Arylthiosemicarbazones as antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2016.07.014

Based on a screening process, we targeted substituted thiosemicarbazone as potential antileishmanial agents. Our objective was to identify the key structural elements contributing to the anti-parasite activity that might be used for development of effective drugs. A series of 32 compounds was synthesized and their efficacy was evaluated against the clinically relevant intracellular amastigotes of Leishmania donovani. From these, 22 compounds showed EC₅₀values below 10?μM with the most active derivative (compound 14) showing an EC₅₀of 0.8?μM with very low toxicity on two different mammalian cell lines. The most relevant structural elements required for higher activity indicate that the presence of a fused bicyclic aromatic ring such as a naphthalene bearing an alkyl or an alkoxy group substituent are prerequisites. Owing to the easy synthesis, high activity and low toxicity, the most active compounds could be considered as a lead for further development.

Full text of DOI:10.1016/j.ejmech.2016.07.014

Accepted Manuscript
Arylthiosemicarbazones as antileishmanial agents
José Ignacio Manzano, Florent Cochet, Benjamin Boucherle, Verónica Gómez-Pérez,
Ahcène Boumendjel, Francisco Gamarro, Marine Peuchmaur
PII:
S0223-5234(16)30562-1
10.1016/j.ejmech.2016.07.014
EJMECH 8732
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2016
Revised Date: 7 July 2016
Accepted Date: 8 July 2016
Please cite this article as: J.I. Manzano, F. Cochet, B. Boucherle, V. Gómez-Pérez, A. Boumendjel,
F. Gamarro, M. Peuchmaur, Arylthiosemicarbazones as antileishmanial agents, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.014.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Arylthiosemicarbazones as Antileishmanial agents
José Ignacio Manzanoa,‡, Florent Cochetb,c,‡, Benjamin Boucherleb,c, Verónica Gómez-
Péreza, Ahcène Boumendjelb,c, Francisco Gamarroa,ǁ, Marine Peuchmaura,b,ǁ,
*
aInstituto de Parasitología y Biomedicina ‘López-Neyra’, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud,
Avda. del Conocimiento s/n, 18016 Armilla, Granada, Spain.
bUniv. Grenoble Alpes, Département de Pharmacochimie Moléculaire DPM UMR 5063, 38041 Grenoble, France.
cCNRS, DPM UMR 5063, 38041 Grenoble, France.
‡These authors contributed equally.
ǁ
Equal contribution of senior authors.
*Corresponding author Tel.: +33476635295; fax: +33476635298.
E-mail address: Marine.Peuchmaur@univ-grenoble-alpes.fr
ABSTRACT
Based on a screening process, we targeted substituted thiosemicarbazone as potential
antileishmanial agents. Our objective was to identify the key structural elements contributing to
the anti-parasite activity that might be used for development of effective drugs. A series of 32
compounds was synthesized and their efficacy was evaluated against the clinically relevant
intracellular amastigotes of Leishmania donovani. From these, 22 compounds showed EC50
values below 10 µM with the most active derivative (compound 14) showing an EC50 of 0.8 ꢀM
with very low toxicity on two different mammalian cell lines. The most relevant structural
elements required for higher activity indicate that the presence of a fused bicyclic aromatic ring
such as a naphthalene bearing an alkyl or an alkoxy group substituent are prerequisites. Owing
to the easy synthesis, high activity and low toxicity, the most active compounds could be
considered as a lead for further development.
Keywords: Leishmania; arylthiosemicarbazone.
1. Introduction
Leishmaniasis, a neglected disease caused by protozoan parasites of the genus Leishmania, is
a major health problem, mainly in developing countries, since it is endemic in 98 countries and
affects more than 12 million people with more than 2 million new cases reported per year.[1,2]
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The World Health Organization classified leishmaniasis as a major tropical disease, ranking
second only after malaria. This pathogenic disease presents four major syndromes, including
cutaneous, mucocutaneous, visceral, post-kala azar dermal leishmaniasis and is caused by at
least 20 different Leishmania species.[3] The life cycle of Leishmania parasite is characterized
by two distinct morphologies: the promastigote form, flagellated and found in the alimentary tract
of the female sand fly vector and the amastigote intracellular form, as the most relevant clinical
form found inside the host mammalian cells.[4]
Since no effective vaccines are currently available against leishmaniasis, chemotherapy
remains the only mean to treat the disease. However, existing treatments are inadequate due to
route of administration, side effects, cost, duration of treatment, and they are threatened by
resistance issues and therapeutic failure.[5,6] Therefore, the demand for new, effective,
affordable and better tolerated drugs is needed.
The first-line treatments consist of pentavalent anti-monials, amphotericin B and its liposomal
formulation AmBisome, paromomycin and miltefosine. Current research interest is mainly
directed towards the discovery and the optimization of novel chemical entities or reworking
existing drugs. Among the recently identified scaffolds, acridines, imidazoles, benzodiazepines,
chromenes, coumarins, chalcones and steroids were identified as potential scaffolds for the
design of new drugs candidate for treating leishmaniasis.[7-10] Despite the poor understanding
of relevant targets in these parasites, some of them are reported, including DNA
topoisomerases, protein kinases, folate biosynthesis and cysteine protease.[8,10] In this regard,
thiosemicarbazones were previously described as inhibitors of cysteine proteases which could
potentially be used to treat protozoan infections such as leishmaniasis.[11-16] So far, the
available data needs to be strengthened by investigating large series of compounds and
withdrawing pertinent structure-activity relationship.
With the aim to find more active compounds against Leishmania but non-toxic on mammalian
cells and to shed more light on the structure-activity relationship aspect, we screened an in
house library of thiosemicarbazone derivatives (Figure 1) against intracellular amastigotes of
Leishmania donovani (HU3 strain). This first primary screening highlighted that non polar
compounds were the most promising. Considering the results obtained with this first series, we
decided to synthesize new thiosemicarbazone derivatives to optimize the selectivity index (SI,
EC50 MRC-5 / EC50 parasite) of these series.
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Figure 1. General structures of the first series of thiosemicarbazone screened.
2. Results and discussion
In order to refine the SAR study, we mainly focused our synthesis on two sub-families of
thiosemicarbazones: the N-alkyl indole derivatives and the non-polar bicyclic compounds. For
both sub-families, the last step of the synthesis is a condensation of an arylaldehyde with the
thiosemicarbazide in the presence of acetic acid in ethanol (Scheme 1). The non-commercial
arylaldehyde were prepared through an alkylation reaction. The N-alkyl indole carboxaldehyde
1a-h were obtained as previously described[17] by treatment of an indole carboxaldehyde with a
halogenoalkane, in the presence of NaH in DMF. The alkoxy arylaldehydes 2a-o were
conventionally synthesized starting from hydroxyl naphthalene carboxaldehyde and
halogenoalkane in presence of a base (K2CO3 or NaH depending on the alkyl group introduced).
Following the same synthetic procedure and starting from [1,1’-biphenyl]-4,4’-
dicarboxaldehyde,[18] one bis-thiosemicarbazide 34 was synthesized to include it in the
biological evaluation. Finally, 32 new compounds were obtained and fully characterized
previous to the evaluation of their activity and toxicity.
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O
H
N
i
NH2
Ar
N
Ar
H
S
commercial or
prepared (1a-h, 2a-o)
arylaldehyde
3-34
R'
R'
ii
N
N
H
R
R
R''
1a-h
R or R' = CHO
O
O
iii or iv
HO
RO
2a-o
Scheme 1. Synthesis of thiosemicarbazone derivatives 3-34. Reagents and conditions: (i)
thiosemicarbazide, EtOH, AcOH, reflux, 10 h, 21-88%; (ii) R”X (X = Br or Cl), NaH 60% in oil, dry DMF, rt,
18-72 h, 41-99%;[17] (iii) for R = alkyl: K2CO3, RX (X = I or Br), acetone, reflux, 4-24 h, 37-92%; (iv) for R
= MOM: MOMBr, NaH 60%, dry DMF, 0°C then rt 1-3 h, 68-76% .
It is well established that the clinically relevant form of Leishmania is the intracellular amastigote
form that proliferate inside the mammalian cells. Therefore, the series of compounds was
evaluated against intracellular amastigotes of L. donovani (HU3 strain). Additionally, we
evaluated the toxicity of the investigated compounds on two mammalian cell lines: MRC-5
(human fetal fibroblast) and THP-1 (acute monocyclic leukemia) cells. The obtained results are
shown in Table 1.
As described in the introduction, our first preliminary screening pointed out the importance of
polarity that seems to be unfavorable for the antileishmanial activity. In the other hand, available
literature reports dealing with semicarbazones as antileishmanial agents or acting on targets
involved in the parasite spread revealed that the presence of aromatic moieties is often
synonymous of higher activity. Therefore, we focused our SAR study on apolar or compounds
with moderate polarity (Table 1). A first global analysis of the results highlighted that anthracene
4 and phenanthrene 3 thiosemicarbazones are weakly active or relatively toxic compounds.
Most of indole thiosemicarbazones are toxic (compounds 8-11), some possess no
antileishmanial activity (compounds 5-7), whereas compound 12 is active on intracellular
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amastigotes of L. donovani (with a EC50 of 9
ꢀM) and nontoxic on THP-1 cells, even if toxicity is
noticeable on MRC-5 cells, displaying a low SI of 3.4. If we consider the bis-thiosemicarbazone
34, it is both non active on intra-cellular amastigotes of L. donovani and no toxic on MRC-5 and
THP-1 cells.
Among the naphthalene thiosemicarbazone derivatives, it is interesting to point out the great
variability of antileishmanial activity and toxicity depending both on the relative position of
substituent and thiosemicarbazone moiety and the nature of the substituent on the naphthalene.
For instance, compounds 16, 22 and 25 bearing a methoxy group on the naphthalene moeity
present an antileishmanial activity ranging from 4 to more than 10
ꢀM whereas their toxicity vary
from 42 to more than 250 M with compound 25 being the most active, displaying the best SI of
ꢀ
this series (>20). Among all O-alkyl substituent evaluated on the naphthalene structure, it was
observed that the benzyl and the methylcyclohexyl groups seem to enhance the toxicity on
MRC-5 or THP-1 cells (compounds 19, 23, 28 and 31 for the benzyl group and compounds 20
and 24 for methylcyclohexyl group) even if EC50 on intracellular amastigotes of L. donovani is in
the low micromolar range and the SI obtained with these compounds are relatively high (3.7-
22.6), with the exception of compound 24. Regarding the methoxymethyl (MOM) substituent, we
observe that it is not beneficial to the antileishmanial activity: compounds 21, 29 and 32 have an
EC50 values ranging from 6.8 to more than 10 ꢀM. Naphthalene thiosemicarbazides bearing a
methoxy, an ethoxy, a butoxy, a methyl or a fluoro substituent (compounds 13, 14, 15, 18, 26,
27 or 30) generally present a better activity on intracellular amastigotes (0.8-6.5 µM and a
promising SI (19.5>250) due to the weak cytoxicity toward human cells. The most active
compound found so far was compound 14 (EC50 = 0.8 µM that displayed a high SI (>250).
Finally, the disubstituted naphthalene compound 33 show a low toxicity (EC50 > 200
MRC-5 and THP-1 cells), but also its antileishmanial activity is lower (9 M).
ꢀM on
ꢀ
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Table 1
Antileishmanial activity and intrinsic toxicity of the different compounds on MRC-5 and THP-1 cells.
EC50 (µM)
Yielda
(%)
Intracellular
amastigotes L.
donovani
Compd
Ar
MRC-5
cells
THP-1 cells
38 ± 5
SIb
AmBc
0.09 ± 0.01
23 ± 2
260
99
> 10
192 ± 10
> 200
-
3
48
92
90
5.3 ± 0.4
> 10
45 ± 9
> 200
> 200
67 ± 11
> 200
8.5
4
5
6
-
-
> 10
> 200
R = Me
CH2Cyd
89
91
60
84
89
> 10
9 ± 1
> 200
26 ± 2
> 200
27 ± 4
28 ± 5
32 ± 5
30 ± 6
-
7
8
2.8
-
Bn
> 10
175 ± 19
23.0 ± 0.6
> 200
9
3-MeBn
(CH2)3Ph
4.3 ± 0.5
4.1 ± 0.5
5.3
>50
10
11
67
9 ± 1
31 ± 4
> 200
3.4
12
R = Me
98
98
80
1.2 ± 0.2
0.8 ± 0.2
6.5 ± 0.7
37 ± 6
> 200
> 200
32 ± 9
39 ± 3
>200
30.4
> 250
> 30
13
14
15
F
N(Me)2
R = Me
55
97
60
59
41
5.0 ± 0.5
7.2 ± 0.7
2.1 ± 0.4
1.9 ± 0.2
2.2 ± 0.3
42 ± 6
> 200
47 ± 12
> 200
8.4
16
17
18
19
20
Et
> 30
19.5
15.2
13.1
n-Bu
Bn
41 ± 3
29 ± 2
28.85 ±
> 200
27 ± 4
CH2Cy
150 ± 20
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MOM
21
> 10
0.08
126 ± 13
126 ± 6
30 ± 7
33 ± 10
> 250
> 200
-
21
R = Me
44
72
57
63
82
55
85
50
49
53
23
> 10
8.1 ± 0.5
> 10
109 ± 21
39 ± 13
28 ± 2
108 ± 10
30 ± 2
36 ± 4
28 ± 2
> 200
-
22
23
24
25
26
27
28
29
30
31
32
Bn
3.7
-
CH2Cy
R = Me
9.3 ± 0.9
5.4 ± 0.5
1.27 ± 0.07
1.2 ± 0.1
> 10
> 20
> 40
33.2
22.6
-
Et
n-Bu
> 200
43 ± 3
27 ± 2
175 ± 6
86 ± 4
25 ± 3
119 ± 4
Bn
MOM
R = n-Bu
Bn
1.4 ± 0.1
2.6 ± 0.4
6.8 ± 0.1
> 200
61.5
9.7
17.5
28 ± 3
56 ± 16
MOM
O
33
34
78
88
9 ± 1
> 10
>200
> 200
> 200
> 20
-
O
> 200
aYields are given for the last step of the synthesis (condensation of bis-aldehyde and benzofuranone).
bSI = Selectivity Index (EC50 MRC-5 / EC50 parasite).
cAmB has been used as an antileishmanial reference compound.
d-CH2Cy : methylcyclohexyl.
3. Conclusion
In conclusion, we elaborated, evaluated and conducted a relation structure-activity study on
thiosemicarbazone bearing either a naphthyl or indolyl moieties. Indeed, we identified
compounds with an antileishmanial activity in the low micromolar range. Particularly, we have
observed that naphthalene thiosemicarbazides bearing a methoxy, an ethoxy, a butoxy, a
methyl or a fluoro substituent compounds presented the best activity against L. donovani,
highlighting compound 14, the most active compound of this study with an EC50 of 0.8
ꢀM, low
toxicity in MRC-5 cells and the best SI (>250). These results are in perfect line with specification
7

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needed for development of novel and costly-effective antileishmanial drugs and highlight the
advantage of this series of compounds which will be further evaluated by in vivo studies.
4. Experimental section
4.1. Chemistry
Commercially available reagents and solvents were used without further purification. Reactions
were monitored by thin-layer chromatography (plates coated with silica gel 60 F254 from
Macherey-Nagel). Products were purified with column chromatography on Silica gel 60 (230-
400 mesh from Macherey-Nagel) or by automatic flash chromatography with the Grace device:
Reveleris X2 using Grace Resolv cartridges. Melting points were measured on a Büchi B540
apparatus and are uncorrected. 1H and 13C NMR spectra were recorded at room temperature in
deuterated solvents on a Brüker Avance-400 instrument (at 400 MHz and 100 MHz
respectively). Chemical shifts (
internal standard or relative to the solvent [1H:
(CD3OD) = 3.31 ppm,
(acetone-d6) = 2.05 ppm; 13C:
77.23 ppm, (CD3OD) = 49.00 ppm, (acetone-d6) = 29.84 ppm]. Electrospray ionization ESI
δ
) are reported in parts per million (ppm) relative to TMS as
(DMSO-d6) = 2.50 ppm, (CDCl3) = 7.24 ppm,
(DMSO-d6) = 39.51 ppm, (CDCl3) =
δ
δ
δ
δ
δ
δ
δ
δ
mass spectra were acquired by the Analytical Department of Grenoble University on an Esquire
3000 Plus Bruker Daltonis instrument with a nanospray inlet. Accurate mass measurements
(HRMS) were carried out on a ESI/QTOF with the Waters Xevo G2-S QTof device. HPLC
analyses were performed with an Agilent 1100 series using a diode array detector and a C18
reversed-phase column (Nucleosil C18, Macherey-Nagel, 5
45°C, with a mobile phase composed of A = water and TFA 0.1% and B = MeOH and TFA 0.1%
with a gradient 85:15 to 0:100 A:B over 14 min, 1 mL/min, 10 L injection, detection at 254 nm.
Purity of the reported compounds were 95%.
ꢀm particle size, 125 mm × 3 mm) at
ꢀ
≥
4.1.1. General procedure A for the alkylation of hydroxy-naphtalene-carboxaldehyde
derivatives (2a-e, g-k, m-n). The mixture of hydroxy-naphtalene-carboxaldehyde (1 equiv.),
K2CO3 (1.5 equiv.) and alkyl halide (1.5 equiv.) in acetone (12 mL/mmol) was stirred under
reflux for 4 to 24 h. When TLC showed complete disappearance of the starting materials,
solvent was evaporated under reduced pressure. The residue was purified by column
chromatography on silica gel (cyclohexane/EtOAc 90:10 to 70:30).
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4.1.2. General procedure B for the synthesis of methoxymethyl derivatives from hydroxy-
naphthaldehydes (2f, 2l, 2o). To a solution of hydroxy-naphthaldehyde (1 equiv.) in dry DMF
(1 mL/mmol) was added, at 0°C, sodium hydride (60% dispersion in mineral oil, 2 equiv.). The
reaction mixture was stirred for 15 min. then MOM-Br (2 equiv.) was added and the reaction
mixture was stirred for 1 to 3 h at rt. The reaction was quenched with brine and extracted 3
times with EtOAc. The organic layer was washed twice with ice cold brine, dried over MgSO4,
filtered and evaporated. The residue was purified by flash chromatography on silica gel
(cyclohexane/EtOAc 80:20).
4.1.3. General procedure C for the synthesis of thiosemicarbazide derivatives (3-33 and
35). To a solution of the appropriate aldehyde (1.1 equiv.) in anhydrous ethanol (10 mL/mmol),
was added thiosemicarbazide (1.0 équiv.) and acetic acid (0.2 mL/mmol). The reaction mixture
was refluxed for 10 h and then was cooled to rt. The precipitate was filtered, washed with ether
and recrystallized from ethanol to obtain the corresponding 1-(1-
arylethylidene)thiosemicarbazone compounds.
4-methoxy-1-naphthaldehyde (2a). The crude product was prepared according to general
procedure A starting from 4-hydroxy-1-naphthaldehyde (150 mg, 0.87 mmol, 1.0 equiv.) and
iodomethane. The pure product (133 mg, 82%) was obtained as a colorless oil. Rf 0.39
(cyclohexane/EtOAc 80:20); 1H NMR (400 MHz, CDCl3)
1H), 7.56 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.69 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.88 (d, J = 8.1 Hz,
1H), 8.29-8.34 (m, 1H), 9.25-9.37 (m, 1H), 10.19 (s, 1H); 13C NMR (100 MHz, CDCl3)
ppm
δ ppm 4.07 (s, 3H), 6.88 (d, J = 8.1 Hz,
δ
56.1 (CH3), 103.0 (CH), 122.5 (CH), 124.9 (CH), 125.1 (C), 125.6 (C), 126.5 (CH), 129.6 (CH),
132.0 (C), 139.8 (CH), 160.9 (C), 192.4 (CH); LRMS (ESI+) m/z (%) 209 [M+Na]+ (100).
4-ethoxy-1-naphthaldehyde (2b). The crude product was prepared according to general
procedure A starting from 4-hydroxy-1-naphthaldehyde (200 mg, 1.16 mmol, 1.0 equiv.) and
iodoethane. The pure product (161 mg, 69%) was obtained as a brown solid. Rf 0.60
1
(cyclohexane/EtOAc 70:30); m.p. 75°C; H NMR (400 MHz, CDCl3)
δ ppm 1.60 (t, J = 9.3 Hz,
3H), 4.31 (d, J = 9.3 Hz, 2H), 6.88 (d, J = 10.7 Hz, 1H), 7.57 (ddd, J = 11.1, 9.2, 1.6 Hz, 1H),
7.69 (ddd, J = 11.1, 9.2, 1.9 Hz, 1H), 7.90 (d, J = 10.7 Hz, 1H), 8.37 (m, 1H), 9.29 (m, 1H),
10.20 (s, 1H); 13C NMR (100 MHz, CDCl3)
δ ppm 14.8 (CH3), 64.6 (CH2), 103.7 (CH), 122.7
(CH), 125.0 (CH), 125.1 (C), 125.8 (C), 126.5 (CH), 129.7 (CH), 132.2 (C), 140.0 (CH), 160.5
(C), 192.4 (CH); LRMS (ESI+) m/z (%) 201 [M+H]+ (100).
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4-butoxy-1-naphthaldehyde (2c). The crude product was prepared according to general
procedure A starting from 4-hydroxy-1-naphthaldehyde (100 mg, 0.58 mmol, 1.0 equiv.) and 1-
bromobutane. The pure product (110 mg, 83%) was obtained as a brown solid. Rf 0.64
1
(cyclohexane/EtOAc 70:30); m.p. 60°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.00 (t, J = 7.4
Hz, 3H), 1.52-1.61 (m, 2H), 1.86-1.92 (m, 2H), 4.31 (t, J = 6.3 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H),
7.54-7.61 (m, 1H), 7.67-7.73 (m, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H), 9.22 (d,
J = 8.1 Hz, 1H), 10.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 14.0 (CH3), 19.6 (CH2),
31.2 (CH2), 68.6 (CH2), 103.7 (CH), 122.6 (CH), 124.9 (C), 125.0 (CH), 125.8 (C), 126.4 (CH),
129.6 (CH), 132.1 (C), 139.9 (CH), 160.6 (C), 192.4 (CH); LRMS (ESI+) m/z (%) 229 [M+H]+
(100), 251 [M+Na]+ (73).
4-benzyloxy-1-naphthaldehyde (2d). The crude product was prepared according to general
procedure A starting from 4-hydroxy-1-naphthaldehyde (100 mg, 0.58 mmol, 1.0 equiv.) and
benzyl bromide (0.14 mL, 1.16 mmol, 2.0 equiv.). The pure product (127 mg, 83%) was
obtained as a brown oil. Rf 0.48 (cyclohexane/EtOAc 80:20). Analyses of the compound are in
agreement with the literature data.[19]
4-(cyclohexylmethoxy)-1-naphthaldehyde (2e). The crude product was prepared according to
general procedure A starting from 4-hydroxy-1-naphthaldehyde (100 mg, 0.58 mmol, 1.0 equiv.)
and cyclohexylmethyl bromide (0.16 mL, 1.16 mmol, 2.0 equiv.). The pure product (102 mg,
1
66%) was obtained as a pink solid. Rf 0.61 (cyclohexane/EtOAc 80:20); m.p. 99°C; H NMR
(400 MHz, CDCl3)
7.54-7.61 (m, 1H), 7.66-7.73 (m, 1H), 7.90 (d, J = 7.6 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 9.30 (d,
J = 8.4 Hz, 1H), 10.19 (s, 1H); 13C NMR (100 MHz, CDCl3)
ppm 26.0 (2xCH2), 26.6 (CH2),
δ ppm 1.11-2.06 (m, 11H), 4.04 (d, J = 5.9 Hz, 2H), 6.89 (d, J = 8.1 Hz, 1H),
δ
30.1 (2xCH2), 37.8 (CH), 74.2 (CH2), 103.7 (CH), 122.6 (CH), 124.8 (C), 125.0 (CH), 125.8 (C),
126.4 (CH), 129.6 (CH), 132.1 (C), 140.0 (CH), 160.6 (C), 192.4 (CH); LRMS (ESI+) m/z (%)
269 [M+H]+ (100).
4-(methoxymethoxy)-1-naphthaldehyde (2f). The crude product was prepared according to
general procedure B starting from 4-hydroxy-1-naphthaldehyde (100 mg, 0.58 mmol, 1.0
equiv.). The pure product (95 mg, 76%) was obtained as a colorless oil. Rf 0.50
(cyclohexane/EtOAc 80:20); 1H NMR (400 MHz, CDCl3)
(d, J = 8.1 Hz, 1H), 7.54-7.61 (m, 1H), 7.64-7.74 (m, 1H), 7.86 (d, J = 8.1 Hz, 1H), 8.34 (d, J =
8.4 Hz, 1H), 9.31 (d, J = 8.4 Hz, 1H), 10.19 (s, 1H); 13C NMR (100 MHz, CDCl3)
ppm 56.4
(CH3), 94.3 (CH2), 106.0 (CH), 122.4 (CH), 125.0 (CH), 125.7 (2xC), 126.6 (CH), 129.6 (CH),
δ ppm 3.56 (s, 3H), 5.47 (s, 2H), 7.17
δ
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132.1 (C), 139.2 (CH), 158.3 (C), 192.5 (CH); LRMS (ESI+) m/z (%) 217 [M+H]+ (48), 239
[M+Na]+ (100).
2-benzyloxy-1-naphthaldehyde (2g). This product was prepared according to previously
described procedure.[20]
2-(cyclohexylmethoxy)-1-naphthaldehyde (2h). The crude product was prepared according to
general procedure A starting from 2-hydroxy-1-naphthaldehyde (50 mg, 0.29 mmol, 1.0 equiv.)
and cyclohexylmethyl bromide (0.08 mL, 0.58 mmol, 2.0 equiv.). The pure product (21 mg, 37%)
1
was obtained as a brown solid. Rf 0.63 (cyclohexane/EtOAc 90:10); m.p. 69°C; H NMR (400
MHz, CDCl3)
δ ppm 1.01-1.96 (m, 11H), 3.99 (d, J = 5.8 Hz, 2H), 7.25 (d, J = 8.8, 0.8 Hz, 1H),
7.39 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.59 (ddd, J = 8.5, 6.8, 1.2 Hz, 1H), 7.74 (dd, J = 8.1, 1.2
Hz, 1H), 8.01 (dd, J = 8.1, 1.2 Hz, 1H), 9.26 (dd, J = 8.8, 0.8 Hz, 1H), 10.93 (s, 1H); 13C NMR
(100 MHz, CDCl3)
δ ppm 25.9 (2xCH2), 26.5 (CH2), 30.0 (2xCH2), 38.1 (CH), 75.1 (CH2), 113.7
(CH), 116.9 (C), 124.8 (CH), 125.1 (CH), 128.3 (CH), 128.5 (C), 130.0 (CH), 131.8 (C), 137.7
(CH), 164.1 (C), 192.3 (CH); LRMS (ESI+) m/z (%) 291 [M+Na]+ (100).
6-ethoxy-2-naphthaldehyde (2i). The crude product was prepared according to general
procedure A starting from 6-hydroxy-2-naphthaldehyde (200 mg, 1.16 mmol, 1.0 equiv.) and
iodoethane. The pure product (206 mg, 88%) was obtained as a yellow solid. Rf 0.57
(cyclohexane/EtOAc 75:25). Analyses of the compound are in agreement with the literature
data.3
6-butoxy-2-naphthaldehyde (2j). The crude product was prepared according to general
procedure A starting from 6-hydroxy-2-naphthaldehyde (100 mg, 0.58 mmol, 1.0 equiv.) and 1-
bromobutane. The pure product (104 mg, 79%) was obtained as an orange solid. Rf 0.72
(cyclohexane/EtOAc 70:30). Analyses of the compound are in agreement with the literature
data.[21]
6-benzyloxy-2-naphthaldehyde (2k). The crude product was prepared according to general
procedure A starting from 6-hydroxy-2-naphthaldehyde (100 mg, 0.58 mmol, 1.0 equiv.) and
benzyl bromide (0.14 mL, 1.16 mmol, 2.0 equiv.). The pure product (121 mg, 80%) was
obtained as a white solid. Rf 0.66 (cyclohexane/EtOAc 70:30). Analyses of the compound are in
agreement with the literature data.[21]
11

ACCEPTED MANUSCRIPT
6-(methoxymethoxy)-2-naphthaldehyde (2l). The crude product was prepared according to
general procedure B starting from 6-hydroxy-2-naphthaldehyde (100 mg, 0.58 mmol, 1.0
equiv.). The pure product (91 mg, 73%) was obtained as a white solid. Rf 0.56
(cyclohexane/EtOAc 80:20). Analyses of the compound are in agreement with the literature
data.[22]
1-butoxy-2-naphthaldehyde (2m). The crude product was prepared according to general
procedure A starting from 1-hydroxy-2-naphthaldehyde (100 mg, 0.58 mmol, 1.0 equiv.) and 1-
bromobutane. The pure product (122 mg, 92%) was obtained as a brown oil. Rf 0.67
(cyclohexane/EtOAc 90:10); 1H NMR (400 MHz, CDCl3)
1.54 (m, 2H), 1.86-1.98 (m, 2H), 4.20 (t, J = 6.5 Hz, 2H), 7.55-7.66 (m, 3H), 7.77-7.83 (m, 2H),
8.24 (d, J = 8.2 Hz, 1H), 10.60 (s, 1H); 13C NMR (100 MHz, CDCl3)
ppm 14.1 (CH3), 19.4
δ ppm 1.04 (t, J = 7.4 Hz, 3H), 1.48-
δ
(CH2), 32.5 (CH2), 79.1 (CH2), 122.8 (CH), 123.5 (CH), 124.6 (CH), 125.5 (C), 126.9 (CH), 128.4
(2xC), 128.5 (CH), 129.5 (CH), 138.3 (C), 190.1 (CH); LRMS (ESI+) m/z (%) 173 [M-Bu]+ (100),
229 [M+H]+ (91).
1-benzyloxy-2-naphthaldehyde (2n). This product was prepared according to previously
described procedure.[20]
1-(methoxymethoxy)-2-naphthaldehyde (2o). The crude product was prepared according to
general procedure B starting from 1-hydroxy-2-naphthaldehyde (100 mg, 0.58 mmol, 1.0
equiv.). The pure product (86 mg, 68%) was obtained as a white solid. Rf 0.52
(cyclohexane/EtOAc 80:20). Analyses of the compound are in agreement with the literature
data.[23]
2-(phenanthren-9-ylmethylene)hydrazinecarbothioamide (3), 2-(anthracen-9-
ylmethylene)hydrazinecarbothioamide (4). These products were prepared according to
previously described procedure.[24]
2-((1-methyl-1H-indol-2-yl)methylene)hydrazinecarbothioamide (5), 2-((1-methyl-1H-indol-5-
yl)methylene)hydrazinecarbothioamide (6), 2-((1-methyl-1H-indol-3-
yl)methylene)hydrazinecarbothioamide (7). These products were prepared according to
previously described procedure.[25]
2-((1-(cyclohexylmethyl)-1H-indol-3-yl)methylene)hydrazinecarbothioamide (8). The crude
product was prepared according to general procedure C starting from N-(cyclohexylmethyl)-
12

ACCEPTED MANUSCRIPT
indole-3-carboxaldehyde[17] 1d (100 mg, 0.44 mmol, 1.1 equiv.). The pure product (113 mg,
1
91%) was obtained as a yellow solid. Rf 0.33 (cyclohexane/EtOAc 60:40); m.p. 207°C; H NMR
(400 MHz, DMSO-d6)
7.20-7.26 (m, 1H), 7.40 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 8.01 (s, 1H), 8.21 (d, J =
7.8 Hz, 1H), 8.27 (s, 1H), 11.15 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm 49.2 (CH2), 110.4
δ ppm 0.90-1.84 (m, 11H), 4.02 (d, J = 7.2 Hz, 2H), 7.09-7.17 (m, 1H),
δ
(CH), 110.5 (C), 120.9 (CH), 122.4 (CH), 122.7 (CH), 124.7 (C), 127.1 (2xCH2), 127.5 (CH),
128.5 (2xCH2), 133.9 (CH2), 136.7 (C), 137.4 (CH), 140.1 (CH), 176.4 (C); LRMS (ESI+) m/z
(%) 315 [M+H]+ (100), 337 [M+Na]+ (32); HRMS (ESI+) m/z calc. for C17H23N4S 315.1643, found
315.1632.
2-((1-benzyl-1H-indol-3-yl)methylene)hydrazinecarbothioamide (9). The crude product was
prepared according to general procedure C starting from N-benzyl-indole-3-carboxaldehyde[17]
1e (100 mg, 0.45 mmol, 1.1 equiv.). The pure product (72 mg, 60%) was obtained as a white
solid. Rf 0.23 (cyclohexane/EtOAc 60:40); m.p. 202°C, lit. 203-204°C; 9 1H NMR (400 MHz,
DMSO-d6)
1H), 7.50 (d, J = 8.1 Hz, 1H), 7.99 (s, 1H), 8.03 (s, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.30 (s, 1H),
11.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm 49.3 (CH2), 110.6 (CH), 110.8 (C), 121.0
δ ppm 5.44 (s, 2H), 7.09-7.16 (m, 1H), 7.17-7.22 (m, 1H), 7.22-7.34 (m, 5H), 7.42 (s,
δ
(CH), 122.5 (CH), 122.8 (CH), 124.6 (C), 127.2 (2xCH), 127.6 (CH), 128.6 (2xCH), 134.0 (CH),
136.9 (C), 137.5 (C), 140.2 (CH), 176.6 (C); LRMS (ESI+) m/z (%) 309 [M+H]+ (100), 331
[M+Na]+ (85); HRMS (ESI+) m/z calc. for C17H17N4S 309.1174, found 309.1136.
2-((1-(3-methylbenzyl)-1H-indol-3-yl)methylene)hydrazinecarbothioamide (10). The crude
product was prepared according to general procedure C starting from N-(3-methylbenzyl)-
indole-3-carboxaldehyde[17] 1f (100 mg, 0.40 mmol, 1.1 equiv.). The pure product (108 mg,
1
84%) was obtained as a yellow solid. Rf 0.30 (cyclohexane/EtOAc 60:40); m.p. 197°C; H NMR
(400 MHz, DMSO-d6)
J = 8.1 Hz, 1H), 7.98 (s, 1H), 8.03 (s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 8.30 (s, 1H), 11.17 (s, 1H);
13C NMR (100 MHz, DMSO-d6)
ppm 38.0 (CH3), 51.8 (CH2), 110.1 (2xC), 110.5 (2xCH), 120.7
δ ppm 2.24 (s, 3H) , 5.39 (s, 2H), 7.01-7.24 (m, 6H), 7.43 (s, 1H), 7.50 (d,
δ
(2xCH), 122.4 (2xCH), 122.6 (2xCH), 124.3 (2xC), 134.3 (CH), 137.3 (C), 140.3 (CH), 176.4
(C); LRMS (ESI+) m/z (%) 323 [M+H]+ (100); HRMS (ESI+) m/z calc. for C18H19N4S 323.1330,
found 323.1334.
2-((1-(3-phenylpropyl)-1H-indol-3-yl)methylene)hydrazinecarbothioamide (11). The crude
product was prepared according to general procedure C starting from N-(3-phenylpropyl)-indole-
3-carboxaldehyde[17] 1g (100 mg, 0.38 mmol, 1.1 equiv.). The pure product (102 mg, 89%) was
13

ACCEPTED MANUSCRIPT
1
obtained as an orange solid. Rf 0.57 (cyclohexane/EtOAc 60:40); m.p. 81°C; H NMR (400
MHz, DMSO-d6) ppm 2.15-2.22 (m, 2H), 2.63 (t, J = 7.5 Hz, 2H), 4.05 (t, J = 7.5 Hz, 2H), 7.12
(s, 1H), 7.17 (d, J = 7.0 Hz, 2H), 7.24 (s, 1H), 7.25-7.40 (m, 7H), 8.16 (s, 1H), 8.29 (s, 1H),
10.90 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm 31.2 (CH2), 32.3 (CH2), 45.4 (CH2), 110.1
δ
δ
(CH), 110.4 (C), 120.8 (CH), 122.4 (CH), 122.7 (CH), 124.5 (C), 125.9 (CH), 128.2 (2xCH),
128.3 (2xCH), 133.6 (CH), 136.9 (C), 140.3 (CH), 141.1 (C), 176.5 (C); LRMS (ESI-) m/z (%),
335 [M-H]- (100), 371 [M+Cl]- (63); HMRS (ESI-) m/z calc. for C19H19N4S 335.1330, found
335.1346.
2-((1-butyl-7-methyl-1H-indol-3-yl)methylene)hydrazinecarbothioamide (12). The crude product
was prepared according to general procedure C starting from N-butyl-7-methylindole-3-
carboxaldehyde[17] 1h (100 mg, 0.47 mmol, 1.1 equiv.). The pure product (91 mg, 67%) was
1
obtained as a white solid. Rf 0.34 (cyclohexane/EtOAc 60:40); m.p. 193°C; H NMR (400 MHz,
DMSO-d6)
4.34 (t, J = 7.2 Hz, 2H), 6.93-7.09 (m, 2H), 7.38 (s, 1H), 7.79 (s, 1H), 8.01 (s, 1H), 8.08 (d, J =
7.6 Hz, 1H), 8.25 (s, 1H), 11.15 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm 13.5 (CH3), 19.2
δ ppm 0.90 (t, J = 7.4 Hz, 3H), 1.21-1.37 (m, 2H), 1.66-1.77 (m, 2H), 2.68 (s, 3H),
δ
(CH3), 19.3 (CH2), 33.9 (CH2), 48.1 (CH2), 110.0 (2xC), 120.4 (CH), 120.9 (CH), 120.9 (C),
125.7 (CH), 135.3 (CH), 135.3 (C), 140.2 (CH), 176.5 (C); LRMS (ESI-) m/z (%) 287 [M-H]-
(100), 323 [M+Cl]- (26); HMRS (ESI-) m/z calc. for C15H19N4S 287.1330, found 287.1335.
2-((4-methylnaphthalen-1-yl)methylene)hydrazinecarbothioamide (13). The crude product was
prepared according to general procedure C starting from 4-methyl-1-naphthaldehyde (200 mg,
1.17 mmol, 1.1 equiv.). The pure product (254 mg, 98%) was obtained as a white solid. Rf 0.37
1
(cyclohexane/EtOAc 60:40); m.p. 254°C; H NMR (400 MHz, DMSO-d6)
δ ppm 2.68 (s, 3H),
7.43 (d, J = 7.8 Hz, 1H), 7.58-7.70 (m, 2H), 7.95 (s, 1H), 8.10 (dd, J = 7.4, 7.4 Hz, 2H), 8.25 (s,
1H), 8.39 (d, J = 7.8 Hz, 1H), 8.89 (s, 1H), 11.42 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm
δ
19.4 (CH3), 123.3 (CH), 124.9 (CH), 125.6 (CH), 126.0 (CH), 126.4 (CH), 126.9 (CH), 127.5 (C),
130.5 (C), 132.2 (C), 136.7 (C), 141.3 (CH), 177.8 (C); LRMS (ESI-) m/z (%) 242 [M-H]- (100);
HMRS (ESI-) m/z calc. for C13H12N3S 242.0752, found 242.0764.
2-((4-fluoronaphthalen-1-yl)methylene)hydrazinecarbothioamide (14). The crude product was
prepared according to general procedure C starting from 4-fluoro-1-naphthaldehyde (200 mg,
1.15 mmol, 1.1 equiv.). The pure product (251 mg, 98%) was obtained as a yellow solid. Rf 0.36
1
(cyclohexane/EtOAc 60:40); m.p. 260°C; H NMR (400 MHz, DMSO-d6)
δ ppm 7.42 (dd, J = 8.5,
5.8 Hz, 1H), 7.66-7.74 (m, 1H), 7.77 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H), 8.02 (s, 1H), 8.13 (d, J = 8.3
14

ACCEPTED MANUSCRIPT
Hz, 1H), 8.24 (dd, J = 8.5, 5.8 Hz, 1H), 8.29 (s, 1H), 8.43 (d, J = 8.3 Hz, 1H), 8.86 (s, 1H), 11.45
(s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 109.8 (d, J = 20 Hz, CH), 120.6 (d, J = 6 Hz, CH),
122.8 (d, J = 16 Hz, C), 123.3 (CH), 126.0 (d, J = 4 Hz, C), 126.5 (d, J = 9 Hz, CH), 126.9 (CH),
128.4 (CH), 131.8 (d, J = 5 Hz, C), 140.3 (CH), 158.9 (d, J = 253 Hz, C), 177.9 (C); LRMS (ESI-
) m/z (%) 246 [M-H]- (100); HMRS (ESI-) m/z calc. for C12H9N3FS 246.0501, found 246.0501.
2-((4-(dimethylamino)naphthalen-1-yl)methylene)hydrazinecarbothioamide (15). This product
was prepared according to previously described procedure.[26]
2-((4-methoxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (16). The crude product was
prepared according to general procedure C starting from 2a (130 mg, 0.70 mmol, 1.1 equiv.).
The pure product (89 mg, 55%) was obtained as a yellow solid. Rf 0.14 (cyclohexane/EtOAc
1
90:10); m.p. 237°C; H NMR (400 MHz, acetone-d6)
δ ppm 4.08 (s, 3H), 7.05 (d, J = 8.2 Hz,
1H), 7.41 (s, 1H), 7.56 (ddd, J = 8.3, 6.8, 1.2 Hz, 1H), 7.64 (ddd, J = 8.3, 6.8, 1.2 Hz, 1H), 7.74
(s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 8.29-8.34 (m, 1H), 8.64 (d, J = 8.5 Hz, 1H), 8.86 (s, 1H), 10.36
(s, 1H); 13C NMR (100 MHz, acetone-d6)
δ ppm 56.3 (CH3), 105.0 (CH), 122.9 (C), 123.3 (CH),
124.5 (CH), 126.4 (C), 126.5 (CH), 128.6 (CH), 129.3 (CH), 132.8 (C), 143.4 (CH), 158.2 (C),
180.2 (C); LRMS (ESI+) m/z (%) 179 (100), 282 [M+H]+ (23), 282 [M+Na]+ (83); HRMS (ESI+)
m/z calc. for C13H14N3OS 260.0858, found 260.0852.
2-((4-ethoxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (17). The crude product was
prepared according to general procedure C starting from 2b (100 mg, 0.50 mmol, 1.1 equiv.).
The pure product (120 mg, 97%) was obtained as a yellow solid. Rf 0.41(cyclohexane/EtOAc
1
70:30); m.p. 240°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.47 (t, J = 7.1 Hz, 3H), 4.27 (q, J =
7.1 Hz, 2H), 7.02 (d, J = 8.1 Hz, 1H), 7.55 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.66 (ddd, J = 8.3, 6.9,
1.3 Hz, 1H), 7.82 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H) 8.17 (s, 1H), 8.25 (d, J = 9.1 Hz, 1H), 8.44 (d,
J = 9.1 Hz, 1H), 8.77 (s, 1H), 11.30 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 14.5 (CH3),
63.8 (CH2), 105.1 (CH), 121.4 (C), 122.2 (CH), 123.2 (CH), 124.8 (C), 125.5 (CH), 127.8 (CH),
128.0 (CH), 131.3 (C), 142.1 (CH), 155.8 (C), 177.5 (C); LRMS (ESI+) m/z (%) 274 [M+H]+
(100); HRMS (ESI+) m/z calc. for C14H16N3OS 274.1014, found 274.1021.
2-((4-butoxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (18). The crude product was
prepared according to general procedure C starting from 2c (60 mg, 0.26 mmol, 1.1 equiv.). The
pure product (43 mg, 60%) was obtained as a yellow solid. Rf 0.45 (cyclohexane/EtOAc 70:30);
1
m.p. 196°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.00 (t, J = 7.4 Hz, 3H), 1.47-1.64 (m, 2H),
15

ACCEPTED MANUSCRIPT
1.78-1.94 (m, 2H), 4.23 (t, J = 6.3 Hz, 2H), 7.05 (d, J = 8.4 Hz, 1H), 7.67 (ddd, J = 8.4, 6.9, 1.4
Hz, 1H), 7.67 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.85 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.19 (s, 1H),
8.25 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 8.4 Hz, 1H), 8.77 (s, 1H), 11.32 (s, 1H); 13C NMR (100
MHz, DMSO-d6)
δ ppm 13.7 (CH3), 18.9 (CH2), 30.7 (CH2), 67.8 (CH2), 105.1 (CH), 121.4 (C),
122.2 (CH), 123.3 (CH), 124.9 (C), 125.5 (CH), 127.8 (CH), 128.0 (CH), 131.3 (C), 142.1 (CH),
155.9 (C), 177.4 (C); LRMS (ESI+) m/z (%) 101 (100), 302 [M+H]+ (27), 324 [M+Na]+ (37);
HRMS (ESI+) m/z calc. for C16H20N3OS 302.1327, found 302.1335.
2-((4-(benzyloxy)naphthalen-1-yl)methylene)hydrazinecarbothioamide (19). The crude product
was prepared according to general procedure C starting from 2d (127 mg, 0.48 mmol, 1.1
equiv.). The pure product (87 mg, 59%) was obtained as a brown solid. Rf 0.20
1
(cyclohexane/EtOAc 80:20); m.p. 217°C; H NMR (400 MHz, DMSO-d6)
δ ppm 5.39 (s, 2H),
7.17 (d, J = 8.3 Hz, 1H), 7.34-7.40 (m, 1H), 7.41-7.48 (m, 2H), 7.55-7.62 (m, 3H), 7.69 (ddd, J =
8.4, 6.8, 1.4 Hz, 1H),7.87 (s, 1H), 8.10 (d, J = 8.3, 1H), 8.20 (s, 1H), 8.30 (dd, J = 8.4, 0.9 Hz,
1H), 8.45 (d, J = 8.4 Hz, 1H), 8.79 (s, 1H), 11.33 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm
69.7 (CH2), 105.9 (CH), 121.8 (C), 122.3 (CH), 123.2 (CH), 125.0 (C), 125.7 (CH), 127.6
(2xCH), 127.7 (CH), 127.9 (CH), 128.0 (CH), 128.6 (2xCH), 131.4 (C), 136.8 (C), 141.8 (CH),
155.4 (C), 177.5 (C); LRMS (ESI-) m/z (%) 334 [M-H]- (100); HRMS (ESI-) m/z calc. for
C19H16N3OS 334.1014, found 334.1016.
2-((4-(cyclohexylmethoxy)naphthalen-1-yl)methylene)hydrazinecarbothioamide (20). The crude
product was prepared according to general procedure C starting from 2e (90 mg, 0.34 mmol,
1.1 equiv.). The pure product (43 mg, 41%) was obtained as a gray solid. Rf 0.18
1
(cyclohexane/EtOAc 80:20); m.p. 210°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.09-1.96 (m,
11H), 4.02 (d, J = 5.8 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 7.54-7.60 (m, 1H), 7.63-7.69 (m, 1H),
7.84 (s, 1H), 8.05 (d, J = 8.3 Hz, 1H), 8.18 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.45 (d, J = 8.4 Hz,
1H), 8.76 (s, 1H), 11.31 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 25.3 (2xCH2), 26.0
(CH2), 29.3 (2xCH2), 37.2 (CH), 73.1 (CH2), 105.1 (CH), 121.3 (C), 122.1 (CH), 123.3 (CH),
124.9 (C), 125.6 (CH), 127.8 (CH), 128.1 (CH), 131.4 (C), 142.1 (CH), 155.9 (C), 177.5 (C);
LRMS (ESI+) m/z (%) 342 [M+H]+ (100), 364 [M+Na]+ (69); HRMS (ESI+) m/z calc. for
C19H24N3OS 342.1640, found 342.1629.
2-((4-(methoxymethoxy)naphthalen-1-yl)methylene)hydrazinecarbothioamide (21). The crude
product was prepared according to general procedure C starting from 2f (97 mg, 0.45 mmol, 1.1
equiv.). The pure product (27 mg, 21%) was obtained as a yellow solid. Rf 0.12
16

ACCEPTED MANUSCRIPT
1
(cyclohexane/EtOAc 80:20); m.p. 199°C; H NMR (400 MHz, acetone-d6)
δ ppm 3.53 (s, 3H),
5.50 (s, 2H), 7.20 (d, J = 8.2 Hz, 1H), 7.44 (s, 1H), 7.58 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.65
(ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.77 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 8.35 (d, J = 8.3 Hz, 1H),
8.63 (d, J = 8.3 Hz, 1H), 8.88 (s, 1H), 10.39 (s, 1H); 13C NMR (100 MHz, acetone-d6)
δ ppm
56.6 (CH3), 95.5 (CH2), 108.4 (CH), 123.4 (CH), 123.9 (C), 124.4 (CH), 126.6 (CH), 128.8 (C),
128.5 (CH), 128.7 (CH), 133.0 (C), 143.1 (CH), 155.6 (C), 180.1 (C); LRMS (ESI+) m/z (%) 290
[M+H]+ (100), 312 [M+Na]+ (23); HRMS (ESI+) m/z calc. for C14H16N3O2S 290.0963; found
290.0952.
2-((2-methoxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (22). This product was
prepared according to previously described procedure.[27]
2-((2-(benzyloxy)naphthalen-1-yl)methylene)hydrazinecarbothioamide (23). The crude product
was prepared according to general procedure C starting from 2g (80 mg, 0.31 mmol, 1.1
equiv.). The pure product (67 mg, 72%) was obtained as a yellow solid. Rf 0.26
1
(cyclohexane/EtOAc 80:20); m.p. 142°C; H NMR (400 MHz, CDCl3)
δ ppm 5.25 (s, 2H), 6.39
(s, 1H), 7.11 (s, 1H), 7.22-7.49 (m, 5H), 7.49-7.64 (m, 2H), 7.69-7.98 (m, 3H), 8.64 (s, 1H), 8.83
(d, J = 8.8 Hz, 1H), 9.43 (s, 1H); 13C NMR (100 MHz, CDCl3)
ppm 71.8 (CH2), 114.1 (CH),
δ
114.6 (C), 124.6 (CH), 125.0 (CH), 127.8 (2xCH), 126.5 (CH), 126.6 (CH), 128.8 (CH), 129.0
(2xCH), 129.4 (C), 131.4 (C), 133.3 (CH), 136.3 (C), 142.0 (CH), 157.7 (C), 178.5 (C); LRMS
(ESI+) m/z (%) 336 [M+H]+ (50), 358 [M+Na]+ (100); HRMS (ESI+) m/z calc. for C19H18N3OS
336.1171, found 336.1162.
2-((2-(cyclohexylmethoxy)naphthalen-1-yl)methylene)hydrazinecarbothioamide (24). The crude
product was prepared according to general procedure C starting from 2h (50 mg, 0.19 mmol,
1.1 equiv.). The pure product (33 mg, 57%) was obtained as a yellow solid. Rf 0.35
1
(cyclohexane/EtOAc 80:20); m.p. 181°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.06-2.02 (m,
11H), 3.97 (d, J = 6.1 Hz, 2H), 7.00 (d, J = 8.3 Hz, 1H), 7.52-7.58 (m, 1H), 7.60-7.65 (m, 1H),
7.86 (s, 1H), 8.02 (d, J = 8.3 Hz, 1H), 8.17 (s, 1H), 8.21-8.27 (m, 1H), 8.43 (d, J = 8.4 Hz, 1H),
8.75 (s, 1H), 11.30 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 27.0 (2xCH2), 27.6 (CH2),
30.8 (2xCH2), 39.3 (CH), 76.1 (CH2), 114.8 (CH), 115.7 (C), 125.1 (CH), 126.3 (CH), 129.1
(CH), 129.6 (CH), 130.5 (C), 132.6 (C), 134.0 (CH), 143.9 (CH), 159.4 (C), 178.5 (C); LRMS
(ESI+) m/z (%) 342 [M+H]+ (100); HRMS m/z (%) calc. for C19H24N3OS 342.1640, found
342.1639.
17

ACCEPTED MANUSCRIPT
2-((6-methoxynaphthalen-2-yl)methylene)hydrazinecarbothioamide (25). The crude product was
prepared according to general procedure C starting from commercially available 6-méthoxy-2-
naphthaldehyde (100 mg, 0.54 mmol, 1.1 equiv). The pure product (88 mg, 63%) was obtained
1
as a white solid. Rf 0.31 (cyclohexane/EtOAc 60:40); m.p. 233°C; H NMR (400 MHz, DMSO-
d6)
Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 8.03 (s, 2H), 8.12 (dd, J = 8.8, 1.6 Hz, 1H), 8.17 (s, 1H), 8.22
(s, 1H), 11.48 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm 55.3 (CH3), 106.3 (CH), 119.0
δ ppm 3.88 (s, 3H), 7.19 (dd, J = 9.2, 2.8 Hz, 1H), 7.35 (d, J = 2.8 Hz, 1H), 7.81 (d, J = 8.8
δ
(CH), 123.6 (CH), 127.2 (CH), 128.1 (C), 128.7 (CH), 129.7 (C), 129.8 (CH), 135.2 (C), 142.6
(CH), 158.1 (C), 177.8 (C); LRMS (ESI-) m/z (%) 258 [M-H]- (100); HMRS (ESI-) m/z calc. for
C13H14N3OS 260.0858, found 260.0865.
2-((6-ethoxynaphthalen-2-yl)methylene)hydrazinecarbothioamide (26). The crude product was
prepared according to general procedure C starting from 2i (100 mg, 0.49 mmol, 1.1 equiv.).
The pure product (110 mg, 82%) was obtained as a white solid. Rf 0.37 (cyclohexane/EtOAc
1
60:40); m.p. 240°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.39 (t, J = 6.9 Hz, 3H), 4.14 (q, J =
6.9 Hz, 2H), 7.17 (dd, J = 8.9, 2.2 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H),
7.85 (d, J = 8.9 Hz, 1H), 7.98-8.05 (m, 2H), 8.10 (dd, J = 8.7, 1.2 Hz, 1H), 8.17 (s, 1H), 8.19 (s,
1H), 11.45 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 14.6 (CH3), 63.2 (CH2), 107.0 (CH),
119.2 (CH), 123.6 (CH), 127.1 (CH), 128.1 (C), 128.7 (CH), 129.7 (C), 129.8 (CH), 135.2 (C),
142.6 (CH), 157.4 (C), 177.8 (C); LRMS (ESI-) m/z (%) 272 [M-H]- (100); HMRS (ESI-) m/z calc.
for C14H14N3OS 272.0858, found 272.0854.
2-((6-butoxynaphthalen-2-yl)methylene)hydrazinecarbothioamide (27). The crude product was
prepared according to general procedure C starting from 2j (76 mg, 0.34 mmol, 1.1 equiv.). The
pure product (47 mg, 55%) was obtained as a white solid. Rf 0.37 (cyclohexane/EtOAc 70:30);
1
m.p. 206°C; H NMR (400 MHz, DMSO-d6)
δ ppm 0.96 (t, J = 7.2 Hz, 3H), 1.43-1.55 (m, 2H),
1.68-1.87 (m, 2H), 4.10 ( t, J = 6.5 Hz, 2H), 7.18 (dd, J = 8.9, 2.5 Hz, 1H), 7.35 (d, J = 2.5 Hz,
1H), 7.79 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 8.02 (m, 2H), 8.10 (dd, J = 8.9, 2.5 Hz,
1H), 8.17 (s, 1H), 8.19 (s, 1H), 11.45 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 13.7 (CH3),
18.7 (CH2), 30.7 (CH2), 67.3 (CH2), 107.0 (CH), 119.2 (CH), 123.6 (CH), 127.1 (CH), 128.0 (C),
128.7 (CH), 129.6 (C), 129.8 (CH), 135.2 (C), 142.6 (CH), 157.5 (C), 177.8 (C); LRMS (ESI+)
m/z (%) 302 [M+H]+ (3), 324 [M+Na]+ (5); HRMS (ESI-) m/z calc. for C16H18N3OS 300.1171,
found 300.1173.
18

ACCEPTED MANUSCRIPT
2-((6-(benzyloxy)naphthalen-2-yl)methylene)hydrazinecarbothioamide (28). The crude product
was prepared according to general procedure C starting from 2k (88 mg, 0.33 mmol, 1.1 equiv.).
The pure product (87 mg, 85%) was obtained as a white solid. Rf 0.10 (cyclohexane/EtOAc
1
80:20); m.p. 219°C; H NMR (400 MHz, DMSO-d6)
δ ppm 5.24 (s, 2H), 7.27 (dd, J = 8.9, 2.4 Hz,
1H), 7.32-7.38 (m, 1H), 7.39-7.45 (m, 2H), 7.46 (d, J = 2.4 Hz, 1H), 7.49-7.56 (m, 2H), 7.80 (d, J
= 8.9 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 8.03 (s, 1H), 8.05 (s, 1H), 8.12 (dd, J = 8.6, 1.5 Hz, 1H),
8.18 (s, 1H), 8.20 (s, 1H), 11.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 69.6 (CH2),
107.9 (CH), 119.5 (CH), 123.9 (CH), 127.4 (CH), 128.0 (2xCH), 128.1 (CH), 128.4 (C), 128.7
(2xCH), 128.9 (CH), 130.0 (C), 130.1 (CH), 135.1 (C), 136.8 (C), 142.5 (CH), 157.2 (C), 177.8
(C); LRMS (ESI-) m/z (%) 334 [M-H]- (100); HRMS (ESI-) m/z calc. for C19H16N3OS 334.1014,
found 334.1014.
2-((6-(methoxymethoxy)naphthalen-2-yl)methylene)hydrazinecarbothioamide (29). The crude
product was prepared according to general procedure C starting from 2l (90 mg, 0.42 mmol, 1.1
equiv). The pure product (55 mg, 50%) was obtained as a white solid. Rf 0.50
1
(cyclohexane/EtOAc 80:20); m.p. 192°C; H NMR (400 MHz, DMSO-d6)
δ ppm 3.43 (s, 3H),
5.33 (s, 2H), 7.28 (dd, J = 8.9, 2.5 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H),
7.91 (d, J = 8.8 Hz, 1H), 8.04 (s, 1H), 8.05 (s, 1H), 8.12 (dd, J = 8.8, 2.4 Hz, 1H), 8.18 (s, 1H),
8.21 (s, 1H), 11.46 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 55.7 (CH3), 93.8 (CH2), 109.9
(CH), 119.3 (CH), 123.6 (CH), 127.3 (CH), 128.6 (C), 128.7 (CH), 129.9 (CH), 130.1 (C), 134.8
(C), 142.3 (CH), 155.4 (C), 177.8 (C); LRMS (ESI+) m/z (%) 290 [M+H]+ (100); HRMS (ESI+)
m/z calc. for C14H16N3O2S 290.0963; found 290.0953.
2-((1-butoxynaphthalen-2-yl)methylene)hydrazinecarbothioamide (30). The crude product was
prepared according to general procedure C starting from 2m (100 mg, 0.44 mmol, 1.1 equiv.).
The pure product (57 mg, 49%) was obtained as a white solid. Rf 0.53 (cyclohexane/EtOAc
1
70:30); m.p. 190°C; H NMR (400 MHz, DMSO-d6)
δ ppm 1.06 (t, J = 7.4 Hz, 3H), 1.55-1.67 (m,
2H), 1.90-1.95 (m, 2H), 4.03 (t, J = 6.7 Hz, 2H), 7.52-7.57 (m, 2H), 7.60 (d, J = 8.7 Hz, 1H),
7.79-7.86 (m, 1H), 7.89 (d, J = 8.7 Hz, 1H), 8.0.8-8.16 (m, 1H), 8.34 (s, 1H) 8.25 (s, 1H), 8.50
(s, 1H), 9.40 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 13.9 (CH3), 18.8 (CH2), 31.7 (CH2),
76.3 (CH2), 121.9 (CH), 123.1 (CH), 123.1 (C), 124.0 (CH), 126.6 (CH), 127.2 (CH), 127.7 (C),
128.1 (CH), 135.1 (C), 138.2 (CH), 154.8 (C), 177.9 (C); LRMS (ESI+) m/z (%) 324 [M+Na]+
(100), 302 [M+H]+ (37); HRMS (ESI+) m/z calc. for C16H20N3OS 302.1327, found 302.1335.
19

ACCEPTED MANUSCRIPT
2-((1-(benzyloxy)naphthalen-2-yl)methylene)hydrazinecarbothioamide (31). The crude product
was prepared according to general procedure C starting from 2n (60 mg, 0.23 mmol, 1.1
equiv.). The pure product (37 mg, 53%) was obtained as a yellow solid. Rf 0.26
1
(Cyclohexane/EtOAc 80:20); m.p. 191 °C; H NMR (400 MHz, acetone-d6)
δ ppm 5.15 (s, 2H),
7.37-7.52 (m, 4H), 7.55-7.61 (m, 2H), 7.61-7.66 (m, 2H), 7.72 (d, J = 8.8 Hz, 1H), 7.92-7.98 (m,
2H), 8.16-8.21 (m, 1H), 8.22 (d, J = 8.8 Hz, 1H), 8.74 (s, 1H), 10.63 (bs, 1H); 13C NMR (100
MHz, acetone-d6)
δ ppm 78.8 (CH2), 123.2 (CH), 123.6 (CH), 124.6 (C), 125.5 (CH), 127.6
(CH), 128.3 (CH), 129.0 (C), 129.1 (2xCH), 129.2 (2xCH), 129.4 (2xCH), 136.8 (C), 137.9 (C),
139.2 (CH), 155.8 (C), 180.3 (C); LRMS (ESI+) m/z (%) 358 [M+Na]+ (100); HRMS (ESI+) m/z
calc. for C19H18N3OS 336.1171, found 336.1163.
2-((1-(methoxymethoxy)naphthalen-2-yl)methylene)hydrazinecarbothioamide (32). The crude
product was prepared according to general procedure C starting from 2o (84 mg, 0.39 mmol,
1.1 equiv.). The pure product (26 mg, 23%) was obtained as a yellow solid. Rf 0.15
1
(cyclohexane/EtOAc 80:20); m.p. 173°C; H NMR (400 MHz, acetone-d6)
δ ppm 3.66 (s, 3H),
5.26 (s, 2H), 7.48 (bs, 1H), 7.55-7.62 (m, 2H), 7.70 (d, J = 8.8 Hz, 1H), 7.89-7.95 (m, 1H), 7.97
(bs, 1H), 8.17-8.20 (m, 1H), 8.21 (d, J = 8.8 Hz, 1H), 8.71 (s, 1H), 10.63 (s, 1H); 13C NMR (100
MHz, acetone-d6)
δ ppm 58.4 (CH3), 102.3 (CH2), 123.6 (2xCH), 124.6 (C), 124.5 (CH), 127.5
(CH), 128.3 (CH), 129.0 (CH), 129.3 (C), 136.7 (C), 139.8 (CH), 154.9 (C), 180.5 (C); LRMS
(ESI+) m/z (%) 290 [M+H]+ (95), 312 [M+Na]+ (100); HRMS (ESI+) m/z calc. for C14H16N3O2S
290.0963; found 290.0959.
2-((4,7-dimethoxynaphthalen-1-yl)methylene)hydrazinecarbothioamide (33). The crude product
was prepared according to general procedure C starting from 4,7-Dimethoxy-1-naphthaldehyde
(200 mg, 0.93 mmol, 1.1 equiv.). The pure product (209 mg, 78%) was obtained as a brown
solid. Rf 0.32 (cyclohexane/EtOAc 60:40); m.p. 221°C; 1H NMR (400 MHz, DMSO-d6)
δ ppm
3.91 (s, 3H), 4.00 (s, 3H), 6.91 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.77 (s, 1H), 7.84 (s,
1H), 8.02 (d, J = 8.2 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.17 (s, 1H), 8.77 (s, 1H), 11.24 (s, 1H);
13C NMR (100 MHz, DMSO-d6)
δ ppm 55.3 (CH3), 56.1 (CH3), 102.8 (CH), 103.0 (CH), 117.7
(CH), 120.1 (C), 120.8 (C), 124.2 (CH), 129.1 (CH), 133.3 (C), 142.9 (CH), 157.1 (C), 158.9 (C),
177.8 (C); LRMS (ESI-) m/z (%) 288 [M-H]- (100); HMRS (ESI-) m/z calc. for C14H14N3O2S
288.0807, found 288.0803.
2,2'-([1,1'-biphenyl]-4,4'-diylbis(methanylylidene))bis(hydrazinecarbothioamide) (34). The crude
product was prepared according to general procedure C starting from [1,1’-biphenyl]-4,4’-
20

ACCEPTED MANUSCRIPT
dicarboxaldehyde[18] (100 mg, 0.48 mmol, 1.1 equiv.) and thiosemicarbazide (2.0 equiv.). The
pure product (137 mg, 88%) was obtained as a yellow solid. Rf 0.18 (cyclohexane/EtOAc
1
70:30); m.p. > 320°C (decomposition); H NMR (400 MHz, DMSO-d6)
δ ppm 7.77 (d, J = 8.3 Hz,
4H), 7.90 (d, J = 8.3 Hz, 4H), 8.05 (s, 2H), 8.09 (s, 2H), 8.23 (s, 2H), 11.48 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) ppm 126.8 (4xCH), 127.9 (4xCH), 133.6 (2xC), 140.4 (2xC), 141.7
δ
(2xCH), 178.0 (2xC); LRMS (ESI-) m/z (%) 355 [M-H]- (100); HRMS (ESI-) m/z calc. for
C16H15N6S2 355.0800, found 355.0802.
4.2. Biology
4.2.1. Leishmania culture conditions
Leishmania donovani MHOM/ET/67/HU3 with luciferase gene integrated into the parasite
genome[28] were grown at 28°C in RPMI 1640-modified medium (Invitrogen) supplemented
with 20% heat-inactivated fetal bovine serum (iFBS, Invitrogen) with 100 µg/ml of hygromycin B.
4.2.2. Cell lines culture and determination of cellular toxicity
Human myelomonocytic cell line THP-1 were grown at 37ºC and 5% CO2 in RPMI-1640
supplemented with 10% iFBS, 2 mM glutamate, 100 U/mL penicillin and 100 ꢀg/mL
streptomycin. 3x104 cells/well in 96-well plates were differentiated to macrophages with 20
ng/mL of PMA treatment for 48 h followed by 24 h of culture in fresh medium.[29]
MRC-5 cells, a SV-40 transformed human fetal lung fibroblast cell line, were maintained at 37ºC
and 5% CO2 in DMEM supplemented with 10% iFBS, 100 U/mL penicillin and 100 ꢀg/mL
streptomycin. Cells were harvested by treatment with 0.05% (w/v) trypsin plus 0.48 mM EDTA
for 5 min, diluted to 4x104 cell/ml in 96-well plates and incubated at 37ºC and 5% CO2 before
toxicity assay.[30]
Cellular toxicity of all compounds was determined using the colorimetric MTT-based assay after
incubation at 37°C for 72 h in the presence of incr easing concentrations of compounds. The
results are expressed as EC50 values, as the concentration of compound that reduce cell growth
by 50% versus untreated control cells.
4.2.3. Susceptibility analysis in intracellular L. donovani amastigotes
The susceptibility of intracellular L. donovani amastigotes to synthesized compounds was
determined using the Luciferase Assay System Kit (Promega, Madison, Wis.) as previously
described.[30] Briefly, macrophage-differentiated-THP-1 cells were plated at a density of 3x104
macrophages/well in 96-well white polystyrene microplates and were infected at a
21

ACCEPTED MANUSCRIPT
macrophage/parasite ratio of 1:10 with late-stage promastigotes. Infected cell cultures were
incubated with different compound concentrations in RPMI 1640 medium plus 10% iFBS at
37°C with 5% CO 2 for 72 h and then lysed. Luminescence intensity was measured as indicative
of the intracellular parasite growth, as previously described.[29] Additionally, Selectivity Indices
(SI) of compounds were subsequently calculated on the basis of their antiparasitic activities
(EC50 parasite) and host cell cytotoxicity (EC50 MRC-5 cells), according to the formula:
EC50(MRC-5) / EC50 parasite.
Acknowledgements
The authors wish to acknowledge the support from the ICMG Nanobio Platform, Grenoble on
which the NMR and MS were performed.
This work was supported by the Spanish Grants Proyecto de Excelencia, Junta de Andalucía,
Ref. CTS-7282 (to F.G.), SAF2012-34267 (to F.G.), FEDER funds from EU to F.G.
The authors are grateful to ANR (Agence Nationale pour la Recherche) for financial support
(Labex Arcane (ANR-11-LABX-0003-01)).
Appendix A. Supplementary data
Supplementary data (1H and 13C NMR spectra) related to this article can be found at
http://dx.doi.org/
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List of captions
Figure 1. General structures of the first series of thiosemicarbazone screened.
Scheme 1. Synthesis of thiosemicarbazone derivatives 3-34. Reagents and conditions: (i)
thiosemicarbazide, EtOH, AcOH, reflux, 10 h, 21-88%; (ii) R”X (X = Br or Cl), NaH 60% in oil, dry DMF, rt,
18-72 h, 41-99%;[17] (iii) for R = alkyl: K2CO3, RX (X = I or Br), acetone, reflux, 4-24 h, 37-92%; (iv) for R
= MOM: MOMBr, NaH 60%, dry DMF, 0°C then rt 1-3 h, 68-76% .
Table 1
Antileishmanial activity and intrinsic toxicity of the different compounds on MRC-5 and THP-1 cells.
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Highlights
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-
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32 arylthiosemicarbazones were synthetized and characterized;
Compounds waere evaluated against intracellular amastigotes of Leishmania donovani;
High activity and low toxicity are reported, SI>250 for the most promising compound.