Synthesis and Evaluation of Antileishmanial and Cytotoxic Activity of Benzothiopyrane Derivatives

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.


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][3][4][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 replacement of the oxygen atom (O) by a sulfur atom (S) it may cause significant changes in the pharmacology activity since the changes in electron-donating potential and the size of sulfur cause solubility [6], polarity, hydrogen bonding and metabolism [7] modifications. In a previous paper, we have reported that acyl hydrazone derivatives of chromanone and thiochromanone have good in vivo antileishmanial activity with effective concentration (EC50) values of 20.1 and 25.0 µM respectively, in addition to anti-inflammatory and wound-healing properties [8]. 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 [9,10]. 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 µM. Ester and amide derivatives of thiochroman-4-ones were also include in this study; the 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.

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], PPh3-catalyzed domino reactions [15] for the synthesis of chroman derivatives and the domino oxa-Michael aldol reaction [16]. The latter approach seemed likely to succeed with the mercapto counterpart.

Scheme 1. Synthesis of 2-mercaptobenzaldehyde.
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 3 or 4. Thus, disulfide 4 was allowed to react with activated alkenes 6a-e in the presence of 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) and triphenylphosphine, whereby thin layer chromatography (TLC) of the reaction mixture showed various spots with similar Rf values. 1 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 3 and the excess of the starting materials. 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 3 or 4. Thus, disulfide 4 was allowed to react with activated alkenes 6a-e in the presence of 1,8diazabicyclo [5.4.0]undec-7-ene (DBU) and triphenylphosphine, whereby thin layer chromatography (TLC) of the reaction mixture showed various spots with similar Rf values. 1 H-NMR analysis of these crude mixtures revealed that this set of spots corresponds to the stereoisomers of thiochromanols 7ae (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 PPh3 and the excess of the starting materials.

Scheme 2. Synthesis of 2H-thiochromanols derivatives 7a-e.
To obtain 2H-thiochromenes 8a-e (Scheme 3), the mixtures of diastereomeric thiochromanols 7a-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.
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,b,d 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 at δ 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 13 C-NMR δ 184.6 characteristic of a ketone group indicate that the compound exists only as its keto form 9a.
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 13 C-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.

Scheme 2. Synthesis of 2H-thiochromanols derivatives 7a-e.
To obtain 2H-thiochromenes 8a-e (Scheme 3), the mixtures of diastereomeric thiochromanols 7a-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 PPh 3 .

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 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,b,d 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 at δ 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 13 C-NMR δ 184.6 characteristic of a ketone group indicate that the compound exists only as its keto form 9a.
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 13 C-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.

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. The above results led us to think that compound 5 formed in situ could react with suitably activated alkenes to yield the addition product; thus, we carried out the reaction of 3 in the presence of cinnamaldehydes 12a-d using 12M HCl. Thus, substituted 2-phenyl-2H-thiochromene-3carbaldehydes 13a-d in moderate to good yields were effectively produced (Scheme 5). The above results led us to think that compound 5 formed in situ could react with suitably activated alkenes to yield the addition product; thus, we carried out the reaction of 3 in the presence of cinnamaldehydes 12a-d using 12M HCl. Thus, substituted 2-phenyl-2H-thiochromene-3-carbaldehydes 13a-d in moderate to good yields were effectively produced (Scheme 5).

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. The above results led us to think that compound 5 formed in situ could react with suitably activated alkenes to yield the addition product; thus, we carried out the reaction of 3 in the presence of cinnamaldehydes 12a-d using 12M HCl. Thus, substituted 2-phenyl-2H-thiochromene-3carbaldehydes 13a-d in moderate to good yields were effectively produced (Scheme 5). 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.

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 AlCl3 or SnCl4. Similarly 4-oxo-thiochroman-2-carboxylic acids 20a-d were obtained by reacting thiophenols 18a-d with furan-2,5-dione (maleic anhydride, 19) in the presence of triethylamine and with subsequent treatment with AlCl3 (Scheme 7). 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. Scheme 6. Synthesis of (aryl-2H-thiochromen-3-yl)-methanones from chalcones.

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 AlCl3 or SnCl4. Similarly 4-oxo-thiochroman-2-carboxylic acids 20a-d were obtained by reacting thiophenols 18a-d with furan-2,5-dione (maleic anhydride, 19) in the presence of triethylamine and with subsequent treatment with AlCl3 (Scheme 7). 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. 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.

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-c were prepared by reaction of ethyl 4-oxothiochromane-2carboxylate with acyl hydrazides 25a-c in the presence of acetic acid (Scheme 10). 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) 2 and dimethyl formamide as catalyst (DMF) and aqueous sodium hydroxide.

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-c were prepared by reaction of ethyl 4-oxothiochromane-2carboxylate with acyl hydrazides 25a-c in the presence of acetic acid (Scheme 10).

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-c were prepared by reaction of ethyl 4-oxothiochromane-2-carboxylate with acyl hydrazides 25a-c in the presence of acetic acid (Scheme 10).

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-c were prepared by reaction of ethyl 4-oxothiochromane-2carboxylate with acyl hydrazides 25a-c in the presence of acetic acid (Scheme 10).

Resolution of Enantiomers of 4-oxo-thiochroman-2-carboxylic Acid Using Brucine
To establish the difference in antileishmanial activity of the enantiomers of 4-oxo-thiochroman-2-carboxylic acid (20a) we attempted the chiral resolution by diastereomeric salt formation through reaction with optically pure (−) -brucine 27 as a chiral selector (Scheme 11). Thus, 20a 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 (−) -brucine and an enantiomerically enriched sample of 4-oxo-thiochroman-2-carboxylic acid (+)-20a with specific rotation [α] 25 D = +35.6. 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 of enantiomers with specific rotation of [α] 25 D = −10.8 for (−)-20a. We did not determine absolute configuration nor enantiomeric excess.

Resolution of Enantiomers of 4-oxo-thiochroman-2-carboxylic Acid Using Brucine
To establish the difference in antileishmanial activity of the enantiomers of 4-oxo-thiochroman-2-carboxylic acid (20a) we attempted the chiral resolution by diastereomeric salt formation through reaction with optically pure (−) −brucine 27 as a chiral selector (Scheme 11). Thus, 20a 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 (−) -brucine and an enantiomerically enriched sample of 4-oxo-thiochroman-2-carboxylic acid (+)-20a with specific rotation [α] 25 D = +35.6. 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 of enantiomers with specific rotation of [α] 25 D = −10.8 for (−)-20a. We did not determine absolute configuration nor enantiomeric excess.

Resolution of Enantiomers of 4-oxo-thiochroman-2-carboxylic Acid Using Brucine
To establish the difference in antileishmanial activity of the enantiomers of 4-oxo-thiochroman-2-carboxylic acid (20a) we attempted the chiral resolution by diastereomeric salt formation through reaction with optically pure (−) −brucine 27 as a chiral selector (Scheme 11). Thus, 20a 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 (−) -brucine and an enantiomerically enriched sample of 4-oxo-thiochroman-2-carboxylic acid (+)-20a with specific rotation [α] 25 D = +35.6. 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 of enantiomers with specific rotation of [α] 25 D = −10.8 for (−)-20a. We did not determine absolute configuration nor enantiomeric excess.

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 EC 50 and LC 50 values of 0.05 µM and 56.8 µM, respectively, was used as a control.

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.

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.

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.

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.

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.

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

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 EC 50 < 20 µM, those with marginal activity had an EC 50 between 20-40 µM and the inactive compounds have EC 50 > 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,b,c all having electron-withdrawing groups. The above seems to indicate 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 EC 50 28.5 and 53.2 µM, unlike the compounds that we have previously analyzed [8], which seems to indicate that the 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.

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 (60F 254, 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, Germany). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded using Bruker 300 and 400 spectrometers (300 and 400 MHz for 1H, 75, and 100 MHz for 13 C). Chemical shifts were reported relative to internal tetramethyl silane (δ 0.00 ppm) for 1 H, and CDCl 3 (δ 77.0 ppm) for 13
(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.  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 3 was added. The 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 2 Cl 2 (3 × 25 mL) three times. The combined organic layers were dried over anhydrous Na 2 SO 4 , the residue after solvent evaporation the mixture was filtered through a silica gel 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.  13