Synthesis and evaluation of antileishmanial and cytotoxic activity of benzothiopyrane derivatives

Article abstract of DOI:10.3390/molecules25040800

In continuation of our efforts to identify promising antileishmanial agents based on the chroman scaffold, we synthesized several substituted 2H-thiochroman derivatives, including thiochromenes, thichromanones and hydrazones substituted in C-2 or C-3 with carbonyl or carboxyl groups. Thirty-two compounds were thus obtained, characterized, and evaluated against intracellular amastigotes of Leishmania (V) panamensis. Twelve compounds were active, with EC50 values lower than 40 μM, but only four compounds displayed the highest antileishmanial activity, with EC50 values below 10 μM; these all compounds possess a good Selectivity Index > 2.6. Although two active compounds were thiochromenes, a clear structure-activity relationship was not detected since each active compound has a different substitution pattern.

Full text of DOI:10.3390/molecules25040800

molecules
Article
Synthesis and Evaluation of Antileishmanial and
Cytotoxic Activity of Benzothiopyrane Derivatives
Cristian Ortiz 1 , Fernando Echeverri
1
, Sara Robledo 2 , Daniela Lanari , Massimo Curini ,
3
3
1
,
1,
Wiston Quiñones * and Esteban Vargas *
1
Química Orgánica de Productos Naturales, Instituto de Química, Facultad de Ciencias Exactas y Naturales,
Universidad de Antioquia, calle 70, No. 52–21, Medellín A. A 1226, Columbia;
cristian.ortizb@udea.edu.co (C.O.); fernando.echeverri@udea.edu.co (F.E.)
PECET-Facultad de Medicina, Universidad de Antioquia, calle 70 No. 52–21, Medell
sara.robledo@udea.edu.co
Dipartimento di Scienze Farmaceutiche, Università di Perugia, Via del Liceo, 1, 06123 Perugia, Italy;
daniela.lanari@unipg.it (D.L.); massimo.curini@unipg.it (M.C.)
2
3
ín A. A 1226, Columbia;
*
Correspondence: esteban.vargasc@udea.edu.co (E.V.); wiston.quinones@udea.edu.co (W.Q.);
Tel.: +5-742-196-596 (E.V. & W.Q.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Andrew Tsotinis and Ioannis Papanastasiou
Received: 7 December 2019; Accepted: 30 January 2020; Published: 12 February 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: In continuation of our efforts to identify promising antileishmanial agents based on
the chroman scaffold, we synthesized several substituted 2H-thiochroman derivatives, including
thiochromenes, thichromanones and hydrazones substituted in C-2 or C-3 with carbonyl or carboxyl
groups. Thirty-two compounds were thus obtained, characterized, and evaluated against intracellular
amastigotes of Leishmania (V) panamensis. Twelve compounds were active, with EC50 values lower
than 40
µM, but only four compounds displayed the highest antileishmanial activity, with EC50
values below 10
µM; these all compounds possess a good Selectivity Index > 2.6. Although two active
compounds were thiochromenes, a clear structure-activity relationship was not detected since each
active compound has a different substitution pattern.
Keywords: Leishmania; thiochromenes; benzothiopyrans; thiochroman-4-ones; synthesis;
leishmaniasis; cytotoxicity
1
. Introduction
Cutaneous leishmaniasis (CL) is a group of skin infections caused by intracellular protozoa
belonging to the genus Leishmania and transmitted by Lutzomyia and Phlebotomus sand flies. CL is a
poverty-associated disease considered by the World Health Organization as one of the 17 neglected
diseases due to a lack of interest by the pharmaceutical industry to develop new drugs and a wide
distribution around the world. More than 310 million people at risk, and about one million new cases
are occurring annually. CL cases occur mainly in Afghanistan, Algeria, Brazil, Colombia, Iran, and
Syria [1].
To date, a few drugs are available against leishmaniasis, including pentavalent antimonials,
amphotericin B, and miltefosine. However, existing treatments are unsatisfactory because of their high
cost, toxicity, need for prolonged treatment, and reduced efficacy, therefore, there is an urgent need to
develop new molecules with novel modes of action against this disease.
The chroman (benzopyran) moiety is considered a privileged scaffold in medicinal chemistry since
it exhibits a broad range of biological activities such as antiemetic, anti-hypertensive, anti-malarial, and
insecticidal activities, among others [
2–5]. From a bioisosteric point of view, replacement of the oxygen
atom (O) by a sulfur atom (S) it may cause significant changes in the pharmacology activity since the
Molecules 2020, 25, 800; doi:10.3390/molecules25040800
www.mdpi.com/journal/molecules

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changes in electron-donating potential and the size of sulfur cause solubility [6], polarity, hydrogen
bonding and metabolism [
derivatives of chromanone and thiochromanone have good in vivo antileishmanial activity with
effective concentration (EC ) values of 20.1 and 25.0 M respectively, in addition to anti-inflammatory
]. Besides, acyl hydrazone derivatives of thiochroman-4-ones have
been reported as potent inhibitors of cysteine proteases which has implications in the treatment of
Chagas’ disease [ 10]. A SAR study involving thiochroman-4-one derivatives found that compounds
containing 1,1-dioxo-2-aryl-4H-thiochromen-4-one scaffolds displayed good antileishmanial activity
below 10 M. Ester and amide derivatives of thiochroman-4-ones were also include in this study; the
7] modifications. In a previous paper, we have reported that acyl hydrazone
µ
50
and wound-healing properties [
8
9,
µ
amides tend to form intermolecular and intramolecular hydrogen bonds with other groups [11] and
esters are used for masking polar moieties and taking advantage of the availability of endogenous,
non-specific esterases, release the molecule inside the cell [12].
Herein, we report the synthesis of a library of compounds bearing a benzothiopyran moiety,
including thiochromenes, thiochromanones and acyl hydrazones with a combination of fragments
possessing a substituted carbonyl group in C-2 or C-3 and their in vitro anti-leishmanial and
cytotoxic activities.
2
. Results
2
.1. Synthesis, Dehydration and Oxidation of Thiochromanols
In our search for methods for the synthesis of 2H-thiochromene-like compounds, we first explored
the synthetic methodologies used for their oxygenated counterparts. Several methodologies have been
proposed, which include, among others, the Petasis reaction [13] of salicylaldehydes, tandem Michael
additions [14], PPh -catalyzed domino reactions [15] for the synthesis of chroman derivatives and
3
the domino oxa-Michael aldol reaction [16]. The latter approach seemed likely to succeed with the
mercapto counterpart.
Synthesis of the precursor 2-mercaptobenzaldehyde (mercaptosalicylaldehyde, Scheme 1) started
from 2-chlorobenzaldehyde (
procedure [17] using 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) under solvent-free conditions leading
to high yields of the desired 2-(tert-butylthio)benzaldehyde ( ). Treatment of with a mixture of
HBr/acetic acid in dimethyl sulfoxide (DMSO) gave the 2,2’-dithiodibenzaldehyde ( ). Following
the work of Humphrey and Hawkins [18], compound was next allowed to react with PPh to give
-mercaptobenzaldehyde (5), which readily oxidizes in air to give again disulfide 4.
1) and tert-butylthiol (2), following a modification of the Chemburkar
3
3
4
4
3
2
Scheme 1. Synthesis of 2-mercaptobenzaldehyde.

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Considering the tedious procedure to obtain
5
and its relatively easy oxidation in the presence
of air, we decided to explore the synthesis of thiochromene compounds directly from aldehydes
or . Thus, disulfide was allowed to react with activated alkenes 6a in the presence of
3
4
4
–e
1
,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and triphenylphosphine, whereby thin layer chromatography
1
(TLC) of the reaction mixture showed various spots with similar Rf values. H-NMR analysis of these
crude mixtures revealed that this set of spots corresponds to the stereoisomers of thiochromanols 7a
–e
(Scheme 2). Due to the difficulty in separating the mixture of stereoisomers and the inconvenience
of using these mixtures in biological tests, we decided to use these compounds as a mixture in the
following reactions. The reaction crude was passed through a chromatography column to remove the
DBU and PPh and the excess of the starting materials.
3
Scheme 2. Synthesis of 2H-thiochromanols derivatives 7a–e.
To obtain 2H-thiochromenes 8a (Scheme 3), the mixtures of diastereomeric thiochromanols 7a–e
–e
were dehydrated by heating in the presence of acid (Amberlyst 15 or HCl) or even by simple direct
heating of the mixture containing the DBU and PPh3.
Scheme 3. Synthesis of 2H-thiochromenes 8a–e, ketones 9a,b,d and the enol forms 10a,b,d.

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In the search for compounds bearing a thiochroman-4-one moiety, thiochromanols 7a
–
e
were
treated with oxidants like pyridinium chlorochromate (PCC) or Dess-Martin Periodinane reagent
DMP, Scheme 3). Oxidation of 7c and 7e did not give the expected products On the other hand,
compounds 7a gave oxidation products which could tautomerize and were analyzed by NMR to
determine in which tautomeric form they exist.
NMR spectra of the oxidation product of 7a showed only one set of signals in which the protons
4.18 (dd, J = 11.6, 3.7 Hz, 1H), 3.67 (dd, J = 13.5, 11.7 Hz, 1H) and 3.57
3.40 (m, 1H) and the 13C-
NMR 184.6 characteristic of a ketone group indicate that the compound exists only as its keto form 9a
(
.
,b,d
at
δ
−
δ
.
In the case of the oxidation of 7b, the NMR spectra showed two sets of signals, indicating the
existence of two tautomeric forms. In the keto form 9b, proton H5 appears as a doublet at 8.17 ppm,
J = 7.9 Hz, while in the enol form 10b this proton signal is shifted at 7.89, d, J = 7.7 Hz, both signals
integrating for 0.2 and 1.0, respectively, the same ratio was can be calculated with the protons of
the methoxy group of the ester. A calculation of peak-intensity showed that this tautomeric mixture
consisted of 83% of the enol form and 17% of the keto form.
A similar analysis for the oxidation 7d showed no proton at C3, and the 13C-NMR resonance at
δ
195.4 and 174.0 is not consistent with a diketo compound 9d, which indicates that it exists only as
an enol compound 10d. It can be assumed that this enol form was stabilized by the intramolecular
hydrogen bond.
2
.2. Alternative Synthesis of 2H-Thiochromene Compounds
Taking into account that S-tert-butyl groups are cleaved under acidic conditions [19,20], treatment
of compound
3 with hydrochloric acid (HCl) or p-toluenesulfonic acid (PTSA) gave a dimeric
hemithioacetal 11 (Scheme 4), previously reported by Dickmann [21]. Formation of compound 11 must
happen through the formation of 2-mercaptobenzaldehyde (5), followed by dimerization.
Scheme 4. Proposed structure of the dimeric hemithioacetal 11.
The above results led us to think that compound
activated alkenes to yield the addition product; thus, we carried out the reaction of
cinnamaldehydes 12a using 12M HCl. Thus, substituted 2-phenyl-2H-thiochromene-3-carbaldehydes
3a–d in moderate to good yields were effectively produced (Scheme 5).
5
formed in situ could react with suitably
3
in the presence of
–
d
1
Scheme 5. Direct synthesis of 2H-thiochromenes starting from 3 and cinnamic aldehydes.

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Similarly, trans-chalcone reacted with
3
in the presence of 4-toluenesulfonic acid in toluene to give
phenyl-(2-phenyl-2H-thiochromen-3-yl)-methanone (15a) in 46% yield (Scheme 6). Acid-catalyzed in
situ formation of the chalcones followed by reaction with 2-(tert-butylthio)-benzaldehyde in a one-pot
procedure also resulted in the formation of the desired aryl-2H-thiochromen-methanones 15b–c.
Scheme 6. Synthesis of (aryl-2H-thiochromen-3-yl)-methanones from chalcones.
2
.3. Synthesis of 4-oxo-thiochroman-2-Carboxylic Acid and Its Derivatives
Derivatives of thiochroman-4-one are typically prepared by conjugate additions of thiophenols
to
α,β-unsaturated carboxylic acids followed by treatment with a strong dehydrating agent or by
formation of the corresponding acid chlorides followed by treatment with a Lewis acid like AlCl or
3
SnCl . Similarly 4-oxo-thiochroman-2-carboxylic acids 20a
–
d
were obtained by reacting thiophenols
with furan-2,5-dione (maleic anhydride, 19) in the presence of triethylamine and with subsequent
treatment with AlCl (Scheme 7).
4
1
8a–d
3
Scheme 7. Synthesis of 4-oxothiochroman-2-carboxylic acid.
In order to get a set of compounds that allow us to perform a SAR analysis to establish the
influence of the functional groups and the length of the side chains we prepared a set of esters and
amides of 4-oxo-thiochroman-2-carboxylic acids.

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Esters of 4-oxo-thiochroman-2-carboxylic acid 22a were prepared by reaction with the
corresponding alcohols 21a using dicyclohexylcarbodiimide (DCC) as a coupling agent and
4-dimethylaminopyridine (DMAP) as a catalyst (Scheme 8).
–
d
O
O
S
DCC/DMAP
ACN
+
HO
O
OH
S
n
n
O
O
2
0a
21a: n = 1
22a: n = 1
2
2
2
1b: n = 3
1c: n = 5
1d: n = 9
2
2
2
2b: n = 3
2c: n = 5
2d: n = 9
Scheme 8. Esters of 4-oxo-thiochroman-2-carboxylic acid.
Amides 24a
–
c
were prepared by reacting the corresponding amines 23a–c with
4-oxo-thiochroman-2-carboxylic acid 20a under Schotten-Baumann conditions (Scheme 9) using
a biphasic system containing in the organic layer, oxalyl chloride (COCl) and dimethyl formamide as
2
catalyst (DMF) and aqueous sodium hydroxide.
Scheme 9. Synthesis of amides of 4-oxo-thiochroman-2-carboxylic acid.
2
.4. Synthesis of Acyl Hydrazone Derivatives
As it was mentioned before, compounds having an acyl hydrazone moiety have shown
good antiparasitic activities. According to this we decided to prepare some derivatives with
this structural feature. Acyl hydrazone derivatives 26a were prepared by reaction of ethyl
-oxothiochromane-2-carboxylate with acyl hydrazides 25a in the presence of acetic acid (Scheme 10).
–
c
4
–c
Scheme 10. Synthesis of acyl hydrazine derivatives.

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2
.5. Resolution of Enantiomers of 4-oxo-thiochroman-2-carboxylic Acid Using Brucine
To establish the difference in antileishmanial activity of the enantiomers of
-oxo-thiochroman-2-carboxylic acid (20a) we attempted the chiral resolution by diastereomeric salt
4
formation through reaction with optically pure (
−
) -brucine 27 as a chiral selector (Scheme 11). Thus,
2
0a was allowed to react with ( ) -brucine 27 in a mixture of THF/water 4:1 at room temperature
−
overnight, to yield a mixture of diastereomeric salts. Attempts were made to crystallize the mixture
by slow evaporation of different solvents (acetone, THF, water, ethanol and ethyl acetate). Finally,
crystalline salts were obtained after evaporation of ethyl acetate. Crystals were separated from mother
liquors and were subjected to acid hydrolysis to yield the salt of (
enriched sample of 4-oxo-thiochroman-2-carboxylic acid (+)-20a with specific rotation [
−
) – brucine and an enantiomerically
25
α
]
= +35.6.
D
After evaporating the ethyl acetate from mother liquors, we obtained an enriched sample of the less
crystalline diastereoisomeric salt. Acid hydrolysis of the residue of the mother liquors yield a mixture
25
of enantiomers with specific rotation of [
configuration nor enantiomeric excess.
α
]
=
−
10.8 for ( )-20a. We did not determine absolute
−
D
Scheme 11. Chiral resolution of 4-oxothiochroman-2-carboxylic acid 20a.
Enantiomerically enriched samples of butyl 4-oxothiochromane-2-carboxylates (+)-22b with
2
5
-
25
[
α] = +32.4 and (
−
)
22b [α]D
enantiomerically enriched samples of 4-oxo-thiochroman-2-carboxylic acid (+)-20a or (
Scheme 12).
=
−
8.3, respectively, were obtained by reaction of butanol with
D
−)-20a
(
Scheme 12. Enantiomerically enriched butyl 4-oxothiochromane-2-carboxylates.

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2
.6. Antileishmanial and Cytotoxic Activities
The synthesized compounds were evaluated for their in vitro antileishmanial and cytotoxic
activities (Table 1), following the methods of Pulido et al. [22] Amphotericin B, with EC50 and LC50
values of 0.05 µM and 56.8 µM, respectively, was used as a control.
Table 1. In vitro antileishmanial and cytotoxic activities.
Thiochromenes
R1
General structure
S
R2
Compound
R1
R2
H
EC50 (µM) 1
6.1 ± 4.6
126.0 ± 15.5
> 2.6
LC50 (µM) 1
4.0 ± 0.6
351.5 ± 47.5
6.0 ± 0.4
25.7 ± 4.6
21.2 ± 3.4
SI 2
0.7
8
a
CN
8
b
COOCH3
COCH3
COCH3
COCF3
H
2.8
8
c
C H
6
< 2.3
3.1
5
8
d
(CH ) CH
8.3 ± 0.8
29.6 ± 6.2
2 4
3
8
e
C H
6
0.7
5
General structure
Compound
R
EC50 (µM) 1
> 23.8
LC50 (µM) 1
54.7 ± 3.2
9.6 ± 0.4
160.8 ± 27.9
23.9 ± 2.4
SI 2
< 2.3
0.3
1
3a
3b
3c
3d
C H
6
5
1
p-C H -F
29.7 ± 4.4
37.0 ± 5.6
9.2 ± 2.7
6
4
1
p-C H -Cl
4.3
6
4
1
o-C H -NO
2
2.6
6
4
General structure
Compound
R1
H
R2
EC50 (µM) 1
43.9 ± 11.6
37.9 ± 2.8
LC50 (µM) 1
> 608.9
SI 2
< 13.9
2.6
1
5a
5b
5c
H
1
Cl
Cl
CF3
97.2 ± 14.2
151.3 ± 18.1
1
NO2
38.1 ± 11.3
4.0
Thiochroman-4-ones
General structure

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Table 1. Cont.
Thiochromenes
Compound
R1
R2
EC50 (µM) 1
LC50 (µM) 1
770.5 ± 49.7
485.0 ± 45.4
51.0 ± 9.0
SI 2
7.2
3.6
4.1
9
a
CN
H
H
107.7 ± 34.3
133.6 ±27.0
12.5 ± 3.3
9
b + 10b
0d
COOCH3
COCH3
1
(CH2)4CH3
General structure
Compound
R1
R2
R3
EC50 (µM) 1
414.1 ± 58.5
LC50 (µM) 1
132.8 ± 67.7
SI 2
2
0a
H
H
OH
0.3
3
517.1 ±
20b
F
H
OH
210.1 ± 78.7
0.1
3
007.5
2
0c
0d
2a
2b
2c
2d
4a
4b
OCH3
H
H
OCH3
H
OH
OH
493.6 ± 324.4
277.0 ± 100.9
> 68.5
76.3 ± 8.6
90.4 ± 16.4
211.1 ± 0.7
231.3 ± 44.5
213.3 ± 47.9
140.5 ± 20.7
157.0 ± 9.7
147.6 ± 45.4
0.2
2
0.3
2
H
OCH CH
< 3.1
< 1.2
< 2.1
< 1.0
0.7
2
3
2
H
H
O(CH ) CH
> 189.3
2
3
3
3
3
2
H
H
O(CH ) CH
> 102.7
2
5
2
H
H
O(CH ) CH
> 143.6
2
9
2
H
H
NH(CH ) CH 216.2 ± 2.5
2
5
3
2
H
H
NH(CH2)11CH3
> 80.0
< 1.8
NH-o-C H -
6
4
2
4c
H
H
> 101.0
118.7 ± 10.9
< 1.2
CH3
2
0a (+)
0a (-)
2b (+)
2b (-)
H
H
H
H
H
H
H
H
OH
OH
505.3 ± 132.6
53.5 ± 10.8
368.0 ± 24.2
53.0 ± 10.8
> 48.1
> 0.1
0.6
2
33.0 ± 1.5
74.9 ± 52.8
28.8 ± 4.5
2
O(CH ) CH
0.2
2
3
3
3
2
O(CH ) CH
0.5
2
3
Acyl hidrazones
General structure
Compound
R1
R2
EC50 (µM) 1
LC50 (µM) 1
15.4 ± 0.2
15.2 ± 0.2
SI 2
0.5
2
6a
C H
6
OCH CH
28.5 ± 6.3
53.2 ± 41.0
5
2
3
26b
C H N
OCH CH
0.3
5
4
2
3
1
156.6 ±
2
6c
CONH2
OH
> 188.6
< 6.1
3
39.6
1
standard deviation of the half-maximum concentration in µ
M. 2 Selectivity
Results reported as the mean value
±
Index (SI) = LC50/EC50. Bold data represent compounds with high activity against amastigotes of L. (V) panamensis.
3
ND = not determined because the compounds were inactive at the maximum concentration evaluated.

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3
. Discussion
Leishmaniasis is a disease that threatens and affects almost 20% of the world’s population,
but, there are very few effective drugs available for its treatment, so new molecules are urgently
needed. However, this task is a priority for the pharmaceutical industry, so the affected countries
must seek their own alternatives. Recently we reported the in vivo leishmanicidal activities of
compounds bearing a benzothiopyran scaffold and also the acyl hydrazones derived from chroman
and thiochromans [8,23]. To optimize their effects, in this work we prepared thirty-two compounds,
including thiochromenes, thichroman-4-ones and acyl hydrazones substituted in C-2 or C-3 with
carbonyl/nitrile or carboxyl groups.
The compounds can be grouped into three types, according to their activity. The most active were
those with EC50 < 20
µM, those with marginal activity had an EC50 between 20–40
µM and the inactive
compounds have EC50 > 40. The most active compounds include three the thiochromenes 8a
,
8d 13d
,
and compound 10d. Substituents at C-2 and C-3 in the former compounds do not have a structural
relationship between them. Compound 8a with a nitrile at C-3 is the most active, but, surprisingly 8b
with a methyl ester is practically inactive, although the methyl ester is considered a nitrile isostere [24].
Compounds 8d and 10d with an alkyl chain at C-2 showed high leishmanicidal activity, and
changing the alkyl chain for a phenyl group resulted in a decrease in anti-leishmanial activity with an
increase in the cytotoxicity. Although none of the compounds bearing a phenyl or aryl group at C-2
showed high anti-leishmanial activity most of the 2-arylthiochromenes displayed a marginal activity,
indicating promising compounds for further optimization. On the other hand, if the C-3 methyl ketone
is substituted by phenyl or aryl groups, the cytotoxicity decreases while maintaining marginal activities
between 37.9–43.9 µM.
Thiochromenes of type C-3-formyl, 13a
,b,c with aryl groups in C-2 have a range of activity similar
to compounds 8e and 15a all having electron-withdrawing groups. The above seems to indicate
,b,c
that steric factors and pi interactions are involved in the interaction of these compounds with a putative
receptor of Leishmania amastigote.
On the other hand, the importance of a C-3 carbonyl substituent is demonstrated in the series of
compounds 20a–d, 22a–d and 24a–c all bearing the thiochroman-4-one scaffold without substituents
at C-3, which were practically inactive. The previous esters and amides were prepared thinking of
performing a SAR study to analyze the effect of chain size on leishmanicidal and cytotoxic activity, but
all these compounds showed very low activity.
Surprisingly, two acyl hydrazones 26a and 26b showed a marginal activity EC50 28.5 and 53.2
µM,
unlike the compounds that we have previously analyzed [ ], which seems to indicate that the
8
substitution by a C-2 carbonyl of a thiochroman-4-one interferes with its mechanism of action against
amastigotes of L. panamensis parasites.
In conclusion, in search of new chemotherapeutic agents against leishmaniasis based on the
benzothiopyran moiety, we synthesized thirty-two structurally diverse compounds. These compounds
possess relevant in vitro antileishmanial activity with moderate cytotoxicity. Compounds 8d and 10d
which differ only in the hydroxy group at C-4, were the most promising compounds among this library
with good antiparasitic activity and Selectivity Index. Biological studies with Leishmania parasite
enzymes need to be performed to identify the potential targets. Structural modification on ring A
,
of 8d and 9d, 10d could enhance the antileishmanial activity or reduce cytotoxicity. Overall, 8d, 9d,
and 10d represents potential hits in the search for new chemotherapeutic agents for the treatment
of leishmaniasis.

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4
4
4
. Materials and Methods
.1. Chemistry
.1.1. General Information
All commercially available reagents and solvents were obtained from commercial suppliers and
used without further purification. The reaction progress was monitored with thin layer chromatography
on silica gel TLC aluminum sheets (60F254, Merck, Darmstadt, Germany). The melting points were
determined using a Mel-Temp apparatus (Electrothermal, Staffordshire, UK) and are uncorrected.
FTIR spectra were obtained on a Bruker Alpha FTIR spectrometer (Bruker Optic GmbH, Ettlingen,
1
13
Germany). H and C nuclear magnetic resonance (NMR) spectra were recorded using Bruker 300
13
and 400 spectrometers (300 and 400 MHz for 1H, 75, and 100 MHz for C). Chemical shifts were
1
13
reported relative to internal tetramethyl silane (
δ 0.00 ppm) for H, and CDCl (δ 77.0 ppm) for C.
3
HRMS was obtained using Q-TOF quadrupole/orthogonal mass spectrometry (Waters, Milford, MA,
+
+
USA) in positive mode (reported as [M + H] or [M + Na] ).
4
.1.2. Synthesis of the Benzothiopyranes and Derivatives
2
-(tert-Butylthio)benzaldehyde (3)
In a screw-capped flask equipped with a stir bar, DMSO (2.0 mL, 0.028 mol, 4 equivalent) and
potassium hydroxide (KOH; 470 mg, 8.4 mmol,1.1 equivalent) were mixed under nitrogen and stirred
at room temperature. After 30 min, the KOH was crushed with a spatula, and 2-methyl-2-propanethiol
(
(
610
810
µ
L, 0.0106 mol, 1.5 eq) was added, and the mixture was stirred for 20 min. 2-Chlorobenzaldehyde
L, 7.2 mmol, 1.0 equivalent) was added, and the reaction was heated to 110 C for 90 min. After
◦
µ
that, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (2
The combined organic layers were washed with water (30 mL) and brine (30 mL), dried (anhydrous
Na SO ), and concentrated on a rotary evaporator the residue was purified by chromatography using
×
30 mL).
2
4
a mixture of 10% ethyl acetate in hexanes to afford 2-(tert-butylthio)benzaldehyde 15 (980 mg, 70%
1
yield) along with some unreacted 2-chlorobenzaldehyde. H-NMR (300 MHz, CDCl ) 10.78 (s, 1H),
3
7
.98 (d, J = 4.3 Hz, 1H), 7.62–7.50 (m, 1H), 7.45–7.30 (m, 1H), 1.30 (s, 9H). GC-MS: m/z (%) = 194 (27)
+
[M ], 161 (8), 138 (100), 77 (5).
0
2
,2 -Disulfanediyldibenzaldehyde (4)
A 25-mL round-bottom flask containing 2-(tert-butylthio)benzaldehyde (3, 194 mg, 1.0 mmol,
1
equivalent) was placed in an ice bath. Acetic acid (343
µL, 6 mmol, 6.0 equivalents), 48% aqueous
HBr (343
µ
L, 3 mmol, 3.0 equivalents) and DMSO (73 L, 1.0 mmol, 1.0 equivalent) were then added to
µ
the resulting cooled mixture. The reaction mixture was then allowed to warm to room temperature
and then was stirred overnight. The reaction mixture, containing a solid precipitate, was diluted
with cold water (2 mL), filtered, dried and purified by column chromatography using 20% ethyl
acetate in hexanes as eluent to afford 2,2-disulfanediyldibenzaldehyde (
4
). Yield 73%. White solid.
◦
1
m.p.: 149–151 C. H-NMR (300 MHz, CDCl )
δ 10.19 (s, 2H), 7.85 (dd, J = 7.5, 1.4 Hz, 2H), 7.75 (d,
3
13
J = 7.8 Hz, 2H), 7.47 (td, J = 7.7, 1.6 Hz, 2H), 7.37 (dd, J = 10.6, 4.2 Hz, 2H). C-NMR (75 MHz, CDCl )
3
+
δ
192.1, 140.2, 135.0, 134.5, 134.0, 126.8, 126.5. IR
ν
: 1664, 1582, 1555. GC-MS: m/z (%) = 274 (14) [M ],
+
1
37 (100), 109 (54). HRMS-ESI (m/z): calcd. for C H O S [M + Na] 297.0014, found: 297.0025.
14
10
2 2
2
-Mercaptobenzaldehyde (5)
In a 25 mL round bottom flask, 2,2-disulfanediyldibenzaldehyde (
4, 177 mg, 0.65 mmol, 1
equivalent), was dissolved in a mixture of DMF (5.4 mL), MeOH (5.4 mL) and of water (3.0 mL; all
solvents were deoxygenated before use) then tripheylphosphine (253 mg, 0.975 mmol, 1.5 equivalent)
was added, and the mixture stirred a room temperature for 30 min. After that the reaction mixture was

Molecules 2020, 25, 800
12 of 22
diluted with water (20 mL) and extracted with deoxygenated diethyl ether (2
×
30 mL). The combined
organic layers were washed with deoxygenated water (30 mL) and brine (30 mL), dried (anhydrous
Na SO ), and concentrated on a rotary evaporator the residue was purified by chromatography using
2
4
a mixture of 10% ethyl acetate in hexanes to afford 2-mercaptobenzaldehyde (5), in 73% yield. During
the whole process, the presence of air in the mixture was avoided to prevent oxidation back to the
1
disulfide 4. H-NMR (300 MHz, CDCl3)
δ
10.02 (s, 1H), 7.73 (dd, J = 8.5, 7.7 Hz, 1H), 7.38 – 7.20 (m,
3
H), 5.48 (s, 1H).
2
H-Thiochromene-3-carbonitrile (8a)
In a round-bottom flask were placed 2,2-disulfanediyl-dibenzaldehyde (4, 137 mg, 0.5 mmol,
1
equivalent), acrylonitrile 58 mg (0.55 mmol, 2.2 equivalents) and DBU 84 mg (0.55 mmol, 1.1
◦
equivalents) under nitrogen, and the mixture was heated with stirring at 80 C for 24 h, After cooling
to room temperature, the crude reaction mixture was then purified by column chromatography over
silica gel using hexane/EtOAc (5:1) as eluent to afford 7a as a white solid (77 mg, 45%). Alternatively,
2
H-thiochromene-3-carbonitrile (7a) was prepared by a different methodology, as follows:
Step 1. In a screw-capped flask, 2,2-disulfanediyl-dibenzaldehyde ( , 70 mg, 0.25 mmol, 1
4
equivalent); triphenylphosphine 131 mg (0.5 mmol, 2 equivalents) were dissolved in THF (5 mL),
the mixture was stirred at room temperature under an argon atmosphere. After 20 min an excess
of acrylonitrile (80 mg, 1.5 mmol, 6 equivalents) was added and after another 10 min of reaction a
TLC plate showed the formation of 2-mercaptobenzaldehyde (
5) and the mixture of stereoisomers of
4-hydroxy-thiochromane-3-carbonitrile. The reaction mixture is stirred overnight, and after that time,
the only products are the 4-hydroxythiochromane-3-carbonitriles.
Step 2. The mixture of 4-hydroxythiochromane-3-carbonitriles is heated overnight at reflux in
dichloroethane with 20% mol of Amberlyst 15 after that, the solvent was evaporated on a rotary
evaporator and diluted in the minimum amount of CH Cl and purified by column chromatography
2
2
using a mixture of 20% ethyl acetate in hexanes as eluent to afford 2H-thiochromene-3-carbonitrile 8a
◦
1
in 68% yield (calculated from the dimer). Mp. 96–98 C. H-NMR (400 MHz, CDCl )
δ
7.30
142.4, 132.9, 131.1,
ν: 2207, 1615, 1435, 753. HRMS-ESI (m/z): calcd. for
−
7.19
3
(
m, J = 7.5 Hz, 2H), 7.20 – 7.10 (m, 3H), 3.57 (s, 2H). 13C-NMR (101 MHz, CDCl3)
30.3, 130.3, 127.7, 126.5, 118.5, 103.9, 26.0. IR
δ
1
+
C H NS [M + H] 174.0372, found: 174.0380.
10
7
Methyl 2H-thiochromene-3-carboxylate (8b)
In a screw-capped flask, 2,2-disulfanediyldibenzaldehyde (
4, 137 mg, 0.5 mmol, 1 equivalent),
methylacrylate (130 mg, 1.5 mmol, 3.0 equivalents) and DBU (230 mg, 1.5 mmol, 3.0 equivalents) were
◦
mixed under an argon atmosphere and then heated to 80 C for 24 h, After cooling to room temperature
the reaction mixture was dissolved with CH Cl and poured directly onto a chromatography column
2
2
that was eluted using 10% ethyl acetate in hexanes to afford methyl 2H-thiochromene-3-carboxylate
◦
1
1
(
7
1
8b) in 48% yield as a yellowish solid with mp. 34–35 C. H-NMR (400 MHz, CDCl )
δ
7.55 (s, H),
7.16 (m, 3H), 7.13 (d, J = 7.4 Hz, 3H), 3.84 (s, 3H), 3.73 (s, 2H). 13C-NMR (100 MHz, CDCl3)
66.4, 137.3, 134.0, 131.3, 130.6, 130.2, 127.1, 125.8, 123.0, 52.2, 24.0. IR : 2952, 1703, 1434, 1235, 751.
HRMS-ESI (m/z): calcd. for C H O S [M + H] 207.0474, found: 207.0185.
3
.30
−
δ
ν
+
11
10
2
1
-(2-Phenyl-2H-thiochromen-3-yl)ethan-1-one (8c)
In a 10 mL round bottom flask equipped with a reflux condenser were added
(E)-4-phenylbut-3-en-2-one (146 mg, 1.0 mmol) and 2-(tert-butylthio)benzaldehyde (3, 250 mg, 1.3 mmol)
and to this mixture was added concentrated hydrochloric acid (12M, 2.0 mL) and the mixture was
◦
placed in a preheated oil bath at 110 C and held for two hours. Thereafter water (10 mL) was
added and the mixture was extracted with dichloromethane (3
×
15 mL), the combined organic
layers were washed with saturated NaHCO solution. After drying over Na SO , the solvent was
3
2
4
evaporated, and the residue was purified by column chromatography to yield 106 mg (40%) of

Molecules 2020, 25, 800
13 of 22
◦
1
1
(
(
-(2-phenyl-2H-thio-chromen-3-yl)ethan-1-one 27, as a yellowish solid. Mp: 115–116 C. H-NMR
400 MHz, CDCl3) 7.67 (s, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.25 (dd, J = 3.9, 2.8 Hz, 1H), 7.21 – 7.13
m, 5H), 5.36 (d, J = 1.6 Hz, 1H), 2.49 (s, 3H). 13C-NMR (101 MHz, CDCl3)
196.5, 142.0, 137.4, 134.0,
32.8, 131.1, 130.7, 130.4, 128.5, 127.6, 127.6, 126.5, 125.7, 38.8, 25.6. IR : 3031, 1656, 1622, 1196, 900.
HRMS-ESI (m/z): calcd. for C H OS [M + H] 267.0838, found: 267.0345.
δ
δ
1
ν
+
17
14
1
-(2-Pentyl-2H-thiochromen-3-yl)ethan-1-one (8d)
In a 10 mL round bottom flask were added 2,2-disulfanediyldibenzaldehyde (4, 140 mg, 0.5
mmol), 2-Nonanone (145 mg, 1.0 mmol) and triphenylphosphine (262 mg, 1.0 mmol) dissolved in
THF (5 mL) and the mixture was stirred under an argon atmosphere at room temperature overnight.
Column chromatography of the crude residue after solvent removal gave 195 mg (70%) of a mixture of
stereoisomers of 1-(4-hydroxy-2-pentylthiochroman-3-yl)ethan-1-one. Later 140 mg of the isomeric
alcohols was dissolved in THF (5 mL containing Amberlyst 15 (20 % mol) in a 10 mL round bottom
flask equipped with a reflux condenser and the mixture was heated to reflux overnight during 20 h.
After cooling to room temperature the reaction mixture was dissolved with CH Cl and the crude was
2
2
purified by column chromatography over silica gel using hexane/EtOAc (9:1) as eluent to afford 40 mg
1
(
29%) of 1-(2-pentyl-2H-thiochromen-3-yl)ethan-1-one (8d) as a yellowish oil. H-NMR (400 MHz,
CDCl3)
δ
7.45 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.34
−
7.24 (m, 2H), 7.20 (td, J = 7.3, 1.4 Hz, 1H), 4.11 (dd,
1.15 (m, 6H), 0.88 (t, J = 6.8 Hz, 3H.
196.8, 136.4, 133.3, 130.8, 130.6, 128.1, 125.5, 36.2, 34.4, 31.2, 25.7, 25.5,
2.5, 14.1. Note: the signals at 130.6 and 133.3 each correspond to two overlapped peaks. IR : 2927,
635, 1407, 1388, 756. HRMS-ESI (m/z): calcd. for C H OS [M + H] 261.1308, found: 261.1320.
J = 8.0, 5.6 Hz, 1H), 2.52 (s, 3H), 1.47 (dt, J = 10.8, 3.7 Hz, 2H), 1.35
−
13
C-NMR (75 MHz, CDCl3)
δ
2
1
ν
+
16
20
4
-Oxo-thiochromane-3-carbonitrile (9a)
According with Step 1 of the procedure for the synthesis of compound 8a we prepared a mixture
of stereoisomers of 4-hydroxythiochromane-3-carbonitrile, then this mixture (191 mg, 1.0 mmol)
was dissolved in dichloromethane (5.0 mL) and mixed with the Dess-Martin periodinane reagent
(430 mg, 1 mmol). After 1 h of stirring at room temperature the crude mixture was purified by
column chromatography over silica gel using hexane/EtOAc (4:1) as eluent to afford the desired
◦
1
4
-oxothiochromane-3-carbonitrile (9a, 60 mg, 30%) as a white solid with mp: 79–81 C. H-NMR
1
(300 MHz, CDCl3)
δ
H NMR (300 MHz, CDCl3)
δ
8.17 (dd, J = 8.0, 1.4 Hz, 1H), 7.61–7.44 (m, 1H),
7
.42–7.21 (m, 2H), 4.18 (dd, J = 11.6, 3.7 Hz, 1H), 3.67 (dd, J = 13.5, 11.7 Hz, 1H), 3.57–3.40 (m, 1H).
13
C-NMR (75 MHz, CDCl3)
δ 184.6, 141.0, 134.7, 130.5, 128.9, 127.7, 126.0, 115.1, 41.8, 29.4. IR ν: 2918,
+
2
256, 1678, 1582, 1430. HRMS-ESI (m/z): calcd. for C H NOS [M + Na] 212.0141, found: 212.0153.
10
7
Methyl 4-oxo-thiochromane-3-carboxylate (9b, 10b)
In a screw-capped flask, 2,2-disulfanediyl-dibenzaldehyde (4, 70 mg, 0.25 mmol, 1 equivalent) and
triphenylphosphine (131 mg (0.5 mmol, 2 equivalents), were dissolved in THF (5 mL), and the mixture
was stirred at room temperature under an argon atmosphere. After 20 min an excess of acrylonitrile
(
80 mg, 1.5 mmol, 6 equivalents) was added and after another 10 min of reaction a TLC plate showed
the formation of 2-mercaptobenzaldehyde ( ) and a mixture of 3-cyano-4-hydroxy-2H-thiochroman
5
isomers. The reaction mixture was stirred overnight, and after that, the only products are the mixture
of stereoisomers of 3-cyano-4-hydroxy-2H-thiochroman. A portion of the above mixture (112 mg)
was dissolved in dichloromethane (5.0 mL) and mixed with the Dess-Martin periodinane reagent
(
430 mg, 1 mmol). After 1 h of stirring at room temperature, the crude mixture was purified by column
chromatography to afford 40 mg (36%) of the desired methyl 4-oxo-thiochromane-3-carboxylate as a
◦
white solid with mp: 85–86 C. Methyl 4-oxo-thiochromane-3-carboxylate (9b) exists in solution in
equilibria with its tautomeric methyl 4-hydroxy-2H-thiochromene-3-carboxylate enol form 10b. The
1
H-NMR spectrum in CDCl revealed the that the ratio of tautomers 9b and 10b was 1:5. Proton H5
3
appears as a doublet at 8.17 ppm, J = 7.9 Hz, the proton H5 of the enol form has a chemical shift of

Molecules 2020, 25, 800
14 of 22
7
.89 d, J = 7.7 Hz the integration areas are 0.2 and 1.0 respectively, which indicates that the enol form
corresponds to 83% of the mixture. The same ratio can be calculated with the protons of the methoxy
1
a
b
a
group of the ester. H-NMR (300 MHz, CDCl )
δ
12.71 (s), 8.17 (d, J = 7.9 Hz), 7.89 (d, J = 7.7 Hz),
3
b
a,b
(m), 3.89 a (s), 3.85 (s), 3.76 a (s,), 3.38 (dd, J = 13.5, 3.6 Hz) ( is the
171.6, 165.9, 137.2, 131.0, 129.8, 129.2, 127.3,
: 3012, 2951, 1718, 1681, 1645, 1608,
b
b
a
7
.44 (t, J = 7.6 Hz), 7.37–7.19
enol form, is the keto form); C-NMR (75 MHz, CDCl3)
26.8, 125.6, 125.3, 93.7, 77.5, 77.1, 76.7, 54.0, 52.7, 52.1, 23.3; IR
583, 1553, 1437. HRMS-ESI (m/z): calcd. for C H O S [M + Na] 245.0243, found: 245.0254.
b
13
δ
1
ν
+
1
11
10
3
1
-(4-Hydroxy-2-pentyl-2H-thiochromen-3-yl)ethan-1-one (10d)
In a 10 mL round bottom flask were added 2,2-disulfanediyldibenzaldehyde 17, (140 mg, 0.5 mmol),
2-nonanone, (145 mg, 1.0 mmol) and triphenylphosphine (262 mg, 1.0 mmol), and the mixture
was stirred under an argon atmosphere at room temperature overnight. Column chromatography
of the crude residue after solvent removal gave 195 mg (70%) of a mixture of stereoisomers of
1-(4-hydroxy-2-pentylthiochroman-3-yl)ethan-1-one (10d). Next, 90 mg of this mixture were dissolved
in dichloromethane (5.0 mL) and mixed with the Dess-Martin periodinane reagent (192 mg, 0.45 mmol)
and water (1.0 mL). After stirring for 1 h at room temperature the crude mixture was purified by
column chromatography to afford 55 mg (60%) of the desired product as a yellowish oil which exists
1
in solution only as its enol form 1-(4-hydroxy-2-pentyl-2H-thiochromen-3-yl)ethan-1-one. H-NMR
(400 MHz, CDCl3)
δ
7.97 (ddd, J = 7.9, 1.4, 0.5 Hz, 1H), 7.36–7.25 (m, 2H), 7.22 (ddd, J = 7.9, 7.0,
1
1
1
.6 Hz, 1H), 3.67 (dd, J = 9.6, 5.0 Hz, 1H), 2.31 (s, J = 1.8 Hz, 3H), 1.77–1.65 (m, 1H), 1.63–1.43 (m, 2H),
1
3
.36–1.09 (m, 5H), 0.85 (t, J = 7.0 Hz, 3H). C-NMR (101 MHz, MeOH)
δ 195.4, 174.0, 135.8, 132.0, 129.5,
28.2, 127.7, 125.5, 108.8, 39.6, 36.4, 31.1, 26.6, 24.4, 22.5, 14.0. IR : 2928, 2856, 1634, 1593, 1545, 1380.
ν
+
HRMS-ESI (m/z): calcd. for C H O S [M + Na] 299.1076, found: 299.1088.
16
20
2
6
H,12H-6,12-Epoxydibenzo[b,f][1,5]dithiocine (11)
In a 10 mL round bottom flask equipped with a reflux condenser containing concentrated
hydrochloric acid (12M, 3.0 mL) was added 2-(tert-butylthio)benzaldehyde (
This mixture was placed in a preheated oil bath at 110 C and held for 2 h, after which water (10 mL)
3
, 582 mg, 3.0 mmol).
◦
was added and the mixture was extracted with dichloromethane (3
×
15 mL) and the combined
organic layers were washed with saturated NaHCO solution. After drying over Na S0 , the solvent
3
2
4
was evaporated, and the residue was purified by column chromatography over silica gel using
hexane/EtOAc (4:1) as eluent to yield 282 mg, (72%) of 6H,12H-6,12-epoxydibenzo[b,f][1,5]dithiocine
◦
1
with m.p.: 160–161 C. H-NMR (300 MHz, CDCl )
δ 7.36–7.31 (m, 1H), 7.17 –7.05 (m, 3H), 6.40 (s, 1H).
3
13
C-NMR (75 MHz, CDCl3)
GC-MS: m/z (%) = 258 (40) [M ], 153 (100), 121 (15), 77 (28).
δ
132.7, 129.0, 128.3, 127.9, 125.2, 74.7. IR
ν
: 3051, 1433, 1262, 1083, 956, 726.
+
4
.1.3. General Procedure for the Synthesis of 2-aryl-2H-thiochromene-3-carbaldehydes 13a–d
In a 10 mL round bottom flask equipped with a reflux condenser were added the corresponding
cinnamaldehyde (1.0 mmol) and 2-(tert-butylthio)benzaldehyde (3, 1.3 mmol) and to this mixture was
added concentrated hydrochloric acid (12M, 2.0 mL). Then, the mixture was placed in a preheated
◦
oil bath at 110 C and held for two hours, after which water (10 mL) was added and the mixture
was extracted with dichloromethane (3
×
15 mL), and the combined organic layers were washed with
saturated NaHCO solution. After drying over Na SO , the solvent was evaporated, and the residue
3
2
4
was purified by column chromatography over silica gel using hexane/EtOAc (4:1) as eluent to yield the
corresponding 2-aryl-2H-thiochromene-3-carbaldehyde 13a–d.
2
-Phenyl-2H-thiochromene-3-carbaldehyde (13a)
Starting from cinnamaldehyde, according to the general procedure described above,
2
-phenyl-2H-thiochromene-3-carbaldehyde (13a, 175 mg, 70%) was obtained as a yellowish solid with
◦
1
mp: 195–196 C. H-NMR (200 MHz, CDCl ) δ 9.70 (s, 1H), 7.55 (s, 1H), 7.43 (m, 1H), 7.35–7.19 (m, 5H),
3

Molecules 2020, 25, 800
15 of 22
7
.10 (s, 1H), 4.80 (s, 1H). IR
ν
: 3040, 2820, 1660, 1622, 1139. HRMS-ESI (m/z): calcd. for C H OS [M +
16
12
+
Na] 275.0501, found: 275.0510.
2
-(4-Fluorophenyl)-2H-thiochromene-3-carbaldehyde (13b)
Starting from 4-fluorocinnamaldehyde (150 mg, 1.0 mmol) according to the general procedure
described above 2-(4-fluorophenyl)-2H-thiochromene-3-carbaldehyde (13b, 160 mg, 59% yield) was
◦
1
obtained as a yellow solid with m.p.: 96–98 C. H-NMR (400 MHz, CDCl )
δ
9.66 (s, 1H), 7.47 (s, 1H),
3
7
.42 (d, J = 7.6 Hz, 1H), 7.35–7.25 (m, 2H), 7.24–7.12 (m, 3H), 6.86 (dd, J = 12.6, 4.8 Hz, 2H), 5.19 (s, 1H).
1
3
C-NMR (101 MHz, CDCl3)
δ 191.3, 162.5 (d, J = 246.5 Hz), 145.4, 137.7, 134.9, 134.0, 132.1, 131.3, 129.9,
1
8
28.4 (d, J = 8.2 Hz), 127.9, 126.2, 115.7 (d, J = 21.6 Hz), 37.8. IR ν: 3050, 2826, 1665, 1625, 1503, 1139,
45. HRMS-ESI (m/z): calcd. for C H FOS [M + Na] 293.0407, found: 293.0416.
16 11
+
2
-(4-Chlorophenyl)-2H-thiochromene-3-carbaldehyde (13c)
Starting from 4-chlorocinnamaldehyde (174 mg, 1.0 mmol) according to the general procedure
described above 2-(4-chlorophenyl)-2H-thiochromene-3-carbaldehyde (13c, 250 mg, 87% yield) was
◦
1
obtained as a yellow solid with m.p.: 119–120 C. H-NMR (400 MHz, CDCl )
1
δ
9.67 (s, 1H), 7.48 (s,
H), 7.42 (d, J = 7.4 Hz, 1H), 7.34–7.25 (m, 2H), 7.21 (td, J = 7.3, 1.1 Hz, 1H), 7.17–7.11 (m, 4H), 5.17
s, 1H). 13C-NMR (101 MHz, CDCl3)
191.1, 145.3, 140.3, 140.1, 134.5, 133.6, 132.0, 131.1, 129.7, 128.8,
: 2845, 1667, 1627, 1580. 1138, 761. HRMS-ESI (m/z): calcd. for C H ClOS
3
(
δ
1
27.9, 127.7, 126.1, 37.7. IR
ν
16
11
+
[M + Na] 309.0111, found: 309.0120.
2
-(2-Nitrophenyl)-2H-thiochromene-3-carbaldehyde (13d)
Starting from 2-nitrocinnamaldehyde (1.0 mmol) according to the general procedure described
above 2-(4-chlorophenyl)-2H-thiochromene-3-carbaldehyde (13d, 410 mg, 69% yield) was obtained
◦
1
as a yellow solid with m.p.: 130–132 C. H-NMR (400 MHz, CDCl3)
δ
9.66 (d, J = 1.7 Hz, 1H), 8.01
(
d, J = 7.1 Hz, 1H), 7.67 (s, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.35 (p, J = 7.5 Hz, 2H), 7.29 (d, J = 7.5 Hz,
13
1
1
1
H), 7.26–7.15 (m, 2H), 7.10 (d, J = 7.1 Hz, 1H), 6.01 (s, 1H). C-NMR (101 MHz, CDCl3)
δ
190.7, 147.1,
: 2992, 1661,
46.4, 135.8, 133.9, 133.5, 133.2, 132.3, 131.2, 129.2, 128.5, 128.4, 127.8, 126.2, 125.9, 33.4. IR
ν
+
623, 1519, 1142, 740. GC-MS: m/z (%) = 297 (4) [M ], 280 (43), 251 (60), 235 (44), 221 (100.). HRMS-ESI
+
(m/z): calcd. for C H NO S [M + Na] 320.0352, found: 320.0362.
16
11
3
Phenyl(2-phenyl-2H-thiochromen-3-yl)methanone (15a)
In a 10 mL round bottom flask equipped with a reflux condenser were added chalcone (1 mmol),
2-(tert-butylthio)benzaldehyde (3, 1.5 mmol) and p-toluenesulfonic acid monohydrate (60 mg) in
toluene (2 mL). The reaction mixture was stirred at reflux for 2 h and progress of the reaction was
monitored with TLC. Upon the consumption of the chalcone, the reaction mixture was directly subjected
to column chromatography over silica gel using hexane/EtOAc (5:1) as eluent to give the desired
◦
1
compound as a yellow solid (45 mg, 46%) m.p.:. 151–153 C. H-NMR (400 MHz, CDCl )
δ
7.71 (dd,
J = 8.3, 1.3 Hz, 2H), 7.57 (ddd, J = 6.7, 2.7, 1.3 Hz, 1H), 7.51–7.44 (m, 2H), 7.41 (s, 1H), 7.33–7.18 (m, 8H),
.14 (tdd, J = 7.7, 5.9, 1.6 Hz, 1H), 5.49 (s, 1H). 13C-NMR (101 MHz, CDCl3)
195.9, 142.1, 140.2, 138.3,
3
7
δ
1
1
33.4, 132.9, 132.1, 131.3, 131.0, 130.6, 129.4, 128.9, 128.6, 127.9, 127.8, 126.8, 125.9, 40.4. IR
ν
: 3126, 2918,
+
640, 1596, 1488, 1087, 756. HRMS-ESI (m/z): calcd. for C H OS [M + H] 329.0995, found: 329.1002.
22
16
4
.1.4. General Procedure for the Synthesis of
(
4-chlorophenyl)(2-(aryl)-2H-thiochromen-3-yl)methanones 15b,c
In a 10 mL round bottom flask equipped with a reflux condenser were mixed
4
’-chloro-acetophenone (155 mg, 1.0 mmol) and the appropriate substituted benzaldehyde (1.0 mmol)
with Amberlyst-15 (250 mg) in toluene (5 mL). The reaction mixture was stirred at room temperature for
h, and the progress of the reaction was monitored with TLC until the formation of the corresponding
chalcone was complete. Afterward 2-(tert-butylthio)benzaldehyde (194 mg, 1.0 mmol) was added,
2
3

Molecules 2020, 25, 800
16 of 22
◦
and the mixture was allowed to react for another 2 h at 110 C and then cooled to room temperature
and subjected to column chromatography over silica gel using hexane/EtOAc (5:1) as eluent to afford
the desired (4-chlorophenyl)(2-(aryl)-2H-thiochromen-3-yl)-methanones 15b,c.
(4-Chlorophenyl)(2-(4-(trifluoromethyl)phenyl)-2H-thiochromen-3-yl)methanone (15b)
Following the general procedure described above, starting from 4-(trifluoromethyl)-benzaldehyde
◦
(
175 mg, 1 mmol) the desired 15b was obtained (200 mg, 46%) as a yellow solid m.p.: 105–106 C.
1
H-NMR (400 MHz, CDCl3)
J = 8.2 Hz, 2H), 7.28 (t, J = 7.5 Hz, 3H), 7.18 (ddd, J = 7.2, 5.3, 3.7 Hz, 1H), 5.43 (s, 1H). C-NMR
75 MHz, CDCl3) 194.4, 145.6, 141.0, 138.7, 136.1, 132.33, 132.26, 131.7, 131.2, 130.8, 130.2, 129.9 (q,
δ 7.66 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.5 Hz, 4H), 7.43 (s, 1H), 7.37 (d,
13
(
δ
J = 32.7 Hz), 129.0, 127.9, 127.0, 126.3, 125.8 (q, J = 3.9 Hz), 123.8 (q, J = 270.4 Hz), 40.0. IR
ν
: 2952, 1612,
+
1
585, 1323, 1114. HRMS-ESI (m/z): calcd. for C H ClF OS [M + Na] 453.0298, found: 453.0304.
23
14
3
(4-Chlorophenyl)(2-(4-nitrophenyl)-2H-thiochromen-3-yl)methanone (15c)
Following the general procedure described above, starting from 4-nitrobenzaldehyde (152 mg,
◦
1
1
mmol) the desired 15c (180 mg, 45%) was obtained as a yellow solid, m.p.: 93–94 C. H-NMR
(
3
400 MHz, CDCl3)
δ
8.07 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.52–7.38 (m, 5H), 7.34–7.25 (m,
H), 7.22–7.16 (m, 1H), 5.42 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ
13
C-NMR (101 MHz, CDCl3) δ 194.3,
148.9, 147.4, 141.3, 138.9, 136.3, 135.9, 132.0, 131.9, 131.4, 130.8, 130.2, 129.1, 128.1, 127.6, 126.6, 124.2,
4
+
0.1. IR
ν
: 3026, 2849, 1656, 1607, 1585, 1515, 1338, 821. HRMS-ESI (m/z): calcd. for C H ClNO S [M
22
14
3
+
Na] 408.0456, found: 408.0461.
4
.1.5. General Procedure for the Synthesis of the 4-oxo-thiochromane-2-carboxylic acids 20a–d
A round bottom flask equipped with a magnetic stirrer was loaded with a mixture of maleic
anhydride (1.718 g, 17.5 mmol) and a slight excess of thiophenols (19.3 mmol, 1.1 equivalents) in
acetonitrile dry and then triethylamine (10 drops) was slowly added. The reaction flask was closed
◦
with a glass-stopper and stirred at 50 C for 2 h. The reaction was quenched at room temperature,
◦
the solvent was removed under reduced pressure, and the black oily residue was cooled at 0 C in
bath ice, and redissolved with dry DCM, after which a significant excess of AlCl was added. The
3
mixture reaction was stirred at room temperature overnight. After the reaction was completed as
determined by TLC, the mixture reaction was treated with a cold solution of hydrochloric acid (5%)
and extracted with CH Cl (3
×
25 mL) three times. The combined organic layers were dried over
2
2
anhydrous Na SO , the residue after solvent evaporation the mixture was filtered through a silica gel
2
4
column using as mobile phase hexanes/ethyl acetate with 5 % of acetic acid as an additive (80:20 v/v) to
give pure compounds 20a–d in 55–70% global yield.
4
-Oxothiochromane-2-carboxylic acid (20a)
◦
1
White solid, m.p.: = 151–152 C. H-NMR (300 MHz, DMSO-d )
δ
7.95 (dd, J = 7.9, 1.5 Hz, 1H),
.49 (td, J = 7.6, 1.5 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 4.39 (dd, J = 6.1, 4.3 Hz, 1H),
.19–2.97 (m, 2H). 13C-NMR (75 MHz, DMSO)
192.63, 172.14, 138.99, 134.25, 130.52, 128.37, 127.82,
(cm ) = 2918.37, 1695.44, 1681.12, 881.15, 768.35. HRMS-ESI (m/z): calcd. for
C H O S [M + H] 209.0267, found 209.0282.
6
7
3
1
δ
−
1
25.94, 41.71, 41.26. IR ν
+
10
8
3
6
-Fluoro-4-oxo-thiochromane-2-carboxylic acid (20b)
◦
1
White solid, m.p.: = 135–137 C. H-NMR (300 MHz, DMSO-d )
δ 7.66 (d, J = 11.0 Hz, 1H), 7.44 (s,
6
13
1
1
2
H), 7.23 (t, J = 8.8 Hz, 1H), 4.40 (t, J = 5.1 Hz, 1H), 3.10 (t, J = 4.9 Hz, 2H). C-NMR (75 MHz, DMSO) δ
91.92, 172.33, 162.10, 158.86, 134.47, 127.97, 122.07, 116.53, 114.27, 41.62, 40.92. IR ν (cm ) = 3498.15,
895.35, 1723.95, 1684.65, 805.21, 238.57. HRMS-ESI (m/z): calcd. for C H O FS [M + H] 227.0173,
−
1
+
10
7
3
found 227.0173.

Molecules 2020, 25, 800
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6
-Methoxy-4-oxo-thiochromane-2-carboxylic acid (20c)
◦
1
White solid, m.p.: = 165–166 C H-NMR (300 MHz, DMSO-d )
δ
7.45 (d, J = 2.8 Hz, 1H), 7.27
6
13
(
d, J = 8.7 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 3.77 (s, 3H), 3.15–2.96 (m, 2H). C-NMR (75 MHz,
DMSO)
(
δ
192.60, 172.21, 157.64, 131.43, 129.80, 129.28, 122.05, 111.46, 55.83, 41.70, 40.44. IR
ν
+
−
cm ) = 2888.72, 1714.93, 1626.73, 881.75, 807.73. HRMS-ESI (m/z): calcd. for C H O S [M + H]
11 10 4
1
2
39.0373, found 239.0387.
7
-Methoxy-4-oxo-thiochromane-2-carboxylic acid (20d)
◦
1
White solid, m.p.: = 162–164 C H-NMR (300 MHz, acetone-d )
δ
7.99 (d, J = 8.6 Hz, 1H), 6.83 (d,
6
J = 2.4 Hz, 1H), 6.83–6.78 (m, 1H), 4.36 (dd, J = 6.4, 4.7 Hz, 1H), 3.88 (s, 3H), 3.08 (dd, J = 5.6, 3.5 Hz, 2H).
1
3
C NMR (75 MHz, acetone-d )
δ 190.56, 170.91, 163.51, 141.17, 130.44, 124.13, 112.67, 110.55, 55.29,
6
−1
4
1.97, 40.89. IR
ν
(cm ) = 3419.98, 2906.14, 1698.81, 1671.88, 870.22, 824.56. HRMS-ESI (m/z): calcd. for
+
C H O S [M + H] 239.0373, found = 239.0375.
1
1
10
4
4
2
.1.6. General Procedure for the Synthesis of the Esters from 4-oxo-thiochromane-2-carboxylic acids
2a–d
A
mixture of 4-oxo-thiochromane-2-carboxylic acid
(20a
,
100 mg, 0.48 mmol),
N,N
´
dicyclohexylcarbodiimide (DCC, 150 mg) and small amount of 4-dimethylaminopyridine (DMAP)
◦
were dissolved in acetonitrile (10 mL) and heated under microwave irradiation at 70 C for 20 min,
then 1.3 equivalents of the corresponding aliphatic alcohol were added. After that the reaction vial
was sealed and heated to 70 C for 40 min. When the reaction was completed as determined by TLC,
◦
the reaction mixture was diluted with ethyl acetate and washed with brine (2
×
20 mL). The combined
organic layers were dried over anhydrous Na SO , and the residue after solvent evaporation was
2
4
purified by flash chromatography using hexanes/ethyl acetate (80:20 v/v) to give 22a–d in 65–80% yield.
Ethyl 4-oxo-thiochromane-2-carboxylate (22a)
1
Yellowish oil. H-NMR (300 MHz, CDCl )
δ 8.14 (d, J = 9.2 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H),
3
13
7
.31–7.19 (m, 2H), 4.27–4.12 (m, 3H), 3.21 (d, J = 6.5 Hz, 1H), 1.25 (t, J = 7.1 Hz, 3H). C-NMR (75 MHz,
CDCl3) 192.24, 169.85, 138.45, 133.71, 130.45, 128.85, 127.37, 126.22, 125.77, 77.55, 77.13, 76.70, 62.27,
2.34, 41.34, 13.97. IR
δ
−1
4
ν
(cm ) = 2981.39, 1731.29, 1684.23, 761.18. HRMS-ESI (m/z): calcd. for
+
C H O S [M + H] 237.0580, found 237.0592.
12
12
3
Butyl 4-oxo-thiochromane-2-carboxylate (22b)
1
Yellowish oil. H-NMR (300 MHz, CDCl )
δ
8.14 (dd, J = 7.9, 1.6 Hz, 1H), 7.43 (ddd, J = 8.0, 7.2,
3
1
1
1
6
7
.6 Hz, 1H), 7.34–7.18 (m, 2H), 4.24–4.04 (m, 3H), 3.21 (dd, J = 5.6, 1.6 Hz, 2H), 1.66–1.45 (m, 2H),
.40–1.22 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). 13C-NMR (75 MHz, CDCl3)
192.18, 169.94, 141.93, 138.49,
37.47, 133.68, 132.32, 130.47, 129.25, 129.14, 128.84, 128.69, 128.30, 127.36, 125.74, 77.55, 77.12, 76.70,
δ
−
1
7.08, 66.06, 42.46, 41.34, 30.38, 19.16, 18.94, 13.71, 13.63. IR
ν (cm ) = 2960.28, 2933.24, 1731.63, 1683.60,
+
58.76. HRMS-ESI (m/z): calcd. for C H O S [M + Na] 287.0712, found 287.0709.
14
16
3
Hexyl 4-oxo-thiochromane-2-carboxylate (22c)
Yellowish oil. 1H-NMR (300 MHz, CDCl3)
.28–7.16 (m, 2H), 4.14 (q, J = 6.5, 5.8 Hz, 3H), 3.20 (d, J = 5.8 Hz, 2H), 1.57 (p, J = 6.4 Hz, 3H), 1.25
δ
8.13 (d, J = 7.9 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H),
7
(
s, 6H), 0.88 (t, J = 6.0 Hz, 3H). 13C-NMR (75 MHz, CDCl3)
δ
192.14, 169.95, 138.48, 133.67, 130.45,
−1
1
1
28.81, 127.33, 125.72, 66.33, 42.42, 41.30, 31.31, 28.34, 25.35, 22.49, 14.01. IR ν (cm ) = 3056.06, 2959.62,
+
730.43, 1684.96, 738.80. HRMS-ESI (m/z): calcd. for C H O S [M + Na] 315.1025, found 315.1005
16
20
3

Molecules 2020, 25, 800
18 of 22
Decyl 4-oxo-thiochromane-2-carboxylate (22d)
1
Yellowish oil. H-NMR (300 MHz, CDCl )
δ
8.14 (dd, J = 7.9, 1.5 Hz, 1H), 7.43 (td, J = 7.7, 1.6 Hz,
3
1
1
1
1
H), 7.31–7.20 (m, 2H), 4.19–4.11 (m, 3H), 3.25–3.18 (m, 2H), 1.58 (d, J = 6.6 Hz, 2H), 1.27 (d, J = 5.7 Hz,
13
6H), 0.95–0.87 (m, 3H). C-NMR (75 MHz, CDCl )
δ
192.15, 169.94, 138.50, 133.67, 130.47, 128.85,
3
27.34, 125.73, 77.53, 77.11, 76.68, 66.36, 42.45, 41.32, 31.91, 29.53, 29.48, 29.33, 29.17, 28.40, 25.70, 22.71,
−
ν (cm ) = 2954.83, 2925.62, 1734.22, 1686.36, 759.28. HRMS-ESI (m/z): calcd. for C H O S
20 28 3
1
4.16. IR
+
[M + H] 349.1832, found 349.1864
4
.1.7. General Procedure for the Synthesis of the -4-oxo-thiochromane-2-carboxamides 24a–c
To a mixture of 4-oxo-thiochromane-2-carboxylic acid (20a, 1.0 g, 4.8 mmol) in anhydrous DCM
◦
(
5 mL), at 0 C was added a small amount of DMF, followed by oxalyl chloride (10.0 mmol). Once
the effervescence stopped, the solution was quenched to room temperature for 2 h. FT-IR analyses
determined that the acid chloride was formed as a brownish oil since Fermi resonance was observed.
Next, a solution of amine (10.0 mmol) in DCM (5 mL) and NaOH (2.0 M, 5 mL) was slowly added to
the initial mixture, under Schotten-Baumann conditions and stirred to room temperature overnight to
give to corresponding amides. When the reaction was completed as determined by TLC, the reaction
mixture was diluted with ethyl acetate and washed with brine (2
×
20 mL). The combined organic
layers were dried over anhydrous Na SO , and the residue after solvent evaporation was purified by
2
4
flash chromatography using hexanes/ethyl acetate (80:20 v/v) as eluent to give pure molecules 3i
0–35% yield.
–k in
3
N-hexyl-4-oxo-thiochromane-2-carboxamide (24a)
◦
1
White solid. m.p.: 128–129 C. H-NMR (300 MHz, CDCl )
δ
8.13 (d, J = 7.9 Hz, 1H), 7.44 (t,
3
J = 7.6 Hz, 1H), 7.33–7.22 (m, 2H), 6.56 (t, J = 5.9 Hz, 1H), 4.05 (t, J = 5.4 Hz, 1H), 3.48 (dd, J = 16.8, 6.5 Hz,
1
3
7
H), 3.36–3.05 (m, 3H), 1.41 (p, J = 7.1 Hz, 2H), 1.23 (td, J = 12.1, 10.4, 5.6 Hz, 6H), 0.87 (t, J = 6.6 Hz,
13
H). C-NMR (75 MHz, CDCl3)
7.11, 76.68, 43.57, 41.75, 40.07, 31.39, 29.29, 26.31, 22.52, 14.04. IR
δ 192.52, 168.50, 138.06, 133.66, 131.04, 129.19, 127.47, 126.00, 77.53,
−1
ν
(cm ) = 3331.59, 2926.30, 2859.39,
+
1
658.69, 755.77. HRMS-ESI (m/z): calcd. for C H NO S [M + H] 292.1366, found 292.1393.
16
21
2
N-dodecyl-4-oxo-thiochromane-2-carboxamide (24b)
◦
1
White solid, m.p.: 125–127 C. H-NMR (300 MHz, CDCl )
δ
8.14 (d, J = 7.9 Hz, 1H), 7.44 (t,
J = 7.6 Hz, 1H), 7.34–7.22 (m, 2H), 6.50 (d, J = 7.2 Hz, 1H), 4.05 (t, J = 5.4 Hz, 1H), 3.49 (dd, J = 16.7,
.5 Hz, 1H), 3.37–3.08 (m, 3H), 1.49–1.14 (m, 19H), 0.97–0.86 (m, 3H). 13C-NMR (75 MHz, CDCl3)
92.44, 168.41, 138.00, 133.66, 131.08, 129.24, 127.48, 126.04, 77.52, 77.09, 76.67, 43.59, 41.75, 40.09, 31.96,
3
6
δ
1
2
2
3
−
1
9.68, 29.68, 29.61, 29.53, 29.40, 29.40, 29.34, 29.24, 29.24, 26.66, 22.74, 14.18. IR
979.88, 2913.42, 1686.63, 779.84. HRMS-ESI (m/z): calcd. for C H NO S [M + H] 376.2305, found
ν (cm ) = 3365.75,
+
22
33
2
76.2314.
4
-Oxo-N-(o-tolyl)thiochromane-2-carboxamide (24c)
◦
1
Yellowish solid, m.p.: 144–146 C. H-NMR (300 MHz, CDCl )
δ
7.61 (s, 1H), 7.40 (d, J = 7.8 Hz,
3
1
H), 6.95 (d, J = 7.9 Hz, 1H), 6.71 (t, J = 7.7 Hz, 1H), 6.64–6.36 (m, 5H), 6.33 (d, J = 7.3 Hz, 1H), 3.48 (d,
J = 5.5 Hz, 1H), 2.88 (dd, J = 17.1, 4.9 Hz, 1H), 2.45 (dd, J = 16.9, 4.0 Hz, 1H), 1.33 (s, 3H). 13C- NMR
75 MHz, CDCl3) 192.00, 166.95, 137.28, 134.97, 133.87, 131.26, 130.54, 129.57, 129.51, 129.41, 127.64,
(
1
1
δ
−
1
26.82, 126.64, 126.46, 125.71, 122.97, 77.54, 77.11, 76.69, 44.11, 41.51, 17.43. IR
ν
(cm ) = 3261.48,
+
673.35, 1650.48, 748.51. HRMS-ESI (m/z): calcd. for C H NO S [M + H] 298.0896, found 298.0919
17
15
2
4
.1.8. General Procedure for the Synthesis of Thiochromane Hydrazones
Ethyl 4-oxo-thiochromane-2-carboxylate (22a, 500 mg) was dissolved in anhydrous methanol
(25 mL). The mixture was heated at reflux for 12 h with the corresponding hydrazide (1.6 mmol)

Molecules 2020, 25, 800
19 of 22
and glacial acetic acid (100
µL). When the reaction was completed, as determined by the resulting
precipitate, the acyl hydrazones were collected by filtration and washed with methanol to give pure
products 26a–c as white solids in 90–98% yield.
Ethyl 4-(2-benzoylhydrazineylidene)thiochromane-2-carboxylate (26a)
◦
1
White solid m.p.: 170–171 C. H-NMR (300 MHz, DMSO-d ) δ 11.20 (s, 1H), 8.77 (d, J = 5.3 Hz,
6
3
4
H), 8.15 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 5.1 Hz, 2H), 7.29 (tt, J = 15.7, 7.4 Hz, 4H), 4.38 (dd, J = 6.7,
.5 Hz, 1H), 4.08 (q, J = 7.1 Hz, 3H), 3.22 (dd, J = 17.5, 4.6 Hz, 1H), 1.13 (q, J = 8.2, 7.1 Hz, 4H). 13C-NMR
(
1
2
75 MHz, DMSO)
δ
170.41, 163.11, 152.94, 150.62, 141.41, 133.54, 131.33, 130.66, 128.21, 127.05, 126.27,
−1
22.39, 61.85, 40.72, 40.58, 40.44, 40.17, 39.89, 39.61, 39.34, 39.05, 30.28, 14.32. IR ν (cm ) = 3173.40,
978.57, 1718.42, 1652.27, 754.99, 734.77. HRMS-ESI (m/z): calcd. for C H N O S [M + H] 355.1111,
19 18 2 3
+
found 355.1135.
Ethyl 4-(2-isonicotinoylhydrazineylidene)thiochromane-2-carboxylate (26b)
◦
1
White solid, m.p.: 190–192 C. H-NMR (300 MHz, DMSO-d )
δ
10.97 (s, 1H), 8.14 (s, 1H), 7.88
d, J = 7.4 Hz, 2H), 7.55 (dt, J = 14.8, 7.2 Hz, 3H), 7.25 (d, J = 18.9 Hz, 3H), 4.38 (t, J = 5.5 Hz, 1H),
.08 (q, J = 7.2 Hz, 2H), 1.12 (t, J = 7.1 Hz, 3H). 13C-NMR (75 MHz, DMSO)
170.43, 164.52, 134.38,
34.37, 133.29, 132.09, 131.61, 130.34, 128.79, 128.46, 128.18, 126.92, 126.21, 61.81, 30.11, 14.33. IR
(
[
6
(
4
1
δ
ν
−
cm ) = 3145.28, 2947.51, 1717.64, 1651.30, 759.40, 708.68. HRMS-ESI (m/z): calcd. for C H N O S
18 17 3 3
M + H] 356.1063, found 356.1096.
1
+
4
-(2-Carbamoylhydrazineylidene)thiochromane-2-carboxylic acid (26c)
◦
1
White solid, m.p.: 238 C. H-NMR (300 MHz, DMSO-d )
δ
9.44 (s, 1H), 8.73 (s, 1H), 8.23 (d,
6
J = 7.8 Hz, 1H), 7.21 (d, J = 4.0 Hz, 2H), 7.14 (dq, J = 8.3, 4.3 Hz, 1H), 6.58 (s, 2H), 4.18 (dd, J = 6.8,
1
3
4
δ
3
2
.7 Hz, 1H), 3.12 (dd, J = 17.6, 7.0 Hz, 1H), 2.99 (dd, J = 17.6, 4.9 Hz, 1H). C-NMR (75 MHz, DMSO-d6)
−1
171.99, 157.60, 141.08, 132.46, 132.16, 129.08, 128.17, 126.80, 126.10, 41.16, 30.31. IR
237.11, 2906.14, 1713.23, 1645.36, 757.97, 736.96. HRMS-ESI (m/z): calcd. for C H N O S [M + H]
ν
(cm ) = 3469.94,
+
11
11
3
3
66.0594, found 266.0595.
4
.2. Resolution of the racemic acids and derivatives
4
-Oxothiochromane-2-carboxylic acid (22a, 1.0 g) dissolved in Tetrahydrofuran (THF)/water (5 mL,
ratio 4:1), was mixed with brucine (2 equivalents). The reaction mixture was overnight stirred at
room temperature. The solvent was evaporated under reduced pressure, and the resulting solid was
re-dissolved in hot ethyl acetate. A crystal phase was generated which was filtered off resulting in two
new fractions (solid and liquid diastereomers). Both fractions were subjected to acid extraction (10%
HCl) The resultant mixture was quenched with saturated brine (20 mL) and was extracted with hot
ethyl acetate (10 mL) three-times. The organic layers were combined, washed with brine, and dried
with anhydrous Na SO . After concentrating in vacuo the resultant residue was purified by flash
2
4
chromatography (ethyl acetate:n-hexane = 1:10) to give pure (+)-20a in 10% yield (specific rotation
2
5
25
[
α] = 35.6) as well (
−
)-20a in 45% yield ([α]D
)-20a, were esterified according with general methodology before described in
the general procedure for the synthesis of the esters from 4-oxothiochromane-2-carboxylic acids to give
= 10.8). The enantiomerically enriched acids mentioned
−
D
above, (+)-20a and (
−
25
compounds (+)-22b (9% yield, specific rotation [α] = 32.4) and (
)-22b (85% yield, specific rotation
−
D
2
5
[
α] = −8.3).
D
4
.3. Biological Activity
4
.3.1. Cytotoxic Activity
Cytotoxicity of the compounds was evaluated on human monocytes (U-937 ATCC CRL-1593.2)
in an exponential growth phase and adjusted at 1 105 cells/mL in RPMI-1640 enriched with 10%
×

Molecules 2020, 25, 800
20 of 22
fetal bovine serum (FBS). One hundred microliters of cell suspension were dispensed in each well of a
6-wells microplate, and then 100 L of 200-50-12.5 and 3.125 g/mL concentration of each compound
9
µ
µ
or standard drug (amphotericin B) were added dissolved in PBS with 0.5% DMSO. Cell exposed to
◦
compounds or standard drugs were incubated 72 h at 37 C and 5% of CO . Cytotoxic activity of each
2
compound was determined according to the effect on the cell viability by the MTT microenzymatic
method in which 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is reduced to a purple
formazan product by mitochondrial enzyme succinate dehydrogenase. Thus, 10 µL/well of MTT
solution (5 mg/mL) was added to each well of exposed and unexposed cells, and plates were incubated
◦
at 37 C, 5% CO during 3 h. The reaction was stopped by adding 100
µL/well of isopropanol with
2
5
0% and 10% of SDS (sodium dodecyl sulfate). The concentration of formazan was determined
spectrophotometrically at 570 nm (Varioskan, Thermo, Waltham, MA, USA) and intensity of color
absorbance) was registered as O.D. Cells exposed to control drug (amphotericin B) were used as
(
control for toxicity (positive control) while cell incubated in the absence of any compound or drug were
used as control for viability (negative control). Non-specific absorbance was corrected by subtracting
absorbance (O.D) of the blank. Determinations were done by triplicate in at least two independent
experiments [24].
4
.3.2. Antileishmanial Activity
Antileishmanial activity of compounds was determined according to the ability of the compound
to reduce the infection by L. panamensis parasites. For this, the antileishmanial activity was tested
on intracellular amastigotes of L. panamensis transfected with the green fluorescent protein gene
(
MHOM/CO/87/UA140-EGFP strain) [22]. Briefly, U-937 human cells at a density of 3
×
105 cells/mL
in RPMI 1640 and 0.1 g/mL of PMA (phorbol-12-myristate-13-acetate) were dispensed on 24-wells
µ
microplate and then infected with stationary phase growing L. panamensis promastigotes in 15:1
◦
parasites per cell ratio. Plates were incubated at 34 C and 5% CO for 3 h, and then cells were
2
washed twice with phosphate buffer solution (PBS) to eliminate not internalized parasites. Fresh
RPMI-1640 was added into each well (1 mL), and plates were incubated again. After 24 h of infection,
the RPMI-1640 medium was replaced by fresh culture medium containing each compound at four
◦
serial dilutions (50, 12.5, 3.125 and 0.78
µ
g/mL) and plates were then incubated at 37 C and 5% CO
2
during 72 h, then, cells were removed from the bottom plate with 100
solution. The cells were centrifuged at 1100 rpm during 10 min at 4 C, the supernatant was discarded,
and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm for 10 min at 4 C. Cells
µ
L of EDTA/trypsin (250 mg)
◦
◦
were washed two times employing PBS, as previously, and after the last wash, the supernatant was
discarded, and cells were suspended in 500 µL of PBS.
Cells were analyzed by flow cytometry employing a flow cytometer (FC 500MPL, Cytomics,
Beckman Coulter. Pasadena, CA, USA) reading at 488 nm (exciting) and 525 nm (emitting) over an
argon laser and counting 10,000 events. Infected cells were determined according to the events for
green fluorescence (parasites). All determinations for each compound and standard drug were carried
out by triplicate, in two experiments. Infected cells exposed to control drug (amphotericin B) were used
as the control for antileishmanial activity (positive control), while infected cells incubated in the absence
of any compound or drug were used as another control for infection (negative control). Nonspecific
fluorescence was corrected by subtracting the fluorescence of unstained cells. Determinations were
done by triplicate in at least two independent experiments [25,26].
4
.3.3. Statistical Analysis
Cytotoxicity was determined according to viability and mortality percentages obtained for each
experimental condition (synthesized compounds, amphotericin B, and culture medium). Results were
expressed as the mean lethal concentrations (LC ), the concentration necessary to kill 50% of cells,
50
calculated by the parametric method of linear regression that permits doses-response analysis (Probit
analysis) [25].

Molecules 2020, 25, 800
21 of 22
Initially, viability percentages were calculated by Equation (1), where the O.D of control well,
corresponds to 100% of viability:
!
O.D. exposed cells
O.D. unexposed cells
%
Viability =
× 100
(1)
Then, the percentage of cell growth inhibition was calculated using Equation (2):
%
Inhibition = 100 − (% Viability)
(2)
The toxicity was defined according to LC50 values, using the follow scale: Toxic; LC50 < 100
moderately toxic; LC50 > 100 µM and < 200 µM and potentially nontoxic; LC50 > 200 µM.
µM;
The antileishmanial activity was determined according to the reduction of the percentage of
fluorescent parasites determined according to the median fluorescence intensity (MFI) obtained for each
experimental condition by cytometry. The parasite values for each concentration of compound were
calculated by Equation (3), where the % of parasites in control well, corresponds to 100% of parasites:
!
MFI exposed parasites
MFI unexposed parasites
%
Parasites =
× 100
(3)
Then, the inhibition percentage was calculated with Equation (4):
%
Inhibitors o f Parasites = 100 − (% Parasites)
(4)
Results of antileishmanial activities were expressed as the median effective concentrations (EC )
50
measured by Probit method. The activity of each compound was established according to EC50 values
as: high activity: EC50 < 25
EC50 > 100 µM.
µM; moderate activity: EC50 > 25 µM and < 100 µM and low activity:
Author Contributions: Conceptualization, W.Q. and E.V.; methodology, D.L.; M.C.; E.V. and C.O. bioassays, S.R.;
writing—review and editing, E.V., F.E.; project administration, W.Q. and E.V. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by COLCIENCIAS and Universidad de Antioquia project code 111571249860,
contract 061-2016.
Acknowledgments: Authors thank to COLCIENCIAS and Universidad de Antioquia for funding the project.
Esteban Vargas thanks COLCIENCIAS for a fellowship in the Program Doctorados Nacionales 567. Cristian Ort
íz
thanks Universidad de Antioquia for funding the Master program.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
DNDi. About Leishmaniasis–DNDi. Available online: https://www.dndi.org/diseases-projects/leishmaniasis/
accessed on 17 April 2018).
Emami, S.; Ghanbarimasir, Z. Recent advances of chroman-4-one derivatives: Synthetic approaches and
bioactivities. Eur. J. Med. Chem. 2015, 93, 539–563. [CrossRef] [PubMed]
Keri, R.S.; Budagumpi, S.; Pai, R.K.; Balakrishna, R.G. Chromones as a privileged scaffold in drug discovery:
A review. Eur. J. Med. Chem. 2014, 78, 340–374. [CrossRef] [PubMed]
Welsch, M.E.; Snyder, S.A.; Stockwell, B.R. Privileged scaffolds for library design and drug discovery. Curr.
Opin. Chem. Biol. 2010, 14, 347–361. [CrossRef] [PubMed]
Johnson, A.T.; Wang, L.; Standeven, A.M.; Escobar, M.; Chandraratna, R.A.S. Synthesis and biological activity
of high-affinity retinoic acid receptor antagonists. Bioorg. Med. Chem. 1999, 7, 1321–1338. [CrossRef]
Lima, L.L.M.; Barreiro, E.J. Bioisosterism: A Useful Strategy for Molecular Modification and Drug Design.
Curr. Med. Chem. 2005, 12, 23–49. [CrossRef]
(

Molecules 2020, 25, 800
22 of 22
7
8
9
1
.
.
.
Davies, D.J.; Faust, R.; Garratt, P.J.; Marivingt-Mounir, C.; Kathryn, D.; Teh, M.T.; Sugden, D. Binding affinity
and biological activity of oxygen and sulfur isosteres at melatonin receptors as a function of their hydrogen
bonding capability. Bioorg. Chem. 2004, 32, 1–12. [CrossRef]
Upegui, Y.; Rios, K.; Quiñones, W.; Echeverri, F.; Archbold, R.; Murillo, J.D.; Torres, F.; Escobar, G.; Vélez, I.D.;
Robledo, S.M. Chroman-4-one hydrazones derivatives: Synthesis, characterization, and in vitro and in vivo
antileishmanial effects. Med. Chem. Res. 2019, 28, 2184–2199. [CrossRef]
Siles, R.; Chen, S.-E.; Zhou, M.; Pinney, K.G.; Trawick, M.L. Design, synthesis, and biochemical evaluation of
novel cruzain inhibitors with potential application in the treatment of Chagas’ disease. Bioorg. Med. Chem.
Lett. 2006, 16, 4405–4409. [CrossRef]
0. Song, J.; Jones, L.M.; Kumar, G.D.K.; Conner, E.S.; Bayeh, L.; Chavarria, G.E.; Charlton-Sevcik, A.K.;
Chen, S.-E.; Chaplin, D.J.; Trawick, M.L.; et al. Synthesis and Biochemical Evaluation of Thiochromanone
Thiosemicarbazone Analogues as Inhibitors of Cathepsin L. ACS Med. Chem. Lett. 2012, 3, 450–453.
[
CrossRef]
1. Vastag, G.; Apostolov, S.; Matijevi c´ , B.; Djakovi c´ -Sekuli c´ , T. Structure-interaction relationship study of N
(4-phenylsubstituted) cyanoacetamides by multivariate methods. J. Chemom. 2016, 30, 210–216. [CrossRef]
1
-
1
2. Lavis, L.D. Ester Bonds in Prodrugs. ACS Chem. Biol. 2008, 3, 203–206. [CrossRef] [PubMed]
1
3. Wang, Q.; Finn, M. 2H-Chromenes from salicylaldehydes by a catalytic petasis reaction. Org. Lett. 2000, 2,
4
063–4065. [CrossRef] [PubMed]
1
1
1
1
4. Lu, D.; Li, Y.; Gong, Y. Organocatalytic Asymmetric Tandem Michael Addition Hemiacetalization: A Route
−
to Chiral Dihydrocoumarins, Chromanes, and 4 H -Chromenes. J. Org. Chem. 2010, 75, 6900–6907. [CrossRef]
5. Meng, X.; Huang, Y.; Zhao, H.; Xie, P.; Ma, J.; Chen, R. PPh3-catalyzed domino reaction: A facile method for
the synthesis of chroman derivatives. Org. Lett. 2009, 11, 991–994. [CrossRef] [PubMed]
6. Lanari, D.; Rosati, O.; Curini, M. A solvent-free protocol for the synthesis of 3-formyl-2H-chromenes via
domino oxa Michael/aldol reaction. Tetrahedron Lett. 2014, 55, 1752–1755. [CrossRef]
7. Chemburkar, S.R.; Anderson, D.G.; Reddy, R.E. Efficient Method for Synthesis of 2-Acetylbenzo(b)thiophene
and Its Derivatives, the Key Synthons for 5-Lipoxygenase Inhibitors. Synth. Commun. 2010, 40, 1887–1894.
[
CrossRef]
8. Humphrey, R.E.; Hawkins, J.M. Reduction of Aromatic Disulfides with Triphenylphosphine. Anal. Chem.
964, 36, 1812–1814. [CrossRef]
1
1
1
2
9. Albericio, F.; Isidro-llobet, A.; Mercedes, A. Review Amino acid protecting. Chem. Rev. 2009, 109, 2455–2504.
0. Bryan, C.S.; Braunger, J.A.; Lautens, M. Efficient Synthesis of Benzothiophenes by an Unusual
Palladium-Catalyzed Vinylic C S Coupling. Angew. Chemie Int. Ed. 2009, 48, 7064–7068. [CrossRef]
1. Dickman, D.A.; Chemburkar, S.; Konopacki, D.B.; Elisseou, E.M. Oxidative Cleavage of Aryl or Alkyl tert-Butyl
Sulfides with Dimethyl Sulfoxide/Hydrobromic Acid to Form Symmetrical Aryl or Alkyl Disulfides. Synthesis
2
2
(Stuttg). 1993, 1993, 573–574. [CrossRef]
2. Pulido, S.A.; Muñoz, D.L.; Restrepo, A.M.; Mesa, C.V.; Alzate, J.F.; V
é
lez, I.D.; Robledo, S.M. Improvement of
the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of
antileishmanial drugs. Acta Trop. 2012, 122, 36–45. [CrossRef] [PubMed]
23. Vargas, E.; Echeverri, F.; Upegui, Y.A.; Robledo, S.M.; Quiñones, W. Hydrazone derivatives enhance
antileishmanial activity of thiochroman-4-ones. Molecules 2018, 23, 70. [CrossRef] [PubMed]
24. Fleming, F.F.; Yao, L.; Ravikumar, P.C.; Funk, L.; Shook, B.C. Nitrile-Containing Pharmaceuticals: Efficacious
Roles of the Nitrile Pharmacophore. J. Med. Chem. 2010, 53, 7902–7917. [CrossRef] [PubMed]
25. Taylor, V.M.; Cedeño, D.L.; Muñoz, D.L.; Jones, M.A.; Lash, T.D.; Young, A.M.; Constantino, M.H.; Esposito, N.;
Vélez, I.D.; Robledo, S.M. In vitro and in vivo studies of the utility of dimethyl and diethyl carbaporphyrin
ketals in treatment of cutaneous leishmaniasis. Antimicrob. Agents Chemother. 2011, 55, 4755–4764.
2
6. Finney, D.J. Statistical method in biological assay, 3rd ed.; Charles Griffin & Company: London, UK, 1978; ISBN
0
852642520.
Sample Availability: Samples of the compounds are available from the authors upon request.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(