Synthesis and Identification of Pentathiepin-Based Inhibitors of Sporothrix brasiliensis

Sporothrix brasiliensis is the causative agent of zoonotic sporotrichosis in Brazil and is currently referred to as the most virulent species among those of clinical importance within the genus. Sporotrichosis is an emergent disease that has come to the forefront over two decades with a recent hot spot of sporotrichosis infection emerging in the state of Rio de Janeiro. The source of these infections is now at epidemic proportions with more than 4000 cases reported in Rio de Janeiro, Brazil, alone since 1998. We developed a focused library of a rare pentathiepin ring system and identified a potent substitution pattern that yielded compounds 21 and 22. These compounds were more potent than itraconazole which is the current standard of care for sporotrichosis.


Introduction
Sporotrichosis is an emergent disease that has come to the forefront over two decades after being first described in 1898 [1,2]. A recent hot spot of Sporothrix infection has emerged in the state of Rio de Janeiro, Brazil [3]. The source of these infections is now at hyperendemic proportions with more than 4000 cases reported in this region since 1998 [3][4][5].
Sporothrix species are usually non-pathogenic environmental fungi that are closely related to decaying wood, plants, and soil [6]. The majority of human and animal infections occur when the epidermis is damaged, allowing plant matter/soil, along with the fungus, to enter into the body [7]. The incidence of zoonotic infection is on the rise with feline transmission of Sporothrix which has been identified as the likely source of the spike in cases within Rio de Janeiro [3][4][5]8]. Sporothrix brasiliensis has been demonstrated to be a highly successful mammal pathogen, and it is related to zoonotic transmission from infected cats in Brazil [9].
Feline sporotrichosis has a varied range of clinical presentation, but it is frequently a severe condition with the development of disseminated skin lesions and respiratory involvement in infected patients [11].
Infected cats are the most important source of Sporothrix transmission in the Brazilian scenario due to the high burden of yeasts in their lesions. Zoonotic transmission usually occurs through their scratches and bites [3,10]. Although the treatment of feline sporotrichosis is shown to efficiently reduce the fungal burden in lesions of cats contributing to the control of zoonotic transmission, it remains a challenge for veterinary practitioners [12]. Itraconazole frequently in association to potassium iodide is still the treatment of choice, but therapeutic failure, the occurrence of adverse effects, and recrudescence of lesions are described, raising the need for alternative therapeutic options [10].
Several small anti-fungal molecules (1)(2)(3)(4)(5)(6)(7)(8) have been reported (Figure 1), targeting different routes to treat fungal infections. The fungal cells, unlike mammalian cells, are encased in a carbohydrate-containing cell wall which has been used as a target to reduce mammalian toxicity and to target specifically the fungus [13]. The fungal cell wall has been targeted with a series of chalcone and quinoline derivatives (1-3) with varying degrees of success [14][15][16].

Initial Investigation
To investigate pentathiepin as a viable treatment for sporotrichosis, we first screened a symmetric thiophene derivative (13) against six isolates of S. brasiliensis from skin lesions of infected cats. The antifungal susceptibility testing was based on the reference broth microdilution method according to the M38-A2 CLSI guidelines [51] with a few modifications to improve minimal inhibitory concentration (MIC) determination for S. brasiliensis [52]. We screened 13 at two concentrations (4 and 8 µg/mL) to determine if there was any inhibition of fungal growth. We evaluated MIC to suppress both 100% and 50% of the growth of the strains. At the higher concentration, 100% inhibition of growth was observed in all six isolates with the isolate 8607 presenting a slightly higher sensitivity and being the only isolate to respond to this compound at the lower concentration (Table 1).

Synthesis of Pentathiepin Analogs
We decided to prepare a small array of pentathiepins (13, 16, and 18-24) (Scheme 1). The older methods for preparation of this advanced heterocycle included using an activated sulfur source, such as disulfur dichloride (S2Cl2) or trisulfur dichloride (S3Cl2) or even directly with elemental sulfur (S8), and adding these to an ortho-dithiol, usually under harsh conditions [53][54][55][56][57]. However, a C-H activated route utilizing the 1,4-diaza-bicyclo(2.2.2)octane (DABCO) sulfur monochloride complex enables formation of pentathiepins in one step from commercially available reagents (Scheme 1) [58,59]. The two thiophene analogs (13 and 18) were furnished by treating Hünig's base and N,Ndibenzylethanamine, respectively, with DABCO and S2Cl2 for 48 h followed by refluxing with triethylamine for a further 2 h to afford 13 and 18 in good and acceptable yields, respectively [32,60]. The pyrrole derivatives were explored using a series of substituted pyrrole ring systems and treating

Initial Investigation
To investigate pentathiepin as a viable treatment for sporotrichosis, we first screened a symmetric thiophene derivative (13) against six isolates of S. brasiliensis from skin lesions of infected cats. The antifungal susceptibility testing was based on the reference broth microdilution method according to the M38-A2 CLSI guidelines [51] with a few modifications to improve minimal inhibitory concentration (MIC) determination for S. brasiliensis [52]. We screened 13 at two concentrations (4 and 8 µg/mL) to determine if there was any inhibition of fungal growth. We evaluated MIC to suppress both 100% and 50% of the growth of the strains. At the higher concentration, 100% inhibition of growth was observed in all six isolates with the isolate 8607 presenting a slightly higher sensitivity and being the only isolate to respond to this compound at the lower concentration (Table 1).

Synthesis of Pentathiepin Analogs
We decided to prepare a small array of pentathiepins (13, 16, and 18-24) (Scheme 1). The older methods for preparation of this advanced heterocycle included using an activated sulfur source, such as disulfur dichloride (S 2 Cl 2 ) or trisulfur dichloride (S 3 Cl 2 ) or even directly with elemental sulfur (S 8 ), and adding these to an ortho-dithiol, usually under harsh conditions [53][54][55][56][57]. However, a C-H activated route utilizing the 1,4-diaza-bicyclo(2.2.2)octane (DABCO) sulfur monochloride complex enables formation of pentathiepins in one step from commercially available reagents (Scheme 1) [58,59]. The two thiophene analogs (13 and 18) were furnished by treating Hünig's base and N,N-dibenzylethanamine, respectively, with DABCO and S 2 Cl 2 for 48 h followed by refluxing with triethylamine for a further 2 h to afford 13 and 18 in good and acceptable yields, respectively [32,60]. The pyrrole derivatives were explored using a series of substituted pyrrole ring systems and treating with DABCO and S 2 Cl 2 for 48 hours to access 16, 19, and 20 in good yield [61]. Treatment of N-methylpyrrolidine under analogous conditions allowed access to 21 and 23 in a one-pot reaction. However, under the same conditions, N-isopropylpyrrolidine produced exclusively 22 with the corresponding asymmetric product not observed [62][63][64]. This was followed by a final analog (24) synthesized by treating N-methylindole with the DABCO sulfur monochloride complex at zero degrees to furnish the final product in excellent yield (70%) [64,65]. with DABCO and S2Cl2 for 48 hours to access 16, 19, and 20 in good yield [61]. Treatment of Nmethylpyrrolidine under analogous conditions allowed access to 21 and 23 in a one-pot reaction. However, under the same conditions, N-isopropylpyrrolidine produced exclusively 22 with the corresponding asymmetric product not observed [62][63][64]. This was followed by a final analog (24) synthesized by treating N-methylindole with the DABCO sulfur monochloride complex at zero degrees to furnish the final product in excellent yield (70%) [
Encouraged by our early results, we screened the same set of isolates with two unsymmetrical pentathiepin derivatives (23 and 24) (Table 3). We found antifungal activity across all isolates with both 23 and 24. Encouraged by our early results, we screened the same set of isolates with two unsymmetrical pentathiepin derivatives (23 and 24) (Table 3). We found antifungal activity across all isolates with both 23 and 24.

Discussion
Sporotrichosis is still a neglected disease. It has reached a significant number of cases in Rio de Janeiro region due to the fact of its uncontrolled zoonotic spread from infected cats. However, not only has the expansion of the disease been identified in other regions in Brazil and Argentina, Encouraged by our early results, we screened the same set of isolates with two unsymmetrical pentathiepin derivatives (23 and 24) (Table 3). We found antifungal activity across all isolates with both 23 and 24.

Discussion
Sporotrichosis is still a neglected disease. It has reached a significant number of cases in Rio de Janeiro region due to the fact of its uncontrolled zoonotic spread from infected cats. However, not only has the expansion of the disease been identified in other regions in Brazil and Argentina,

Discussion
Sporotrichosis is still a neglected disease. It has reached a significant number of cases in Rio de Janeiro region due to the fact of its uncontrolled zoonotic spread from infected cats. However, not only has the expansion of the disease been identified in other regions in Brazil and Argentina, worldwide emergence is clear, whether related or not to zoonotic transmission. Outbreaks have been described on different continents and infected immunosuppressed patients are at serious risk. In addition, Sporothrix brasiliensis is the causative agent of zoonotic sporotrichosis in Brazil and is currently referred to as the most virulent species among those of clinical importance within the genus.
The sub-micro molar potencies of the pentathiepins coupled with previous reports of moderate toxicities suggests a low-risk path towards the development of a candidate compound for targeting sporotrichosis in cats and potentially humans [32]. The two asymmetric pentathiepins (23 and 24) both show improvements over the current standard of care (itraconazole). While the symmetrical analogs showed only weak activity, increasing the electron withdrawing nature of the substituents (methyl (16) versus chloro (21)) did increase anti-fungal potency. There is increasing evidence that the pentathiepin functionality itself is not a toxic motif and that rather the electronic contributions pendant arms are what drives non-specific toxicity. The pentathiepin functionality and other high-density sulfur heterocycles provide an exciting opportunity for the development of new clinically relevant compounds highlighted by 23 and 24. The pentathiepin functionality, while still under-explored, has the potential to generate a pre-clinical candidate for treating sporotrichosis in vivo.

Isolate Collection
The isolates were previously collected from cats with sporotrichosis seen at the Laboratory of Clinical Research on Dermatozoonosis in Domestic Animals (Lapclin-Dermzoo), Evandro Chagas National Institute of Infectious Diseases (INI), Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil. The strains were initially recovered from the exudate of ulcerated lesions or secretions from nasal cavities that were collected using a sterile swab and cultured on Sabouraud dextrose agar and Mycobiotic Agar (Difco), incubated at 25 • C and observed over 4 weeks for fungal growth. Suspected isolates were sub-cultivated on brain heart infusion agar medium (Difco) at 37 • C, and dimorphism was demonstrated by conversion to the yeast-like form. Then, the isolates were stored in sterile distilled water in the Mycology Laboratory of INI until used.

General Procedures for Screening
The compounds (13,16,(18)(19)(20)(21)(22)(23)(24) were tested using the broth microdilution technique in accordance with the respective reference protocols of the CSLI (Clinical and Laboratory Standards Institute). The reading of the plates was performed in wells with 100% inhibition of fungal growth (all isolates). All isolates were previously identified by the T3B-fingerprint technique as S. brasiliensis, and their antifungal susceptibilities to itraconazole are known [66]. Repetitions of the test were performed to confirm each result.

General Procedure to Afford Compounds 13 and 18
Disulfur dichloride (100 mmol) was added dropwise at −15 • C to −20 • C to a stirred solution of DABCO (100 mmol) in chloroform (40 mL). The mixture was stirred at 0 • C for 48 h. The corresponding amine (200 mmol) was added, and the mixture was refluxed for 2 h, filtered, and the solvents were evaporated. The residue was separated by column chromatography (light petroleum and then light petroleum-CH 2 Cl 2 mixtures) to afford products 13 and 18.