Abstract
Malaria is a serious infectious disease caused by protozoan parasites in tropical and subtropical regions. Even inhabitants of temperate zones are exposed to the danger of malaria infection because of travel and global warming. Novel, effective, safe, and inexpensive drugs are required to treat malaria and contribute to the global goal of eradication. A search for new antimalarial agents has been performed by the synthesis of new benzo[a]phenoxazines, followed by biological evaluations. The derivative SSJ-183 (5), having a 4-aminopyridine group, showed an IC50 value against Plasmodium falciparum of 7.6 nM and a selectivity index of >7300. Cure was achieved by three oral doses of 5 at 100 mg/kg to mice infected with the Plasmodium berghei ANKA strain. The safety of 5 was supported by acute toxicity testing in mice with single doses up to 2000 mg/kg po, chromosome aberration test, in vitro as well as in vivo micronucleus tests, and phototoxicity studies in mice. Thus, 5 is a promising candidate as a new antimalarial agent.
Keywords: Antimalarial activity, benzo[a]phenoxazine, Plasmodium falciparum, Plasmodium berghei, oral administration
In tropical and subtropical regions, malaria is one of the most perilous infectious diseases caused by protozoan parasites. Each year, 500 million cases of malaria occur, and nearly 1 million people die, the majority of whom are young children and pregnant women.1−4 Because of global warming, even inhabitants of temperate zones are in danger of exposure to malaria infection. No vaccine is currently available for malaria, and the resistance of the protozoa to clinically used chemotherapeutic agents is increasingly common. Thus, novel, effective, safe, and inexpensive drugs are highly desired to control malaria.
We have searched new hit compounds against malaria parasites from our compound library, taking into account the π-delocalized lipophilic cation (DLC) hypothesis5 in which hydrophobic cations containing delocalized π-electrons are accumulated into mitochondria and inhibit metabolic activity. According to the working hypothesis, the rhodacyanine derivative MKT-077 (1)6 (Figure 1) was first identified as a potential lead compound possessing potent in vitro activity against Plasmodium falciparum.7 However, very poor efficacy was observed in vivo against Plasmodium berghei. Further searches based on the combinatorial synthetic approach8 provided various in vivo active rhodacyanine derivatives.9,10 Among them, SSJ-127 (2) showed cure of P. berghei-infected mice with subcutaneous administration,11 but the rhodacyanine derivatives did not exhibit any oral activity. Phenoxazinium derivatives, a type of DLC exemplified by 3 and 4, displayed potent in vitro activity and oral efficacy.12−15 However, cytotoxicity was observed at low micromolar concentrations in several phenoxazinium derivatives.12,14 Because the electrophilicity of the carbon atom at the 1 position in the phenoxazinium skeleton would be troublesome, we examined the addition of a benzene ring to the phenoxazinium framework. Thus, we have prepared and evaluated various benzo[a]phenoxazine derivatives. The corresponding salts of benzo[a]phenoxazines are a variant of DLC candidates. Herein, we disclose the details of the discovery and testing of SSJ-183 (5), having high in vitro and in vivo antimalarial activity when administered to P. berghei-infected mice via the oral route.
Figure 1.
Structures of previous examples of antimalarial compounds and the candidate 5.
A number of benzo[a]phenoxazine derivatives have been prepared by known methods (Scheme 1).16−20 As a typical example, the synthesis of 5 is shown in Scheme 1. Namely, the nitrate salt of 6 (X = NO3)16 was reacted with 4-aminopyridine for 24 h in refluxing ethanol. Chromatographic purification of the product on silica gel revealed 5, mp 247−248 °C, in 38% yield. Notably, 5 is very stable as a free amine for a long period under ambient conditions. All tested compounds were prepared by the same method as described above. When 5 was converted into a salt by treatment with hydrochloric acid as usual, the elemental analysis of the salt, mp >300 °C, indicated that the molecular ratio of 5 and hydrogen chloride must be 1:2.
Scheme 1. Synthesis of Benzo[a]phenoxazine 5 and Corresponding Quaternary Salts.
Furthermore, the quaternary salts 25 and 26 were prepared by treatment of 5 with alkyl halides in acetonitrile under refluxing conditions. The physical and spectral data of new compounds 5 and 11−26, prepared by the similar method, are recorded in the Supporting Information.
We evaluated compounds 5−26 (Figures 1 and 2) for their in vitro activity (IC50) against P. falciparum K1, a strain resistant to chloroquine and pyrimethamine, and determined the cytotoxicity to mammalian cells. They were further screened by in vivo efficacy against P. berghei strain NK-65 via the oral route (po) according to established protocols (Table 1).10 For the in vivo screening, test compounds were formulated in 5% glucose and 10% ethanol solution or in 7% Tween 80 and 3% ethanol solution to provide a dose of 100 mg/kg. The resulting suspensions were administered to female ICR mice as a single oral dose.
Figure 2.
Structures of tested benzo[a]phenoxazine derivatives.
Table 1. Evaluation of Benzo[a]phenoxazines: In Vitro Activity against P. falciparum K1, Cytotoxicity toward L-6 Myoblasts, and in Vivo Activity against P. berghei NK-65 (n = 3 Mice).
| IC50 (μM)a |
|||||
|---|---|---|---|---|---|
| compound | P. falciparum K1 | cytotoxicity L-6 | selectivityb | inhibition (%)c | MSD |
| 6; X = Cl | 0.606 | 3.82 | 6.3 | ND | ND |
| 7 | 0.868 | 65.9 | 76.0 | 15 | 6.3 |
| 8 | 0.0156 | 43.14 | 2765 | 15 | 6.6 |
| 9 | 0.191 | 164.9 | 863 | 0 | 6.3 |
| 10 | 0.232 | 123.2 | 531 | 0 | 6.3 |
| 11 | 0.063 | 179.8 | 2855 | 14.7 | 6.3 |
| 12 | 0.027 | 67.9 | 2515 | 81 | 10.3 |
| 13 | 0.1125 | 167.9 | 1493 | 11.4 | 6.3 |
| 14 | 0.005 | 13.6 | 2734 | 3.3 | 6.3 |
| 15 | 0.015 | 15.8 | 1052 | >99.9 | 8.3 |
| 16 | 0.023 | 117.0 | 5088 | 62.7 | 10.0 |
| 5 | 0.0076 | 55.7 | 7334 | >99.9 | 14.6 |
| 17 | 0.0081 | 165.1 | 20390 | >99.9 | 10.0 |
| 18 | 0.05 | 67.1 | 1342 | 15.3 | 6.3 |
| 19 | 0.195 | 63.8 | 327 | 5.6 | 6.3 |
| 20 | 0.029 | 96.9 | 3342 | 97.9 | 7.3 |
| 21 | 0.18 | 86.6 | 481 | 99.7 | 13.0 |
| 22 | 0.017 | 14.8 | 887 | >99.9 | 15.7 |
| 23 | 0.038 | >190.2 | >5007 | 12.7 | 6.3 |
| 24 | 0.011 | >190.2 | >17290 | 14.2 | 6.6 |
| 25 | 0.009 | 1.56 | 174 | 22.0 | 6.6 |
| 26 | 0.024 | 0.75 | 31 | 39 | 7.3 |
| chloroquine | 0.019−0.066 | NDd | ND | >99.9 | 12.6 |
Mean of two independent assays.
Calculated as IC50 for L-6 cells/IC50 for P. falciparum.
Parasitemia was determined on day 4 after infection. The difference between the mean value of the control group and those of treated groups is calculated and expressed as a percent relative to the control group. Single oral administration (100 mg/kg) was given on day 1.
ND, not determined.
Benzo[a]phenoxazinium 6,16 having no substituent at the 6-position, showed weak activity against P. falciparum K1. The activity of Nile red (7) possessing an oxygen substituent was also weak, while the introduction of a nitrogen functionality increased the potency and reduced the IC50 value of Nile blue A (8) to 0.0156 μM. Although a low cytotoxicity was observed for 8, the in vivo activity was poor. The phenyl and tolyl derivatives 9(16) and 10(16) showed low activities in both in vitro (IC50: 0.191 and 0.232 μM) and in vivo tests. Higher activities were achieved in the in vitro test of compounds possessing hetero aromatic rings on the nitrogen at the 6-position. Therefore, several derivatives 11−14 were prepared and evaluated, but their in vivo efficacies were unsatisfactory. Improved in vivo efficacies were observed for the compounds 15, 16, and 5 carrying a pyridine ring. Among them, 5 exhibited the best activity: an IC50 value of 0.0076 μM against P. falcipaurm K1, an IC50 value of 55.7 μM in the cytotoxicity test, a selectivity of 7334, and >99.9% inhibition of P. berghei NK-65. In addition to the inhibition of parasitemia, the mean survival days (MSD) after a single dose were extended to 14.6 days as compared to approximately 6 days for an untreated control. Very similar results were obtained, when single oral doses of 100 mg/kg were administered to NMRI female mice infected with P. berghei ANKA strain21 in three independent experiments (n = 3 mice per experiment, average inhibition = 99%, and average MSD = 16; data not shown).
Although numerous analogues were prepared and assessed, only the 4-aminopyridine derivatives are presented here. Thus, the dimethyl derivative 17 showed potent activity but a shorter survival as compared to 5. With longer substituents at the 9-position, 18 and 19 were less active. Morpholine compound 20 displayed good activity. Furthermore, substitution of a methyl group at the 11-position improved the safety, and two derivatives 21 and 22 provided good in vivo efficacy. Because the hydrochlorides 21 and 22 gave similar in vivo activities as that of 5, benzo[a]phenoxazine is apparently absorbed through the gut as the hydrochloride when administered po. The absence of cytotoxicity of compounds 23 and 24, having a bromine atom on the A ring, was encouraging; however, both showed low in vivo activity, possibly due to their poor solubility and poor oral bioavailability. On the other hand, high cytotoxicity was observed for the quaternary salts 25 and 26.
To gain additional information on the in vivo efficacy of 5, we carried out a dose−response experiment in NMRI female mice infected with P. berghei GFP ANKA strain21 (Table 2). High efficacy was observed by the po route with cures achieved by oral administration of three daily doses of 100 mg/kg.
Table 2. In Vivo Results for 5 Orally Administrated to n = 3 Mice/Dose Once Daily for Three Consecutive Days to P. berghei GFP ANKA Strain (Data from 2 to 3 Independent Experiments).
| mg/kg | inhibition (%)a | MSD (% of cured animals) |
|---|---|---|
| 3 × 100 | >99.9 | >30.0 (100%) |
| 3 × 30 | 99 | 27.2 (78%) |
| 3 × 10 | 26 | 4.0b |
Determined on day 4 postinfection.
Mice were euthanized on day 4 based on a lack of sufficient efficacy.
In other evaluations with compound 5, we detected no lethality at doses up to 2000 mg/kg po using 20 mice. Furthermore, no effects were found at 1000 μM (− and + S9) in a chromosome aberration test, at 2.0 μM (− and + S9) in an in vitro micronucleus test and at 1000 mg/kg × 2 in an in vivo rat micronucleus test. No phototoxicity was detected in mice dosed at 300 mg/kg po. In binding assays against 80 receptors, only two human recombinant receptors, A3 and D3, were inhibited ∼80% with 1 μM, and no inhibitions were noted with other receptors. The selectivity was further supported by ≥1000-fold higher IC50 values of 5 against three other protozoal parasites (36000 nM for Trypanosoma brucei rhodesiense, 11300 nM for Trypanosoma cruzi, and 6500 nM for Leishmania donovani) as compared to P. falciparum strain K1. Interestingly, the deep purple/blue color of the compound formulation was not detected in the urine, eyes, and organs of mice treated with a single oral dose of 100 mg/kg, although the prototype molecule, methylene blue, in this class stains tissues and urine. In vitro and in vivo activities of 5 are much better than those of methylene blue. Furthermore, no hemolysis was proved in blood taken from G6PD-deficient patient at Jichi Medical University, and the details will be discussed in the future.
In vivo pharmacokinetic studies in rats were carried out according to the reported procedure (Figure 3).21 After oral dosing, 5 had a bioavailability of approximately 30%. After iv administration, the terminal half-life was approximately 5.5 h, and 5 demonstrated a high volume of distribution and high clearance.
Figure 3.
Plasma concentration vs time profile of 5 in male Sprague−Dawley rats following intravenous (2.8 mg/kg, filled symbols) and oral (21.5 mg/kg, open symbols) administration.
In summary, in a search for novel antimalarial agents, new benzo[a]phenoxazines were synthesized and subjected to biological evaluation. The derivative 5 possessing the 4-aminopyridine moiety showed an IC50 value against P. falciparum of 7.6 nM and a selectivity index of >7300. Cure was achieved with three daily oral doses to mice infected with P. berghei ANKA strain at a dosage of 100 mg/kg. The safety of 5 was demonstrated by a single dose toxicity test, the highest dose being 2000 mg/kg, a chromosome aberration test, in vitro and in vivo micronucleus tests, and a phototoxicity test. Binding assays against 80 receptors supported a high selectivity. Urine and eyes were not colored during treatment, and no color was detected in organs after 7 days of administration. On the basis of the above findings, compound 5 appears to be a promising candidate for further testing. We have also developed a more efficient synthesis of 5, and a report of this new synthesis as well as the investigation of the mechanism of action of 5 will be published in due course.
Acknowledgments
We thank Professor Terumi Nakajima and Professor Toshio Honda, Hoshi University, and Dr. Hiroyuki Togashi and Seiki Sakanoue, Synstar Japan Co., Ltd., for their encouragement. We also thank Professor Hiroyuki Matsuoka, Jichi Medical University, for in vitro work using blood from a G6PD patient.
Supporting Information Available
General synthetic procedure, melting point, and spectral data for all new tested compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
This study was supported by the Creation and Support Program for Start-ups from Universities, Japan Science Technology Agency (JST), and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO).
Supplementary Material
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