Symmetrical Pyridinium-Phanes and –Diazacyclophanes — Promising Heterocyclic Scaffolds for the Development of Anti-Leishmanial Agents

There is an urgent need for better drugs for a more successful fight against leishmaniasis, one of the most important neglected diseases caused by the parasite Leishmania . We have recently synthesized several symmetrical pyridinium compounds belonging to two dif‐ ferent series: bis-pyridinium and bis-quinolinium acyclic structures and bis-pyridinium diazacyclophanes derivatives. The first series of bis-pyridinium derivatives have been found to display activity against promastigotes and intracellular amastigotes of Leishma‐ nia donovani and Leishmania major , with EC 50 values lower than 1 μM. The majority of compounds show a similar behavior in both Leishmania species, being slightly more active against intracellular amastigotes of L. major . The series of bis-pyridinium diazacyclo‐ phanes can be considered as rigid analogues of the previous bis-cationic ones. The activi‐ ty of these compounds has also been evaluated against promastigotes and intracellular amastigotes of L. donovani and L. major . All the diazacyclophanes are more active against L. major , with EC 50 values of between 1 and 17 μM in intracellular amastigotes, and in some cases they present a higher selectivity index than the reference anti-leishmanial drugs such as amphotericin B and miltefosine. In conclusion, these bis-quaternary com‐ pounds represent promising candidates as potential therapeutic agents against leishma‐ niasis.


Introduction
Leishmaniasis is a major group of neglected tropical diseases caused by the protozoan parasite Leishmania. Currently it affects 12 million people in 98 countries, and around 350 million people worldwide are at risk of infection [1]. Leishmaniasis is responsible for a variety of pathologies that have been classified in three main clinical manifestations including cutaneous (CL), mucocutaneous (MCL), and visceral (VL) leishmaniasis, ranging from self-healing cutaneous lesions to fatal visceral infection [2].
All Leishmania species are digenetic parasites that exist as both insect vector (promastigotes) and mammalian forms (intracellular amastigotes). The digenetic life cycle of Leishmania consists of flagellated, motile, extracellular promastigote form that proliferates in the midgut of phlebotomine sand fly family vectors, which infect mammalian host and transform into the non-motile, intracellular amastigote form that resides in phagolysosomes of macrophages and other reticuloendothelial cells.
Since an effective vaccine against leishmaniasis is not available, chemotherapy is at present the only effective way to treat all forms of the disease. The recommended first-line therapies for leishmaniasis include pentavalent antimonials such as sodium stibogluconate and meglumine antimoniate, amphotericin B (AmB), paromomycin, and miltefosine ( Figure 1), all of which have different types of limitations including toxicity, price, efficacy, and emerging resistance [3], which emphasizes the importance of developing new drugs against leishmaniasis. Pentamidine [1,5-bis(4-amidinophenoxy)pentane] is an aromatic diamidine ( Figure 1) widely used for the treatment of sleeping sickness caused by Trypanosoma brucei [4]. It was used as a second-line drug against VL in cases of antimony failure, but its use against leishmaniasis is now limited to the treatment of some forms of CL in South America [5]. Pentamidine acts at the mitochondrial level of the parasite by accumulating within the mitochondria and binding to DNA, thus interfering with the replication and transcription [6]. Novel diamidine derivatives with improved pharmacokinetic properties have been under development in recent years [7,8].
New diamidine and choline-derivative dications have been developed recently in order to find new drugs with improved activity against leishmaniasis and lower toxicity [9][10][11][12] (Figure 2).
Chemistry is a science on which all the other sciences are based. An understanding of biology requires knowledge of chemistry. The majority of the leishmaniasis reviews are concentrating on the biology of the processes and very little on the chemistry. We would like to fill this gap and we will focus on the chemical structures that could be useful to the medicinal chemists working in this important area of research.
Here we present the anti-leishmanial activity of a set of symmetrical bis-pyridinium compounds with cyclic or acyclic structures. Both types of compounds can be named according to the IUPAC nomenclature for phanes, a method based on assembling names that describe component parts of a complex structure.

Symmetrical bis-pyridinium compounds
We have previously designed and synthesized a set of symmetrical bis-pyridinium compounds, which consist of a linker and two cationic heads which are 4-substituted pyridinium or quinolinium rings with cyclic or acyclic amino groups, as inhibitors of the human choline kinase (ChoK) ( Table 1), the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (ChoKα1, α2, and β). Specific inhibition of ChoKα has been reported to selectively kill the tumor cells. Ten symmetrical bis-pyridinium and bisquinolinium derivatives were tested for their ability to inhibit human ChoKα2, and VGP-118 and VGP-150 were identified as highly potent choline kinase inhibitors with EC 50 values of 80 nM. Kinetic enzymatic assays indicated a mixed, predominantly competitive, inhibition mechanism for these compounds. These novel compounds showed strong anti-proliferative activity (EC 50 of 1 μM) on the human breast cancer SKBR3 cell line [13].
In addition, these compounds can be considered as structural analogues of pentamidine in which the amidino moiety, which is protonated at physiological pH, has been replaced by a positively charged nitrogen atom as a pyridinium ring. In view of this structural resemblance and with the intention of identifying potential drugs against leishmaniasis, we analyzed the anti-leishmanial activity of these bis-pyridinium derivatives.

Susceptibility analysis in Leishmania lines
The anti-leishmanial activity of the ten choline kinase inhibitors was evaluated against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania major in order to identify the potential hits for further optimization. The cytotoxic effect of these compounds was also investigated on the human monocytic cell line THP-1, the host cell used in the assay with intracellular amastigotes. Selectivity indexes (SI) were calculated as the ratio of the EC 50 (the concentration of compound required to inhibit growth by 50%) for THP-1 to the EC 50 for intracellular amastigotes. Table 2 shows the results, where miltefosine and AmB were used as the reference anti-leishmanial drugs. Most assayed compounds exhibit a specific high activity against promastigotes and intracellular amastigotes of L. major, with EC 50 values between 0.09 and 0.42 μM in amastigotes, except for compounds VGP-106 and VGP-118 (EC 50 13.07 and 6.21 μM, respectively). With regard to L. donovani, all assayed compounds display EC 50 values below 1 μM in promastigotes, except compound VGP-138 (EC 50 2.11 μM).
Although these values are slightly higher in intracellular amastigotes, they are similar to those for the anti-leishmanial drug miltefosine [14].  were calculated by dividing the EC 50 THP-1 by that for intracellular amastigotes. Compound VGP-106 (grey color) was selected for further studies of the mechanism of action. Table 2. Anti-leishmanial activity and toxicity in THP-1 cells of symmetrical bis-pyridinium compounds. a Compound VGP-106 was identified as a representative compound that displayed a potent activity against L. donovani intracellular amastigotes. As the least cytotoxic of the set of compounds assayed for THP-1 cells, it was selected to further elucidate their mechanism of action in this protozoan parasite [14].

Drug susceptibility assay of L. donovani lines overexpressing CEK or EK
Considering that the Leishmania genome includes two homologous enzymes of human ChoK, namely, choline/ethanolamine kinase (CEK) and ethanolamine kinase (EK), we decided to study whether there is a correlation between their ChoK inhibitory activity and anti-leishmanial activity. These proteins can be overexpressed in L. Donovani promastigotes by transfecting the parasites with a plasmid encoding the Leishmania CEK (pXG-CEK) or EK (pXG-EK) genes [14]. The susceptibility of transfected parasites to compound VGP-106 was determined in both promastigotes and intracellular amastigotes. As can be seen from Table 3, there are no significant differences between the EC 50 values of parasites overexpressing CEK or EK enzymes compared to control parasites. These results suggest that the mechanism of action of this compound in Leishmania is independent of the aforementioned enzymes [14]. If this were not the case, overexpression of these enzymes would have resulted in an increase in the EC 50 value.

Symmetrical bis-pyridinium diazacyclophanes
Rigidification is a commonly used strategy to increase the activity of a drug or to reduce its side effects. A cyclophane is a hydrocarbon consisting of an aromatic unit (typically a benzene ring) and an aliphatic chain that forms a bridge between two non-adjacent positions of the aromatic ring.
We have synthesized a new family of symmetrical bis-pyridinium diazacyclophanes designed as cyclic analogues of previously reported acyclic bis-pyridinium derivatives, by cyclization through the exocyclic nitrogen atoms at position 4 of the pyridinium moiety via linker 2, which leads to the diazacyclophane targets ( Figure 3) [15]. These compounds have been evaluated against L. major and L. donavani. This new compounds are symmetrical bis-pyridinium derivatives which differ from each other in the upper and lower spacers. Four different spacers were used: two are phenyl-p-diylmethylene and phenyl-m-diylmethylene linkers, and the other two are aliphatic, such as the 1,5-pentanediyl and 3-oxa-1,5-pentanediyl moieties. At least one of the two spacers in every cyclophane is an aliphatic linker ( Table 4).
The final compounds were synthesized according to Scheme 1. Dipyridines 1 and 2 were prepared from commercially available diamines and 4-bromopyridine in the presence of phenol under argon atmosphere, as previously described [11]. A reaction involving phenol as proton donor, solvating agent, and source of phenoxide ion is envisaged, as outlined in Scheme 1 [16]. As a reaction medium, phenol reduces both the reaction time and temperature of the halogen-replacement reactions. The novel dipyridines (3 and 4) were prepared from commercially available pentane-1,5diamine and bis-2-(aminoethyl)ether, and following the same synthetic protocol previously reported [11].
Cyclophanes were obtained by cyclization of dipyridines 1-4 and the dibromide derivatives in acetonitrile, according to our reported procedures [11]. The reaction was carried out by adding 4 mM solution of the dibromide drop by drop to the dipyridine in acetonitrile at the reflux temperature of the mixture for a period of 10-12 days, which favors the cyclization step and avoids the intermolecular reaction [17]. In order to shorten the reaction time, microwave was used.  quinolinium cyclophanes [18][19][20] needed to be purified by tedious reverse-phase preparative HPLC because conventional purification methods failed to give analytically pure samples for biological testing, despite having been obtained under high-dilution conditions (1-2 mM). In our case, this represents a great advantage for the accessibility of such an interesting class of compounds (Scheme 2).

Anti-leishmanial activity
The final nine cyclophanes were tested as anti-leishmanial agents against promastigotes and intracellular amastigotes of L. donovani and L. major [15]. The results are shown in Table 5, where miltefosine and AmB were used as reference drugs.
All assayed compounds exhibit activity against promastigotes and intracellular amastigotes of L. major and L. donovani, being more active in L. major, with EC 50 values lying in the range 1 and 17 μM in amastigotes. Compounds VGP-310, VGP-318, VGP-334, VGP-340, and VGP-352 display EC 50 values below 1 μM against promastigotes of L. major, an activity 100-fold higher than that obtained in promastigotes of L. donovani. However, the differences in activity decrease in the amastigote forms, because some of these compounds are less active in amastigotes than in promastigotes of L. major and more active in amastigotes than in promastigotes of L. donovani. a Parasites were grown for 72 h at 28 °C (promastigotes) or 37 ºC (intracellular amastigotes) in the presence of increasing concentrations of compounds. THP-1 cells were grown for 72 h at 37 ºC, in the presence of increasing concentrations of compounds. Promastigote and THP-1 viability was determined using an MTT-based assay. Number of intracellular amastigotes was determined by nuclear staining. AmB, and miltefosine were used as standard anti-leishmanial agents.
Data are means ± SD of three independent determinations. b Selectivity indexes [SI] were calculated by dividing the EC 50 THP-1 by that for intracellular amastigotes. Compound VGP-318 (grey color) was selected for further studies of the mechanism of action. Table 5. Anti-leishmanial activity and toxicity in THP-1 cells of symmetrical bis-pyridinium diazacyclophanes a .
In general, from a structural point of view, compounds with two aliphatic linkers show better activity against promastigotes of L. major than compounds with an aromatic linker. However, the presence of an aromatic spacer increases the activity in intracellular amastigotes relative to the activity in promastigotes, except for VGP-310. This could be due to the higher lipophilicity of these structures that allowed a better penetration into THP-1 cells. Nevertheless, most compounds displayed higher activity in intracellular amastigotes than in promastigotes of L. donovani. Regarding the aliphatic linker, the presence of an oxygen atom in the linker did not involve significant differences in the activity. All diazacyclophanes exhibited very low toxicity against THP-1 cells (EC 50 values between 128 and 220 μM) and some of them evince a higher selectivity index than the reference compounds.
Compound VGP-318 was chosen as a representative compound to further investigate the mechanism of action of this new family of compounds [15]. This compound shows promising activity against intracellular amastigotes of L. major (EC 50 1.3 ± 0.3 μM), with a selectivity index (122) higher than those of AmB (51) and miltefosine (30). It is also the most active diazacyclophane derivative against intracellular amastigotes of L. donovani (EC 50 7.6 ± 0.2 μM).

Drug susceptibility assay of L. donovani lines overexpressing CEK or EK
As we have previously published that other bis-pyridinium diazacyclophanes were ChoK inhibitors and active anti-proliferative drugs [11], we performed a sensitivity test for VGP-318 in promastigotes and intracellular amastigotes of L. donovani overexpressing the Leishmania enzymes CEK or EK. The sensitivity for VGP-318 is similar in both promastigotes and intracellular amastigotes overexpressing CEK and EK versus control parasites ( Table 6). This result suggests that the anti-leishmanial activity of these compounds is not related to the CEK and EK enzymes [15].

Effect of VGP-318 on Leishmania metabolism
In order to investigate the anti-leishmanial mechanism of action of compound VGP-318, we focused the studies on the energetic metabolism of Leishmania promastigotes [15]. First, the effect of the compound on intracellular ATP levels was analyzed by the bioluminescence assay, which generates a luminescent signal proportional to the amount of ATP. In L. major, this assay showed a rapid decrease in the intracellular ATP levels which depends on the compound concentration ( Figure 4A). However, no effect was observed on L. donovani after incubation with 30 μM for 3 h ( Figure 4B). The decrease in the ATP levels may be caused mainly by an effect on the ATP synthesis or a release of the intracellular ATP due to the permeabilization of plasma membrane. However, under conditions that decrease 95% of the ATP relative to control (30 μM for 3 h), no sign of plasma membrane alteration was observed ( Figure 5), showing that the drop of free intracellular ATP is not due to disruption of the plasma membrane and suggesting that this may be due to a defect in the ATP synthesis.

Figure 5.
Compound VGP-318 does not alter the plasma membrane potential in Leishmania lines. Promastigotes were incubated with 30 μM of compound VGP-318 for 3 h and then treated with 2 μM of the specific plasma membrane potential probe DIBAC4(3) for 10 min at 28 ºC. Untreated parasites were used as control, and treatment with 10 μM CCCP was used as 100% depolarization of the plasma membrane potential. Data are means ± SD of three independent experiments. Significant differences were determined using Student's t-test (*p< 0.01).
In Leishmania, the ATP is mainly synthesized by mitochondrial oxidative phosphorylation [21]. The anti-parasitic activity of many drugs, such as pentamidine and miltefosine, is mediated by an alteration of the mitochondrial membrane potential (∆Ψ m ) [22,23]. To determine whether the intracellular ATP decay was associated with an effect of VGP-318 on the mitochondria, the variation of its electrochemical potential was monitored in parasites incubated with VGP-318 using the JC-1 fluorescent marker. L. major parasites incubated for 1 h with 0.2 μM of VGP-318 (conditions where there was 50% decay in the ATP levels) showed a significant decrease in JC-1 ratio compared with untreated parasites (Figure 6), evidencing a depolarization of the mitochondrial potential. However, in L. donovani promastigotes non-significant depolarization was observed after treatment with 30 μM of VGP-318 for 3 h (Figure 6). The depolarization of the ∆Ψ m in L. major promastigotes suggests that this compound may cause damage in the mitochondria, leading to a fall in the intracellular ATP levels and the death of parasites. a similar behavior in both species, being slightly more active against L. major amastigotes. All the cyclophanes are more active against promastigotes and amastigotes of L. major than L. donovani, although with a lower potency than the acyclic derivatives. However, in contrast to the variable toxicity of the acyclic compounds [14], all cyclophanes exhibit very low toxicity against mammalian cells THP-1 and some of them evince a higher safety margin than wellknown anti-leishmanial drugs such as AmB and miltefosine [15].
Although we have studied certain aspects of the mechanism of action of these compounds [14,15], it has not been determined any key target on which they are operating, which would be decisive for the rational design of new structures. Future work should be directed to carry out studies to elucidate the metabolism, pharmacokinetics, and mechanism of action of these compounds. On the other hand, it would be interesting to conduct a screening of a large number of symmetrical bis-pyridinium compounds that allows us to study structure-activity relationships. In any case, additional experiments are necessary for evaluating the toxicity and potency of these compounds by in vivo assays.

Note
Some parts of this chapter have been previously published in references [14,15].