**6. Synthetic drugs**

No vaccine candidates for leishmaniasis are currently under animal or clinical trials. Thus, new and effectives drugs should be investigated. In the last years, several drugs have been studied to find potential new drugs with desirable characteristics. Leishmaniasis is expanding in developed countries and in North America and Europe, which has alarmed health authorities worldwide [106]. Although leishmaniasis is treatable, it is difficult to control due to the absence of an effective vaccine, the adaptation of the vector and reservoir hosts to human environments, and the emergence of resistant lineages [107]. The first-line chemotherapy drugs available are pentavalent antimonials [108], whereas pentamidine and amphotericin B are second-line therapies, but these are associated with limited effectiveness, a long-term treatment, toxicity, and significant side effects [109]. Consequently, there is an urgent need to discover new drugs that are effective against leishmaniasis [110].

The development of drugs directed at new targets such as parasite enzymes represents another approach in the search for new antileishmanial drugs. Arginase is a recently described target for the treatment of leishmaniasis. The enzyme is localized in the glycosome, a subcellular organelle found in some trpypanosomatids such as *Leishmania*. Inhibition of the arginase pathway causes inhibits the polyamine biosynthetic pathway, resulting in antileishmanial

Other enzyme systems investigated as potential targets for antileishmanial drug action include nitric oxide synthase, DNA topoisomerase, trypanothione redutase, superoxide dismutase enzymes, and hypoxanthine-guanine phosphoribosyltransferase [74]; heme oxygenase-1 [87]; ribose 5-phosphate isomerase B [88]; dihydroorotate dehydrogenase [89]; ornitine decarbox‐ ylases [62]; Abl family kinases and phosphoinositide 3-kinase γ [90-93]; and spermidine

A total of 154 peptidases were detected in the *Leishmania major* genome, including serine, cysteine, aspartic, threonine, and metallopeptidases. The cysteine peptidase-specific inhibitor K11777 has shown that these peptidases are necessary for parasite growth [96]. Even though some inhibitors have been developed but failed to kill parasites, peptidases are promising

Carbonic anhydrases (CAs, EC 4.2.1.1) are a new target that are starting to be studied for *Leishmania*. CAs are metalloenzymes that catalyze CO2 hydration to bicarbonate and protons, and five CA classes have been identified: α, β, γ, δ, and ζ. The reaction catalyzed by CAs is essential in the regulation of acid-base balance in organisms [100, 101]. A β-carbonic anhydrase was recently cloned and characterized from *Leishmania donovani chagasi*, and enzyme-specific

The death of the parasite by inhibiting an enzyme or pathway essential for parasite survival and non-essential for the host requires the exploration of differences between these pathways or enzymes [103]. Thus, new *Leishmania* molecules should be studied and the possibility of developing rational and more effective drugs with less harmful side effects for the host investigated. Finally, new *Leishmania* chemotherapeutic targets and new approaches to the

No vaccine candidates for leishmaniasis are currently under animal or clinical trials. Thus, new and effectives drugs should be investigated. In the last years, several drugs have been studied to find potential new drugs with desirable characteristics. Leishmaniasis is expanding in developed countries and in North America and Europe, which has alarmed health authorities worldwide [106]. Although leishmaniasis is treatable, it is difficult to control due to the absence of an effective vaccine, the adaptation of the vector and reservoir hosts to human environments, and the emergence of resistant lineages [107]. The first-line chemotherapy drugs available are pentavalent antimonials [108], whereas pentamidine and amphotericin B are second-line therapies, but these are associated with limited effectiveness, a long-term treatment, toxicity,

activity [86].

synthase [94, 95].

targets [97-99].

**6. Synthetic drugs**

inhibitors were tested against *Leishmania* [102].

362 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

development of drugs should be considered [104, 93, 105].

Some compounds have been studied and regarded as promising new drugs. The stilbene trans-3,4',5-trimethoxy-3'-amino-stilbene (TTAS) showed a LD50 of 2.6 lg/mL against *Leishma‐ nia infantum* with low toxicity. The action mechanism is the disruption of the mitochondrial membrane potential and the ability to block *Leishmania* parasites during the G2/M phase of cell cycle [111].

*N*-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxamide has been tested against *Leishmania amazonensis* and ultrastructural alterations, depolarization of the mitochondrial membrane with loss of cell membrane integrity, and increased formation of mitochondrial superoxide anions were detected, indicating that this compound induced mitochondrial dysfunction [111].

Besides the lack of an effective vaccine against leishmaniasis, in some East African regions, up to 40% of patients with visceral leishmaniasis are co-infected with HIV, which complicates the treatment. Peptidase inhibitors, used to treat HIV-infected individuals are a new route that needs more studies. HIV-1 protease inhibitors such as Indinavir, Saquinavir, and others have been tested to treat leishmaniasis and inhibition of parasite growth has been reported at high drugs concentrations [9].

Other synthetic antileishmanial compounds are currently being developed an evaluated for therapeutic use. β-carbolines from various natural and synthetic sources have shown diverse biological activities. A total of 22 compounds were synthesized and tested *in vitro* against *Leishmania donovani*, out of which six compounds (4, 5, 10, 11, 19, and 22) showed more activity than the standard miltefosine (IC50 = 12.07 ± 0.82 lM), with compound 4 being the most potent (IC50 = 2.16 ± 0.26 lM) [112]. Moreover, a semi-synthetic berberine analogue, 5,6-didehydro-8,8 diethyl-13-oxodihydroberberine chloride showed nanomolar level potency against *in vitro* models of leishmaniasis, malaria, and trypanosomiasis, as well as activity in an *in vivo* visceral leishmaniasis model [113]. Tamoxifen is a synthetic estrogen that has been successfully used to prevent recurrence of breast cancer in women who are estrogen-receptor positive. Miguel *et al.* (2008) [114] reported the leishmanicidal activity of tamoxifen *in vitro* using BALB/c mice infected with *L. amazonensis* and treated with this compound for 15 days.

Diospyrin, a bis-naphthoquinone isolate from the tree *Diospyros montana* and its semisyntheitic derivatives showed inhibitory activity against *Leishmania* spp. The di-epoxide derivative of diospyrin (D17) was more effective against *L. donovani* promastigotes than diospyrin. The same derivative tested in *L.donovani* BHU1216 selectively inhibited intracellular amastigotes. Computational docking studies demonstrated that D17 could inhibit *L donovani* ornithine decarboxylase but not trypanothione reductase [115].

Therapeutic approaches using drugs that act on structures of vital importance to the parasite but absent or sufficiently different in their hosts have been explored by several research groups. Indotecan and AM13-55, are TopIB poisons with indenoisoquinoline structure. Both com‐ pounds were tested against *L infantum* and the results compared with paromomycin, a leishmanicidal drug. The tests were done on a murine BALB/c model of splenocytes infected with *L. infantum*. The results showed that Indotecam reduced more than 80% of the parasite burden of the spleen and liver, indicating that this compound is a potential drug against visceral leishmaniasis [116].

TiO2@Ag nanoparticles (TiAg-Nps) produce reactive oxygen species (ROS), which have an antimicrobial effect, including antileishmanial effects on *Leishmania tropica* and *Leishmania infantum* promastigotes and amastigotes, mainly non-visible light-exposed TiAg-Nps [117]. Twenty-four porphyrin precursors and derivatives were evaluated against *Trypanosoma brucei*, *L. donovani*, and *Plasmodium* sp. The perforine 4i derivative showed the best activity against *T. brucei* with a MEC value of 6.25 mM, but the compound was not active against intramacrophage amastigotes of *L. donovani* [118].

Another approach used in studies is the combination of drug therapies aimed at finding the most effective and secure one. Pam3Cys (an in-built immunoadjuvant and TLR2 ligand) and miltefosine were combined and the resulting combination was evaluated. All experiments were done in BALB/c mouse. Parasitic inhibition significantly increased in groups treated with combinations of the drugs compared to groups receiving miltefosine and Pam3Cys separately. Moreover, increased production of Th1 cytokines, RNS, ROS, and H2O2, as well as increased phagocytosis were observed during the study of immunological alterations [119].

Several aromatic/heterocyclic sulfonamides and 5-mercapto-1,3,4-thiadiazoles were recently investigated against *L. donovani* CAs. The sulfonamides were medium potency-weak inhibi‐ tors, but some heterocyclic thiols inhibited the enzyme with KIs in the range of 13.4–52 nM. Microscopic studies revealed cell swelling and structural alterations on the flagellar pocket such as presence of vacuoles. Autophagic vacuoles that cause intracellular damages and parasite death, and accumulation of intracytoplasmic electron-dense granules were also induced by the inhibitors. These result suggest that β-CA from *Leishmania* is a potential new antileishmanial drug target [102].
