Synthesis and Evaluation of Novel 2,2-Dimethylthiochromanones as Anti-Leishmanial Agents

Within this work, we describe the design and synthesis of a range of novel thiochromanones based on natural products reported to possess anti-leishmanial action, and their synthetic derivatives. All compounds were elaborated via the key intermediate 2,2,6-trimethoxythiochromanone, which was modified at the benzylic position to afford various ester, amine and amide analogues, substituted by chains of varying lipophilicity. Upon testing in Leishmania, IC50 values revealed the most potent compounds to be phenylalkenyl and haloalkyl amides 11a and 11e, with IC50 values of 10.5 and 7.2 μM, respectively.


Introduction
Leishmaniasis refers to a spectrum of diseases due to infection with one of a number of protozoal species within the genus Leishmania, transmitted through the bite of infected sandflies. Historically, it has been widespread in tropical climates across many continents. In humans, Leishmania parasites replicate intracellularly and patients classically present with either visceral or cutaneous disease [1]. Available treatments suffer from disadvantages including toxicity, formulation challenges and a lack of oral dosage forms. Of particular concern, resistance to all common agents, namely pentavalent antimonials, pentamidine, miltefosine, paromomycin and amphotericin B has been documented [2]. Therefore, novel approaches to prevent and treat Leishmania require an ongoing research focus. In this context, small molecule therapies remain affordable and druggable approaches, with significant anti-parasitic activity demonstrated for several compounds, encompassing natural products such as oxygenated heterocycles and alkenylphenols, and their synthetic isosteres ( Figure 1). Examples include the chromanone uniflorols 1-2 of Calea spp, which inhibited L. major promastigote growth by 55-89% between 25-100 µg/mL [3]. Synthetic derivatization of these compounds with the aim of improving stability and activity resulted in the synthesis of 3, which inhibited axenic amastigotes and intracellular amastigotes of L. infantum with IC 50 values of 25.3 and 24.6 µM respectively [4]. This compound is notable for possessing a lipophilic aminoalkyl chain para to the chromanone oxygen. Considering this structural scaffold, parallels emerge to the observations of Varela et al. [5], who prepared a range of synthetic phenolic derivatives based on the alkenylphenol gibbilimbol B, 4, a natural product isolated from Piper malacophyllum by de Oliveira and colleagues [6], who noted that it possessed leishmanicidal activity. Subsequent papers by Varela and colleagues have expanded the structure-activity relationships around this compound, with that it possessed leishmanicidal activity. Subsequent papers by Varela and colleagues have expanded the structure-activity relationships around this compound, with the aim of improving selectivity and solubility, while maintaining anti-parasitic activity. Of the various analogues prepared to date, methyl ether 5, bearing a para-octanoyl chain, displayed high activity against amastigotes of both L. infantum and Trypanosoma cruzi, with IC50 values of 1.3 and 5.8 μM respectively [7]. In addition to these structures, other groups have probed the anti-leishmanial activity of related synthetic heterocycles. One notable example is the thiochromanone class ( Figure 2), the sulfur analogues of chromanones such as 1-3. Thiochromanones represent an interesting group from a medicinal chemistry perspective, with examples known to display anti-fungal [8], anti-cancer [9] or anti-trypanosomal [10] activities. In the context of Leishmania, certain thiochromanone derivatives containing either semicarbazone, thiosemicarbazone or triazine nitrile warheads were developed as specific cysteine protease B inhibitors [11]. In more recent work, Vargas and colleagues [12] prepared several thiochromanones modified at ring positions 2 and 6. Upon testing against L. (V) panamensis, active compounds such as 6 were those bearing a vinyl sulfone moiety and a phenyl moiety at C-2, with EC50 values < 10 μM and a selectivity index of over 100 for some compounds. Within this series of compounds, activity decreased upon removal of either the double bond or the sulfone moiety. The same group later published further results on the activity of acyl hydrazone derivatives of thiochromanones against the same leishmanial species [13]. Such derivatization significantly enhanced anti-leishmanial activity, with semicarbazone and thiosemicarbazone derivatives of thioflavanone displaying the highest activities, with 7 displaying an EC50 value of 5.1 µ M, with low cytotoxicity. Thiochroman hydrazides without C-2 substitution were separately evaluated by Zapata et al. [14], who studied topical application of 8 co-formulated with saponins, and this combination proved very effective against parasite survival (L. braziliensis & L. pifanoi) and infectivity. Most recently, Ortiz et al. [15] published an evaluation of the activity of various thiochromenes, thichromanones and hydrazones with C-2 or C-3 carbonyl or carboxyl substitutions against intracellular amastigotes of L. (V) panamensis. A number of compounds, notably of structural type 9, had EC50 values below 10 µ M, but clear structure-activity relationships were not discerned. In addition to these structures, other groups have probed the anti-leishmanial activity of related synthetic heterocycles. One notable example is the thiochromanone class ( Figure 2), the sulfur analogues of chromanones such as 1-3. Thiochromanones represent an interesting group from a medicinal chemistry perspective, with examples known to display anti-fungal [8], anti-cancer [9] or anti-trypanosomal [10] activities. In the context of Leishmania, certain thiochromanone derivatives containing either semicarbazone, thiosemicarbazone or triazine nitrile warheads were developed as specific cysteine protease B inhibitors [11]. In more recent work, Vargas and colleagues [12] prepared several thiochromanones modified at ring positions 2 and 6. Upon testing against L. (V) panamensis, active compounds such as 6 were those bearing a vinyl sulfone moiety and a phenyl moiety at C-2, with EC 50 values < 10 µM and a selectivity index of over 100 for some compounds. Within this series of compounds, activity decreased upon removal of either the double bond or the sulfone moiety. The same group later published further results on the activity of acyl hydrazone derivatives of thiochromanones against the same leishmanial species [13]. Such derivatization significantly enhanced anti-leishmanial activity, with semicarbazone and thiosemicarbazone derivatives of thioflavanone displaying the highest activities, with 7 displaying an EC 50 value of 5.1 µM, with low cytotoxicity. Thiochroman hydrazides without C-2 substitution were separately evaluated by Zapata et al. [14], who studied topical application of 8 co-formulated with saponins, and this combination proved very effective against parasite survival (L. braziliensis & L. pifanoi) and infectivity. Most recently, Ortiz et al. [15] published an evaluation of the activity of various thiochromenes, thichromanones and hydrazones with C-2 or C-3 carbonyl or carboxyl substitutions against intracellular amastigotes of L. (V) panamensis. A number of compounds, notably of structural type 9, had EC 50 values below 10 µM, but clear structure-activity relationships were not discerned. Given the interesting activities of the chromanones and thiochromanones described in the literature against Leishmania species, we resolved to prepare thio analogues of the known anti-leishmanial chromanones 1-3 ( Figure 3). We envisaged ester 10 and amide 11 derivatives, which incorporate either a benzylester or benzylamido linkage para to the thiochromanone sulfur. We also sought to vary the amido functionality by replacing it with a more basic alkylamino chain, as seen in 12. These compounds represent both sulfur analogues of chromanone 3 and also derivatives of the potent gibbilimbol ether 5, within a thiochromanone framework. To investigate the impact of variant oxidation states of sulfur, we envisaged compounds 13 and 14.

Chemistry
Construction of thiochromanone skeletons may follow one of a number of approaches, including Pd-catalyzed carbonylative heteroannulation of iodothiophenols with allenes and carbon monoxide, base-catalyzed cyclization of β-halopropanoic acids with arylthiophenols, base-catalyzed condensation of β-propiolactone with 2-ethylthiophenol followed by acid-promoted cyclization, and intramolecular Friedel−Crafts acylation with Lewis acids or methanesulfonic acid [16]. A recently described one-pot procedure employs a microwave-assisted protocol for the synthesis of 2-/3-methylthiochroman-4-ones by superacid-catalyzed alkylation followed by cyclic acylation [17]. However, preparation of the more hindered 2,2-dimethyl-substituted analogues is more challenging than either un-or mono-substituted C-2 analogues, and protocols such as base-catalyzed cyclization afforded very low yields in our preliminary experiments. Attempts at superacid-catalyzed reactions were entirely unsuccessful. Ultimately, we utilised the established Friedel-Crafts heterocyclisation of commercial thiophenol 15 with 3,3-dimethylacrylic acid in methanesulfonic acid [18], to afford thiochromanone 16 (Scheme 1). Oxidation using persulfate afforded the benzylic aldehyde 17, easily separable from the side-product sulfoxide. Bioreduction using Daucus carota cleanly afforded 18, which was Given the interesting activities of the chromanones and thiochromanones described in the literature against Leishmania species, we resolved to prepare thio analogues of the known anti-leishmanial chromanones 1-3 ( Figure 3). We envisaged ester 10 and amide 11 derivatives, which incorporate either a benzylester or benzylamido linkage para to the thiochromanone sulfur. We also sought to vary the amido functionality by replacing it with a more basic alkylamino chain, as seen in 12. These compounds represent both sulfur analogues of chromanone 3 and also derivatives of the potent gibbilimbol ether 5, within a thiochromanone framework. To investigate the impact of variant oxidation states of sulfur, we envisaged compounds 13 and 14.  Given the interesting activities of the chromanones and thiochromanones described in the literature against Leishmania species, we resolved to prepare thio analogues of the known anti-leishmanial chromanones 1-3 ( Figure 3). We envisaged ester 10 and amide 11 derivatives, which incorporate either a benzylester or benzylamido linkage para to the thiochromanone sulfur. We also sought to vary the amido functionality by replacing it with a more basic alkylamino chain, as seen in 12. These compounds represent both sulfur analogues of chromanone 3 and also derivatives of the potent gibbilimbol ether 5, within a thiochromanone framework. To investigate the impact of variant oxidation states of sulfur, we envisaged compounds 13 and 14.

Chemistry
Construction of thiochromanone skeletons may follow one of a number of approaches, including Pd-catalyzed carbonylative heteroannulation of iodothiophenols with allenes and carbon monoxide, base-catalyzed cyclization of β-halopropanoic acids with arylthiophenols, base-catalyzed condensation of β-propiolactone with 2-ethylthiophenol followed by acid-promoted cyclization, and intramolecular Friedel−Crafts acylation with Lewis acids or methanesulfonic acid [16]. A recently described one-pot procedure employs a microwave-assisted protocol for the synthesis of 2-/3-methylthiochroman-4-ones by superacid-catalyzed alkylation followed by cyclic acylation [17]. However, preparation of the more hindered 2,2-dimethyl-substituted analogues is more challenging than either un-or mono-substituted C-2 analogues, and protocols such as base-catalyzed cyclization afforded very low yields in our preliminary experiments. Attempts at superacid-catalyzed reactions were entirely unsuccessful. Ultimately, we utilised the established Friedel-Crafts heterocyclisation of commercial thiophenol 15 with 3,3-dimethylacrylic acid in methanesulfonic acid [18], to afford thiochromanone 16 (Scheme 1). Oxidation using persulfate afforded the benzylic aldehyde 17, easily separable from the side-product sulfoxide. Bioreduction using Daucus carota cleanly afforded 18, which was

Chemistry
Construction of thiochromanone skeletons may follow one of a number of approaches, including Pd-catalyzed carbonylative heteroannulation of iodothiophenols with allenes and carbon monoxide, base-catalyzed cyclization of β-halopropanoic acids with arylthiophenols, base-catalyzed condensation of β-propiolactone with 2-ethylthiophenol followed by acid-promoted cyclization, and intramolecular Friedel−Crafts acylation with Lewis acids or methanesulfonic acid [16]. A recently described one-pot procedure employs a microwave-assisted protocol for the synthesis of 2-/3-methylthiochroman-4-ones by superacid-catalyzed alkylation followed by cyclic acylation [17]. However, preparation of the more hindered 2,2-dimethyl-substituted analogues is more challenging than either un-or mono-substituted C-2 analogues, and protocols such as base-catalyzed cyclization afforded very low yields in our preliminary experiments. Attempts at superacid-catalyzed reactions were entirely unsuccessful. Ultimately, we utilised the established Friedel-Crafts heterocyclisation of commercial thiophenol 15 with 3,3-dimethylacrylic acid in methanesulfonic acid [18], to afford thiochromanone 16 (Scheme 1). Oxidation using persulfate afforded the benzylic aldehyde 17, easily separable from the side-product sulfoxide. Bioreduction using Daucus carota cleanly afforded 18, which was esterified with appropriate acids using the coupling agent 1-ethyl-3-(3 -dimethylaminopropyl)carbodiimide hydrochloride (EDC) under basic conditions. esterified with appropriate acids using the coupling agent 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC) under basic conditions. For amide series 11 (Scheme 2), benzylic bromination of 16 with N-bromosuccinimide (NBS) afforded 19 in good yield, analogous to the conditions employed for the chromanone analogue [4]. Nucleophilic substitution with sodium azide proceeded smoothly to azide 20, which was reduced either under a Staudinger protocol or via zinc powder in aqueous media to afford amine 21. Amidation of the primary amine nitrogen was achieved using EDC coupling with the appropriate acid under basic conditions. In parallel, amine series 12 was prepared (Scheme 3) via substitution of bromide synthon 19 in refluxing acetonitrile, or by first oxidising 16 to aldehyde 17 before reductive amination with sodium triacetoxyborohydride (STAB). Direct coupling of the bromide with primary amines proceeded almost instantaneously, yet required careful stoichiometric control and subsequent chromatography to avoid residual contaminating amine. On the other hand, particularly for the higher-boiling alkylamines, reductive amination proved more favourable, with the stable intermediate imine 22 being purified prior to regioselective reduction with one equivalent of sodium borohydride. For amide series 11 (Scheme 2), benzylic bromination of 16 with N-bromosuccinimide (NBS) afforded 19 in good yield, analogous to the conditions employed for the chromanone analogue [4]. Nucleophilic substitution with sodium azide proceeded smoothly to azide 20, which was reduced either under a Staudinger protocol or via zinc powder in aqueous media to afford amine 21. Amidation of the primary amine nitrogen was achieved using EDC coupling with the appropriate acid under basic conditions. For amide series 11 (Scheme 2), benzylic bromination of 16 with N-bromosuccinimide (NBS) afforded 19 in good yield, analogous to the conditions employed for the chromanone analogue [4]. Nucleophilic substitution with sodium azide proceeded smoothly to azide 20, which was reduced either under a Staudinger protocol or via zinc powder in aqueous media to afford amine 21. Amidation of the primary amine nitrogen was achieved using EDC coupling with the appropriate acid under basic conditions. In parallel, amine series 12 was prepared (Scheme 3) via substitution of bromide synthon 19 in refluxing acetonitrile, or by first oxidising 16 to aldehyde 17 before reductive amination with sodium triacetoxyborohydride (STAB). Direct coupling of the bromide with primary amines proceeded almost instantaneously, yet required careful stoichiometric control and subsequent chromatography to avoid residual contaminating amine. On the other hand, particularly for the higher-boiling alkylamines, reductive amination proved more favourable, with the stable intermediate imine 22 being purified prior to regioselective reduction with one equivalent of sodium borohydride. In parallel, amine series 12 was prepared (Scheme 3) via substitution of bromide synthon 19 in refluxing acetonitrile, or by first oxidising 16 to aldehyde 17 before reductive amination with sodium triacetoxyborohydride (STAB). Direct coupling of the bromide with primary amines proceeded almost instantaneously, yet required careful stoichiometric control and subsequent chromatography to avoid residual contaminating amine. On the other hand, particularly for the higher-boiling alkylamines, reductive amination proved more favourable, with the stable intermediate imine 22 being purified prior to regioselective reduction with one equivalent of sodium borohydride.

Computational Study
Simple molecular descriptors were calculated using the Molinspiration online property calculation toolkit (http://www.molinspiration.com, accessed on 1 February 2021). Results are shown in Table 1.

Computational Study
Simple molecular descriptors were calculated using the Molinspiration online property calculation toolkit (http://www.molinspiration.com, accessed on 1 February 2021). Results are shown in Table 1.

Computational Study
Simple molecular descriptors were calculated using the Molinspiration online property calculation toolkit (http://www.molinspiration.com, accessed on 1 February 2021). Results are shown in Table 1.

Pharmacological Activity
Selected compounds were evaluated for their leishmanicidal ability as previously described [21], through examining their activity on L. infantum axenic amastigotes, and the results are expressed in Table 2 as IC 50 values. In parallel, evaluation of the cytotoxicity of test compounds was performed by MTT assay using the J774A.1 macrophage cell line. Results are presented as CC 50 values.
To obey Lipinski's rule, orally active drugs should have: (a) no more than 5 hydrogen bond donors (n-OHNH); (b) no more than 10 hydrogen acceptors (n-ON); (c) an octanolwater partition coefficient (log P) not >5; and (d) a molecular weight <500 Da. Of our synthesised compounds, seven fulfilled Lipinski's rule, with six each showing one violation. Rotatable bond count (nRotb), molecular volume (Vol.) and topological polar surface area (TPSA) were also calculated for the compounds. Molecular volume is a function of molecular weight and structure and considers all accessible conformations available to the molecule under physiological conditions, and alongside TPSA, is used to predict drug transport properties. Drugs of poor bioavailability and absorption have high TPSA values. Our compounds had TPSA values ranging from 29 to 65, with compound 12c, the thio isostere of 3 showing predicted improved bioavailabilty with a lower TPSA score.
Compounds 10a and 10b are ester derivatives of previously synthesised chromanone esters, and while both compounds showed activity against L. infantum, styryl ester 10a was three-fold more active, with an IC 50 of 34.4 µM. As the ester moiety of these molecules is susceptible to hydrolysis, we prepared series 11, a group of amides. A comparison of the activity of 10a and 11a revealed that the amide analogue was over three times more active than the ester against L. infantum, perhaps reflecting the expected better stability profile. However, piperamide 11c was devoid of activity. Within amides 11, extending the lipophilic chain of 11a by two carbons, as in 11b, halved activity (10.5 vs. 19.9 µM). While a linear octanoyl chain in 11d reduced activity 5-fold compared to the styryl derivative, the haloalkyl unit in 11e resulted in the most active compound (IC 50 7.2 µM), superior to the phenylalkenyl derivative 11a. Within the alkylamide compounds 12a-d, extension of the lipophilic carbon chain by two atoms each time correlated with an increase in activity, with the decylamine derivative 12d having an IC 50 of 12.4 µM. Compound 12c (IC 50 19.5 µM) represents the thio isostere of the previously reported 3, which inhibited axenic amastigotes of L. infantum with an IC 50 of 25.3 µM, suggesting that the sulfur atom results in a moderate improvement in activity. However, we must note the cytotoxicity of some of the compounds, notably 11b and alkylamines 12, which although showing interesting activity against axenic amastigotes, were comparably cytotoxic to macrophages. Structures with similarity to compounds 4 and 5 may have effects as membrane disruptors; this needs further exploration within our series of compounds to try and optimise promising anti-leishmanial activity but without appreciable toxicity. Additional work should involve exploration of likely mechanisms of action, such as inhibition of pteridine reductase 1 (PTR1), a proposed mechanism of action of analogous chroman-4-one derivatives [22,23]. In conclusion, compounds 11a and 11e represent interesting compounds, with notable anti-leishmanial activity and good selectivity.

Chemistry
All required chemicals, solvents, and reagents were purchased from Sigma-Aldrich (Arklow, Ireland) and were of reagent grade. Reaction progress was monitored on precoated thin layer chromatographic aluminum sheets (silica gel 60 F254, Merck, Carrigtwohill, Ireland), and TLC visualization was done using a UV lamp. Fourier transform infrared spectra were carried out with neat film coated samples on diamond using a Nicolet TM iS TM 10 FT-IR spectrophotometer (Thermo Fisher, Dublin, Ireland). Significant absorption peak (νmax) values are given in cm −1 . 1 H-and 13 C-NMR spectra were recorded on an Avance 400 spectrometer (Bruker, Rheinstetten, Germany) at 400 MHz and 100 MHz, respectively, in CDCl 3 and CD 3 OD, using tetramethylsilane (TMS) as the internal standard (Spectra available in Supplementary Materials). Chemical shift values are given on the δ (ppm) scale, with signals described as follows: s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), br. (broad signal), m (multiplet), and coupling constants (J) expressed in Hz. Mass spectral analyses were recorded using a LCT Premiere XE (ESI-TOF MS) instrument (Waters, Dublin, Ireland). All calculated exact mono isotopic mass distributions were calibrated against internal reference standards. (16) This compound was prepared and characterized as previously described [17]. Yield: 33%.  (18) To 17 (70 mg, 0.032 mmol) in DMF (1 mL) was added distilled water (50 mL) and freshly cut slices of D. carota (10 g). The resulting mixture was stirred vigorously at room temperature for 72 h. The reaction was filtered, and the filtrate washed with ethyl acetate (50 mL). The water/ethyl acetate mixture was separated, and the ethyl acetate extract dried over Na 2 SO 4 . The crude orange oil was purified by flash column chromatography (pet. ether/EtOAc 7:1) to afford alcohol 18, (54 mg, 76%). 1   Compound 16 (1.55 g (7.5 mmol) was dissolved in cyclohexane (25 mL). To this stirred solution was added N-bromosuccinimide (2.66 g, 14.9 mmol) and a catalytic quantity of dibenzoylperoxide, and the reaction mixture was stirred at 100 • C for 6 h. Upon completion, the solvent was removed in vacuo, and the residue purified by flash chromatography (pet. ether/EtOAc 10:1) to give 19 as a golden oil (1.072 g, 51%), which solidified on standing. IR ν max (neat) 1676 cm

6-(Hydroxymethyl)-2,2-dimethylthiochroman-4-one
To a solution of 17 (100 mg, 0.45 mmol) in tetrahydrofuran (10 mL) was added n-butylamine (33 mg, 0.45 mmol) and sodium triacetoxyborohydride (144 mg, 0.68 mmol). To the stirred suspension was added acetic acid (20 µL, 0.35 mmol). The reaction was stirred under a N 2 atmosphere at room temperature overnight. The reaction mixture was quenched by adding aqueous saturated NaHCO 3 , and the product was extracted with EtOAc (3 × 30 mL). The EtOAc extract was dried (Na 2 SO 4 ), and the solvent was evaporated to give the crude imine as a clear oil (84 mg, 67%). 1  Reduction of 22a-d with one equivalent of sodium borohydride in methanol for one hour at 0 • C to room temperature afforded 12a-d in quantitative yield.