Synthesis of Bisquinolines, Napthythyridines and Pyronaridine to Elucidate the Mechanism of Antimalarial Drug Action

In recent years Plasmodium falciparum has become resistant to many clinically used antimalarial agents. This reinforces the need to develop Novel antimalarial drugs. This study details the synthesis of improved antimalarials based on bisquinolines. The synthesised molecules were studied for their potential activity, both in silico and in-vitro and in an attempt to understand the structural determinants of their antimalarial activity. Further in-vivo studies were performed on a variety of quinoline based compounds to determine their possible mode/s of action, possibly associated with their interaction with haem and haem derived complexes. The synthesis of various bisquinolines, and consequently, the development of an extra thermodynamic pharmacophore model that has allowed identification of a potent, soluble, orally bioavailable antimalarial bisquinoline, meta-quine (N,N-bis(7-chloroquinolin-4-yl)benzene-1,3-diamine) (dihydrochloride), which is active against Plasmodium berghei in-vivo (oral ID50 25 μmol/kg) and multidrug-resistant Plasmodium falciparum K1 in-vitro (0.17 μM). Meta-quine shows strong affinity for the putative antimalarial receptor, haem at pH7.4 in aqueous DMSO. In an effort to find compounds superior to meta-quine, the de-halogeno-amination/SNAr Displacement reaction in N-methylpyrollidinone was used to construct both a fluorinated analogue of RO 47-7737, and an inactive 4-aminoquinoline/ mefloquine hybrid, and observed lack of antimalarial activity of this compound against Plasmodium berghei infection in mice. The latter Structure determined by X-ray crystallography, revealed hydrogen bonding to solvent (methanol). The site and order of protonation of these quinolines was investigated using quantum mechanics calculations (Courtesy of Prof Drew, Reading University). Results suggested that protonation was impeded at the endocyclic nitrogen in mefloquine due to electronic rather than steric factors and the molecule was protonated preferentially at the piperidine side chain in Mefloquine. In contrast, both mono- and bis-4-aminoquinolines were protonated at the endocyclic quinoline nitrogen. This suggested that trifluoromethyl groups buttressing the endocyclic quinolinyl nitrogen was detrimental to 4-aminoquinoline receptor binding, but not quinoline methanols; suggesting that these two classes of molecules act by different mechanisms/binding modes within the acidic vacuole of Plasmodia. 4-Hydroxy-1,5-naphthyridine (SN 13,639), a key intermediate in the production of certain azasubstituted antimalarials, is normally synthesised by a multistep route comprising cyclization, de-esterification and decarboxylation. An unexpected and direct formation of 4-hydroxy-1,5-naphthyridine by Gould-Jacob cyclization in refluxing diphenyl ether was found. This occurred under microwave conditions, to produce ethyl 4-hydroxy-1,5-naphthyridine-3-carboxylate, which spontaneously underwent a rare retro-fries rearrangement to form 4-hydroxy-1,5-naphthyridine, which also identified the transition state involved. The product was unambiguously characterised by X-ray crystallography and 2D-NMR experiments. Various adducts and solvated forms were examined by DFT experiments. An examination of the large downfield shifts observed for the molecule in TFA-d were rationalised by proposing protonation at N-5 accompanied by the formation of a novel hydrogen bonded complex with the solvent. Similar interactions may occur between naphthyridines and the acidic side chain of the haem receptor. Although antimalarial activity of 4-hydroxy-1,5-naphthyridine is weak against multi-drug resistant strains of Plasmodia, the compound allows rapid access to various known antimalarial naphthyridines and could act as fluorescent probe of drug action. Pyronaridine (4-[(7-chloro-2-methoxybenzo[b][1,5]naphthyridin-10-yl)amino]-2,6-bis[(pyrrolidin-1-yl)methyl]phenol). The first European synthesis of pyronaridine 4-(7-Chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-ylamino)-2,6-bis-pyrrolidin-1-ylmethyl-phenol (15), a potent antimalarial, from commercially available starting materials, namely 2,4-dichlorobenzoic acid and 2-methoxy-5- aminopyridine. The copper mediated Ullmann synthesis of 4-chloro-2-(methoxy-3-pyridylamino) benzoic acid in n-pentanol was accompanied by the formation of its pentyl ester complicating isolation and purification. An alternative Ullmann-Goldberg procedure was developed using DMF, both successfully replaced the n-pentanol and also produced the pyridylamino-benzoic acid under anhydrous, oxygen free, conditions, in which cyclodehydration was followed by dehydrochlorination using anhydrous POCl3, the desired product 7,10-dichloro-2-methoxy-benzo [b][1,5] naphthyridine was accompanied by an unexpected (and undesired) trichlorinated product in which the methoxy group had been displaced to form 2,7,10-trichloro-benzo [b][1,5] naphthyridine. The trichlorinated molecule was the dominant product (>50%) if the reaction was continued beyond four hours as confirmed by GC-MS spectrometry. Subsequent SNAr reaction whereby the halogen situated at the 10 position in 7,10-dichloro-2-methoxy-benzo [b][1,5] naphthyridine was displaced by p-aminophenol produced 4-(7-chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-yl(amino)-phenol as a bright yellow solid, as opposed to a brown solid as reported by Zheng et al. (1982). The presence of oxygen during the reflux period proved detrimental and rapidly oxidized the material to various compounds including a red-brown product identified as the corresponding quinone-imine. The target substance was produced by refluxing the air sensitive 4-(2,7-dichloro-benzo [b][1,5] naphthyridin-10-ylamino)-phenol with excess pyrrolidine and aqueous formaldehyde (Mannich Reaction) under argon. Pyronaridine was isolated as a moderately air sensitive free base using flash column chromatography on silica developed with methanol: ammonia (9:1, v/v under argon), and proved identical to an authentic sample when examined by a variety of analytical techniques (NMR, HRMS, HPLC-MS and TLC). The solid was, however, stable as the known yellow tetraphosphate salt. Hence, a superior, enhanced reliable synthetic procedure, but lower in overall yield (9-19%), when compared to the original method (33%) published in the Chinese language (Zheng et al. 1982) has been developed. Our spectroscopic (NMR and accurate MS) confirms that the published structure coincides with the data presented herein. The biological activity of pyronaridine, pyracrine and the aromatic head group was determined in-vivo. The side chain/head group was completely inactive at the doses tested whereas both pyracrine and pyronaridine were highly active against the Plasmodium berghei drug sensitive strain N/13/1A/4/20 and are suitable for further development.