WO2024249604A2 - Covalent inhibitors as anti-malarial agents - Google Patents

Abstract

The present application generally relates to compounds, pharmaceutical compositions and method to treat a patient with malaria related disease or Plasmodium infection. Particularly the method compromises the step of administering a therapeutically effective amount of the disclosed compounds or compositions, with or without one or more anti-infective agents, to the patient in need of relief from said infection. Methods of uses and composition matters are within the scope of this disclosure.

Description

COVALENT INHIBITORS AS ANTI-MALARIAL AGENTS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This Application claims the benefit of US provisional application 63/470,664, filed on June 2, 2023. The contents of which are expressly incorporated herein by reference in its entirety into this present disclosure.

TECHNICAL FIELD

[0002] The present application relates generally to compounds and pharmaceutical formulations of compounds and methods to treat a patient with malaria. Particularly the method compromises the step of administering a therapeutically effective amount of the compounds, with or without one or more anti-infective agents, to the patient in need of relief from said infection. More particularly the present disclosure relates to the identification of covalent fragment inhibitors for Plasmodium falciparum UCHL3 with anti-malarial efficacy.

BACKGROUND AND BRIEF SUMMARY

[0003] This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.

[0004] Malaria continues to be a major burden on global health, responsible for 619,000 deaths in 2021. The causative agent of malaria is the eukaryotic parasite Plasmodium. Resistance to artemisinin-based combination therapies (ACTs), the current first-line treatment for malaria, has emerged in Asia, South America, and more recently Africa, where >90% of all malaria-related deaths occur. This has necessitated the identification and investigation of novel parasite proteins and pathways as antimalarial targets, including components of the ubiquitin proteasome system.

[0005] In 2021, according to the World Health Organization World Malaria Report (2022) there were an estimated 247 million cases of malaria worldwide, resulting in 619,000 deaths. Malaria is caused by the eukaryotic protozoan parasite, Plasmodium, which is spread through the bite of an infected Anopheles mosquito. Of the five species of Plasmodium that cause disease in humans, P. falciparum is the most prevalent and the deadliest. The current first-line treatment for P. falciparum malaria are artemisinin-based combination therapies (ACTs), which consist of a short-lived artemisinin that rapidly reduces parasite biomass paired with a longer-lived partner drug to clear remaining parasites. Partial resistance to artemisinin has emerged in Asia, South America, and Africa. (Imwong et al., 2020; Ashley et al., 2014; Tumwebaze et al., 2022) This has spurred a call to action by the global community to address the significant need for novel therapeutic targets and molecular scaffolds for antimalarial drug discovery.

[0006] The ubiquitin-proteasome system (UPS) is conserved in eukaryotes, responsible for regulating several vital cell processes such as cell-cycle progression, protein trafficking, signaling, DNA repair, and protein quality control. (Komander et al., 2012) Ubiquitin is attached to substrate proteins through a series of El ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligating enzymes, and sometimes E4 ubiquitin chain elongation factors. (Komander et al., 2009) The type and length of ubiquitin linkage(s) dictates substrate fate. (Akutsu et al., 2016) Monoubiquitin and polyubiquitin chains can be edited or removed from substrate proteins via deubiquitinases (DUBs), which are also responsible for generating free ubiquitin from ubiquitin precursors. Mevissen et al., 2017; Komander et al., 2009)

[0007] In humans, the UPS is an established target for cancer, and several components of the UPS including the proteasome, E3 ubiquitin ligases, and DUBs have been validated as therapeutic targets. (Myung et al., 2001; Park et al., 2020) The UPS has also been gaining increasing interest as a therapeutic target in malaria. Proteasome inhibitors have been shown to kill parasites at the liver, blood, and mosquito stages, as well as block parasite transmission to mosquitos. (Gantt et al., 1998; Czesny et alk., 2009; Kirkman et al., 2018; Li et al., 2014) Multiple classes of Plasmodium- specific proteasome inhibitors have now been developed, several of which have been shown to overcome artemisinin resistance. (Yoo et al., 2018; LaMonte et al., 2017; Li et al., 2016) In addition, several classes of mammalian DUB inhibitors were demonstrated to have antimalarial activity against Plasmodium in vitro and in vivo. (Simwela et al., 2021) [0008] Here, we describe Plasmodium falciparum deubiquitinase ubiquitin C-terminal hydrolase L3 (PfUCHL3) as one such target. We carried out a high-throughput screen with covalent fragments and identified eight scaffolds that selectively inhibit the plasmodial UCHL3, but not human UCHL3 or the closely related human UCHL1. After assessing toxicity in human cells, we identified four promising hits and demonstrated their efficacy against asexual P. falciparum blood stages and P. berghei sporozoite stages. The present disclosure provides for the identification of covalent fragment inhibitors for Plasmodium falciparum UCHL3 with anti-malarial effica The present application relates generally to a method to treat a patient with malaria. In particular, malaria due to Plasmodium infection. [0009] Therefore, the present disclosure provides for compounds and method for treating a patient of symptoms of malarial infection compromising the step of administering a therapeutically effective amount of a compound of the disclosure to the patient in need thereof. The present disclosure provides for pharmaceutical formulation comprising the disclosed compounds with a pharmaceutically acceptable carrier, diluent, excipient or salt thereof.

[0010] The present disclosure further provides for a method that compromises the step of administering a therapeutically effective amount of the disclosed compound, with or without one or more anti-malarial or anti-infective agents, to the patient in need thereof.

[0011] The present disclosure provides for a compound having the formula:

[0012]

[0013] or a pharmaceutically acceptable salt thereof, [0014] where A is optionally substituted 1 - 2 C,

[0015] X is CH or N,

[0016] Y is an optionally substituted 1 - 6 alkyl,

[0017] Z is optionally substituted N,

[0018] and R is an optional substituent.

[0019] In particular, the disclosure provides for where Z is N substituted by 1 - 6 alkyl or 1 - 6 aryl. In a further aspect, the disclosure provides for where R is not present as a substitutent.

[0020] The present disclosure provides for a compound having formula (I) where R is selected from the group consisting of optionally substituted alkyl, phenyl, optionally substituted benzyl, optionally substituted benzoyl, optionally substituted C3-C8 cycloalkyl, optionally substituted CH2-(C3-C8 cycloalkyl), optionally substituted 5-6 membered heterocyclyl, and optionally substituted CH2-(5-6 membered heterocyclyl).

[0021] Thus the present disclosure provides for a compound

[0023] where R2 is optionally substituted 1 - 6 alkyl, S, N, or halo.

[0024] The present disclosure provides for a compound having formula

[0025]

[0026]

[0027] The present disclosure provides for a pharmaceutical composition comprising a compound as disclosed above, and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof.

[0028] The present disclosure provides for a pharmaceutical composition comprising a compound as disclosed above, and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof, for the treatment of Plasmodium falciparum deubiquitinase ubiquitin C- terminal hydrolase L3 (PfUCHL3) mediated disease, in a patient in need thereof.

[0029] The present disclosure provides for a pharmaceutical composition comprising a compound as disclosed above, and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof, for the treatment of plasmodium infection, in a patient in need thereof.

[0030] The present disclosure provides for a pharmaceutical composition comprising a compound as disclosed above, and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof, for the treatment of malaria, in a patient in need thereof.

[0031] The present disclosure provides for a pharmaceutical composition comprising a compound selected from the group consisting of:

[0032]

thereof. [0039] The present disclosure provides for the use of the compositions described above for the treatment of Plasmodium falciparum dcubiquitinasc ubiquitin C-tcrminal hydrolase L3 (PfUCHL3) mediated disease. The compounds and formulations of the disclosure are useful for mediating the activity of Plasmodium falciparum deubiquitinase ubiquitin C- terminal hydrolase L3 (PfUCHL3) or similar enzyme found in any other organism. As such the compounds and formulations of the present disclosure are useful as therapeutic agents for treating a patient in need thereof where Plasmodium falciparum deubiquitinase ubiquitin C- terminal hydrolase L3 (PfUCHL3) or similar enzymatic homolog is mediating a disease state. [0040] The pharmaceutical formulations of the disclosure can comprise one or more of the disclosed compounds or be combined or used in combination with other therapeutic or ameliorative agents.

[0041] In some illustrative embodiments, the present disclosure relates to a method for treating a patient of a Plasmodium infection compromising the step of administering a therapeutically effective amount of a UCHL3 inhibitor to a patient in need of relief from said infection.

[0042] In another embodiment, the disclosure provides for methods of treatment wherein the pharmaceutical compositions arc administered orally. In another embodiment of the present disclosure, the pharmaceutical compositions of the disclosure are suitable for administration intravenously, intradermally, intramuscularly, and intra cerebrospinal administration.

[0043] In some illustrative embodiments, the present disclosure relates to a method for treating a patient compromising the step of administering a therapeutically effective amount of a compound as shown in Table 1 (and shown above), together with one or more other anti- malarial agents, such as quinine, ACTs, UPS DUBs, to the patient in need of relief from said infection.

[0044] The present disclosure provides for methods for treating a patient infected by Plasmodium comprising the step of administering a therapeutically effective amount of a compound of Table 1, together with one or more pharmaceutically acceptable carriers, diluents, and excipients, to the patient in need of relief from said infection. [0045] In another aspect, the present disclosure provides for a pharmaceutical composition comprising a compound described above and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof.

[0046] And so the present disclosure provides for a method for treating a patient of a Plasmodium infection compromising the step of administering a therapeutically effective amount of the compound as described above, to a patient in need thereof. It is further provided that this can be done with one or more pharmaceutically acceptable carriers, diluents, and excipients, to the patient in need of relief from said infection. It is further provided for by the present disclosure that the patient may have a malarial infection, and/or suffering from malaria.

[0047] In another embodiment the present disclosure relates to methods for treating a patient in need thereof comprising administering a DUB (deubiquitinase) inhibitor to the patient.

[0048] In particular, a method for treating a patient with malaria comprising administering a pharmaceutically effective amount of a DUB inhibiter, where the DUB inhibitor is effective against Plasmodium’ s pre-erythrocytic stages.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and other objects, features, and advantages of the present disclosure will become more apparent when taken in conjunction with the following drawings, and wherein:

[0050] Fig. 1A depicts PfUCHL3 inhibition dose-response curves for eight validated acrylamide hits.

[0051] Fig. IB shows TABLE 1 PfUCHL3 high-throughput screen hits and identified compounds.

[0052] Fig. 2A shows representative images from high content imaging. Hoechst (blue) was used to stain for parasite DNA and Mitotracker Deep Red (pink), which stains respirating parasite mitochondria, was used as a viability marker. Scale bar = 50 um.

[0053] Fig. 2B depicts a graph of mean % inhibition +- standard error of the mean (S.E.M.) from 3 biological replicates, each performed in technical duplicates.

[0054] Fig. 3 shows graphs depicting the potency of the lead compounds against P. falciparum asexual blood stages. Fig. 3A depicts IC50 curves against Cam3.II K13 C580Y. Figure 3B shows the mean IC50 values +- S.E.M. for Cam3.II K13 C580Y (blue), Cam3.II K13 WT (grey), and 3D7 strain parasites (teal) from 3 biological replicates.

[0055] Fig. 4 shows UCH0081 anti-plasmodium activity is mediated through covalent inhibition with greater potency than MMV688704. (Fig. 4A) Dose-response curves for UCH0081 , UCH0114, MMV688704 versus asynchronous Cam3.II K13 C580Y parasites. (Fig. 4B) Mean IC50 values ± S.E.M. from 3 biological replicates. Statistical significance was examined using a Mann-Whitney U test. *p < 0.05.

[0056] Fig. 4C shows TABLE 2.

[0057] Fig. 5 depicts a graph of cell viability when tested with the molecules at 500uM. [0058] Fig. 6 depicts a graph of Anti-P. berghei sporozoite activity of UCH0080 (blue), UCH0081 (red), UCH0082 (green), and UCH0083 (purple). The calculated IC50 values for each analog are shown at right.

[0059] Fig. 7 depicts Synthesis Scheme 1.

[0060] Fig. 8 depicts Synthesis Scheme 2. DETAILED DESCRIPTION

[0062] For the purposes of promoting and understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

[0063] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the ail.

[0064] In the present disclosure the term “about” can allow for a degree of variability in a value or range, for example, within 20%, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. In the present disclosure, the term “substantially” can allow for a degree of variability in a value or range, for example, within 80%, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.

[0065] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0066] "Identity," as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. Methods to determine "identity" are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs can be used to determine "identity" between two sequences these programs include but are not limited to, GCG; suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN). The BLAST X program is publicly available from NCBI and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.

[0067] The term “substituted” as used herein refers to a functional group in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, azides, hydroxylamines, cyano, nitro groups, N-oxides, hydrazides, and enamines; and other heteroatoms in various other groups.

[0068] The term “alkyl” as used herein refers to substituted or unsubstituted straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-Cs), or, in some embodiments, from 1 to 6 carbon atoms (Ci-Ce). Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n- pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec -butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. [0069] Combinations and sub-combinations can be formed from the known substituents. For example, a substituent can be selected from the group consisting of optionally substituted phenyl, optionally substituted benzyl, optionally substituted benzoyl, optionally substituted C3-C8 cycloalkyl, (preferably optionally substituted C5-C6 cycloalkyl) optionally substituted CH2-(C3-C8 cycloalkyl) (preferably optionally substituted CH2-(C5-C6 cycloalkyl), optionally substituted 5-6 membered heterocyclyl, and optionally substituted CH2-(5-6 membered heterocyclyl).

[0070] The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain and branched divalent alkenyl and cycloalkenyl groups having from 2 to 20 carbon atoms(C2-C20), 2 to 12 carbons (C2-C12), 2 to 8 carbon atoms (C2-C8) or, in some embodiments, from 2 to 4 carbon atoms (C2-C4) and at least one carbon-carbon double bond. Examples of straight chain alkenyl groups include those with from 2 to 8 carbon atoms such as -CH=CH-, -CH=CHCH2-, and the like. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and the like.

[0071] An alkynyl group is the fragment, containing an open point of attachment on a carbon atom that would form if a hydrogen atom bonded to a triply bonded carbon is removed from the molecule of an alkyne. The term “hydroxyalkyl” as used herein refers to alkyl groups as defined herein substituted with at least one hydroxyl (-OH) group.

[0072] The term “cycloalkyl” as used herein refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. In some embodiments, cycloalkyl groups can have 3 to 6 carbon atoms (C3-C6). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like.

[0073] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a “formyl” group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atoms bonded to the carbonyl group. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0074] The term “aryl” as used herein refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons (C6-C14) or from 6 to 10 carbon atoms (C6-C10) in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono- substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein.

[0075] The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0076] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec -butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure arc substituted therewith.

[0077] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)₃ wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialky larylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0078] The term “amino group” as used herein refers to a substituent of the form -NH2, -NHR, -NR2, -NR A. wherein each R is independently selected, and protonated forms of each, except for - NR₃ ⁺, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0079] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. [0080] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, polyhalo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1 -dichloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH₃)2 and the like.

[0081] The term “optionally substituted,” or “optional substituents,” as used herein, means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different. When using the terms “independently,” “independently are,” and “independently selected from” mean that the groups in question may be the same or different. Certain of the herein defined terms may occur more than once in the structure, and upon such occurrence each term shall be defined independently of the other.

[0082] The compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.

[0083] Similarly, the compounds described herein may include geometric centers, such as cis, trans, E, and Z double bonds. It is to be understood that in another embodiment, the invention described herein is not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. It is also to be understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other double bonds.

[0084] As used herein, the term “salts” and “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.

[0085] Pharmaceutically acceptable salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts arc found in Remington’s Pharmaceutical Sciences, 17th cd., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference.

[0086] The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may 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.

[0087] As used herein, the term “administering” includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0088] Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like. Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intra urethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration. [0089] Illustrative means of parenteral administration include needle (including micronccdlc) injectors, ncedlc-frcc injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.

[0090] The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

[0091] It is to be understood that in the methods described herein, the individual components of a co-administration, or combination can be administered by any suitable means, contemporaneously, simultaneously, sequentially, separately or in a single pharmaceutical formulation. Where the co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

[0092] The term ‘‘therapeutically effective amount” as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit, /risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder: activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill. [0093] Dependin g upon the route of admini stration, a wide range of permissible dosages are contemplated herein, including doses falling in the range from about 1 pg/kg to about 1 g/kg. The dosages may be single or divided, and may administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like. In each of these cases it is understood that the therapeutically effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.

[0094] In addition to the illustrative dosages and dosing protocols described herein, it is to be understood that an effective amount of any one or a mixture of the compounds described herein can be determined by the attending diagnostician or physician by the use of known techniques and/or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0095] The term “patient” includes human and non-human animals such as companion animals (dogs and cats and the like) and livestock animals. Livestock animals are animals raised for food production. The patient to be treated is preferably a mammal, in particular a human being.

[0096] The present disclosure provides for a compound having the formula:

or a pharmaceutically acceptable salt thereof, where X is CH or N, and R is an optional substituent.

[0097] The present disclosure further provides for where R is selected from the group consisting of optionally substituted alkyl, phenyl, optionally substituted benzyl, optionally substituted benzoyl, optionally substituted C3-C8 cycloalkyl, optionally substituted CH2- (C3-C8 cycloalkyl), optionally substituted 5-6 membered heterocyclyl, and optionally substituted CH2-(5-6 membered heterocyclyl).

[0098] In a further aspect, the present disclosure provides for a compound having the formula:

where R2 is optionally substituted 1 - 6 alkyl, S, N, or halo.

[0099] In a another aspect of the disclosure, it is provided for a compound having the formula:

[0100] In another aspect of the disclosure teaches pharmaceutical compositions comprising a compound of Formula I, Formula II and as above, and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof.

[0101] The disclosure provides for the use of the compound and pharmaceutical composition as disclosed for the treatment of deubiquitinase ubiquitin C-terminal hydrolase L3 (PIUCHL3) mediated disease, in a patient in need thereof. For the treatment of plasmodium infection mediated disease, in a patient in need thereof. For the treatment of malaria, in a patient in need thereof. [0102] In another aspect of the disclosure, pharmaceutical composition comprising a compound selected from the group consisting of

and a pharmaceutically acceptable carrier, diluent, excipient, or salt thereof is provided for.

[0103] The disclosed pharmaceutical formulations can be used for the treatment of malaria, Plasmodium infection, and related disease states.

[0104] The disclosure further provides for methods for treating a patient in need of therapy for Plasmodium infection, comprising administering a compound or formulation as described above to the patient in need thereof. A method for treating a patient in need of therapy for malaria, comprising administering a therapeutically effective among of a compound or formulation described above to a patient in need thereof.

[0105] In some illustrative embodiments, the present disclosure relates to a method for treating a patient compromising the step of administering a therapeutically effective amount of a compound shown above, together with one or more other anti-malarial agents, such as quinine, ACTs, UPS DUBs, to the patient in need of relief from said infection. [0106] The present disclosure may be better understood in light of the following nonlimiting compound examples and method examples.

[0107]

[0108] COMPOUNDS

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0119] EXAMPLES

[0120] High-throughput screening.

[0121] Here, we investigate P. falciparum ubiquitin C-terminal hydrolase L3 (PfUCHL3;

PF3D7_1460400) as a potential antimalarial target. PfUCHL3 is a cysteine protease that was shown to have both deubiquitinase and deneddylase activity in P. falciparum. However, little is known regarding the natural substrate(s) of this enzyme within the parasite. PfUCHL3 is expressed in ookinetes, oocysts, gametocytes, and throughout asexual development.

Researchers were able to create transgenic parasites overexpressing wild type PfUCHL3, but not a catalytically dead mutant, suggesting that the enzyme may be essential in asexual blood stages.20 PfUCHL3 shares only 36% sequence identity with human UCHL3 (hUCHL3) and crystal structures of the human and parasite enzymes reveal differences in the active site that could be exploited to selectively target PfUCHL3. By conducting a virtual structure-based screen of the pathogen box from Medicines for Malaria Venture, one group recently identified two putative PfUCHL3 inhibitors that have anti-malarial activity against asexual blood stage parasites. To our knowledge, no other PfUCHL3 inhibitors have been reported and we sought to further explore this DUB as a potential anti-malarial therapeutic target. [0122] To this end, we performed a high-throughput screen on a library of fragments that contain cysteine reactive electrophiles. We identified eight covalent-fragment inhibitors of PfUCHL3 that have selectivity over human UCHL3 (hUCHL3) and the closely related human DUB, UCHL1 (hUCHLl). Four molecules were found to exhibit low toxicity to human HEK293 cells and have anti-parasitic activity against P. falciparum asexual blood stages and P. berghei sporozoite stages. The results of this study suggest targeting PfUCHL3 may be a viable anti-plasmodial therapeutic approach.

[0123] To begin, we screened a 1700-member covalent fragment library from Enamine (Kyiv, Ukraine) containing diverse scaffolds and four known cysteine electrophiles: activated nitriles, epoxides, chloroacetamides and acrylamides. For the primary PfUCHL3 screen, we utilized the fluorescence-based ubiquitin rhodamine- 110 (Ub-Rho) enzymatic activity assay .27 lodoacetamide served as a positive control while DMSO served as the negative control. The Z’ -factor analysis for the controls was determined to be 0.76 indicating the assay exhibited a sufficiently wide window between signal-to-noise to reliably identify hit molecules.28 Fragments were first screened in singlet at a single-concentration of 500 pM, with a 1 h preincubation prior to the addition of Ub-Rho substrate. Percent inhibition was determined compared to a DMSO control set to 100% activity. The hit criteria were set at 90% inhibition of P1UCHL3, which yielded 76 hits (Figure SI A). The 76 hits were then validated in triplicate against P1UCHL3 in the same assay at which point 12 did not validate and were triaged leaving 64 to move forward (Figure SIB). The 64 hits represented two electrophile classes: 9 acrylamides and 55 chloroacetamides. We prioritized the acrylamide hits, due to acrylamide being a pharmaceutically acceptable cysteine electrophilic moiety targeting many human kinases.29-32 Eight of the nine were commercially available as dry powders and evaluated for off-target inhibition against the human DUB orthologs, hUCHLl and hUCHL3. These molecules were evaluated in dose-response assays against PfUCHL3, hUCHL3, and hUCHLl to determine half-maximal inhibitory concentrations (Figure 1 and Table 1). [0124] Of the eight hits, UCH0080, UCH0082, UCHOO83, UCH0084 and UCH0087 all displayed sub- 100 pM (23 - 90 pM range) potency against PfUCHL3, with UCH0080 and UCH0087 being the most potent. Six of the eight compounds did not exhibit any inhibition of hUCHL3 even up to 500 pM. Only UCHOO83 and UCH0084 displayed sub-500 pM hUCHL3 potency, with IC50 values of 282 pM and 180 pM, respectively. No compound displayed inhibitory activity against hUCHLl at 500 pM. At the hit stage, the high- throughput screen produced validated PfUCHL3 hits with a range of activity against the desired target and varying selectivity against the two closest human DUB off-targets.

[0125] Molecules were next evaluated for efficacy in P. falciparum asexual blood-stage parasites. Asynchronous Cam3.II C580Y parasites were exposed to a single dose (500 pM) of UCH0080, UCH0081, UCH0082, UCHOO83, UCH0084, UCH0085, UCH0086, or UCH0087 and parasite viability was assessed in the following replication cycle at 72 h by high content imaging . Parasite DNA was stained with Hoechst 33342, and respirating parasite mitochondria were detected with MitoTracker™ Deep Red FM. RBCs were visualized by Brightfield and by staining with Wheat Germ Agglutinin Alexa Fluor™ 488 (Figure S2). Of the eight compounds evaluated, six displayed >99% inhibitory activity at 500 pM (Figure 2). The other two molecules, UCH0086 and UCH0087, displayed < 10% inhibitory activity and were deprioritized from further evaluation (Figure 2).

[0126] Dose response assays were then conducted with UCH0080, UCH0081, UCH0082, UCH0083, UCH0084, and UCH0085 against Cam3.II K13 C580Y, Cam3.II KI 3 WT, and 3D7 strain parasites. These parasite strains have numerous differences genetically and consequently in drug resistance profiles (Table SI). Specifically, Cam3.II parasites have mutations in the known drug resistance genes dhfr (PF3D7_0417200), dhps

(PF3D7_0810800), and pfert (PF3D7_0709000), which confer resistance to the antimalarials pyrimethamine, sulfadoxine, and chloroquine, respectively. In addition, Cam3.II K13 C58OY parasites harbor an additional mutation in kelch 13 (K13; PF3D7_1343700), which confers resistance to artemisinin.33 Asynchronous parasites were exposed to a range of compound concentrations from 0.5 pM to 500 pM and parasite viability was assessed 72 h later (Figure 3).

[0127] Analogs provided a range in IC50 values from 2 pM to 180 pM for the six tested molecules, with some compounds exhibiting pronounced parasite strain- specific effects (Figure 3 and Table 2). For example, sensitivity to UCH0082 appeared to be dependent on KI 3 genotype, as KI 3 C580Y parasites displayed 2-fold higher IC50 values than KI 3 WT parasites (Cam3.II K13 C58OY: 106.6 pM vs Cam3.II K13 WT: 50.0 pM and 3D7: 54.9 pM, p = 0.0159). In contrast, sensitivity to UCH0084 and UCH0085 appeared to be background specific. Relative to 3D7 parasites, Cam3.II parasites had 3.5-fold reduced susceptibility to UCH0084 (Cam3.II K13 C58OY: 51.4 pM and Cam3.II K13 WT: 57.0 pM vs 3D7: 15.6 pM, p = 0.0159 and p = 0.0286) and 7-fold reduced susceptibility to UCH0085 (Cam3.II K13 C58OY: 146.1 pM and Cam3.II K13 WT: 140.0 pM vs 3D7: 21.2 pM, p = 0.0159 and p = 0.0286; Figure 3B).

[0128] Of the six analogs, UCH0081 exhibited the greatest potency with an IC50 of 4.1 pM for Cam3.II K13 C58OY, 2.6 pM for Cam3.II K13 WT, and 2.3 pM for 3D7 (Cam3.II K13 C580Y vs Cam3.II K13 WT p = 0.0159 and 3D7 p = 0.0464; Figure 3B). To assess whether the inhibition exhibited by UCH0081 was dependent on a covalent inhibition mechanism we synthesized the non-electrophilic analog UCH0114 (Scheme SI) in which the acrylamide was replaced with an isosteric ethyl amide moiety, then evaluated the potency against asynchronous Cam3.II K13 C580Y (Figure 4 and Table 2). The antimalarial activity for UCH0114 was abrogated and we were unable to determine an IC50 value (Figure 4 and Table 2), indicating that the mode of action for UCH0081 is likely covalent . Additionally, we compared UCH0081 activity to MMV688704, which was recently identified as a putative PfUCHL3 inhibitor.26 Notably, under the assay conditions used here, UCH0081 was eightfold more potent than MMV688704 (IC50 value = 32.8 pM, p = 0.0357; Figure 4 and Table 2).

[0129] To de-risk scaffolds, the toxicity of the top six compounds was evaluated in human embryonic kidney 293 (HEK293) cells using CellTiter-Glo® assay (Promega, Madison, WI). For this assay, cells were treated with 500 pM of each inhibitor, as well as doxyrubicin as a positive control, and viability was assessed 24 h later. Cell viability was quantified by luminescence and normalized to the DMSO control set at 100% viability. Results indicated that four of the six molecules - UCH0080, UCH0081, UCH0082, UCH0083 - displayed low toxicity to the mammalian cell line with greater than 80% cell viability at 500 pM. In contrast, UCH0084 and UCH0085 each demonstrated < 50% cell viability at the same concentration after 24 h incubation (Figure 5). Based on these results, UCH0080, UCH0081 , UCH0082, and UCH0083 were prioritized for additional antiparasitic evaluation against the sporozoite stage of P. berghei.

[0130] The parasite’s clinically asymptomatic development in the liver is a desirable target for chemotherapy aimed at disease prophylaxis. Therefore, activity of the four prioritized analogs were tested for activity in blocking sporozoite infection of and development within hepatocytes, using the rodent-infective P. berghei as a model. All molecules were effective in inhibiting parasite growth in the human hepatoma cell line HepG2 at non-toxic concentrations (Figure 6). UCH0081 showed the greatest efficacy (IC50 of 6.4 pM) which was comparable to the efficacy against the asexual blood stage of P. falciparum. This was followed by UCH0080 (IC50 of 49 pM). UCH0082 and UCH0083 displayed comparable EC50 of 80 pM and 87 pM, respectively.

[0131] PfUCHL3 represents a potential anti-malarial therapeutic target and few inhibitors have been reported to date. In this study we conducted a high-throughput screen to identify covalent fragment hits against the recombinant PfUCHL3 enzyme. The 1,700-molecule fragment library yielded eight hits representing unique scaffolds with IC50 values < 350 pM, with the majority at IC50 values below 120 pM. The fragment library contains a mixture of four known cysteine-reactive electrophiles - activated nitriles, epoxides, chloroacetamides, and acrylamides - yet it was observed that PfUCHL3 enzyme displayed a preference for the chloroacetamide and the acrylamide electrophile classes. Despite some chloroacetamide hits displaying better activity we chose to move forward with the acrylamide hits because this electrophile is more stable in pharmacologic assays and is a common warhead used in several FDA-approved covalent kinase inhibitors; thus, improving the potential for translation of these hits into in vivo models. Hits were subjected to secondary assays for assessment of activity versus two human DUB homologs, hUCHL3 and hUCHLl and displayed generally good selectivity with only two molecules exhibiting less than 500 pM potency against hUCHL3.

[0132] These eight molecules were then evaluated for activity against P. falciparum asexual blood stages at a single dose of 500 pM. Two of the hits did not possess antiparasitic activity and were triaged. The remaining six were tested in dose-response assays against three parasite strains. IC50 values ranged from 2 pM to 200 pM across all strains tested. Analog UCH0081 exhibited the most potent anti-Plasmodium activity in the 2 - 4 pM against the three strains tested. Additionally, this analog exhibited comparable potency in blocking sporozoite stage infection of P. berghei in a hepatocyte model. The mode of action for UCH0081 is believed to be mediated through the covalent modification of the intracellular target based on the lack of efficacy for the non-electrophilic control analog UCH0114 against P. falciparum. It should be noted that the antiparasitic activity is 30-fold more potent for UCH0081 compared to the in vitro inhibition of PIUCHL3, a trend that would suggest that either there are multiple targets for UCH0081 that include PfUCHL3 or that the antiparasitic activity for UCH0081 is through covalent inhibition of a different target altogether. The remaining analogs all exhibited anti-Plasmodium activity that was less potent that the PIUCHL3 activity, as would be expected if PIUCHL3 is indeed the target. Due to the lack of a PfUCHL3 antibody it is difficult to confirm on target engagement of these molecules with PfUCHL3 through standard ubiquitin activity-based probe assays.34-37 Experiments using both chemoproteomics and resistant mutant isolation followed by genome wide sequencing approaches are currently in progress to assess intracellular- target engagement for all molecules.

[0133] Notably, activity of UCH0084 and UCHOO85 appeared to be highly dependent on parasite background. Multidrug-sensitive 3D7 parasites displayed IC50 values of approximately 20 pM, while multidrug-resistant Cam3.II parasites had 2- to 7-fold higher IC50 values to these compounds. Note that Cam3.II parasites have mutations in the known drug resistance genes dhfr, dhps, mdrl , pfert, and kl3 that are common in clinical isolates from Southeast Asia, as well as mutations numerous unstudied genes which may contribute to antimalarial drug resistance. These data demonstrate the importance of using multidrugresistant parasites for drug discovery purposes, as parasites in the field may already be highly resistant to certain compounds, even if these compounds have high potency against multidrug sensitive parasites.

[0134] Our work is the first in vitro demonstration of the activity of DUB inhibitors against the Plasmodium’s pre-erythrocytic stages (sporozoites and liver stages). These stages are the targets of causal prophylactic strategies that aim to eradicate or substantially decrease parasite burden before the infection turns symptomatic and parasites form transmissible gametocytes in the bloodstream. It is crucial for controlling malaria caused by P. vivax and P. ovale as these species form hypnozoites that stay dormant in the liver and when reactivated cause disease relapse. Since there are no diagnostic tests for liver stage infection, drugs that prevent sporozoite infection and/or liver stage development will be crucial for malaria eradication.

[0135] In conclusion, the team reports a covalent fragment-based high-throughput screening approach that identified inhibitors that are selective for PfUCHL3 over two closely related human DUB orthologs. Hits were validated for in vitro PfUCHL3 activity, did not display appreciable toxicity against human cells at high concentrations and displayed antiPlasmodium activity against both the asexual blood stage of P. falciparum and the sporozoite hepatocyte stage. Future studies will focus on confirming on-target engagement for the hits against PfUCHL3 in the parasites. Nonetheless, these hits represent viable starting points for medicinal chemistry optimization with the potential to be translated into new anti-malarial therapeutics.

[0136] Materials and Methods

[0137] Chemistry

[0138] General; Molecules were purchased as dry powders from Enamine, LLC (Kyiv, Ukraine) as the following catalog numbers: UCH0080 (Z1938266317), UCH0081 (Z1938228867), UCH0082 (Z2285377613), UCHOO83 (Z3024762640), UCH0084 (Z2698956550), UCH0085 (Z3488584795), UCH0086 (Z3405283705), UCH0087 (Z1938166344). All molecules were provided with a certificate of analysis from Enamine that confirmed the mass and HPLC purity were >95% with the exception of UCHOO83 being 90% pure. Our group carried our own characterization and purity analysis of the dry powders using 1H NMR, mass spectrometry and HPLC and are provided in the Supporting Information. 1H NMR spectra were recorded on Bruker DRX500 in DMSO-d6 or CDC13 with or without the internal standard of TMS at 0.05% v/v. Chemical shifts (8) reported below are as parts per million (ppm) and the coupling constants are reported as follows: s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, h=hextet, hept=heptet, dd=doublet of doublets, ddd=doublet of doublet of doublets, m=multiplet. The purity of all final compounds was >95% purity as assessed by HPLC. Final compounds were analyzed on an Agilent 1200 series chromatograph. The chromatographic method used was ThermoScientific Hypersil GOLDC18 column; UV detection wavelength=254nm; flow rate=1.0 mL/min; solvent=acetonitrile/water for reverse phase gradient of 5% ACN to 95% ACN over 9 minutes. Both organic and aqueous mobile phases contain 0.1% v/v trifluoroacctic acid. The mass spectrometer used is an Advion CMS-L Compact Mass Spectrometer with an APCI ion source using both typical or low temperature low fragmentation settings. Samples are submitted for analysis using the atmospheric solids analysis probe (ASAP). Compounds were prepared according to the protocols in supporting information.

[0139] Synthesis

[0140] Scheme 1

[0141] Synthetic procedure for UCH0114

[0142] 4-chloro-l,6-dimethyl-lH-pyrazolo[3,4-d]pyrimidine (1). A suspension of the l,6-dimethyl-l,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (1.00 g, 6.10 mmol, 1 eq) in phosphorous oxychloride (10 mL) was heated under reflux (110°C) for 12 h. After 12 hours, the excess POC13 was distilled off to result in 1 (490mg, 2.68mmol, 44.1%) isolated as a yellow solid was moved directly to the next step. APCI-MS(+) m/z: 183.0, 185.0 (3:1) [M + H]+, [M + H + 2]+.

[0143] l,6-dimethyl-4-(piperazin-l-yl)-lH-pyrazolo[3,4-d]pyrimidine (2). A mixture of 1 (120 mg, 1 Eq, 0.66 mmol), piperazine (57 mg, 0.66 mmol, 1 eq), sodium iodide (10 mg, 0.1 mmol, 1 eq) and triethylamine (92 pL, 0.66 mmol, leq) in isopropylalcohol (5 mL) was heated under reflux for 2 h. After cooling the reaction mixture was diluted with water, extracted with DCM, washed with brine, and crystallized from ethanol-water to afford 2 (140 mg, 0.60 mmol, 91.7 %). APCI-MS(+) m/z: 233.1 [M + H]+.

[0144] l-(4-(l,6-dimethyl-lH-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-l-yl)propan-l- one (UCH0114). Intermediate 2 (50 mg, 0.22 mmol, 1 eq) was dissolved in THE (1mL) and stirred at 0 °C. To this was added triethylamine (45 μL, 0.32 mmol, 1.5 eq) followed by propionyl chloride (21 pL, 0.24 mmol, 1.1 eq). The reaction was allowed to warm to room temperature and stirred for 18 hours. The reaction was then poured onto water, extracted with ethyl acetate, washed with brine, dried over sodium sulfate, filtered and concentrated. The crude product was then purified via normal phase flash chromotography (1-10% MeOH/DCM gradient) to afford UCH0114 (27 mg, 0.09 mmol, 43%) as a white solid. 1H NMR (500 MHz, DMSO-d6 ppm) 5 8.20 (s, 1H), 3.98 (t, J = 5.5 Hz, 2H), 3.91 (t, J = 5.6 Hz, 2H), 3.87 (s, 3H), 3.63 (d, J - 7.4 Hz, 4H), 2.44 (s, 3H), 2.37 (q, J - 7.4 Hz, 2H), 1.02 (t, J = 7.4 Hz, 3H).13C NMR (800 MHz, DMSO-d₆, ppm) 5 172.0, 163.9, 156.7, 155.2, 133.0, 98.4, 44.4, 40.4, 33.8, 26.3, 25.9, 9.6. APCI-MS(+) m/z: 289.2 [M + H]+. HPLC retention time: 5.425 min. HPLC purity 100%.

[0145] Scheme 2

[0150]

[0151]

[0152] Diethyl 2-((l -cyanoprop- l-en-2-yl)amino)malonate (3). To a suspension of an E/Z mixture of 3-aminobut-2-enenitrile (3 g, 35.1 mmol, 1 eq) in MeOH (60 mL) was added diethyl 2-aminomalonate hydrochloride (7.900 g, 37.33 mmol, 1.02 eq). The resulting mixture was stirred at room temperature for 5 h. The reaction was diluted with ethyl acetate, washed with brine, dried over sodium sulfate and concentrated in vacuo. The resulting material was purified via flash chromatography (0-20% EtOAc/Hex) to afford off-white solid as 3 (7.96 g, 33.1 mmol, 91 %). APCI-MS(+) m/z: 241.1 [M + H]+.

[0153] Methyl (6-methyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pynmidin-2-yl)carbamate (5).

[0154] To a round bottom vial was added 4 (2.68 g, 16 mmol, 1 eq) in MeOH (40 mL), and l,3-bis(methoxycarbonyl)- 2-methyl-2-thiopseudourea (3.74 g, 18 mmol, 1.1 eq) was added followed by AcOH (4.6 mL). The mixture stirred at room temperature overnight and became a thick paste. To the reaction mixture was added 45 mL of NaOMe in MeOH (25%), and stirring was continued at room temperature for 2 h. The mixture was neutralized with AcOH, and the solid was collected by filtration and washed well with water and dried under vacuum to afford 5 (2.60 g, 12.6 mmol, 79 %) as an off-white powder and carried into the next step without characterization.

[0155] 2-Amino-6-methyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (6).

[0156] To a 200 mL round-bottomed flask was added 5 (2.0 g, 9.0 mmol, 1 eq) suspended in 1 N NaOH (70 mL). The reaction mixture was heated at 55°C for 3 h. The resulting solution was cooled in an ice bath and neutralized with AcOH. The precipitated solid was collected by filtration, washed with brine, and dried in vacuo to afford 6 a white solid. APCI-MS(+) m/z: 165.1 [M + H]+. [0157] N-(6-methyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide (7). [0158] To a 40 mL vial was added 6 (300 mg, 1.83 mmol, 1 cq,) suspended in 5 mL of dichloroethane. Then pivaloyl chloride (450 pL, 3.65 mmol, 2 eq), DMAP (33.5 mg, 0.27 mmol, 0.15 eq), and TEA (586 p.L, 4.20 mmol, 2.3 eq) were added. The mixture was stirred overnight at 50°C. The resulting mixture was cooled, diluted with dichloromethane, washed with brine, dried over sodium sulfate, and concentrated in vacuo. Flash chromatography was performed with 9:1 ethyl acetate/hexanes to afford 7 (270 mg, 1.09 mmol, 60%) as a white solid: APCI-MS(+) m/z: 249.2 [M + H]+.

[0159] N- 4-Chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide (8). [0160] To a 50mL round-bottomed flask was added 8 (200 mg, 806 pmol, 1 eq) suspended in 4 mL of phosphorus oxychloride. The reaction mixture was heated at reflux with stirring in an anhydrous atmosphere for 3 h. The dark-orange solution was allowed to cool to room temperature and concentrated in vacuo. Water (10 mL) was then added to the residue at 0°C with vigorous stirring to give an exothermic reaction. Concentrated aqueous ammonium hydroxide was added to attain pH 5 to give a precipitate, which was collected by filtration, washed with water, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography with 1-10% MeOH/DCM to afford 8 (173 mg, 0.65 mmol, 80%) as a white solid: APCI-MS(+) m/z: 266.8, 268.8 (3:1) [M + H]+, [M + H + 2]+.

[0161] N-(4-chloro-5-(4-chlorobenzyl)-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2- yl)pivalamide (MMV688704). To a mixture of NaH (11.7 mg, 487 pmol, 1.3 eq) and anhydrous DMF (10 mL) at 0 °C was added dropwise 8 (100 mg, 375 pmol, 1 eq) in DMF (3 mL), and the mixture was stirred for 20 min. P-Chlorobenzyl bromide (92.4 mg, 450 pmol, 1.2 eq) was added, and the mixture was stirred at 0 °C for 3 h. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by reversed phase flash column chromatography (5-95% ACN/H2O) to afford MMV688704 (53 mg, 0.14 mmol, 36%) as a white power. 1H NMR (500 MHz, DMSO-d6, ppm) 3 9.94 (s, 1H), 7.39 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 6.56 (s, 1H), 5.70 (s, 2H), 2.43 (s, 3H), 1.21 (s, 9H).13C NMR (800 MHz, DMSO-d6 , ppm) δ 176.3, 153.2, 150.5, 149.1, 140.1, 137.4, 132.3, 129.2, 127.7, 121.6, 101.8, 47.1, 40.4, 27.3, 13.3. APCI-MS(+) m/z: 390.9, 393.0 (3:1) [M + H]+, [M + H + 2]+. HPLC retention time: 8.213 min. HPLC purity 100%. [0162] Characterization of molecules tested

[0163] N-(l-(2-(dimcthylamino)cthyl)-lH-indol-6-yl)acrylamidc (UCH0080). 1H NMR

(500 MHz, DMSO-d6, ppm) δ 10.09 (s, 1H), 8.02 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 3.1 Hz, 1H), 7.09 (dd, J = 8.4, 1.9 Hz, 1H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.31 (t, J = 3.9 Hz, 1H), 6.21 (dd, J = 17.0, 2.1 Hz, 1H), 5.69 (dd, J = 10.1, 2.1 Hz, 1H), 4.14 (t, J = 6.7 Hz, 2H), 2.56 (t, J = 6.7 Hz, 2H), 2.14 (s, J = 9.2 Hz, 6H). APCI-MS(+) m/z: 258.2 [M + H]+. HPLC retention time: 0.715 min. HPLC purity 100% (recorded by Enamine in Certificate of Analysis).

[0164] 1 -(4-( 1 ,6-dimethyl- 1 H-pyrazolo [3 ,4-d]pyrimidin-4-y l)piperazin- 1 -y l)prop-2-en- 1 - one (UCH0081) 1H NMR (500 MHz, DMSO-d6 , ppm) 3 8.20 (s, 1H), 6.78 (dd, J = 16.7, 10.4 Hz, 1H), 6.13 (dd, J = 16.6, 2.4 Hz, 1H), 5.71 (dd, J = 10.4, 2.4 Hz, 1H), 3.94 (s, 2H), 3.84 (s, 3H), 3.73 (s, 2H), 3.68 (s, 2H), 3.47 (s, 2H), 2.42 (s, 3H). APCI-MS(+) m/z: 287.1 [M + H]+. HPLC retention time: 5.396 min. HPLC purity 99.8%.

[0165] N-(2-(4-methylpiperazin-l-yl)-5-(trifluoromethyl)benzyl)acrylamide (UCH0082). 1H NMR (500 MHz, DMSO-d6 , ppm) δ 8.62 (s, 1H), 7.54 (d, J = 5.8 Hz, 1H), 7.44 (s, 1H), 7.22 (d, J = 8.4 Hz, 1H), 6.33 - 6.24 (m, 1H), 6.11 (dd, J = 17.1, 2.2 Hz, 1H), 5.62 (dd, J = 10.2, 2.2 Hz, 1H), 4.40 (d, J = 5.9 Hz, 2H), 2.88 (s, 4H), 2.50 (s, 3H), 2.23 (s, 4H). APCL MS(+) m/z: 328.2 [M + H]+. HPLC retention time: 6.378 min. HPLC purity 96.5%.

[0166] N-(2-methoxy-6-(4-methylpiperazin-l-yl)pyridin-3-yl)acrylamide (UCH0083). 1H NMR (500 MHz, DMSO-d6 , ppm) δ 9.29 (s, 1H), 7.93 (d, J = 8.5 Hz, 1H), 6.55 (dd, J = 17.0, 10.2 Hz, 1H), 6.28 (d, J = 8.6 Hz, 1H), 6.13 (dd, J = 17.0, 2.1 Hz, 1H), 5.62 (dd, J = 10.2, 2.2 Hz, 1H), 3.82 (s, 3H), 3.42 - 3.35 (m, 4H), 2.35 (s, 4H), 2.17 (s, 3H). APCI-MS(+) m/z: 277.1 [M + H]+. HPLC retention time: 0.594 min. HPLC purity 90.4% (recorded by Enamine in Certificate of Analysis).

[0167] N-methyl-N-(2-(8-methyl- 1,3,4, 5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)-2- oxoethyl)acrylamide (UCH0084). 1H NMR (500 MHz, DMSO-d6, ppm) 8 10.81 - 10.72 (m, 1H), 7.21 - 7.10 (m, 2H), 6.83 (t, J = 8.7 Hz, 1H), 6.53 (ddd, J = 16.5, 10.2, 3.8 Hz, 1H), 6.06 (ddd, J = 19.2, 16.6, 2.6 Hz, 1H), 5.67 (dd, J = 10.4, 2.5 Hz, 1H), 4.58 (dd, J = 9.6, 5.5 Hz, 2H), 4.51 (d, J = 8.0 Hz, 1H), 4.33 (d, J = 8.7 Hz, 1H), 3.81 (d, J = 5.9 Hz, 1H), 3.77 - 3.67 (m, 1H), 3.03 (d, J = 2.8 Hz, 1H), 2.82 (s, 2H), 2.50 (s, 2H), 2.33 (d, J = 2.5 Hz, 1H), 2.31 (s, 1H). APCI-MS(+) m/z: 31 1.9 [M + H]+. HPLC retention time: 7.321 min. HPLC purity 95.7%.

[0168] N-(2-morpholinobenzo[d]oxazol-6-yl)acrylamide (UCH0085). 1H NMR (500 MHz, DMSO-ok, ppm) 8 10.18 (s, 1H), 7.96 (d, J = 1.6 Hz, 1H), 7.25 - 7.19 (m, 2H), 6.39 (dd, J = 17.0, 10.1 Hz, 1H), 6.21 (dd, J = 17.0, 2.0 Hz, 1H), 5.71 (dd, J = 10.1, 2.0 Hz, 1H), 3.68 (dd, J = 5.8, 4.0 Hz, 4H), 3.56 - 3.49 (m, 4H). APCI-MS(+) m/z: 274.1 [M + H]+. HPLC retention time: 6.070 min. HPLC purity 96.7%.

[0169] N-(2-(2-phenyl-lH-imidazol-l-yl)ethyl)acrylamide (UCH0086). 1H NMR (500 MHz, DMSO-cL, ppm) 8 8.25 (br s, 1H), 7.54 (d, J = 6.6 Hz, 1H), 7.43 - 7.38 (m, 3H), 7.26 (d, J = 1.3 Hz, 1H), 6.96 (s, 1H), 6.10 (dd, J = 17.1, 10 Hz, 1H), 6.00 (dd, J = 17.1, 2.2 Hz, 1H), 5.53 (dd, J = 10.1, 2.3 Hz, 1H), 4.08 (t, J = 6.4 Hz, 2H), 3.40 (q, J = 6.3 Hz, 3H). APCI- MS(+) m/z: 242.2 [M + H]+. HPLC retention time: 0.691 min. HPLC purity 97.1% (recorded by Enamine in Certificate of Analysis).

[0170] N-((4'-((lH-imidazol-l-yl)methyl)-[l,T-biphenyl]-2-yl)methyl)acrylamide (UCH0087). 1H NMR (500 MHz, DMSO-d6 , ppm) 8 9.30 (d, J = 6.3 Hz, 1H), 8.48 (d, J = 5.9 Hz, 1H), 7.79 (d, J = 8.6 Hz, 2H), 7.44 - 7.29 (m, 7H), 7.17 (d, J = 7.4 Hz, 1H), 6.03 (dd, J = 17.1, 2.4 Hz, 1H), 5.58 - 5.52 (m, 1H), 5.44 (d, J = 6.2 Hz, 2H), 4.35 (t, J = 6.7 Hz, 2H), 4.20 (d, J = 5.8 Hz, 2H), 2.89 (d, J = 6.6 Hz, 3H). APCI-MS(+) m/z: 318.2 [M + H]+. HPLC retention time: 6.534 min. HPLC purity 99.3%.

[0171]

[0172] Recombinant protein expression and purification

[0173] hUCHLl, hUCHL3 and PfUCHLl pGEX-6P-l plasmids were purchased from Genscript. The plasmids were transformed into Rosetta (DE3) E. coli cells (New England Biolabs) per manufacturer’s instructions. Starter cultures were grown overnight at 37°C with shaking at 225rpm. Ten milliliters of starter culture was inoculated into IL LB media containing 100μg/mL ampicillin. The cultures were grown at 37°C with shaking at 225rpm until OD=0.8. At this point the cultures were cold shocked and induced with 500μL of IM IPTG. The induced cultures were grown at 17°C for 16 hours with shaking at 225 rpm. The bacterial cells were then pelleted via centrifugation at 4000xg and resuspended in lysis buffer (IxPBS). The resuspended cultures were the lysed via sonication with the addition of lysozyme. The lysed cells were then clarified via centrifugation at 14000xg for 1 hour. The resulting supernatant was loaded onto a Glutathione Sepharose column (Cytiva) equilibrated with lx PBS. After the flow through was collected, the protein was eluted with elution buffer containing 5mM glutathione. The GST tag was cleaved overnight using Precision Protease and the resulting untagged protein was subjected to a subtraction column to remove the cleaved GST tag. This resulting protein sample was concentrated using Amicon Ultra Centrifugal Filters and further purified by size-exclusion chromatography (SEC) on an S100 column using running buffer (50 mM Tris, 150 mM NaCl, and 1 mM DTT, pH 7.6). Fractions that contained the protein of interest were concentrated, flash frozen and held at -80 °C for future experimental use.

[0174] PfUCHL3 high-throughput screen

[0175] PfUCHL3 was expressed and purified as noted above. Covalent Fragment library obtained from Enamine. PfUCHL3 enzyme assay buffer is 50 mM Tris, pH 7.6, 0.5 mM EDTA, 0.1 w/v BSA. Using a black 384 well plate, PfUCHL3 was diluted to a stock concentration of 0.025 nM (Final concentration in well is 0.01 nM). Ubiquitin-rhodamine 110 (250 pM, Boston Biochem) was diluted to 1000 nM for final concentration in well of 400 nM. First, 0.25 pL of the fragment (100 mM stock, final concentration in well 500 pM) or DMSO (Control) was added to the well followed by 9.75 pL buffer. Then 20 pL of 2.5x protein solution was added to each well. This was allowed to incubate for 1 hour at room temperature. Then 20 pL of Ub-Rho stock solution was added for a final volume of 50pL and immediately read on a Biotek Synergy Neo 2. Hits were defined as molecules that had a percent inhibition greater than or equal to 90%, data analyzed using Microsoft Excel and GraphPad Prism 9.

[0176] Biochemical Inhibition Assays of PfUCHL3, hUCHL3, and hUCHLl.

[0177] Reactions were performed in black 384 well plates (Fisher 12566624) in a final volume of 50pL. PfUCHL3 was diluted in reaction buffer (50mM Tris, 0.5mM EDTA, 5mM DTT, 0.1% (w/v) BSA, pH 7.6) to a concentration of 0.025nM, for a final concentration in well of O.OlnM (UCHL1, final concentration in well InM, hUCHL3 final concentration in well O.lnM). To each well was added 10 pL of 5x fragment hit in reaction buffer (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, OpM) and 20pL of protein solution. This was allowed to incubate at room temperature for Ihr. Reactions were initiated by the additional 20pL of 250nM Ub-Rho (Boston Biochem U-555, Final concentration in well 100 nM). Reactions were read immediately for 20 minutes on a Synergy Neo 2. Biochemical IC50valucs were calculated using GraphPad Prism 9 and the standard deviation was calculated over three independent experiments.

[0178] Mammalian Cell Toxicity Assay

[0179] Human embryonic kidney 293T (HEK293T) cells were seeded in 96-well plates at 10,000 cells per well with indicated compounds at 500pM. DMSO served as vehicle control. After 24 hours, Promega CellTiter-Glo reagent was added, and the cells were incubated with reagent for 30 minutes at 37°C before reading luminescence at 37°C using a Biotek Synergy Neo 2 plate reader. Assay was run in triplicate and cell viability was normalized to vehicle control; data analyzed using GraphPad Prism 9.

[0180] P. falciparum asexual blood stage assays

[0181] Cam3.II K13 C580Y, Cam3.II K13 WT, and 3D7 parasites were used. Routine parasite culture and dose response assays to determine half-maximal inhibitory concentrations (IC50) were performed as previously described.38 For the initial screening for antimalarial activity, Cam3.II K13 C580Y parasites were treated with 500 pM of each compound for 72 h in technical duplicates. Antimalarial activity was determined using high content imaging as previously described, 38 except that Wheat Germ Agglutinin Alexa Fluor™ 488 Conjugate (Thermo Fisher Scientific, Waltham, MA) was used to visualize red blood cells instead of CellMask™ Orange Plasma membrane Stain. DHA was purchased from Sigma-Aldrich (St. Eouis, MO) and was solubilized in DMSO. The final concentration of DMSO used in all asexual blood stage assays did not exceed 0.5%. At least three independent biological replicates were performed for each experiment. Statistical significance for IC50 values was examined using a Mann-Whitney U test with GraphPad Prism 9.

[0182] P. berghei Liver Stage Assay

[0183] Mosquito rearing and sporozoite production

[0184] Anopheles stephensi mosquitoes were purchased from the Seattle Children's Hospital. They were maintained and infected with P. berghei as previously described.39 [0185] HepG2 Cell line culture and Liver stage assay

[0186] HepG2 cells were cultured in a standard tissue culture incubator (37°C, 5% CO2) with Dulbecco's Modified Eagle Medium (DMEM) supplemented with L-glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS) (v/v) (Sigma- Aldrich), and 1 % penicillinstreptomycin (Thermo Fisher Scientific). Cells were seeded at a density of 3x104 in a 96- well microplate (F bottom, uClear, white; Greiner bio-one, Ref# 655098) for 24 h after which firefly luciferase expressing P. berghei (Pb-Luc) sporozoites (1.5x104 per well) and test compounds were added to the plate. Plates were centrifuged for 10 min at 330 g to facilitate rapid sporozoite invasion and placed at 37°C. Cells were washed twice with fresh culture media after 3 h to remove sporozoites that had not invaded and then incubated for another 48 h in compound-containing media. Parasite load was quantified using the Spectra Max iD5 multi-mode microplate reader (Molecular Devices) for bioluminescence measurement. Each well was treated with 100 uL One-Gio reagent (Promega, reconstituted following the manufacturer’s instructions). Data were normalized to vehicle treatment (1% DMSO) and dose response curves of the average of three biological replicates were generated in GraphPad Prism version 9 (GraphPad Software Inc.).

[0187] Analogs Based on UCH0081

[0188] Test against Plasmodium falciparum

P4K0015

[0194] Pf IC50 = 75 M

[0195] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.

[0196] While the disclosure has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0197] It is intended that the scope of the present methods and apparatuses be defined by the following claims. However, it must be understood that this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the ail that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

Claims

WE CLAIM:

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, where A is optionally substituted 1 - 2 C,

X is CH or N,

Y is an optionally substituted 1 - 6 alkyl,

Z is optionally substituted N, and R is an optional substituent.

2. The compound of claim 1 where Z is N substituted by 1 - 6 alkyl or 1 - 6 aryl.

3. The compound of claim 2 having the formula selected from the group consisting of

4. The compound of claim 1 where R is selected from the group consisting of optionally substituted alkyl, phenyl, optionally substituted benzyl, optionally substituted benzoyl, optionally substituted C3-C8 cycloalkyl, optionally substituted CH2-(C3-C8 cycloalkyl), optionally substituted 5-6 membered heterocyclyl, and optionally substituted CH2-(5-6 membered heterocyclyl).

5. The compound of claim 2 having the formula:

where R2 is optionally substituted 1 - 6 alkyl, S, N, or halo.

6. The compound of claim 3 having the formula:

7. A pharmaceutical composition comprising a compound of claim 1, and a pharmaceutically acceptable carrier, excipient, diluent or salt thereof.

8. A pharmaceutical composition comprising a compound selected from the group consisting of

and a pharmaceutically acceptable carrier, diluent, excipient, or salt thereof.

9. A pharmaceutical composition of claim 7 or 8, for the treatment of Plasmodium infection.

10. A pharmaceutical composition of claim 7 or 8, for the treatment of malaria.

11. A method for treating a patient in need of therapy for Plasmodium infection, comprising administering a compound of claim 1 to the patient in need thereof.

12. A method for treating a patient in need of therapy for malaria, comprising administering a therapeutically effective among of a compound of claim 1 to a patient in need thereof.

13. A method for treating a patient in need of therapy for Plasmodium infection, comprising administering a therapeutically effective among of a composition of claim 7 or 8 to a patient in need thereof.

14. A method for treating a patient in need of therapy for malaria, comprising administering a therapeutically effective among of a composition of claim 7 or 8 to a patient in need thereof.