JP7742093B2 - Malaria prevention medication - Google Patents

Description

The present invention relates to a drug for preventing and treating malaria.

Malaria, one of the world's three major infectious diseases along with HIV/AIDS and tuberculosis, is a parasitic disease caused by the malaria parasite Plasmodium. It is a typical mosquito-borne infection and is transmitted by the blood-sucking Anopheles mosquito. There are five species of malaria parasites that infect humans, but Plasmodium falciparum, which can cause severe and fatal diseases such as cerebral malaria, is the most widespread in the world, mainly in Africa.
More than 200 million new cases of malaria occur annually, primarily in developing countries in tropical and subtropical regions, and 420,000 people die from malaria. In endemic countries, malaria slows economic development, which in turn leads to malaria epidemics, creating a vicious cycle. Malaria and development are inextricably linked, and eradicating malaria will break this vicious cycle and enable sustainable development. For this reason, the Sustainable Development Goals (SDGs) adopted at the United Nations Summit in September 2015 include preventing the spread of malaria, along with tuberculosis and HIV/AIDS, as goals to be achieved by 2030.

Malaria parasites have two hosts: humans and mosquitoes, and in humans, they parasitize the liver and red blood cells. They adapt to each environment by changing their morphology and metabolism within the host and parasite. In particular, parasitic parasites within red blood cells (blood-forming parasites) are closely related to the pathology of malaria, and drugs that inhibit the growth and proliferation of blood-forming parasites are expected to have a significant therapeutic effect.

Various efforts are being made worldwide to eliminate malaria. Two effective methods are widely used to eliminate vector mosquitoes: the distribution of insecticide-treated bed nets and indoor spraying with residual insecticides. Furthermore, early treatment of malaria can prevent deaths.
The current first-choice drug is artemisinin combination therapy, which combines an artemisinin derivative with one of the following drugs: lumefantrine, mefloquine, piperaquine, amodiaquine, or fansidar. This treatment quickly eliminates the parasite and relieves malaria symptoms. Artemisinin combination therapy is currently used as the first-choice treatment in all endemic countries, including those in Africa, and its effectiveness has led to a decline in malaria-related deaths since the mid-2000s.

However, malaria parasites resistant to artemisinin have appeared in the Mekong River basin in Southeast Asia, and their distribution is gradually expanding. No new antimalarial drugs with the same effectiveness as artemisinin have yet been approved, and the spread of resistance will pose a major socioeconomic problem, not only in malaria control but also in all endemic regions, not just in Africa, where 90% of malaria patients are concentrated. Therefore, there is a need to develop drugs that selectively inhibit the growth and proliferation of malaria parasites.

Meanwhile, some of the present inventors have discovered and reported that an amide derivative of differentiation-inducing factor-1 (DIF-1), a differentiation-inducing factor from slime mold that induces stalk cell differentiation in slime molds and is isolated as a degradation product of this factor, has the ability to induce cellular slime mold differentiation, inhibit tumor cells, and promote sugar metabolism (Patent Document 1, Non-Patent Document 1).

Patent No. 5476650

Kikuchi H, Ishiko S, Oshima Y, Gokan N, Hosaka K, & Kubohara Y. Biological activities of novel derivatives of DIF-1 isolated from Dictyostelium. Biochem. Biophys. Res. Commun. 377(3), 1012-1017. (2008).

The objective of the present invention is to provide a new malaria preventive and therapeutic drug that inhibits the proliferation of malaria parasites.

The inventors therefore investigated the effect of the aforementioned DIF-1 amide derivatives on malaria parasites, the effects of which on malaria parasites were completely unknown, and found that they have a strong growth inhibitory effect, and also exhibit growth inhibitory effect on artemisinin-resistant malaria parasites, leading to the completion of the present invention.

That is, the present invention provides the following inventions [1] to [8].
[1] A preventive and therapeutic drug for malaria, comprising as an active ingredient a compound represented by the following general formula (1) or a salt thereof:

(wherein ^R1 represents an alkyl group having 1 to 10 carbon atoms, ^R2 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent, ^R3 represents a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent, or ^R2 and ^R3 may together with the nitrogen atom form a cyclic amino group, and ^X1 and ^X2 independently represent a halogen atom)
[2] The preventive and therapeutic drug for malaria according to [1], wherein R ¹ is an alkyl group having 1 to 5 carbon atoms.
[3] The preventive and therapeutic drug for malaria according to [1] or [2], wherein R ² represents a hydrogen atom or a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms which may have a substituent, and ^{R 3} represents a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms which may have a substituent, or R ² and R 3 together with the nitrogen atom may form a cyclic amino group having 2 to 8 carbon atoms.
[4] The therapeutic agent for malaria parasite infection according to [1] or [2], wherein R ² represents a hydrogen atom or a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, and R ³ represents a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, or R ² and R ³ together with the nitrogen atom may form a cyclic amino group having 2 to 8 carbon atoms.
[5] A growth inhibitor for malaria parasites, comprising a compound represented by the following general formula (1) or a salt thereof as an active ingredient:

(wherein ^R1 represents an alkyl group having 1 to 10 carbon atoms, ^R2 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent, ^R3 represents a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent, or ^R2 and ^R3 may together with the nitrogen atom form a cyclic amino group, and ^X1 and ^X2 independently represent a halogen atom)
[6] The growth inhibitor of malaria parasites according to [5], wherein R ¹ is an alkyl group having 1 to 5 carbon atoms.
[7] The growth inhibitor of malaria parasites according to [5] or [6], wherein R ² represents a hydrogen atom or a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms which may have a substituent, and R ³ represents a linear, branched or cyclic alkyl group having 1 to 12 carbon atoms which may have a substituent, or R ² and R ³ together with the nitrogen atom may form a cyclic amino group having 2 to 8 carbon atoms.
[8] The growth inhibitor of malaria parasites according to [5] or [6], wherein R ² represents a hydrogen atom or a linear, branched, or cyclic alkyl group having 1 to 12 carbon atoms, and R ³ represents a linear, branched, or cyclic alkyl group having 1 to 12 carbon atoms, or R ² and R ³ together with the nitrogen atom may form a cyclic amino group having 2 to 8 carbon atoms.

The compound represented by the general formula (1) or a salt thereof has excellent inhibitory activity against the proliferation of malaria parasites, and also exhibits inhibitory activity against artemisinin-resistant malaria parasites, making it useful as a preventive or therapeutic drug for malaria.

FIG. 1 shows the effect of compound (1) on the proliferation of malaria parasite 3D7.

The active ingredient of the malaria preventive/therapeutic drug and malaria parasite growth inhibitor of the present invention is a compound represented by the general formula (1) (hereinafter also referred to as compound (1)) or a salt thereof. Compound (1) can be synthesized according to the method described in Patent Document 1.
As described in Patent Document 1 and Non-Patent Document 1, this compound (1) is known to have the effects of inducing cellular slime mold differentiation, inhibiting tumor cells, and promoting glucose metabolism, but its effect on malaria parasites is not known at all.

In the general formula (1), R ¹ represents an alkyl group having 1 to 10 carbon atoms.
Here, examples of the alkyl group include linear, branched, and cyclic alkyl groups. A linear or branched alkyl group having 1 to 5 carbon atoms is preferred, and an alkyl group having 1 to 3 carbon atoms is more preferred. Specific examples of ^R1 include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and an n-pentyl group, with a methyl group, an ethyl group, an n-propyl group, and an isopropyl group being more preferred.

^R2 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent.
Here, the hydrocarbon group having 1 to 20 carbon atoms, which may have a substituent, is preferably a linear, branched, or cyclic alkyl group having 1 to 12 carbon atoms, which may have a substituent, and more preferably a linear, branched, or cyclic alkyl group having 1 to 12 carbon atoms. Here, examples of groups that can be substituted on the hydrocarbon group include an alkoxycarbonyl group, a carboxyl group, a hydroxy group, and an alkoxy group.
Preferred examples of ^R2 include a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, and an n-dodecyl group.

^R3 represents a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent.
Here, the hydrocarbon group having 1 to 20 carbon atoms, which may have a substituent, is preferably a linear, branched, or cyclic alkyl group having 1 to 12 carbon atoms, which may have a substituent, and more preferably a linear, branched, or cyclic alkyl group having 1 to 12 carbon atoms. Here, examples of groups that can be substituted on the hydrocarbon group include an alkoxycarbonyl group, a carboxyl group, a hydroxy group, and an alkoxy group.
Preferred examples of ^R3 include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-dodecyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.

^R2 and ^R3 may combine with the nitrogen atom to form a cyclic amino group. Examples of such a cyclic amino group include cyclic amino groups having 2 to 7 carbon atoms, such as a pyrrolidino group, a piperidino group, and a heptacycloimino group.

^X1 and ^X2 independently represent a halogen atom. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, with a chlorine atom, a bromine atom, and an iodine atom being preferred.

Specific preferred examples of compound (1) include the following compounds:

Salts of compound (1) are not limited as long as they are pharmaceutically acceptable, and examples include metal salts such as sodium, potassium, magnesium, and calcium, and ammonium salts. Compound (1) may also be a hydrate. Furthermore, if compound (1) has an asymmetric carbon atom in its structure, its optical isomers are also included.

As will be shown in the Examples below, compound (1) or a salt thereof has an excellent inhibitory effect on the proliferation of malaria parasites, and also exhibits an inhibitory effect on the proliferation of artemisinin-resistant malaria parasites, and is therefore useful as a preventive or therapeutic drug for malaria.
Examples of target malaria parasites include malaria parasites (Plasmodium genus parasites) that infect primates and rodents. Examples of malaria parasites that infect humans, which belong to primates, include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, preferably Plasmodium falciparum. Examples of malaria parasites that infect monkeys, which belong to primates, include Plasmodium knowlesi. Furthermore, examples of malaria parasites that infect rodents include Plasmodium berghei and Plasmodium vinckei, and examples of malaria parasites that infect mice, which belong to rodents, include Plasmodium berghei, Plasmodium chabaudi, and Plasmodium yoelii.

The malaria preventive and therapeutic drugs of the present invention include not only medicines that completely cure symptoms caused by infection with malaria parasites, but also medicines that ameliorate symptoms and medicines that prevent the onset of symptoms. Furthermore, the malaria parasite growth inhibitors of the present invention include drugs that reduce the number of malaria parasites, drugs that suppress an increase in the number (growth) of malaria parasites, drugs that inactivate malaria parasites, and the like. In the present invention, "inactivating malaria parasites" includes reducing or suppressing the ability of malaria parasites to reproduce, and an example of a means of inactivation is killing them.

The malaria preventive/therapeutic drug and malaria parasite growth inhibitor of the present invention are administered to animals infected with malaria or animals that have developed malaria. Such animals include, for example, primates and rodents; specific examples of primates include humans, chimpanzees, and monkeys, and specific examples of rodents include mice. In addition to animals infected with or that have developed malaria, as described above, the drugs can also be administered prophylactically to animals that may be susceptible to malaria infection.

A pharmaceutical containing compound (1) or a salt thereof can be prepared into various pharmaceutical preparations by mixing the compound or a salt thereof with a pharmaceutically acceptable carrier or as is according to a known method commonly used in the production of pharmaceutical preparations. Such pharmaceutical preparations (dosage forms) can be, for example, tablets (including sugar-coated tablets and film-coated tablets), powders, granules, capsules (including soft capsules), liquids, injections, suppositories, sustained-release preparations, etc., and can be administered orally or parenterally (e.g., topically, rectally, intravenously, etc.).
Examples of pharmaceutically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents in liquid preparations. Furthermore, conventional additives such as preservatives, antioxidants, coloring agents, sweeteners, adsorbents, and wetting agents can also be used appropriately and in appropriate amounts, if necessary.
Examples of excipients include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose, etc. Examples of disintegrants include starch, carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethylstarch, L-hydroxypropylcellulose, etc.
Examples of solvents include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil, etc. Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, and D-mannitol. Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Examples of soothing agents include benzyl alcohol. Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Examples of antioxidants include sulfites, ascorbic acid, α-tocopherol, etc.

Furthermore, the pharmaceutical of the present invention may be used in combination with antimalarial drugs other than compound (1) or other drugs.
The content of compound (1) or a salt thereof in the pharmaceutical composition of the present invention is 0.01 to 100% by mass of the total preparation.
The dose of compound (1) or a salt thereof varies depending on the subject, symptoms, body weight, administration method, etc., and is not particularly limited. In general, the dose is preferably 0.1 to 5 g, more preferably 0.1 to 1 g, and even more preferably 0.1 to 0.5 g per day for a patient (body weight 60 kg).

The present invention will now be described in more detail using examples, but the present invention is not limited to these examples.

Example 1
The following compounds were synthesized according to the method described in Patent Document 1.

The above compounds (hereinafter also referred to as DIF amides) were all colorless amorphous solids. These DIF amides were dissolved in DMSO to form 10 mM solutions and stored at -20°C.

(Existing antimalarial drugs as control)
For drug testing, the existing antimalarial drug chloroquine was used as a positive control. Chloroquine was dissolved in ultrapure water. The artemisinin drug resistance assay system was excluded from the control because it uses a special method.

Example 2 (Study of antimalarial activity of DIF amide)
(Culture strains used)
Here, we used the cultured Plasmodium falciparum strain 3D7 and the artemisinin-resistant strains MRA1236 and MRA1240 (which are also resistant to chloroquine).

(Screening for antimalarial activity)
Protozoan growth rates were compared in the presence of DIF amide (10 μM) or 0.1% DMSO (control). Protozoan hematocrit was adjusted to 2% and infection rate to 0.05%, and they were cultured at 37°C in a gas mixture (90% _N , 5% _O , 5% _CO ) for 72 hours. The media used were RPMI 1640 (GIBCO), hypoxanthine, Gentamicin Reagent Solution (GIBCO), Sodium Bicarbonate Solution, AlbuMAX1 (GIBCO), and standard Malaria Complete Medium (MCM) supplemented with deionized serum (donated by the Japanese Red Cross Society). The number of parasites was measured by ELISA using HRP-2, a malaria-specific antigen. When 10 μM DIF amide inhibited proliferation by 40% or more, it was determined that the compound had protozoan proliferation inhibitory activity.
The results are shown in Figure 1. As can be seen from Figure 1, the DIF amide was found to have antiproliferative activity.

Example 3 (Evaluation of antimalarial activity based on _IC50 value)
Three types of Plasmodium falciparum strains were cultured in the presence of DIF amide diluted in seven stages with DMSO, and the growth inhibitory effect was examined. The growth inhibitory effect was evaluated by the number of parasites. The culture conditions and the method for measuring the number of parasites were the same as in Example 2. The _IC50 value was determined using the Sigmoid Emax method. The results are shown in Table 1.

The _{IC50 values} of DIF1[A+3], DIF-1[A+4], and Amide4 were 0.5-2.05 μM. These values nearly meet the 1-2 μM target for selecting lead compounds as antimalarial drugs. MRA1236 and MRA1240 are artemisinin-resistant culture strains, and Amide4 showed growth effects on the artemisinin-resistant strains similar to those on the susceptible strains.

Claims (4)

A preventive and therapeutic drug for malaria, comprising as an active ingredient a compound represented by the following general formula (1) or a salt thereof.
(wherein ^R1 represents an alkyl group having 1 to 10 carbon atoms, ^R2 represents a hydrogen atom or a linear, branched, or cyclic alkyl group having 5 to 12 carbon atoms , ^R3 represents a linear, branched, or cyclic alkyl group having 5 to 12 carbon atoms , or ^R2 and ^R3 may together with the nitrogen atom form a cyclic amino group having 5 to 8 carbon atoms , and ^X1 and ^X2 independently represent a halogen atom) 2. The preventive and therapeutic drug for malaria according to claim 1, wherein R ¹ is an alkyl group having 1 to 5 carbon atoms. A growth inhibitor of malaria parasites, comprising as an active ingredient a compound represented by the following general formula (1) or a salt thereof:
(wherein ^R1 represents an alkyl group having 1 to 10 carbon atoms, ^R2 represents a hydrogen atom or a linear, branched, or cyclic alkyl group having 5 to 12 carbon atoms , ^R3 represents a linear, branched, or cyclic alkyl group having 5 to 12 carbon atoms , or ^R2 and ^R3 may together with the nitrogen atom form a cyclic amino group having 5 to 8 carbon atoms , and ^X1 and ^X2 independently represent a halogen atom) 4. The agent for inhibiting the proliferation of malaria parasites according to claim 3 , wherein R ¹ is an alkyl group having 1 to 5 carbon atoms.