Synthesis and Evaluation of Naphthyridine Compounds as Antimalarial Agents

Malaria is the best known protozoal disease, caused by one of four species of the sporazoa type --Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The malaria parasite has now become resistant to the best anti-malarial drugs, and can now be found in at least 100 tropical and subtropical countries. Worldwide, malaria infects 300-600 million people and kills about three million in a year.¹

Primaquine (Figure 1) is the drug of choice for the radical cure of P. vivax malaria for over 40 years.² Primaquine mode of action could be based in the capacity of primaquine to bind to the parasite’s DNA and modify its properties.³ Primaquine, however, like all its predecessors, is by no means nontoxic. Among the serious side effects it could produce are acute haemolysis in patients with certain types of glucose 6-phosphate dehydrogenase deficiency, methaemoglobinaemia and severe gastrointestinal disturbances.⁴

Figure 1.

Over the years, several attempts have been made to improve the therapeutic index of primaquine through modification of its chemical structure. Introduction of 4-methyl and 5-phenoxy^{5, 6} or alkoxy groups⁷ have produced analogs with much superior tissue and blood schizonticidal activity. However, toxicity studies have shown that these analogs also have a greater potential of producing methemoglobin.⁸ Tafenoquine (also known as WR 238605), is a primaquine analogue and is underdevelopment for the treatment of relapsing malaria.⁹ However, tafenoquine would also cause hemolysis in persons with G6PD deficiency, and must not be used during pregnancy because of its potential effect on the fetus.¹⁰ Therefore, further research in the discovery and development of novel molecule entities as safe, effective, and low cost replacement for primaquine is urgently needed.

Early mechanistic studies of primaquine have established that primaquine hemotoxicity was due to metabolites of the drug, rather than the parent compound. Phenolic metabolites of primaquine have been identified in the biological fluids of experimental animals and have long been suggested as candidate hemotoxic species.^{11, 12, 13} These compounds were shown to induce a variety of oxidative effects in both normal and G6PD-deficient red cells, including stimulation of hexose monophosphate shunt activity, hemoglobin oxidation and glutathione depletion.^{14, 15, 16} Both 5-hydroxyprimaquine and 5-hydroxy-6-desmethylprimaquine are direct-acting hemolytic agents. These mechanistic studies have suggested that 5-hydroxyprimaquine forms a redox pair with its p-quinoneimine analog and that the 5,6-diphenolic metabolite forms a redox pair with either an o-quinone or a p-quinoneimine. It has also been suggested that the hemotoxicity of primaquine is associated with arylhydroxylamine metabolites.¹⁷ Primaquine is known to undergo N-dealkylation (oxidative deamination) in humans to yield 6-methoxy-8-aminoquinoline (MAQ). It has been concluded that MAQ could be converted to the corresponding 8-hydroxylamino metabolite (i.e., MAQ-NOH) and that this metabolite could be capable of inducing methemoglobinemia and hemolytic damage by forming a redox pair with its nitroso analogue.¹⁷

As continued efforts in antimalarial drug discovery at our laboratory, novel primaquine analogues were designed and synthesized. These compounds (1-12) are shown in Figure 2. Lower toxicity will be achieved by reducing the tendency to form chemically reactive and toxic intermediates and metabolites. To achieve this goal, the following strategies are employed: (I) Replacing the quinoline moiety with a [1,5]naphthyridine core to prevent hydroxylation at C-5 position.^{18, 19} Toxic phenolic metabolites arise when C-5 position of the quinoline ring becomes oxidized and this process can be made unfavorable by blocking the C-5 position of the quinoline ring. Naphthyridine closely resembles quinoline, yet possesses higher oxidation potential due to the introduction of the second nitrogen on the aromatic system. Although a naphthyridine analogue of primaquine has previously been synthesized,^{18, 19} there was no systemic evaluation of naphthyridine analogues of primaquine to date. (II) Introducing a second substitution at the α position of the aliphatic side chain so that oxidative deamination will be disfavored. These compounds (1-12) now bear a dimethyl group at the α position, or the αposition is part of a new cyclopropane unit. (III) Compounds 7-12 also bear a substitution at position 2 of the aromatic ring. It has been reported that introducing an alkyl or alkoxy group at position 2 of the aromatic ring reduces the toxicity of a compound (induction of methemoglobin).^{20, 21}

Figure 2.

Scheme 1 describes the synthesis of compound 1. Compounds 2-6 were synthesized in similar routes. The synthesis began with 6-methoxy-pyridin-3-ylamine (13). Condensation of 13 with propane-1,2,3-triol under strong acidic condition²² furnished 2-methoxy-[1,5]-naphthyridine (14), which underwent oxidation with 3-chloroperbenzoic acid, to give 2-methoxy-[1,5]-naphthyridine 1-oxide (15). Compound 15 then reacted with phosphorus(III) oxychloride,²³ to furnish 4-chloro-2-methoxy-[1,5]naphthyridine (16). After deprotonation with sodium hydride, 2-(4-amino-4-methyl-pentyl)-isoindole-1,3-dione (17) reacted with compound 16 in a S_N2 type mode, which is followed by removal of the protective group on the terminal amine, to furnish N⁴-(2-methoxy-[1,5]naphthyridin-4-yl)-4-methyl-pentane-1,4-diamine (1).^{24, 25}

Scheme 1.

The synthesis of compounds 7-12 follows a different route. Scheme 2 describes the synthesis of compound 7. Compounds 8-12 were synthesized in similar procedures. Condensation of 3-amino-pyridine-2-carboxylic acid ethyl ester (18) with diethyl malonate followed by cyclization and decarboxylation afforded [1,5]-naphthyridine-2,4-diol (19), which then reacted with phosphorus oxychloride, to furnish 2,4-dichloro-[1,5]-naphthyridine (20). Acid methanolysis of 20 gave 4-chloro-2-methoxy-[1,5]-naphthyridine (21) as the major isomer and 2-chloro-4-methoxy-[1,5]naphthyridine (22) as the minor one. The synthesis of 21 is a modified methods of McCaustland et al.¹⁹ Compound 7 was then synthesized from intermediate 21, using the method described above.²⁶

Scheme 2.

In vitro antimalarial activities of compounds 1-12 as IC₅₀ values for the inhibition of chloroquine-resistant P. falciparum strain W2 were determined (Table 1), using previously reported protocols.^{27, 28} Most compounds in this class exhibited potent antimalarial activity (IC₅₀ <= 0.11 μM), superior to that of chloroquine (IC₅₀ = 0.31 μM).

Table 1.

In vitro (P. falciparum) and in vivo (P. berghei and P. yoelii nigeriensis) antimalarial studies^a

Compound	IC50 for P. falciparum W2(μM)	LD50(mg/kg)b	ED50 (mg/kg)c	ED50 (mg/kg)d	TIe
1	0.065	130	1.8	2.1	72
2	0.077	155	0.95	0.89	163
3	0.11	167	0.33	0.28	506
4	0.057	145	1.6	1.4	91
5	0.072	161	0.88	1.0	183
6	0.10	175	0.29	0.38	603
7	0.033	>200	1.6	2.0	>125
8	0.021	>200	1.5	1.7	>133
9	0.045	>200	0.72	0.65	>278
10	0.058	>200	0.75	0.68	>267
11	0.047	>200	0.31	0.41	>645
12	0.055	>200	0.26	0.23	>769
PQ	>10	55	ND	ND	ND
CQ	0.31	70	2.0	ND	35

^aAntimalarial activities were determined after i.p. administration of the compounds once daily for 4 days to infected mice. The first drug administration began 1 h after i.p. inoculation with malarial strain. PQ: Primaquine. CQ: Chloroquine. ND: Not determined.

^bAcute toxicity was determined after one single drug injection into four non-infected random-bred Swiss mice and expressed as the 50% lethal dose (LD₅₀), which corresponds to the dose leading to deaths of 50% of the subjects 10 days after the drug injection.

^cThe 50% efficient dose (ED₅₀), which is the dose leading to 50% parasite (P. berghei) growth inhibition compared to the control (treated with an equal volume of vehicle), was evaluated from a plot of activity (expressed as a percentage of the control) versus the log dose.

^dThe parasite is P. yoelii nigeriensis.

^eTI (Therapeutic Index) were calculated on the basis of the acute LD₅₀/ED₅₀(P. berghei) ratios.

The target compounds were also evaluated for the blood-schizontocidal antimalarial activity against P. berghei (sensitive strain) and P. yoelii nigeriensis (multidrug-resistant strain) in a rodent model (Table 1), using previously reported protocols.²⁹

The objective of this investigation was to prepare less toxic analogue of primaquine. As mentioned earlier, primaquine and related metabolites are capable of inducing methemoglobinemia and hemolytic damage. Therefore, in vivo MetHb-inducing properties estimation of compounds 1-12 was carried out in Mastomys coucha (Table 2), a rodent animal model using previously reported protocols.³⁰ Primaquine was used as the standard drug for the test.

Table 2.

In vivo methemoglobin (MetHb) toxicity estimation in Mastomys coucha.^a

Compound	% of MetHb
1	0.10
2	-0.12
3	-0.15
4	0.10
5	-0.12
6	-0.10
7	0.13
8	-0.11
9	-0.10
10	0.095
11	-0.12
12	0.14
PQ	3.4
Vehicle	-0.14

^aCompound was administered at a dose of 15 mg/kg per day for 3 days. Data are mean±SEM for each group of animals (set of three experiments). PQ: primaquine.

Replacing the quinoline moiety in primaquine with 1,5-naphthyridine clearly offers advantages. Primaquine itself is not active against P. falciparum strains in vitro. However, all new compounds (1-12) exhibited potent in vitro antimalarial activity against chloroquine-resistant P. falciparum strain W2. It is not clear why this series of compounds are effective against P. falciparum while primaquine, the compound on which the work is based, is ineffective. It is believed that changing the structure of the aromatic core of primaquine will change the mode of action, hence rendering the compound active in vitro against P. falciparum. Previous studies have also found that analogues with an alkyl substitution at position 2 of the aromatic ring possess in vitro antimalarial activity against P. falciparum.²¹

Compounds 1-12 have also shown excellent in vivo blood-schizontocidal antimalarial activity against both P. berghei (sensitive strain) and P. yoelii nigeriensis (multidrug-resistant strain). They were over three times less toxic than the parent compound primaquine, as measured by the in vivo LD₅₀. These compounds did not show any increase in MetHb, whereas primaquine induced 3.4% increase in MetHb. Replacing the α,α-dimethyl group in compounds 1-3 with a bioisosteric cyclopropyl group gives compounds 4-6. This modification did not significantly alter the efficacy and toxicity of a compound. However, introducing a substitution at position 2 of the naphthyridine ring further reduces the toxicity, while preserving the desired antimalarial activity.

Variation of the side chain substitution at the position 6 of the naphthyridine ring did not have profound effect on their toxicity. However, an increase in the lipophilicity of a compound has consequently improved the efficacy. For example, when the methyl group in compound 1 is replaced by the more lipophilic isopropyl group as in compound 3, efficacy is increased by six-fold, while toxicity is more or less flat.

In conclusion, new primaquine analogues were designed and synthesized. Some of these compounds (3, 6, 11, 12) possess a therapeutic index over ten times superior to that of the commonly used antimalarial drug chloroquine. These compounds, as well as the underlying design rationale, may find usefulness in the discovery and development of new antimalarial drugs.