Thiadiazine Derivatives as Antiprotozoal New Drugs

2.1. 3,5 disubstituted-tetrahydro-2H-1,3,5-thiadiazine-2-thione (Mono-THTT)

The most used procedure to obtain the THTT derivatives is the reaction of the appropriate amine 1 with carbon disulfide 2 and potassium hydroxide 3, to give the dithiocarbamate potassium salt 4 (which was not isolated), followed by cyclocondensation with formaldehyde 5 and the selected amino acids [9-11, 13-15], pseudo peptides [9-11, 13-18], and amines or amino ester (6) [12] able to provide the nitrogen at 5th position of the thiadiazine ring in fair to moderate or good yields. In the first step of these synthetic procedures, water is employed as a protic polar solvent in order to stabilize the dithiocarbamate intermediate 4, in the second step pH 7-8 phosphate buffer was used (Scheme 1) [9-18].

This methodology allowed the synthesis of 78 new mono-THTT derivatives to bunch up in at least seven series (Fig. 2). The series synthesized differ as to the nature (lipophilic or hydrophilic) of substituent at 3rd position. Some series bear an aromatic (furfuryl [9,11], benzyl [11] and D or L deacylated chloramphenicol [12]) and alkyl or cycloalkyl (ethyl, butyl, octyl, dodecyl, cyclopropyl and cyclohexyl) [9-11, 13-18] moiety, all of them lipophilic group; for other series the starting amines were hydrophilic (carboxyalkyl) group [9].

Another synthetic method used to obtain these compounds was the solid phase synthesis of 3-(5-carboxypentyl)-5-substituted tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives (Serie I). 6-Amino-n-hexanoic acid was attached via its C-terminal to hydroxymethyl polystyrene using a ‘SASRIN’ linker. The bound amino acid was transformed into the corresponding dithiocarbamate 8 followed by cyclization in the presence of formaldehyde and the corresponding free amino acids to afford 3-(5´-carboxypentyl)-5-substituted tetrahydro-2H-1,3,5-thiadiazin-2-thiones (9). The final products I were cleaved from the resin and obtained in moderate yields due to low solubility of the corresponding free amino acids in 1,4 dioxan (Scheme 2).

Although its use is limited by two factors: the possibility for the starting amines to be properly functionalized for the efficient coupling to the resin and the low solubility of the amino acids in the solvent employed. The development of this methodology would allow the generation of combinatorial library for THTT compounds.

2.2. Alkyl-linked bis-(2-thioxo-[1,3,5] thiadiazinan-3-yl) carboxylic acids (bis-THTT)

The synthetic methods for obtaining bis-THTT have been much less reported in the bibliography than the synthetic route for mono-THTT [19-22]. The general procedure is very similar to the one used for mono-THTT. The bis-THTTs were always obtained using diamines 10 and the amounts of all reagents were duplicated. In the first step the diamine reacts with carbon disulfide 2 in the presence of potassium hydroxide 3 to obtain the expected bisdithiocarbamate salt 11. The addition of formaldehyde 5 and the corresponding amine or aminoacid (6) to 11 resulted in the cyclocondensation in a slightly alkaline medium (phosphate buffer, pH 7-8) to generate, after treatment with HCl 15%, the desired bis-THTT (Scheme3) [19-21].

This procedure allowed the synthesis of 43 new bis-THTT derivatives to bunch up in at least four series (Fig. 3). All compounds were obtained in moderate to good yields except those of series IX, probably due to the use of a bulky diamine (2,2-dimethyl-1,3-propanediamine) and the resulting steric hindrance at the cyclization stage.

Recently, our group explored the feasibility of synthesis of new bis-THTT using more complex polyamines as linkage other than the initially reported diamines [22]. The N4-benzyl polyamine 14 was previously synthesized according to a methodology reported by O'Sullivan et al. [23] via a protection-deprotection strategy using ethyl trifluoroacetate as the selective protective group for primary amines in the presence of secondary amines. The synthetic route leading to derivatives of serie XII from the benzylated spermidine was similar to the one described above (Scheme 4).

The spermidyl linked bis-THTT series (XII) were obtained as solids in moderate yields.

The experimental procedure used to obtain the THTT derivatives (mono and bis-THTT) is simple, allowing a wealth of molecular diversity depending on the nature of groups attached to both nitrogens (N-3 and N-5) of the heterocycle.

2.3. Proposed Reaction Mechanism for the Generation of the Thiadiazine Ring

According to the authors, the formation of the thiadiazinane ring is performed via a one pot domino reaction between the pre-formed DTC, formaldehyde and the amino acid component. Despite being considered a multi-component reaction, the reactants are added in a stepwise fashion. Undoubtedly, one of the least explored aspects regarding the synthesis of THTT has been the study of the reaction pathway from the corresponding DTC. In one approach (Scheme 5a) the preformed DTC 4 is allowed to react simultaneously with formaldehyde and the corresponding amine to produce [substituted(aminomethyl)methanethionyl methylidenazanium 12 [20]. This species possesses two different reactive centers in the same molecular backbone, a protonated imine and a thiosulfanylmethylamino group. The intramolecular addition of the secondary amino group to the carbon atom of the methylidene moiety leads to the THTT ring. The formation of the THTT ring via a {[hydroxymethyl (substituted) carbamothioyl] sulfanyl}methanol intermediate 13 [13] was proposed (Scheme 5b). This process involves in situ generation of 13 from the corresponding DTC 4 and formaldehyde (5) followed by condensation with a primary amine. The isolation and characterization of an analogue of 13, via a crystallization process induced by the presence of KOH, was reported [13].

Recently, we have reported a preliminary DFT study aimed at predicting the probable cyclization mechanism of the thiadiazinane-2-thione from an intermediate of type 13 [24]. Based on experimental facts and DFT studies, a probable cyclization route to the THTT ring from the corresponding {[hydroxymethyl(substituted) carbamothioyl] sulfanyl}methanol intermediate 13 in aqueous medium is proposed. Notably, water not only contributes to the reaction as a mere solvent, but also plays an active role in the reaction mechanism. As far as we know, this is the first computational study on the formation of the thiadiazinane-2-thione ring.

4.1. Biological Activity of Mono-THTT Derivatives

In 1999, we reported for the first time the antiprotozoan properties of 34 mono-THTT derivatives (Fig. 6) corresponding to the series I, II and III against Trichomonas vaginalis and Trypanosoma cruzi [9]. One year later, we published the study on the decomposition products of their mono-THTT and their anticancer properties (Fig. 5) [10].

All mono-THTT derivatives studied showed significant in vitro antiprotozoan activity (both anti-trichomonas and anti-trypanosoma) at the highest dose tested (100 µg/mL), except for compound L-IIIe. (Fig. 6). The results obtained at 10 and 1µg/mL (Fig. 7) showed that the nature of substituents not afforded important differences in the trichomocidal activity (%R) of the three series studied, since most of the compounds lose their trichomonacidal activity at 10 µg/mL. This fact seems to indicate that the lipophilic character of R₁ does not influence the in vitro trichomonacidal activity significantly. Only two compounds of series III (D-IIIj and IIIm) maintain their efficacy at 10 µg/mL and again IIIm was the most active compound at 1 µg/mL (%R=59), with a trichomonacydal effect similar to that of Metronidazole at 0.1 µg/mL (%R=52). None of the compounds studied were more active than Metronidazole (Fig. 6). The most active compounds, L-IIId, D-IIIj and IIIm, that were assayed in vivo, showed a reduction in the pathogenecity index, and this was mainly true for compound IIIm [9].

Compounds of series II and III showed trypanosomicidal activity, both at 100 and 10 µg/mL, whilst compound of series I only showed cytostatic activity at 10 µg/mL. This fact indicated that the lipophilic substituents at the N-3 were better than hydrophilic ones for obtaining active compounds against T. cruzi. Compounds IIc, L-IId, DL-IId, DL-IIf, IIIm and L-IIIn maintained trypanosomicidal activity at 1 µg/mL showing a higher activity than Nifurtimox. We have found more compounds with trypanosomicidal than trichomonacidal activity (Fig. 6) [9].

Taking into account the highest citotoxic activities showed by some compounds of the series I, II and III in a previous work [9], we studied the anticancer properties of mono-THTT derivatives (Fig. 5). This study has been carried out by using cytotoxicity assays against HeLa, HT-29 and Hep G2 cells. The decomposition products of thiadiazinthione IIIm have been studied and their anticancer properties evaluated (Fig. 7) [10].

The results of this study could lead to the conclusion that most of the mono-THTT derivatives showed noticeable cytotoxic properties against HeLa and HT-29 but did not do so against Hep G2 cells. The compounds of series I and II were, in general, less cytotoxic than those of serie III, none of them showed a IC₅₀ lower than 10 ìmol against any cell line. Compound IIIo, detected in the decomposition of compound IIIm and presumingly in one of the other thiadiazinones, were less cytotoxic than IIIm. The nature of R₁ modulates the cytotoxicity of these compounds. Compounds bearing the aromatic furfuryl moiety (Series III) yielded the most interesting thiadiazinones. However, the nature of R₂ influences the cytotoxic activity to a lesser grade. The (S) enantiomer showed more cytotoxic properties than its (R) isomer. Derivative (S) IIIj, bearing furfuryl and L-asparagine moieties, yielded the most interesting compound which is a candidate for a future anticancer study [10]. These results allowed the study through QSAR methodology of mono-THTT derivatives using the novel hybrid index pMRχ [30].

With the aim to increase the antiprotozoan activity spectra, our group reported new biological assays to study the non-specific toxicity and anti-amastigote activity of 18 of these compounds [31]. Cytotoxicity assays of 24 mono-THTT were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, therafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives, (S)-IId was selected to be assayed in vivo, because of its remarkable trypanocidal properties. Nifurtimox and benzidazole were used as reference drugs. All of the compounds were highly toxic at 100 µg/mL for macrophages and a few of them maintained this cytotoxicity even at 10 µg/mL. Of the derivatives assayed against amastigotes, Ik and (S)-IId showed an interesting activity, which was held even at 1 µg/mL. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after the administration of (S)-IId to infected mice [31].

In 2004 and 2005 ten mono-THTT derivatives (Fig. 8) were tested in vitro for antiparasitic effects against both extracellular promastigotes and intracellular amastigotes of Leishmania amazonensis [32,33]. The compounds were found to be active against the amastigote form of the parasite, inhibiting parasite growing, from 10 to 89%, at a concentration of 100 µg/mL. This activity suggests that thiadiazine derivatives could be considered as potential antileishmanial compounds (Fig. 9) [32].

These results confirmed that the THTT represent a group of compounds with significant in vitro activity against L. amazonensis and suggested that some of them could be considered for further study as new therapeuthic alternatives [32,33].

All evaluated compounds caused an irreversible inhibition of the promastigote growth either after 1h of treatment with 10 µg/mL or after 24 h with 1 µg/mL (Fig. 10). However, the compounds exhibited high toxicity and produced inhibition of the phagocytosis in the murine host cell [33]. The mono–THTT tested exhibited a strong activity against L. amazonensis at low concentrations.

4.2. Biological Activity of Bis-THTT Derivatives

It has been reported that bis-thiadiazines have antifungical, antimicrobial and antiprotozoal activity [19-22, 34]. Specifically with antiprotozoal activities three series of bis-THTT had been reported [21, 22, 34]. The in vitro activity of compounds belonging to series VIII and IXI (Fig. 11) against Leishmania donovani, Trypanosoma brucei rhodesiense, and Plasmodium falciparum was studied. From these results it may be observed (Fig. 12) that the best activity profiles were found against T. b. rhodesiense. It is intereresting that the activity against T. b. rhodesiense seems to be favoured for compounds having linear aminoacidic residues as substituents in N-5 of the THTT ring. Despite exerting a notable activity against T. b. rhodesiense, derivatives from series VIII appeared to be more citotoxic than the analogs of series IX (Fig. 13) [34].

The in-vitro activity against L. donovani, T. cruzi, T. b. rhodesiense , and P. falciparum of some compounds of series XII, was compared with these activities for compounds of series VIII (Fig. 14 and Fig. 15) [22]. For comparative purposes, structures VIII and XII were changed only in the nature of their connective backbone. In the bis-THTT derivatives in both series that had the same aminoacid residue (R₂), the displayed protozoocidal profile against the three parasitic lines assayed was almost the same. However, higher values of therapeutic indexes were found for compounds VIIIa–c. Considering that both series exerted a similar protozoal activity it is possible to assume that structures XII are more cytotoxic than their alkyl tethers analogs [22].