HETEROCYCLES

Review | Regular issue | Vol. 83, No. 8, 2011, pp. 1727-1756
Received, 3rd March, 2011, Accepted, 7th April, 2011, Published online, 8th April, 2011.
DOI: 10.3987/REV-11-706

■ Discovery of New Anti-Protozoan Agents Having Novel Mode of Action

Masataka Ihara*

Research Centre of Medicinal Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan

Abstract

Malaria, leishmaniasis, African sleeping sickness (African trypanosomiasis) and Chagas disease (American trypanosomiasis) are caused by different protozoan parasites. Although many people suffer from these diseases in tropical and subtropical areas, efficient medicines against these protozoan diseases are very few or absent. Efforts to develop new drugs against these neglected diseases led us to the discovery of SSJ-127 (62), which cured malaria and African trypanosomiasis mouse models by treatment with injection, SJL-01 (74) as a hit compound for leishmaniasis, and SSJ-183 (109) as a candidate against malaria, respectively. These compounds displayed novel modes of actions different from those of conventional medicines.

CONTENTS
1. Introduction
1.1. Malaria
1.2 Leishmaniasis
1.3 African Sleeping Sickness (African Trypanosomiasis)
1.4 Chagas Disease (American Trypanosomiasis)
1.5. Demands for New Medicines
2. Random Screening
3. Synthesis of Artemisinin Derivative
4. Working Hypothesis for Further Research
5. Rhodacyanines
5.1. Antimalarial Activity
5.2. SSJ-127
5.3. Anti-leishmanial Activity
5.4. SJL-01 as an Anti-visceral Leishmaniasis (VL) Agent
6. Phenoxazinium Salts
7. Benzo[a]phenoxazines
7.1 SSJ-183 as an Anti-malarial Agent
8. Study of the Mode of Action
9. Consideration of a Molecular Model
10. Conclusion

1. INTRODUCTION
A large number of diseases caused by various parasites are known in tropical and subtropical regions. There are three main classes of parasites associated with diseases in humans: protozoa, helminths, and ectoparasites. Malaria, leishmaniasis, African sleeping sickness, and Chagas disease, caused by protozoan parasite infection, are target diseases for a special Program for Research and Training in Tropical Diseases (TDR) executed by World Health Organization (WHO).1 Protozoa are microscopic, one-celled organisms that live in the blood or tissues of humans and are transmitted to other humans by an arthropod vector such as a mosquito or fly. Protozoan diseases are sometimes referred to as neglected diseases, since these are illnesses most often occurring in developing countries. Although many people suffer seriously from these sicknesses, these orphan diseases are frequently ignored by pharmaceutical industries in developed countries. Therefore, aid from not-for-profit public-private organizations such as Medicines for Malaria Venture (MMV), Drugs for Neglected Diseases initiative (DNDi), Institute for Oneworld Health, and so on, are critically needed for the development of new medicines.2 We have worked toward the discovery of new drug candidates against protozoan diseases and would like to describe our ongoing findings. Since protozoan diseases are not common in developed countries, we present here a brief description of them.

1.1 Maralia
In tropical and subtropical regions, malaria is one of the most perilous infectious diseases. Each year, about 500 million cases of malaria occur, and nearly 1 million people die, the majority of them being young children and pregnant women.3-7 Because of global warming, even inhabitants of temperate zones are in danger of exposure to malaria infection. Malaria is caused by protozoan parasites of the genus Plasmodium. Human malaria cases are generally brought about by four species, including P. falciparum, P. vivax, P. ovale and P. malariae.4 Different animals are infected by different species of Plasmodium. For example, P. berghei, P. yoelii and P. chabaudi are known as rodent malaria, and do not infect humans. The most serious forms to humans are caused by P. falciparum and P. vivax. Malaria parasites are transmitted by the bites of female Anopheles mosquitoes. The life cycle of malaria parasites is complex and they multiply within red blood cells of the host. Malaria transmission can be reduced with mosquito nets and insect repellents. No vaccine is currently available, and structures of typical medicines against malaria are shown in Figure 1.

Quinine (1), a cinchona alkaloid, is a well-known antimalarial agent, but its activity against the malaria parasite is not very high. The requirement that this drug be administered by injection is a serious drawback for its use as a tropical medicine. On the other hand, the synthetic compounds, chloroquine (2), mefloquine (3) and pyrimethamine (4) are orally active. Unfortunately, the appearance of parasite strains that are resistant to these drugs has introduced a serious problem in malaria treatment. Artemisinin (5), a sesquiterpene, also known as qinhaosu, has been isolated from Artemisia annua and is active against the drug resistant form of P. falciparum. Treatments containing an artemisinin derivative (artemisinin-combination therapy, ACTs) are standard treatment worldwide for the P. falciparum malaria. On the Thai-Cambodian border, artemisinin is losing its potency due to a drug-resistant form of the malaria parasite.8 Atovaquone (6), an analog of ubiquinone acting on mitochondria, is available as a combination preparation with proguanil, but its cost as an anti-malarial agent is high. Primaquine (7) in combination with quinine or chloroquine is mainly used to treat malaria transmitted by P. vivax. Primaquine remains the only licensed drug that can provide a radical cure of the P. vivax malaria but 7 produces hemolytic anemia in individuals with glucose-6-phosphate deficiency.5

1.2. Leishmaniasis
Leishmaniasis is one of the major target protozoan diseases for WHO. This serious tropical disease is caused by parasitic protozoa of the genus Leishmania.9 Leishmania are obligate intracellular protozoan parasites of macrophages that are transmitted between hosts by the bite of female phlebotomine sand flies and are delivered to a host as animal-infective metacyclic promastigotes along with the saliva of the sand fly and a mucin-rich gel produced by the parasites in the sand fly midgut.10 Leishmaniasis threatens about 350 million people in 88 countries around the world. As many as 12 million patients are currently infected, with over 1 million estimated new cases occurring every year. It manifests mainly in three clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL), of which VL is the most severe form of the diseases. VL, also called Kala-azar or black fever, is a fatal disease with an estimated incidence of 500,000 cases per year. Over 90% of VL cases occur in India, Bangladesh, Nepal, Brazil, and Sudan. Diagnosis and treatment of VL have always been difficult, but recent development of new medicines has improved the situation, although the development of new, more effective drugs with less toxicity is urgently required. Examples of drugs for leishmaniasis are shown in Figure 2.

Although several medicines are available for leishmaniasis, the most widely used drug is pentostam (8) containing pentavalent antimony. Other medicines such as amphotericin B (9), liposomal amphotericin B and paromomycin (10) also require injection administration. Only miltefosine (11) is orally active but the activity is not very high. All agents have individual problems such as toxicity, poor effects, or high cost. Development of other potential compounds including buparvaquone (12) and aminoquinolines are underway.

1.3. African Sleeping Sickness (African Trypanosomiasis)
Human African trypanosomiasis (HAT), also known as sleeping sickness, is an endemic disease of people and animals, caused by protozoa of the species Trypanosoma brucei and transmitted by the tsetse fly. HAT is a fatal disease if untreated. In 2008, this disease caused 48,000 deaths, but it is believed that many cases go unreported. African trypanosomiasis symptoms occur in two stages. The first stage is known as the hemolymphatic phase and if left untreated, the disease moves into the second stage, called the neurological phase. The arsenic compound melarsoprol (13) and eflornithine (14) are used for the second stage of HAT, but they are highly toxic. Pentamidine (15) is a treatment for the first stage, and other compounds including ascofuranone (16) are currently being examined in order to develop more effective medicines.

1.4. Chagas Disease (American Trypanosomiasis)
Chagas disease, also called as American trypanosomiasis, is a tropical disease caused by the flagellate protozoan T. cruzi, transmitted to humans and mammals by the triatomine vector. It is estimated that as many as 8 to 11 million people in Mexico, Central America, and South America are infected with this disease. Chagas disease is a chronic illness leading to death. Benznidazole (17) and nifurtimox (18) are used as treatments, but they have various side effects. Development of a prodrug of ravuconazole (19) is now in progress.

1.5. Demands for New Medicines
There are too few medicines for treating protozoan diseases and the emergence of drug resistant parasites has caused further problems for therapy. The development of new types of medicines having novel chemical frameworks and new modes of action are extremely desired. Oral administration is preferred over injectable drugs due to the poor medical conditions in tropical and subtropical regions. Additionally, the new medicines should be available in large quantities at low cost.
In this context, we began a research program in 1996 that focused on developing new medicines for protozoan diseases. Herein, we present the details of our progress.

2. RANDOM SCREENING
More than 150 compounds were selected from the compound library in our group at Tohoku University and Professor Wataya’s group at Okayama University conducted the screening tests. Inhibitory activity (as IC50 values) against P. falciparum (chloroquine-sensitive FCR-3 strain) and evaluation of the cytotoxicity against FM3A were determined. Of these test compounds, the results of only four compounds (20 – 23) are shown together with the activities of quinine and chloroquine (Table 1). The selectivity of each compound was defined as the ratio of the cytotoxicity / the inhibitory activity.

Racemates of the synthetic alkaloids tylophorine (20) and tubulosine (21) exhibited comparable inhibition activities against P. falciparum as those of quinine and chloroquine, but their cytotoxicities against the mouse FM3A cell were high. As a result, the corresponding selectivity was low. Although the inhibitory activity of 22 was high, the selectivity was poor.11 Since homoprotoberberine derivatives showed reasonable activity with low cytotoxicity, many analogues were synthesized, but the best selectivity recorded by 23 was only 93.12 Since selectivity values >100 were required for a promising hit compound in the screening assay, the random screening methodology was not effective.

3. SYNTHESIS OF ARTEMISININ DERIVATIVE
Since artemisinin (5) is easily available in large quantity from the plant A. annua,13 synthesis of an analogue (28), which can not be readily prepared from the natural product, was investigated as shown in Scheme 1. The intramolecular Diels-Alder reaction of the triene 25, which was stereoselectively synthesized from the alcohol 24, occurred at room temperature to give the cis-octalone 26 in good yield. After conversion into the corresponding trans fused isomer, the irradiation of the product with a tungsten lamp in acetone containing a catalytic amount of methylene blue under atmospheric oxygen, followed by treatment of the formed hydroperoxide 27 with trifluoroacetic acid under atmospheric oxygen,14 provided the desired peroxide 28 in a poor yield. The IC50 value of 28 against P. falciparum was 0.039 µM with a low cytotoxicity toward to FM3A cells, IC22: 24 µM.15 Although 28 gave a potent activity with high selectivity, the synthesis was too costly and would not provide the best method for the protozoan drug.

4. WORKING HYPOTHESIS FOR FURTHER RESEARCH
Malaria, leishmania and trypanosoma parasites are eukaryotes, which possess energy producing, and thus negatively charged organelles such as mitochondria and apicoplasts. Therefore, we focused on a concept proposed by Chen16 in 1988; the accumulation of π-electron-delocalized lipophilic cations (DLCs) in the mitochondrion would lead to destruction of the organelle’s function.
We observed that the IC50 value of the naturally occurring alkaloid MVC (29) against P. falciparum was enhanced from 5.0 µM to 0.13 µM by the transformation of the compound into its corresponding quaternary ammonium compound 30.17

5. RHODACYANINES
5.1. Antimalarial Activity
Based on the above working hypothesis, we searched hit compounds carrying quaternary ammonium cations as DLCs. Typical examples are shown in Table 2. Methylene blue (31) and rhodamine 123 (32) exhibited good activity against chloroquine sensitive P. falciparum, but the selectivities were not adequate. On the other hand, the activity of MKT-077 (33) was high, IC50 0.07 µM, with low cytotoxicity, IC50 15.0 µM, thus, the selectivity was 214.18

Rhodacyanine derivative MKT-077 (33) had been developed as a novel anticancer agent, and it has been subjected to further clinical investigation for the treatment of solid tumors.19, 20 Rhodacyanines 34 consist of three linearly linked heterocyclic groups, in which the two terminal heteroaromatic rings (A and C) flank a rhodanine (4-oxothiazolidine) B-ring. The compounds are double conjugates of two different units, having parts comprised of neutral merocyanine (left, A ring) and cationic cyanine (right, C ring) structures.

The requirement for each of the heterocyclic components of the rhodacyanines as shown in Table 3 was evidenced by the very low antimalarial activities of merocyanines 35 and 36. Cyanines 37 and 38, which have no merocyanine conjugation, display moderate activity against P. falciparum, but their potency is lower than that of 33. Thus, tricyclic-rhodacyanine structures, containing both merocyanine and cyanine conjugation, are required for high antimalarial activity.

Although good activity and selectivity of 33 were observed, 33 showed a very poor efficacy upon in vivo testing using P. berghei. We next studied syntheses of various rhodacyanine derivatives, followed by biological evaluations. Most rhodacyanine derivatives can be prepared from commercially available starting materials within five steps. Generally, all synthetic intermediates as well as the rhodacyanines are easily crystallized compounds and the parallel combinatorial synthesis could be carried out by the combination of three components in one pot as shown in Scheme 2.21

Further variations of rhodacyanines such as 43 and 44 (Figure 7) were prepared and their in vitro antimalarial activities against P. falciparum K1 (chloroquine-resistant strain) as well as their in vivo activities against P. berghei (NK65 strain) in mice were determined.22

Several aza-fused rhodacyanines including the two types of compounds 45 and 46 (Figure 8) were also synthesized and their antimalarial activities were evaluated.23

Synthesis of new types of rhodacyanines having rigid structures, as exemplified by 60 and 61, and their biological evaluations have also been undertaken.24
In vitro activities of some typical examples against P. falciparum K1 (chloroquine-resistant strain) and cytotoxicity toward rat L-6 cells, carried out at the Swiss Tropical and Public Health Institute (Swiss TPH), are shown in Table 4.

The allyl substituted rhodacyanine 47 showed potent inhibitory activity with a lower cytotoxicity; the selectivity was 5,789. The activity of 48 carrying a hydroxyl group was high enough and 49 possessing an amide functionality gave good in vitro activity. Although 50 and 51 having additional conjugation between the A and B rings exhibited somewhat lower activity, the analogues 52 and 53 showed higher activity. Activities of bis-rhodanine derivatives such as 54 and 55 were varied with structures of heterocyclic moieties at both edges. High in vitro activities were observed for aza-rhodacyanines 56 and 57, although bis-rhodanine derivatives 58 and 59 showed lower activities. 60 and 61 having rigid structures also gave reasonable activities. Many rhodacyanines showed in vivo efficacy to some extent against P. berghei via injection treatment but cure was difficult. One rhodacyanine derivative, SSJ-127 (62), provided cure in an in vivo study by s.c. treatment as discussed below.

5.2. SSJ-127
SSJ-127 (62) was prepared starting with the benzo[d]thiazol-3-ium segment, as shown in Scheme 3.

Reaction of 2,3-dimethylbenzo[d]thiazol-3-ium tosylate (63) and ethyl isothiocyanate in the presence of triethylamine in pyridine gave the thioamide 64. Without purification, 64 was treated with bromoacetic acid in acetic acid to afford the 4-oxothiazolidine 65. Treatment of 65 with N,N’-diphenylformimidamide in acetic anhydride provided the phenylacetamidomethylenethiazolidine 66. Reaction of 66 with the pyridinium iodide 67 in the presence of triethyamine in acetonitrile, followed by elution of the product through IRA-400 (Cl), gave SSJ-127 (62) in a reasonable yield. Thus, 62 was easily synthesized in five steps from known compounds using a standard synthetic procedure.19
IC50 values of SSJ-127 (62) against P. falciparum K1, Trypanosoma cruzi, T. brucei rhodesiense, Leishmania donovani and L-6 rat myocytes together with the selectivity index (SI) are shown in Table 5. SSJ-127 (62) showed activities at ten nM concentrations against P. falciparum K1 and T. brucei rhodesiense with good selectivity.25

Next, in vivo experiments with SSJ-127 (62) were performed using P. berghei GFP ANKA strain at Swiss TPH and using P. berghei NK 65 strain at Hoshi University. Single subcutaneous (s.c.) administration of 100 mg/kg of 62 to NMRI mice (females) infected with P. berghei GFP ANAK exhibited 95% suppression after 4 days. A positive result, 40% suppression, was also obtained by a single p.o. administration of 100 mg/kg of 62 to the infected mice. A complete cure was observed by the in vivo test dosing 62 at Hoshi University. Namely, three times s.c. administrations of 40 mg/kg/d of 62 to ICR mice (males) infected with P. berghei NK 65 strain provided 99.9% suppression and all treated mice survived until natural death.
A preliminary pharmacokinetic study using male rats, Crl:CD(SD), was carried out by intravenous (i.v.) and s.c. administrations. The results, analyzed by two-compartmental methods using the computer program 3P87, are summarized in Table 6. The s.c. bioavailability of 62 was determined to be excellent.

Furthermore, administration of SSJ-127 (62) resulted in a cure for the in vivo anti-trypanosomal tests against T. brucei brucei S427 in mice carried out at the Center for Basic Research, Kitasato University.26 T. brucei brucei parasite causes animal African trypanosomiasis.

5.3. Anti-leishmanial Activity
Many rhodacyanines exhibited good activity against leishmania parasites. For example, the IC50 value of 68 against L. major causing cutaneous leishmaniasis (CL) was 0.012 µM,27 a value that exceeds that of amphotericin B (9) whose IC50 was 0.14 µM.

Various rhodacyanine derivatives were subjected to the screening test against L. donovani, which causes visceral leishmaniasis (VL). Each candidate was evaluated for its in vitro activity against L. donovani strain MHOM/ET/67/L82 and for its inherent cytotoxicity against L-6 rat skeletal myoblasts (Table 7). MKT-077 (33) exhibited ~ 2-fold greater inhibitory activity as compared with miltefosine (11) and with a selectivity factor of 450. Although SSJ-127 (62) could cure rodent malaria by s.c. administration, the in vitro activity was rather low against Leishmania parasites with low selectivity. Numerous derivatives of 62 having different alkyl substituents on three nitrogen atoms were prepared and their activities were tested. While compound 70 possessed relatively good inhibitory activity as indicated by its low IC50 value, its cytotoxicity was high. In compound 71, replacing the oxazolo[4,5-b]pyridine moiety with benzo[d]thiazole did not improve the activity. In compound 72, substitution of an imine functionality provided better activity and selectivity in the in vitro test. Introduction of a chlorine atom on the benzothiazole C ring (compound 73) enhanced the activity, while substitution of a fluorine atom at the same site resulted in excellent activity in the in vitro test, as well as a high selectivity factor for compound 74, termed SJL-01. The corresponding maleate (75) and mesylate (76) derivatives showed good inhibitory activities. However, in 77, the presence of an alcohol group at the fluorobenzothiazole ring reduced the activity dramatically. The replacement of the left hand benzo[d]thiazole with oxazolo[4,5-b]pyridine gave poor activity (78). Connecting the 1,3-dimethylbenzo[d]imidazole through a nitrogen (compound 79) gave unsatisfactory results in the in vitro test. The same tendencies were observed in the tests of 3,5-dimethylthiazole 80, 3,4-dimethylthiazole 81, and with the methylpyridine derivative 82.28

Macrophage in vitro screening, which is crucial to evaluate anti-leishmania activity, was then applied to several of the most promising rhodacyanines, but most of the compounds showed poor results as indicated by the IC50 value of compound 72 in Table 8. On the other hand, compound 74 showed noteworthy activities, compared with that of miltefosine. Thus, SJL-01 (74) was selected for in vivo evaluation.

5.4. SJL-01 (74) as an Anti-visceral Leishmaniasis (VL) Agent
Synthesis of 74 was carried out as shown in Scheme 4. Reaction of rhodanine 83 with N,N’-diphenyl formamidine in DMF, followed by treatment with acetic anhydride gave 84, which was reacted with

N-methyl-2-methylbenzothiazolium salt 63 to provide merocyanine 85. After formation of 86, obtained by the reaction of 85 with methyl p-toluenesulfonate, condensation of 86 with 87 in the presence of triethylamine, followed by treatment with hydrochloric acid, furnished 74. The desired compound can be easily synthesized in excellent yield in six steps, affording a crystalline compound. The highly pure product, mp 274.5-275.6 oC, was obtained by simple crystallization.28
On the basis of the results of the above in vitro macrophage assays, compound 74 was further evaluated by in vivo testing using L. donovani strain HU3 in female BALB/c mice, performed at the London School of Hygiene & Tropical Medicine. Since preliminary studies showed that no bioavailability was obtained by s.c. administrations of 74, the in vivo studies were carried out via i.v. administration. Excellent activities, ~ 95% inhibition, were observed by the 5-times treatments with compound 74 at dosages ranging from 1.3 - 12.5 mg/kg (Table 9). Even at a dose as low as 0.2 mg/kg x 5 of 74 gave 16.13% inhibition, suggesting the existence of a dose dependent activity. The activity of compound 74 is much better than that of the conventional medicines, pentostam (8) and amphotericin B (9), and comparable to that of liposomal amphotericin B.28

Preclinical and clinical development of 74 aimed at leishmaniasis is important for the therapy of this neglected disease. In vitro receptor binding assays were carried out against 80 receptors and very small inhibitions were observed at 1.0 µM except in the cases of four receptors, BZD (rat heart) (72%), M1 (human recombinant) (60%), M2 (human recombinant) (80%) and M4 (human recombinant) (73%). Furthermore, the in vitro micronucleus test of 74 showed a negative result at 2 µM in the absence of S9 (a supplemented rat liver homogenate fraction) and 31.3 µM in the presence of S9, while negative results were obtained at 5.19 µM (-S9) and at 3.97 µM (+S9) by the chromosomal aberration test. It was concluded that 74 did not demonstrate mutagenic potential in the in vitro cell mutation assay. Furthermore, it was concluded that 74 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity. The in vitro acute effects of 74 on the hERG K+ channel current, recorded from stably transfected HEK-293 cells, were evaluated at nominal concentrations ranging from 1 µM up to 10 µM. Thus, compound 74 is very hopeful candidate for VL.

6. PHENOXAZINIUM SALTS
Oral administration is important for antimalarial agents. We found that phenoxazinium salts exhibited high efficacy via oral administration (p.o.),29 and excellent bioavailabilities of phenoxazinium salts were gained by p.o.30 However, the development of 3,7-bis(dialkylamino)phenoxazinium derivatives as medicines was difficult because these compounds typically were purified with the aid of zinc chloride.31 We found that symmetric and asymmetric 3,7-bis(dialkylamino)phenoxazinium derivatives with high purity could be obtained chromatography followed by large-scale crystallization.32
Some synthetic intermediates can be prepared easily by the palladium-catalyzed reaction.33 3,7-bis(Dialkylamino)phenoxazinium salts 90 were obtained by the reaction of 3-N,N-dialkylaminophenols 88 and N,N-dialkyl-3-methoxy-4-nitrosoanilines 89 in acidic solutions, and then purified by chromatography with a short silica gel column.

Antiprotozoal and cytotoxic activities of the 3,7-bis(dialkylamino)phenoxazinium salts are shown in Table 10. Comparison of the IC50 value of 95 with the IC50 values of the other compounds suggests that the presence of a morpholino group might increase the polarity and hydrophilicity of those compounds, thereby decreasing their activities. However, the introduction of a morpholino group substantially reduced the toxicity, which suggests that increased polarity and hydrophilicity result in decreased toxicity. This phenomenon was evidenced by the high toxicity and low selectivity of 97, in which long alkyl chain led to increased lipophilicity of the compound. Taking into consideration activity and toxicity, the compounds with a short alkyl chain (methyl and ethyl) are good antiprotozoal candidates, whereas the introduction of long alkyl chains or morpholino groups is not recommended. Symmetrical and asymmetrical structures did not have different activities, but a bulky wing structure affected both toxicity and efficacy. We hypothesize that the active center of these drugs might be the central tricyclic moiety and that a planar conformation of this structure is essential. Thus, the introduction of bulky groups might increase toxicity or decrease activity. Most phenoxaziniums showed good activities against P. falciparum. It was noteworthy that phenoxazinium derivatives had potent activity against T. cruzi.34

As compared to the phenoxazinium ions, the phenothiazinium ions such as methylene blue (31) displayed potent in vitro activity against P. falciparum K1 but their cytotoxicities were generally high.35

7. BENZO[a]PHENOXAZINES
Although phenoxazinium salts showed good efficacy in in vivo tests by oral administration, the toxicity study gave unsatisfactory results. Since the electrophilicity of the carbon atom at the 1 position in the phenoxazinium skeleton would be troublesome, we considered the effect of the addition of a benzene ring to the phenoxazinium framework. We therefore prepared and evaluated various benzo[a]phenoxazine derivatives. The corresponding salts of benzo[a]phenoxazines are a variant of DLC candidates but they would exist as a free base, non-DLC form, in the mammalian body. A number of benzo[a]phenoxazine derivatives were prepared by known methods36-40 and then subjected to the screening test. The results of some benzo[a]phenoxazines against P. falciparum K1, cytotoxicity toward L-6 myoblasts, and in vivo activity against P. berghei NK-65 are summarized in Table 11.

Benzo[a]phenoxazinium 98, having no substituent at the 6 position, showed weak activity against P. falciparum K1. The activity of Nile red (99) 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 (100) to 0.0156 µM. Although a low cytotoxicity was observed for 100, the in vivo activity was poor. The phenyl and tolyl derivatives 101 and 102 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 103 – 106 were prepared and evaluated but their in vivo efficacies were unsatisfactory. Improved in vivo efficacies were observed for the compounds 107, 108 and 109 carrying a pyridine ring, with 109 exhibiting the best activity among these three compounds. In addition to the inhibition of parasitemia, the mean survival days (MSD) after a single dose of 109 were extended to 14.6 days compared to approximately 6 days for an infected 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 strain in three independent experiments.
Although numerous analogues were prepared and assessed, only the 4-aminopyridine derivatives are presented here. Although the dimethyl derivative 110 showed potent activity, it produced shorter survival compared to 109. With longer substituents at the 9 position, 111 and 112 were less active. Morpholine compound 113 displayed good activity. Furthermore, substitution of a methyl group at the 11 position improved the safety and two derivatives 114 and 115 provided good in vivo efficacy. The hydrochlorides 114 and 115 gave similar in vivo activities as that of 109, suggesting that benzo[a]phenoxazine is apparently absorbed through the gut as the hydrochloride when administered p.o. Absence of cytotoxicity of compounds 116 and 117, 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.41
On the basis of the above findings including other factors such as ease of preparation and toxicity, SJL-183 (109) was selected for the further study.

7.1. SSJ-183 (109) as an Anti-malarial Agent
SSJ-183 (109) can be prepared from the commercially available 118 in one step (Scheme 6). Notably, the compound 109 is very stable as the free amine for a long period under ambient conditions. An alternative and more effective synthetic method of 109 has been elaborated.42

SSJ-183 (109) exhibited potent activity: an IC50 value of 0.0076 µM against P. falciparum K1, IC50 of 55.7 µM in the cytotoxicity test, selectivity of 7334, and >99.9% inhibition of P. berghei NK-65. To gain additional information on the in vivo efficacy of 109, we carried out a dose response experiment in NMRI female mice infected with P. berghei GFP ANKA strain (Table 12). High efficacy was observed by the p.o. route with cures achieved by oral administration of three daily doses of 100 mg/kg.

In other evaluations with compound 109, we detected no lethality at doses up to 2,000 mg/kg p.o. using 20 mice. Furthermore, no effects were found at 1,000 µM (- and + S9) in a chromosome aberration test, at 2.0 µM (- and + S9) in an in vitro micronucleus test and at 1,000 mg/kg x 2 in an in vivo rat micronucleus test. No phototoxicity was detected in mice dosed at 300 mg/kg p.o. In binding assays against 80 receptors, only two human recombinant receptors, A3 and D3, were inhibited ~80% at 1 µM and no inhibitions were noted with other receptors. The selectivity was further supported by ≥1000-fold higher IC50 values of 109 against three other protozoal parasites (36 µM for T. brucei rhodesiense, 11.3 µM for T. cruzi and 6.5 µM for L. donovani) 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 109 are much better than those of methylene blue. Furthermore, no hemolysis was observed in blood taken from G6PD deficient patient at Jichi Medical University.
In vivo pharmacokinetic studies in rats were carried out according to the reported procedure (Figure 10).43 After oral dosing, 109 had a bioavailability of approximately 30%. After i.v. administration, the terminal half life was approximately 5.5 h and 109 demonstrated a high volume of distribution and high clearance.

8. STUDY OF THE MODE OF ACTION
Rhodacyanines were accumulated into a special organelle, which was stained with fluorescence. Visualization of the fluorescent organelle showed it to be in close proximity to mitochondria.44 SSJ-127 (62) was detected in mouse malaria parasites using fluorescence imaging in vitro and in the experimentally administered model. Selective accumulation of 62 in an organelle was observed in all blood stages of live malaria parasites. The organelle was clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 (62) accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways.45
Further studies for the determination of the 3-D structure of receptors against our compounds are now underway.

9. CONSIDERATION OF A MOLECULAR MODEL
CPS models for two compounds, SJL-01 (74) and SSJ-183 (109), are illustrated in Figure 11. The two molecules are similar in having two planes composed of heteroaromatic rings that are intersected by some degree of an angle, resulting in their asymmetric shapes. These phenomena may be important in creating the specific biological properties of the molecule.

10. CONCLUSION
According to the working hypothesis based on DLC, several hopeful candidates have been discovered. Thus, rhodacyanine derivative SSJ-127 (62) cured rodent malaria (P. berghei) and T. bucei brucei infected mouse models. Fluorinated rhodacyanine SJL-01 (74) showed extraordinary efficacy against L. donovani in the in vivo test via i.v. administration. The activity of 74 was much better than or comparable to those of conventional medicines, which are toxic and expensive. Furthermore, benzo[a]phenoxazine derivative SSJ-183 (109) possessing the 4-aminopyridine moiety showed an IC50 value against P. falciparum of 7.6 nM and a selectivity index of >7,300. Cure was achieved with three daily oral doses to mice infected with P. berghei ANKA strain. Benzo[a]phenoxazines are not DLCs. Preliminary biological experiments clearly demonstrated that the target organelle for our compounds is not the mitochondrion but an organelle specific to protozoan parasites. Thus, high safety could be expected for these compounds.
Further extensions of these studies are now in progress.

ACKNOWLEDGEMENTS
The author expresses gratitude to co-workers whose names are written in the references and particularly to the collaborators at Hoshi University, Dr. Jian-Feng Ge (presently, Soochow University), Dr. C. Arai, Dr. Jun Lu, Dr. Khanitha Pudhom (presently, Chulalongkorn University), Dr. Mei Yang, Dr. Nasser S. M. Ismail (presently, Ain Shams University), Dr. Abu Bakar Md., and Yuki Mizukawa for their valuable contributions. He also thanks Professor Reto Brun, Marcel Kaiser and Dr. Sergio Wittlin at Swiss TPH, Vanessa Yardley at London School of Hygiene & Tropical Medicine, and Professor Susan A. Charman at Monash University for their kind assistance. Furthermore, Dr. Isamu Itoh, Dr. Hiroyuki Togashi and Seiki Sakanoue of Synstar Japan Co., Ltd., and Professor Terumi Nakajima and Professor Toshio Honda of Hoshi University are specially acknowledged for their encouragement. These studies were supported by a Grant-in-Aid for Scientific Research on Priority Areas and for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology, the Creation and Support Program for Start-ups from Universities, and Adaptable and Seamless Technology Transfer Program through Target-driven R&D from Japan Science and Technology Agency (JST) and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO).

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