WO2010036908A1

WO2010036908A1 - Use of benzoxazole compounds in the treatment of malaria

Info

Publication number: WO2010036908A1
Application number: PCT/US2009/058401
Authority: WO
Inventors: Wanjun Zheng; Mark Spyvee; Fabian Gusovsky; Sally T. Ishizaka
Original assignee: Eisai R&D Management Co Ltd
Current assignee: Eisai R&D Management Co Ltd
Priority date: 2008-09-26
Filing date: 2009-09-25
Publication date: 2010-04-01
Anticipated expiration: 2011-03-26
Also published as: BRPI0919816A2

Abstract

The present invention provides methods of preventing or treating malaria using benzoxazole compounds. The present invention further provides pharmaceutical compositions including an effective amount of the benzoxazole compounds for the prevention or treatment of malaria.

Description

USE OF BENZOXAZOLE COMPOUNDS IN THE TREATMENT OF MALARIA

BACKGROUND OF THE INVENTION

[0001] Malaria constitutes one of the most devastating global health problems in human history. Infection with malarial parasites affects more than 300 million people annually, killing over one million children. (Snow et al. Nature 434: 214-217 (2005) and

J Health Sci. 5:58-62 (1998)). The pathogenesis of malaria is multifactorial, and serious sequalae can result from three primary pathophysiological events: (i) red blood cell destruction; (ii) adhesion of infected erythrocytes to the capillary veins; and (iii) an excessive pro-inflammatory response. Excessive pro-inflammatory response is responsible for sepsis- like signs and symptoms such as rigors, headache, chills, spiking fever, sweating, vasodilatation and hypoglycemia. (Clark et al. Malaria Journal 5 (2006); Stevenson et al. Nat. Rev. Immunol. 4:169-180 (2004) and Schofield et al. Nature Reviews Immunology 5:722-735 (2005)). Cerebral malaria is a severe neurological complication of malarial infection and is a major cause of acute non-traumatic encephalopathy in tropical countries. (Idro et al. Lancet Neurol. 4: 827-840 (2005)).

[0002] Toll-like receptors (TLRs) are germline encoded receptors of the innate immune system that sense the presence of microbes. (Medzhitov. Nat. Rev. Immunol. 1 :135-145 (2001)). TLRs recognize and are activated by a variety of microbial products such as lipopolysaccharide (LPS, endotoxin) from gram-negative bacteria and viral nucleic acids. More recently, it has been shown that TLRs also recognize glycolipids and DNA derived from protozoan parasites, including Plasmodium, a genus of parasites that cause malaria. (Campos et al. J. Immunol. 167:416-423 (2001); Gazzinelli et al. Nat. Rev. Immunol. 6:895- 906 (2006); Krishnegowda et al. Journal of Biological Chemistry 280(9):8606-8616 (2005); Coban, et al. J Exp. Med 201(1): 19-25. (2005); and Parroche et al. Proc. Natl Acad Sci USA 104(6): 1919-1924 (2007)). There is a growing body of literature suggesting that TLRs are central mediators of pro-inflammatory responses during malaria (Medzhitov. New Engl. J Med. 343(5):338-344 (2000)) and that TLR antagonists may be useful in the treatment of malaria. In particular, TLR9 has been shown to have a role in recognizing Plasmodium DNA and eliciting pro-inflammatory cytokines during malaria (Parroche et al. Proc. Natl. Acad Sci USA 104(6):1919-1924 (2007)). Studies have indicated that TLR9 seems to mediate some of the pathogenic events during acute episodes of malaria (Coban et al. Int. Immunol. 19(1):67- 79(2007); Mockenhaupt FP, J Infect Dis 194(2): 148-8 (2006)). SUMMARY OF THE INVENTION

[0003] Embodiments of the present invention provide a method of preventing or treating malaria including administering to a subject a compound as described herein in an effective amount.

[0004] Embodiments of the present invention further provide pharmaceutical compositions including an effective amount of a compound as described herein for the prevention or treatment of malaria.

[0005] Embodiments of the present invention also provide the use of a compound as described herein for the manufacture of a medicament for the prevention or treatment of malaria.

[0006] Other embodiments of the present invention are disclosed herein and discussed in greater detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0007] Figure 1 shows results of pro-inflammatory cytokine suppression in splenocytes of C57BL/6 mice with Compound 23 nine days after Plasmodium chabaudi (P chabaiidi) infection and daily administration of 0 mg/kg. 20 mg/kg or 60 mg/kg Compound 23. beginning one day prior to infection, as described in Example 8. Panel A shows the effect of Compound 23 on IL- 12 production. Panel B shows the effect of Compound 23 on TNF-α production. Panel C shows the effect of Compound 23 on INF-γ production. Panel D shows the effect of Compound 23 on IL-10 production. Panel E shows the effect of Compound 23 on monocyte chemotactic protein- 1 (MCP-I) production. Panel F shows the effect of Compound 23 on IL-6 production.

[0008] Figure 2 shows results of serum cytokine suppression in C57BL/6 mice with Compound 23 nine days after P chabaudi infection and daily administration of 0 mg/kg. 20 mg/kg or 60 mg/kg Compound 23, beginning one day prior to infection, as described in Example 9. The effect of Compound 23 on serum levels of INF-γ. TNF-α. MCP-I and IL-10 are shown.

[0009] Figure 3 shows the lack of a significant effect on body temperature in C57BL/6 mice after dosing of Compound 23 administered orally at 60 mg/kg, as described in Example 10.

[0010] Figure 4 shows the lack of a significant effect on body weight in C57BL/6 mice after dosing of Compound 23 administered orally at 60 mg/kg. as described in Example 10. [0011] Figure 5 shows the lack of a significant effect on parasitemia in C57BL/6 mice after dosing of Compound 23 administered orally at 60 mg/kg. as described in Example 10.

[0012] Figures 6 through 9 show the results of inhibition of in vivo priming of TLR responses in C57BL/6 mice after dosing of Compound 23 administered orally at 60 mg/kg one week after infection and daily administration of Compound 23. beginning one day prior to infection, as described in Example 11. INF-γ (Figure 6), IL- 12 (Figure 7). nitric oxide

(NO) (Figure 8) and IL-IO (Figure 9) were measured.

[0013] Figure 10 shows the lack of a significant effect on body weight in C57BL/6 mice after dosing of compound 23 administered orally at 120 mg/kg/day as described in Example

13. Part A.

[0014] Figure 11 shows the lack of a significant effect on parasitemia in C57BL/6 mice after dosing of Compound 23 administered orally at 120 mg/kg/day as described in Example

13. Part A.

[0015] Figure 12 shows the significant effect on survival of C57BL/6 mice after dosing of Compound 23 administered orally at 120 mg/kg/day as described in Example 13. Part B.

[0016] Figure 13 shows the inhibition of in vivo priming of LPS response in C57BL/6 mice after dosing of Compound 23 administered orally at 120 mg/kg/day beginning one day prior to infection until day seven post-infection as described in Example 13. Part C. Mice were sacrificed on day 8 post-infection and IFNγ responses to LPS were evaluated in splenocyte cultures.

[0017] Figure 14 shows a significant improvement in clinical symptoms/signs in

C57BL/6 mice after dosing of Compound 23 administered orally at 120 mg/kg/day, beginning one day prior to infection until day 12 post-infection, as described in Example 13.

Part D.

[0018] Figure 15 shows a significant decrease in numbers of vascular lesions in the central nervous system of C57BL/6 mice after dosing of Compound 23 administered orally at

120 mg/kg/day beginning one day prior to infection until day 9 post-infection, when mice were sacrificed for histopathological analysis. The graph on the right shows histological quantification of vascular lesions in mice treated with vehicle or Compound 23. as described in Example 13. Part E.

[0019] Figure 16 shows the impairment of cytokine production in CDl Ic" splenic dendritic cells (DCs) by Compound 23 in mice infected with P berghei ANKA (1 x 10E5

PbA iPvBCs). as described in Example 14. DETAILED DESCRIPTION A. Definitions

[0020] "Treatment", "treat", and "treating" refer to reversing, alleviating, or inhibiting the progress of a disorder or disease as described herein. Moreover, as used herein, "treatment" of a subject includes the application or administration of a compound of the invention described herein to a subject, or application or administration of the compound to a cell or tissue from a subject, who has malaria or a malaria-related disease or condition, has a symptom of such a disease or condition, or is at risk of (or susceptible to) such a disease or condition, with the purpose of curing, healing, alleviating, relieving, altering, remedying. ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission: diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating: improving a subject's physical or mental well-being: or. in some situations, preventing the onset of a more severe form or symptom of the disease or condition. The treatment or amelioration of symptoms can be based on objective or subjective parameters: including the results of a physical examination, a psychiatric evaluation, or a diagnostic test such as peripheral smear study for malarial parasite (MP Test), a Quantitative Buffy Coat (QBC) Test, fluorescence microscopy of parasite nuclei stained with acridine orange, a Rapid Diagnostic Test (RDT), a rapid dipstick immunoassay, a Polymerase Chain Reaction (PCR) assay, or another test known in the art.

[0021] In one embodiment, the term "treating" includes improving a symptom of malaria such as improvement or relief from confusion, coma, neurologic focal signs, severe anemia, and/or respiratory difficulties caused by or associated with malaria. In another embodiment, "treating" includes a reduction in the number of parasites as determined by one or more assays, e.g.. a reduced number of parasites per microliter as tested in a thick smear Malarial Parasite (MP) Test or another test known in the art.

[0022] "Prevention", "prevent", and "preventing" refer to eliminating or reducing the incidence or onset of a disorder or disease as described herein, as compared to that which would occur in the absence of the measure taken.

[0023] "Effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation. and/or the like. An "effective amount" can further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and/or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" can designate an amount that maintains a desired physiological state. i.e.. reduces or prevents significant decline and/or promotes improvement in the condition of interest. An "effective amount" can further refer to a therapeutically effective amount. [0024] "Subject", as used herein, means a mammalian subject (e.g , dog. cat. horse, cow. sheep, goat, monkey, etc.). and particularly human subjects (including both male and female subjects, and including neonatal, infant, juvenile, adolescent, adult, and geriatric subjects, further including pregnant subjects and further including various races and ethnicities including, but not limited to. white, black. Asian, American Indian and Hispanic). [0025] As used herein, the term "a pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic and organic acid salts of compounds of the invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative acid salts include acetate, adipate. aspartate, benzoate. besylate. bicarbonate/carbonate, bi sulphate/sulphate, borate, camsylate. citrate, cyclamate, edisylate. esylate. formate, fumarate. gluceptate. gluconate, glucuronate. hexafluorophosphate. hibenzate. hydrochloride/chloride, hydrobromide/bromide. hydroiodide/iodide. isethionate. lactate, malate. maleate. malonate. mesylate, methylsulphate. naphthylate. 2-napsylate. nicotinate. nitrate, orotate. oxalate, palmitate. pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate. saccharate. stearate, succinate, tannate, tartrate, tosylate. trifluoroacetate and xinafoate salts. In one embodiment, the pharmaceutically acceptable salt is a hydrochloride/chloride salt.

[0026] As used herein, "mean parasitemia" refers to the average of percent parasitemia of a group of mice at each time point. Percent parasitemia refers to the percentage of infected red blood cells (iRBCs) divided by the number of total RBCs counted in blood smears from each mouse (x 100) at different time points post infection.

B. Compounds

[0027] Some of the compounds described herein can comprise one or more asymmetric centers, and thus, the compounds can exist in various isomeric forms, e.g.. stereoisomers and/or diastereomers. When the orientation of a bond around a chiral center is not specified in a formula, it is to be understood that the formula encompasses every possible isomer such as geometric isomer, optical isomer, stereoisomer and tautomer based on asymmetric carbon, which can occur in the structures of the inventive compounds and mixtures of such isomers. [0028] As noted above, the present invention provides a method of treating malaria in a subject, comprising administering to the subject an effective amount of a compound having a structure selected from:

or a pharmaceutically acceptable salt thereof.

[0029] In another embodiment, the compound used in the present invention is selected from the group consisting of Compound 13. Compound 16. Compound 30. Compound 34. and Compound 35 or a pharmaceutically acceptable salt thereof. In another embodiment, the compound is selected from the group consisting of Compound 23. Compound 26. and Compound 30 or a pharmaceutically acceptable salt thereof. In yet another embodiment, the compound is Compound 23 or a pharmaceutically acceptable salt thereof.

C. Synthesis of Compounds

[0030] Compounds described herein can be prepared by a sequence of reactions. Methods of synthesis of specific compounds are shown in the examples provided below.

D. Pharmaceutical Compositions

[0031] In one embodiment, the present invention is a pharmaceutical composition comprising the compound. In another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable earner. The term "pharmaceutically acceptable carrier" as used herein refers to any substance, not itself a therapeutic agent, used as a vehicle for delivery of a therapeutic agent to a subject.

[0032] Administration of an effective amount of the compounds described herein includes administration of the compounds as formulated for oral, parenteral, inhalation spray, topical, rectal, nasal, sublingual, buccal, vaginal or implanted reservoir administration, etc

E. Methods of prevention and treatment

[0033] Methods of the present invention include administration of compounds described herein to prevent or treat malaria. In one embodiment, the compound is selected from the group consisting of Compound 13. Compound 16. Compound 22. Compound 23. Compound 26. Compound 27. Compound 30. Compound 34. and Compound 35. or a pharmaceutically acceptable salt thereof. In a particular embodiment, the compound is selected from the group consisting of Compound 23. Compound 26. and Compound 30 or a pharmaceutically acceptable salt thereof. In another embodiment, the compound is Compound 23 or a pharmaceutically acceptable salt thereof.

[0034] As used herein "malaria" refers to a clinical syndrome caused by infection with Plasmodium species. Currently, P falciparum and P vivax account for the majority of human malaria cases. If not treated early and effectively, infection with P falciparum may lead to single or multi-organ failure and severe malaria. Infection with P vivax may result in less severe disease: however, there may be periodic relapses at approximately 6-9 month intervals, due to reactivation of parasites (hypnozoites) that remain dominant in the liver after initial infection.

[0035] As used herein "severe malaria" refers to malaria cases in which the subject may show one or more of the following signs or symptoms as defined by the World Health Organization (WHO): prostration, impaired consciousness (coma, cerebral malaria), respiratory distress, multiple convulsions, circulatory collapse, pulmonary edema, bleeding, jaundice, hemoglobinuria, severe anemia, hypoglycemia, acidosis, renal impairment, hyperlactatemia and hyperparasitemia. As used herein "cerebral malaria" refers to severe P falciparum infection associated with coma, seizures and/or nonresponsiveness. [0036] In some embodiments of the present invention, the malaria is severe malaria. In some other embodiments of the present invention, the malaria is cerebral malaria. [0037] According to further embodiments of the present invention, the compounds described herein can be used to treat subjects who present with high parasitemia, i.e., hyperparasitemia. In some embodiments, the compounds described herein can be used to treat subjects who have shown treatment failure with conventional antimalarial treatments. In further embodiments, the compounds described herein can be used to prevent further development of additional signs and/or severity of malaria.

[0038] In some embodiments, the compounds described herein can be co-administered with an additional antimalarial compound and/or additional agent for reversing (i.e.. treating) antimalarial resistance, i.e.. a combination therapy. The additional antimalarial compound and/or additional agent for reversing antimalarial resistance can be one or more of the following classes of drugs: quinolines. folic acid antagonists, sulfonamides and antibiotics. In some embodiments, the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is at least one of the following: chloroquine. hydrox} chloroquine. amodiaquine, sulfadoxine. pyrimethamine, mefloquine, artemisinin. artemether. artesunate. dihydroartemismin. artemotil. lumefantrine. primaquine, atovaquone. proguanil. chlorproguanil. dapsone. quinine, quinidine. quinacrine. tetracycline, doxycycline and/or clindamycin. In particular embodiments, the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is an artemisinin-derived compound. As used herein an "artemisinin-derived compound" is artemisinin or a natural or synthetic derivative thereof. Examples of artemisinin-derived compounds include, but are not limited to, artesunate. artemether. artemotil. dihydro artemisinin and artemisinin. In some embodiments the artemisinin-derived compound is artesunate.

[0039] In some embodiments, the compounds described herein are administered prior to. concurrently with or subsequent to the administration of the additional antimalarial agent and/or the agent for reversing antimalarial resistance.

[0040] Administration of the compounds described herein "prior to" administration of the additional agents refers to administering the compounds described herein to a subject prior to initial treatment with the additional antimalarial agent and/or the agent for reversing antimalarial resistance, or prior to administration of the additional agents during a treatment protocol that includes administering the compounds described herein and the additional agents to a subject population that is at risk or afflicted with malaria, severe malaria, cerebral malaria and/or high parasitemia.

[0041] With concurrent administration, the compounds described herein and the additional agents are administered at the same point in time or immediately following one another. In general, the compounds described herein and the additional agents are administered at times sufficiently close that the results observed are relatively indistinguishable from those achieved when they are administered at the same point in time. [0042] Methods of the present invention include administering the compounds described herein to subjects by any suitable route, including orally (inclusive of administration via the oral cavity and further includes administration via an orogastric feeding tube), parenterally, by inhalation spray, topically, transdermally, rectally (for example, via suppository), nasally (including a nasogastric feeding tube), sublingually, buccally. vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial. intrasternal. intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compounds are administered orally, parenterally. rectally. transdermally or by inhalation spray. [0043] Compounds are administered to the subjects in an effective amount. An effective amount is generally 0.01 mg/kg to 500 mg/kg body weight per day. In some embodiments, compounds provided in pharmaceutically acceptable compositions may be formulated so that a dosage of between 0.01 mg/kg to 120 mg/kg body weight per day of a compound described herein can be administered to a patient receiving the compound. In certain embodiments, the compounds described herein are provided in a composition formulated to provide a dosage of between 0.01 mg/kg and 100 mg/kg. In other embodiments, the compounds described herein are provided in a composition formulated to provide a dosage of between 0.1 mg/kg and 25 mg/kg or between 5 mg/kg and 60 mg/kg.

[0044] In some embodiments, the effective dose is between about 5 and 250 mg/kg. between about 10 and 200 mg/kg. or between about 20 and 120 mg/kg. In some embodiments, effective dosages include 5 mg/kg. 10 mg/kg. 20 mg/kg. 25 mg/kg. 40 mg/kg. 50 mg/kg. 60mg/kg. 75 mg/kg. 100 mg/kg, 120 mg/kg and 150 mg/kg. Dosage forms can be in the form, e.g., of tablets or capsules, and the effective dose may be provided in one or more tablets, capsules or the like, and be provided once a day or throughout the day at intervals, e.g.. of 4. 8 or 12 hours. Tablets or capsules, for example, could contain, e.g.. 10, 25. 50. 75. 100, 150. 200 mg of compound. Liquid formulations could also be prepared so that any dosage could readily and conveniently be dispensed.

[0045] In some embodiments, the invention is a pharmaceutical formulation comprising an amount of the compounds described herein in a single or multiple dosage form. The amount of the compound in a dosage form will vary depending upon the host treated, and the particular route of administration.

[0046] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age. body weight, general health, gender, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound described herein in the composition will also depend upon the particular compound in the composition.

[0047] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. A comparator compound is used in several of the examples, below. The formula of the comparator is as follows:

EXAMPLE 1 Synthesis of Compound 40

Compound 39 Compound 40

[0048] Compound 40. As depicted above, l-bromo-3-chloropropane (7.3 mL. 0.074 mol) was added to a stirring mixture of Compound 39 (5.62 g. 0.0247 mol) and potassium carbonate (10 g, 0.074 mol) in DMSO (50 mL) at room temperature and the resulting mixture was stirred for 4 hours and thin-layer chromatography (TLC) showed the reaction was completed. The mixture was then poured into water (200 mL). extracted three times in 150 niL ethyl acetate (EtOAc), dried over Na₂SO₄, filtered and concentrated. Vacuum chromatography (10% to 30% EtOAc/hexane) of the residue gave a white solid product Compound 40 (8.10 g. 86%). Crystallization in hexane/EtOAc gave a white pure solid product (6.40 g) and mother liquor with minor impurities (1.6Og). ¹H NMR (400 MHz, CD₃OD) δ 8.11 (d, J=9.1 Hz. 2H). 7.55 (d. J=8.8 Hz. IH). 7.27 (d, J=2.3 Hz, IH). 7.11 (d. J=9.1 Hz. 2H). 7.00 (dd. J=2.3, 8.8 Hz. IH). 4.22 (t. J=5.9 Hz. 2H), 4.19 (t. J=6.15, 2H), 3.78 (tt. J=6.4 Hz. 4H). 2.30 (dt, J=I .76. 1.76 Hz. 4H). For preparation of starting compound Compound 39. see J Med Chem 2004, 47, 5021-5040.

[0049] The same reaction was performed with isomers of Compound 39. Isomers Compound 47 and Compound 48 were prepared via the synthetic procedure in J Med Chem 2004. 47. 5021-5040.

Compound 47 Compound 48

EXAMPLE 2 Synthesis of Compound 23

Compound 40 Compound 23

[0050] Compound 23. Referring to the scheme above, a mixture of Compound 40 (8.40 g. 0.0221 mol) produced as described in Example 1 and pyrrolidine (7.4 mL, 0.088 mol) in ethanol (50 mL) was heated at 100 ⁰C for 12 hours in a sealed container. TLC (20% triethylamine in methanol (Et₃NMeOH)) showed a single new spot and mass spectroscopy showed one single desired peak of 450 (M+H). The mixture was cooled to room temperature, concentrated and the excess pyrrolidine was removed by azeotropic concentration with toluene (three times in 50 mL). The residue was dissolved in ethanol (EtOH). filtered and concentrated. The residual solid was crystallized in MeOH/EtOAc to give pure product Compound 23 (7.88 g. 68%). ¹H NMR (400 MHz, CD₃OD) δ 8.12 (d, J=8.9 Hz, 2H). 7.57 (d. J=8.8 Hz. IH). 7.28 (d, J=2.2 Hz. IH), 7.12 (d, J=8.9 Hz. 2H), 7.01 (dd. J=2.2, 8.8 Hz, IH). 4.18 (t. J=5.86 Hz. 2H). 4.15 (t. J=6.15 Hz, 2H). 3.07-2.94 (m. 12H). 2.15 (m. 4H)₅ 1.96 (m, 8H); MS (ES") m/z 450.4.

EXAMPLE 3 Synthesis of Compound 30

Compound 40 Compound 30

[0051] Compound 30. Referring to the scheme above, a mixture of Compound 40 (13.4 g. 0.0352 mol) produced as described in Example 1 and piperidine (14 mL. 0.14 mol) in ethanol (100 mL. 2 mol) was heated at 100 ⁰C for 18 hours in a sealed container. TLC (10% Et₃NMeOH) showed a single spot and MS showed one single desired peak of 478 (M+H). The mixture was concentrated and the excess piperidine was removed by azeotropic concentration with toluene (three times in 50 mL). The residue was dissolved in water with addition of MeOH. and then IN NaOH was added until no further precipitate was formed with adequate stirring. The mixture was concentrated to remove any residual MeOH. filtered and washed with water. The collected free form solid (16.55 g. 34.7 mmol after dried under vacuum) was dissolved in warm MeOH (100 mL) and treated with 70 mL of IN HCl (70 mmol) to form a di-HCl salt and the mixture was concentrated to dryness. The residual solid was crystallized in MeOH/EtOAc to give a pure product Compound 30 (16.0 g, 82%). ¹H NMR (400 MHz. CD₃OD) δ 8.13 (d. J=8.8 Hz. 2H). 7.58 (d. J=8.8 Hz. IH). 7.29 (d. J=2.3 Hz. IH). 7.14 (d. J=8.8 Hz. 2H). 7.02 (dd. .7=2.3. 8.8 Hz. IH). 4.21 (t. J=5.57 Hz. 2H), 4.19 (t. J=5.57 Hz. 2H). 3.59 (m. 4H). 3.34 (m, 4). 2.99 (m. 4h). 2.28 (tt. j=2.3, 5.6 Hz. 4H). 2.00- 1.50 (m. 12H): MS (ES^") m/z 478.5.

EXAMPLE 4 Synthesis of Compound 34

Cl^'

OBn- >J

OBn- V OH

O ^~O σ

Compound 43 Compound 44 Compound 45

Compound 46 Compound 34

[0052] Compound 34. Referring to the scheme above, a mixture of 4-aminoresorsinol hydrochloride (5.3 g. 33 mmol). 4-benzyloxybenzoic acid (5.0 g, 22 mmol). 1- hydroxybenzotriazole (5.9 g. 44 mmol), benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (23 g. 44 mmol) and N.N-diisopropylethylamine (19 mL. 1 10 mmol) in dimethylformamide (DMF) (50 mL) was stirred at O⁰C to room temperature overnight. After concentration to remove DMF. the reaction mixture was purified by silica gel chromatography (20% to 80%EtOAc/hexane. EtOAc and 30%MeOH/EtOAc) to give desired amide Compound 41 (or/and ester. 7.3 g. 95%).

[0053] A solution of Compound 41 (7.3 g. 22 mmol) in acetic acid (90 mL) was heated at 120 C for 3 hours. The reaction mixture was cooled to room temperature and concentrated. The residue was purified by silica gel chromatography (10% to 30% EtOAc/hexanes) to give the desired product Compound 42 (2.0 g. 29%) as light yellow solid. ¹H NMR (400 MHz. CD₃OD) δ 8.09 (d. J=9.1 Hz. 2H). 7.47-7.4 (m. 6H). 7.16 (d. J=9.1 Hz. 2H), 7.02 (d. J=2.4 Hz. IH). 7.02 (dd. J=2.4. 8.5 Hz. IH). 5.18 (s. 2H): MS (ES^") m/z 316.4. [0054] A mixture of Compound 42 (1.77 g. 5.58 mmol). l-bromo-3-chloropropane (1.6 mL. 17 mmol) and potassium carbonate (2.7 g. 20 mmol) in 10 mL DMSO at room temperature was stirred for 2.5 hours until the reaction was completed by TLC. The reaction was quenched by addition of water and then extracted five three times with EtOAc. The combined organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was crystallized from ethyl acetate-hexane to give Compound 43 as a light solid (1.93 g. 88%). [0055] To a suspension of Compound 43 (1.10 g, 2.8 mmol) in 40 mL of ethanol was added 5 mL of dimethylamine at -78 ⁰C. The mixture was sealed in a sealed tube and heated at 100 ⁰C for 10 hours. The mixture was concentrated to give Compound 44 (1.1 g. 98%). The product was carried on to next step reaction. [0056] A mixture of Compound 44 (1.1 g, 2.7 mmol) and palladium hydroxide (7 mg) in 32 mL of methanol and 5 mL of dichloromethane was stirred under a hydrogen balloon at room temperature over night. The mixture was filtered through celite and the filtrate was concentrated to give crude product Compound 45. Assuming a theoretical yield of 0.85 g. and the product was carried on to the next step directly without further purification. [0057] A mixture of Compound 45 (200 mg, 0.64 mmol), l-bromo-3-chloropropane (0.32 mL. 3.2 mmol) and potassium carbonate (470 mg. 3.4 mmol) in 4 mL DMSO at room temperature was stirred for 2 hours until the reaction was completed by TLC. The reaction was quenched by addition of water and then extracted four times with EtOAc. The combined organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by preparative TLC (4% Et3N in 5% MeOH/THF) to give Compound 46 (170 mg. 68%). [0058] A mixture of Compound 46 (30 mg. 0.08 mmol). N.N,N^'-trimethyl-1.3-propane- diamine (50 mg. 0.43 mmol) in 2 mL of ethanol under a sealed tube was heated at 100 ⁰C for 10 hours. The mixture was concentrated and the residue was purified by HPLC to give 8 mg of Compound 34 as a white solid (8 mg, 20%). ¹H NMR (400 MHz. CD₃OD) δ 8.10 (d. J=8.8 Hz. 2H). 7.55 (d. J=8.8 Hz IH). 7.25 (d, J=2.3 Hz. IH), 7.10 (d. J=8.8 Hz. 2H). 7.00 (dd. J=2.3. 8.8 Hz. IH). 4.14 (t. J=5.9 Hz. 2H). 4.10 (t, J=6.2Hz. 2H). 2.69 (m. 4H). 2.56 (m. 4H). 2.39 (s. 12H). 2.36 (s. 3H). 2.04 (m. 4H). 1.77 (m. 2H); MS (ES^") m/z+tt 469.55.

EXAMPLE 5 Exemplary Compounds

[0059] The following analogs in Table 1 below have been prepared. These compounds may be prepared following the procedure for the preparation of Compound 23 (Example 2) and/or Compound 30 (Example 3) using the corresponding dichloroalkanes and the side chain amines identified in the table.

Table 1. Exemplary Compounds

NMR Data for Select Compounds in Table Above:

Compound 27: ¹H NMR (400 MHz. CD₃OD) δ 8.13 (d. J=9.2 Hz. 2H), 7.58 (d. J=8.8 Hz. IH), 7.29 (d. J=2.4 Hz. IH). 7.13 (d, J=9.2 Hz. 2H). 7.03 (dd. J=2.2. 8.6 Hz. IH). 4.20 (m. 4H). 3.60-3.10 (m. 12H). 2.98 (s. 6H), 2.93 (s. 12H). 2.25 (m. 8H).

Compound 35: ¹H NMR (400 MHz. CD₃OD) δ 8.10 (d, J=8.8 Hz, 2H). 7.55 (d. J=8.8 Hz. IH). 7.25 (d. J=2.4 Hz. IH). 7.10 (d. J=8.8 Hz. 2H). 6.99 (dd. J=2.4. 8.8 Hz. IH). 4.10 (m. 4H). 3.60 (m. 8H). 2.65 (m. 8H). 2.35 (m, 18H). 2.02 (m. 4H).

EXAMPLE 6 In Vitro Biological Activity

[0060] A. Primary Assay: The primary assay involved transfecting human embryonic kidney epithelial (HEK) cells with TLR9 to assay for antagonism of TLR9 activity. More specifically. HEK293 fibroblast cells (ATCC No. CRP- 1573. American Type Culture Collection. Manassas. VA) were transfected with pcDNA3.1D/V5-His-TOPO^® plasmid (Invitrogen. Carlsbad. CA) encoding human TLR9 (directly inserted as a Taq polymerase- amplified PCR product) and firefly luciferase under the control of three NF-kB binding sites contained in the E-selectin-1 promoter, which was cloned into a pGL3 -Enhancer Vector (Promega Corp. Madison. WI). The compounds listed in Table 1. above, were tested in this assay. Each compound was added to cells 30 min. before stimulation with Oligo CpG 2006. a synthetic phosphorothioate oligonucleotide (Hartmann et al. J Immunol. 164:1617-24

(2000)). with nucleotide sequence 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3' (SEQ ID

NO:1). The cells were incubated overnight at 37 ⁰C. The luciferase substrate Steady-Glo^®

(Promega) was added to the wells, and luminescence was measured to determine the extent of

TLR9-driven gene activation. The IC₅₀ of the comparator was found to be 0.16 micromolar, and the IC₅₀ for the remaining compounds was less than 0.04 micromolar. as shown in Table

1. The IC₅₀ is defined as the concentration of compound that suppresses the luminescence to

50% of that observed in the absence of compound, i.e., full stimulation.

[0061] B. Secondary Assay: The same compounds tested in the primary assay were tested in the same way as in that assay, with the following differences. The plasmid used to transfect the HEK cell line encoded for TLR7 instead of TLR9. and R848 was used to stimulate the cells instead of Oligo CpG 2006. The results of this assay are also shown in

Table 2. For each compound tested the IC₅₀ from the HEK/TLR9 activation assay, above, was significantly lower than the IC₅₀ from the HEK/TLR7 assay, indicating a greater specificity for TLR9 than for TLR7.

[0062] Table 2 below shows the compounds tested and results obtained from the primary and secondary in vitro assays described and discussed above.

Table 2. In Vitro Assay Results

EXAMPLE 7

In Vivo Biological Activity: Short Term CpG Stimulation

[0063] Short Term CpG Stimulation Assay: Compounds identified as having potency in the HEK/TLR9 assay described in Example 1 , above, were tested in an in vivo short term CpG stimulation assay. Mice were orally administered 20 mg/kg of compound in water, and challenged within 90 minutes by subcutaneous (s.c.) injection of 30 micrograms of Oligo CpG 1668. a synthetic phosphorothioate oligonucleotide (Krieg et al. Nature. 374:546-9 (1995)), with nucleotide sequence 5'-TCC ATG ACG TTC CTG ATG CT-3' (SEQ ID NO:2). Two hours later serum was taken and assayed for IL-6. The results of this assay are shown in Table 3, below. Results from assays repeated on different dates are given as multiple numbers separated by commas. Table 3. CpG Stimulation Assay

[0064] Table 3 shows that at least 69% suppression of IL-6 was observed after oral administration of Compound 23. Compound 30. or Compound 26. At least 75% suppression of IL-6 was observed after oral administration of Compound 23 and Compound 30. No suppression was observed after administration of two of the compounds tested. Compound 14 and Compound 20. despite the high levels of suppression exhibited in the in vitro assay described above.

EXAMPLE 8

Ex Vivo Biological Activity: Spontaneous Splenocyte Cytokine Levels [0065] C57BL/6 mice were infected intraperitoneally (i.p.) with 10³ P. chabaiidi-infccted red blood cells (RBCs) that had been harvested at day eight or nine of infection and stored frozen in 10% glycerol. Infected mice were treated orally with Compound 23 given daily at dosages of 0 mg/kg. 20 mg/kg. 60 mg/kg or 120 mg/kg. On day 9 post-infection, spleens were removed. Splenocytes were obtained by macerating spleens through a nylon mesh, and erythrocytes were lysed with ice-cold isotonic solution (155 mM NH₄Cl. 10 niM KHCO₃. 100 mM EDTA; pH 7.4). Splenocytes were suspended in RPMI 1640. 5% fetal calf serum, 1% gentamycin at concentration of 5 x 10⁶ cells/ml and cultured in 24-well plates for 48h without stimulation. Cytokine levels were measured in supernatant by using a cytometric bead array (CBA) kit (BD Biosciences, Mississauga. Ontario. Canada), according to the manufacturer's protocol. Drug treatment rendered a dose-dependent suppression of spontaneous production of INF-γ. TNFα, IL-10 and IL- 12. The results in Figure 1 show that there was no significant effect on MCP-I.

EXAMPLE 9

In Vivo Biological Activity: Serum Cytokine Levels

[0066] C57BL/6 mice were infected with 10³ P chabaiidi infected RBCs and then treated orally with Compound 23 given daily at dosages of 0 mg/kg. 20 mg/kg or 60 mg/kg. Nine days post- infection, the level of four cytokines (TNFα. IFN -γ, ILlO and MCP-I) was determined in the serum of P chabaiidi infected mice. Cytokine levels were measured in serum by using a CBA kit. Figure 2 shows that levels of IFN-γ. TNFα. and IL-10 were decreased by drug treatment "while there was no effect on MCP-I.

EXAMPLE 10 In Vivo Biological Activity: Effects on Clinical Parameters and Parasitemia

[0067] C57BL/6 mice were infected with 10⁵ P chabaiidi infected RBCs and then treated orally with Compound 23 given daily at a dosage of 60 mg/kg. The mice were followed every three days for parasitemia, body weight and body temperature. Figures 3 through 5 show that Compound 23 at 60 mg/kg had no significant effect on these clinical parameters or parasitemia.

EXAMPLE 11 In Vivo Biological Activity: Inhibition of Pro-Inflammatory Priming Effects

[0068] C57BL/6 mice were infected i.p. with 10³ pRBCs. One week post infection and daily oral treatment with Compound 23 at 60 mg/kg, or vehicle. Spleens were aseptically removed and macerated through a nylon mesh. After erythrocytes were lysed with ice-cold isotonic solution, the splenocytes were suspended in RPMI. 10% fetal calf serum. 1% gentamicin at density of 2.5 x 10⁶ cells/ml and cultured in 48-well plate in the presence of TLRs ligands (LPS (lμg/ml). Pam3cysk4ser (lμg/ml). Poly:IC (lOOμg/ml). CL075 (0.5 μg/ml), CpG DNA (1 μg/ml), or malaria extract (100 μg/ml). for 48 h. Cytokines or nitric oxide (NO) were measured by CBA (Pro-Inflammatory Cytokines from Beckton & Dickson, USA) and Griess method, respectively. The results of these studies are shown in Figures 6 through 9. It can be observed that upregulation of IFN-γ production upon TLR stimulation in malaria-infected mice is marked, and is markedly reversed by administration of Compound 23 during the period of infection. This indicates that the drug blocks induction of TLR hypersensitivity in malaria infection.

EXAMPLE 12 In vivo Biological Activity: Malaria-mediated Priming and LPS-induced Shock

[0051] C57BL/6 mice were injected (i.p.) with 10⁵ P chabaudi AS infected RBCs. Infected mice v%ere then treated with either vehicle or Compound 23 at a daily dosage of 120 mg/kg. After 7 days of infection, mice were challenged with low doses of LPS. Mortality was evaluated 12 and 24 hours after LPS inoculation. The results of this experiment are shown in Table 4A. below. Results are from one experiment with 4 mice per group. Table 4A shows suppression of mortality observed after oral administration of Compound 23. The study was repeated with additional mice per group and the results are shown in Table 4B.

Table 4A. In vivo Activity Results

9? Table 4B. In vivo Activity Results

In vivo Biological Activity: Effects of Compound 23 or Vehicle on P. bershβi ANKA Infected C57BL/6 Mice

[0052] C57BL/6 mice were treated daily with either vehicle or 120 mg/kg Compound 23 ("Cmpd 23" or "C23") before, and for 12 days post, infection with 10⁵ P. berghei ANKA infected RBCs. 23 mice were treated with vehicle and 21 mice were treated with Compound 23. The following experiments were conducted during the course of the study:

EXAMPLE 13-PART A In vivo Biological Activity: Effects on body weight and Parasitemia

[0053] Mice were evaluated every 3 days during disease course for body weight and parasitemia. Figure 10 and Figure 11 show the lack of a significant effect on body weight or parasitemia by administration of Compound 23 in C57BL/6 mice.

EXAMPLE 13— PART B In vivo Biological Activity: Survival

[0054] Survival was monitored daily. Figure 12 shows pooled survival data from 3 independent experiments that yielded similar results. A significant effect on survival of C57BL/6 mice was observed upon administration of Compound 23. EXAMPLE 13— PART C In vivo Biological Activity: Inhibition of Pro-Inflammatory Priming Effects

[0055] On day 9 post infection spleen cells were harvested and cultivated in the presence of LPS for 48 hours. Levels of IFNγ were measured in supernatants by ELISA (R&D Systems. Minneapolis. MN) and the results are shown in Figure 13.

EXAMPLE 13— PART D In vivo Biological Activity : Inhibition of Cerebral Malaria Associated Symptoms

[0056] Cerebral malaria associated symptoms were evaluated every day during acute disease. Mice treated with vehicle which survived infection (•); mice treated with vehicle which succumbed to infection (A); mice treated with Compound 23 which survived infection (o). mice treated with Compound 23 which succumbed to infection (Δ). Figure 14 shows data from 3 independent experiments that yielded similar results; Significant improvement on clinical symptoms/signs in C57BL/6 mice were observed upon treatment with Compound 23.

EXAMPLE 13— PART E In vivo Biological Activity: Inhibition of Cerebral Malaria Associated Symptoms

[0057] Histological analysis of brain sections of C57BL/6 mice treated with vehicle or Compound 23 sacrificed on the ninth day after infection was performed on two mice in each treatment group. Brains were removed and fixed for histological analysis. Representative sections from areas around the blood vessels of cerebrum after hematoxylin and eosin HE are shown in Figure 15. A significant decrease in numbers of vascular lesions in the cerebrum was observed upon treatment with Compound 23. The graph on the right shows histological quantification of vascular lesions in mice treated with vehicle or Compound 23.

EXAMPLE 14 In vivo Biological Activity: Effects of Compound 23 on Cytokine Production in

P. bershei ANKA Infected C57BL/6 Mice

[0058] The effect of Compound 23 on cytokines produced by CDl lc^τ splenic dendritic cells (DCs) was evaluated employing the fluorescence-activated cell sorting (FACS) assay for intracellular cytokine staining. At 8 days p.i. after i.p. administration of 1 x 10E5 PbA iRBCs. spleens from mice treated with vehicle (n=4) and mice treated with Compound 23 (120mg/kg/day) (n=4) were harvested and CDl Ic" splenic DCs were isolated using CDl Ic microbeads (Miltenyi Biotec). Non-infected mice (n=2) were used as a control group. Cells (5 x 10E5) were Fc blocked with 2.4G2 mAb (BD Pharmingen San Diego, CA) and labeled with FITC-conjugated anti-CD 1 Ic, PE-conjugated anti mouse CD40, CD86 and CD80 mAbs (BD Pharmingen). A nonrelated IgG mAb was used as a control for staining specificity. For intracellular analysis of cytokines, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Pharmingen) and stained intracellularly using PE-conjugated anti- mouse TNFα, IL12p40/p70 or IL6 mAbs (BD Pharmingen). Analysis of intracellular staining of cytokines in the CDl Ic^1" splenic DCs is shown in Figure 16. The frequency of CDl lc^τ splenic DCs producing TNFα, IL12p40/p70 and IL6 was significantly increased at day 8 p.i. in infected mice. The production of all three cytokines was impaired in DCs from PbA infected mice treated with Compound 23.

Claims

We claim:

1. A method of preventing or treating malaria in a subject, comprising administering to the subject an effective amount of a compound selected from the group consisting of:

Compound Y.

Compound 16:

Compound 22;

Compound 23:

Compound 26;

Compound 27:

Compound 30:

Compound 34: and

Compound 35,

or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the compound is selected from the group consisting of:

Compound 30;

Compound 23;

Compound 26,

or a pharmaceutically acceptable salt thereof.

3. The method of claim 1 , wherein the compound is

Compound 23, or a pharmaceutically acceptable salt thereof.

4. The method as in one of claims 1-3. wherein the malaria is severe malaria.

5. The method as in one of claims 1-3. wherein the malaria is cerebral malaria.

6. The method as in one of claims 1-5. wherein the compound is co-administered with an additional antimalarial compound and/or additional agent for reversing antimalarial resistance.

7. The method of claim 6. wherein the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is selected from the group consisting of quinolines, folic acid antagonists, sulfonamides and antibiotics.

8. The method of claim 6. wherein the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is an artemisinin-derived compound.

9. The method of claim 8, wherein the artemisinin-derived compound is artesunate.

10. The method of claim 6. wherein the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is chloroquine, hydroxychloroquine, amodiaquine, sulfadoxine, pyrimethamine, mefloquine, artemisinin, artemether. artesunate, dihydroartemisinin. artemotil, lumefantrine. primaquine, atovaquone. proguanil. chlorproguanil. dapsone. quinine, quinidine. quinacrine, tetracycline, doxycycline and clindamycin.

11. The method as in one of claims 6-10, wherein the compound is administered prior to, concurrently with or subsequent to the administration of the additional antimalarial agent and/or the agent for reversing antimalarial resistance.

12. A pharmaceutical composition comprising an effective amount of a compound for the prevention or treatment of malaria, wherein the compound is selected from the group consisting of:

Compound 13:

Compound 16:

Compound 22:

Compound 23;

Compound 26:

Compound 27:

Compound 30:

Compound 34; and

Compound 35.

or a pharmaceutically acceptable salt thereof.

13. The pharmaceutical composition of claim 12. wherein the compound is selected from the group consisting of:

Compound 30:

Compound 23: and

Compound 26. or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical composition of claim 12, wherein the compound is

Compound 23,

or a pharmaceutically acceptable salt thereof.

15. Use of a compound selected from the group consisting of the following:

Compound 13;

Compound 22;

Compound 23 :

Compound 26;

Compound 27:

Compound 30;

Compound 34; and

Compound 35,

or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of malaria.

16. Use of a compound according to claim 15. wherein the malaria is severe malaria.

17. Use of a compound according to claim 15, wherein the malaria is cerebral malaria.

18. Use of a compound according to any of one of claims 15-17 in combination with an additional antimalarial compound and/or additional agent for reversing antimalarial resistance.

19. Use of a compound according to claim 18, wherein the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is selected from the group consisting of quinolines, folic acid antagonists, sulfonamides and antibiotics.

20. Use of a compound according to claim 18, wherein the additional antimalarial compound and/or additional agent for reversing antimalarial resistance is an artemisinin- derived compound.