citotoxidade em z morio

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Drug design and synthesis of new N-substituted-thienopyridine based on 
2-aminothiophene derivatives as antileishmanial agents☆
Vitória Gaspar Bernardo a,b,1, Rodrigo Santos Aquino de Araújo a,c,1, Natália Barbosa de Mélo a, 
Ricardo Eduardo Pereira Coutinho a, Julyanne Maria Saraiva de Sousa d, 
Maria Gabrielly Gonçalves da Silva Sousa d, Natália Ferreira de Sousa c, 
Wadja Feitosa dos Santos Silva e, Paulo Fernando da Silva Santos-Júnior e,f, 
Teresinha Gonçalves da Silva b, Eduardo René Pérez González g, 
Edeildo Ferreira da Silva Junior e, Marcus Tullius Scotti c, 
Anuraj Nayarisseri h, Klinger Antonio da Franca Rodrigues d,i, 
Francisco Jaime Bezerra Mendonça-Junior a,c,*
a Laboratory of Synthesis and Drug Delivery, Department of Biological Sciences, State University of Paraíba, João Pessoa 58071-160, Brazil
b Postgraduate Program in Therapeutic Innovation, Biological Sciences Center, Federal University of Pernambuco, Recife 50670-420, Brazil
c Postgraduate Program in Natural and Synthetic Bioactive Products, Federal University of Paraíba, João Pessoa 58051-900, Brazil
d Infectious Disease Laboratory, Campus Ministro Reis Velloso, Federal University of Parnaíba Delta, Parnaíba 64202-020, Brazil
e Group of Research on Chemical and Biological Chemistry, Institute of Chemistry and Biotechnology, Federal University of Alagoas, 57072-970 Maceió, Alagoas, Brazil
f Laboratory of Organic Synthesis and Medicinal Chemistry, Post-Graduation Program in Natural Sciences and Biotechnology, Education and Health Center, Biology and 
Chemistry Academic Unit, Federal University of Campina Grande (UFCG), 58175-000 Cuité, Paraíba, Brazil
g Fine Organic Chemistry Lab, School of Sciences and Technology, São Paulo State University (UNESP), 19060-080 Presidente Prudente, São Paulo, Brazil
h In Silico Research Laboratory, Eminent Biosciences, 91-A, Sector-A, Mahalakshmi Nagar, Indore 452010, Madhya Pradesh, India
i Department of Pathology, Dom Delgado University City, Federal University of Maranhão, São Luiz 65065-545, Brazil
A R T I C L E I N F O
Keywords:
2-Aminothiphene
Thienopyridine
Antileishmanial agents
ADMET properties
Molecular docking
Acute toxicity in Zophobas morio
A B S T R A C T
Leishmaniasis is a neglected tropical disease with limited and often toxic treatment options. This study explores 
the antileishmanial potential of N-substituted thieno[2,3-c]pyridine derivatives through combined in vitro and in 
silico approaches. Initially, N-Boc-thieno[2,3-c]pyridine (2a–i) and N-Boc-thieno[2,3-b]pyridine (1a–f) de-
rivatives were synthesized and evaluated against Leishmania amazonensis, L. braziliensis, L. infantum, and RAW 
264.7 macrophages. Only N-Boc-thieno[2,3-c]pyridine derivatives with substituted nitrogen at position 6 (2b, 
2c, 2f, 2g) were active (IC₅₀to those of the reference drug amphotericin B, but with a 
selectivity index (SI) more than 250 times higher, making it a very 
promising drug candidate for leishmaniasis treatment. Additionally, the 
physicochemical and the pharmacokinetic (ADME) prediction studies 
indicated that compound 3f presents favorable drug-like properties, not 
violating Lipinski’s rule and Weber parameters, presenting a high like-
lihood of being absorbed by the gastrointestinal tract, and low suscep-
tibility to cross the blood-brain barrier (BBB). These findings suggest 
that analog 3f is a potential drug candidate for leishmaniasis treatment, 
or at least it is an interesting lead candidate for further optimization, and 
elaboration of new drug libraries.
Molecular docking analyses demonstrated that N-substituted thieno 
[2,3-c]pyridine derivatives possess promising binding profiles, exhibit-
ing more favorable MolDock scores than their respective co-crystallized 
ligands across the three targets investigated: Nucleoside Diphosphate 
Kinase (NDK), Dihydroorotate Dehydrogenase (DHODH), and Sterol 
14α-Demethylase (CYP51). The host–guest complexes were further sta-
bilized by multiple and specific interactions with catalytic residues in 
the active sites of these enzymes, including the heme-binding pocket in 
CYP51, reinforcing their potential as enzyme inhibitors. Complementary 
molecular dynamics (MD) simulations revealed that the ligand–protein 
complexes exhibited low and stable Root Mean Square Deviation 
(RMSD) values over 100 ns, indicating significant conformational sta-
bility. Furthermore, the Radius of gyration (Rg) profiles showed that 
most complexes underwent a modest yet consistent level of structural 
compaction, suggesting enhanced structural integrity upon ligand 
binding. Root Mean Square Fluctuation (RMSF) analyses further 
demonstrated that several derivatives reduced the local flexibility of 
residues—particularly those associated with the catalytic regions of the 
target proteins—supporting the hypothesis of tight and specific binding.
Taken together, these findings strongly highlight the potential of N- 
substituted thieno[2,3-c]pyridine derivatives as promising scaffolds for 
further development of novel antileishmanial agents.
4. Experimental procedures
4.1. Chemistry section
Chemicals used for the synthesis were reagent grade purchased from 
Sigma-AldrichⓇ (St. Louis, MO, USA) and were used without purifica-
tion. Reactions were monitored by thin-layer chromatography (TCL). 
TLC plates were examined under 254 and 360 nm ultraviolet lights. 
Melting points were measured with a fusiometer FisatomⓇ, model 431D. 
Purity was determined by a HPLC Shimadzu model LC-20A Prominence, 
with a Promosil C18 column from Agela Technologies using a binary 
mobile phase containing acetonitrile/water in the ratio of 85/15. 
(chromatograms are available in supplementary material).1H and 13C 
NMR were recorded on a BrukerⓇ Advance 100 or 400 MHz spec-
trometer and on a BrukerⓇ Advance 125 or 600 MHz spectrometer in 
DMSO‑d6 or CDCl3 solutions. Chemical shifts (ẟ) are recorded in parts 
per million (ppm) downfield from TMS as internal standard. Coupling 
constants (J) were expressed in Hertz (Hz). High-Resolution Mass 
Spectrometry data were obtained on a mass spectrometer MicrOTOF-Q 
BrukerⓇ equipped with ESI in positive ionization source, scan range 
between 50 and 1000 m/z. All the spectra are available in the supple-
mentary data file.
4.2. General synthesis procedure
The synthesis procedure for Gewald adducts (1, 2, and 3) was per-
formed by adding their respective cyclic ketones (N-Boc-3-piperidone, 
N-Boc-4-piperidone, or N-benzyl-4-piperidone) (1 eq., 0.02 mol), sulfur 
(1 eq.) and malononitrile (1 eq.) in a round-bottom flask, followed by the 
addition of ethanol (20 mL). The reaction flask was then placed in an ice 
bath, under stirring, for the dropwise addition of morpholine (1 eq.). 
After dripping, the reaction remained stirring in an ice bath for 30 min 
and then at room temperature for an additional 4 h. The reactions were 
monitored by thin-layer chromatography. Adducts 1, 2, and 3, after 
formation, precipitated in the ethanolic medium and were therefore 
purified by filtration.
Solids of 1, 2, and 3 (0.1 g, 1 eq.) were used, without further puri-
fication, for condensation reactions with aromatic aldehydes 
(substituted indole and benzyl aldehydes) (0.7 eq.). The addition of 
Gewald adducts and their respective aromatic aldehydes in a round- 
bottom flask was followed by the addition of ethanol (4 mL) and the 
dripping of a catalytic amount of acetic acid (10 drops). The reaction 
flask was then kept under stirring at room temperature overnight. The 
condensation reactions were monitored by thin-layer chromatography. 
The products formed (1a-f, 2a-i, and 3a-i) precipitated in the ethanolic 
medium and were therefore initially purified by filtration. When 
necessary, additional purification was performed by recrystallization in 
ethanol.
Compound 2a had their structure confirmed by comparison with 
NMR spectra data available in the literature (23). All the other com-
pounds were characterized and are described below.
4.2.1. 7-N-(Carboxylate tert-butyl ester)-2-(((5′-Bromo-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitrile 
(1a)
Yield: 43.7 %, Appearance: Yellow solid, Rf: 0.42 (6:4 – Hexane/ 
Ethyl acetate), mp.: 223–224 ◦C, Purity by HPLC = 99.90 %. Molecular 
Formula: C22H21BrN4O2S. Molecular Mass: 484.0569 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 12.18 (s, 1H, NH (1′)), 8.79 (s, 1H, N––CH (15)), 
8.57 (d, J = 1.9 Hz, 1H, CHAr(4′)), 8.16 (s, 1H, CHAr (2′)), 7.49 (d, J =
8.6 Hz, 1H, CHAr (7′)), 7.40 (dd, J = 8.6, 2.0 Hz, 1H, CHAr(6′)), 3.79 (s, 
2H, CH2 (6)), 2.63 (t, J = 6.2 Hz, 2H, CH2 (4)), 1.98 (s, 2H, CH2 (5)), 
1.50 (s, 9H, CH3 (11–13)). 13C NMR APT: (100 MHz, DMSO‑d6): δ 
156.42, 153.93, 137.09, 136.59, 130.17, 126.71, 126.39, 124.80, 
119.24, 115.39, 114.87, 114.78, 114.49, 102.17, 82.50, 45.05, 28.27, 
22.27, 21.41. LC/MS (ESI): Calculated to [M + H]+: 485.0629 g/mol; 
Found to [M + H]+: 485.0609 g/mol.
4.2.2. 7-N-(Carboxylate tert-butyl ester)-2-((4′-nitrobenzylidene)amino)- 
4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitrile (1b)
Yield: 53.5 %, Appearance: Orange solid, Rf: 0.64 (7:3 – Hexane/ 
Ethyl acetate), mp.: > 300 ◦C, Purity by HPLC = 99.93 %. Molecular 
Formula: C20H20N4O4S. Molecular Mass: 412.1205 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 8.80 (s, 1H, N––CH (15)), 8.35 (d, J = 8.8 Hz, 
2H, CHAr(3′, 5′)), 8.14 (d, J = 8.8 Hz, 2H, CHAr(2′, 6′)), 3.81 (s, 2H, CH2 
(6)), 2.65 (t, J = 6.2 Hz, 2H, CH2 (4)), 2.15 (s, 2H, CH2 (5)), 1.51 (s, 9H, 
CH3 (11–13)). 13C NMR APT (100 MHz, DMSO‑d6): δ 155.70, 152.14, 
151.75, 149.21, 141.38, 134.69, 130.04, 124.63, 120.74, 114.49, 
107.54, 83.10, 45.17, 28.20, 22.14, 21.08. LC/MS (ESI): Calculated to 
[M + H]+: 413.1205 g/mol; Found to [M + H]+: 413.1281 g/mol.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
15 
4.2.3. 7-N-(Carboxylate tert-butyl ester)-2-(((1H-indol-3′-yl)methylene) 
amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitrile (1c)
Yield: 35.3 %, Appearance: Yellow solid, Rf: 0.59 (6:4 – Hexane/ 
Ethyl acetate), mp.: 210–211 ◦C, Purity by HPLC = 99.88 %. Molecular 
Formula: C22H22N4O2S. Molecular Mass: 406.1463 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 12.04 (s, 1H, NH (1′)), 8.79 (s, 1H, N––CH (15)), 
8.39 (d, J = 7.0 Hz, 1H, CHAr (4′)), 8.13 (s, 1H, CHAr (2′)), 7.52 (dd, J =
6.9, 1.1 Hz, 1H, CHAr (7′)), 7.26 (pd, J = 7.1, 1.3 Hz, 2H, CHAr (5′, 6′)), 
3.78 (s, 2H, CH2 (6)), 2.62 (t, J = 6.1 Hz, 2H, CH2 (4)), 1.97 (s, 2H, CH2 
(5)), 1.51 (s, 9H, CH3 (11–13)). 13C NMR APT (100 MHz, DMSO‑d6): δ 
157.04, 154.39, 152.13, 137.91, 136.46,129.76, 124.96, 123.91, 
122.48, 122.12, 119.16, 115.55, 115.06, 112.85, 101.59, 82.46, 45.04, 
28.28, 22.24, 21.43. LC/MS (ESI): Calculated to [M + H]+: 407.1463 g/ 
mol; Found to [M + H]+: 407.1526 g/mol.
4.2.4. 7-N-(Carboxylate tert-butyl ester)-2-(((5′-methoxy-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitrile 
(1d)
Yield: 35.6 %, Appearance: Yellow solid, Rf: 0.48 (6:4 – Hexane/ 
Ethyl acetate), mp.: 220–222 ◦C, Purity by HPLC = 99.82 %. Molecular 
Formula: C23H24N4O3S. Molecular Mass: 436.1569 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 11.91 (s, 1H, NH (1′))), 8.78 (s, 1H, N––CH 
(15)), 8.04 (s, 1H, CHAr (2′)), 8.04 (s, 1H, CHAr (4′)), 7.40 (d, J = 8.8 Hz, 
1H, CHAr (7′)), 6.89 (dd, J = 8.8, 2.6 Hz, 1H, CHAr (6′)), 3.81 (s, 3H, 
OCH3), 3.78 (s, 2H, CH2 (6), 2.62 (t, J = 6.1 Hz, 2H, CH2 (4)), 1.97 (s, 
2H, CH2 (5)), 1.51 (s, 9H, CH3 (11–13)). 13C NMR APT (100 MHz, 
DMSO‑d6): δ 156.89, 155.76, 153.83, 152.09, 136.05, 132.54, 129.73, 
125.84, 118.91, 115.59, 114.93, 113.71, 113.50, 104.35, 101.93, 82.44, 
55.59, 45.05, 28.29, 22.23, 21.44. LC/MS (ESI): Calculated to [M + H]+: 
437.1569 g/mol; Found to [M + H]+: 437.1640 g/mol.
4.2.5. 7-N-(Carboxylate tert-butyl ester)-2-((3′-nitrobenzylidene)amino)- 
4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitrile (1e)
Yield: 57.8 %, Appearance: Yellow solid, Rf: 0.41 (8:2 – Hexane/ 
Ethyl acetate), mp.: 201–204 ◦C, Purity by HPLC = 99.90 %. Molecular 
Formula: C20H20N4O4S. Molecular Mass: 412.1205 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 8.88 (s, 1H, N––CH (15)), 8.73 (s, 1H, CHAr (2′)), 
8.35 (t, J = 9.2 Hz, 2H, CHAr(4′, 6′)), 7.82 (t, J = 8.0 Hz, 1H, CHAr (5′)), 
3.81 (s, 2H, CH2 (6)), 2.66 (t, J = 6.1 Hz, 2H, CH2 (4)), 1.99 (s, 2H, CH2 
(5)), 1.52 (s, 9H, CH3 (11–13)). 13C NMR APT (100 MHz, DMSO‑d6): δ 
156.19, 151.95, 148.86, 137.44, 134.90, 134.23, 131.08, 126.31, 
123.29, 120.60, 114.45, 106.77, 100.31, 99.99, 45.18, 28.29, 22.26, 
21.22. LC/MS (ESI): Calculated to [M + H]+: 413.1205 g/mol; Found to 
[M + H]+: 413.1277 g/mol.
4.2.6. 7-N-(Carboxylate tert-butyl ester)-2-(((6′-cyano-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitrile 
(1f)
Yield: 69.3 %, Appearance: Yellow solid, Rf: 0.35 (6:4 – Hexane/ 
Ethyl acetate), mp.: 229–230 ◦C, Purity by HPLC = 99.88 %. Molecular 
Formula: C23H21N5O2S. Molecular Mass: 431.1416 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 12.47 (s, 1H, NH (1′)), 8.83 (s, 1H, N––CH (15)), 
8.50 (d, J = 8.2 Hz, 1H, CHAr (4′)), 8.35 (s, 1H, CHAr (2′)), 8.04 (s, 1H, 
CHAr (7′)), 7.61 (dd, J = 8.2, 1.4 Hz, 1H, CHAr (5′)), 3.78 (s, 2H, CH2 (6)), 
2.62 (t, J = 6.1 Hz, 2H, CH2 (4)), 1.97 (s, 2H, CH2 (5)), 1.50 (s, 9H, CH3 
(11–13)). 13C NMR APT (100 MHz, DMSO‑d6): δ 156.07, 153.84, 
152.13, 139.16, 136.78, 130.50, 128.09, 124.87, 123.17, 120.48, 
119.32, 117.77, 115.33, 115.19, 105.37, 102.56, 82.56, 45.01, 28.27, 
22.20, 21.38. LC/MS (ESI): Calculated to [M + H]+: 432.1416 g/mol; 
Found to [M + H]+: 432.1475 g/mol.
4.2.7. 6-N-(Carboxylate tert-butyl ester)-2-((4′-Nitrobenzylidene)amino)- 
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (2b)
Yield: 89.0 %, Appearance: Dark yellow solid, Rf: 0.32 (6:4 – Hex-
ane/Ethyl acetate), mp.: 223–226 ◦C, Purity by HPLC = 99.85 %. Mo-
lecular Formula: C20H20N4O4S. Molecular Mass: 412.1205 g/mol. 1H 
NMR (600 MHz, DMSO‑d6): δ 8.84 (s, 1H, N––CH (15)); 8.39 (d, 2H, J =
7.62 Hz, CHAr (3′, 5′)); 8.22 (d, 2H, J = 7.62 Hz, CHAr (2′, 6′)); 4,58 (s, 
2H, CH2 (7)); 3.66 (s, 2H, CH2 (4)); 2.70 (m, 2H, CH2 (5)); 1.44 (s, 9H, 
CH3 (11–13)). 13C NMR DEPT (150 MHz, DMSO‑d6): δ 164.13, 160.02, 
159.71, 154.29, 149.84, 140.70, 134.21, 130.77, 124.65, 116.17, 
114.02, 107.84, 80.17, 28.50, 24.40. LC/MS (ESI): Calculated to [M +
H]+: 413.1205 g/mol; Found to [M + H]+: 413.1256 g/mol.
4.2.8. 6-N-(Carboxylate tert-butyl ester)-2-(((1H-indol-3′-yl)methylene) 
amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (2c)
Yield: 44.0 %, Appearance: Yellow solid, Rf: 0.41 (6:4 – Hexane/ 
Ethyl acetate), mp.: 231–233 ◦C, Purity by HPLC = 99.89 %. Molecular 
Formula: C22H22N4O2S. Molecular Mass: 406.1463 g/mol. 1H NMR 
(600 MHz, DMSO‑d6): δ 12.15 (sl, 1H, NH (1′)); 8.74 (s, 1H, N––CH 
(15)); 8.38 (d, 1H, J = 7.26 Hz, CHAr (4′)); 8.23 (s, 1H, CHAr (2′)); 7.53 
(d, 1H, J = 3.24 Hz, CHAr (7′)); 7.29 (m, 2H, CHAr (5′, 6′); 4.50 (s, 2H, 
CH2 (7)); 3.65 (s, 2H, CH2 (4)); 2.65 (m, 2H, CH2 (5)); 1.44 (s, 9H, CH3 
(11–13)). 13C NMR DEPT (150 MHz, DMSO‑d6): δ 164.66, 156.56, 
154.33, 138.00, 137.58, 132.75, 126.10, 125.05, 124.10, 122.50, 
122.39, 115.05, 114.82, 112.95, 102.47, 80.04, 28.51, 24.46. LC/MS 
(ESI): Calculated to [M + H]+: 407.1463 g/mol; Found to [M + H]+: 
407.1526 g/mol.
4.2.9. 6-N-(Carboxylate tert-butyl ester)-2-(((5′-methoxy-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile 
(2d)
Yield: 36.0 %, Appearance: Yellow solid, Rf: 0.44 (6:4 – Hexane/ 
Ethyl acetate), mp.: 253–255 ◦C, Purity by HPLC = 99.87 %. Molecular 
Formula: C23H24N4O3S. Molecular Mass: 436.1569 g/mol. 1H NMR 
(600 MHz, DMSO‑d6): δ 12.02 (sl, 1H, NH (1′)); 8.72 (s, 1H, N––CH 
(15)); 8.15 (s, 1H, CHAr (2′)); 8.03 (s, 1H, CHAr (4′)); 7.41 (d, 1H, J =
8.64 Hz, CHAr (7′)); 6.90 (d, 1H, J = 8.58 Hz, CHAr (6′)); 4.50 (s, 2H, CH2 
(7)); 3.82 (s, 3H, OCH3); 3.65 (s, 2H, CH2 (4)); 2.65 (m, 2H, CH2 (5)); 
1.44 (s, 9H, CH3 (11–13)). 13C NMR DEPT (150 MHz, DMSO‑d6): δ 
164.48, 156.02, 156.00, 154.33, 137.23, 132.62, 132.51, 125.96, 
115.14, 114.46, 113.87, 113.62, 104.57, 102.80, 80.04, 55.66, 28.51, 
24.45. LC/MS (ESI): Calculated to [M + H]+: 437.1569 g/mol; Found to 
[M + H]+: 437.1634 g/mol.
4.2.10. 6-N-(Carboxylate tert-butyl ester)-2-((3′-nitrobenzylidene)amino)- 
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (2e)
Yield: 46.0 %, Appearance: Dark yellow solid, Rf: 0.60 (6:4 – Hex-
ane/Ethyl acetate), mp.: 213–216 ◦C, Purity by HPLC = 99.84 %. Mo-
lecular Formula: C20H20N4O4S. Molecular Mass: 412.1205 g/mol. 1H 
NMR (600 MHz, DMSO‑d6): δ 8.89 (s, 1H, N––CH (15)); 8.79 (s, 1H, 
CHAr (2′)); 8.41 (t, 2H, J = 7.56 Hz, CHAr (4′, 6′)); 7.86 (t, 1H, J = 7.86 
Hz, CHAr (5′)); 4.58 (s, 2H, CH2 (7)); 3.66 (s, 2H, CH2 (4)); 2.70 (m, 2H, 
CH2 (5)); 1.44 (s, 9H, CH3 (11–13)). 13C NMR DEPT (150 MHz, 
DMSO‑d6): δ 160.14, 159.93, 154.30, 148.79, 136.78, 135.34, 134.03, 
131.22, 127.11, 124.08, 114.07, 107.30, 80.16, 28.50, 24.40. LC/MS 
(ESI): Calculated to [M + H]+: 413.1205 g/mol; Found to [M + H]+: 
413.1264 g/mol.
4.2.11. 6-N-(Carboxylate tert-butyl ester)-2-(((6′-cyano-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile 
(2f)
Yield: 65.0 %, Appearance: Yellow solid, Rf: 0.30 (6:4 – Hexane/ 
Ethyl acetate), mp.: 242–244 ◦C, Purity by HPLC = 98.60 %. Molecular 
Formula: C23H21N5O2S. Molecular Mass: 431.1416 g/mol. 1H NMR 
(600 MHz, DMSO‑d6): δ 12.56 (sl, 1H, NH (1′)); 8.77 (s, 1H, N––CH 
(15)); 8.49 (d, 1H, J = 7.98 Hz, CHAr (4′)); 8.45 (s, 1H, CHAr (2′)); 8.04 (s, 
1H, CHAr (7′)); 7.63 (d, 1H, J = 8.41 Hz, CHAr (5′)); 4.51 (s, 2H, CH2 (7)); 
3.65 (s, 2H, CH2 (4)); 2.65 (s, 2H, CH2 (5)); 1.43 (s, 9H, CH3 (11–13)). 
13C NMR DEPT (150 MHz, DMSO‑d6): δ 163.80, 156.26, 154.32, 140.23, 
136.88, 132.95, 128.18, 126.96, 125.15, 123.18, 120.34, 117.85, 
116.16, 114.86, 114.82, 105.64, 103.39, 80.07, 28.51, 24.44. LC/MS 
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
16 
(ESI): Calculated to [M + H]+: 432.1416 g/mol; Found to [M + H]+: 
432.1469 g/mol.
4.2.12. 6-N-(Carboxylate tert-butyl ester)-2-(((5′-cyano-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile 
(2g)
Yield: 55.0 %, Appearance: Yellow solid, Rf: 0.23 (6:4 – Hexane/ 
Ethyl acetate), mp.: 280–282 ◦C, Purity by HPLC = 99.87 %. Molecular 
Formula: C23H21N5O2S. MolecularMass: 431.1416 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 12.54 (sl, 1H, NH (1′)); 8.77 (s, 1H, N––CH 
(15)); 8.74 (m, 1H, CHAr (4′)); 8.39 (s, 1H, CHAr (2′)); 7.70 (dd, 1H, J =
8.40, 0.44 Hz, CHAr (6′)); 7.65 (dd, 1H, J = 8.40, 1.56 Hz, CHAr (7′)); 
4.50 (s, 2H, CH2 (7)); 3.65 (t, 2H, J = 5.52 Hz, CH2 (4)); 2.65 (s, 2H, CH2 
(5)); 1.44 (s, 9H, CH3 (11–13)). 13C NMR DEPT (125 MHz, DMSO‑d6): δ 
163.58, 155.91, 154.28, 139.74, 139.05, 132.84, 127.40, 127.14, 
126.87, 124.87, 120.61, 114.89, 114.85, 114.42, 104.46, 103.75, 80.06, 
28.48, 24.41. LC/MS (ESI): Calculated to [M + H]+: 432.1416 g/mol; 
Found to [M + H]+: 432.1484 g/mol.
4.2.13. 6-N-(Carboxylate tert-butyl ester)-2-(((7′-azo-1H-indol-3′-yl) 
methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile 
(2h)
Yield: 46.0 %, Appearance: Dark yellow solid, Rf: 0.24 (6:4 – Hex-
ane/Ethyl acetate), mp.: 250–252 ◦C, Purity by HPLC = 99.62 %. Mo-
lecular Formula: C21H21N5O2S. Molecular Mass: 407.1416 g/mol. 1H 
NMR (600 MHz, DMSO‑d6): δ 12.65 (sl, 1H, NH (1′)); 8.72 (s, 1H, N––CH 
(15)); 8.64 (d, 1H, J = 7.80 Hz, CHAr (4′)); 8.38 (d, 1H, J = 4.36 Hz, CHAr 
(6′)); 8.36 (s, 1H, CHAr (2′)); 7.33 (dd, 1H, J = 7.56, 4.80 Hz, CHAr (5′)); 
4.51 (s, 2H, CH2 (7)); 3.54 (s, 2H, CH2 (4)); 2.65 (m, 2H, CH2 (5)); 1.44 
(s, 9H, CH3 (11–13)). 13C NMR DEPT (150 MHz, DMSO‑d6): δ 163.91, 
156.46, 154.32, 150.32, 145.32, 137.45, 132.87, 130.56, 118.55, 
117.51, 114.86, 113.43, 103.30, 80.04, 28.51, 24.43. LC/MS (ESI): 
Calculated to [M + H]+: 408.1416 g/mol; Found to [M + H]+: 408.1486 
g/mol.
4.2.14. 6-N-(Carboxylate tert-butyl ester)-2-((2′-nitrobenzylidene)amino)- 
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (2i)
Yield: 44.0 %, Appearance: Dark yellow solid, Rf: 0.58 (6:4 – Hex-
ane/Ethyl acetate), mp.: 215–218 ◦C, Purity by HPLC = 99.80 %. Mo-
lecular Formula: C20H20N4O4S. Molecular Mass: 412.1205 g/mol. 1H 
NMR (400 MHz, DMSO‑d6): δ 8.96 (s, 1H, N––CH (15)); 8.21 (dd, 1H, J 
= 7.60, 1.20 Hz, CHAr (3′)); 8.16 (dd, 1H, J = 8.08, 0.96 Hz, CHAr (6′)); 
7.92 (t, 1H, J = 7.40 Hz, CHAr (5′)); 7.92 (dt, 1H, J = 7.92, 1.40 Hz, CHAr 
(4′)); 4.58 (s, 2H, CH2 (7)); 3.65 (t, 2H, J = 5.36 Hz, CH2 (4)); 2.69 (m, 
2H, CH2 (5)); 1.43 (s, 9H, CH3 (11–13)). 13C NMR DEPT (125 MHz, 
DMSO‑d6): δ 160.09, 158.07, 154.22, 149.78, 134.53, 134.21, 133.36, 
131.34, 129.98, 129.33, 125.43, 113.95, 107.47, 80.13, 28.47, 24.40. 
LC/MS (ESI): Calculated to [M + H]+: 413.1205 g/mol; Found to [M +
H]+: 413.1264 g/mol.
4.2.15. 6-N-(Benzylidene)-2-(((5′-bromo-1H-indol-3′-yl)methylene) 
amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3a)
Yield: 64.0 %, Appearance: Yellow solid, Rf: 0.15 (7:3 – Hexane/ 
Ethyl acetate), mp.: 202–204 ◦C, Purity by HPLC = 99.87 %. Molecular 
Formula: C24H19BrN4S. Molecular Mass: 474.0514 g/mol. 1H NMR (400 
MHz, DMSO‑d6): δ 12.27 (sl, 1H, NH (1′)); 8.71 (s, 1H, N––CH (16)); 8.57 
(d, 1H, J = 1.96 Hz, CHAr (4′)); 8.25 (d, 1H, J = 2.96 Hz, CHAr (7′)); 7.50 
(d, 1H, J = 8.6 Hz, CHAr (2′)); 7.41 (dd, 1H, J = 2.0, 2.0 Hz, CHAr (6′)); 
7.33 (m, 4H, CHAr (10, 11, 13, 14); 7.29 (m, 1H, CHAr (12)); 3.70 (s, 2H, 
CH2 (7)); 3.55 (s, 2H, CH2 (8)); 2.79 (m, 2H, CH2 (4)); 2.67 (m, 2H, CH2 
(5)). 13C NMR APT (100 MHz, DMSO‑d6): δ 24.40, 49.15, 51.37, 60.98, 
114.19, 114.97, 115.04, 124.85, 126.57, 126.77, 127.62, 128.79, 
128.79, 129.27, 129.27, 132.59, 136.65, 138.10, 155.82, 163.59. LC/ 
MS (ESI): Calculated to [M + H]+: 475.0514 g/mol; Found to [M + H]+: 
475.0584 g/mol.
4.2.16. 6-N-(Benzylidene)-2-((4′-nitrobenzylidene)amino)-4,5,6,7- 
tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3b)
Yield: 48.0 %, Appearance: Yellow solid, Rf: 0.38 (7:3 – Hexane/ 
Ethyl acetate), mp.: 170–172 ◦C, Purity by HPLC = 96.38 %. Molecular 
Formula: C22H18N4O2S. Molecular Mass: 402.1150 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 8.80 (s, 1H, N––CH (16)); 8.37 (dt, 2H, J = 2.0, 
1.68, 1.8, 2.08 Hz, CHAr (2′, 5′)); 8.19 (dt, 2H, J = 2.08, 1.84, 1.76, 2.12 
Hz, CHAr (2′, 6′)); 7.36 (m, 4H, CHAr (10, 11, 13, 14)); 7.29 (m, 1H, CHAr 
(12)); 3.72 (s, 2H, CH2 (7)); 3.63 (s, 2H, CH2 (8)); 2.80 (m, 2H, CH2 (4)); 
2.70 (m, 2H, CH2 (5)). 13C NMR APT (100 MHz, DMSO‑d6): δ 24.23, 
48.91, 51.40, 60.82, 107.95, 114.23, 124.66, 124.66, 127.65, 128.81, 
128.81, 129.26, 129.26, 130.66, 130.66, 132.77, 134.05, 138.44, 
140.75, 149.68, 159.11, 159.49. LC/MS (ESI): Calculated to [M + H]+: 
403.1150 g/mol; Found to [M + H]+: 403.1234 g/mol.
4.2.17. 6-N-(Benzylidene)-2-(((1H-indol-3′-yl)methylene)amino)-4,5,6,7- 
tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3c)
Yield: 75.0 %, Appearance: Yellow solid, Rf: 0.24 (7:3 – Hexane/ 
Ethyl acetate), mp.: 183–185 ◦C, Purity by HPLC = 99.84 %. Molecular 
Formula: C24H20N4S. Molecular Mass: 396.1409 g/mol. 1H NMR (400 
MHz, DMSO‑d6): δ 12.14 (sl, 1H, NH (1′)); 8.71 (s, 1H, N––CH (16)); 8.40 
(dd, 1H, J = 2.32, 1.76 Hz, CHAr (5′)); 8.22 (d, 1H, J = 2.88 Hz, CHAr 
(2′)); 7.52 (dt, 1H, J = 1.36, 4.92, 0.28, 0.76 Hz, CHAr (6′)); 7.35 (m, 4H, 
CHAr (10, 11, 13, 14)); 7.28 (m, 3H, CHAr (12, 4′, 7′)); 3.70 (s, 2H, CH2 
(7)); 3.54 (s, 2H, CH2 (8)); 2.79 (m, 2H, CH2 (4)); 2.66 (m, 2H, CH2 (5)). 
13C NMR APT (100 MHz, DMSO‑d6): δ 24.41, 49.18, 51.40, 61.00, 
102.52, 112.95, 114.79, 115.30, 122.36, 122.52, 124.08, 125.02, 
127.30, 127.61, 128.79, 128.79, 129.26, 129.26, 132.53, 137.45, 
138.58, 156.18, 164.16. LC/MS (ESI): Calculated to [M + H]+: 
397.1409 g/mol; Found to [M + H]+: 397.1492 g/mol.
4.2.18. 6-N-(Benzylidene)-2-(((5′-methoxy-1H-indol-3′-yl)methylene) 
amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3d)
Yield: 58.0 %, Appearance: Yellow solid, Rf: 0.23 (7:3 – Hexane/ 
Ethyl acetate), mp.: 211–213 ◦C, Purity by HPLC = 99.91 %. Molecular 
Formula: C25H22N4OS. Molecular Mass: 426.1514 g/mol. 1H NMR (400 
MHz, DMSO‑d6): δ 12.01 (sl, 1H, NH (1′)); 8.70 (s, 1H, N––CH (16)); 8.13 
(d, 1H, J = 3.08 Hz, CHAr (4′)); 8.04 (d, 1H, J = 2.52 Hz, CHAr (7′)); 7.41 
(d, 1H, J = 8.8 Hz, CHAr (2′)); 7.35 (m, 4H, CHAr (10, 11, 13, 14)); 7.29 
(m, 1H, CHAr (12)); 6.91 (dd, 1H, J = 2.56, 2.6 Hz, CHAr (6′)); 3.82 (s, 
3H, OCH3); 3.69 (s, 2H, CH2 (7)); 3.53 (s, 2H, CH2 (8)); 2.78 (m, 2H, CH2 
(4)); 2.65 (m, 2H, CH2 (5)). 13C NMR APT (100 MHz, DMSO‑d6): δ 24.39, 
49.18, 51.42, 55.61, 61.01, 102.85, 104.46, 113.62, 113.85, 114.66, 
115.38, 125.93, 127.19, 127.60, 128.78, 128.78, 129.27, 129.27, 
132.27, 132.57, 137.05, 138.56, 155.61, 155.94, 163.97. LC/MS (ESI): 
Calculated to [M + H]+: 427.1514 g/mol; Found to [M + H]+: 427.1571 
g/mol.
4.2.19. 6-N-(Benzylidene)-2-((3′-nitrobenzylidene)amino)-4,5,6,7- 
tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3e)
Yield: 51.0 %, Appearance: Yellow solid, Rf: 0.49 (7:3 – Hexane/ 
Ethyl acetate), mp.: 163–165 ◦C, Purity by HPLC = 99.79 %. Molecular 
Formula: C22H18N4O2S. Molecular Mass: 402.1150 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 8.84 (s, 1H, N––CH (16)); 8.76 (t, 1H, J = 1.92, 
1.8 Hz, CHAr (2′)); 8.39 (m, 2H, CHAr (4′, 6′)); 7.84 (t, 1H, J = 8.0, 7.96 
Hz, CHAr (5′)); 7.36 (m, 4H, CHAr (10, 11, 13, 14)); 7.29 (m, 1H, CHAr 
(12)); 3.72 (s, 2H, CH2 (7)); 3.62 (s, 2H, CH2 (8)); 2.80 (m, 2H, CH2 (4)); 
2.69 (m, 2H, CH2 (5)). 13C NMR APT (100 MHz, DMSO‑d6): δ 24.24, 
48.93, 51.38, 60.85, 107.40, 114.28, 123.98, 127.02, 127.64, 128.80, 
128.80, 129.26, 129.26, 131.19, 132.20, 133.84, 135.28, 136.79, 
138.45, 148.69, 159.31, 159.59. LC/MS (ESI): Calculated to [M + H]+: 
403.1150 g/mol; Found to [M + H]+: 403.1216 g/mol.
4.2.20. 6-N-(Benzylidene)-2-(((6′-cyano-1H-indol-3′-yl)methylene) 
amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3f)
Yield: 55.0 %, Appearance: Yellow solid, Rf: 0.11 (7:3 – Hexane/ 
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
17 
Ethyl acetate), mp.: 247–249 ◦C, Purity by HPLC = 97.91 %. Molecular 
Formula: C25H19N5S. MolecularMass: 421.1361 g/mol. 1H NMR (400 
MHz, DMSO‑d6): δ 12.53 (s, 1H, NH (1′)); 8.75 (s, 1H, N––CH (16)); 8.49 
(dd, 1H, J = 0.32, 0.4 Hz, CHAr (7′)); 8.43 (s, 1H, CHAr (2′)); 8.04 (m, 1H, 
CHAr (5′)); 7.63 (dd, 1H, J = 1.4, 1.4 Hz, CHAr (4′)); 7.33 (m, 4H, CHAr 
(10, 11, 13, 14)); 7.28 (m, 1H, CHAr (12)); 3.69 (s, 2H, CH2 (7)); 3.54 (s, 
2H, CH2 (8)); 2.78 (m, 2H, CH2 (4)); 2.66 (m, 2H, CH2 (5)). 13C NMR 
APT (100 MHz, DMSO‑d6): δ 24.37, 49.12, 51.38, 60.99, 103.44, 
105.54, 114.83, 115.07, 117.86, 120.41, 123.19, 125.12, 127.61, 
128.15, 128.79, 128.79, 129.25, 129.25, 132.71, 136.83, 138.54, 
140.12, 155.81, 163.29. LC/MS (ESI): Calculated to [M + H]+: 
422.1361 g/mol; Found to [M + H]+: 422.1430 g/mol.
4.2.21. 6-N-(Benzylidene)-2-(((5′-cyano-1H-indol-3′-yl)methylene) 
amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3g)
Yield: 59.0 %, Appearance: Yellow solid, Rf: 0.31 (1:1 – Hexane/ 
Ethyl acetate), mp.: 213–215 ◦C, Purity by HPLC = 99.29 %. Molecular 
Formula: C25H19N5S. Molecular Mass: 421.1361 g/mol. 1H NMR (400 
MHz, DMSO‑d6): δ 12.54 (s, 1H, NH (1′)); 8.76 (s, 1H, N––CH (16)); 8.76 
(d, 1H, J = 2.0 Hz, CHAr (4′)); 8.38 (s, 1H, CHAr (2′)); 7.70 (dd, 1H, J =
0.48, 0.56 Hz, CHAr (7′)); 7.65 (dd, 1H, J = 1.56, 1.68 Hz, CHAr (6′)); 
7.34 (m, 4H, CHAr (10, 11, 13, 14)); 7.28 (m, 1H, CHAr (12)); 3.69 (s, 2H, 
CH2 (7)); 3.55 (s, 2H, CH2 (8)); 2.78 (m, 2H, CH2 (4)); 2.67 (m, 2H, CH2 
(5)). 13C NMR APT (100 MHz, DMSO‑d6): δ 24.38, 49.13, 51.38, 60.99, 
103.79, 104.41, 114.43, 114.91, 115.06, 120.65, 124.87, 126.86, 
127.44, 127.62, 128.35, 128.79, 128.79, 129.26, 129.26, 132.68, 
138.53, 138.85, 139.73, 155.53, 163.07. LC/MS (ESI): Calculated to [M 
+ H]+: 422.1361 g/mol; Found to [M + H]+: 422.1418 g/mol.
4.2.22. 6-N-(Benzylidene)-2-(((7′-azo-1H-indol-3′-yl)methylene)amino)- 
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3h)
Yield: 77.0 %, Appearance: Yellow solid, Rf: 0.11 (7:3 – Hexane/ 
Ethyl acetate), mp.: 255–258 ◦C, Purity by HPLC = 96.31 %. Molecular 
Formula: C23H19N5S. Molecular Mass: 397.1361 g/mol. 1H NMR (400 
MHz, DMSO‑d6): δ 12.61 (sl, 1H, NH (1′)); 8.72 (s, 1H, N––CH (16)); 8.63 
(dd, 1H, J = 1.68, 1.68 Hz, CHAr (6′)); 8.39 (dd, 1H, J = 1.68, 1.64 Hz, 
CHAr (5′)); 8.36 (s, 1H, CHAr (2′)); 7.34 (m, 5H, CHAr (10–11, 13–14, 4′)); 
7.29 (m, 1H, CHAr (12)); 3.71 (s, 2H, CH2 (7)); 3.56 (s, 2H, CH2 (8)); 2.80 
(m, 2H, CH2 (4)); 2.67 (m, 2H, CH2 (5)). 13C NMR APT (100 MHz, 
DMSO‑d6): δ 24.38, 49.16, 51.39. 60.97, 103.35, 113.40, 115.12, 
117.48, 118.56, 127.62, 128.02, 128.80, 128.80, 129.27, 129.27, 
130.57, 132.68, 137.34, 138.57, 145.32, 150.30, 156.11, 163.42. LC/ 
MS (ESI): Calculated to [M + H]+: 398.1361 g/mol; Found to [M + H]+: 
398.1429 g/mol.
4.2.23. 6-N-(Benzylidene)-2-((2′-nitrobenzylidene)amino)-4,5,6,7- 
tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3i)
Yield: 45.0 %, Appearance: Yellow solid, Rf: 0.66 (7:3 – Hexane/ 
Ethyl acetate), mp.: 156–158 ◦C, Purity by HPLC = 98.40 %. Molecular 
Formula: C22H18N4O2S. Molecular Mass: 402.1150 g/mol. 1H NMR 
(400 MHz, DMSO‑d6): δ 8.93 (s, 1H, N––CH (16)); 8.19 (dd, 1H, J =
1.32, 1.36 Hz, CHAr (3′)); 8.14 (dd, 1H, J = 1.08, 1.12 Hz, CHAr (6′)); 
7.92 (td, 1H, J = 0.52, 0.64, 0.68 Hz, CHAr (5′)); 7.82 (td, 1H, J = 1.48, 
1.36, 1.48 Hz, CHAr (4′)); 7.36 (m, 4H, CHAr (10, 11, 13, 14)); 7.28 (m, 
1H, CHAr (12)); 3.71 (s, 2H, CH2 (7)); 3.63 (s, 2H, CH2 (8)); 2.80 (m, 2H, 
CH2 (4)); 2.70 (m, 2H, CH2 (5)). 13C NMR APT (100 MHz, DMSO‑d6): δ 
24.20, 48.89, 51.35, 60.75, 107.53, 114.13, 125.42, 127.63, 128.79, 
128.79, 129.26, 129.29, 129.38, 129.94, 132.49, 133.27, 134.07, 
134.50, 138.47, 149.74, 157.49, 159.55. LC/MS (ESI): Calculated to [M 
+ H]+: 403.1150 g/mol; Found to [M + H]+: 403.1209 g/mol.
4.3. Biological section
4.3.1. In vitro anti-Leishmania and cytotoxicity assays
4.3.1.1. Parasite and cell cultures. The in vitro tests utilized Leishmania 
(Leishmania) amazonensis (IFLA/BR/67/PH8), Leishmania (Vianna) bra-
ziliensis (MHOM/BR/1975/M2903), Leishmania (Leishmania) infantum 
(MHOM/BR/1974/PP75). These leishmania cell lines were obtained 
from Oswaldo Cruz Foundation (FIOCRUZ)/Brazil. The promastigote 
forms were cultivated in Schneider’s insect medium at 26 ◦C in a BOD 
incubator. Schneider’s insect medium was supplemented with 20 % fetal 
bovine serum (FBS) and 1 % penicillin (10,000 UL/mL) and strepto-
mycin (10 mg/mL) (complete Schneider). Next, we differentiated them 
from promastigote forms by changing the conditions to Schneider’s in-
sect medium supplemented with 5 % FBS, pH 4.6, and a temperature of 
32 ◦C to obtain axenic amastigote forms. RAW 264.7 macrophages were 
cultured using DMEM medium supplemented with 10 % FBS, 1 % 
penicillin 10,000 UL/mL, and 10 mg/mL streptomycin (complete 
DMEM) and incubated at 37 ◦C and 5 % CO2.
4.3.1.2. Antileishmanial activity of promastigote and axenic amastigote 
forms of Leishmania sp. and determination of cytotoxicity in macrophages 
RAW 264.7. The in vitro antileishmanial activity assays against the 
promastigotes and axenic amastigotes of Leishmania sp. as well as the 
cytotoxicity evaluation in RAW 264.7 macrophages of compounds 1a-f, 
2a-i and 3a-i, and positive control (amphotericin B) were performed 
according to the procedures described previously.23,24 Briefly, com-
pounds, and AmpB were applied in serial concentrations to a 96-well 
plates containing each of the cells (promastigotes and amastigotes of 
Leishmania sp. and RAW 264.7 macrophages) in their appropriate cul-
ture media. Each plate was incubated for a specified time, and then MTT 
was added to each well for colorimetric evaluation of growth inhibition 
and/or macrophage viability. All tests were performed in triplicate.
4.3.2. In vivo acute toxicity in model of Zophobas morio
4.3.2.1. Animals and population rearing. Initially, the Zophobas morio 
larvae were commercially obtained by Agro Nutripet® (Minas Gerais, 
Brazil), CONAMA license number: 237, MAPA registration number: 
13015207. Then, after 100 days our own colony of Z. morio coleoptera 
was in-house stablished, in which the larvae (600 ± 50 mg and 41 ± 4 
mm in length) were kept in plastic organizer boxes with dimensions of 
40 × 30 × 8 cm, with chayote and courgetti as water sources and chick 
starter feed (ad libitum) in a closed room with ambient temperature 
25 ◦C ± 2 ◦C, and photoperiod of 8 h light 16 h dark. Throughout the 
rearing process, pieces of chayote and courgetti were included to supply 
moisture to the larvae and reduce cannibalistic behavior. Under this 
feeding regimen, the larval stage typically spans 90 to 95 days before 
pupation begins. For the experiment, larvae aged 85 to 90 days, corre-
sponding to the 15th or 16th instar, were selected and used for all ex-
periments after 2–3days of acclimatization. Then, larvae weighing 600 
to 650 mg, displaying a light and uniform coloration with no dark or 
grayish markings, were selected and allocated into pre-established 
groups for the experiment. Our procedures are in accordance with 
other studies found in the literature.57,58
4.3.2.2. Acute toxicity assessment in Zophobas morio larvae. In order to 
determine the safety profile of compounds 3c, 3f, and 3g by performing 
acute toxicity assessment, ten Z. morio larvae were used for each con-
centration tested, in duplicate, including the negative control (only 
vehicle). Initially, all larvae were anesthetized under low temperature 
(~4 ◦C) for 10 min. As vehicle was used a mixture of phosphate buffered 
saline (PBS) pH 7.4, dimethylsulfoxide (DMSO), and Kolliphor® 
(detergent), in a 8.9:1:0.1 ratio, respectively. Then, the compounds were 
screened in doses of 0.2, 20.0, 200.0, and 2000 mg/kg, diluted in the 
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
18 
aforementionedvehicle solution. All doses were inoculated in a volume 
of 25 μL, using a Hamilton® syringe (model 702 N, volume 25 μL, needle 
size 22 s ga (bevel tip), needle L 51 mm), in the second sternite visible 
above the legs, in the ventral region. Post-inoculation, these larvae were 
incubated at room temperature (25 ◦C ± 2 ◦C) in Petri dishes containing 
breeding diet. Then, these were monitored up to seven days post- 
inoculation. Thus, visual inspection was performed to check for the 
presence of response to physical stimuli (by forceps) and/or presence of 
melanization.58 In this context, if mortality was one or fewer, testing 
proceeded at a higher dose. In cases where two or three larvae died, the 
same dose was reassessed with 10 individuals. If eight or more of them 
died, testing resumed at a lower dose; if mortality ranged from four to 
six, the LD₅₀ value was considered confirmed. Finally, this workflow is in 
accordance with the study of Brai et al., 2023.33
4.3.2.3. Hemolymph extraction and cell viability assessment by light 
microscopy. Prior to hemolymph extraction, the larvae were anes-
thetized by cold exposure, remaining for 10 min in a refrigerator at 
approximately 4 ◦C. Then, the proleg was punctured to a depth of 2–3 
mm using a 22-gauge needle, and the exuding hemolymph was drawn 
with a pipette adjusted to 50 μL (based on Senior & Titball, 2021.37
Thus, hemolymph (3 μL) of insects injected with 3 μL PBS pH 7.4 was 
collected 1 h after inoculation of vehicle (for negative control) and 
compounds tested at LD50 value. Then, 6 μL of diluted hemolymph were 
mixed with 6 μL of blue trypan solution 0.4 % (Sigma-Aldrich) to 
measure the number of hemocytes in 1 μl of hemolymph on a Olen® LED 
light microscopy (frequency: 50–60 Hz and magnification range: 
1600×). For the determination of viability of the hemocytes in the he-
molymph probe, it was calculated for 1000 hemocytes in the microscopy 
slide. These procedures were adapted from other studies found in the 
literature.59
4.4. In silico studies
4.4.1. Physicochemical and pharmacokinetic (ADME) predictions
In silico tests for predicting physicochemical, and pharmacokinetic 
(ADME) predictions parameters were performed using the Osiris Prop-
erty Explorer software (https://www.organicchemistry.org/prog/peo/) 
and SwissADME software (http://swissadme.ch/index.php), where the 
following physicochemical parameters were evaluated: topological 
surface area (TPSA), number of rotatable bonds (nRoTB) (Weber’s rule), 
water solubility (LogS), octanol/water partition coefficient (LogP), 
molecular weight (MW), and number of hydrogen bond acceptor (HBA) 
and hydrogen bond donor (HBD) groups (Lipinski’s rule).
4.4.2. Molecular docking
The three-dimensional structures of proteins - Nucleoside Diphos-
phatase Kinase (NDK) from Leishmania amazonensis (PDB: 5GO1),49
Dihydroorotate dehydrogenase from Leishmania Viannia braziliensis 
(PDB: 4WZH)51 and 14-alpha demethylase (CYP51) from Leishmania 
infantum (PDB: 3L4D)53 were obtained from the Protein Data Bank 
(PDB) library (https://www.rcsb.org/, Accessed on: September 6, 
2025).
The active sites of proteins Dihydroorotate dehydrogenase from 
Leishmania Viannia braziliensis (PDB: 4WZH) and 14-alpha demethylase 
(CYP51) from Leishmania infantum (PDB: 3L4D) were identified through 
the template function with the co-crystallized ligand, which corresponds 
respectively to the piperazine ethanesulfonic acid (EPE) derivative for 
the enzyme Leishmania Viannia braziliensis (PDB: 4WZH) and the com-
pound fluconazole for the enzyme 14-alpha demethylase (CYP51) from 
Leishmania infantum (PDB: 3L4D), using the redocking process and 
determining Root Mean Square Deviation (RMSD) values for prior 
validation of these targets, adopting a limit value ≤2.0 Å. For the 
enzyme Nucleoside Diphosphatase Kinase (NDK) from Leishmania ama-
zonensis (PDB: 5GO1), the active site was identified through Molecular 
Pockets predictions through the platform DoGSiteScorer (https://prote 
ins.plus/, accessed on: September 6, 2025).60. This tool employs a Dif-
ference of Gaussian (DoG) method to detect and analyze cavities on the 
surface of proteins, which are potential binding sites for ligands.61,62 As 
this enzyme does not have a co-crystallized ligand, the compound 
Auranofin was used as a control parameter for the simulation.63
The structures of compounds 2b, 2c, 2f, 2g, 3c, 3f, 3g (which pre-
sented IC50 values ≤10.0 μM in the biological assay performed), 
Amphotericin B and Auranofin submitted to molecular Docking simu-
lations using the Molegro Virtual Docker software, version 6.0.1 
(MVD).64 Previously these compounds were designed in the software in 
the Marvin Sketch v. 19.18 (https://chemaxon.com/marvin) and saved 
as .sdf files, then the conformational search and minimization of the 
compounds under study were performed in the Spartan 14 software 
(https://www.wavefun.com/spartan-version-history) using the molec-
ular mechanics method,65 and immediately afterwards a second mini-
mization was carried out using the semi-empirical method Recife Model 
1 (RM1),66 The most stable conformation of each compound was sub-
jected to molecular docking. All water molecules were excluded from 
the enzyme structures. The enzyme and compound structures were 
prepared using the default parameter settings of the software package 
(ligand evaluation: internal ES, internal H-bond, Sp2-Sp2 torsions, all 
verified; number of runs: 10; algorithm: MolDock SE; maximum in-
teractions: 1500; maximum population size: 50; runs: 30; maximum 
steps: 300; distance factor between neighbors: 1.00; maximum number 
of returned poses: 5).
The docking procedure was performed using GRID with a radius of 
18 Å and a resolution of 0.30 to cover the ligand-binding site of each 
protein. The grid box was applied to the center of the target site. The 
MolDock scoring algorithm was used, along with the MolDock search 
algorithm.67 Molegro Virtual Docker generated five poses for each 
molecule in the active site of each protein. The most stable pose, that is, 
the one with the lowest interaction energy, was selected and imported 
into Discovery Studio Visualizer v20.1.0.19295 - BIOVIA software was 
used (https://discover.3ds.com/discovery-studio-visualizer-download) 
for visual inspection.
4.4.3. Molecular dynamics (MD) simulations
Molecular Dynamics simulations were performed using GROMACS 
2024.2,68,69 with topologies of compounds 2C, 2F, 3C, 3F (which pre-
sented IC50 values ≤5.0 μM in the biological assay performed), 
Amphotericin B, Auranofin and co-crystallized ligands generated on the 
ATB platform using the GROMOS54a7 force field (https://atb.uq.edu.au 
/index.py, Accessed on: September 06, 2025). The system was solvated 
with the SPC water model in a cubic box,70 energy-minimized, heated to 
300 K, and equilibrated at 1 ATM for 100 ns e 50.000.000 steps. RMSD, 
RMSF and energy metrics of the C-α atoms were calculated, and the 
corresponding graphs were generated using the Grace software 
(http://plasma-gate.weizmann.ac.il/Grace/, Accessed on: September 
06, 2025).
CRediT authorship contribution statement
Vitória Gaspar Bernardo: Writing – original draft, Methodology, 
Investigation. Rodrigo Santos Aquino de Araújo: Writing – original 
draft, Formal analysis, Conceptualization. Natália Barbosa de Mélo: 
Methodology, Investigation. Ricardo Eduardo Pereira Coutinho: 
Methodology, Investigation. Julyanne Maria Saraiva de Sousa: Vali-
dation, Methodology, Investigation. Maria Gabrielly Gonçalves da 
Silva Sousa: Methodology, Investigation. Natália Ferreira de Sousa: 
Investigation, Methodology, Writing – review & editing. Wadja Feitosa 
dos Santos Silva: Funding acquisition, Investigation, Methodology. 
Paulo Fernando da Silva Santos-Júnior: Investigation, Methodology, 
Writing – review & editing. Teresinha Gonçalves da Silva: Validation, 
Supervision, Resources, Methodology, Funding acquisition. Eduardo 
René Pérez González: Writing– review & editing, Validation. Edeildo 
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
19 
https://www.organicchemistry.org/prog/peo/
http://swissadme.ch/index.php
https://www.rcsb.org/
https://proteins.plus/
https://proteins.plus/
https://chemaxon.com/marvin
https://www.wavefun.com/spartan-version-history
https://discover.3ds.com/discovery-studio-visualizer-download
https://atb.uq.edu.au/index.py
https://atb.uq.edu.au/index.py
http://plasma-gate.weizmann.ac.il/Grace/
Ferreira da Silva Junior: Investigation, Methodology, Writing – review 
& editing. Marcus Tullius Scotti: Methodology, Supervision, Valida-
tion, Writing – review & editing. Anuraj Nayarisseri: Methodology, 
Validation, Writing – review & editing. Klinger Antonio da Franca 
Rodrigues: Writing – review & editing, Validation, Supervision, Fund-
ing acquisition, Formal analysis. Francisco Jaime Bezerra Mendonça- 
Junior: Writing – review & editing, Project administration, Funding 
acquisition, Formal analysis, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial 
interests or personal relationships that could have appeared to influence 
the work reported in this paper.
Acknowledgements
This study was funded by the Conselho Nacional de Desenvolvimento 
Científico e Tecnológico (CNPq) from Brazil, grant number #316996/ 
2023-8. This study was financed in part by the Coordenação de Aper-
feiçoamento de Pessoal de Nível Superior (CAPES), finance code 001. 
This work was also funded by CAPES through: Institutional Postdoctoral 
Program - PIPD-1/2024, process number 88887.177732/2025-00 for 
RSAA. This study was funded by Paraíba State Research Founda-
tion—FAPESQ-PB through the PRONEX program, grant number #030/ 
2023. The authors would like to thank the NAPRMN from UFAL, the 
LMCA from UFPB, and the CM-ACQuimBio from UEPB for the acquisi-
tion of the NMRs and mass spectra.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. 
org/10.1016/j.bmc.2025.118475.
Data availability
Data will be made available on request.
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	Drug design and synthesis of new N-substituted-thienopyridine based on 2-aminothiophene derivatives as antileishmanial agents
	1 Introduction
	2 Results and discussion
	2.1 Synthesis
	2.2 In vitro evaluation
	2.3 In vivo acute toxicity evaluation in model of Zophobas morio
	2.3.1 Cell viability assessment by light microscopy
	2.4 In silico predictions
	2.4.1 Physicochemical and pharmacokinetic (ADME) predictions
	2.4.2 Molecular docking
	2.4.3 Molecular dynamics simulations
	3 Conclusions
	4 Experimental procedures
	4.1 Chemistry section
	4.2 General synthesis procedure
	4.2.1 7-N-(Carboxylate tert-butyl ester)-2-(((5′-Bromo-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyrid ...
	4.2.2 7-N-(Carboxylate tert-butyl ester)-2-((4′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitr ...
	4.2.3 7-N-(Carboxylate tert-butyl ester)-2-(((1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑car ...
	4.2.4 7-N-(Carboxylate tert-butyl ester)-2-(((5′-methoxy-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyr ...
	4.2.5 7-N-(Carboxylate tert-butyl ester)-2-((3′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyridine-3‑carbonitr ...
	4.2.6 7-N-(Carboxylate tert-butyl ester)-2-(((6′-cyano-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-b]pyrid ...
	4.2.7 6-N-(Carboxylate tert-butyl ester)-2-((4′-Nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitr ...
	4.2.8 6-N-(Carboxylate tert-butyl ester)-2-(((1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑car ...
	4.2.9 6-N-(Carboxylate tert-butyl ester)-2-(((5′-methoxy-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyr...
	4.2.10 6-N-(Carboxylate tert-butyl ester)-2-((3′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonit ...
	4.2.11 6-N-(Carboxylate tert-butyl ester)-2-(((6′-cyano-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyri ...
	4.2.12 6-N-(Carboxylate tert-butyl ester)-2-(((5′-cyano-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyri ...
	4.2.13 6-N-(Carboxylate tert-butyl ester)-2-(((7′-azo-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridi ...
	4.2.14 6-N-(Carboxylate tert-butyl ester)-2-((2′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonit ...
	4.2.15 6-N-(Benzylidene)-2-(((5′-bromo-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitri ...
	4.2.16 6-N-(Benzylidene)-2-((4′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3b)
	4.2.17 6-N-(Benzylidene)-2-(((1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3c)
	4.2.18 6-N-(Benzylidene)-2-(((5′-methoxy-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonit ...
	4.2.19 6-N-(Benzylidene)-2-((3′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3e)
	4.2.20 6-N-(Benzylidene)-2-(((6′-cyano-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitri ...
	4.2.21 6-N-(Benzylidene)-2-(((5′-cyano-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitri ...
	4.2.22 6-N-(Benzylidene)-2-(((7′-azo-1H-indol-3′-yl)methylene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile ...
	4.2.23 6-N-(Benzylidene)-2-((2′-nitrobenzylidene)amino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3‑carbonitrile (3i)
	4.3 Biological section
	4.3.1 In vitro anti-Leishmania and cytotoxicity assays
	4.3.1.1 Parasite and cell cultures
	4.3.1.2 Antileishmanial activity of promastigote and axenic amastigote forms of Leishmania sp. and determination of cytotox ...
	4.3.2 In vivo acute toxicity in model of Zophobas morio
	4.3.2.1 Animals and population rearing
	4.3.2.2 Acute toxicity assessment in Zophobas morio larvae
	4.3.2.3 Hemolymph extraction and cell viability assessment by light microscopy
	4.4 In silico studies
	4.4.1 Physicochemical and pharmacokinetic (ADME) predictions
	4.4.2 Molecular docking
	4.4.3 Molecular dynamics (MD) simulations
	CRediT authorship contribution statement
	Declaration of competing interest
	Acknowledgements
	Appendix A Supplementary data
	Data availability
	Referencessignificant burden of disease and therapeutic limita-
tions of SbV, these medicines were originally developed for leishmani-
asis, reflecting the poor investment in leishmaniasis therapeutic 
innovation. Amphotericin B, paromomycin, and pentamidine are anti-
microbials, while miltefosine, the only oral drug approved for leish-
maniasis, was developed for topical administrations in cutaneous 
cancers.16 Despite the repurposing, the majority of clinical evidence that 
corroborates these drug applications in leishmaniasis has low quality, 
according to the second edition of the Guideline for the Treatment of 
Leishmaniasis in the Americas.17
This scarcity of effective, safe, and innovative treatment options 
classifies leishmaniasis as a neglected tropical disease (NTD). Its elimi-
nation is part of the United Nations Sustainable Development Goal 3, 
which aims to end epidemics of NTDs by promoting equitable access to 
effective therapies.18 In this context, the development of new chemical 
entities with leishmanicidal potential is of paramount importance.
In turn, 2-aminothiophene derivatives (2-AT) stand out among the 
privileged structures that have demonstrated great potential for the 
design of new antileishmanial drug candidates, which are also the main 
class of molecules of interest to our research group. This class of com-
pounds is described by a pharmacophore which allows simple and low- 
cost synthesis along with high atom economy, and enables generating 
molecules and/or synthetic intermediates with great chemical di-
versity19,20 and broad biological and pharmacological applications.21,22
In one of our most recent publications, we described the Hit-to-Lead 
(H2L) optimization process carried out over the last 10 years with these 
2-AT, 23 which has allowed us to obtain several compounds with anti-
leishmanial activity at submicromolar level and with therapeutic safety, 
as well as the compound SB-83, which presents oral bioavailability and 
superior in vivo antileishmanial activity to the reference drug 
(Glucantime®).24,25
In some of these studies and based on Structure–Activity Relation-
ship (SAR) and Quantitative Structure-Activity Relationship studies, we 
have shown that the substitution pattern of the radicals or cycles linked 
at the C4 and C5 positions of the thiophene ring exerts great influence on 
pharmacomodulation.23,26,27 One of the findings of our most recent 
publication23 was the observation that replacing the cycloalkyl moiety 
at C4–C5 by a substituted-piperidinyl ring (affording thieno[2,3-c]pyr-
idines]) could increase the antileishmanial activity of these 2-AT against 
L. amazonensis, as observed with compound 6-(tert-butyl) 3-ethyl 2- 
amino-4,7-dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxylate (21) 
(IC50 value = 3.1 and 2.9 μM against promastigotes e amastigotes, 
respectively) (Fig. 1).
Rodriguez and colleagues also described potent anti-Leishmania 
major activity of tetrahydrothieno[2,3-c]pyridine derivatives, with 
compound Ethyl-2-(2-fluoro-3-(trifluoromethyl)benzamido)-5,5,7,7- 
tetramethyl-4,5,6,7-tetrahydrothieno[2,3-c]-pyridine-3-carboxylate 
(5D) (Fig. 1) being the most promising, presenting an IC50 value = 0.09 
+ 0.02 μM against promastigotes, and with inhibition of the prolifera-
tion of intracellular amastigotes higher than 75 % at 0.63 μM, and 99 % 
at 10 μM.28
In this context, this work aims to contribute to the rational drug 
design and synthesis of new therapeutic alternatives inspired on the N- 
substituted-thienopyridine based on 2-aminothiophene scaffold which 
could overcome the limitations of current treatments and reduce the 
global health burden of leishmaniasis. In the present study, we have 
focused on evaluating pharmacomodulation based on a phenotypic 
approach promoted by a series of N-substituted-thienopyridine ana-
logues designed and synthesized to be active against Leishmania species. 
The compounds obtained through the chemical variability of the pyr-
idinyl nitrogen position, and its substitution pattern were evaluated in 
silico and in vitro against L. amazonensis, L. braziliensis, and L. infantum, 
and against three key enzymes of the parasite Nucleoside Diphosphatase 
Kinase (NDK) from L. amazonensis, Dihydroorotate dehydrogenase from 
L. (V.) braziliensis, Sterol 14-alpha demethylase (CYP51) from L. infan-
tum. Their cytotoxicity was also evaluated in vitro in RAW 264.7 
Fig. 1. Chemical structure of thieno[2,3-c]pyridine derivatives with promising 
anti-Leishmania activity.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
2 
macrophages, and the acute toxicity of the most promising compounds 
was evaluated in vivo in model of Zophobas morio.
2. Results and discussion
2.1. Synthesis
To synthesize the N-substituted-thienopyridine based on 2-amino-
thiophene derivatives 1a-f, 2a-i and 3a-i evaluated in this study, we 
initially used the well-established, one pot three components coupling 
reaction between malononitrile, elemental sulfur, and a N-substituted- 
piperidone (ketone). The Gewald reaction19,29 subsequently afforded 
the Gewald adducts, compounds 2-amino-N-Boc-thieno[2,3-b]pyridine- 
3‑carbonitrile (1) and 2-amino-N-substituted-thieno[2,3-c]pyridine- 
3‑carbonitrile derivatives (2 and 3) in good yields (Scheme 1).
These adducts were reacted with substituted aromatic aldehydes 
(substituted -benzaldehydes and -3-indole-carboxaldehydes) to obtain 
the planned final compounds (1a-f, 2a-i and 3a-i – Scheme 1) by 
condensation reaction as described previously.24 All new compounds 
had their purity by HPLC (higher than 96 %), and their physicochemical 
properties determined. Their chemical structure were unequivocally 
confirmed by Nuclear Magnetic Resonance (13C and 1H NMR) and by 
High Resolution Mass Spectra (HRMS) (all spectra and chromatograms 
are available in Supplementary Data).
The ketone used in the Gewald reaction for the synthesis of com-
pound 1 was the N-Boc-3-piperidone, which would allow obtaining a 
mixture of two isomers with the presence of the N-Boc in positions 5 or 
7. However, the reaction proved to be regioselective and only the 7-N- 
Boc isomer (thieno[2,3-b]pyridine) was observed and isolated, showing 
that the α-methylene carbonyl at position 2 (between the carbonyl and 
nitrogen) are much more acidic.
The 1H and 13C NMR of compound 1 confirm the regioselectivity 
based on the multiplicity and chemical shift patterns of the methylene 
hydrogens, and the chemical shifts of their respective carbons. The 
presence of one triplet can be observed in the 1H NMR at 2.38 ppm (J =
6.2 Hz) and two multiplets at 1.87 and 3.64, indicating the existence of 
only one methylene neighboring the piperidinyl nitrogen atom. The 13C 
NMR confirms these findings, evidencing the presence of methylene 
carbons at 21.92, 22.41 and 44.73 ppm.
For the series of compounds 1a-f and 2a-i, the diagnostic signals 
observed in the 1H NMR spectra for all compounds were the singlet close 
to 1.50 ppm integrating for 9 hydrogens, referring to the tert-butyl 
group, and the singlet close to 8.80 ppm integrating for 1 hydrogen, 
referring to the imine group, thus characterizing successful coupling 
between the 2-amino and the substituted aromatic aldehydes. For the 
series 3a-i, in addition to the singlet related to the imine signal at around 
8.80 ppm, 4 signals related to pairs of methylene hydrogens are also 
observed between 2.65 and 3.71 ppm, being two singlets and two 
multiplets.
After unequivocal structural confirmation, compounds 1a-f, 2a-i and 
3a-i, were assessed for their potential in vitro antileishmanial activity.
2.2. In vitro evaluation
The antileishmanial activity was evaluated in vitro against three 
different Leishmania species (Leishmania amazonensis,L. braziliensis, and 
L. infantum) using the MTT assay, and amphotericin B as a positive 
control. Cytotoxicity was assessed with RAW 264.7 macrophages. The 
highest concentration evaluated in the antileishmanial assays was 10.0 
μM, and compounds that failed to inhibit parasite growth below this 
concentration were considered inactive. These results allowed us to 
calculate the selectivity index (SI) of N-substituted-thienopiperidine 1a- 
f, 2a-i and 3a-i analogs.
First, compounds 1a-f (thieno[2,3-b]pyridine) and 2a-i (thieno[2,3- 
c]pyridine), presenting N-Boc substituent at position 7 and 6 respec-
tively, were evaluated against the promastigote forms of the three 
Leishmania species, and the active compounds had their cytotoxicity and 
selectivity indices determined (Table 1).
As summarized in Table 1, four compounds showed antipromastigote 
activity against the Leishmania species (IC50 lower than 10 μM). All of 
Scheme 1. Reactional scheme for obtaining the N-substituted-thienopyridine derivatives, compounds 1a-f, 2a-i and 3a-i.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
3 
these active compounds belong to the same series of compounds, series 2 
(thieno[2,3-c]pyridine analogs), which has the N-Boc moiety substituted 
at position 6. The change of the N-Boc group from position 6 to position 
7 (compounds of series 1a-f) results in a complete loss of anti-
promastigote activity, indicating that the 7-N-Boc position is unfavor-
able for the antileishmanial activity of these analogs.
In analyzing the substitution pattern of series 2a-i and its relation-
ship with the antipromastigote activity profile, it is observed that 3 of 
the 4 active compounds (compounds 2c, 2f and 2g) possess an indole 
ring linked to the amino group of the thiophene ring, while only one 
phenyl analog, compound 2b, was active. The positive contribution of 
the indole ring linked at 2-amino portion of 2-aminothiophene de-
rivatives for the anti-Leishmania activity of these analogues has also been 
observed by Rodrigues et al.24
With regard to the favorable substitution pattern for the anti- 
promastigote activity and with greater safety among the analogs with 
indole ring (series 2a-i), it is observed that the most active compounds 
and those with the highest selectivity index (SI) are the same. Despite 
being active, none of them showed antipromastigote activity compara-
ble to amphotericin B (IC50 between 0.32 and 0.84 μM). Nevertheless, all 
active thieno[2,3-c]pyridine analogues presented a superior safety 
profile to amphotericin B by up to more than 60 times, representing an 
important step in the process of drug discovery for leishmaniasis 
treatment.
The most active and safest analogs were those possessing a strongly 
electron-withdrawing group (carbonitrile (–CN)) at positions 6′ (2f) and 
5′ (5g). For 2f, the IC50 and SI values ranged from 2.11 μM/130.1 (for L. 
infantum) to 4.73 μM/58.0 (for L. amazonensis). The change in the car-
bonitrile position from the position 6′ to 5′ (analog 5g) led to losses in 
activity and safety in the order of half to one third. Thus, the IC50 and SI 
values observed for 5g ranged from 6.93 μM/40.2 (for L. infantum) to 
9.38 μM/29.7 (for L. amazonensis).
The unsubstituted indole analog (2c) was the second compound with 
the best activity and safety profile, presenting IC50 and SI values ranging 
from 4.79 μM/78.9 (for L. brasiliensis) to 6.28 μM/60.2 (for 
L. amazonensis).
Among the inactive indole compounds, we observe compound 2h 
(1H-pyrrolo[2,3-b]pyridine derivative), which is an isostere of 2c, as 
well as the 5′-halogenated compound 2a (5′-Br) and the compound 2d 
with an electron-donating group (5′-OMe).
In turn, only 2b (4′-nitro-phenyl) was active among the analogues 
containing a phenyl ring linked to the 2-amino group of thiophene. Its 
positional isomers, compounds 2e (3′-nitro-phenyl) and 2i (2′-nitro- 
phenyl), were inactive at the highest concentration evaluated. Unlike 
the indole-containing derivatives, compound 2b was only active against 
the promastigote forms of L. brasiliensis (IC50 = 9.62/SI = 23.4) and L. 
infantum (IC50 = 9.27/SI = 24.3), being inactive against L. amazonensis.
Despite the great efforts of the scientific community to identify po-
tential biological targets for 2-AT derivatives, evaluation of the anti-
leishmanial potential of thiophene derivatives continues to have an 
almost exclusively a phenotypic approach. In one of the most recent 
studies which sought to identify potential targets for thieno[2,3-c]pyr-
idine derivative through chemogenomic screenings by Cos-Seq, the au-
thors were unable to observe the production of high levels of resistance 
by cosmids, which reinforces the evidence for the absence of a specific 
target.30 Borsari et al.31 also observed a similar result with a benzo-
thiophene derivative in Leishmania spp.
The lack of knowledge of the biological target(s) of these compounds 
and the impossibility of conducting Structure-Based Drug Design studies 
(especially Molecular Docking on the real target) make it difficult to 
unequivocally state the reasons why the change in the position of the N- 
Boc group from position 6 (series 2a-i) to position 7 (series 1a-f) results 
in the complete loss of antileishmanial activity. We believe that this loss 
of activity may be related to both steric and electronic factors (inter-
ference of the non-bonding electron pair of nitrogen with the electron 
cloud of the thiophene ring), which may be hindering or preventing 
drug-receptor recognition related to the antileishmanial activity.
Based on these findings, and especially considering that only series 
2a-i (thieno[2,3-c]pyridine analogs) containing the piperidine nitrogen 
at position 6 showed promising antileishmanial potential, the series 3a- 
i, which contains an N-benzyl radical replacing the N-Boc group, was 
synthesized and then had its antipromastigote activity and cytotoxicity 
profile evaluated using the same procedures performed with the previ-
ous series. The results can be seen in Table 2.
The evaluation results of the antipromastigote activity and cytotox-
icity observed for the series 3a-i confirm the same pharmacomodulation 
profile observed for series 2a-i, with the exception of the loss of anti- 
promastigote activity of the 4′-nitro-phenyl analogue (3b). The only 
three active compounds in series 3a-i show exactly the same substitution 
pattern in the indolic moiety (i.e. the analogs substituted with carbon-
itrile in positions 6′ (3f) and 5′ (3g), and the unsubstituted indole (3c)).
The order of efficiency and safety was also maintained, being 3f > 3c 
> 3g; however, replacing the N-Boc portion by N-benzyl promoted a 2- 
to 3-fold increase in the antipromastigote activity for analogues 3c (IC50 
ranged from 1.67 to 3.72 μM) and 3f (IC50 ranged from 0.93 to 1.96 μM), 
in addition to increasing the safety of analog 3f by 2 times (SI values 
ranged from 107.4 to 226.5).
These results, especially those observed for the most active com-
pound (3f), bring its antipromastigote activity profile closer to that of 
Table 1 
Evaluation of the antipromastigote activity (IC50), cytotoxicity (CC50) and 
selectivity index (SI) of 1a-f and 2a-i analogs.
Compounds IC50 CC50 SI
LA LB LI RAW 264.7
1a > 10.0 > 10.0 > 10.0 – ​
1b > 10.0 > 10.0 > 10.0 – ​
1c > 10.0 > 10.0 > 10.0 – ​
1d > 10.0 > 10.0 > 10.0 – ​
1e > 10.0 > 10.0 > 10.0 – ​
1f > 10.0 > 10.0 > 10.0 – ​
2a > 10.0 > 10.0 > 10.0 ​ ​
2b > 10.0 9.62 9.27 225.95 23.4–24.3
2c 6.28 4.79 5.53 378.27 60.2–78.9
2d > 10.0 > 10.0 > 10.0 – ​
2e > 10.0 > 10.0 > 10.0 – ​
2f 4.73 2.78 2.11 274.70 58.0–130.1
2g 9.38 7.95 6.93 279.05 29.7–40.2
2h > 10.0 > 10.0 > 10.0 – ​
2i > 10.0 > 10.0 > 10.0 – ​
Amphotericin B 0.84 0.32 0.400.83 1.0–2.5
LA = Leishmania (Leishmania) amazonensis; LB = Leishmania (Viannia) brazil-
iensis; LI = Leishmania (Leishmania) infantum. IC50 and CC50 values were pre-
sented in μM.
Table 2 
Evaluation of the antipromastigote activity (IC50), cytotoxicity (CC50) and 
selectivity index (SI) of 3a-i analogs.
Compounds IC50 CC50 SI
LA LB LI RAW 264.7
3a > 10.0 > 10.0 > 10.0 – ​
3b > 10.0 > 10.0 > 10.0 – ​
3c 3.72 1.67 1.73 184.38 49.5–110.4
3d > 10.0 > 10.0 > 10.0 – ​
3e > 10.0 > 10.0 > 10.0 – ​
3f 1.96 0.93 1.15 210.67 107.4–226.5
3g > 10.0 8.27 9.10 262.72 28.8–31.7
3h > 10.0 > 10.0 > 10.0 – ​
3i > 10.0 > 10.0 > 10.0 – ​
Amphotericin B 0.84 0.32 0.40 0.83 1.0–2.5
LA = Leishmania (Leishmania) amazonensis; LB = Leishmania (Viannia) brazil-
iensis; LI = Leishmania (Leishmania) infantum. IC50 and CC50 values were pre-
sented in μM.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
4 
the reference drug amphotericin B, but with a cytotoxicity profile 
improved by more than 250 times, making 3f a very promising drug 
candidate.
Next, the three most active compounds (3c, 3fc and 3g) were eval-
uated against the amastigote forms of the three Leishmania species to 
expand the investigation of the antileishmanial activity of these ana-
logues. The results can be seen in Table 3.
According to the data in Table 3, two of the three compounds (3c 
(unsubstituted indole) and 3f (6′-CN-indole)) evaluated for their anti-
amastigote potential were able to inhibit the growth of the amastigote 
forms of the three Leishmania species up to the maximum concentration 
evaluated (10 μM).
Once again, the compound with the best activity profile was the one 
containing the 6′-cyano-indole substituent (3f). It presented IC50 values 
of 0.83, 1.12, and 1.13 μM, respectively, against L. brasiliensis, 
L. infantum, and L. amazonensis, and was on average 2 to 4 times less 
potent than the reference drug Amphotericin B. However, as previously 
mentioned, it had a selectivity index many times higher, making 3f a 
good drug candidate for antileishmanial therapy.
The IC50 and CC50 values found for analogue 3f exceed the values 
observed for the best thieno[2,3-c]pyridine identified in our previous 
study23 (compound 21 – Fig. 1) and demonstrate the progress in the hit- 
to‑lead optimization process.
To contribute to the future design of new analogues, and considering 
that Quantitative Structure-Activity Relationship (QSAR) studies were 
not carried out, we use the Craig plot32 ((2D visualization graph between 
the Hammett σ (electronic effect) and Hansch π (hydrophobicity) pa-
rameters) to predict functional groups with the greatest potential for 
increasing or maintaining antileishmanial activity.
Based on the Craig Plot, it can be observed that active compounds 
have a substitution pattern containing substituents with a negative π 
value, while compounds with a positive π value are inactive, suggesting 
that decreased hydrophobicity appears to be an important parameter for 
activity. In this context, the use of substituents belonging to the left 
quadrants as amine (–NH2), sulfonamides (–SO2NH2), carboxylic acid 
(–COOH) and hydroxyl (–OH) is something that should be considered in 
the substituent choice.
2.3. In vivo acute toxicity evaluation in model of Zophobas morio
Since compounds 3c, 3f, and 3g were found to be the most active in 
this study, we proceeded for in vivo acute toxicity evaluation by using 
Zophobas morio (Coleoptera, Tenebrionidae) as animal model, adopting 
the method already developed by Brai et al 2023,33 which used Tenebrio 
larvae. In general, this animal model is considered to be easier to 
manage and provide reproducible results.
For our experiments, we considered injections of 25 μL of vehicle or 
diluted compounds for screening due to their body weight (600–650 
mg). As vehicle was used a mixture of phosphate buffered saline (PBS) 
pH 7.4, dimethylsulfoxide (DMSO), and Kolliphor® (detergent). In total, 
300 larvae were distributed into control and test groups (10 larvae per 
dose in duplicates). After administration (25 μL per larva), Z. morio 
larvae were monitored daily for seven days. During this stage, their 
behavior was additionally observed to detect if the tested compounds 
could interfere within their feeding or photophobia, limiting the 
reproducibility of these experiments.
As a result, it was observed that the vehicle solution was perfectly 
tolerated in the negative control group, with a 100 % survival rate. 
Besides, behavior alterations or even melanization signs were not 
observed for the Z. morio larvae. Regarding the tested compounds, in 
doses ranging from 0.2 to 2000 mg/kg, it was verified that none of these 
compounds could interfere with the behavior of Z. morio larvae.
Acute toxicity was determined following the Organization for Eco-
nomic Cooperation and Development (OECD) guidelines for acute 
toxicity of chemicals, which the lethal dose for 50 % (LD50) of compound 
3g was determinated to be 200 mg/kg, whereas for compounds 3c and 
3f presented a LD50 value of 2000 mg/kg, being determined at 6th and 
7th days post-inoculation (dpi), respectively. For both of these com-
pounds, the larvae demonstrated melanization signs (browning in the 
posterior end of the abdomen and thoracic regions) immediately 
observed after administration of their LD50 (Fig. 2). It is known that the 
melanization in insects is self-defense process as an immune response to 
the entry of bacteria or chemical agents,34,35 including in Z. morio.36
With respect to compounds 3c and 3f, their LD50 values suggest that 
these compounds display a great safety profile, considering their 
activity-toxicity relationship.
2.3.1. Cell viability assessment by light microscopy
Trypan blue has been proved to be effective in distinguishing viable 
from non-viable cells in insect hemolymph. This procedure is particu-
larly relevant in studies of immunocompetence and cytotoxicity in 
coleopteran hemocytes, where the presence of lipid inclusions can 
interfere with visual interpretation.37,38
The evaluation of cell viability using trypan blue staining revealed 
hemocytes of Z. morio. In the negative control group, most of the iden-
tified hemocytes (at 400× magnification) showed intact cytoplasm and 
no strong intracellular staining, indicating high cellular viability 
(Fig. 3a). Only a small fraction displayed blue staining, suggesting 
compromised plasma membrane integrity in a minor proportion of cells. 
Thus, the results confirm the applicability of trypan blue as a reliable 
marker of viability in Z. morio hemocytes, in which demonstrated that 
the negative control did not cause meaningful toxicity in the larvae up to 
7th dpi.
Additionally, to prove the toxic effects of the compounds tested, 
exhibiting the melanization process, the evaluation of cell viability using 
trypan blue staining revealed a total of 38.9 % hemocytes displaying low 
or no intracellular blue staining and preserved cytoplasmic appearance, 
consistent with viable cells, whereas 61.1 % hemocytes showed appre-
ciable blue uptake, indicating compromised plasma membrane integrity 
Table 3 
Evaluation of the antiamastigote activity (IC50), of the three most active com-
pounds (3c, 3f and 3g) against Leishmania (L.) amazonensis (LA), L. (V.) brazil-
iensis and L. (L.) infantum. IC50 values are presented in μM.
Compounds IC50
LA LB LI
3c 7.26 2.47 3.69
3f 1.13 0.83 1.12
3g > 10.0 > 10.0 > 10.0
Amphotericin B 0.6 0.24 0.25
Fig. 2. Post-inoculation melanization process in Zophobas morio larvae within 
acute toxicity assays observed for compounds 3c and 3f. In a: initial morphology 
of the larvae immediately post-inoculation; In b: mild melanization process observed 
one day post-inoculation(dpi); In c: advanced melanization process observed 4 dpi; 
In d: necrosed larva 6 dpi.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
5 
(Fig. 3b). Finally, trypan-stained cells were able to determine the toxic 
effects of compounds at their LD50 values.
2.4. In silico predictions
2.4.1. Physicochemical and pharmacokinetic (ADME) predictions
All N-substituted-thienopyridine analogues (1a-f, 2a-i and 3a-i) had 
their physicochemical parameters and their pharmacokinetic profiles 
predicted in silico with SwissADME and Osíris Property Explorer 
software.
The SwissADME software enabled generating the Bioavailability 
Radar graph, which provided the druglikeness (DL) profiles of the studied 
compounds based on a set of properties favorable to their bioavail-
ability. For this purpose, the program uses the following properties: 
lipophilicity, size, polarity, solubility, flexibility, and saturation.39 The 
BOILED-Egg graph, generated via SwissADME, enabled predicting pas-
sive gastrointestinal absorption (HIA) characteristics and blood-brain 
barrier (BBB) permeability, as well as the possibility of the compounds 
acting as P-glycoprotein substrates (P-gp).39
The use of Osíris Property Explorer enabled comparing similarities 
between the synthesized derivatives and known drugs. It did so by 
comparing the presence of functional groups and/or consistent physical 
properties based on a list of approximately 5300 fragments of distinct 
substructures in order to trace their druglikeness profiles.40 This profile, 
together with cLogP, LogS, molecular weight, and toxicity parameters, 
enabled generating the drugscore (DS).
Our analysis of the physicochemical parameters indicates that all 
synthesized analogs (1a-f, 2a-i and 3a-i), except 3a, proceed to Ro-5 
(Lipinski’s rule) and Weber parameters: Octanol/water partition coef-
ficient (LogP)compounds with key enzymatic targets involved in 
the Leishmania metabolic pathways, thereby providing insights into 
possible mechanisms underlying their anti-leishmanial activity.
For this purpose, we selected the following targets: (i) Nucleoside 
Diphosphate Kinase (NDK) from L. amazonensis (PDB ID: 5GO1), an 
enzyme essential for nucleotide metabolism and parasite survival;49,50
(ii) Dihydroorotate Dehydrogenase (DHODH) from L. (V.a) braziliensis 
(PDB ID: 4WZH), a mitochondrial enzyme responsible for catalyzing the 
conversion of dihydroorotate to orotate in the de novo pyrimidine 
biosynthesis pathway, which is critical for parasite survival and repli-
cation;51,52 and (iii) Sterol 14α-Demethylase (CYP51) from L. infantum 
(PDB ID: 3L4D), a cytochrome P450 enzyme involved in the biosynthesis 
of ergosterol, the primary sterol component of the Leishmania plasma 
membrane.53,54
Docking analyses were performed using the MolDock scoring algo-
rithm to estimate the binding affinities based on calculated energy in-
teractions between the ligands and their respective targets. The docking 
score values obtained for the active thienopyridine derivatives against 
each of the selected enzymes are summarized in Table 04.
According to Table 4, the enzymatic targets Dihydroorotate dehy-
drogenase from L. (V.) braziliensis (PDB: 4WZH) and sterol 14-alpha 
demethylase (CYP51) from L. infantum (PDB: 3L4D) were validated 
through redocking procedure. The Root Mean Square Deviation (RMSD) 
values obtained were 0.217 and 0.225 Å, respectively, which are within 
the acceptable threshold (≤ 2.0 Å), thus confirming that there are no 
structural differences between the co-crystallized ligand and the most 
stable pose.
All N-substituted thienopyridine analogs exhibited negative docking 
scores across the three evaluated targets, indicating favorable binding 
interactions. Notably, these compounds presented more negative Mol-
Dock scores compared to the reference ligands, including Auranofin in 
NDK (PDB ID: 5GO1) (Edock = − 105.189); Piperazine ethanesulfonic 
acid-derived in DHODH (PDB ID: 4WZH) (Edock = − 33.670); and Flu-
conazole in CYP51 (PDB ID: 3L4D) (Edock = − 73.246).
In Nucleoside Diphosphatase Kinase (PDB: 5GO1) the complex 
formed by the test compound 2c (− 155.371) demonstrated the highest 
binding affinity among the test ligands, second only to the positive 
control Amphotericin B (− 155.787), and followed by compounds 2f 
(− 152.744), 3f (− 150.392), 2 g (− 149.929), 3c (− 143.829) and refer-
ence compound Auranofin (− 105.189).
The 2D molecular interaction maps (Fig. 5) reveal that most test li-
gands established key interactions with His117, a residue essential for 
catalytic activity.49 This interaction manifested as either a conventional 
hydrogen bond (e.g., dashed line in dark green in compound 2g) or a 
carbon–hydrogen bond (e.g., dashed line in light green in compounds 
2c, 2f, 3c, and 3f). Additional hydrogen bonds were observed with 
essential residues such as Asp120, Arg104, Gly118, and Glu128. Hy-
drophobic interactions (pink dashed lines) were also observed with 
Phe59, Tyr51, and Leu54, all contributing to binding stabilization. 
Compounds 2c, 2f, 3c, and 3f stand out for forming multiple simulta-
neous hydrogen bonds, which may enhance target affinity. Interestingly, 
the positive control (PC) Amphotericin B exhibited unfavorable in-
teractions (red dashed lines) with residue Arg104, while the reference 
ligand (RL) Auranofin primarily engaged in conventional hydrogen 
bonding, corroborating the pharmacophoric relevance of the observed 
binding mode. Taken together, these results support that the test ligands 
share crucial residues for enzymatic activity, particularly His117, 
highlighting their potential as NDK inhibitors.
For the mitochondrial enzyme Dihydroorotate dehydrogenase (PDB: 
4WZH), the positive control Amphotericin B (PC) once again demon-
strated the most favorable binding score (− 139.928), followed by 
compounds 3c (− 95.343), 3g (− 94.105), 2g (− 93.005), 3f (− 91.541), 
2f (− 83.979), 2c (− 67.388), and 2b (− 58.191). The co-crystallized 
ligand (CL) exhibited a comparatively lower binding score (− 33.670), 
serving as a reference point for docking accuracy.
The 2D molecular interaction maps (Fig. 6) showed that the test 
compounds consistently formed hydrogen bonds (green dashed lines) 
with catalytically relevant residues such as Pro242 (e.g., 2b, 3f, 3g), 
Leu8 (e.g., 2b, 2c, 3c, 3f, 3g), and Ile7 (2f). Moreover, π-alkyl hydro-
phobic interactions (light pink dashed lines) with Leu245 were 
frequently observed across most ligands, (e.g., 2b, 2c, 2f, 2g, 3f, 3g), 
facilitating anchorage within the active site. Compounds 3c and 3g also 
present additional interactions with residues Cys241 and Gly6, sug-
gesting improved polar complementarity and enhanced binding poten-
tial. The positive control Amphotericin B (PC) formed multiple hydrogen 
bonds with Asp303, Gly6, Arg306, Pro242, Gln189, and Leu8, albeit 
with some unfavorable contacts involving Ile7. The interaction profile 
of the co-crystallized ligand derived from piperazine ethanesulfonic acid 
(CL) closely resembled that of the tested compounds, affirming the 
validity of the docking protocol and reinforcing the hypothesis of target 
engagement by the thienopyridine derivatives.
For the sterol 14-alpha demethylase (CYP51) (PDB: 3L4D), the most 
favorable docking scores were obtained for compounds 3g (− 125.596) 
and 2g (− 124.968), followed by 2f (− 109.940), 2c (− 104.946), 2b 
Fig. 4. Commercial drugs containing a thieno[2,3-c]pyridine and thieno[3,2-c]pyridine ring.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
7 
(− 100.627), Amphotericin B (− 97.079), 3f (− 94.636), 3c (− 93.586), 
and the co-crystallized ligand Fluconazole (− 73.246). The 2D molecular 
interaction maps (Fig. 7) showed that the test compounds established 
critical interactions at the heme-binding site,53 including hydrogen 
bonds (green dashed lines) with residues such as Tyr102 (3c), Ala290 
(2c, 2f), Met459 (3c), and Met357 (2g, 3g). Hydrophobic interactions 
(pink and purple dashed lines) were also observed with Leu 355 (e.g., 
2b, 2c, 2f, 2g, 3c, 3f, 3g), Met 459 (e.g., 2g, 3f, 3g), Leu 129 (2f), Ala 
290 (e.g., 2b, 2c, 2f, 2g, 3c, 3f, and 3g), Met 105 (e.g., 2g, 2f, 2g), Phe 
109 (e.g., 2b and 3g), Tyr 102 (e.g., 2g, 3c, 3f, 3g) and Tyr 115 (e.g., 2c, 
2f and 3f) (HEME site), which are essential for complex stabilization at 
the active site. Compounds 2c and 2f stand out for forming multiple 
interactions with residue Ala290, a key residue known to be central to 
CYP51 inhibition. These interactions including to the conventional 
hydrogen bond (dark green dashed line) with a length of 1.86 Å and 
π-alkyl interactions (light pink dashed line) in compound 2c and con-
ventional hydrogen bond (dark green dashed line) with a length of 2.81 
Å, π-alkyl interaction (light pink dashed line) and π–σ interaction (pur-
ple dashed line) in compound 2f, suggesting greater affinity for the 
active site. Notably, π‑sulfur interactions (orange dashed lines) were 
identified between the thiophene moiety and residue Phe289 (2c, 2f, 2g, 
3g), as well as between the índole moiety substituted with carbonitrile 
in positions 6′ (3f) and residue Met459, further supporting binding 
relevance at the heme coordination site.
The positive control Amphotericin B (PC) showed several hydrogen 
interactions such as the carbon‑hydrogen interaction (dashed line in 
Table 4 
Score and Root Mean Square Deviation (RMSD) values according the MolDock Score algorithm for 2b, 2c, 2f, 2g, 3c, 3f and 3g analogs in Nucleoside Diphosphatase 
Kinase (NDK) from L. amazonensis (PDB: 5GO1), Dihydroorotate Dehydrogenase(DHODH) from L. braziliensis (PDB: 4WZH), and Sterol 14α-Demethylase (CYP51) 
from L. infantum (PDB ID: 3L4D).
ID NDK DHODH CYP-51
MolDock 
Score
RMSD MolDock 
Score
RMSD MolDock 
Score
RMSD
2b –
–
− 58.191
0.217
− 100.627
0.225
2c ¡155.371 − 67.388 − 104.946
2f − 152.744 − 83.979 − 109.94
2g − 149.929 − 93.005 ¡124.968
3c − 143.829 ¡95.343 − 93.586
3f − 150.392 − 91.541 − 94.636
3g – − 94.105 ¡125.596
Amphotericin B ¡155.787 ¡139.928 − 97.079
Co-crystallized − 105.189 − 33.670 − 73.246
Caption: The compounds with the lowest energy score are highlighted in bold.
Fig. 5. 2D molecular interaction maps of compounds (2c), (2f), (2g), (3c), (3f), Positive Control (PC) Amphotericin B, Reference Ligand (RL) Auranofin with the 
target Nucleoside Diphosphatase Kinase (NDK) from L. amazonensis (PDB: 5GO1). Interactions: Conventional hydrogen interaction (dark green line), Carbon‑hy-
drogen interaction and π-donor hydrogen bond (light green line), π-anion and π-cation (orange line), π-π T-shaped and π-π stacked (dark pink line), Alkyl and π-alkyl 
(light pink line), Sulfur-X (yellow line), π–σ (purple line), Unfavorable interaction (red line). Residues: Leu (Leucine), Phe (Phenylalanine), Tyr (Tyrosine), Asn 
(Asparagine), Glu (Glutamic acid), Asp (Aspartic acid), Arg (Arginine), Gly (Glycine), His (Histidine), Lys (Lysine) and Met (Methionine). (For interpretation of the 
references to colour in this figure legend, the reader is referred to the web version of this article.)
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
8 
light green) through residue Ala 290 and the conventional hydrogen 
interactions (dark green dashed line) through Ala 286 and Met 387, as 
well as hydrophobic interactions of the alkyl type (dashed line in light 
pink) through residues Met 369, Phe 104, Val 101, Val 212, Cys 422, Phe 
415 (2 interactions) and Ile 349, confirming its engagement with the 
HEME site.
The co-crystallized ligand Fluconazole (CL) served as a reference for 
validation of the docking protocol, exhibiting consistent π-alkyl in-
teractions (dashed line in light pink) through residues Ala286 and 
Ala290 which were recapitulated by most N-substituted-thienopyridine 
test compounds, further validating the docking results.
Collectively, these findings indicate that the N-substituted thieno-
pyridine derivatives exhibit robust binding affinities and conserved 
interaction patterns across all three enzymatic targets. Their ability to 
engage catalytically essential residues, particularly within the active 
sites of NDK, DHODH, and CYP51, suggests promising inhibitory po-
tential and supports their candidacy for further optimization as anti- 
leishmanial agents.
2.4.3. Molecular dynamics simulations
Molecular dynamics (MD) simulations were performed for the pro-
tein–ligand complexes involving the most promissing compounds (2c, 
2f, 3c and 3f) in association with each of the targets: NDK (PDB ID: 
5GO1); DHODH (PDB ID: 4WZH); and CYP51 (PDB ID: 3L4D). These 
compounds were selected based on their low IC₅₀ values ≤5 μM observed 
in the anti-leishmanial assays, as well as their more favorable (i.e., 
lower) docking scores compared to the co-crystallized ligands in 
DHODH and CYP51 and the reference ligand Auranofin in NDK.
The Fig. 8 presents the RMSD (Root Mean Square Deviation) profiles 
of the protein backbone atoms for each of the target proteins: (A) NDK 
from L. amazonensis, (B) DHODH from L. (V.) braziliensis, and (C) CYP51 
from L. infantum, in complex with the respective N-substituted-thieno-
pyridine compounds over the course of 100 ns of MD simulation, aiming 
to assess the conformational stability of the protein–ligand complexes.
For NDK (Fig. 8A), the systems comprising the free enzyme (black 
line), the positive control Amphotericin B (brown line), and compound 
2c (red line) exhibited the highest RMSD values (≈0.5–0.6 nm), sug-
gesting greater conformational fluctuations. Conversely, the complex 
with compound 3c (blue line) showed the lowest RMSD (≈0.25–0.30 
nm), indicating enhanced structural stability. This was followed by the 
complex with Auranofin (gray line), while compounds 2f (green line) 
and 3f (yellow line) displayed intermediate RMSD values.
For DHODH (Fig. 8B), all systems converged to stable RMSD values 
ranging from 0.3 to 0.5 nm. Among these, the 2f complex (green line) 
demonstrated the lowest RMSD values, indicative of greater conforma-
tional stability. The positive control Amphotericin B (brown line) also 
Fig. 6. 2D molecular interaction maps of compounds (2b), (2c), (2f), (2g), (3c), (3f), (3g), Positive Control (PC) Amphotericin B, Co-crystallized ligand (CL) 
piperazine ethanesulfonic acid with the target Dihydroorotate dehydrogenase from L. (V.) braziliensis (PDB: 4WZH). Interactions: Conventional hydrogen interaction 
(dark green line), Carbon‑hydrogen interaction and π-Donor Hydrogen bond (light green line), Alkyl and π-alkyl (light pink line), π-π stacked (dark pink line), Sulfur- 
X (yellow line), π-lone pair (lime green line), Unfavorable interaction (red line). Residues: Tyr (Tyrosine), Leu (Leucine), Pro (Proline), Gly (Glycine), Ile (Isoleucine), 
Gln (Glutamine), Ser (Serine), Cys (Cysteine), Asp (Aspartic acid), Arg (Arginine) and Lys (Lysine). (For interpretation of the references to colour in this figure legend, 
the reader is referred to the web version of this article.)
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
9 
maintained low deviations, suggesting that both ligands may contribute 
to a more stable protein conformation during simulation.
Regarding CYP51 (Fig. 8C), with the exception of the 2c complex 
(green line), all remaining systems exhibited low RMSD values 
(≈0.25–0.40 nm), reflecting favorable dynamic stability. Complexes 
formed with 2c (red line), 3c (blue line), and 3f (yellow line) presented 
the lowest mean RMSD values, implying the establishment of more 
stable and persistent interactions at the binding site, which may corre-
late with enhanced binding affinity and potential biological activity.
The Fig. 9 presents the RMSD profiles of the ligands in complexes 
with the protein targets: (A) NDK from L. amazonensis; (B) DHODH from 
L. (V.) braziliensis; and (C) CYP51 from L. infantum over 100 ns of mo-
lecular dynamics (MD) simulation. This analysis was conducted to assess 
the positional stability and persistence of the ligands within the 
respective active sites, even under conditions influenced by explicit 
solvation, temperature, and pressure variations. In general, the ligands 
reached stability after the first 10–20 ns, maintaining limited fluctua-
tions around constant RMSD value throughout the remainder of the 
simulation time. This behavior suggests that the ligands remained 
consistently bound within the binding pocket, with no significant evi-
dence of dissociation or displacement, even under dynamic physico-
chemical conditions. These findings support the structural robustness of 
the ligand–protein interactions and indicate the thermodynamic favor-
ability of the complexes during prolonged simulation periods.
In NDK (Fig. 9A), compound 2f (green line) and the reference ligand 
Auranofin (gray line) exhibited the lowest RMSD values (≈0.1–0.2 nm), 
indicating greater conformational stability and persistent binding within 
the active site. Conversely, Amphotericin B (brown line) and compound 
3f (yellow line) showed larger positional fluctuations, suggesting higher 
molecular mobility and potentially less stable interactionswith the 
target.
In DHODH (Fig. 9B), the complexes with compounds 2c (red line), 2f 
(green line), and the co-crystallized ligand derived from piperazine 
ethanesulfonic acid (gray line) displayed the lowest RMSD variations, 
consistent with a stable binding conformation. In contrast, Amphoter-
icin B (brown line) exhibited the highest RMSD values (≈0.4–0.5 nm), 
reflecting greater instability and suggesting less favorable interactions 
with this enzyme.
For CYP51 (Fig. 9C), all ligand–protein complexes demonstrated 
relatively stable RMSD trajectories, with compound 2c (red line) and the 
co-crystallized ligand Fluconazole (gray line) showing the lowest RMSD 
deviations, indicative of consistent occupation and stabilization within 
the active site. In contrast, Amphotericin B (brown line) again exhibited 
increased fluctuations, suggesting greater mobility and potentially 
weaker interactions.
Collectively, these results underscore the enhanced stability of N- 
substituted thienopyridine derivatives, particularly compounds 2c (red 
line) and 2f (green line), across the three Leishmania targets. Their 
reduced RMSD values in ligand-centric analyses indicate favorable 
conformational persistence within the active sites under dynamic 
Fig. 7. 2D molecular interaction maps of compounds (2b), (2c), (2f), (2g), (3c), (3f), (3g), Positive Control (PC) Amphotericin B, Co-crystallized Ligand (CL) 
Fluconazole with the target Sterol 14-alpha demethylase (CYP51) from Leishmania infantum (PDB: 3L4D). Interactions: Carbon‑hydrogen interaction (light green 
line), π–π T-shaped and π-π stacked (dark pink line), Alkyl and π-alkyl (light pink line), Conventional Hydrogen interaction (dark green line), π-σ (purple line) and 
π‑sulfur (orange line). Residues: Ile (Isoleucine), Ala (Alanine), Phe (Phenylalanine), Cys (Cysteine), Thr (Threonine), Leu (Leucine), Tyr (Tyrosine), Met (Methi-
onine) and Val (Valine). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
10 
simulation conditions, including solvent effects and variations in tem-
perature and pressure. These findings are in line with the previously 
reported protein RMSD data, further supporting their potential as 
promising enzyme modulators in antileishmanial drug discovery.
Fig. 10 depicts the Radius of Gyration (Rg) profiles of the studied 
protein–ligand complexes. The Rg profile is a metric used to assess the 
overall compactness and structural integrity of the protein throughout 
the molecular dynamics (MD) simulations. Variations in Rg values are 
indicative of conformational changes in the protein backbone that may 
be induced by ligand binding or by environmental factors such as sol-
vent dynamics, temperature, and pressure.
For NDK (Fig. 10A), the Rg values of the complexes varied 
throughout the simulation without complete overlap among the 
different systems. Notably, compound 2c (red line), the free enzyme 
(black line), and the reference ligand Auranofin (gray line) exhibited the 
highest Rg values (≈1.65–1.67 nm), suggesting a tendency toward less 
compact or more open conformational states. In contrast, other com-
plexes displayed lower and more stable Rg values, indicative of 
increased structural compaction.
In DHODH (Fig. 10B), Rg values were consistently observed in the 
range between 1.85 and 1.95 nm across all complexes. A gradual 
downward trend in Rg was noted during the simulation period, indi-
cating a progressive compaction of the protein structure. This effect was 
more pronounced in the complexes formed with 2c (red line), 3c (blue 
line), 3f (yellow line), and the co-crystallized ligand derived from 
piperazine ethanesulfonic acid (CL) (gray line), supporting the notion 
that ligand binding may promote structural tightening of the enzyme.
For CYP51 (Fig. 10C), the Rg values ranged between 2.20 and 2.35 
nm. Among the systems analyzed, the complexes with compounds 2f 
(green line), 3c (blue line), and 3f (yellow line) exhibited the lowest 
average Rg values, suggesting a tendency toward more compact and 
stable conformational states under simulation conditions.
Taken together, the Rg data indicate that several of the N-substituted 
thienopyridine derivatives, particularly compounds 3c (blue line) and 3f 
(yellow line), promote higher levels of structural compaction in the 
target proteins across all three Leishmania species. This enhanced 
compactness may be associated with greater global conformational 
stability of the protein–ligand complexes, further supporting the struc-
tural integrity and potential functional relevance of these interactions.
Fig. 11 presents the Root Mean Square Fluctuation (RMSF) profiles 
for each amino acid residue in the protein–ligand complexes under 
investigation. RMSF analysis allows the evaluation of local flexibility 
Fig. 8. Metrics of all C-α atoms of the complexes under study - Root Mean Square Deviation - Protein (RMSD - Protein). Targets: (A) Nucleoside Diphosphatase Kinase 
(NDK) from L. amazonensis (PDB: 5GO1); (B) Dihydroorotate dehydrogenase from L. (V.) braziliensis (PDB: 4WZH) and (C) Sterol 14-alpha demethylase (CYP51) from 
L. infantum (PDB: 3L4D). Complexes: Enzyme (black line); 2c + Enzyme (red line); 2f + Enzyme (green line); 3c + Enzyme (blue line); 3f + enzyme (yellow line); 
Positive Control (PC) Amphotericin B + Enzyme (brown line) and Co-crystallized Ligand (CL) or Reference Ligand (RL) + Enzyme (gray line). (For interpretation of 
the references to colour in this figure legend, the reader is referred to the web version of this article.)
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
11 
and the dynamic behavior of individual residues during the molecular 
dynamics (MD) simulations, thus providing insights into regions of 
structural mobility that may influence binding interactions or overall 
protein stability.
In NDK (Fig. 11A), increased flexibility was observed at the N- and C- 
terminal regions, specifically at residues Met1, Tyr150, and Glu151, 
where RMSF values exceeded 3.0 nm. These fluctuations suggest local-
ized conformational mobility; however, it is noteworthy that none of 
these residues are located within the enzyme’s active site, and therefore 
are unlikely to directly influence ligand binding or catalytic function.
For DHODH Fig. 11B, pronounced fluctuations were detected at 
residues Ile203, Asp204, Val205, Glu206, Thr207, Glu208, Ser209, 
Val210, Lys213, Lys215, Gln216, and Met312, all of which are compo-
nents of the enzyme’s active site region. RMSF values in these residues 
reached or exceeded the 3.0 nm threshold in some complexes, indicating 
increased local mobility that may affect ligand binding stability and 
enzymatic function. These findings suggest that certain ligands may 
induce flexibility within catalytically relevant regions, potentially 
influencing inhibitory efficiency.
Regarding CYP51 (Fig. 11C), only residue Gly29 displayed RMSF 
values ≥3.0 nm across all studied complexes. Importantly, this residue is 
not part of the active site or the heme-binding region, suggesting that the 
observed fluctuation is unlikely to interfere with the ligand–protein 
interaction or enzymatic activity.
Taken together, the RMSF analysis reinforces that compound 3c is 
associated with reduced residue-level mobility, particularly within or 
near catalytically important regions of the target proteins. This reduced 
flexibilitymay contribute to the enhanced structural stability of the 
corresponding protein–ligand complexes, in agreement with the RMSD 
and Radius of Gyration (Rg) data, and further supports the potential of 
3c as a promising candidate for target modulation in Leishmania spp.
Table 5 presents the calculated interaction energies derived from 
Coulombic (electrostatic) forces and Lennard-Jones potentials, which 
represent van der Waals and hydrophobic dispersion interactions be-
tween the ligands and their respective protein targets.55,56 These pa-
rameters are fundamental for understanding the nature and stability of 
the ligand–protein complexes formed by the N-substituted thienopyr-
idine derivatives with the three Leishmania enzymes: NDK, DHODH and 
CYP51.
It was observed that, for the NDK target, the reference ligand 
Fig. 9. Metrics of all C-α atoms of the complexes under study - Root Mean Square Deviation - Ligand (RMSD - Ligand). Targets: (A) Nucleoside Diphosphatase Kinase 
(NDK) from L. amazonensis (PDB: 5GO1); (B) Dihydroorotate dehydrogenase from L. (V.) braziliensis (PDB: 4WZH) and (C) Sterol 14-alpha demethylase (CYP51) from 
L. infantum (PDB: 3L4D). Complexes: 2c + Enzyme (red line); 2f + Enzyme (green line); 3c + Enzyme (blue line); 3f + enzyme (yellow line); Positive Control (PC) 
Amphotericin B + Enzyme (brown line) and Co-crystallized Ligand (CL) or Reference Ligand (RL) + Enzyme (gray line). (For interpretation of the references to colour 
in this figure legend, the reader is referred to the web version of this article.)
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
12 
Auranofin exhibited the highest electrostatic (Coulomb) interaction 
energy, with a value of − 214.472 kJ⋅mol− 1, followed by compound 3f 
(− 120.346 kJ⋅mol− 1), suggesting a strong polar interaction profile. In 
contrast, the Lennard-Jones energy contribution was most pronounced 
for the positive control Amphotericin B (− 245.309 kJ⋅mol− 1), followed 
by compound 2f (− 177.017 kJ⋅mol− 1), indicating dominant van der 
Waals and hydrophobic interactions in these complexes.
Regarding DHODH, the co-crystallized ligand derived from pipera-
zine ethanesulfonic acid presented the most significant Coulombic 
interaction (− 147.854 kJ⋅mol− 1), followed by compound 3f (− 102.349 
kJ⋅mol− 1). Once again, Amphotericin B yielded the highest Lennard- 
Jones interaction energy (− 162.735 kJ⋅mol− 1), with compound 3f 
Fig. 10. Radius of gyration (Rg). Targets: (A) Nucleoside diphosphatase kinase (NDK) from L. amazonensis (PDB: 5GO1); (B) Dihydroorotate dehydrogenase from 
L. (V.) braziliensis (PDB: 4WZH) and (C) Sterol 14-alpha demethylase (CYP51) from L. infantum (PDB: 3L4D). Complexes: Enzyme (black line); 2c + Enzyme (red line); 
2f + Enzyme (green line); 3c + Enzyme (blue line); 3f + enzyme (yellow line); Positive Control (PC) Amphotericin B + Enzyme (brown line) and Co-crystallized 
Ligand (CL) or Reference Ligand (RL) + Enzyme (gray line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web 
version of this article.)
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
13 
close behind (− 140.224 kJ⋅mol− 1), reinforcing the ability of this com-
pound to engage in stabilizing non-covalent hydrophobic interactions 
within the binding site.
Finally, for CYP51, Amphotericin B also presented the highest elec-
trostatic contribution (− 51.6751 kJ⋅mol− 1), followed by compound 2c 
(− 49.2817 kJ⋅mol− 1), suggesting a meaningful role of polar interactions 
in these complexes. For the Lennard-Jones component, Amphotericin B 
once again displayed the strongest interaction (− 302.05 kJ⋅mol− 1), with 
compound 2f also exhibiting a high value (− 227.316 kJ⋅mol− 1), which 
may contribute significantly to the overall binding affinity and complex 
stability.
Collectively, these results underscore the relevance of both 
electrostatic and hydrophobic interactions in ligand binding, and high-
light compounds 2f, 2c, and 3f as promising candidates due to their 
energetically favorable interaction profiles across multiple targets.
3. Conclusions
In summary, we successfully designed and synthesized new series of 
N-substituted-thienopyridine derivatives based on the 2-aminothio-
phene scaffold, which differed from each other due to the presence of 
the pyridine nitrogen at positions 6 and 7, as well as by the presence of 
different N-substitution patterns.
The pharmacomodulation evaluation based on the phenotypic 
Fig. 11. Root mean square fluctuation (RMSF). Targets: (A) Nucleoside diphosphatase kinase (NDK) from L. amazonensis (PDB: 5GO1); (B) Dihydroorotate dehy-
drogenase from L. (V.) braziliensis (PDB: 4WZH) and (C) Sterol 14-alpha demethylase (CYP51) from L. infantum (PDB: 3L4D). Complexes: Enzyme (black line); 2c +
Enzyme (red line); 2f + Enzyme (green line); 3c + Enzyme (blue line); 3f + enzyme (yellow line); Positive Control (PC) Amphotericin B + Enzyme (brown line) and 
Co-crystallized Ligand (CL) or Reference Ligand (RL) + Enzyme (gray line). (For interpretation of the references to colour in this figure legend, the reader is referred 
to the web version of this article.)
Table 5 
Contributions of Coulomb and Lennard-Jones energies (kJ.mol− 1) for the title compounds in Nucleoside Diphosphatase Kinase (NDK) from L. amazonensis (PDB: 
5GO1), Dihydroorotate Dehydrogenase (DHOD) from L. braziliensis (PDB: 4WZH), and Sterol 14α-Demethylase (CYP51) from L. infantum (PDB ID: 3L4D).
Compound NDK DHOD CYP-51
Coulomb Energy Lennard-Jones Energy Coulomb Energy Lennard-Jones Energy Coulomb Energy Lennard-Jones Energy
2c − 55.2416 − 163.23 − 75.3724 − 104.518 ¡49.2817 − 214.291
2f − 55.0432 − 177.017 − 31.7385 − 115.202 − 31.3415 − 227.316
3c − 51.9303 − 140.905 − 81.0408 − 124.022 − 34.1743 − 212.858
3f − 120.346 − 151.832 − 102.349 − 140.224 − 32.7041 − 193.726
Amphotericin B − 107.323 ¡245.309 − 98.4386 ¡162.735 ¡51.6751 ¡302.05
Co-crystallized ¡214.472 − 110.54 ¡147.854 − 81.2237 − 14.4184 − 154.189
Caption: The compounds with the lowest energy score are highlighted in bold.
V.G. Bernardo et al. Bioorganic & Medicinal Chemistry 132 (2026) 118475 
14 
approach against the three Leishmania species allowed us to verify that 
the position of the pyridine nitrogen is essential for the antileishmanial 
activity, and only compounds with thieno[2,3-c]pyridine scaffold (with 
the nitrogen at position 6) result in analogs with antipromastigote ac-
tivity. A new series of analogues (3a-i) was subsequently synthesized by 
replacing the N-Boc group with the N-benzyl radical. Our investigation 
of the antileishmanial potential of this series showed that this modifi-
cation promoted a 2- to 3-fold increase in the antipromastigote activity, 
in addition to increasing safety by up to 2-fold.
Compounds 3c and 3f were the most promising due to the fact that 
they also exhibited significant antiamastigote activity against all three 
Leishmania species, associated to great safety profile, without causing 
significant cytotoxic effects in vitro on RAW 264.7 macrophages, and 
toxic effects in vivo on Zophobas morio larvae (LD50 = 2000 mg/kg). 
However, compound 3f (containing a 6′-cyano-indole substituent linked 
to the 2-amino position) stands out for its IC50 values below 2.0 μM, 
comparable