Biological evaluation and structure activity relationship of 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole derivatives as anti-leishmanial agents

Graphical abstract Anti-leishmanial activity EC50 (µM). L. infantum: promastigotes 1.59 and axenic amastigotes 1.4. L. donovani: promastigotes 0.9, axenic amastigotes 1.4 and intracellular amstigotes 1.3.


Introduction
Leishmaniasis is caused by intracellular protozoan Leishmania spp parasites and is considered as one of the most neglected tropical diseases. Among various clinical forms of leishmaniasis, visceral leishmaniasis is the most severe, affecting internal organs like bone marrow, liver and spleen. VL is also known as kala-azar and is lethal if untreated in over 95% of cases. VL caused by Leishmania donovani and Leishmania infantum [1,2]. Leishmaniasis control is majorly dependent upon chemotherapy that includes decade old drugs, due to no effective vaccines. Antimonial drugs such as sodium stibogluconate and meglumineantimoniate have been used for the last 70 years, however increasing incidence of resistance has been reported [3]. Anti-leishmanial drugs such as amphotericin B, diamidine, pentamidine and paromomycin has been restricted by cost, toxicity and resistance [4,5]. Miltefosine is originally developed as anti-cancer agent, has been approved by FDA for leishmaniasis treatment [6]. Miltefosine has restricted use in pregnancy and has high risk of resistance due to long half-life (150 h). Clinical efficacy of miltefosine has been decreased in countries like India, where it is used extensively [7]. Based upon these collective facts, there is an increased need of new anti-leishmanial agents with good therapeutic profiles.

Cytotoxicity evaluation
Initially all these reported β-carboline derivatives were evaluated for cytotoxicity against HeLa cell lines at 500 μM by an Alamar blue assay, which showed most of these compounds were non-toxic to HeLa cells at tested concentration. Compounds which showed cytotoxicity against HeLa cells were further evaluated to determine cytotoxic concentration (CC 50 ) value. Among these reported β-carboline derivatives, compound 7o and 7p showed cytotoxicity (CC 50 29.7 and 27.3 μM, respectively) against HeLa cells.

Anti-leishmanial screening
Anti-leishmanial activity of these analogues was determined by evaluating their inhibition activity against both promastigote, amastigote forms of Leishmania infantum and Leishmania donovani strains. Compounds were screened against promastigote forms of the Leishmania strains to determine their effective concentration (EC 50 ) values. Finally, compounds which exhibited potent inhibition of promastigote (EC 50 < 20 µM) were further evaluated against amastigote forms of respective strain using established axenic and intracellular amastigote assay methodologies. Anti-leishmanial drugs miltefosine and pentamidine were used as standards for comparison purpose.
Structure Activity Relationship (SAR). Among these piperzinyl-β-carboline derivatives, un-substituted proto compound displayed significant anti-leishmanial activity against both promastigote and amastigotes of L. infantum. A series of β-carboline derivatives with different ortho-para and meta directing groups on various positions of phenyl were studied to determine their effect on anti-leishmanial activity. Substitution of ortho-para directing methyl group on ortho position of phenyl ring has increased anti-leishmanial activity significantly against both forms, whereas methyl substitution on para position has reduced the activity. Ortho-para directing groups such as methoxy and chloro on para substitution strongly enhanced the anti-leishmanial activity, while ortho substitution lessen the activity and meta substitution drastically decreased the activity. Meta directing nitro and fluoro group substitution on para position decreased the activity considerably and fluoro substitution on ortho position severely decreased the activity. Dual substitution on ortho and meta position with chloro declined the activity. Moreover, phenyl ring replacement with pyridine and benzyl ring has adversely affected the anti-leishmanial activity. SAR studies suggested that, meta position of phenyl ring is highly susceptible, substitution with any group drastically reduced the activity. Ortho-para directing methoxy, chloro groups on para position and methyl group on ortho position favored the activity. Phenyl ring replacement with heterocyclic rings is not recommended for anti-leishmanial activity against L. infantum.
Structure Activity Relationship (SAR). Structure activity relationship study suggests that, substitution or replacement of phenyl ring has significant effect on anti-leishmanial activity as well as cytotoxicity of these analogues. Among these analogues, un-substituted phenyl derivative displayed significant inhibition of promastigotes, axenic amastigote and intracellular amastigotes of L. donovani. Ortho-para directing methyl group substitution on ortho and para position enhanced the potency against axenic amastigotes and intracellular amastigotes of L. donovani significantly, while on para substitution anti-promastigote activity altered marginally. Methoxy, chloro group substitution had strong impact on anti-leishmanial activity of these analogues, position of substitution is vital in their inhibition activity. Ortho substitution of methoxy group showed excellent increase in inhibition potency against promastigotes and both amastigote forms of L. donovani. While methoxy substitution on ortho position slightly increased the anti-leishmanial activity, meta substitution resulted in drastic decline in activity. Chloro substitution on para and meta position has significantly increased the inhibition potency against tested forms, while on ortho substitution has marginally affected the activity. Meta directing nitro and fluoro substitution on para position haven't showed any considerable effect on inhibition potency of these analogues. However, fluoro substitution on ortho position and chloro di-substitution (ortho and meta) significantly decreased the anti-leishmanial potency. Replacement of phenyl ring with benzyl ring has significantly enhanced the activity. Even though pyridyl replacement resulted in increased anti-leishmanial activity, it also enhanced the cytotoxicity of these analogues. SAR studies suggested that, ortho-para directing methoxy group on para position, chloro group on para and meta position and benzyl replacement was recommended for anti-leishmanial activity of these analogues against L. donovani.
Through the present study, we have identified new lead molecules with potent anti-leishmaninal activity against L. infantumand L. donovani. Structure activity relationship studies would be utilised to optimize the lead molecules to generate clinically effective against both drug sensitive and resistance strains of L. infantumand L. donovani.  Exact mechanism of action of these molecules for thier potential activity is needs to be evaluated. We hypothesize based upon literature reports, that these molecules may exhibit the observed potent activity through interaction with nucleic acids.

Conclusion
In summary, we have successfully applied molecular hybridization technique to design piperazinyl-β-carboline-3-carboxamide derivatives as anti-leishmanial agents. Designed molecules were synthesized and evaluated for anti-leishmanial activity against L. infantum and L. donovani. Analogues displayed significant inhibition of promastigote forms (EC 50 < 20 μM) were further evaluated against amastigote forms of respective species. Most of these reported analogues displayed potent anti-leishmanial activity (promastigote and amastigote forms) against the both tested strains with better selectivity index. Compounds such as, 7d, 7g and 7c wereexhibited potent inhibition of promastigote (EC 50 1.59, 1.47 and 3.73 µM respectively) and amastigote (EC 50 1.4, 1.9 and 2.6 µM respectively) of L. infantum than standard drugs miltefosine, pentamidine. SAR studied suggested that, ortho-para directing methoxy, chloro groups substitution on para position as well as methyl group on ortho position favored the anti-leishmanial activity against L. infantum.
More interestingly, most of these compounds showed potent antileishmanial activity against L. donovani. Especially, compound 7d displayed potent inhibition of L. donovani promastigotes, axenic amastigotes and intracellular amastigotes with EC 50 0.91, 0.9 and 1.3 µM respectively. Analogues 7n, 7g and 7h were exhibited potent inhibition activity against all tested forms of L. donovani. SAR studies suggested that, ortho-para directing methoxy group on para position, chloro group on para and meta position were recommended for anti-leishmanial activity against L. donovani. Although, pyridyl replacement has increased the inhibition activity as well cytotoxicity and benzyl replacement has enhanced the anti-leishmanial activity with better selectivity.

Chemistry
All solvents and reagents purchased from Sigma or Merck companies are used as received without further purification. Solvent system used throughout the experimental work for running Thin Layer Chromatography (TLC) was ethyl acetate and hexane mixture (6:4) in order to monitor the reaction. Column chromatography was performed using silica gel (100-200 mesh, SRL, India) as stationary phase and mixture of ethyl acetate and hexane as mobile phase. Melting points are uncorrected and were determined in open capillary tubes on a Precision Buchi B530 (Flawil, Switzerland) melting point apparatus containing silicon oil. IR spectra of the synthesized compounds were recorded using FT-IR spectrophotometer (Shimadzu IR Prestige 21, India). 1 H NMR spectra were recorded on a Bruker DPX-400 spectrometer (Bruker India Scientific Pvt. Ltd., Mumbai) using TMS as an internal standard (chemical shifts in d, ppm). Elemental analysis was performed on Vario EL III M/s Elementar C, H, N and S analyzer (ElementarAnalysensysteme GmbH, Germany). The ESMS were recorded on MICROMASS Quattro-II LCMS system (Waters Corporation, Milford, USA).

General procedure for the preparation of 7
To a stirred solution of (6) (0.29 g, 0.001 mol) in dry THF, HOBt (0.16 g, 0.012 mol) and EDCI HCl (0.23 g, 0.012 mol) were added and continued stirring for 30 min. To the reaction mixture, substituted phenylpiperazine (0.001 mol) was added under ice cold temperature and the reaction mixture was further stirred at room temperature for 6 h. After completion of reaction as monitored by TLC, solvent was evaporated under vacuum. Reaction mixture was extracted with ethyl acetate (2 × 20 mL), collected organic layer was dried over Na 2 SO 4 and concentrated under vacuum to get (7a-p).

Cytotoxicity assay
HeLa cell cytotoxicity studies were carried out as described previously. Briefly, the cells were cultured in DMEM supplemented with 10% fetal calf serum and 2 mM L-glutamine. Cells were plated at initial cell concentration of 2 × 10 4 cells/well and incubated with the compounds for ∼65 h prior to addition of Alamar Blue solution for further 5 h [36].

Intra macrophage L. donovani (amastigote) assay
THP-1 (human monocytic leukemia) cells were a kind gift from Dr Susan Wylie (Dundee) and maintained in minimal essential medium plus 10% (vol/vol) FBS. An intracellular Leishmania assay using LdBOB amastigotes were performed utilizing PMA differentiated THP-1 cells infected overnight with axenic amastigotes, prior to compounds being added and incubated for further 72 h, subsequent microscopy-based readout was used to determine EC 50 values [38]. Briefly, THP-1 cells (20,000 per well, 200 µL) were plated into 96 well plates in presence of 10 nM PMA and incubated at 37°C in 5% CO 2 atmosphere for 75 h. The adhered cells were then washed with phosphate buffered saline supplemented with 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% (w/v) bovine serum albumin and using LdBOB amastigotes added to all wells at a multiplicity of infection of 5 (100,000 amastigotes per well) and incubated for 18 h. Any remaining extracellular amastigotes were removed and the adhered cells were washed with the same supplemented PBS solution as above. Pre-aliquoted compounds in a serial dilution were added from another plate, such that the total well volume was 200 μL and incubated for further 72 h. Cells were fixed with 100% methanol, stained with Giemsa and examined microscopically. Number of intracellular amastigotes (∼200 macrophages per well) were determined and the percentage infection was established compared to an untreated control (100%) allowing EC 50 values to be calculated [38].

Conflicts of interest
There are no conflicts of interest declared by the authors.