**2. Metal compounds as a new generation of leishmanicidal agents. Design strategies**

#### **2.1. Metal-based drugs for leishmaniasis**

Currently, there is no vaccine against leishmaniasis yet, either purely organic or containing metals. Disease treatment relies solely on chemotherapy. After an intensive revision of the literature, we have found a wide range of metal-containing compounds that are currently used to treat different varieties of leishmaniasis or present a strong antileihsmanial activity and hence potential to be part of a new generation of chemotherapeutic agents with high efficacy and minimal toxicity for the patient. All of them are described in detail in this section.

#### *2.1.1. Pentavalent antimonials: a (still) unbeatable classic in leishmanicidal therapy*

Antimony-based compounds started to be used a century ago. Trivalent antimonials -Sb(III) were first used, e.g. tartar emetic, which was first reported for treatment of cutaneous leish‐ maniasis in 1913. But the high toxicity of Sb(III) compounds and their unstability in tropical climate, led to discovery of pentavalent antimonials –Sb(V)- and in 1920 the Sb(V) compound urea stibamine emerged as an effective agent against visceral leishmaniasis (kala azar) while being less toxic than the trivalent antimonials.

Other advantages of using metal compounds are their pronounced selectivity for selected parasites biomolecules compared to the host biomolecules,[7] and the possibilities they offer to targeted therapies as targeting molecules may be reversibly appended and prodrugs can be developed to deliver highly reactive metal specia in the parasite target while minimising non-

In the last years, nanotechnology has revolutioned the medicine field by opening novel and promising approaches for drug design, in particular regarding use of nanoparticles (1-100 nm) as drug delivery vehicles. Despite being liposomes and polymeric particles the most investi‐ gated systems to deliver antiparasitic drugs, metal nanoparticles have also emerged as interesting alternative carriers. [8] Furthermore, use of nanoforms of antiparasitic metals like antimony and selenium as alternatives to molecular forms [9,10] has also been recently

In summary, there is a clear need for research in this largely neglected area of medicinal chemistry that is tropical parasitic diseases, and use of metal complexes as possible chemo‐ therapeutic agents arises as a very attractive alternative to tackle this immense problem. However, despite the obvious potential of metal complexes as diagnostic and chemothera‐ peutic agents, few pharmaceutical or chemical companies have serious in-house research programs that address these important bioinorganic aspects of medicine, which contrast

The following sections will focus on diverse examples of metal compounds with current or

**2. Metal compounds as a new generation of leishmanicidal agents. Design**

Currently, there is no vaccine against leishmaniasis yet, either purely organic or containing metals. Disease treatment relies solely on chemotherapy. After an intensive revision of the literature, we have found a wide range of metal-containing compounds that are currently used to treat different varieties of leishmaniasis or present a strong antileihsmanial activity and hence potential to be part of a new generation of chemotherapeutic agents with high efficacy and minimal toxicity for the patient. All of them are described in detail in this section.

Antimony-based compounds started to be used a century ago. Trivalent antimonials -Sb(III) were first used, e.g. tartar emetic, which was first reported for treatment of cutaneous leish‐ maniasis in 1913. But the high toxicity of Sb(III) compounds and their unstability in tropical climate, led to discovery of pentavalent antimonials –Sb(V)- and in 1920 the Sb(V) compound urea stibamine emerged as an effective agent against visceral leishmaniasis (kala azar) while

*2.1.1. Pentavalent antimonials: a (still) unbeatable classic in leishmanicidal therapy*

tremendously with the case of purely organic drugs.

468 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

potential applications for leishmaniasis treatment.

**2.1. Metal-based drugs for leishmaniasis**

being less toxic than the trivalent antimonials.

specific interactions.

reported.

**strategies**

**Figure 2.** Structures of antimalarial drug chloroquine and antitrypanocidal drug benznidazole, and their respective metallo-derivatives, which show enhanced antiparasitic properties.

Nowadays pentavalent antimonials still constitute the first-line treatment for leishmaniasis. The most commonly used organic salts of Sb(V) are sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime). See figure 3.

However, a significant increase in clinical resistance has been reported for this class of drugs in recent years. In some parts of the world like North East India, the percentage of cases of resistance development is so high (up to 65%) that these drugs are becoming obsolete.

Although acquired resistance is the most limiting factor for the application of pentavalent antimonials, these drugs present other important drawbacks such as low efficacy for some forms of leishmaniasis and toxic effects (e.g. cardiotoxicity, pancreatitis, anemia and leucope‐ nia). Their toxicity is aggravated by usually required long periods of therapy (up to 4-6 weeks).

Even though antimonials have been in clinical use against leishmaniasis for more than 60 years, their molecular and cellular mechanisms of action are not well understood yet. [11] What is clear is that to be active, antimony has to enter the host cell, cross the phagolysomal membrane and act against the intracellular parasite. By analogy to pentavalent arsenate, it has been

**Figure 3.** Chemical structure of sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime).

suggested that they enter the cell via a phosphate transporter. Two main models have been proposed to explain the mechanism of action of pentavalent antimonials (Figure 4):

*Prodrug model.* Recent studies suggest that antimony compromises the thiol redox potential of the cell by inducing efflux of intracellular thiols and by inhibiting trypanothione reductase. Because Sb(III) is highly active against both stages of the parasite, extra- and intracellular on one hand, and Sb(V) is active mostly against amastigotes on the other, it is generally accepted that Sb(V) needs to be reduced to Sb(III) in order to be active. However, the site and the mechanism of reduction are unclear. Recent results suggest that activation occurs inside macrophages as well as inside parasites (amastigotes). [12] Both reduced glutathione (GSH) and reduced trypanothione (T(SH)2) have been found to be responsible for non-enzymatic reduction of Sb(V) to Sb(III). Other studies have suggested the participation of a parasitespecific enzyme, namely thiol-dependent reductase (TDR1), in the reduction process of Sb(V) to Sb(III). Recent crystal structure studies display the mechanism of *Leishmania* trypanothione reductase (TR) inhibition by Sb(III). These studies show that trivalent antimony binds the protein active site with high affinity, and strongly inhibits enzyme activity. Metal binds directly to Cys52, Cys57, Thr335 and His461, thereby blocking hydride transfer and trypanothione reduction. Also evidence suggests that the active specia Sb(III) may interact with zinc-finger proteins by binding Cys residues. The interaction with TR would affect the metabolism of T(SH)2 and induce rapid efflux of intracellular T(SH)2 and GSH in *Leishmania* cells. [13] Moreover, the lowering of concentration of intracellular trypanothione in its reduced form T(SH)2, increases the chances for oxidative damage and decreases the disposal of reducing equivalents for DNA synthesis. Sereno *et al*. found that Sb(III) induces DNA fragmentation after treating amastigotes of *L. infantum* at low concentrations of drug, which suggests appearance of late events of apoptosis.[14]

*Active Sb(V) model*. According to this model, Sb(V) would present intrinsic anti-leishmanial activity. It has been shown that sodium stibogluconate, but not Sb(III), specifically inhibits type I DNA topisomerase by binding the enzyme, thus inhibiting unwinding and cleavage.[15] Formation of Sb(V) complexes with ribonucleosides has been reported, which would be kinetically favored in acidic biological compartments. Moreover, stability constants are consistent with the formation of such a complex in the vertebrate host following treatment with pentavalent antimonial drugs. It is hypothesized that formation of this complex might act as an inhibitor of the *Leishmania* purine transporters or that, once inside the parasite, this complex interferes with the purine salvage pathway. [16] The formation of these complexes with ribonucleosides might explain as well the depletion of ATP and GTP, as reported previously with sodium stibogluconate. [17]

When antimonials fail, amphotericin B and pentamidine are the recommended second-line treamtent for visceral, cutaneous and mucocutaneous leishmaniasis.However, they are not fully effective either and, additionally,produce toxic side effects. On the other hand, new formulations such as liposomal amphotericin B have been found to be very effective, but its high cost limits its availability to patients.

suggested that they enter the cell via a phosphate transporter. Two main models have been

*Prodrug model.* Recent studies suggest that antimony compromises the thiol redox potential of the cell by inducing efflux of intracellular thiols and by inhibiting trypanothione reductase. Because Sb(III) is highly active against both stages of the parasite, extra- and intracellular on one hand, and Sb(V) is active mostly against amastigotes on the other, it is generally accepted that Sb(V) needs to be reduced to Sb(III) in order to be active. However, the site and the mechanism of reduction are unclear. Recent results suggest that activation occurs inside macrophages as well as inside parasites (amastigotes). [12] Both reduced glutathione (GSH) and reduced trypanothione (T(SH)2) have been found to be responsible for non-enzymatic reduction of Sb(V) to Sb(III). Other studies have suggested the participation of a parasitespecific enzyme, namely thiol-dependent reductase (TDR1), in the reduction process of Sb(V) to Sb(III). Recent crystal structure studies display the mechanism of *Leishmania* trypanothione reductase (TR) inhibition by Sb(III). These studies show that trivalent antimony binds the protein active site with high affinity, and strongly inhibits enzyme activity. Metal binds directly to Cys52, Cys57, Thr335 and His461, thereby blocking hydride transfer and trypanothione reduction. Also evidence suggests that the active specia Sb(III) may interact with zinc-finger proteins by binding Cys residues. The interaction with TR would affect the metabolism of T(SH)2 and induce rapid efflux of intracellular T(SH)2 and GSH in *Leishmania* cells. [13] Moreover, the lowering of concentration of intracellular trypanothione in its reduced form T(SH)2, increases the chances for oxidative damage and decreases the disposal of reducing equivalents for DNA synthesis. Sereno *et al*. found that Sb(III) induces DNA fragmentation after treating amastigotes of *L. infantum* at low concentrations of drug, which suggests

*Active Sb(V) model*. According to this model, Sb(V) would present intrinsic anti-leishmanial activity. It has been shown that sodium stibogluconate, but not Sb(III), specifically inhibits type I DNA topisomerase by binding the enzyme, thus inhibiting unwinding and cleavage.[15]

proposed to explain the mechanism of action of pentavalent antimonials (Figure 4):

**Figure 3.** Chemical structure of sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime).

appearance of late events of apoptosis.[14]

470 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

#### *2.1.2. Metal complexes of organic drugs: Following the metal-drug synergism approach*

Metal-drug synergism has led to several attempts to develop new potent antiparasitic agents. This approach involves combination of a compound of known antiparasitic activity and a metal in a single molecule. One example is complexation of antileishmanial drug pentamidine with Rh(I) and Ir(I) to form binuclear complexes of general formula [M2(L2)(pentamidine)]2+, where L2= 1,5-cyclooctadiene (COD), 1,3,1,5-cyclooctatetraene (COT) or (CO)2. Some of these com‐ pounds were found to be more active than the uncomplexed drug pentamidine isethionate. The complex [Ir(COD)(pentamidine)][BPh4] exhibits the same *in vitro* activity as free pentam‐ idine, but its *in vivo* activity reaches 23% and 32% of parasite suppression for *L. donovani* and *L. major*, respectively, under conditions where pentamidine isethionate is inactive. The related compound [Ir2(COT)2(pentamidine)][alizarin red]2 showed to be at least twice as active as pentamidine isethionate against amastigotes of *L. donovani* and synergistic effect was observed when this complex was administered in combination with pentamidine, amphotericin B or paromomycin. [18]

Other metal-drug synergy-based strategies make use of diverse chemotherapeutic targets such as sterol 14-demethylases by attaching azole-type sterol biosynthesis inhibitors (SBIs) such as clotrimazole (CTZ) and ketoconazole (KTZ), to a metal-containing fragment. For example, compound [Ru(η<sup>6</sup> -p-cymene)Cl2(CTZ)] shows an enhancement of the activity of CTZ by a factor of 110 against *L. major* promastigotes, resulting in low nanomolar lethal doses. In addition, this Ru(II) compound does not exhibit any appreciable toxicity toward human osteoblasts when assayed up to 7.5 µM, which translates into excellent selectivity indexes higher than 500. This compound also significantly inhibited the proliferation of intracellular amastigotes of *L. major* in infected intraperitoneal mouse macrophages (IC70=29 nM). *In vivo* testing and detailed mechanistic studies of these ruthenium–CTZ complexes are currently in progress.[19] Likewise, a series of Ru(II) complexes with KTZ have recently been synthesized: *cis,fac*-[RuCl2(DMSO)3(KTZ)], *cis*-[RuCl2(bipy)(DMSO)(KTZ)], [Ru(η<sup>6</sup> -p-cymene)Cl2(KTZ)], [Ru(η<sup>6</sup> -p-cymene)(en)(KTZ)][BF4]2, [RuII(η<sup>6</sup> -pcymene)(bipy)(KTZ)][BF4]2, and [Ru(η<sup>6</sup> -pcymene)(acac)(KTZ)][BF4]. They showed a marked increase of the activity against promasti‐ gotes and intracellular amastigotes of *L. major* when compared with free KTZ or with similar ruthenium compounds not containing KTZ. Interestingly, selectivity of some of these com‐ pounds toward *Leishmania* parasites in relation to normal human cells was also higher than selectivities of the individual constituents of the drug. Hydrolysis of the chloride ligands to form cationic aqua species appears to be a prerequisite for biological activity, and dissociation of KTZ probably occurs but only on further interactions of the active species with biomolecules within the parasite cell. Authors relate the antiparasitic activity to a combination of the SBI action of dissociated KTZ and the ability of the nitrogen-containing ligands on the remaining ruthenium fragment to promote interactions with DNA through hydrogen bonding or by π– stacking interactions. [20]

Other metal ions like Pt(II), Rh(I) or Os(III) have been used to obtain organometallic com‐ pounds with ligands derived from benzothiazole, a compound of which some derivatives have shown promising antimicrobial, antifungus and antiparasite activity. The obtained com‐ pounds were active against promastigotes and amastigotes of *L. donovani* by targeting different biochemical pathways: [*cis*-[Pt(da)(2,5-dihydroxybenzenesulfonate)2] (da = 1,2-diaminocyclo‐ hexane), [Ru(CO)2(2-aminobenzothiazole)], [Ru(CO)2(2-methylbenzothiazole)], [21] and a series of dithiocarbamate complexes with formula [Os(L)] where L= nitroimidazole, dinitroi‐ midazole, benznidazole. Osmium complexes clearly inhibited DNA, RNA and protein synthesis, as well as enzymatic activities of succinate dehydrogenase, malate dehydrogenase and pyruvate kinase. [22]

Apart from vanadium compounds ability to exert different insulin-mimetic and antidiabetic effects, it has been recently proved that vanadium also offers interesting chemical and biochemical properties for the development of antiparasitic drugs. Noleto *et al.* combined the oxovanadium(IV) core with the antileishmanial compound galactomannan (GMPOLY), isolated from southern Brazil lichen *Ramalina celastri*. [23] Complexation highly increased leishmanicidal effect of galactomannan on amastigotes of *L. amazonensis* infecting peritoneal macrophages. This effect of GMPOLY on amastigotes could be attributed to the activation of the nitric oxide pathway. Nitric oxide is secreted by macrophages in response to IFN-γ (interferon γ) stimulation and it is regulated by tyrosine phosphatase events. Since the effect detected for GMPOLY oxovanadium(IV) complex occurred at concentrations where GMPOLY was non active, authors suggested the involvement of oxovanadium(IV) ion in the anti-parasite action.

#### *2.1.3. Targeting cysteine proteases*

Rh(I) and Ir(I) to form binuclear complexes of general formula [M2(L2)(pentamidine)]2+, where L2= 1,5-cyclooctadiene (COD), 1,3,1,5-cyclooctatetraene (COT) or (CO)2. Some of these com‐ pounds were found to be more active than the uncomplexed drug pentamidine isethionate. The complex [Ir(COD)(pentamidine)][BPh4] exhibits the same *in vitro* activity as free pentam‐ idine, but its *in vivo* activity reaches 23% and 32% of parasite suppression for *L. donovani* and *L. major*, respectively, under conditions where pentamidine isethionate is inactive. The related compound [Ir2(COT)2(pentamidine)][alizarin red]2 showed to be at least twice as active as pentamidine isethionate against amastigotes of *L. donovani* and synergistic effect was observed when this complex was administered in combination with pentamidine, amphotericin B or

Other metal-drug synergy-based strategies make use of diverse chemotherapeutic targets such as sterol 14-demethylases by attaching azole-type sterol biosynthesis inhibitors (SBIs) such as clotrimazole (CTZ) and ketoconazole (KTZ), to a metal-containing fragment. For example,

factor of 110 against *L. major* promastigotes, resulting in low nanomolar lethal doses. In addition, this Ru(II) compound does not exhibit any appreciable toxicity toward human osteoblasts when assayed up to 7.5 µM, which translates into excellent selectivity indexes higher than 500. This compound also significantly inhibited the proliferation of intracellular amastigotes of *L. major* in infected intraperitoneal mouse macrophages (IC70=29 nM). *In vivo* testing and detailed mechanistic studies of these ruthenium–CTZ complexes are currently in progress.[19] Likewise, a series of Ru(II) complexes with KTZ have recently been synthesized:

cymene)(acac)(KTZ)][BF4]. They showed a marked increase of the activity against promasti‐ gotes and intracellular amastigotes of *L. major* when compared with free KTZ or with similar ruthenium compounds not containing KTZ. Interestingly, selectivity of some of these com‐ pounds toward *Leishmania* parasites in relation to normal human cells was also higher than selectivities of the individual constituents of the drug. Hydrolysis of the chloride ligands to form cationic aqua species appears to be a prerequisite for biological activity, and dissociation of KTZ probably occurs but only on further interactions of the active species with biomolecules within the parasite cell. Authors relate the antiparasitic activity to a combination of the SBI action of dissociated KTZ and the ability of the nitrogen-containing ligands on the remaining ruthenium fragment to promote interactions with DNA through hydrogen bonding or by π–

Other metal ions like Pt(II), Rh(I) or Os(III) have been used to obtain organometallic com‐ pounds with ligands derived from benzothiazole, a compound of which some derivatives have shown promising antimicrobial, antifungus and antiparasite activity. The obtained com‐ pounds were active against promastigotes and amastigotes of *L. donovani* by targeting different biochemical pathways: [*cis*-[Pt(da)(2,5-dihydroxybenzenesulfonate)2] (da = 1,2-diaminocyclo‐ hexane), [Ru(CO)2(2-aminobenzothiazole)], [Ru(CO)2(2-methylbenzothiazole)], [21] and a series of dithiocarbamate complexes with formula [Os(L)] where L= nitroimidazole, dinitroi‐ midazole, benznidazole. Osmium complexes clearly inhibited DNA, RNA and protein

*cis,fac*-[RuCl2(DMSO)3(KTZ)], *cis*-[RuCl2(bipy)(DMSO)(KTZ)], [Ru(η<sup>6</sup>


472 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment





paromomycin. [18]

compound [Ru(η<sup>6</sup>

stacking interactions. [20]

[Ru(η<sup>6</sup>

Cysteine proteases have been found to play multiple roles in parasitic life cycles includ‐ ing nutrition, host invasion, protein processing, and evasion of the host immune re‐ sponse. In fact, there is an abundance of data to suggest that parasite cysteine proteases represent valid drug targets. For example, it was shown that an inhibitor of cathepsin Blike cysteine protease of *L. major*, cpB, inhibited parasite growth *in vitro* and ameliorated the pathology associated with a mouse model of leishmaniasis. [24] Since cysteine proteas‐ es found in *Leishmania* and *T. cruzi* have similarities to mammalian cathepsins B and L, the latter ones have been used as models to study the bioactivity of diverse metallic com‐ pounds. Cyclometallated gold, palladium, and rhenium derivatives have displayed cathepsin B inhibitory ability against cathepsin B and also similar order activity against the corresponding parasite enzyme cpB. These compounds have also shown growth inhibi‐ tion of extracellular promastigotes of *L. major*, *L. mexicana* and *L. donovani* (see figure 5 and table 1). [25]

#### *2.1.4. Vanadium compounds and their interaction with protein tyrosine phosphatases*

Peroxovanadium compounds have shown potent inhibitors of protein tyrosine phosphatases and inducers of antileishmania effects like ROS and NO. Treatment of infected mice with bisperoxovanadium-1,10-phenanthroline or bis-peroxovanadiumpicolinate completely controlled progression of leishmaniasis in a NO-dependent manner. After injection, com‐ pounds rapidly triggered expression of inducible NO synthase in liver of mice infected with *L. major*. *In vivo* functional and immunological events associated with this peroxovanadium protective process have been identified. More recently, three dinuclear triperoxovanadate complexes, two mononuclear diperoxovanadate complexes with aminoacids or dipeptides as ancillary ligands and bis-peroxovanadate have been tested for their ability to kill *Leishmania* parasites *in vitro*, being K[VO(O2)2(H2O)] the most potent one. Combined administration of the latter with sub-optimal doses of sodium antimony gluconate on BALB/c mice experimentally

**Figure 5.** A) Structures of metal complexes with antileishmanial activity via inhibition of parasite cysteine proteases: (**1**) diaceto [2-[(2-pyridinyl-κ*N*)methyl] phenyl-κ*C*] gold(I), (**2**) aceto [2,6-bis [(butylthio-κ*S*)methyl]-phenyl-κ*C*] palladium(II), (**3**) (*p*-methoxyphenylthiolato-*S*) [2,6-bis[(mercapto-κ*S*)methyl] pyridine-κ*N*1] oxorhenium(V), (**4**) (2(1*H*)-pyridinethiona‐ to-κ*S*<sup>2</sup>)[2,6-bis [(mercapto-κ*S*)-methyl] pyridine-κ*N*1] oxorhenium(V), (**5**) chloro [2,2'-(thio-κ*S*)bis [ethanethiolato-κ*S*)]] ox‐ orhenium(V), (**6**) (methanethiolato) [2,2'-(thio-κ*S*)bis [ethanethiolato-κ*S*)]] oxorhenium(V). B) Hypothetical model of oxorhenium(V) complex 3 binding to the active site cysteine of cathepsin B. Adapted from Ref. [25].


**Table 1.** Inhibitory effect of metal compounds 1-6 against mammalian cathepsin B and cathepsin B-like cystein protease of *L. major*. Results expressed as IC50 (µM). [25]

infected with antimony resistant *L. donovani* was highly effective in reducing the organ parasite burden. The effect was mainly associated with generation of ROS and nitrogen species that could kill intracellular parasites.

#### *2.1.5. DNA-metallointercalators are not only to fight cancer*

As the metabolic pathways of kinetoplastid parasites are similar to those of tumor cells, it has been proposed that compounds which efficiently interact with DNA in an intercalative mode could also show anti-trypanosomatid activity. [26] Based on this hypothesis, some work has been carried out on design of metallointercalators as anti-leishmania drugs, including metals of pharmacological interest. It has been found that certain DNA intercalating drugs which have potent trypanocidal action, such as ethidium, acriflavine, and ellipticines, inhibit the DNA topoisomerases. These enzymes thus may represent another potential target for DNAintercalating trypanocidal metallodrugs.

DNA-intercalating metal complexes with potential leishmanicidal activity are generally made up of metals of known clinical application such as platinum, copper, silver and gold with planar polyaromatic ligands such as dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dpq (dipyr‐ ido[3,2-a:2',3'-h]quinoxoline). Figure 6. Copper complexes with dppz and dpq ligands, [Cu(L)n(NO3)2-n](NO3)n where L = dppz or dpq (Fig. 6) have shown activity against *Leishmania braziliensis* (causative of the muco-cutaneous mode of the disease), and it has been demon‐ strated that their action is related to their ability to interact with DNA. [Cu(dppz)2](NO3)2 was the most effective complex in this series, and the activity order was [Cu(dppz)2] (NO3)2>[Cu(dppz)(NO3)](NO3) > [Cu(dpq)2](NO3)2>[Cu(dpq)(NO3)](NO3). [27]

Among the most effective complexes is [Au(dppz)2]Cl3. This complex induced a dose depend‐ ent antiproliferative effect with a minimal inhibitory concentration (MIC) of 3.4 nM and lethal doses LD26 of 17 nM at 48 h. This strong *in vitro* activity against *L. mexicana* could be related to their ability to interact with DNA through an intercalative mode. Also, preliminary ultra‐ structural studies using transmission electron microscopy carried out with treated parasites at a sublethal concentration (IC7 = 0.34 nM for 24 h) showed polynucleated cells with DNA fragmentation and drastic disorganization of the mitochondria. [28]

Several years ago, a DNA metallointercalator (2,2':6'2''-terpyridine) platinum showed a remarkable antileishmanial activity, causing complete growth inhibition of *Leishmania donovani* amastigotes at 1 µM concentration.[29] This complex exploits simultaneous DNA intercalation of terpyridine and platinum(II) binding to the enzyme active site. The highest activity against *L. donovani* was found for the case of *p*-bromophenyl substituents in 4' terpyridine position, and NH3 as the ancillary hydrolysable ligand.[18]

Various DNA-intercalating organic ligands, have also been bound to vanadium ions. Although the potentiality of vanadium compounds in medicinal chemistry and medicinal applications has been extensively explored, work on vanadium compounds for treatment of some parasitic diseases of high incidence in human health has only arisen in a systematic way in recent years. [30] Benítez *et al.* obtained a series of oxovanadium complexes combining the aromatic planar polycyclic system 1,10-phenanthroline (phen) and tridentate salicylaldehyde semicarbazone derivatives as ligands, [VO(L1 -2H)(phen)] and [VO(L2 -2H) (phen)], where L1 = 2-hydroxyben‐ zaldehyde semicarbazone and L2 = 2-hydroxy-3-methoxybenzaldehyde semicarbazone. These compounds were active against *Leishmania* parasites showing low toxicity on mammalian cells. In addition, they showed cytotoxicicity on human promyelocytic leukemia HL-60 cells with

infected with antimony resistant *L. donovani* was highly effective in reducing the organ parasite burden. The effect was mainly associated with generation of ROS and nitrogen species that

**Figure 5.** A) Structures of metal complexes with antileishmanial activity via inhibition of parasite cysteine proteases: (**1**) diaceto [2-[(2-pyridinyl-κ*N*)methyl] phenyl-κ*C*] gold(I), (**2**) aceto [2,6-bis [(butylthio-κ*S*)methyl]-phenyl-κ*C*] palladium(II), (**3**) (*p*-methoxyphenylthiolato-*S*) [2,6-bis[(mercapto-κ*S*)methyl] pyridine-κ*N*1] oxorhenium(V), (**4**) (2(1*H*)-pyridinethiona‐ to-κ*S*<sup>2</sup>)[2,6-bis [(mercapto-κ*S*)-methyl] pyridine-κ*N*1] oxorhenium(V), (**5**) chloro [2,2'-(thio-κ*S*)bis [ethanethiolato-κ*S*)]] ox‐ orhenium(V), (**6**) (methanethiolato) [2,2'-(thio-κ*S*)bis [ethanethiolato-κ*S*)]] oxorhenium(V). B) Hypothetical model of

**Compound 1 2 3 4 5 6 Cat B** 1.29 0.40 6.51 0.12 0.0088 1.26 **L. Major cpB** 1.7 2.1 1.0 0.07 0.2 "/> 10

**Table 1.** Inhibitory effect of metal compounds 1-6 against mammalian cathepsin B and cathepsin B-like cystein

oxorhenium(V) complex 3 binding to the active site cysteine of cathepsin B. Adapted from Ref. [25].

could kill intracellular parasites.

protease of *L. major*. Results expressed as IC50 (µM). [25]

474 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

**Figure 6.** Structures of dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dpq (dipyrido[3,2-a:2',3'-h]quinoxoline).

IC50 values of the same order of magnitude as cisplatin. Their interaction with DNA was demonstrated and studied by different techniques, suggesting that this biomolecule could be one of the potential targets for activity either in parasites or in tumor cells. [31]

#### *2.1.6. Zinc sulphate against cutaneous leishmaniasis: The privilege of simplicity*

Since zinc sulphate administered orally has been used in the last decades in medicine and dermatology, [32] then its use as an oral therapy for cutaneous leishmaniasis has appeared recently as an important addition to the armamentarium of antileishmanial drugs.

*In vitro* sensitivities of *L. major* and *L. tropica* strains to zinc were reported to be higher than those to pentavalent antimony, and these data were confirmed on mice. Zinc sulphate was also delivered intralesionally with success in cutaneous leishmaniasis. It is been suggest‐ ed that oral zinc might not only affect directly to the parasite but also to macrophages function. Also it could have immunomodulatory effect (including T-lymphocytes), and help wound-healing. [33]

More recently, zinc sulphate was orally administered to Iraqi patients suffering from parasi‐ tologically confirmed cutaneous leishmaniasis. The species was not identified but it is known that only *L. major* and *L. tropica* are present in Iraq. This salt showed very promising cure rates (96.9%) against cutaneous leishmanaisis in a 45-days treatment with oral daily doses of 10 mg/ kg. After a comparative study between oral zinc sulfate and meglumine antimoniate in the treatment of cutaneous leishmaniasis, it was suggested that systemic antimonial injections in cutaneous leishmaniasis treatment were better than zinc sulphate but oral administration of zinc sulphate makes it cheaper, more convenient its consumption, and nearly close cure percentage to systemic meglumine antimoniate injections without serious side effects. However at the moment zinc sulphate therapeutic effects should be confirmed by a greater sample volume. [34] Nevertheless, reported studies suggest that antileishmanial effect of zinc may result, partially or entirely, from inhibition of enzymes that are necessary for the parasites' carbohydrate metabolism and virulence. [35]

#### *2.1.7. Selenium and the key role of antioxidants in disease*

IC50 values of the same order of magnitude as cisplatin. Their interaction with DNA was demonstrated and studied by different techniques, suggesting that this biomolecule could be

Since zinc sulphate administered orally has been used in the last decades in medicine and dermatology, [32] then its use as an oral therapy for cutaneous leishmaniasis has appeared

*In vitro* sensitivities of *L. major* and *L. tropica* strains to zinc were reported to be higher than those to pentavalent antimony, and these data were confirmed on mice. Zinc sulphate was also delivered intralesionally with success in cutaneous leishmaniasis. It is been suggest‐ ed that oral zinc might not only affect directly to the parasite but also to macrophages function. Also it could have immunomodulatory effect (including T-lymphocytes), and help

More recently, zinc sulphate was orally administered to Iraqi patients suffering from parasi‐ tologically confirmed cutaneous leishmaniasis. The species was not identified but it is known that only *L. major* and *L. tropica* are present in Iraq. This salt showed very promising cure rates (96.9%) against cutaneous leishmanaisis in a 45-days treatment with oral daily doses of 10 mg/ kg. After a comparative study between oral zinc sulfate and meglumine antimoniate in the treatment of cutaneous leishmaniasis, it was suggested that systemic antimonial injections in cutaneous leishmaniasis treatment were better than zinc sulphate but oral administration of zinc sulphate makes it cheaper, more convenient its consumption, and nearly close cure percentage to systemic meglumine antimoniate injections without serious side effects. However at the moment zinc sulphate therapeutic effects should be confirmed by a greater sample volume. [34] Nevertheless, reported studies suggest that antileishmanial effect of zinc may result, partially or entirely, from inhibition of enzymes that are necessary for the parasites'

one of the potential targets for activity either in parasites or in tumor cells. [31]

**Figure 6.** Structures of dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dpq (dipyrido[3,2-a:2',3'-h]quinoxoline).

recently as an important addition to the armamentarium of antileishmanial drugs.

*2.1.6. Zinc sulphate against cutaneous leishmaniasis: The privilege of simplicity*

476 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

wound-healing. [33]

carbohydrate metabolism and virulence. [35]

Selenium is an important and potent antioxidant in cells. Selenium compounds like selenites and selenates have strong inhibitory effects particularly on mammalian tumor cell growth. What is more, the nutritional deficiency of this essential trace metal may inhibit initiation and post-initiation phases of chemically induced mammary carcinogenesis and expression of some viruses, and it is important for optimal functioning of the immune system. [36]

Compounds of this metal have been reported to control human malaria if used in combination with vitamin E. [37] *In vitro* studies have shown that sodium selenite can inhibit *Leishmania donovani* growth although the mechanism of action is not clear yet. [38] Some authors have suggested that selenium has an important role in the pathophysiologic processes of cutaneous leishmaniasis, and that decreasing levels of this metal may be a host defense strategy of the organism against cutaneous leishmaniasis infection. Lack of selenium leads to a decrease of GSH-Px enzyme activity (it degrades H2O2), leading to increased amounts of hydroperoxides to kill protozoa as a host defense strategy.

#### *2.1.8. Triazolopyrimidines and their metal complexes: Mimicking the nature*

Triazolopyrimidines are purine analogues that have attracted much pharmaceutical interest during last decades. The most widely known derivative is the simple molecule Trapidil or Rocornal, a clinically used antiischemic and cardiatonic agent which acts as a platelet-derived growth factor (PDGF) antagonist and as a phosphodiesterase inhibitor. [39] This family of compounds have also found interesting applications as antipyretic, analgesic and antiinflammatory, herbicidal, fungicidal agents with about 200 relevant patents. For example, 2 arenesulfonamido triazolopyrimidines were tested as leishmanicides showing some of them similar *in vitro* activity than pentamidine against *L. donovani* (Figure 7).

**Figure 7.** Structures of triazolopyrimidine drugs: a) the anticoagulant drug Trapidil; b) a series of leishmanicidal deriv‐ atives.[39]

The biological activity of this family of organic compounds has led to investigating their coordination chemistry with the aim to develop new drugs with enhanced leishmanicidal activity and selectivity towards the parasites. Recently our group developed a series of transition metal complexes containing 1,2,4-triazolo[1,5-a]pyrimidines with high antiprolifer‐ ative activity and extremely high selectivity indexes (see section 3). Studies revealed that apart from being all of them active *in vitro* against both extracellular and intracelular forms of *L. infantum* and *L. braziliensis*, these compounds are not toxic towards the host cells and are effective at lower concentrations than the drug used as a reference, Glucantime.[40] In the following section, we will present a case study in which our latest findings of our research with triazolopyrimidine metal complexes are described.

#### *2.1.9. Nanoparticles: a promise for the future*

A vast array of intriguing nanoscale particulate systems capable of targeting different cells and extracellular elements in the body to deliver drugs, genetic materials and diagnostic agents have been developed in the last years. Currently, antiparasitic delivery via nanosized particles is at the forefront of the research in this area. Liposomes and polymeric nanoparticles are the best studied nanosystems for evaluating antileishmania activity of compounds like ampho‐ tericin B or pentamidine. [41]

But nanosized metal particles are also emerging as promising antiparasitic agents. In recent studies it was determined that metal nanoparticles possess effective antimicrobial activities due to their unique properties and large surface areas. Moreover, metal nanoparticles are capable of producing reactive oxygen species (ROS), which would be able to kill parasites and other infectious agents.

Use metal of metal nanoparticles against *Leishmania* has followed two main approaches:

**a.** As antiparasitic drug carriers. Nano-bioconjugate gold has recently been conceived as a stratagem against macrophage-infested leishmanial infections. One example is the functionalization of gold nanoparticles with the flavonoid quercetin, reported as one of the most powerful leishmanicidal among all plan flavonids tested so far. [8] This flavonoid inhibits synthesis of parasite DNA by inhibition of topoisomerase II mediated lineariza‐ tion of kDNA. Quercetin in addition can chelate iron and then limit availability of this metal for ribonucleotide reductase during DNA synthesis. On the other hand, gold nanoparticles as such can cause impairments in parasite oxygen metabolism.

Quercetin functionalized gold nanoparticles showed to be effective against *L. donovani* promastigotes and amastigotes. They were also effective against drug resistant strains with a very high selectivity index. A synergistic effect was considered by the authors as a possible reason for the higher activity of the nanoconjugate related to the free quercetin.

**b.** As antiparasitic administration nanoforms. Because of the larger surface area of nano‐ particles, they are more reactive and thus chemotherapeutic properties of a metal with antiparasitic activiy would be enhanced for its nanoform.

Selenium, for example, is a bioactive metal as it has antioxidant, cancer preventing, and antiviral activities. [37] Beheshti *et al.* prepared biogenic selenium nanoparticles, in this case, biosynthesized by *Bacillus sp*. MSh-1 and tested their *in vitro* and *in vivo* activity against *Leishmania major*. The particles showed antiproliferative activity against promastigote and amastigote forms of *L. major* and limited localized cutaneous leishmaniasis in animal model. These results present this kind of particles as novel therapeutic agents for treatment of the localized lesions typical of cutaneous leishmaniasis. However further studies are needed to investigate the mechanism of action of these Se NPs.[9]

Antimony sulfide NPs (Sb2S5), obtained also by green synthetic methods, proved to be effective on proliferation of promastigote forms of *L. infantum* and can induce apoptosis in promastigotes. [10]

The capability of metal nanoparticles to generate ROS and their potential use as leishmanicidal agents have also been explored. This is the case of silver nanoparticles, which have shown to be able to produce high amounts of ROS independently of the host cells. *In vitro* effects of AgNPs against promastigotes and amastigotes of *Leishmania tropica* were investigated. In order to increase the amount of ROS that are generated, AgNPs were irradiated with UV light which enhanced their antileishmanial effects without affecting host cells. [42]

#### **2.2. Strategies for the design of new metal-based leishmanicidal drugs**

To address the need for new, cost-effective metal-based leads for chemotherapy of leishma‐ niasis, different strategies of structure-based drug design have been applied so far. Four main strategies may be identified along revision in section 2.1:

#### *2.2.1. Antitumoral activity implies antiparasitic activity*

This strategy is based on the knowledge that highly-proliferative cells such as kinetoplastid parasites and tumor cells show metabolic similarities that lead in many cases to a correlation between antitrypanosomal and antitumor activities.[4] In this sense, use of metal complexes which have previously shown antitumoral activity, or synthesis of new metal complexes with ligands bearing activity could be a promising approach towards development of new agents against protozoa like *Leishmania*. A good correlation between antitumor and trypanostatic properties of several metal-based drugs has already been observed.

#### *2.2.2. Metal-drug synergism approach*

following section, we will present a case study in which our latest findings of our research

A vast array of intriguing nanoscale particulate systems capable of targeting different cells and extracellular elements in the body to deliver drugs, genetic materials and diagnostic agents have been developed in the last years. Currently, antiparasitic delivery via nanosized particles is at the forefront of the research in this area. Liposomes and polymeric nanoparticles are the best studied nanosystems for evaluating antileishmania activity of compounds like ampho‐

But nanosized metal particles are also emerging as promising antiparasitic agents. In recent studies it was determined that metal nanoparticles possess effective antimicrobial activities due to their unique properties and large surface areas. Moreover, metal nanoparticles are capable of producing reactive oxygen species (ROS), which would be able to kill parasites and

Use metal of metal nanoparticles against *Leishmania* has followed two main approaches:

nanoparticles as such can cause impairments in parasite oxygen metabolism.

reason for the higher activity of the nanoconjugate related to the free quercetin.

antiparasitic activiy would be enhanced for its nanoform.

investigate the mechanism of action of these Se NPs.[9]

Quercetin functionalized gold nanoparticles showed to be effective against *L. donovani* promastigotes and amastigotes. They were also effective against drug resistant strains with a very high selectivity index. A synergistic effect was considered by the authors as a possible

**b.** As antiparasitic administration nanoforms. Because of the larger surface area of nano‐ particles, they are more reactive and thus chemotherapeutic properties of a metal with

Selenium, for example, is a bioactive metal as it has antioxidant, cancer preventing, and antiviral activities. [37] Beheshti *et al.* prepared biogenic selenium nanoparticles, in this case, biosynthesized by *Bacillus sp*. MSh-1 and tested their *in vitro* and *in vivo* activity against *Leishmania major*. The particles showed antiproliferative activity against promastigote and amastigote forms of *L. major* and limited localized cutaneous leishmaniasis in animal model. These results present this kind of particles as novel therapeutic agents for treatment of the localized lesions typical of cutaneous leishmaniasis. However further studies are needed to

**a.** As antiparasitic drug carriers. Nano-bioconjugate gold has recently been conceived as a stratagem against macrophage-infested leishmanial infections. One example is the functionalization of gold nanoparticles with the flavonoid quercetin, reported as one of the most powerful leishmanicidal among all plan flavonids tested so far. [8] This flavonoid inhibits synthesis of parasite DNA by inhibition of topoisomerase II mediated lineariza‐ tion of kDNA. Quercetin in addition can chelate iron and then limit availability of this metal for ribonucleotide reductase during DNA synthesis. On the other hand, gold

with triazolopyrimidine metal complexes are described.

*2.1.9. Nanoparticles: a promise for the future*

478 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

tericin B or pentamidine. [41]

other infectious agents.

Perhaps one of the most popular strategies to develop new antiparasitic drugs consists on using an established antiparasitic drug as scaffold for the inclusion of a metal centre, either via direct coordination to the drug or by binding a metal complex. This way an enhancement of the drug pharmacological properties is pursued and resistance mechanism might be circum‐ vented. See section 2.1.2.

#### *2.2.3. Delivery nanovehicles*

In finding innovative parasite-specific formulations, established but deficient drugs might be optimised by using drug delivery systems, in order to enhance their efficiency and reduce negative side effects at relatively low cost. Antiparasitic efficacy of drugs already in clinical use might be significantly improved by the adaptation of a new drug formulation. Use of nanocarriers to deliver established metal-based drugs such as antimonials would be both costeffective and the quickest way to produce effective results. New drug formulations like liposomes for other drugs like amphotericin B (Ambisome) have been successfully developed for treating visceral leishmaniasis.

On the other hand, use of metal-based nanosystems as drug carriers, e.g. noble metal nano‐ particles, might provide additional advantages such as the possibility for diagnosis by imaging techniques and the combined effect of producing ROS as it is the case for silver. ROS can induce oxidative stress, DNA damage, alkylation of target proteins and eventually apoptosis of the parasite.

In order to inhibit *Leishmania* parasites with a ROS-based treatment, these oxygen derivatives must be produced in a physical way rather than in an enzymatic way that can be blocked by parasites. Metal nanoparticles are able to produce high amounts of ROS, as they are more reactive than the corresponding bulk metal (see example of AgNPs in section 2.1.9).

Nanocarriers also offer the possibility to specifically target the parasites by attaching appro‐ piate targeting molecules onto their surface. This way side effects to the host would be minimised. In addition, drug delivery vehicles such as nanoparticles allow prompt interactions with biomolecules present within as well as on the surface of the cell and may be tuned into different sizes to get the optimal uptake rate and blood circulation times of the drug.

#### *2.2.4. Specifically targeted drugs: Metal inhibitors of parasite enzymes and DNA-binders*

Recent advancements in molecular biology have identified a few parasite targets that are likely to be very sensitive to metal-based compounds. These targets usually are enzymes, some of them bearing free thiols at their active sites that manifest a high propensity to react with soft Lewis acids, i.e. metal ions such as Ag(I), Au(III) or Zn(II). Therefore these parasite targets will be susceptible to strong and selective inhibition by this kind of metals. This is the case of dithiol reductases like trypanothione reductase (T(SH)2), which have been shown to play a key role in the *Leishmania* metabolism (see Section 2.1.1) and therefore constitute primary targets for metal compounds. Cysteine proteases, such as cathpesin L-like or cathepsin B-like, are another example of proteins with thiol-containing active sites and thus responsive to inhibition by metal compounds. Inhibitors that would effectively target both types of cysteine proteases in *Leishmania*, while maintaining some selectivity versus homologous host enzymes, would be ideal drug leads.

Regarding DNA interaction, previous studies have shown that DNA-binding metal com‐ pounds such as cisplatin display antiparasitic activity. These findings along with the obser‐ vation that many antiparasitic drugs bind to DNA, have led to propose that in general every DNA interacting compound is potentially active against parasites.

Therefore DNA-intercalating molecules have been used as ligands to form metal complexes showing antiparasitic activity. Intercalating ligands are usually polyaromatic systems with two or more donor atoms in close disposition to "chelate" metal ions. These ligands would not only be responsible for interaction of the metal compound with DNA but also they could act as carriers of the metal, increasing interaction of complexes with DNA by minimizing exposure of metal to inactivating cellular nucleophiles such as thiols.
