal/mol
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respectively. (B) Hydrophobic (green) regions of compounds 6AMD (pIC50 = 3.37) and
AC02 (pIC50 = 4.97) generated at -0.2 kcal/mol.
Figure 7. (A) Interactions between the active site of pyruvate kinase of L. mexicana and
ligand ABH and compound ACS01. (B) Interactions between the active site of pyruvate
kinase of L. mexicana and ligands ACS01 to ACS04. The distance between carbonitrile and
asparagine 152 increases with the number of carbons of the fused ring of thiophene. (C)
Interactions between the active site of pyruvate kinase of L. mexicana and ligands ACS05 and
ACS06.
Figure Legends for Supporting Information
Figure 1. NMR 1H of compound ACS01
Figure 2. NMR 13C of compound ACS01
Figure 3. IR of compound ACS01
Figure 4. HRMS of compound ACS01
Figure 5. NMR 1H of compound ACS02
Figure 6. NMR 13C of compound ACS02
Figure 7. IR of compound ACS02
Figure 8. HRMS of compound ACS02
Figure 9. NMR 1H of compound ACS03
Figure 10. NMR 13C of compound ACS03
Figure 11. IR of compound ACS03
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Figure 12. HRMS of compound ACS03
Figure 13. NMR 1H of compound ACS04
Figure 14. IR of compound ACS04
Figure 15. HRMS of compound ACS04
Figure 16. NMR 1H of compound ACS05
Figure 17. NMR 13C of compound ACS05
Figure 18. IR of compound ACS05
Figure 19. HRMS of compound ACS05
Figure 20. NMR 1H of compound ACS06
Figure 21. NMR 13C of compound ACS06
Figure 22. IR of compound ACS06
Figure 23. HRMS of compound ACS06
Figure 24. NMR 1H of compound ACS07
Figure 25. NMR 13C of compound ACS07
Figure 26. IR of compound ACS07
Figure 27. HRMS of compound ACS07
Figure 28 (Graph S1). Growth inhibition of the promastigote forms of Leishmania (L.)
amazonensis in presence of different concentrations of moieties 2-amino-tiophen and 6,9-
diclhoro-2-methoxi-acridine.
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Figure 29 (Graph S2). Growth inhibition of the promastigote forms of Leishmania
amazonensis in presence of different concentrations of hybrids derivatives thiophene-acridine
serial ACS.
Figure 30 (Graph S3). Growth inhibition of the promastigote forms of Leishmania (L.)
amazonensis in presence of different concentrations of the reference drugs: Glucantime®,
Trivalent antimonial and Amphotericin B.
Conflict of Interest
The authors declare that there was no competing interests.
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Table 1. Data on 50% growth inhibition of the parasite population (IC50±SEM), cellular
viability (HC50) and selectivity index (SI).
Compounds
ACS
n
-
R
SI
IC50 (M)
HC50 (M)
>1000
-
229.46±10.78
10.66±4.31
87.68±4.00
181.76±5.46
94.28±11.78
66.40±8.91
>4.35
>93.80
>11.40
>5.50
>10.60
>15.06
>11.95
>11.25
>2.33
NE
-
-
>1000
AC02
1
2
3
4
2
3
2
1
1
2
3
4
2
3
2
-
-C≡N
-C≡N
-C≡N
-C≡N
>1000
5CN
>1000
6CN
>1000
7CN
>1000
8CN
CO2C2H5 83.62±7.72
CO2C2H5 88.87±12.97
>1000
6EST
>1000
7EST
CONH2
-C≡N
-C≡N
-C≡N
-C≡N
-C≡N
429.02±18.67
>500
>1000
6AMD
ND
ACT01
9.60±3.19
10.95±3.96
32.26±5.50
68.97±4.85
>1000
>104.16
>91.32
>30.99
>14.49
>37.66
>31.44
>19.46
>1
ACS01
>1000
ACS02
>1000
ACS03
>1000
ACS04
CO2C2H5 26.55±3.98
CO2C2H5 31.80±5.86
>1000
ACS05
>1000
ACS06
CONH2
51.37±13.14
>1000
>1000
ACS07
-
-
-
>1000
Glucantime
Trivalent Antimony
Amphotericin B
-
14.77±0.52
0.19±0.09
565.62±68.99
24.25±2.52
38.29
-
127.63
ND – not determined; NE – not estimated
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Table 2. Antileishmanial activity of thiophene-acridine compound ACS01 and ACS02 on
promastigote forms of L. (L.) amazonensis, both sensitive and resistant to the trivalent
antimony.
IC50 (±SEM)
Sensitive strain
14.04±1.46
Resistant strain
14.83±0.44
Compound
ACS01
16.05±1.38
16.36±1.72
ACS02
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