Conformation-Guided Analogue Design Identifies Potential Antimalarial Compounds through Inhibition of Mitochondrial Respiration

Abstract

The synthesis of a 2-methyl-substituted analogue of the natural product, neopeltolide, is reported in an effort to analyze the importance of molecular conformation and ligand-target interactions in relation to biological activity. The methyl substitution was incorporated via highly diastereoselective ester enolate alkylation of a late-stage intermediate. Coupling of the oxazole sidechain provided 2-methyl-neopeltolide and synthetic neopeltolide via total synthesis. The substitution was shown to maintain the conformational preferences of its biologically active parent compound through computer modeling and NMR studies. Both compounds were shown to be potential antimalarial compounds through the inhibition of mitochondrial respiration in P. falciparum parasites.

Introduction

Polyketide natural products possess a number of structural features designed to influence molecular conformation, which are imparted through the evolution of their producing microorganisms and the associated polyketide synthase (PKS) genes, proteins, and pathways. We and others have shown that these highly-substituted molecules preferentially adopt a limited set of conformations complementary to their respective active sites which is essential for biological activity.¹ More recently, Wender and colleagues have shown that protein binding of multiple low energy conformers can help rationalize disparate biological activity in bryostatin-PKC interactions.² Therefore, it is clear that a detailed understanding of conformational preferences can guide the design of bioactive natural product analogues as an essential complement to classic structure-activity relationships.

In 2007, Wright and co-workers reported the isolation of a new polyketide natural product, neopeltolide 1, from a deep-water sponge off the north Jamaican coast.³ Neopeltolide consists of a 14-membered macrolactone containing a 2,4,6-trisubstituted tetrahydropyran and six stereocenters in total. It also possesses an oxazole-containing side chain identical to that of the structurally-related polyketide leucascandrolide A 2 (Figure 1). Subsequent biological studies found neopeltolide to be a potent inhibitor of both the fungal pathogen Candida albicans and a variety of cancer cell lines in vitro. Later work performed by the Floreancig group showed that neopeltolide is considerably less active towards a variant of the MCF-7 breast cancer cell line, suggesting that it demonstrates cell line selectivity rather than general cytotoxicity.⁴ Neopeltolide’s potent biological activity has attracted significant attention from the synthetic community.⁵ Our group’s previous work on neopeltolide consisted of a formal synthesis in 15 steps overall, utilizing ether transfer methodology, previous developed in our laboratory.⁶

Figure 1.

Structures of neopeltolide and leucascandrolide A.

Since our initial synthetic efforts, analogue programs from a number of laboratories have established a relatively comprehensive SAR profile for neopeltolide within cancer cell lines.^4,7–13 In summary, the side chain is essential for antiproliferative activity, as is the absolute configuration of the tetrahydropyran ring. The C9, C11 and C13 substituents are somewhat amenable to epimerization or omission with relatively minimal impact on biological activity, though the C13 n-propyl tail is required to achieve low nanomolar inhibition. Floreancig and co-workers also found that alterations to the C8–C9 region, particularly hydroxylation of C8, are generally well-tolerated.^4,10 Herein we report an effort to utilize conformation analysis to design a C2-substituted analogue, its preparation via X total synthesis, and the evaluation of the compound in relation to neopeltolide in an assay shown to identify potential antimalarial agents.

Results and discussion

The initial assignment of neopeltolide’s relative stereochemistry was complicated by multiple low energy solution conformers. NMR analysis provided NOESY cross peaks unlikely to fit a single conformation and thus, chemical synthesis was necessary to unambiguously confirm the relative and absolute stereochemical configuration of the natural product.^14,15 Preliminary conformational searches, performed in our lab, found two low energy conformations that could explain the much of the observed NMR data, which could potentially be controlled by the orientation of the s-cis ester linkage relative the plane of the macrocycle. We hypothesized that substitution at the C2 position could attenuate the observed rotation of this functional group through allylic strain, similar to what we observed in our work with C14-substituted epothilone analogues.¹⁶ It is worth mentioning that there are a number of structurally-related polyketide natural products that have 2-methyl substitution including dolastatin 19,¹⁷ ausiride A,¹⁸ and the glycosidically-diverse phorbasides¹⁹ and callipeltosides²⁰ (Figure 2). Not only do all of the them have a relative stereochemistry similar to that the analogue proposed here but also a higher oxidation state at C3. It is likely that this 2-substitution is beneficial to their stability due to a propensity towards these structures to undergo pyran-opening elimination as we observed in our total synthesis of the C2-unsubstituted macrolide, lyngbyaloside C.²¹

Figure 2.

Pyran-containing 14-membered Macrolides with 2-Methyl Substitution.

In order to assess the effect of C2-substitution on the conformational preferences of the macrolide ring, 50,000 step Monte Carlo search generated a library of conformers ordered by energy (see supporting information for details). The potential energy surface was graphically displayed by a series of polar coordinate maps, one for each torsional within the macrocycle, as we have described previously.²² Much to our surprise, the computationally predicted torsional preferences associated with C2-subsitution were not significantly affected by C2-methylation or stereochemical configuration (Figure 3). Regardless, since a late-stage macrolactone intermediate was readily available from our previous work, we proceeded with synthetic studies.

Figure 3. Conformational Analysis of Macrolactone Analogues.

Polar coordinate map presentation of computer-generated potential energy surface (energy vs torsional angle).

Macrolactone 3 was prepared as previously described from our laboratory.⁶ As the C2 protons are directly adjacent to the ester carbonyl we envisioned that a simple enolate alkylation could be achieved on the completed macrolide. High diastereoseletivity might be achievable if the intermediate enolate geometry was formed exclusively. This would likely be affected by thermodynamic constraints on of the twelve-membered ring which contains a number of torsions constrained in an anti-relationship. However, a diastereorandom alkylation would fortuitously provide two, stereocomplementary analogues for comparative studies.

After quantitative protection of the C5 hydroxyl as a TBS ether, exposure to 5 equivalents of NaHMDS and 6 equivalents of methyl iodide gave 4 as a single diastereomer in 82% yield (Scheme 1). Proton NMR showed the disappearance of one of the C2 protons and the introduction of a new doublet of quartets, with the doublet portion possessing a coupling constant consistent with a vicinal coupling constant consistent with an anti-periplanar orientation between the C2 and C3 protons (9.8 Hz). This indicates that the methyl group was installed pseudo-equatorial to the macrolide ring, which would be the (R)-2-methyl product. As predicted computationally, NMR-guided conformational analysis of the modified macrolactone 4 provided no evidence for an influence of the newly installed methyl group on the broader conformational preferences of the macrocycle (supporting information).

Scheme 1.

Stereoselective Enolate Alkylation on the Macrolactone.

The exclusive formation of 4 supports the exclusive formation of a single enolate geometry. Selectivity in enolate alkylation within macrolides has been elegantly described in the seminal work of Still.²³ Allylic strain on both sides of the intermediate enolate provides exposure of a single face leading to stereoselective peripheral alkylation.

Completion of a neopeltolide standard and 2-methylneopeltolide was accomplished by attachment of the oxazole sidechain using a strategy developed by Panek.¹⁵ We synthesized the oxazole side chain 7 using the route developed by the Leighton group in their synthesis of leucascandrolide A.²⁴ Coupling of the side chain to the neopeltolide macrolide 3 and the 2-methyl analogue 4 was accomplished using a Still-Gennari olefination to give neopeltolide 1 and the (R)-2-methyl analogue 6 (Scheme 2). Spectroscopic data of our synthetic neopeltolide matched data described by Panek and co-workers and the analogue 6 had the expected spectroscopic differences.

Scheme 2.

Sidechain Appended to (R)-2-methyl Neopeltolide

In addition to the biological assays establishing its antifungal and antiproliferative activity, Kozmin and co-workers have reported that neopeltolide, along with leucascandrolide A, specifically binds to complex III (cytochrome bc₁) of the mitochondrial electron transport chain (ETC).²⁵ The P.falciparum bc₁ complex is the target of the clinically used antimalarial drug, atovaquone, but the emergence of resistance has initiated research to identify alternatives.²⁶ Malaria remains a significant threat to global health resulting in approximately 200 million clinical cases and over 1 million deaths per year.²⁷ Thus, the Wirth lab recently developed an assay platform for target identification and mode of action studies of mitochondrial respiration in malaria parasites utilizing the Seahorse Bioscience XFe24 Extracellular Flux Analyzer.²⁸

Neopeltolide 1 and the 2-methyl analogue 6 were evaluated using this assay (Figure 4A). Within error, 2-methyl neopeltolide showed similar inhibitor activity (Figure 4B). Antimycin A, a known specific inhibitor of ETC complex III,²⁹ is shown as a positive control at a single dose (Figure 4C). No extracellular acidification rate (ECAR) reduction was observed with these compounds (data not shown). However, it is also known that reduction of oxygen consumption rate (OCR) by antimycin A can be restored by chemically reducing cytochrome c, and indeed, an increase in OCR was observed for all three compounds after injection of a reducing reagent; a mixture of N,N,Nʹ,Nʹ-tetramethyl-p-phenylene diamine dihydrochloride (TMPD) and ascorbate (Figure 4D). On the basis of these observations and our previous efforts, we can conclude that neopeltolide and its 2-methylanalogue are potential inhibitors of malaria parasites through inhibition of mitochondrial respiration.

Figure 4. Inhibition of Glycolysis and Mitochondrial Respiration in Malaria Parasites.

XFe24 analyzer monitoring of glycolysis and mitochondrial respiration in P. falciparum. Dose-responsive OCR reduction is observed for synthetic neopeltolide (A), synthetic 2-methyl-neopeltolide (B), and known cytochrome b inhibitor antimycin A (C). Freed schizonts were exposed to three doses of test compounds or antimycin A (1 μM) in unbuffered RPMI (100%: OCR values before compound addition; 0%: OCR values of antimycin A treatment). All data represent means ± SD (n = 3), and one representative analysis of two or three bioreplicates is shown. An increase in OCR after injection of a reducing reagent; N,N,Nʹ,Nʹ- tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and ascorbate (D).²⁸

Conclusions

Conformational analysis was utilized in the design of 2-methylsubstituted analogues of neopeltolide. The target was prepared by a diastereoseletive enolate alkylation on a late-stage intermediate and completion of the synthetic neopeltolide along with its 2-methyl congener was accomplished by addition of the oxazole sidechain. Surprisingly, C2-methyl substitution was shown to have a limited effect on conformational preferences and both compounds were found to be potent inhibitors of mitochondrial respiration in malarial parasites. The experiments confirm that neopeltolide and a designed analogue maintain their targeting cytochrome bc₁ in malarial parasites and have the potential to act as potent antimalarial compounds as a complement to their known antifungal and antiproliferative activity.