Simplified tricyclic model of quassinoids with in vitro antiparasitic activity

This is a report of the synthesis of BCD ring partial analogs of quassinoids, and the evaluation of their biological activity so as to elucidate minimal functional requirements as potential antimalarial and leishmanicidal agents.


Introduction
Many infections are caused by protozoan parasites.2][3][4] Malaria is the world´s second major killer after tuberculosis.The most deadly of the four Plasmodium species that causes human malaria is the protozoan parasite Plasmodium falciparum. 5he development of new compounds for the treatment of these diseases is based on the available drugs which are few, inadequate in terms of efficiency, and often toxic.The synthesis of new chemotherapeutic agents requires a suitable selection of the molecular target that will be used in the compounds design.
Natural products have been and still are the main source of structurally diverse compounds as well as the main source of small molecule drugs. 6amaderine X chaparrinone orinocinolide quassin Quassinoids are complex natural triterpenes with a high degree of oxygenation.Their diverse biological activities, such as antitumoral, antifeedant, insecticidal, antimalarial, among others, motivated investigation related to their organic chemistry and pharmacology. 7Many of these natural products, isolated from plants, such as chaparrinone, 8 dihydroxyeuricomanol, 9 samaderines X and Z, orinocinolide, excelsite, and quassin, [10][11][12][13] (Figure 1), have shown antimalarial activity.Owing to the wide spectrum of biological activities shown by this family of natural products, we postulate that their structure can be used as a privileged scaffold to prepare new bioactive compounds.Recently, we have been working on the search of new synthetic compounds with antiparasitic activity.Using 1,4-diene 2 as key intermediates in synthetic sequences, 14 we have been able to prepare different saudin model compounds and A/B rings of ouabain.Following the same strategy, we envisioned that the same intermediates could also be useful to prepare simplified quassinoid analogs.In particular, in this opportunity, we were able to apply our experience on 1,4-diene to obtain tricyclic acetals and lactones as mimetics of the B/C/D cyclic system of the natural product and study if that could fill the minimum structural features to produce activities in the antiparasitic assay.The proposed structures hold different positions where it should be possible to introduce diversity over rings C and D and also on the axial substituent of the ring fusion, Scheme 1.The products can be prepared following our reported procedure 14d that has the 1,4-dienol 2 as key intermediate and that is the product of the reductive alkylation of α-tetralone, followed by the selective reduction of the ketone.Scheme 1. Retrosynthetic analysis for the target compounds prepared.
The selective ketone reduction of 1 leads to the dienols 2a-b.α-alcohols were obtained when L-Selectride  was used; while β-alcohols were generated with NaBH4, MeOH, at -78 o C.These dienols were converted to the corresponding bromoacetals 7 which have been the starting materials for obtaining the tricyclic compounds 4 and 8 by radical cyclizations with NaBH3CN, AIBN, and Bu3SnCl in tBuOH.The presence of the products 8 has demonstrated that the reactivity of the C3a-C4 double bond towards radical attack is directed by the stereochemistry of the alcohol.Therefore the reaction gives exclusively six membered rings via a regioselective 6exo-trig closure. 14,15This outcome was rationalized assuming that the conformer that leads to a chair-axial-chair transition state is responsible for the before mentioned stereocontrol.
We have been able to extend the cyclization reaction toward a cyanation of the incipient radical using tBu-isocyanide as intermolecular radical trap.Thus, the reaction performed in a ratio of tBuNC : substrate 7a of 10:1, led stereoselectively to the β-cyano 10.In this case the cyclization followed the same ring closure stereochemical behavior, and the radical intermediate reacted with the isocyanide by the opposite face of the new formed bond.
The conversions from acetals to lactones 10 → 11 and 8 → 9 have been accomplished employing the hydrolysis reaction with 6M HCl, THF/H2O, 40 o C and further oxidation of corresponding acetals with Jones reagent in acetone.
These prepared substructures of quassinoids were screened in vitro for their antimalarial and antileishmanial activities (Table 1).
As a result of this screening only the compound 8a showed activity on the chloroquine sensitive Plasmodium falciparum strain (D6 clone) with an IC50 value of 550 ng/mL and 870 ng/mL on the chloroquine resistant (W2 clone).Under the same experimental conditions chloroquine exhibited an IC50 value lower than 9.7 and 148.5 ng/mL against D6 and W2 clones respectively.
The presence of an acetal group at C2 and a methyl group at C9b on D ring of 8a (R = Me, R 1 = OEt) seems of pivotal importance, whereas a small structural variation of the acetal function 8c (R 1 = Ac) or 8d (R 1 = Bn) induced the total loss of antimalarial activity.Similarly, the relative lactone 9a obtained from the oxidation of 8e was equally ineffective against Plasmodium falciparum strains.Conversely 8d (R 1 = Bn) that has proven to be ineffective against malaria, displayed activity against L. donovani with an IC50 value of 13 µg/mL.The incorporation of larger acetal substituents 8d (R 1 = Bn) at C2 had a better result than the acetyl group in compound 8c.The lack of antileishmanial activity of product 8c could be a consequence of the ester hydrolysis by an esterase producing the hemiketal 8e that is inactive, but that hypothesis should be studied further.The comparison of the antiparasitic activity developed by 8a with the inactive compound 8b against Plasmodium falciparum strains, shows an interesting input on the requirement of the oxygen function on the E ring, Figure 1.The introduction of the methoxy methyl group at C9b which mimics a tetrahydrofuran, suggested that E ring might not be needed for bioactivity of this core of quassinoids.
In general, the antimalarial activity cannot be ascribed to the C5-carbonyl group of the α,βunsaturated ketone 6 (C5-C=O, R = Me) of the C-ring because it was an activity inhibitor structural feature.The structural modifications introduced at C6a, C6 (4a, 4d), and at C4 (10)  along with its corresponding lactone 11 showed no contribution for the activity against Plasmodium falciparum and Leishmania donovani.The transformation of 8a into 9 gave a lactone group on D-ring, present in bruceantin 16 and its related compounds already described as antimalarials.9a was active for L. donovani with an IC50 of 19 µg/mL, but it was inactive for antimalarial activity on Plasmodium falciparum.All these synthetic compounds were noncytotoxic to the Vero cells at the maximum concentration tested (4.75 g/mL).This result is not negligible due to one of the main drawbacks of quassinoids is their cytotoxicity although they have high potency as antimalarial compounds.

Conclusions
We synthesized two series of compounds (R = Me, R = CH2OMe) as substructures of quassinoids for the understanding of structure-antiparasitic activity relationships.This exploratory study allowed us to validate the method in order to discover the minimal tricyclic structure having bioactivity.In this way the above-mentioned compounds are the starting point to obtain new analogs for improving presented activities.

Experimental Section
General.All chemicals and solvents are commercially available and were used after distillation or treatment with drying agents.Melting points are uncorrected. 1H (300 MHz) and 13 C NMR (75.13 MHz) spectra were recorded in CDCl3 (with TMS for 1 H and chloroform-d for 13 C as the internal standard).Numbering of compounds is according to tricyclic structure as we have shown in Scheme 2.
General procedure for bromoketal generation 1,2-dibromoethyl ether (1.1 eq) was added to a solution of the alcohol 2b (1.1 eq) with N,Ndimethylaniline (1.5 eq) in dichloromethane (10 mL) at 0 °C.The solution was stirred at room temperature over 3 h and then 1,2-dibromoethyl ether (1.1 eq) and N,N-dimethylaniline (1.5 eq) were added.The mixture was stirred overnight and cold sat.NaHCO3 (20 mL) was added.The reaction mixture was extracted three times with dichloromethane (3 × 15 mL), water (1 × 10 mL) and dried (Na2SO4).A stirred solution of the dienone (1 mmol) in THF (5 mL/mmol) at -78 °C was treated with 2 mmol of a 1.0 M solution of L-selectride ® in THF.After 1.5 h 2 mL/mmol of 1.0 M aqueous solution NaOH was added, followed by of 0.6 mL/mmol 30% H2O2.The heterogeneous solution was stirred for 2 h at 45 °C.The combined organic extract was washed with water (30 mL), dried (Na2SO4), filtered and the solvent removed under reduced pressure.The crude residue was purified by column chromatography.

In vitro antimalarial and antileishmanial assays
Antimalarial activity of the compounds 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. 18The 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 37 °C for 48 h.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.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 leishmania promastigotes culture (2 × 10 6 cell/mL) to get the final concentrations of 40, 8 and 1.6 µg/mL.The plates were incubated at 26 °C for 72 h and growth of leishmania promastigotes was determined by Alamar blue assay. 19Pentamidine 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. 20IC50 value for each compound was computed from the growth inhibition curve.

Table 1 .
Antiparasitic activities of synthetic substructures of quassinoids NA: inactive, NC: no cytotoxicity, NT: not tested; SI-Selectivity index= IC50 against vero cells/IC50 against parasite; The highest concentration tested for antimalarial activity and vero cell cytotoxicity is 4760 ng/mL.The highest concentration tested against L. donovani promastigotes is 40 μg/mL.*Values are mean + S.D. of three observations.