Parallel solution synthesis of lavendustin analogs as antileishmanial agents

A concise parallel synthesis of 12 lavendustin analogs is described. Starting with differently substituted benzaldehydes, a two-step sequence involving reductive amination and N-benzylation accelerated by microwaves was performed to provide the compounds in excellent yields. The compounds have been assayed in vitro showing activity against Leishmania donovani , the causative agent of visceral leishmaniasis


Introduction
Leishmaniasis is a parasitic disease caused by protozoan haemoflagellates of the genus Leishmania, which has more than 30 known species, with 20 of those species being pathogenic towards humans. 1 There are three main forms of the disease: cutaneous, muco-cutaneous and visceral, the latter being the most severe form, fatal in almost 100% of the cases if left untreated.Visceral leishmaniasis (VL) has major public health implications in the Indian subcontinent (Bangladesh, India and Nepal), East Africa (Ethiopia, Kenya and Sudan) and Latin America (Brazil).It has been estimated that this diseases has 500,000 cases per annum and affects mostly the poorest and most marginalized communities living in primarily rural areas.Of the annual cases, 60% occur in about 109 districts of India, Bangladesh and Nepal, where about 150 million people are at risk of developing VL. 2 Visceral leishmaniasis is primarily caused by Leishmania donovani and affects the spleen, liver, lymph nodes and bone marrow.
Pentavalent antimonial compounds, sodium stibogluconate (Pentosam) and meglumine antimoniate (Glucantime) have been used for many decades as the main choice in antileishmanial chemotherapy. 3Some drawbacks associated with these drugs are their high toxicities, variable efficacy, intravenous administration in high dosages and an increasing number of reports of drug resistance, especially in endemic areas. 4O Recently, Pentamidine and Amphotericin B has been added as alternative drugs but, for different reasons, i.e. high prices and undesirable side effects and difficult availability, are limited. 5Miltefosine, a new drug for the treatment of visceral leishmaniasis went to Phase IV trials in public and private clinics in India few years ago, 6 and has been recommended for visceral disease in India and in Ethiopia, and for the cutaneous form of the disease in Colombia and Bolivia. 7Paromomycin, was originally isolated from Streptomyces krestomuceticus in the 1950s, and studied as an antibiotic.This compound has recently passed a multi-center phase III study in India, demonstrating high efficacy, affordability, and presenting itself as a safe treatment. 8These results prompted the Drug Controller General of India (DCGI) to approve paromomycin for the treatment of visceral leishmaniasis. 9Despite the encouraging success obtained thus far, there is a long way to go yet in order to find new and improved drug candidates, to add to the arsenal of oral chemotherapeutics against leishmaniasis and other neglected parasitic diseases. 10uring the last few years, different compounds against several forms of leishmaniasis have been isolated from natural sources or have been designed, synthesized and tested, -i.e. the anticancer tamoxifen, 11 chalcones, 12 bisphosphonates, 13 and polyamines 14 and also many pentamidine analogs. 15ubulin is a globular protein that assembles into microtubules in vivo and in vitro under the proper conditions.Microtubules play critical roles in mitosis, the maintenance of cell shape, cell motility, and organelle transport in eukaryotes.It has been postulated that different compounds have tubulin as a target, including drugs to treat cancer, fungal and helminthal infections.Among those compounds are anthelmintic benzimidazols, like oxfendazole and thiabendazole, and also some herbicidal dinitroanilines such as trifluralin and oryzalin.All of these have shown to possess a phylogenetic selectivity for tubulin.Werbovetz and collaborators, based on those precedents, have proposed and validated that tubulin can also be a target to design new antileishmanial agents. 16Over the last ten years an extensive work has been conducted preparing different libraries of antitubulin herbicidal analogs generating a group of promising drug candidates for antileishmanial chemotherapy. 17Lavendustin A is a natural metabolite that was first isolated by Onoda from Streptomyces griseolavendus in 1989.The initial reported activity of Lavendustin was protein−tyrosine kinase inhibition, but later this compound and its synthetic partial structure have been validated as tubulin polymerization inhibitors. 18s previously stated, tubulin is a validated target for the design of antileishmanial drugs and lavendustin is an inhibitor of tubulin polymerization, we envisioned that this natural product would be a privileged scaffold to design new synthetic analogs with potential activity against leishmaniasis.
Based on the structure of lavendustin, a series of analogs ABC were proposed (Figure 2).The analogs preserve the benzyl and aniline moieties, respectively named rings A and B. We chose to introduce diversity through substitutions on aromatic rings A and B and the number of methylenes groups between ring C and the central nitrogen.
As part of our program to design and synthesize new agents against parasitic diseases, we describe herein the synthesis of a series of 12 N,N-disubstituted anilines and their activities against Leishmania donovani promastigotes.

Synthesis
Based on the general ABC structure, a series of 12 compounds was prepared through 6 common intermediates.A concise synthetic strategy was designed that starts with a reductive amination of the benzaldehydes and the aniline completing the synthesis with the N-alkylation of the secondary amine intermediate.The substituents chosen for rings A and B were benzyloxy and methoxyl groups, in order to prevent oxidative degradation and to simplify the synthesis.
Commercial benzaldehydes and anilines were mixed with NaBH(OAc)3 and AcOH in dichloromethane 19 to produce 6 different substituted anilines.After purification, the necessary intermediates were obtained in 89 % average yield (Scheme 1).

Scheme 1. Reductive amination of substituted aldehydes A.
The last step involved an N-alkylation of the secondary amines AB.This reaction usually works efficiently with several soft nucleophiles.On the other hand, for some nitrogen-containing nucleophiles like aniline derivatives, the benzylation can be extremely slow (1-4 days) even at an elevated temperature.Initially, standard conditions were used, where the amines AB were treated with benzyl bromide in DMF using potassium carbonate as base at 100 o C for 18 hours.Under those conditions, it was observed that reaction mixture contained degradation products, while obtaining the expected tertiary amine ABC after purification only in moderate yields.To improve the yield and decrease reaction time, a microwave accelerated reaction was tested.This reaction was initially conducted under microwave irradiation using the same solvent and base that were used under normal heating, producing the expected acceleration of the reaction, decreasing the reaction time from 18 hours to 10-30 minutes, depending of the substrate.After that, the solvent was also studied considering the microwave energy to heat transferability according to the loss angle (tan ). 20Reactions using different solvents were tested (DMF, THF and toluene), being acetonitrile the one which performed better.With these conditions in hand, the 6 N-substituted anilines were alkylated using benzyl bromide and 2-bromoethyl benzene (phenethyl bromide), using potassium carbonate as base in acetonitrile.The purified 12 lavendustin analogs were obtained with an 85 % average yield.(Table 1).

Biological activity
The lavendustin analogs ABC were tested in vitro against Leishmania donovani promastigotes.The activities of the compounds are shown on Table 1.As shown, most of the compounds were active with the exception of A1B1C2 and A3B1C1 (the maximum concentration tested was 40 g/mL).The range of activity for the remaining ten compounds was narrow, being 6.5 g/mL the most active, and 40 g/mL the less active.The activity (determined as IC50) of the most active compound A1B1C1 was 6.5 g/mL when for the control antileishmanial drugs pentamidine was and 1.7 g/mL, and for Amphotericine B was 0.15 g/mL.Since antitubulin compounds have also shown promising activity against different apicomplexan parasites including the causative agent of malaria, 21 we decided to test the antimalarial activity of the compounds prepared against two different strains of Plasmodium falciparum, one sensitive and another resistant to chloroquine.(D6, Sierra Leone, sensitive; W2, IndoChina, resistant).Unfortunately none of the compounds showed activity below 4.75 g/mL, a value considered inactive compared to the control drugs artemisinin (IC50=5.3ng/mL for D6, IC50=4.9 ng/mL for W2) and chloroquine (IC50=9.5 ng/mL for D6, IC50 =148,5 ng/mL for W2).The cytotoxicity test toward Vero cells revealed that all the compounds were non-cytotoxic at 4.75 g/mL.
A more detailed analysis of the activity of these compounds can be done by grouping into families according to the building block C. (Table 2) In the C1 family, when the B1 derivatives are analyzed, it is clear that increasing the size of the phenol substituents decreased the activity.In the B2 group, this behavior is not observed, having a similar IC50 on average.Moving to the C2 family, similar structural-activity was observed, with activities for B1 analogs that decrease from A1 to A3 with B2 analogs with same IC50 values no matter the phenol subtituent.The only exception is A1B1C2 that was surprisingly inactive.In general terms, the introduction of a second methylene on the C building block had no effect on the antileishmanial activity, not producing any substantial change on the activities besides the effect on A1B1.In summary, the best activity was observed with dimethoxy substitution on the A ring, no oxygenated subtituent on the B ring and N-Benzyl group for the ring C.

Conclusions
A straightforward synthetic route was used to prepare a small collection of compounds introducing a microwave accelerated N-alkylation in very good yields.The antileishmanial activity of this family of 12 lavendustin analogs clearly validated this natural product as a valuable scaffold to prepare new antileishmanial drugs.Further studies will include the preparation of new collections of analogs with halogenated and non-polar substituents on the B ring and different patterns of dimethoxy substitution on the A ring.

Experimental Section
General. 1 H and 13 C NMR spectra were measured on a 400 MHz Bruker Avance DRX or a 300 MHz Bruker Avance II using CDCl3 as solvent.Chemical shifts were reported in ppm downfield from tetramethylsilane (δ) as the internal standard and coupling constants are in hertz (Hz).
Assignments of proton resonances were confirmed by correlated spectroscopy (HH COSY and Heteronuclear Single Quantum Coherence, HSQC) high-resolution mass spectra (HRMS) were recorded on a Micromass spectrometer with lock spray source or Bruker MicroTOF II.
Microwave accelerated reactions were performed in a CEM discovery microwave reactor.All the melting points were determined in open Pyrex capillaries with a Electrothermal 9000 melting point apparatus and are uncorrected.The reaction progress was monitored on precoated silica gel G or GP TLC plates.Spots were visualized under 254 nm UV light and/or by dipping the TLC plate into a solution of 2 mL anisaldehyde and 10 mL glacial acetic acid and 5 mL H2SO4 in 340 mL MeOH followed by heating with a heat gun.Column chromatography was performed with silica gel 60 (230-400 mesh) under a low pressure of nitrogen, using increasing EtOAc-hexane gradients as All the solvents (hexane, ethyl acetate, CH2Cl2, Et2O) were distilled prior to use.All reactions were performed under an atmosphere of nitrogen or argon using oven-dried glassware and standard syringe/septa techniques.CH2Cl2 was dried over P2O5.DMF was distilled from BaO.

General procedure for reductive amination
To a solution of aldehyde (1.0 equiv) in 10 mL of dichloromethane, amine (1.1 equiv), NaBH(AcO)3 (1.4 equiv) and finally acetic acid (1.1 equiv) were added in this order and the reaction mixture was stirred overnight at room temperature.Then, the reaction was quenched by addition of 5 % NaHCO3 (30 mL) and the layers were separated and the aqueous phase was extracted with ether (4 x 10 mL).Combined organic extracts were dried over Na2SO4 and concentrated.The products were purified by column chromatography.

General procedure for microwave N-alkylation
To a solution of amine (1 eq.) in dry acetontrile prepared inside the microwave reaction tube, anh.K2CO3 (1.3 eq.) and benzyl bromide (1.3 eq.) were added.The sealed tube was heated on the microwave reactor using the microwave settings describe below.The reaction mixture was poured 30 mL of brine extracted with dichloromethane (4 x 10 mL).Combined organic extracts were dried over magnesium sulfate and evaporated.The compound was purified by flash column chromatography.Microwave settings: Temp = 150 ºC, Power: 100 W, Run time= 10 min, Hold time = 20 min.Numbering by assignment on NMR-spectra

In vitro antileishmanial and antimalarial assays
Antileishmanial activity of the compounds was tested in vitro on a culture of Leishmania donovani promastigotes (Strain S1).In a 96 well microplate assay the compounds with appropriate dilution were added to the promastigotes culture (2 x 10 6 cell/mL) to get the final concentrations of 40, 8 and 1.6 g/ml.The plates were incubated at 26 C for 72 hours and growth was determined by Alamar blue assay. 22Pentamidine and Amphotericin B were used as the standard antileishmanial agents.All the analogs were simultaneously tested for cytotoxicty on VERO (monkey kidney fibroblast) cells by Neutral Red assay. 23IC50 value for each compound was computed from the growth inhibition curve.Antimalarial activity was determined in vitro on chloroquine sensitive (D6, Sierra Leone) and resistant (W2, IndoChina) strains of Plasmodium falciparum.The 96 well microplate assay is based on evaluation of the effect of the compounds on growth of asynchronous cultures of P. falciparum , determined by the assay of parasite lactate dehydrogenase (pLDH) activity. 24The appropriate dilutions of the compounds were prepared in DMSO or RPMI-1640 medium and added to the cultures of P. falciparum (2% hematocrit, 2% parasitemia) set up in clear flat bottomed 96 well plates.The plates were placed into the humidified chamber and flushed with a gas mixture of 90% N2, 5% CO2 & 5% O2.The cultures were incubated at 37C for 48 hours.Growth of the parasite in each well was determined by pLDH assay using Malstat® reagent.The medium and RBC controls were also set-up in each plates.The standard antimalarial agents, chloroquine and artemisinin, were used as the positive controls while DMSO was tested as the negative control.

Figure 2 .
Figure 2. Lavendustins A and B, and general structure of the designed analogs.

Table 1 .
Antileishmanial activity of ABC products

Table 3 .
Microwave N-alkylation of AB secondary amines