Novel small molecule antimalarial drugs

Abstract

Disclosed herein are methods and compositions for treating and / or preventing Plasmodium falciparum malaria by administering a compound that mimics the binding of anti-Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP).

Description

Application 0312021.00258 NOVEL SMALL MOLECULE ANTIMALARIAL DRUGSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 719,589, filed November 12, 2024, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to compositions and treatments for malaria.BACKGROUND

[0003] Malaria is a life-threatening disease caused by parasites that are transmitted to people through mosquito bites. It remains a major public health challenge, particularly in sub-Saharan Africa.

[0004] There is a critical need for novel approaches that can effectively target the malaria parasite and prevent the disease, especially in regions with high transmission rates and drug-resistant strains.SUMMARY

[0005] Embodiments disclosed herein comprise compositions and methods of treating and / or preventing an infectious disease in a subject in need thereof, the method comprising the administration of a therapeutically effective amount of a compound.

[0006] In some embodiments, the method is a method of treating and / or preventing an infectious disease in a subject in need thereof, the method comprising administration of a therapeutically effective amount of one or more compounds as described herein.

[0007] In some embodiments, the infectious disease is malaria.

[0008] In some embodiments, the infectious disease is Plasmodium falciparum (P. falciparum) malaria.Application 0312021.00258

[0009] In some embodiments, the compound mimics a binding ligand of antiPlasmodium falciparum Glutamic Acid Rich Protein (PfGARP) and / or mimics a binding of one or more anti-pfGARP antibodies.

[0010] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4;A is H, isopropyl,R9and R10are independently H, halogen, OCH3, or CF3;Z is CH2CH2-P-C6H4R11or cyclohexylmethyl;R11is H, halogen, or OCH3;J is isopropyl, isobutyl, or cyclohexylmethyl;Y is C(O), C(NH), or S(O)2; andR12is NH2, CH2-P-C6H4R11or -p-C6H4R11.

[0011] In some embodiments, the compound can have a formula:Application0312021.00258or a pharmaceutically acceptable salt thereof;whereinn is 1-4;R9and R10are independently H, halogen, OCH3, or CF3;Z is CH2CH2-P-C6H4R11or cyclohexylmethyl;R11is H, halogen, or OCH3; andJ is isopropyl, isobutyl, or cyclohexylmethyl.

[0012] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4; andR9and R10are independently H, halogen, OCH3, or CF3.

[0013] In some embodiments, the compound can have a formula:Application 0312021.00258>or a pharmaceutically acceptable salt thereof;whereinn is 1-4; andR9and R10are independently H, halogen, OCH3, or CF3.

[0014] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4; andR9and R10are independently H, halogen, OCH3, or CF3.

[0015] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4;Application 0312021.00258 R9and R10are independently H, halogen, OCH3, or CF3;R12is CH2-P-C6H4R11; andR11is H, halogen, or OCH3.

[0016] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4;R9and R10are independently H, halogen, OCH3, or CF3;R12is p-CeF R11; andR11is H, halogen, or OCH3.

[0017] In some embodiments, the compound can have a formula:whereincombination thereof.Application 0312021.00258 BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts that 3D7-PfGARP-KO infected RBCs fail to traverse microfluidic artificial spleen. A) Microfluidic device with transiting RBCs used for rheologic assay (slit dimensions are 4 x 10 x 5 microns). For comparison, human splenic interendothelial slit dimensions are 1.9 X 0.65 microns. B) Magnetically purified trophozoite and schizont infected RBC, and uninfected RBC were introduced into the microfluidic device under flow conditions designed to mimic the shear stress of the splenic microcirculation. Cells were optically tracked as they transited the device and scored as trapped vs not trapped. All uninfected RBC transited the device. 38% of 3D7-PfGARP-KO infected iRBC became trapped in the device compared to only 12.5% of 3D7 infected iRBC (Chi square, P < 0.001).

[0019] FIG. 2 demonstrates that PfGARP is essential for in vivo survival. NSG mice were treated with human RBC and inoculated with 2 x 1073D7 or 3D7-PfGARP KO parasite infected RBCs. Mice were followed with daily blood films to detect patent infection.

[0020] FIG. 3 depicts a high-throughput screening (HTS) assay for identifying compounds that bind to PfGARP.

[0021] FIG. 4 shows that inhibition of anti-PfGARP antibody binding to PfGARP by ChemBridge hits. 10,000 compounds (10 pM) were screened as 125 mixtures of 80 compounds for inhibition of binding of anti-PfGARP polyclonal Ab to bead-immobilized PfGARP. Results presented for mixtures that resulted in greater than 20% inhibition of anti-PfGARP binding.

[0022] FIG. 5 shows that 579 kills P. falciparum parasites. 3D7 (top) or 3D7 PfGARP KO (bottom) parasites were synchronized to the ring stage and incubated with a dilution series of compounds or media control for 48 hours followed by quantification of parasitemia by pLDH assay. Each dilution was evaluated in quadruplicate and error bars represent standard deviation (SD). The ICso = 1.8 - 4.8 pM for killing of 3D7 parasites. Results representative of >4 independent experiments.Application 0312021.00258

[0023] FIG. 6 shows that 424 kills P. falciparum parasites. 3D7 parasites were synchronized to the ring stage and incubated with a dilution series of compounds or media control for 48 hours followed by quantification of parasitemia by pLDH assay. Each dilution was evaluated in triplicate and error bars represent SD. ICso = 4.4 pM. Results representative of >4 independent experiments.

[0024] FIG. 7 shows that 579 is not cytotoxic to human white blood cells. Human WBC (CD45+) were incubated in the presence of increasing concentrations of 579 or vehicle alone for 48 hours. Cells were stained with 7AAD and annexin V and analyzed by flow cytometry to assess viability (7AAD annexin V‘), necrosis (7AAD+, annexin V-), early apoptosis (7AAD annexin V+), and late apoptosis (7AAD+, annexin V+).

[0025] FIG. 8 shows that 579 does not inhibit the growth of BeWo cells. BeWo cells, a human choriocarcinoma cell line, were grown in the presence of increasing concentrations of 579 or vehicle alone and cell concentration was measured using a crystal violet viability assay, performed 48 hours after addition of drug. Points represent the mean of three independent technical replicates. Bars represent SD.

[0026] FIG. 9 shows 579 and 424 activate parasite apoptosis. Drugs activate mitochondrial membrane depolarization as assessed by JC-1 staining (top), DNA fragmentation as assessed by TUNEL staining (middle), and caspase activation as assessed by Apostat staining (bottom).

[0027] FIG. 10 shows 579 kills P. falciparum parasites. 3D7 or 3D7 PfGARP KO parasites were synchronized to the ring stage and incubated with 100 pM 579 or vehicle control for 48 hours.

[0028] FIG. 11 shows 579 and 424 bind specifically to full length PfGARP by SPR. Drugs were immobilized to a CMS sensorchip and probed with varying concentrations of full length PfGARP on a BiaCore100. Results demonstrate fast on, slow off kinetics and a KD= 29.9 and 24.4 nM.

[0029] FIG. 12 depicts1H-15N HSQC identifies binding signature for 424 to PfGARP. Spectra were collected on a 600MHz Bruker spectrometer at 17°C from samples withApplication 0312021.00258 PfGARP (amino acids 377-560) alone (Red), or in complex with 424 (Blue). Arrows indicate spectral shifts in the PfGARP spectrum which indicate specific drug binding.

[0030] FIG. 13 shows single dose pharmacokinetics (PK) of 579 and 424 in mice. Balb / cJ mice (n=2 per timepoint) were treated intraperitoneally (IP) with a single dose of drug and blood samples collected at the indicated timepoints for analysis of drug levels by LC / MS-MS. Error bars indicate SEM. Desired trough level of 50 pM is indicated by dashed line.

[0031] FIG. 14 shows 579 kills P. falciparum in vivo. 579 was evaluated in the NSG / P. falciparum humanized mouse model. Mice were reconstituted with human RBC, infected with P. falciparum 3D70087 / N9strain parasites and treated with drug or vehicle control (100 mg / kg dosed intraperitoneally (IP every 8 hours for 4 days). Parasitemia was monitored by microscopy.

[0032] FIG. 15 shows multiple compounds from 2741 family kill P. falciparum parasites with low nM activity. 3D7 parasites were synchronized to the ring stage and incubated with a dilution series of compound or media control for 48 hours followed by quantification of parasitemia by flow cytometry. Circles depict means of 3 replicate wells and error bars represent SEM. Results are representative of more than 5 biologically independent experiments.

[0033] FIG. 16 depicts structure of 2741 family compounds with ICso < 55 nM. Scaffold (top) and R groups (bottom) of most active compounds in parasite killing assays.

[0034] FIG. 17 shows 2741-20 kills drug resistant P. falciparum parasites with low nM activity. Dd2 parasites were synchronized to the ring stage and incubated with a dilution series of compound or media control for 48 hours followed by quantification of parasitemia by flow cytometry.

[0035] FIG. 18 shows cytotoxicity of 2741 series compounds. A) E. coli, B) A549, C) human PBMCs, and D) THP-1 cells were treated with 2741 series compounds for 48 hours and then assessed for growth (E. coli and A549) and / or viability (PBMCs and THP-1 ) by flow cytometry staining for 7-AAD (PBMC and THP-1 ), crystal violet staining (A549), or spectrophotometry (E. coli).Application 0312021.00258

[0036] FIG. 19 shows single dose pharmacokinetics (PK) of 2741-19 in Aotus monkeys. Aotus monkeys (n=2) were treated subcutaneously (SC) with a single dose of 3.2 mg / kg drug and blood samples were collected at the indicated timepoints for analysis of drug levels by LC / MS. Error bars indicate SEM. Desired trough level of 1000 nM (~20 times the IC50 for parasite killing in culture) is indicated by dotted line.

[0037] FIG. 20 shows 2741-19 kills P. falciparum in vivo. 2741-19 was evaluated in the NSG / P. falciparum humanized mouse model. Mice were reconstituted with human RBC, infected with P. falciparum 3D70087 / N9strain parasites and treated with drug or vehicle control (5 mg / kg dosed subcutaneously (SC) every 8 hours for 4 days). Parasitemia was monitored by flow cytometry and confirmed by microscopy. Results representative of 3 independent trials.

[0038] FIG. 21 shows cytotoxicity of 2741 series compounds. A) A549, B) human PBMC, C) E. coli, and D) HepG2 cells were treated with 2741-19 for 48 hrs and then assessed for growth (E. coli and A549) and / or viability (PBMCs and THP-1) by flow cytometry staining for 7-AAD (PBMC and HepG2), crystal violet staining (A549), or spectrophotometry (E. coli).

[0039] FIG. 22 shows that 2741-19 inhibits anti-PfGARP binding to PfGARP. rPfGARP was immobilized on beads, incubated with 2741 -19 at 100 pM or vehicle control (1% DMSO in PBS, pH 7.4) for 1 hour at 25 °C, washed three times in PBS-T (0.05% Tween 20 in PBS, pH 7.4), incubated with polyclonal anti-PfGARP (1:4000 in PBS-T) for 1 hour at 25 deg C, and detected with PE conjugated anti-mouse IgG (1:250 in PBS-T). Beads were analyzed on a Luminex 100 bead reader. Incubation with 2741-19 significantly inhibited anti-PfGARP from binding to recombinant PfGARP constructs by 42-74% (all P < 0.0010). Results representative of 3 independent experiments, “aa” is an abbreviation for amino acids.

[0040] FIG. 23 depicts an1H-15N HSQC, which identifies binding of 2741-19 to PfGARP. Spectra were collected on a 500 MHz Bruker spectrometer at 17°C, pH 5.3 from samples with 95 pM PfGARP (amino acids 377-560) alone (Blue), or in complex with 95 pM 2741-19 (Red). Arrows indicate representative changes in the PfGARP spectrum suggesting specific drug binding.Application 0312021.00258

[0041] FIG. 24 shows that PfGARP knockout parasites are resistant to 2741-19 induced killing. Ring stage wild type (3D7) and PfGARP deleted (3D7-PfGARP-KO) P. falciparum parasites (0.3% parasitemia, 2% hematocrit) were incubated with a dilution series of 2741 -19 for 72 hours and parasitemia was enumerated by flow cytometry. 3D7-PfGARP-KO parasites showed significantly lower susceptibility to 2741-19 mediated killing compared to wild type parasites. Results representative of 7 independent experiments.DEFINITION OF TERMS

[0042] "Pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects such as (but not limited to) toxicity, irritation, and / or allergic response commensurate with a reasonable benefit / risk ratio.

[0043] "Patient" or "subject" as used herein includes human, mammals, and veterinary subjects.

[0044] "Mammal" refers to any animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, non-human primates, and any other animal that has mammary tissue.

[0045] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition / disease / infection as well as individuals who are at risk of acquiring a particular condition / disease / infection (e.g., those needing prophylactic / preventative measures).

[0046] "Therapeutic composition" or "pharmaceutical composition" as used herein refers to an agent that may be administered in vivo to bring about a therapeutic and / or prophylactic / preventative effect.

[0047] Administering a therapeutically effective amount or prophylactical ly effective amount is intended to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, condition, and / or infection. The specific amount that is therapeutically effective can be readily determined by the ordinary medical practitionerApplication 0312021.00258 and can vary depending on factors known in the art, such as (but not limited to) the type of condition / disease / infection, the patient's history and age, the stage of the condition / disease / infection, and the co-administration of other agents.

[0048] “Effective amount" refers to an amount of a biologically active molecule or conjugate or derivative thereof, or an amount of a treatment protocol (e.g., an alternating electric field), sufficient to exhibit a detectable therapeutic effect without undue adverse side effects (such as (but not limited to) toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of the inventive concept(s). The therapeutic effect may include, for example but not by way of limitation, preventing, inhibiting, or reducing the occurrence of at least one condition, disease, and / or infection. The effective amount for a subject will depend upon the type of subject, the subject's size and health, the nature and severity of the condition / disease / infection to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0049] "Concurrent therapy" as used herein, is used interchangeably with the terms "concomitant therapy" and "adjunct therapy," and will be understood to mean that the patient in need of treatment is treated or given another drug for the condition / disease / infection in conjunction with the treatments of the present disclosure. This concurrent therapy can be sequential therapy, where the patient is treated first with one treatment protocol / pharmaceutical composition and then the other treatment protocol / pharmaceutical composition, or the two treatment protocols / pharmaceutical compositions are given simultaneously.

[0050] "Administration" and "administering," as used herein, will be understood to include all routes of administration known in the art, including but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal, and intravenous routes, and including both local and systemic applications. In addition, the compositions of the present disclosure (and / or the methodsApplication 0312021.00258 of administration of same) may be designed to provide delayed, controlled, or sustained release using formulation techniques which are well known in the art.

[0051] "Conjoint administration" and "administered conjointly" refer to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered at the same time, within one minute, 2 minutes, 4 minutes, 6 minutes, 10 minutes, 30 minutes, or an hour or 90 minutes of one another. In some embodiments, the different therapeutic compounds can be administered within 1 year of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

[0052] "Treating" refers to administering an agent / element / method to a patient for therapeutic and / or prophylactic / preventative purposes. "Treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), sign(s), diminishment of extent of the deficit, stabilized (i.e. , not worsening) state of a symptom or condition, delay or slowing of onset of symptoms or indications, and an increased lifespan as compared to that expected in the absence of treatment.Application 0312021.00258

[0053] "Small molecule" refers to a chemical that has a molecular weight (MW) of 1000 Daltons (Da) or less in a free form (i.e., as measured in a non-salt form). A reference to a "large molecule" refers to a molecular weight of greater than 1000 Da.

[0054] "Long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent, combination, or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1 , 2, 3, 5, 7 or 10 years, or more.

[0055] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, application (e.g., topical, otic, or ocular), or transplantation. Administration can be accomplished by an implant. In some embodiments, compositions are administered parenterally. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into an artery, vein, lymph node, or organ (e.g., heart, muscle, organ).

[0056] "Decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" does not encompass a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition as comparedApplication 0312021.00258 to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0057] "Prodrug", as used herein, is intended to encompass compounds which, under physiological conditions, are converted into the therapeutically active agents of the present disclosure (e.g., any compound selected from this disclosure). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the disclosed compositions. In certain embodiments, some or all of the compounds selected from this disclosure in a formulation can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester. In some embodiments, a "prodrug" is made by using an absorbing particle that subsequently releases an active form after administration.

[0058] "Increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0059] As used herein, an agent or a therapeutic agent provided to a subject and suspected to be or involved in a treatment can be a small molecule less than 1000 molecular weight or a large molecule not less than 1000 molecular weight including, for example, biologies, oligonucleotides, peptides, systems of large molecules,Application 0312021.00258 oligosaccharides, and larger molecules. Any of the therapeutic agents disclosed herein can be used as or in combination with small molecules and / or large molecules as discussed herein.

[0060] As used herein, a subject may or may not be aware of suffering from malaria or a disease condition.

[0061] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., malaria or related disorder) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more complications related to a condition or a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, suspected as having, or at risk of developing that condition. In another example, the subject has been brought into a treatment situation entirely without the subject's knowledge and / or intent. For example, a subject can obviously be in need of treatment but not be responsive to a treatment, and as described herein the present methods and compositions may be used to help save the subject's life.

[0062] The compositions and methods of the present disclosure may be utilized to treat an individual (or subject) in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasiveApplication 0312021.00258 routes of administration (i.e. , routes, such as injection or implantation, which circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to affect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection, or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0063] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable, and metabolizable carriers that are relatively simple to make and administer.

[0064] "Pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.Application 0312021.00258

[0065] "Pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0066] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos.6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, as well as in patents cited therein.Application 0312021.00258

[0067] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 1 % to about 99% of active ingredient, or from about 5% to about 70%, or from about 10% to about 30%.

[0068] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0069] Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0070] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin,Application 0312021.00258 polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0071] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0072] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectableApplication 0312021.00258 medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0073] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0074] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0075] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0076] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.Application 0312021.00258

[0077] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0078] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0079] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0080] "Parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratumoral, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0081] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol,Application 0312021.00258 polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0082] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chloro-butanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0083] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0084] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradableApplication 0312021.00258 polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0085] For use in the methods of this disclosure, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0086] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0087] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0088] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0089] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desiredApplication 0312021.00258 effect is achieved. By "therapeutically effective amount" is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the disclosure. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art.

[0090] In general, a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0091] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present disclosure, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0092] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.

[0093] In certain embodiments, compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent.

[0094] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the disclosure in the compositions and methods of the present disclosure. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, L-arginine,Application 0312021.00258 benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1 H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoricacid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, I-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, I-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0095] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0096] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents,Application 0312021.00258 sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0097] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0098] The effective amounts can be determined with no more than routine experimentation. For example, effective amounts may range from about 1 ng / kg to about 200 mg / kg, about 1 pg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg. The dosage of a composition can be at any dosage including, but not limited to, about 1 pg / kg. The dosage of a composition may be at any dosage including, but not limited to, about 1 pg / kg, about 10 pg / kg, about 25 pg / kg, about 50 pg / kg, about 75 pg / kg, about 100 pg / kg, about 125 pg / kg, about 150 pg / kg, about 175 pg / kg, about 200 pg / kg, about 225 pg / kg, about 250 pg / kg, about 275 pg / kg, about 300 pg / kg, about 325 pg / kg, about 350 pg / kg, about 375 pg / kg, about 400 pg / kg, about 425 pg / kg, about 450 pg / kg, about 475 pg / kg, about 500 pg / kg, about 525 pg / kg, about 550 pg / kg, about 575 pg / kg, about 600 pg / kg, about 625 pg / kg, about 650 pg / kg, about 675 pg / kg, about 700 pg / kg, about 725 pg / kg, about 750 pg / kg, about 775 pg / kg, about 800 pg / kg, about 825 pg / kg, about 850 pg / kg, about 875 pg / kg, about 900 pg / kg, about 925 pg / kg, about 950 pg / kg, about 975 pg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, or more. In other embodiments, the dosage is 1-500 mg. In some embodiments, the dosage is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg. These doses may be unitary or divided and may be administered one or more times per day. The above dosages are exemplary of the average case, but there can be individual instances inApplication 0312021.00258 which higher or lower dosages are merited, and such are within the scope of this disclosure. In practice, the physician determines therapeutically effective amounts and the actual dosing regimen that is most suitable for an individual subject, which can vary with age, weight, and response of the particular subject.

[0099] The therapeutic agents may be administered once, twice or three times per day for 1 day to the end of life, or for 1 day to 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years, or until the agents cause unacceptable side effects or are no longer useful.

[0100] The patient is monitored for changes in the symptoms. In one embodiment, there is a reduction in the symptoms. In another embodiment, the symptoms remain about the same and there is no evidence of progression. Methods for monitoring and quantifying any symptom change can be carried out by routine methods or by routine experimentation.

[0101] Any change in symptoms may be monitored for 1-36 months or more, e.g., 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months.

[0102] In another embodiment, the patient is monitored for a change in the underlying pathology. In one embodiment, there is a reduction in the underlying pathology. In another embodiment, the underlying pathology remains about the same and there is no evidence of progression.DETAILED DESCRIPTION

[0103] Disclosed herein are compositions and methods for treating infectious diseases. In one embodiment, the infectious disease can include malaria.

[0104] Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. Plasmodium falciparum is the most dangerous of the malaria parasites, responsible for the majority of severe cases and deaths. Despite considerable progress in reducing the global burden of malaria, the disease remains a major public health challenge, particularly in sub-Saharan Africa. The emergence of drug-resistant strains of Plasmodium falciparum hasApplication 0312021.00258 further complicated efforts to control and eliminate malaria, highlighting the urgent need for new and effective therapeutic strategies.

[0105] Current treatments for malaria primarily rely on artemisinin-based combination therapies (ACTs), which have been effective in reducing mortality rates. However, the increasing resistance to artemisinin and its partner drugs poses a significant threat to malaria control efforts. Additionally, the development of a malaria vaccine has been challenging due to the complex life cycle of the parasite and its ability to evade the human immune system. Currently there are malaria parasite strains that are resistant to all known small molecule anti-malarial drug treatments. As a result, there is a critical need for novel approaches that can effectively target the malaria parasite and prevent the disease, especially in regions with high transmission rates and drug-resistant strains.

[0106] In addition to conventional anti-malarial drugs, efforts have been made to develop vaccines targeting specific antigens expressed by the Plasmodium parasite. One such antigen is the Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP), which plays a crucial role in the live cycle of the parasite in the red blood cells (RBCs). Vaccines designed to induce an immune response against PfGARP have shown promise in preclinical studies, but their clinical efficacy remains to be fully compared to any other treatments newly established. Furthermore, the development of effective vaccines against malaria has been hindered by the complex varieties / biology of the parasite and the variability of antigenic targets.

[0107] There is a need for novel small molecule anti-malarial drugs, especially those which can kill malaria parasites that are resistant to all known antimalarial drugs.

[0108] Compositions

[0109] Described herein are novel small molecule compounds for the treatment of malaria.

[0110] In some embodiments, the compound can have a formula:Application 0312021.00258>or a pharmaceutically acceptable salt thereof;whereinn is 1-4;A is H, isopropyl,R9and R10are independently H, halogen, OCH3, or CF3;Z is CH2CH2-P-C6H4R11or cyclohexylmethyl;R11is H, halogen, or OCH3;J is isopropyl, isobutyl, or cyclohexylmethyl;Y is C(O), C(NH), or S(O)2; andR12is NH2, CH2-P-C6H4R11or -p-C6H4R11.

[0111] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinApplication 0312021.00258 n is 1-4;R9and R10are independently H, halogen, OCH3, or CF3;Z is CH2CH2-P-C6H4R11or cyclohexylmethyl;R11is H, halogen, or OCH3; andJ is isopropyl, isobutyl, or cyclohexylmethyl.

[0112] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4; andR9and R10are independently H, halogen, OCH3, or CF3.

[0113] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4; andR9and R10are independently H, halogen, OCH3, or CF3.

[0114] In some embodiments, the compound can have a formula:Application0312021.00258or a pharmaceutically acceptable salt thereof;whereinn is 1-4; andR9and R10are independently H, halogen, OCH3, or CF3.

[0115] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinn is 1-4;R9and R10are independently H, halogen, OCH3, or CF3;R12is CH2-P-C6H4R11; andR11is H, halogen, or OCH3.

[0116] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinApplication 0312021.00258 n is 1-4;R9and R10are independently H, halogen, OCH3, or CF3;R12is p-C6H4R11; andR11is H, halogen, or OCH3.

[0117] In some embodiments, the compound can have a formula:

[0118] In some embodiments, the compound can have a formula:Application0312021.00258Application0312021.00258Application 0312021.00258 2741-15 2741-16Application0312021.00258Application 0312021.00258 2741-27 2741-28Application 0312021.00258 2741-31 2741-32or a pharmaceutically acceptable salt thereof.

[0119] In some embodiments, the compound can have a formula:Application 0312021.00258 2741-19 ana-1 2741-19 ana-2Application 0312021.00258 2741-19 ana-9 2741-19 ana-10Application 0312021.00258 2741-19 ana-17 2741-19 ana-182741-19 ana-19Application 0312021.00258 2741-19 ana-23 2741-19 ana-25or a pharmaceutically acceptable salt thereof.

[0120] In some embodiments, the compound can have a formula:or a pharmaceutically acceptable salt thereof;whereinR9and R10are independently H, halogen, OCH3, or CF3;X is S, 0, or NH.

[0121] In some embodiments, the compound can have a formula:R9and R10are independently H, halogen, OCH3, or CF3;R13is n-butyl, p-bromophenyl, p-chlorophenyl or phenyl;X is S, 0, or NH.

[0122] In some embodiments, the compound can have a formula:Application0312021.00258or a pharmaceutically acceptable salt thereof.

[0123] In some embodiments, the compound is a racemic mixture of stereoisomers, a non-racemic mixture of stereoisomers, an R-enantiomer, or an S-enantiomer.

[0124] In some embodiments, the compound is antiparasitic.

[0125] In some embodiments, the compound can comprise a [4-(2-lmino-4-imidazolidinyl)butyl]-2-imidazolidinimine moiety.

[0126] In some embodiments, the compound can comprise a [4-(2-im inoimidazolidin-4-yl)butyl]imidazolidin-2-imine moiety.

[0127] In some embodiments, the compound can comprise a N-Methyl[2-(1-piperazinyl)ethyl]amine moiety.

[0128] In some embodiments, the compound can comprise a N-methyl[2-(piperazin-1-yl)ethyl]amine moiety.

[0129] In some embodiments, the compound can have a formula:Application 0312021.00258or a pharmaceutically acceptable salt thereof;wherein each of R1, R2, R3, R4, R5, R6, R7, and R8independently comprises a substituent; and wherein each of R1, R2, R3, R4, R5, R6, R7, and R8can be independently interchanged with any other R1, R2, R3, R4, R5, R6, R7, and R8; and wherein different linkers includes a combination of -H, -OH, carbonyl (=0), F, N, -0-, -S-, -NH-, a halogen, methyl (-CH3), t-butyl, -(CH2)n-CH3, wherein independently each n = 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; -O-(CH2)n-CH3, wherein independently each n = 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; -S-(CH2)n-CH3, wherein independently each n = 0, 1 , 2, 3, 4, 5, 6, 7, 8, or 9; -NH-(CH2)n-CH3, wherein independently each n = 0, 1 , 2, 3, 4, 5, 6, 7, 8, or 9.

[0130] In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8can independently comprise any of any of R1, R2, and / or R3shown below:Application 0312021.00258Application 0312021.00258Application 0312021.00258Application 0312021.00258Application 0312021.00258

[0131] In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8independently can comprise R4, R5, and / or R6:Application 0312021.00258Application 0312021.00258Application 0312021.00258

[0132] In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8each can independently comprise one or more of the following non-limiting examples ofApplication 0312021.00258 incorporated functionalities: S-methyl, S-benzyl, hydrogen, S-2-butyl, S-butyl-dimethylamine, S-isobutyl, S-methylsulfinylethyl, R-hydroxymethyl, (R,R)-I hydroxymethyl, S-isopropyl, S-4-hydroxybenzyl,R-methyl, R-benzyl, R-2-butyl, R-butyl-dimethylamine, R-isobutyl, S-hydroxymethyl, (S, S)-1 -hydroxyethyl, R-isopropyl, R-4-hydroxybenzyl, S-butyl, R-butyl, S-propyl, R-propyl, S-2-naphthylmethyl, R-2-naphthylmethyl, S-phenyl, S-cyclohexyl, R-cyclohexyl, S-methyl, S-benzyl, hydrogen, S-2-butyl, S-isobutyl, S-methylsulfinylethyl, R-hydroxymethyl, (R,R)-1-hydroxycthyl, S-isopropyl, S-4-hydroxybenzyl, R-methyl, R-benzyl, R-2-butyl, R-isobutyl, S-hydroxym ethyl, (S, S)-1 -hydroxyethyl, R-isopropyl, R-4-hydroxybenzyl, S-butyl, R-butyl, S-propyl, R-propyl, S-2-naphthylmethyl, R-2-naphthylmethyl, S-phenyl, S-cyclohexyl, R-cyclohexyl, (l-phenyl-cyclopropyl)-methyl, m-tolylethyl, 2-(3-fluoro-phenyl)-ethyl, 2-(3-trifluoromethyl-phenyl)-ethyl, p-tolylethyl, 2-(3-methoxy-phenyl)-ethyl, 2-(4-methoxy-phenyl)-ethyl, 2-(4-ethoxy-phenyl)-ethyl, 2-(4-lsobutyl-phenyl)-propyl, 3,4-dichlorophenethyl, 2-(3,5-bis-trifluoromethyl- phenyl)-ethyl, phenethyl, phenylpropyl, 4-phenylbutyl, butyl, heptyl, isobutyl, 3-methylbutyl, 4-methylpentyl,t-butylmethyl,t-butylethyl, cyclohexyl-methyl, cyclohexyl-ethyl, cyclohexyl-butyl, cycloheptyl-methyl, ethyl, cyclobutyl-methyl, cyclopentyl-methyl, cyclohexyl-propyl, 4-methyl-1-cyclohexylmethyl,4-tert-butyl-cyclohexyl-methyl, adamantan-l-yl-methyl, 3,3-diphenyl-propyl, dicyclohexylethyl, 3-indolylethyl, 1 -napthyl-ethyl, 3-(3,4,5-trimethoxypheny)-propyl, 2-Bicyclo[2.2.1 ]hept-2-yl-ethyl, cyclopentylethyl, 2-ethylbutyl, or some combination thereof.

[0133] In some embodiments, the compound can comprise at least one of a [4-(2-lmino-4-imidazolidinyl)butyl]-2-imidazolidinimine moiety, a [4-(2-iminoimidazolidin-4-yl)butyl]imidazolidin-2-imine moiety, a N-Methyl[2-(1-piperazinyl)ethyl]amine moiety, a N-methyl[2-(piperazin-1-yl)ethyl]amine moiety, any of the above formulas, or some combination thereof.

[0134] In some embodiments, the compound can comprise one or more moieties selected from imidazolidinimine, imidazolidin-imine, piperazinylethylamine, and piperazinylethylamine derivatives, any of the above formulas, or some combination thereof.Application 0312021.00258

[0135] In some embodiments, the [4-(2-iminoimidazolidin-4-yl)butyl]imidazolidin-2-imine moiety can be linked to an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl group.

[0136] In some embodiments, the [4-(2-lmino-4-imidazolidinyl)butyl]-2-imidazolidinimine moiety can be linked to a heteroaryl, aryl, cycloalkyl, or heterocyclic group.

[0137] In some embodiments, the piperazine ring of the N-Methyl[2-(1-piperazinyl)ethyl]amine moiety can be optionally substituted with one or more substituents selected from halo, hydroxyl, alkoxy, amino, alkylamino, and dialkylamino.

[0138] In some embodiments, the compound can comprise a N-methyl[2-(piperazin-1-yl)ethyl]amine moiety, and wherein the N-methyl[2-(piperazin-1-yl)ethyl]amine moiety can be linked to an acyl, carbamoyl, carbamyl, or sulfonyl group.

[0139] Pharmaceutical compositions

[0140] In some embodiments, a pharmaceutical composition for treating and / or preventing Plasmodium falciparum malaria can comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier.

[0141] In some embodiments, the pharmaceutical composition can be formulated for oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, or rectal administration.

[0142] In some embodiments, the pharmaceutical composition can be in a dosage form selected from the group consisting of a tablet, a capsule, a solution, a suspension, an emulsion, a powder, a granule, a suppository, an injection, an infusion, and an implant.

[0143] In some embodiments, a pharmaceutical composition for treating and / or preventing Plasmodium falciparum malaria can comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier, wherein the composition is formulated for oral, intravenous, intramuscular, subcutaneous, transdermal, or rectal administration.Application 0312021.00258

[0144] In some embodiments, the compound has a molecular weight between 100-1000 Daltons, is administered orally, intravenously, intramuscularly, subcutaneously, transdermally, or rectally at a dose of 0.1 -100 mg / kg body weight of the subject and has an ICso value of less than 1 pM against Plasmodium falciparum in vitro.

[0145] In some embodiments, the compound has an IC50 value of less than 1 pM against Plasmodium falciparum.

[0146] Methods

[0147] Certain embodiments disclosed herein include methods for administering a therapeutically effective amount of a small molecule to a subject for the treatment and / or prevention of malaria. The administration of a therapeutically effective amount of a small molecule to the subject may be initiated at any time or phase.

[0148] In some embodiments, any composition described above can be described as a method or any method described below can be described as a composition, and which can be inter-combined with any other embodiment, example, or aspect disclosed herein.

[0149] In some embodiments, the compound can be effective in treating Plasmodium falciparum malaria in the subject. In some embodiments, the small molecule can mimic the binding of anti-Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP) antibodies. The compound can also be effective in preventing the malaria in the subject, suggesting a dual role in both treatment and prevention.

[0150] The subject may be a human, indicating the applicability of the method to human patients. The therapeutically effective amount of the compound may be administered through various routes, including oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, or rectal administration, providing flexibility in delivery methods. Additionally, the administration may occur in different dosage forms, such as tablets, capsules, solutions, suspensions, emulsions, powders, granules, suppositories, injections, infusions, or implants, allowing for tailored treatment regimens. The method may involve the formulation of a pharmaceutical composition comprising theApplication 0312021.00258 compound and a pharmaceutically acceptable carrier, which may facilitate the delivery and effectiveness of the treatment.

[0151] The compound's ability to mimic the binding of PfGARP may play a role in its mechanism of action, potentially disrupting the malaria parasite's lifecycle. The method may offer an approach to managing Plasmodium falciparum malaria, addressing both therapeutic and preventive needs.

[0152] In some embodiments, the method can treat and / or prevent Plasmodium falciparum malaria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound that mimics a binding of anti-Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP) antibodies.

[0153] In some embodiments, the method can be effective in treating Plasmodium falciparum malaria in a subject.

[0154] In some embodiments, the method can be effective in preventing the Plasmodium falciparum malaria in a subject.

[0155] In some embodiments, the malaria can be caused by Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, or some combination thereof.

[0156] In some embodiments, the subject can be a human.

[0157] In some embodiments, the malaria is asymptomatic.

[0158] In some embodiments, the malaria is severe.

[0159] In some embodiments, the malaria is drug resistant.

[0160] In some embodiments, a therapeutically effective amount of the compound can be administered orally, parenterally, intravenously, intramuscularly, subcutaneously, transdermally, rectally, or some combination thereof.

[0161] In some embodiments, a therapeutically effective amount of the compound can be administered in a dosage form selected from the group consisting of a tablet, a capsule, a solution, a suspension, an emulsion, a powder, a granule, a suppository, an injection, an infusion, and an implant.Application 0312021.00258

[0162] In some embodiments, the compound can treat and / or prevent Plasmodium falciparum malaria, the compound comprising a substituted chemical structure that mimics a binding of anti-Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP).

[0163] In some embodiments, the compound can be effective in treating the Plasmodium falciparum malaria.

[0164] In some embodiments, the compound can be effective in preventing the Plasmodium falciparum malaria.

[0165] In some embodiments, the malarial protein can be Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP), and wherein binding of the compound to PfGARP inhibits PfGARP's function in red blood cell invasion, parasite growth, or parasite egress from infected red blood cells.

[0166] In some embodiments, the method further comprises administering of at least one second therapeutic agent to the patient. The method can be in a combination therapy with any other therapeutic agent or any other technique(s).

[0167] In some embodiments, the compound can be used to treat parasites.EXAMPLESExample 1. Malaria drug discovery targeting PfGARP.

[0168] A study was preformed to develop lead-optimized anti-malarial drugs, designed to kill P. falciparum blood stage parasites by binding red blood cell (RBC) surface expressed PfGARP and activating parasite apoptosis and cell death.

[0169] Study Overview

[0170] Identifying of PfGARP as a novel drug target.

[0171] Screening for compounds which inhibit anti-PfGARP binding to PfGARP, followed by validation of hits in in vitro parasite growth inhibition assays.

[0172] Optimizing compounds using structure-activity relationship (SAR) assays and approaches.Application 0312021.00258

[0173] Assessing efficacy / safety of lead compounds (following cytotoxicity studies in vitro and in mice) in a humanized mouse model of P. falciparum.

[0174] Plasmodium falciparum Glutamic Acid Rich Protein (PfGARP) is a ~80 kDa parasite antigen expressed on the exofacial surface of trophozoite-infected RBCs (iRBC) as a target of antibodies that kill parasites. Naturally occurring antibodies to PfGARP demonstrated significant killing of trophozoite stage iRBCs by an apoptosis-like mechanism that involves disruption of mitochondrial membrane potential and food vacuole integrity with activation of caspase-like enzymes. In addition, vaccination of nonhuman primates with PfGARP in Ribi adjuvant or as a lipid encapsulated mRNA resulted in significant protection from P. falciparum challenge.

[0175] Thus, PfGARP was determined to be a high value druggable target based on 1) its surface expression on iRBCs, 2) its absence of amino acid homology with host proteins, 3) its absence of significant sequence variation in over 3,000 field isolates sequenced, 4) the requirement for PfGARP for parasite growth in vivo, and 5) the ability of antibody binding to PfGARP to kill essentially all parasites within 12-24 hours.

[0176] To develop a drug based on PfGARP binding, a total of 10,000 compounds were screened from the 160,000-compound ChemBridge DIVERset library and the 30 million compound CTS-FIU library to identify compounds that inhibit binding of anti-PfGARP antibody (Ab) to PfGARP protein. It was reasoned that compounds which bind to the same region of PfGARP that is targeted by the parasite-lethal anti-PfGARP antibodies would be enriched for effective PfGARP targeting anti-malarial drugs. Screening for compounds which mimic the binding of lethal anti-PfGARP antibodies represents an entirely new, and rationally designed approach to drug discovery for malaria.

[0177] Using this approach, two independent drug libraries (Chem Bridge and CTS-FIU) were screened to identify antimalarial compounds which kill parasite infected RBCs. Hits were subjected to SAR by catalog followed by SAR by synthesis using standard medicinal chemistry-based derivatization followed by down-selection in anti-PfGARP inhibition of binding assays, in vitro parasite killing assays, drug binding, induction of parasite apoptosis, and drug half-lives in pharmacokinetic (PK) studies that suggestApplication 0312021.00258 suitable dosing regimens in humans. During the SAR by synthesis and down-selection steps, priority was placed on improving potency and intravenous (IV) and / or subcutaneous (SC) half-life, in keeping with the central goal of generating a PfGARP targeting antimalarial for parenteral use in severe malaria. The lead compound from the ChemBridge library kills at low pM concentration while the lead compounds from the CTS-FIU library kill malaria parasites at low nanomolar concentrations. The results are discussed below:Results

[0178] Evidence for essentiality PfGARP of for in vivo survival

[0179] The ability to generate a gene knockout (KO) in P. falciparum, while extremely useful for in vitro mechanistic studies, is often over-interpreted as evidence that the gene is dispensable in vivo. However, knockouts result in effects on function and demonstrate that PfGARP is essential for in vivo survival of parasites.

[0180] It was evaluated whether PfGARP-KO parasites altered deformability of RBCs. The ability of PfGARP KO parasites to transit a previously characterized, microfluidics-based "artificial spleen" molded in polydimethylsiloxane was evaluated. Magnetically purified iRBC were introduced into the device under flow conditions designed to mimic the shear stress of the splenic microcirculation and cells were optically tracked as they transited the slits produced by 10 columns in the device. RBC were scored as infected vs uninfected and trapped vs not trapped. All uninfected RBC transited the device. Within the infected cell population, 38% of 3D7-PfGARP- KO iRBC became trapped in the device compared to only 12.5% of 3D7 iRBC (FIG. 1, P < 0.001).

[0181] Furthermore, it was demonstrated that PfGARP expression is necessary for parasite growth in vivo. Using the NSG / P. falciparum model, n=10 NSG mice were treated with human RBCs (1 mL intraperitoneally (IP) daily) for the duration of the experiment. On day 12, mice were infected with 2 x 1073D7 iRBC (N=6) or with 2 x 1073D7-PfGARP KO iRBC (N=4) via tail vein inoculation. Mice were followed with daily blood films for 10 days post-infection. All mice injected with 3D7 parasites developed patent infections within 3 days post inoculation, while zero mice injected with 3D7-PfGARP KO parasitesApplication 0312021.00258 developed patent infections (FIG. 2). On Day 12, mice previously inoculated with 3D7-PfGARP KO parasites were challenged with 2 x 1073D7 parasite infected RBCs. All four mice developed patent infections by day 3 post 3D7 infection- conclusively demonstrating that all the NGS mice were permissible to infection and that successful infection required parasite expression of PfGARP.

[0182] The combined data support the essentiality of PfGARP for parasite survival in vivo.

[0183] Screening of Chem Bridge library

[0184] An inhibition of binding-based high-throughput screening (HTS) compatible assay was developed using a lethal recombinant monoclonal antibody (mAb), monovalent fragment antigen binding (Fab), and polyclonal anti-PfGARP to screen based on inhibition of antibody binding to its target, PfGARP (FIG. 3). This inhibition of binding assay allowed efficient screening of drug-like libraries to discover compounds which mimic the binding of these antibodies to their target, PfGARP, and screen for parasiticidal activity.

[0185] The HTS screening approach was used to screen a subset of the Chem Bridge library. Briefly, PfGARP was coupled to Luminex beads and incubated with mixtures of compounds from the library. The beads were washed and probed with biotinylated anti-PfGARP. The beads were washed again, and antibody binding was detected with streptavidin-PE. In this inhibition of binding assay, low fluorescence values indicated the presence of compound compound(s) in the mixture that specifically bind to PfGARP and inhibit the binding of anti-PfGARP (FIG. 3).

[0186] Using this assay, a 10,000-compound subset of the 160,000 compound ChemBridge library was screened for inhibition of binding between bead-immobilized PfGARP and polyclonal anti-PfGARP antibodies. This subset was arrayed as 125 mixtures of 80 compounds (all with unrelated scaffolds) with each compound at 10 pM. Of the 125 mixtures tested, five mixtures inhibited binding of anti-PfGARP to PfGARP coated beads by more than 20% (FIG. 4). This level of inhibition reflected the high affinity of the polyclonal anti-PfGARP and the fact that the polyclonal antibody recognizesApplication 0312021.00258 multiple epitopes in PfGARP with only a minority of these epitopes being blocked by drug binding.

[0187] Two active mixtures, E7 and H2, were deconvoluted by testing their 160 compounds, arrayed as 16 mixtures of 10 compounds each, in a similar antibody inhibition assay. These mixtures were further deconvoluted and three compounds (ID# 9083816, 9133276, and 9133926) were confirmed in parasite killing as having antiparasitic activity. A limited SAR by catalog campaign around ID# 9083816 was conducted, evaluating 48 analog compounds having the same backbone (black), but varying R groups (red). Two compounds (ID# 579 and ID# 424, ICso 2-20 pM) were identified as having enhanced killing compared to the parent compound in 3D7 parasites and no activity in PfGARP KO parasites (FIG. 5 and FIG. 6) (Note: DMSO concentration was < 0.1 %). Furthermore, ID# 579 was evaluated in INDO (highly chloroquine and quinine resistant) and its ICso was found to be 5-8 pM.

[0188] This result demonstrated both the specificity of the activity of ID# 579 for PfGARP, as well as its lack of general toxicity to eukaryotic cells (3D7-PfGARP-KO cells). Toxicity assessments in human white blood cells (WBC) (FIG. 7), and BeWo cells (a model of human syncytiotrophoblasts at the materno-fetal interface, FIG. 8) showed no loss of viability or proliferative capacity at up to 400 pM, the highest concentration tested, >100 fold higher than the lower estimate of the ICso for parasites.

[0189] Both ID# 579 and ID# 424 induced parasite apoptosis as evidenced by mitochondrial membrane depolarization, DNA fragmentation, caspase activation (FIG. 9), and the crisis-form morphology of drug treated parasites (pyknotic, shrunken, apoptotic appearing parasite remnants, FIG. 10).

[0190] Direct binding of ID# 579 and ID# 424 to its target PfGARP was also demonstrated by surface plasmon resonance (SPR; FIG. 11) with a KD = 29.9 ± 0.8 and 24.8 ± 0.91 nM, respectively. This binding was further confirmed using Nuclear Magnetic Resonance (NMR) spectroscopy Using a series of recombinant PfGARP fragments,1H-15N HSQC NMR experiments were performed using PfGARP with and without ID# 424. These experiments have confirmed predictions that PfGARP was largely an intrinsically disordered protein. In addition, regions of structure were identified as well as specific drugApplication 0312021.00258 binding to a PfGARP fragment encoding amino acids 377-560, confirming the SPR data (FIG. 12).

[0191] An LC / MS assay was developed for these drugs in mouse sera, and single dose pharmacokinetic (PK) studies were performed. Both compounds were delivered intraperitoneally (IP) and a Ti / 20f ~2 hours was observed. This dose can maintain plasma trough levels in mice above 50 pM - a level that is uniformly lethal to parasites in culture using a 100 mg / kg dose with 8-hour dose interval for both drugs (FIG. 13). This dose was well tolerated with no signs of toxicity.

[0192] Lastly, the efficacy of both lead compounds were evaluated in the NSG / P. falciparum humanized mouse model (FIG. 14). Briefly, NSG mice (NOD.Cg- Prkdcscid||2rgtmi v ji / szj;JAX Cat# 005557) were reconstituted with human RBC (huRBC) by daily intraperitoneal (IP) injections with 1 mL of huRBC at 50% hematocrit. On day 12, mice were infected with 2 x 107Pf3D7 infected huRBCs via tail vein injection. On day 15, mice were injected intraperitoneally (IP) with ID# 579 at 100 mg / kg (approximately 2 mg per mouse in 5% DMSO / 95% PEG400 vehicle) or vehicle alone. Mice were treated with drug or vehicle control every 8 hours for 4 days. This dose and schedule were designed to maintain plasma drug levels above 50 pM, as informed by the single dose PK study (FIG.13). ID# 579 showed rapid clearance and complete killing of P. falciparum parasites with no detectible parasitemia after 3 days of drug treatment. This rapid clearance is similar to that observed for oral artesunate, an FDA-approved anti-malarial drug, at 50 and 100 mg / kg in this model (DOI: 10.1128 / AAC.00519-09).

[0193] In summary, using inhibition of anti-PfGARP binding assay, screening of a subset (6.25%) of the ChemBridge 160,000 compound library was performed and 5 mixtures which inhibit anti-PfGARP binding were identified. Two mixtures were deconvoluted to three individual compounds which kill parasites in culture. For one of these compounds (ID# 9083816), a limited SAR by catalog campaign was conducted around this hit and identified two lead-like compounds which selectively kill parasites (SI >100) with an ICso of 2-20 pM by activating parasite apoptosis and have demonstrated strong binding to the PfGARP target and favorable in vivo PK parameters. Preliminary efficacy studies in the NSG / P. falciparum model were conducted, and rapid parasiteApplication 0312021.00258 clearance was observed with no toxicity. These results from the Chem Bridge screen demonstrated the encouraging efficacy of the lead compounds in vivo.

[0194] Screening of CTS-FIU Library

[0195] In addition, the CTS-FIU 30 million compound combinatorial library was screened using the inhibition of binding assay. The CTS-FIU library was arrayed as 92 individual mixtures, each constructed on a unique scaffold. Each scaffold contains several variable positions (R groups) at which a moiety selected from up to 50 unique structures is attached, resulting in remarkable diversity of chemical space. These 92 individual mixtures were screened, and 2 sub-libraries (#2353 and #1664) were identified that inhibited anti-PfGARP binding by more than 20% to PfGARP immobilized on beads..

[0196] Sub-library #2353: Using a positional scanning approach, Library #2353 was constructed on a bis-cyclic guanidine-S-butyl linker backbone and contains three variable positions. R1with 42 possible substitutions, R2with 26 possible substitutions, R3with 42 possible substitutions for a total of 110 sub-libraries and 45,864 compounds. The sublibraries were synthesized and screened. The screening identified antibody inhibition with three R1, three R2, and four R3specific moieties. Using combinations of these moieties, the sub-library was deconvoluted to 36 individual compounds, which were synthesized as individual compounds (designated 2741-1 to -36).

[0197] This approach is illustrated below and includes additional (non-limiting) example step plans for the #2353 sub-library:TRIMS 2353Bis-cyclic guanidine-S-butyl linker libraryEntire library: 45,864 compounds (42 R1x 26 R2x 42 R3)Application 0312021.00258 Step 1: Screen 3 sublibraries, each with a defined R. Total of 110 mixtures (42+26+42)Sublibrary 1 : 42 mixtures with Sublibrary 2: 26 mixtures with Sublibrary 3: 42 mixtures with defined R1but varying R2and R3defined R2but varying R1and R3defined R3but varying R1and R2Step 2: Identify mixtures with most active R groupsStep 3: Synthesize 36 individual compounds representing all possible combinations of these R groupsStep 4: Evaluate each compound in antibody binding inhibition assay and parasite killing and toxicity assays

[0198] In parasite killing assays, 13 of these 36 compounds killed P. falciparum 3D7 infected RBCs with ICso < 100 nM, that 7 of these compounds kill P. falciparum parasites at low nM range , and 3 compounds had ICso < 55 nM (FIG. 15). None show toxicity to mammalian cells at up to 50 pM. Interestingly the 3 most active compounds in parasite killing assays utilize the same R1and R2(FIG. 16), thus supporting the decision to target the SAR synthesis campaign for this family on variants at R3. This family of compoundsApplication 0312021.00258 is highly active against multi-drug-resistant parasites (FIG. 17) and are freely soluble (>100 pM) in aqueous buffers at neutral pH.

[0199] Three of these 13 highly active compounds (2741-17, 19, and -20) were evaluated in extensive parasite killing assays, and cytotoxicity assays in E. coli, human PBMCs, A549 (human lung epithelial), and THP-1 (human monocyte / macrophage). These compounds had ICso for cytotoxicity >50 pM, giving a selectivity index (SI) > 1 ,000 (FIG. 18).

[0200] Since it was demonstrated that 2741 -19 has favorable in vivo PK and showed excellent efficacy in the in vivo NSG / P. falciparum model, a SAR by synthesis campaign was conducted around #2741 -19. 26 variants were synthesized and evaluated in parasite killing assays. Several of the variants demonstrated similar potency (ICso= 50 nM), whilst the majority demonstrated markedly attenuated killing. Importantly, a variant with a 2-carbon linker or a 1 carbon linker between the 2 guanidine groups (instead of the 4-carbon linker in 2741-19) was synthesized. The variant with a 2-carbon linker demonstrated equivalent potency (ICso = 50 nM) as the parent #19, while the variant with a 1 carbon linker had no activity in parasite killing assays.

[0201] An LC / MS-based single dose PK study was conducted for the series lead 2741-19 and found that at a subcutaneous (SC) dose of 3.5 mg / kg, T1 / 2 (distribution) was 1.4 hours and T1 / 2 (elimination) was 38 hours in Aotus monkeys (FIG. 19).

[0202] Next, 2741-19 was evaluated in the in vivo NSG / P. falciparum model. NSG were reconstituted with human RBC and infected with Pf3D7 infected huRBCs and treated with 2741 -19 (5 mg / kg) or vehicle control every 8 hours for 4 days. This dose and schedule were designed to maintain plasma drug levels above 50 nM as informed by the single dose PK studies (FIG. 19). 2741-19 treated mice showed rapid clearance and killing of parasites (FIG. 20).

[0203] Sub-library #1664: CTS-FIU sub-library #1664 contained 29 R1, 27 R2and 40 R3moieties (31,320 compounds) on an N-methyl tri-substituted piperazine scaffold. The 96 sub-libraries (each sub-library holds one Rxmoiety constant and allows the other R groups to vary) were synthesized and screened to identify the key Rxmoieties whichApplication 0312021.00258 inhibit anti-PfGARP from binding PfGARP. All possible combinations of these Rxmoieties were then synthesized and those individual compounds that inhibited anti-PfGARP binding to PfGARP by more than 20% were advanced.

[0204] Sub-library #1664 can, in a non-limiting example, entail:Library 166429 R1, 27 R2, 40 R3Total diversity: 29 x 27 x 40 = 31320Detailed Methods

[0205] Workflow leading up to screening phase:

[0206] Step 1: The individual remaining 150,000 compounds of the ChemBridge library were pooled into 1875 mixtures (80 compounds / mixture) and each mixture was screened (at 10 pM each compound). Deconvolution of active mixtures was done by preparation and testing of less complex mixtures derived from each active parent mixture until individual active compounds was identified. Three iterations pinpointed individual active compounds. Compounds that inhibited anti-PfGARP binding to PfGARP by more than 20% were advanced.

[0207] Step 2: Compounds that advanced from step 1 were tested in parasite killing assays at 100 nM final concentration (< 0.1 % DMSO) using 3D7 strain parasites. The primary outcome was parasite death measured using a high throughput pLDH based assay. Alternative assays included SYBR Green I and flow cytometry. Compounds which killed parasites by >90% compared to media controls were advanced to step 3.

[0208] Step 3: Compounds from step 2 were used in parasite killing assays at 100 nM concentration on 3D7 and 3D7-PfGARP KO parasites. Compounds which killed by >90% in 3D7 and showed a selectivity index (SI) of >100 in comparison to 3D7-PfGARP KO parasites were advanced to step 4. This was both a specificity and preliminary toxicity screen as PfGARP-KO parasites broadly flag eukaryotic anti-metabolites.Application 0312021.00258

[0209] Step 4: Compounds from step 3 were used in parasite killing assays on several different parasite lines using a range of concentrations. Parasite lines included: NF54, ITG4, two field isolates, INDO (chloroquine resistant), and artemisinin resistant parasites. Compounds with ICso < 100 nM for parasite killing in all strains were advanced to step 5.

[0210] Step 5: Compounds from step 4 were preliminarily screened for toxicity in three human cell types including BeWo (syncytiotrophoblast model), HLIVEC, and PBMCs at 25 pM and by Ames test. Additional cell lines used broadly in the drug development literature were added at later stages of toxicity testing 33 (step 10).

[0211] Step 6: Compounds from step 5 with no overt toxicity signals in human cells were evaluated for the induction of parasite apoptosis in 3D7 and 3D7-PfGARP KO parasites. Assays included TUNEL, caspase activation, mitochondrial membrane depolarization, and Ca++ release from the food vacuole. Up to 5 compounds which activate PfGARP-specific apoptosis and show no significant toxicity were advanced to step 7 - the SAR phase.

[0212] SAR phase:

[0213] Step 7: ChemBridge compounds from step 6 were subjected to SAR by catalog to identify active, structurally related compounds outside of the Chem Bridge library. SAR by catalog compounds were evaluated in parasite killing assays on 3D7 and 3D7-PfGARP KO parasites as described in step 3. ChemBridge compounds which killed by >90% in 3D7 and showed a selectivity index (SI) of >100 in comparison to 3D7-PfGARP KO parasites were compared with the hits from the deconvolution of CTS-FIU 1664. The two hits with the lowest ICso and highest SI were advanced to step 7.

[0214] Step 8: The seven most active compounds from CTS-FIU #2741 (all ICso < 100 nM as presented above), and the two optimal compounds from step 7 entered the SAR by synthesis phase (this selection was changed if hits emerging from step 7 were significantly better in terms of ICso, SI, and physicochemical properties such as solubility than the 7 compounds from #2741). The number of potential compounds depended on the chemical properties of the hit and how amenable it is to derivatization. 10-60Application 0312021.00258 derivatives were planned to be synthesized for each of the nine compounds entering step 8 depending on their specific chemistry.

[0215] The seven most active compounds from CTS-FIU 2741 showed a remarkable conservation at R1and R2(FIG. 16). Therefore, the SAR by synthesis approach for this family focused on substitutions at R3. It provided a robust example of SAR by synthesis for the current most active compound, 2741-19 below.

[0216] Step 9: The derivatives synthesized in step 8 were evaluated in parasite killing assays on 3D7 and 3D7-PfGARP KO parasites using a range of concentrations. Each concentration were performed in quadruplicate. Compounds with ICso < 50 nM on 3D7 and SI > 200 (in comparison to 3D7-PfGARP KO parasites) were advanced to step 10.

[0217] Lead selection phase:

[0218] Step 10: Compounds from step 9 were tested in parasite killing assays (using NF54, ITG4, INDO (chloroquine resistant), DD2 (multidrug resistant), two field isolates, and artemisinin resistant parasites). The ten compounds with the lowest ICso for parasite killing in these assays were characterized in mammalian and human cell cytotoxicity experiments using a number of cell lines and methods. Compounds with ICso < 50 nM on 3D7 and Selectivity Index (SI) >200 (in comparison to mammalian and human cells) were advanced to step 11.

[0219] Step 11: Compounds from step 10 were evaluated in Ames (mutagenicity), micronucleus (genotoxicity), microsomal stability, hERG, solubility, plasma protein binding, and whole blood stability assays. Compounds with no evidence of mutagenicity or genotoxicity and with "low" intrinsic microsomal clearance values (typically < 8.6 ul / min / mg protein), high whole blood stability (>80% recovery at 24 hours) and high solubility in aqueous buffers (>100 pg / mL) were prioritized for advancement for mouse toxicity and PK studies prior to evaluation in humanized mouse / P. falciparum treatment studies.

[0220] HTS library screening using antibody inhibition assay: A high-throughput inhibition of binding assay was developed to identify compounds which inhibit the binding of anti-PfGARP to recombinant PfGARP protein. Briefly, in a 96-well filter bottom plate,Application 0312021.00258 mixtures of compounds (80 compounds / mixture, each at 10uM) or control (vehicle alone) were incubated with PfGARP coated beads for 1 hour at RT with mixing. The beads were washed and incubated with biotinylated rec mAb or affinity purified polyclonal anti-PfGARP. Following incubation for 1 hour, the beads were washed and probe with streptavidin-PE, wash and read on a BioPlex.

[0221] SAR: The synthesis strategy was informed by classic design approaches to enhance drug-like properties such as Lipinski's Rule of Five as well as other measures of drug-likeness. In addition, SAR approaches that are known to enhance plasma half-life, such as strategic modifications of the metabolic soft spots, were prioritized. Known techniques included: 1 ) the introduction of halogens, 2) increasing lipophilicity, ±1 , and 3) N-alkylation of amides. As the NMR experiments proceeded, the strategy was also informed by structural models of lead compounds complexed to PfGARP.

[0222] Provided below is an example of a SAR by synthesis campaign around the most active compound, 2741 -19 (FIG. 16). Because of the marked lack of variation in R1and R2for the three most active compounds in the 2741 family, the SAR campaign was focused around substitutions at R3(Note: SAR optimization was also performed for Chembridge hits using similar approaches). In this example, a total of 64 analogs were synthesized below in Scheme 1 of SAR studies for hit to lead optimization.Application 0312021.00258

[0223] Retaining the main backbone (bis cyclic guanidine) and using T-bag technology for the parallel synthesis of large numbers of compounds, a variety of analogs of the identified hits at the 5-10 mg scale (sufficient for initial down-select assays) were synthesized.

[0224] Examples of modifications (depicted by letter in Scheme 1 ) included:

[0225] a) Incorporation of different linkers (derived from commercially available orthogonally protected amino acids such as ornithine, diaminobutyric acid, and diaminopropionic acid) while maintaining the same R groups of the most active identified compounds (up to 15 compounds will be prepared). Additional analogs (up to 6 compounds) were prepared by the incorporation of different R3groups not included in the original library.

[0226] b) Synthesis of analogs which are fragments of the identified most active compounds while keeping the same chemical nature of the R groups. Approximately 12 individual compounds for each substructure were prepared, purified, and tested.

[0227] c) Synthesis of cyclic guanidine tethered guanidine derived from modified homoarginine. Approximately 10 compounds were prepared.

[0228] d) Incorporation of chemical modifications to the cyclic guanidine template that modified properties such as acidity / basicity and / or hydrogen bonding strength to enhance their potency. Cyclic urea and cyclic dihydro-imidazole analogs of the most active compounds were prepared. Up to 10 compounds were prepared for each pharmacophore.

[0229] e) Selective alkylation and / or acylation of the guanidine group of the most active compounds. Up to 10 compounds were prepared.

[0230] f) Synthesis of the stereoisomer (R) of the most active compound (2741-19) derived from D-lysine.

[0231] Importantly, four compounds were synthesized at a 5 mg scale, and these compounds entered the down-select assays. These compounds included: the R and SApplication 0312021.00258 enantiomers of 579 as well as these chiral versions of 579 with an additional carbon between the aromatic ring and the amine.

[0232] NMR studies: In preliminary experiments (FIG. 12), it was shown that direct binding of one of the compounds (424) occurred to structured regions within PfGARP (amino acids 377-560) using1H-15N HSQC. The1H-15N HSQC was repeated on progressively smaller fragments of PfGARP to further define the specific regions of PfGARP necessary for drug binding. When the minimum region necessary for drug binding was defined, the solution structure of the PfGARP-drug complex was solved using a suite of 3D NMR experiments. This structural information was used for the design of improved analogs using a "SAR by NMR" approach. These experiments included: Triple-resonance, NOESY, Filtered-NOESY, RDC, PRE, STD-NMR, WaterLOGSY, Transferred-NOE, ILOEs, and T1 / T2 relaxation measurements. These experiments were repeated on the highly active leads emerging from out CTS-FIU library screen (the 2741 family of compounds). This structural information informed the synthesis of variants with improved binding to PfGARP.

[0233] Parasite killing assays: Parasite growth / killing assays (NF54, ITG4, DD2, two field isolates, chloroquine resistant parasites, and artemisinin resistant parasites (IPC_6261 and CamWT_C580Y)) were performed at several compound doses to quantify their IC50 for parasite killing. pLDH assays were used to quantify parasitemia allowing high throughput quantification of up to 48 compounds / day with a 12-point dilution curve / compound using the robotic pipetting facilities (epMotion). Alternative assays included SYBR Green I and flow-based assays.

[0234] Parasite apoptosis assays: To evaluate if the identified compounds induce an apoptotic-like cell death in treated parasites, a parasite growth assay was performed, using sorbitol synchronized ring-stage parasites incubated for 24 hours at a range of concentrations. Following incubation, parasites were assayed for TUNEL, caspase activation, mitochondrial membrane potential, and Ca++ release.

[0235] Drug binding assays: The strength of binding of compounds to immobilized PfGARP (KD) were quantified by SPR on a BioCorel 00 according to standard methods.Application 0312021.00258

[0236] AMES and micronucleus: These assays were performed according to international standard, published protocols. These assays were performed early in the down select process for hits so compounds with undesirable properties could be quickly screened out, allowing focus on the most promising candidates.

[0237] Microsomal stability: Assessment of drug stability / metabolism in the presence of liver microsomes (both human and mouse) were evaluated using commercially available assays according to manufacturer's instructions (Sigma).

[0238] Plasma and whole blood stability: Assessment of drug stability in human whole blood and plasma was measured according to standard protocols using the LC / MS assays.

[0239] hERG blockade: Lead compounds were assessed for hERG blockade with and without serum according to FDA and CiPA guidance using electrophysiologic methods.

[0240] In vitro cytotoxicity assays: In vitro cytotoxicity was evaluated in several cell types (human PBMCs, BeWo, A549, THP-1, HUVEC, etc.) according to our published protocols as well as in other cell lines most commonly used in drug cytotoxicity studies.

[0241] Solubility assays: Lead compounds were evaluated for solubility at multiple concentrations in a series of aqueous and pharmaceutically acceptable buffers by spectroscopic methods.

[0242] Compound concentration assays: An LC / MS based quantitative assay was developed for each compound entering step 10 of the Lead Selection Phase (necessary for microsomal and blood / plasma stability) using standard methods - these assays were already developed for 579, 424, and 2741-19 using a primary ion for quantification and two daughter ions for verification. The R2for the standard curve for these assays was >99%.

[0243] Selection for drug resistance: Resistance to the lead compounds was induced using protocols previously outlined. Specifically, the MIR (minimum inoculation for resistance) was assessed at a drug concentration of 3x the IC90 (approximately 35 pM for 579 and 150 nM for 2741-19) using 106, 107, 108, and 109initial parasites of both 3D7Application 0312021.00258 and Dd2 strains. Parasites which grew out of these cultures had their drug sensitivity assessed. Note: These assays were performed using 3D7 at an initial inoculum of 2.5 x 108parasites and have been unable to recover parasites when treating with 579 or 424 at 3 x IC90. It was repeated using Dd2.

[0244] Potential cross resistance to marketed antimalarials using the published criteria (IC50 shift of >5-fold results in down prioritization) was also evaluated. For 579, cross-resistance in parasites with demonstrated resistance to chloroquine and quinine (using INDO strain) were evaluated and changes in the IC50 were not observed. As detailed above, cross-resistance with ART resistant strains was evaluated, using the RSA to verify ART resistance prior to the standard killing assay with the compound in question.

[0245] Importantly, the compounds bound to PfGARP, which is located on the exofacial surface of the iRBC. Their site of action does not require internalization into the iRBC. Therefore, enhanced drug efflux, a major mechanism for antimalarial resistance, is not a likely failure mode for PfGARP targeting compounds.

[0246] Testing of lead compounds in humanized mouse model of P. falciparum.

[0247] PfGARP is not expressed by murine parasites. Therefore, compounds which target PfGARP must be tested for efficacy in humanized mouse models infected with Plasmodium falciparum. Eight lead compounds - with optimal PfGARP binding, low toxicity to human cells in culture, long IV half-life, and potent parasite killing properties in culture - were identified and prioritized. Toxicity and PK studies were performed in mice which bracketed the IC100 (in vitro) and assessed efficacy for each of these compounds in a humanized mouse model of P. falciparum.

[0248] A 5-day toxicity study in BALB / c mice was performed (up to n=20, if all have favorable microsomal stability properties) for each compound selected in step 10 of the lead selection phase at 2 doses for each compound. Groups of n=4 mice were injected once daily IV (tail vein) with 10 and 20 mg / kg of each compound in 100 pL of appropriate vehicle. Vehicle selection depended on the particular solubility / stability properties of the compounds but was restricted to selections that are acceptable for IV use in rodents and non-human primates. The lead compounds from the ChemBridge library have beenApplication 0312021.00258 soluble in 5% DMSO, 95% PEG 400 which is acceptable for use in both rodents and nonhuman primates while the lead compounds from the CTS-FIU library are freely soluble in aqueous buffers.

[0249] For 579, the current lead compound from the ChemBridge library, which has a molecular weight of 400 Da and a lower estimate of IC50 of 2 pM in growth assays, these doses represent 100 - 200-fold excess (based on initial blood concentration upon injection) over the predicted IC50 for a mouse with a 2.5 mL blood volume. For the lead compound from the CTS-FIU library, 2741-19 with a molecular weight of 585 Da and an IC50 of 55 nM, these doses represent >3, 000-fold excess over the IC50. If necessary, these doses were modified based on the IC100 determined in the in vitro parasite killing assays with the goal to select a dose that will achieve a drug level that is > 200-fold higher than the predicted IC100.

[0250] Clinical signs of toxicity (alopecia, scaly skin, hunching, ruffled fur, reduced activity, loss of body weight, and mortality) were monitored daily and the maximum tolerated dose (MTD) was defined as the highest dose at which no mortality is observed. Toxicities were scored according to their severities using a 5-point severity scale. Equal numbers of male and female mice were used.

[0251] Murine PK studies: PK studies were performed for each of the lead compounds (up to n=20, if all have favorable toxicity profiles) that shows acceptable toxicity profile (MTD>200 fold expected IC100 dose) 2 doses for each compound. Compounds were evaluated at 10 and 20 mg / kg but values can be modified based on the IC100 determined in the in vitro parasite killing assays with the goal to select doses that will achieve plasma concentrations at IC100, and 1.5 x IC100. Groups of n=2 mice / timepoint can be injected IV (tail vein) with the compound at one of two doses. Male and female mice were used in equal numbers and PK endpoints were analyzed by sex.

[0252] To derive key PK parameters for each compound, the plasma concentrationtime profiles was analyzed and measured in mice with non-com partmental and one compartmental PK models using non-linear regression software (Phoenix WinNonlin 8.1 ). The goodness of fit for model variants was assessed using the AIC, diagnostic plots, variance, and random distribution of residuals. The values for area under the curve (AUC,Application 0312021.00258 total plasma exposure) and other PK parameters such as clearance (CL), volume of distribution (Vd), and half-life (ti / 2) were obtained from the WinNonlin output. To guide interpretation and extrapolation of the murine PK results drug binding to human plasma proteins was measured using ultrafiltration and the LC / MS assay.

[0253] Humanized mouse efficacy studies: A well-established NSG / P. falciparum mouse model reconstituted with human red blood cells was used to evaluate the eight lead candidates emerging from the murine toxicity and PK studies, selecting compounds with lowest toxicity and longest T1 / 2. Four independent trials, each trial evaluated two experimental compounds and one control group, were conducted.

[0254] For each trial, male and female NSG mice (the branded name for NOD.Cg-PrkdcscidH2rgtm1vyil / SzJ, JAX Cat# 005557) were reconstituted daily with 1 mL huRBC at 50% hematocrit via intraperitoneal (IP) injection (huRBC obtained commercially from the RIBC). Twelve days after the start of engraftment, mice (both males and females) were infected by IV injection of 2 x 107Pf3D70087 / N9infected human RBCs.

[0255] On day 3 after infection, mice were randomly allocated to treatments that were administered daily for a standard 4-day trial 29 (n = 5 mice) by IV or SC injection (depending on the PK results) as described below.

[0256] Group 1 - no treatment (infection control), n=5

[0257] Group 2 - Compound 1 at dose / interval designed to achieve compound level at IC100, n=5

[0258] Group 3 - Compound 1 at dose / interval designed to achieve compound level at 1.5 x IC100, n=5

[0259] Group 4 - Compound 2 at dose / interval designed to achieve compound level at IC100, n=5

[0260] Group 5 - Compound 2 at dose / interval designed to achieve compound level at 1.5 x IC100, n=5

[0261] Endpoints - Safety Biomarkers - Reactogenicity, body weight, CBC, and LFTs were assessed daily.Application 0312021.00258

[0262] Efficacy: The primary outcome was parasitemia. Blood samples (via tail vein) were evaluated daily from the day of challenge by flow cytometry and continuing until day 28 post challenge to quantify parasitemia with confirmation of results by microscopy. Animals which resolved their infections post-treatment were followed with daily blood films for 2 weeks and weekly blood films for 1 month to assess recrudescence. RBC chimerism were monitored by flow cytometry using anti-murine erythrocyte TER119 monoclonal antibody (PharMingen, San Diego, CA) and SYTO-16 and then analyzed by flow cytometry.

[0263] Statistical Analyses: Parasitemia, and other continuous endpoints, were assessed for normality and assessed for differences by Students t-Test or Mann Whitney U-tests as appropriate. Sex was evaluated as a potential effect modifier using linear models. The sample size (n=5 / group) was selected. With an outcome measure of % iRBC, an alpha of 0.05, an SD of 0.5%, 5 mice per group, there was 80% power to detect a difference of 1 % in parasitemia between treated versus control groups in a 2-tailed test. For example, 80% powerto detect a difference between Drug Treated and Control groups would be significant if the Drug Treated group had mean parasitemia of 2.9% and the Control group had a mean parasitemia of 4%.

[0264] Summary

[0265] Screened a >30 million compound combinatorial library.

[0266] Identified 2 scaffolds (each with 20-50 thousand variant compounds) which inhibit anti-PfGARP from binding to PfGARP.

[0267] Deconvoluted 2741 scaffold to a family of 36 compounds which all have parasite killing activity with ICso < 100 nM.

[0268] Generated ~20 additional analogs of 2741-19 and three of these analogs retain parasite killing activity.

[0269] Cytotoxicity, genotoxicity, cardiotoxicity, pharmacokinetic, parasite drug resistance, and in vivo efficacy studies of 2741-19 demonstrate favorable properties.Application 0312021.00258 Example 2. Additional Testing of Compounds

[0270] Many compounds discussed above have been synthesized, including 2741-1 to 2741-36, shown in

[0118] , Analogs of 2741-19 (2741-19 ana-1 to ana-25), shown in

[0119] , were synthesized.

[0271] Significant additional data focusing on the lead compound, 2741-19, was generated in Example 1. More than 5 independent parasite killing assays have been performed with each assay having 3-5 replicates per dose and quantified parasitemia using three approaches (microscopy, flow cytometry, and pLDH assay). These experiments were performed in different parasite strains, including 3D7, Dd2, D10, W2, Cam WT_C580Y, and fresh field isolates. All results were concordant with ICso ~40-55 nM as reported in FIG. 15.

[0272] In addition, cytotoxicity testing of 2741-19 has been conducted in both E. coli and HepG2 cells. Viability was assessed by crystal violate staining (HepG2) and proliferation was assessed by spectrophotometry (E. coli). The ICso for cytotoxicity was 76uM for E. coli and 140 pM for HepG2 (FIG. 21), indicating a >1, 500-fold selectivity for drug killing of malaria parasites. The results in HepG2 are particularly important as this human hepatocellular cell line has well developed metabolism pathways and is a standard screening assay for cytotoxicity in drug development programs.

[0273] A mutagenicity assessment of 2741 -19 was also completed via the Ames test. Two strains (TA98 and TA100) were used, with and without S9 metabolic activation.2741-19 demonstrated no mutagenic potential in either strain tested.

[0274] Cardiotoxicity assessments (hERG) for 2741-19 were also completed. The ability of drug to alter hERG cardiac ion channel function, expressed in mammalian cells (HEK-293), was assessed using the electrophysiology platform QPatch HTX. The ICso for hERG channel inhibition was 45-fold higher than the ICso for parasite killing, indicating a wide therapeutic window.

[0275] One-month selection for resistant parasites was completed as follows. Briefly, 109Dd2 strain iRBCs were cultured with 2741-19 at 3 x ICso (150 nM). Media was changed twice a week, and live parasites were enumerated. At 4 weeks of culture, no liveApplication 0312021.00258 parasites were able to be recovered, indicating an MIR > 9 (minimum inoculum for resistance).

[0276] Lastly, two additional NSG mouse studies were completed, using 2741-19 at 5 mg / kg SC twice a day for 4 days according to FIG. 20. In the first experiment, drug exposure was initiated at day 3 post-infection, when all mice were patently infected as assessed by microscopy (FIG. 20). In the second experiment, drug exposure was initiated when each animal's parasitemia exceeded 0.5%. In both experiments, treatment of P. falciparum infected NSG mice resulted in parasite clearance while controls developed high density parasitemia.

[0277] 2741 -19 can be developed even further, as well as other members in the 2741 series, as well as other hit / leads from further library screening, through structured SAR, parasite killing, toxicity, ADME / PK and along the continuum to in vivo testing in the NSG / P. falciparum model.

[0278] Sub-library #1664 can, in a non-limiting example, entail:Library 166429 R1, 27 R2, 40 R3Total diversity: 29 x 27 x 40 = 31320Example 3. 2741-19 Analogs

[0279] Analogs of 2741-19, 2741-19 ana-1 to ana-25, shown in

[0119] , were synthesized. Select ICso values are tabulated below for some 2741-19 analogs:Application 0312021.00258Example 4. 2741-19 specificity for PfGARP

[0280] A study was conducted to determine specificity of binding between PfGARP and 2741-19, providing evidence for the specificity of binding between 2741-19 and PfGARP and specificity of killing between 2741-19 and PfGARP.

[0281] There are two lines of evidence for the specificity of binding between 2741 -19 and PfGARP. Incubation of PfGARP with 2741-19 inhibits the binding of anti-PfGARP to its target PfGARP (FIG. 22), indicating that the drug is binding to its target, PfGARP. This is further demonstrated by showing that a 2D-NMR spectrum for PfGARP shifts with the addition of 2741-19, indicating direct binding (FIG. 23).

[0282] The evidence suggests that the PfGARP-2741-19 interaction is responsible for the parasite killing activity of 2741-19. Parasites with a gene deletion of PfGARP are significantly less susceptible to 2741-19 mediated killing as compared to wild type parasites (FIG. 24). This indicates that PfGARP is necessary for 2741-19 mediated parasite killing. This effect is most pronounced at lower drug concentrations.Example 5

[0283] A 14-year-old female patient with drug-resistant malaria can be treated with a therapeutic oral dosage of compound 2741-19 (below) and makes a full recovery.Application 0312021.00258 Example 6

[0284] A racemic mixture can be formulated into a pharmaceutical composition, containing a therapeutically effective dose, and pharmaceutically acceptable excipients.Example 7

[0285] A 55-year-old male patient with malaria can be administered a therapeutically effective dosage of the below compound intravenously. The malaria can be treated, and the patient can make a full recovery.Example 8

[0286] A 25-year-male traveler is administered the below compound subcutaneously. Malaria can be prevented while the individual is visiting locations with high risk of transmitting malaria.Application 0312021.00258Example 9

[0287] A 37-year-old female patient with malaria caused by Plasmodium falciparum can be administered a therapeutically effective dosage of the below compound intramuscularly. The malaria can be treated, and the patient can make a full recovery. The compound and dosages can be optimized according to the severity of malaria, disease stage, and species of Plasmodium parasite.

[0288] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, theApplication 0312021.00258 numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0289] The terms “a” and “an” and “the” and similar references used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0290] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of any and all Markush groups used in the appended claims.

[0291] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes allApplication 0312021.00258 modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0292] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0293] Furthermore, references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.

[0294] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

Claims

1. Application 0312021.00258 CLAIMS2.What is claimed is:

1. A compound having a formula:

5. 7.or a pharmaceutically acceptable salt thereof;8.wherein9.n is 1-4;10.A is H, isopropyl,11. 13.R9and R10are independently H, halogen, OCH3, or CF3;14.Z is CH2CH2-P-C6H4R11or cyclohexylmethyl;15.R11is H, halogen, or OCH3;16.J is isopropyl, isobutyl, or cyclohexylmethyl;17.Y is C(O), C(NH), or S(O)2; and18.R12is NH2, CH2-P-C6H4R3or -P-C6H4R11.

2. The compound of claim 1 , wherein the compound has a formula:

21. 23.or a pharmaceutically acceptable salt thereof;24.wherein Application 0312021.00258 n is 1-4;25.R9and R10are independently H, halogen, OCH3, or CF3;26.Z is CH2CH2-P-C6H4R11or cyclohexylmethyl;27.R11is H, halogen, or OCH3;28.J is isopropyl, isobutyl, or cyclohexylmethyl;29.Y is C(O), C(NH), or S(O)2.

3. The compound of claim 1 , wherein the compound has a formula:

32. 34.or a pharmaceutically acceptable salt thereof;35.wherein36.n is 1-4; and37.R9and R10are independently H, halogen, OCH3, or CF3.

4. The compound of claim 1 , wherein the compound has a formula:

40. 42.or a pharmaceutically acceptable salt thereof;43.wherein44.n is 1-4; and45.R9and R10are independently H, halogen, OCH3, or CF3.

5. The compound of claim 1 , wherein the compound has a formula:Application0312021.0025848. 50.or a pharmaceutically acceptable salt thereof;51.wherein52.n is 1-4; and53.R9and R10are independently H, halogen, OCH3, or CF3.

6. The compound of claim 1 , wherein the compound has a formula:

56. 58.or a pharmaceutically acceptable salt thereof;59.wherein60.n is 1-4;61.R9and R10are independently H, halogen, OCH3, or CF3;62.R12is CH2-P-C6H4R11; and63.R11is H, halogen, or OCH3.

7. The compound of claim 1 , wherein the compound has a formula:

66. 68.or a pharmaceutically acceptable salt thereof;69.wherein Application 0312021.00258 n is 1-4;70.R9and R10are independently H, halogen, OCH3, or CF3;71.R12is p-C6H4R11; and72.R11is H, halogen, or OCH3.

8. The compound of claim 1, wherein the compound is75. 77.wherein78.

79. combination thereof.

9. The compound of claim 1 , wherein the compound has a formula:Application0312021.0025882.

83. Application0312021.0025885.

86. Application0312021.0025888.

89. Application0312021.0025891.

92. Application0312021.0025894.

95. Application0312021.0025897.

98. Application0312021.00258101.

103.

104. or a pharmaceutically acceptable salt thereof.

10. The compound of claim 1 , wherein the compound has a formula:Application0312021.00258107.

108. acceptable salt thereof.

11. The compound of any one of Claims 1 -10, wherein the compound is a prodrug thereof.

12. The compound of any one of Claims 1-10, wherein the compound is a racemic mixture of stereoisomers, a non-racemic mixture of stereoisomers, an R-enantiomer, or an S-enantiomer.

13. A composition, comprising the compound of any of Claims 1 -12.

14. A pharmaceutical composition, comprising the compound of any of Claims 1-12, and a pharmaceutically acceptable excipient.

15. A method of treating and / or preventing an infectious disease in a subject in need thereof, the method comprising administration of a therapeutically effective amount of one or more compounds selected from any one of Claims 1-12 or the compositions selected from any one of Claims 13 and 14.Application 0312021.00258 16. The method of claim 15, wherein the infectious disease is malaria.

17. The method of claim 16, wherein the malaria is caused by Plasmodium falciparum.

18. The method of claim 15, wherein the compound mimics the binding of antiPlasmodium falciparum Glutamic Acid Rich Protein (PfGARP).

19. The method of claim 15, further comprising an administering of at least one second therapeutic agent to the subject.

20. The compound of any one of Claims 1-10, wherein the compound has an ICso value of less than 1 pM against Plasmodium falciparum.

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