WO2012033878A2 - Compounds and methods for acemif inhibition and the treatment of parasites - Google Patents

Abstract

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores, Hookworms are blood-feeding, intestinal nematode parasites infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a "proof-of-concept" for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A non-steroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analogue that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity. Compounds and methods for inhibiting AceMIF and treating parasitic, including hookworm infections represent additional aspects of the invention.

Description

Compounds and Methods for AceMIF Inhibition and the Treatment of Parasites

Field of the Invention

The present invention relates to compounds which exhibit utility as

inhibitors/modulators of A. ceylanicum macrophage migration inhibitory factor (AceMIF) and their use in the treatment of parasites, especially including hookworm disease and/or symptoms, disease states or conditions caused by hookworms, including Ancylostoma ceylanicum, among others as well as other parasitic infections, including protozoal infections, nematode infections, cestode infections and trematode infections Pharmaceutical

compositions based upon these compounds, along or in combination with other bioactive agents, represent an additional aspect of the invention,

Related Applications and Government Support

This application claims the benefit of priority of United States provisional application no. US61/380,858, filed September 8, 2010, entitled "Structure determination and inhibitors of A. ceylanicum MIF for the treatment of Ancyclostomiasis", the entire contents of which are incorporated by reference herein.

This invention was made with government support under grant nos. ROl AI06 029, RO 1 AI058980, ROl A1042310, F32 AI077267 and T32 NS007136 awarded by the National Institutes of Health. The government retains certain rights in the invention.

Background of the Invention

Hookworms are hematophagous, intestinal nematodes that exact a particularly devastating toll on young children and women of childbearing age by causing severe anemia and protein malnutrition. The majority of human hookworm infections are caused by

Ancylostoma duodenale, A, ceylanicum, and Necator americanus (Bungiro and Cappello, 2004; Hotez, et al., 2004). For each hookworm species, the life cycle begins when eggs are excreted in the feces of an infected individual onto warm, moist soil. The eggs hatch, releasing a first stage hookworm larva (LI), which undergoes successive molts to the infective third (L3) stage. Infectious L3 invade host skin and migrate to the lungs via the vasculature. After breaking out of the alveolar spaces and ascending the bronchial tree, the larvae are coughed up and swallowed by the host. Upon reaching the small intestine, the larvae molt to become adult worms, where they attach to the intestinal mucosa, ingest blood and tissue and begin to produce eggs. In heavily infected individuals with low dietary iron intake, the associated blood loss can rapidly lead to chronic hookworm disease characterized by severe anemia, malnutrition and growth/cognitive delay in children (Stephenson, et al., 2000). Nearly 600 million people are infected by hookworms, virtually all of whom live in resource-limited countries (Bethony, et al., 2006; de Silva, et al., 2003). While treatment for hookworm disease is available, there is concern about drug resistance and the lack of late- stage development of novel therapeutics (Albonico, et al., 2004). Additionally, there are commercial challenges in supporting drug development for this parasitic disease. Drug repositioning is an effective mechanism to meet these challenges if there are currently used drugs that possess anthelminthic activity.

Macrophage migration inhibitory factor (MIF) is a mammalian cytokine involved in innate and adaptive immunity that plays multiple roles in the inflammatory response (Guo, et al., 2009; Roger, et al., 2001 ; Weiser, et al., 1989). MIF functions by activating the

CD74/CD44 receptor complex which signals through a Src kinase, resulting in the phosphorylation of the ER -1/2, production of PGE₂, and counter-regulation of

corticosteroid activity, among other intracellular signaling events (Leng, et al, 2003; Lolis, 2001 ; Shi, et al., 2006). MIF has also been shown to activate the chemokine receptors CXCR2 and CXCR4, and has a role in atherosclerosis (Bernhagen, et al., 2007). MIF is a unique cytokine, in that, in contrast to being de novo expressed in response to cellular stress or inflammatory stimuli, MIF is present in the cytosol and is released upon cellular stimulation (Kleemann, et al., 2000; Merk, et al,, 2009). Also, MIF is expressed in a wide range of mammalian tissue and cell types as well as across a wide range of taxa including both free living and parasitic nematodes (Esumi, et al., 1998 ; Leng, et al., 2003; Sato, et al., 2003; Vermeire, et al, 2008). Finally, structural studies reveal that MIF forms a homotrimer with three catalytic sites, each between two subunits, with structural similarity to two microbial enzymes, 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase (Subramanya, et al., 1996; Sun, et al., 1996; Suzuki, et al,, 1996). MIF has tautomerase activity on "model" substrates such as a 2-carboxy-2,3-dihydroindole-5,6- quinone (X-dopachrome) and hydroxyphenylpyruvate (HPP) (Rosengren, et al., 1997;

Rosengren, et al., 1996). Small molecule binding within the active site of mammalian MIF active site reduces cellular (Lubetsky, et al., 2002; Swope, et al., 1998) and in vivo biological activity, providing a therapeutic effect in a number of mouse models disease including sepsis, colitis, and lupus among others (Crichlow, et al., 2007; Dagia, et al, 2009; Leng, et al,, 201 1)

In our previous work, the cDNA of an MIF homolog from A. ceyl nicum (Ace IF) was cloned and the recombinant protein was expressed, functionally characterized, and its three-dimensional structure determined by X-ray crystallography (Cho, et al., 2007). In vitro experiments revealed AceMIF has tautomerase activity and binds the MIF receptor, CD74, suggesting a role in modulating host immune responses to hookworm infection. Importantly, an inhibitor of human MIF, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), did not inhibit AceMIF tautomerase or chemoattractant activities, suggesting that differences in the enzymatic sites might allow for identification of specific inhibitors of AceMIF.

Recently the issue of repositioning FDA-approved drugs for new indications has gained significant attention due to the time and cost necessary of bringing a novel drug into clinical use (Chong and Sullivan, 2007). Here we report the results of a high throughput screening (HTS) of a clinically active, small molecule library against AceMIF based on the inhibition of tautomerase activity. We tested the effect of each inhibitor in three assays to choose a compound for further therapeutic development: inhibition of (1) catalytic activity, (2) binding to CD74, and (3) AceMIF-mediated monocyte migration. We also examined the toxicity of the compounds in an ex vivo worm-killing assay and recognize it is independent of host responses to infection Analyses of the results allowed us to choose one inhibitor compound with activities in assays 1-3 for repositioning and another inhibitor with activities in all four assays for further structure-activity and crystallographic studies, forming the basis for future structure-based drug design and in vivo studies with a hamster model of hookworm disease (Bungiro, et al., 2001 ; Cappello, et al., 2006; Garside and Behnke, 1989).

Brief Description of the Invention

The present invention relates to compounds which are useful to inhibit AceMIF and for the treatment of hookworms, in animals, especially including humans and domesticated mammals, the causative agent of which is Ancyiostoma duodenaie, A. ceylanicum, and Necalor americanus. In this method an effective amount of a AceMIF inhibitor is administered to a patient or subject in order to inhibit AceMIF and/or treat a parasitic, especially including a hookworm infection as otherwise described herein. A number of compounds are useful in the present invention.

In a first aspect, the present invention is directed to a method for inhibiting AceMIF and/or treating a parasitic infection, especially including a hookworm infection or a symptom or secondary disease state or condition of a parasitic or hookworm infection in a patient in need, the method comprising administering to said patient an effective amount of at least one compound according to the chemical structure:

Where RM is H or a Ci-C₂o optionally substituted alkyl group;

RMI is H or a C1-C3 alkyl group which is optionally substituted with one or two hydroxyl groups;

RM2 and MS are each independently a H, a halogen (F, CI, Br, 1, preferably F or CI), nitro, cyano, hydroxyl, -(CI-y_n-CC^H, (Ci-C |₂) alkyl (preferably C|-C₆ alkyl) which is optionally substituted, -(CH₂)_nOC(0)(C₁-C|o) alkyl ester (oxyacylester) which is optionally substituted, -(CH2) C(0)0-(Ci -Cto) alkyl ester (carboxy ester) which is optionally substituted,

-(CH₂)„C(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted, or a -(CH₂)_n-0-(Ci- C₁₀) alkyl (alkoxy) which is optionally substituted;

RM₄ is H, halogen (F, CI, Br, I, preferably F or CI), nitro, cyano, hydroxyl, (Cj-C₁₂) hydrocarbyl group, preferably a (Ci-C₆) alkyl which is optionally substituted,

-(CH₂)_nOC(O)(Ci-C₁₀) alkyl ester (oxyacylester) which is optionally substituted,

-(CH2)_nC(0)0-(C | -Cio) alkyl ester (carboxy ester) which is optionally substituted, -(CH₂)i,C(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted, or a -(CH₂)_n-0-(C|- Cio) alkyl (alkoxy) which is optionally substituted;

n is 0, 1,2,3, 4 or 5,

or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof, or at least one compound according to the chemical structure:

Where I is OR', NR"R'" or a C|-C₂₀ alkyl group which is optionally substituted or an aryl group (phenyl or naphthyl group) which is optionally substituted;

R' is H, a Ci-C₂₀ alkyl group which is optionally substituted or an aryl group which is optionally substituted;

R" is H or a Ci-C₆ alkyl group which is optionally substituted with a hydroxyl group or together with R"' forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C|-C₃ alkyl group;

R"' is H, a C1-C₂0 alkyl which is optionally substituted (preferably substituted with a 5- or 6- membered heterocyclic group), an optionally substituted phenyl group, or a 5- or 6- membered heterocyclic group which is optionally substituted with one or two C1-C3 alkyl groups, or together with R" forms a 5-or 6-membered heterocyclic group which is optionally substituted with a C_rC₃ alkyl group;

RFI is H, a Ci-C₆ alkyl group which is optionally substituted with a 5-or 6-membered heterocyclic group (preferably a heteroaryl group such as a furan or pyrrole group) which is optionally substituted with a C,-C₃ alkyl group, or a NR^H R^FLA group; R^{F 1} is H or a Ci-C₆ alkyl group (preferably a Q-C3 alkyl group) which is optionally substituted with one or two hydroxyl groups or together with R^{F L A} forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

R^{F L A} is H or a Ci-C₂₀ alkyl (preferably a C1-C3 alkyl group) which is optionally substituted (preferably substituted with a 5- or 6-membered heterocyclic group which is optionally substituted, preferably with a C 1 -C3 alkyl group), an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted (preferably with one or two C 1-C3 alkyl groups), a C(0)R^{F L N} group where R^{H N} is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two Ci-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted (preferably optionally substituted with one or two Cj-C3 alkyl groups), or a heterocyclic group (preferably a 5- or 6-membered heteroaryl group) which is optionally substituted (preferably optionally substituted with one or two C 1 -C3 alkyl groups) or together with R forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C C₃ alkyl group;

RF₂ is H, C|-C₆ alkyl which is optionally substituted, 0-(C|-C₆)alkyl which is optionally substituted, 0-C(0)-(C C₆)alkyl which is optionally substituted, -C(0)-0~(C_rC₆)alkyl which is optionally substituted, -C(0)-(C₍-C₆)alkyl which is optionally substituted, a -C(0)NR^F2R^F2A group, a NR^F2RF^2A group or a -S(0)(0)-R^F2S (sulfonyl) group;

R^FZ is H or a Ci-C₆ alkyl group (preferably a C 1-C3 alkyl group) which is optionally substituted with one or two hydroxyl groups or together with R^F2A forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C|-C₃ alkyl group;

R^F2A is H or a Ci-C₂o alkyl (preferably a C1 -C3 alkyl group) which is optionally substituted (preferably substituted with a 5- or 6-membered heterocyclic group which is optionally substituted, preferably with a C 1-C3 alkyl group), an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted (preferably with one or two C 1 -C3 alkyl groups), a -C(0)R ^' group, or R ^' together with R forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

_RF2N is an optionally substituted Ci-C₆ alkyl group, a phenyl group which is optionally substituted (preferably optionally substituted with one or two C 1 -C3 alkyl groups), or a heterocyclic group (preferably a 5- or 6-membered heteroaryl group) which is optionally substituted (preferably optionally substituted with one or two C1-C3 alkyl groups);

R^F2S is a C_!-C₆ optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted 5- or 6-membered heterocyclic group or a NR^F2SNR^F2SNa group; R^F2SN is H , or a Ci-C₃ alky group which is optionally substituted with one or two hydroxy 1 groups or together with R^['2SNa forms a 5- or 6-membered optionally substituted heterocyclic group;

R^F2SNa is H, an optionally substituted Cj-C₆ alkyl group (preferably an optionally substituted C|-C₃ alkyl group, more preferably a methyl group substituted with a 2-furanyl group which is optionally substituted with one or two methyl groups), an optionally substituted phenyl group or an optionally substituted 5- or 6-membered heterocyclic group (preferably a 2- furanyl group), or R^1'"™ together with R , forms a 5- or 6-membered optionally substituted heterocyclic group;

RF3 is H, a halogen (preferably F or CI, more preferably CI), a Ci-C₆ alkyl which is optionally substituted, 0-(Ci-C₆)alkyl which is optionally substituted, 0-C(0)-(Ci-C₆)alkyl which is optionally substituted, -C(0)-0-(d-C6)aIkyl which is optionally substituted, -C(0)-(d- C₆)alkyl which is optionally substituted, a -C(0)NR^F3R^F3a group or a NR^F3RF^3fl group;

R is H or a Cj-C_fi alkyl group (preferably a Q-C3 alkyl group) which is optionally substituted with one or two hydroxyl groups or together with R^F3a forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1 -C3 alkyl group;

R^F3a is H or a Q-C20 alkyl (preferably a C1 -C3 alkyl group) which is optionally substituted (preferably substituted with a 5- or 6-membered heterocyclic group which is optionally substituted, preferably with a C 1 -C3 alkyl group), an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted (preferably with one or two C1 -C3 alkyl groups), a -C(0)R^F3N group, or R^1'3" together with R^F3 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1 -C3 alkyl group;

R^F3N is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two C[-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups (preferably optionally substituted with one or two C 1 -C3 alkyl groups), a phenyl group which is optionally substituted (preferably optionally substituted with one or two C 1 -C3 alkyl groups), or a heterocyclic group

(preferably a 5 - or 6-membered heteroaryl group) which is optionally substituted (preferably optionally substituted with one or two C 1 -C3 alkyl groups), with the proviso that Rpi, RF₂ and RF3 are not all simultaneously H, or

a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof; or a compound according to the chemical structure:

or a pharmaceutically acceptable salt, solvate or polymorph thereof; or a compound mixture according to the chemical structure:

or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof.

In preferred aspects of the invention, R_M is H, R_{M I} is H, R ₂ and S are both halogens, preferably F or CI, more preferably CI; and R_M4 is C1-C3 alkyl group, preferably a methyl group.

In other preferred aspects of the invention, Rp> is H, OR' where R' is a C1-C3 alkyl group, preferably a methyl group, or a -NH-2-furanyl group, Rpi is a -NH-C¾-2-furanyl group, a N- piperidine or N-morpholine group, a -NHC(0)-2-furanyl group which is optionally substituted with one or two methyl groups, or a -NHC(0)NH-(Ci-C₃) alkyl group (preferably an isopropyl group), R_F2 is preferably H, ~0(Ci-C₆)alkyl, more preferably -OCH3, or a sulfonamide group -S(0)(0)NI-I₂ (sulfonamide) group which is optionally substituted on the nitrogen with a Cj-C₃ alkyl group (preferably a methyl when substituted) and/or an optionally substituted heteroaryl (e.g. -CH₂-2-furanyl group) or an optionally substituted phenyl group (the nitrogen of the sulfonamide preferably contains a methyl group) and Rp₃ is preferably H, -0(Ci-C6)alkyl, more preferably -OCH₃, or a halogen, preferably CI or F, more preferably CI.

The present invention also relates to compounds as disclosed hereinabove. In further alternative embodiments the present invention relates to a pharmaceutical composition comprising an effective amount of at least one compound set forth hereinabove in

combination with a pharmaceutically acceptable carrier, additive and/or excipient and further optionally in combination with a further agent selected from the group consisting of a benzimidazole, especially albendazole or mebendazole, levamisole, pyrantel pamoate, ferrous sulfate, folic acid, vitamin B12 or mixtures of these agents. An immunogenic composition and method of immunizing a subject or patient is also disclosed.

Brief Description of the Figures

Figure 1 shows the results of immunization of hamsters with recombinant AceMIF followed by challenge with Ancylostoma ceylanicum hookworms, Hamsters (5/group) were immunized subcutaneously with 100 μg rAceMIF in the adjuvant alum, while controls received alum only. After boosting twice with 50 μg of rAceMIF, animals were challenged with 100 A. ceylanicum infective larvae and monitored for 40 days. As shown in panel A, immunization of hamsters with rAceMIF was associated with partial protection from hookworm associated growth delay, with significantly higher body weights noted at days 26- 40 post infection (p<0.03), compared to controls. In panel B, immunized animals also exhibited less severe anemia following challenge infection, with higher blood hemoglobin levels from days 23-33 postinfection (p<0.05).

Figure 2 shows the results of an inhibition assay against the enzymatic and biological activity of AceMIF. Representative AceMIF hydroxyphenylpyruvate tautomerase inhibition of A, compound 2 (competitive); B, compound 5 (non-competitive); C, compound 3 (mixed). Non-linear regression of the initial velocity at various substrate and inhibitor concentrations is shown on the top panel, and Lineweaver-Burk plots on the bottom panel for each inhibitor. D, PBMC migration inhibition assay with 8 nM AceMIF in the presence of 10 μΜ compound concentration. Migrated cells were measured by relative fluorescence units (RFU) as described in the Experimental Procedures, The numbers above the bars are j's (in μ ) obtained from the tautomerase inhibition assay. One-letter codes above the numbers represent the type of enzymatic inhibition: C (competitive), NC (non- competitive), and M (mixed). E, Inhibition of CD74-AceMIF interaction by compound 2. Percent of AceMIF bound to immobilized CD74 in the presence of various concentrations of compound 2 is plotted.

Chemical structure of compound 2 is shown next to the plot, The IC₅₀ value was determined based on the plot. F, Toxicity of AceMIF-specific inhibitors was determined by observing the survival of cultured Ancylostoma ceylanicum adult worms. Only three of six compounds revealed worm-killing activity (greater than 15% toxicity) at a dose of ΙΟΟμΜ, It is important to note this is an ad hoc assay that has nothing to do with host receptor binding and was performed only to determine whether compounds also had worm-killing activities.

Figure 3 shows the results of a Na-K-Cl cotransporter ( CC1) flux assay with compound 2 and its structural analogs. Percent inhibition of Rb influx into N CC1 cotransporter-expressed HEK293 cells was measured in presence or absence of the compounds. Bumetanide was used as a positive control. The assay was performed at 10 μΜ and 100 nM compounds (n=3). The flurosemide analogs did not exhibit any NKCC1 flux.

Figure 4 shows a complex crystal structure with compound 2. A, Difference electron density of compound 2 was generated at 3σ omitting the inhibitor in the final structure model. B, Interaction of compound 2 with active site residues. The residues involved in hydrogen- bond (ball and stick) and hydrophobic (spiked hemisphere) interactions are depicted.

Hydrogen-bond distances are present on the green dashed lines. C, Compound 2 is shown on the electrostatic surface of AceMIF in an orientation pointing into the active site. Panel D is a 90° rotation of panel C. In panels C and D the two protomers I and II forming the active site are represented in ribbons. The dotted circle represents a protein site that could be used by novel analogues of the parent molecule to form new interactions increasing the affinity for AceMIF and decreasing the affinity for carbonic anhydrase and the NKCC1 cotransporter.

Figure 5. Complex crystal structure with compound 9, a structural analog of compound 2, Panels A and B were generated in the same manner with those of compound 2 in figure 3. Panel C is the superposition of compound 2 and 9 at the catalytic site. Figure 6, Table 1 shows a number of AceMIF inhibitors which were identified from HTS and structural similarity search. Six AceMIF inhibitors (compounds 1 - 6) were identified from GenPlus small molecule library and seven structural analogs (compounds 7- 13) of compound 2 were additionally searched from the PubChem database. All the compounds were assayed for the inhibition of HPP tautomerase activity (inhibition types: C=competitive, M=mixed, NC=noncompetitive), PBMC migration and CD74 binding of AceMIF, and for worm-killing activity. ^a Maximum percentile inhibition is listed due to the limited solubility of the compound,

Figure 7 shows a representative dose-response study of compound 2 against

AceMIF-induced chemotaxis. The chemoattractant activities of AceMIF in the

presence of compound 2 were measured using human peripheral blood

monocytes (PBMCs) isolated from whole blood by centrifugation. These assays

were carried out in 24-well tissue culture plates utilizing 8.0 μιη cell culture

inserts. The cells were placed in the cell culture insert with various concentration

of compound 2, followed by incubation for 3 h at 37 °C, 5% C02. Cells that

migrated through the membrane were fixed in methanol and stained with

Geimsa, followed by counting under light microscopy. Results are expressed as

the mean number of cells counted per high power field for each of two replicates.

Figure 8 shows the blocked catalytic site of AceMIF by C-terminal residues of an adjacent trimer. In the original crystallization conditions for apo- AceMIF, the carboxyl terminal methionine from one trimer makes interactions with the active site residues of another trimer (PDB ID 20S5) that is due to crystal packing artifacts. Therefore deletion mutants of the C-terminal sequence were made to identify the appropriate mutant for crystallization.

Figure 9 shows a comparison between AceMIF-furosemide (compound 2) and human MIF/ ISO-1 , Superposed inhibitors show their unique occupancy in each protein. Protein and each inhibitor are colored consistently as labeled. A clash between furosemide and the residue Phel08 of human MIF is intended to show from superposition. The comparison of the AceMIF-compound 2 structure with the human MIF/ISO-1 [2] structure reveals the basis of its specificity, as well as that of ISO-1, for their respective proteins, Half of the active site is virtually identical for hMIF and AceMIF. These residues are involved in hydrogen-bond interactions with both the inhibitors. The other half of the active sites is distinct and contributes to the discrimination of the respective inhibitors. These residues are hydrophobic and determine the volume of each active site. In particular, Tyr95 in human MIF makes an aromatic-aromatic interaction with ISO-1 and occupies the space that is available in AceMIF for the furfuryl ring of compound 2. Phel 13 of human MIF also part of the active site and allows only ISO-1 to occupy this space, while Vail 13 of AceMIF increases the volume to accommodate the benzene ring of compound 2. Notably, both proteins have a Met2, but the side chain occupies different positions, with AceMIF Met2 occupying space that would clash with the phenol ring of ISO-1 , When superposed with the compound 2 bound structure, the side chain sulfur of Met2 of apo AceMIF points away from the Nl of compound 2 while that of the bound structure points toward the Nl atom making a 3.49A distance hydrogen bond. This hydrogen bond is within the optimum hydrogen bond distance reported for methionine residues [3].

Figure 10 shows compound 2 interacts differently with carbonic anhydrase and can be modified to lose diuretic property for anti-AceMIF specific inhibition. Independent of the Na- -Cl cotransporter target and mechanism, compound 2 is also a sulfonamide inhibitor of carbonic anhydrase [4], The interconversion of carbon dioxide and bicarbonate by carbonic anhydrase maintains the acidity of blood and tissues. When the carbonic anhydrase is inhibited, the acid-base balance in the blood is disrupted and results in acidosis, another mechanism leading to diuresis. Carbonic anhydrase has been co-crystallized with compound 2 (furosemide) (PDB ID 1Z9Y) and reveals that the sulfonamide is completely buried within the protein, making both covalent (with zinc ion) and non-covalent (with the active site residues) interactions [5], Since there is no space next to the chloride atom and the sulfonamide, chemical modification of these two groups could remove binding to carbonic anhydrase and provide specificity for AceMIF.

Figure 1 1, Table 2 shows the cross-activity of the AceMIF inhibitors to human MIF. Ki values were determined against the HPP tautomerase activity of human MIF. Chemotactic inhibition assay was performed by stimulating PBMCs with 8 nM human MIF in the presence of ΙΟμΜ AceMIF inhibitors, For CD74 interaction assay, human MIF was biotinylated and reacted with immobilized soluble CD74 on an assay plate in the presence of AceMIF inhibitors at maximum solubility. Figure 12, Table 3 shows crystallographic data collection and refinement statistics. A single crystal was used for each structure. *Highest resolution shell.

Detailed Description of the Invention

In accordance with the present invention there may be employed conventional chemical synthetic methods, as well as molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are well-known and are otherwise explained fully in the literature.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those

described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It is to be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set out below. It is understood that in the event a specific term is not defined hereinbelow, that term shall have a meaning within its typical use within context by those of ordinary skill in the art. The term "compound", as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein, Within its use in context, the term generally refers to a single compound comprising a hydrophobic moiety and a linker which is capable of reacting and forming a covalent bond with a fusion protein as otherwise described herein, In certain instances the term may also refer to stereoisomers and/or optical isomers

(including racemic mixtures) or enantiomerically enriched mixtures of disclosed compounds, Compounds which are disclosed are those which are stable and where a choice of

substituents and claim elements is available, the substituent or claim element is chosen such that stable compounds are formed from the disclosed elements and substituents.

The term "patient" or "subject" is used throughout the specification within context to describe an animal, especially including a domesticated mammal and preferably a human, to whom a treatment or procedure, including a prophylactic treatment or procedure is performed. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a domesticated/agricultural animal or human patient of either or both genders.

The term "effective" is used herein, unless otherwise indicated, to describe an amount of a compound or composition which, in context, is used to produce or effect an intended result, whether that result relates to the inhibition of AceMIF or to the inhibition of growth and/or the treatment of hookwork in a patient or subject. The term effective subsumes all other effective amount or effective concenti'ation terms which are otherwise described or used in the present application.

The term "parasite" is used herein to describe any of a variety of parasites which may be treated according to the present invention and includes, for example, protozoa parasites, including Entamoeba histolytica, Trypanosome spp., including Trypanosomes responsible for sleeping sickness, leishmaniasis, Chaga's disease, and nagana (cattle), Giardia spp.,

Cestodes, including tapeworms, such as T. solium, T. saginata, Diphyllobothriwn spp., Hymenolepsis spp., Echinococcus spp., Trematodes, including Paragonimus westermani (lung fluke), Clonorchis sinensis and Fasciola hepatica (liver fluke) and nematodes, including intestinal nematodes such as Trichuris trichiura (threadworm/whipworm),

Enierobius vermicularis (pinworm), Ascaris lumbricoides, Strongyloides stercoralis (Cochin- China diarrhea), tissue nematodes, including Trichinella spp., including T. spirala and T. nativa, Toxocaria canis, Filar ia spp., Wucheria spp. and Onchocera volvulus, as well as the nematodes which are responsible for hookworm infections as otherwise described herein.

The term "AceMlF" is used to describe A. ceylanicum macrophage migration inhibition factor, a protein which is proposed to modulate the growth and/or proliferation of hookworms in a host animal. AceMlF is involved in signaling transduction as a regulator for cell proliferation in Ancylostoma duodenale, A. ceylanicum, and Necator americanus, all causative agents of hookworm disease in animals, especially including domesticated animals and/or human patients. AceMlF has been selected as a potential target for drug development in the therapy of hookworm disease, because AceMlF is believed to be an important modulator of the growth and proliferation of Ancylostoma duodenale, A. ceylanicum, and Necator americanus. AceMlF has tautomerase activity and binds the MIF receptor, CD74, suggesting a role in modulating host immune responses to hookworm infection. Importantly, an inhibitor of human MIF, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), did not inhibit AceMlF tautomerase or chemoattractant activities, indicating that differences in the enzymatic sites may allow for identification of specific inhibitors of AceMlF as potential therapeutic agents for the treatment of hookworm disease.

The term "hookworm disease" "hookworm infection" or "ancyclostomiasis" refers to a parasitic infection by a nematode, for example, of the genus Ancylostoma, in particular Ancyclostoma duodenale and A. ceylanicum, as well as by Necator americanus, as well as A_ braziliense, A. tubaeforme (infecting cats), A. caninum (infecting dogs), Uncinaria stenocephala (infecting both dogs and cats), Oesophagestomum spp., including O. bifurcum and O. dentatum, Hookworm is a parasitic worm, generally transmitted in soil. Hookworms live in the small intestine. Hookworm eggs are passed in the feces of an infected person or animal. If the infected person/animal defecates near bushes, in a garden, or field, or if the feces of an infected person or animal are used as fertilizer, eggs are deposited on soil. They can then mature and hatch, releasing larvae (immature worms). The larvae mature into a form that can penetrate the skin of humans. Hookworm infection is mainly acquired by exposure (e.g. walking barefoot) on contaminated soil. Hookworms can also be transmitted through the ingestion of larvae. Most individuals infected with hookworms have no symptoms, although some have gastrointestinal symptoms, especially persons who are infected for the first time, The most serious effects of hookworm infection are blood loss leading to anemia, in addition to protein loss.

Hookworm infection is generally considered to be asymptomatic, but hookworm may be considered an extremely dangerous infection because its damage is silent and insidious, There are general symptoms that an individual may experience soon after infection. Ground- itch, which is an allergic reaction at the site of parasitic penetration and entry, is common in patients infected with N. americanus. Additionally, cough and pneumonitis may result as the larvae begin to break into the alveoli and travel up the trachea. Once the larvae reach the small intestine of the host and begin to mature, the infected individual will suffer from diarrhea and other gastrointestinal discomfort. However, certain symptoms are related to chronic, heavy-intensity hookworm infections. Major morbidity associated with hookworm is caused by intestinal blood loss, iron deficiency anemia, and protein malnutrition. They result mainly from adult hookworms in the small intestine ingesting blood, rupturing erythrocytes, and degrading hemoglobin in the host. Long-term blood loss can manifest itself physically through facial and peripheral edema; eosinophilia and pica caused by iron deficiency anemia are also experienced by some hookworm-infected patients. Recently, more attention has been given to other important outcomes of hookworm infection that play a large role in public health. It is now more widely accepted that children who suffer from chronic hookworm infection can suffer from growth retardation as well as intellectual and cognitive

impairments. Additionally, recent research has focused on the potential of adverse maternal- fetal outcomes when the mother is infected with hookworm during pregnancy.

The symptoms can be linked to inflammation in the gut stimulated by feeding hookworms, such as nausea, abdominal pain and intermittent diarrhea, and to progressive anemia in prolonged disease: capricious appetite, pica (or dirt-eating), obstinate constipation followed by diarrhea, palpitations, thready pulse, coldness of the skin, pallor of the mucous membranes, fatigue and weakness, shortness of breath and in cases running a fatal course, dysentery, hemorrhages and edema. Any one or more of these hookworm disease symptoms or secondary disease states or conditions may be treated by the compounds according to the present invention.

The term "pharmaceutically acceptable salt" is used throughout the specification to describe a salt form of one or more of the compounds or compositions herein which are presented to increase the solubility of the compound in saline for parenteral delivery or in the gastric juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids well known in the pharmaceutical art. Sodium and potassium salts are preferred as neutralization salts of carboxylic acids containing compositions according to the present invention. The term "salt" shall mean any salt consistent with the use of the compounds according to the present invention. In the case where the compounds are used in pharmaceutical indications, including the treatment of hookworm, the term "salt" shall mean a pharmaceutically acceptable salt, consistent with the use of the compounds as pharmaceutical agents.

The term "pharmaceutically acceptable derivative" is used throughout the

specification to describe any pharmaceutically acceptable prodrug form (such as an ester or ether or other prodrug group) which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound.

The term "coadministration" or "combination therapy" is used to describe a therapy in which at least two active compounds in effective amounts are used to treat a hookworm infection, including symptoms of same and/or secondary disease states and conditions at the same time. Although the term coadministration preferably includes the administi'ation of two active compounds to the patient at the same time, it is not necessary that the compounds be administered to the patient at the same time, although effective amounts of the individual compounds will be present in the patient at the same time, One or more compounds according to the present invention may be coadministered with each other or with one or more traditional agents for use in the treatment of hookworm disease including a

benzimidazole, especially albendazole and mebendazole, as well as levamisole and pyrantel pamoate, or mixtures of these agents. Treatments for anemia as a secondary disease state or condition of hookworm disease may be treated with ferrous sulfate, among other iron- containing agents, as well as folic acid and vitamin B12. The term "hydrocarbyl" refers to a radical containing hydrogen and carbon and which may be optionally substituted as otherwise described herein, Hydrocarbyl groups may be saturated or unsaturated and may be linear, branched, cyclic or aromatic.

The term "alkyl" refers to a fully saturated monovalent hydrocarbyl radical containing cai'bon and hydrogen, and which may be cyclic, branched or a straight chain containing from 1 to 20 cai'bon atoms, from 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-butyl, n-hexyl, n-heptyl, n-octyl, isopropyl, 2-methyl- propyl, cyclopropyl, cyclopropyl methyl, cyclobutyl, cyclopentyl, cyclopentylethyl, cyclohexylethyl and cyclohexyl. Preferred alkyl groups are CpC₆ or C3-C 10 alkyl groups. "Alkylene" when used refers to a fully saturated hydrocarbon which is divalent (may be linear, branched or cyclic) and which is optionally substituted. Substituted alkyl groups may also be alkylene groups according to the present invention. Other terms used to indicate substituent groups in compounds according to the present invention are as conventionally used in the art.

"Aryl" or "aromatic", in context, refers to a substituted or unsubstituted monovalent hydrocarbyl (aromatic) radical having a single ring (e.g., benzene) and can be can be bound to the compound according to the present invention at any position on the ring(s). Other examples of aryl groups, in context, may include 5- or 6-membered heterocyclic aromatic ring systems "heteroaryl" groups having one or more nitrogen, oxygen, or sulfur atoms in the ring (moncyclic) such as imidazole, furyl, pyrrole, pyridyl, furanyl, thiene, thiazole, pyridine, pyrimidine, pyrazine, triazole, oxazole, among others, which may be substituted or unsubstituted as otherwise described herein.

The term "cyclic" shall refer to an optionally substituted carbocyclic or heterocyclic group, preferably a 5- or 6-membered ring. A heterocyclic ring or group shall contain one monocyclic ring containing between 3 and 7 atoms of which up to four of those atoms are other than carbon and are selected from nitrogen, sulfur and oxygen. Carbocyclic and heterocyclic rings according to the present invention may be unsaturated or saturated.

Preferred cyclic groups are phenyl groups which are optionallyl substituted. Other preferred cyclic groups are 5- or 6-membered heteroaryl or heteroaromatic groups. The term "heterocyclic group" as used throughout the present specification refers to an aromatic ("heteroaryl") or non-aromatic cyclic group forming the cyclic ring and including at least one hetero atom such as nitrogen, sulfur or oxygen among the atoms forming the cyclic ring. The heterocyclic ring may be saturated (heterocyclic) or unsaturated

(heteroaryl). Exemplary heterocyclic groups include, for example pyrrolidinyl, piperidinyl, morpholinyl, pyrrole, pyridine, pyridone, pyrimidine, imidazole, thiophene, furan, pyran, thiazole, more preferably pyrrolidinyl, piperidinyl, morpholinyl, pyrrole, pyridine, thiophene, thiazole and even more preferably furyl, 3-methylfuryl, thiazole, piperazinyl, N- methylpiperazinyl, tetrahydropyranyl and 1 ,4-dioxane, among others.

Exemplary heteroaryl moieties which may be used in the present invent ion include for example, pyrrole, pyridine, pyridone, pyridazine, pyrimidine, pyrazine, pyrazole, imidazole, triazole, tetrazole, oxadiazole, sulfur-containing aromatic heterocycles such as thiophene; oxygen-containing aromatic heterocycles such as furan and pyran, and including aromatic heterocycles comprising 2 or more hetero atoms selected from among nitrogen, sulfur and oxygen, such as thiazole, thiadiazole, isothiazole, isoxazole, furazan and oxazole. Further heteroaryl groups may include pyridine, triazine, pyridone, pyrimidine, imidazole, furan, pyran, thiazole.

The term "substituted" shall mean substituted at a carbon (or nitrogen) position within context, hydroxyl, carboxyl, cyano (C≡N), nitro (ΝΌ₂), halogen (preferably, 1, 2 or 3 halogens, especially on an alkyl, especially a methyl group such as a trifluoro methyl), alkyl group (preferably, C|-Cio, more preferably, Ci-C₆), alkoxy group (preferably, Ci-Cio alkyl or aryl, including phenyl and substituted phenyl), ester (preferably, Ci-Cio alkyl or aryl) including alkylene ester (such that attachment is on the alkylene group, rather than at the ester function which is preferably substituted with a CpCio alkyl or aryl group), preferably, Ci-Cio alkyl or aryl, halogen (preferably, F or CI), nitro or amine (including a five- or six-membered cyclic alkylene amine, further including a Ci-Cio alkyl amine or Ci-Qo dialkyl amine which alkyl groups may be substituted with one or two hydroxyl groups), amido, which is preferably substituted with one or two Ci-Cio alkyl groups (including a carboxamide which is substituted with one or two Q-Cio alkyl groups), alkanol (preferably, Ci-Cio alkyl or aryl), or alkanoic acid (preferably, C|-Cjo alkyl or aryl). Preferably, the term "substituted" shall mean within its context of use alkyl, alkoxy, halogen, ester, keto, nitro, cyano and amine (especially including mono- or di- Ci-C₁₀ alkyl substituted amines which may be optionally substituted with one or two hydroxyl groups). Any substitutable position in a compound according to the present invention may be substituted in the present invention, but no more than 3, more preferably no more than 2 substituents and more preferably no more than one substituent is present on a ring. Preferably, the term "unsubstituted" shall mean substituted with one or more H atoms.

Compounds according to the present invention may be used in pharmaceutical compositions having biological/pharmacological activity for the treatment of, for example, hookworm infection or any one or more of the secondary disease states and/or conditions which result from hookworm infection. These compositions comprise an effective amount of any one or more of the compounds disclosed hereinabove, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient. Compounds according to the present invention may also be used as intermediates in the synthesis of compounds exhibiting biological activity as well as standards for determining the biological activity of the present compounds as well as other biologically active compounds.

The compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers. Phaimaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat,

The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically (including transdermally), rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal,

intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneal ly, or intravenously. Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are

conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract, Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. In topical applications, transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable earners include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance

bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

The amount of active compound of the instant invention that may be combined with the earner materials to produce a single dosage form will vary depending upon the host treated, as well as the particular mode of administration. Preferably, the compositions should be formulated so that a dosage of between about 0.001 and 150, preferably about 0.5 to about 25 mg kg of patient/day of the novel compound can be administered to a patient receiving these compositions. It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated.

Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q.I.D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal and suppository administration, among other routes of administration, Enteric coated oral tablets may also be used to enhance bioavailability of the compounds from an oral route of administration. The most effective dosage form will depend upon the pharmacokinetics of the particular agent chosen as well as the severity of disease in the patient. Oral dosage forms are particularly preferred, because of ease of administration and prospective favorable patient compliance.

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose, A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques. The use of these dosage forms may significantly impact the bioavailability of the compounds in the patient. For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients, including those which aid dispersion, also may be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be appropriate for the delivery of free nucleosides, acyl/alkyl nucleosides or phosphate ester pro-drug forms of the nucleoside compounds according to the present invention.

In particularly preferred embodiments according to the present invention, the compounds and compositions are used to treat, prevent or delay the onset of a hookworm infection infections of mammals, In its preferred embodiments, the compounds are used to inhibit AceMIF or to treat hookworm infections in a patient or subject in need thereof or a symptom, secondary disease state or condition of hookworm disease, Preferably, to inhibit AceMIF or to treat, prevent or delay the onset of hookworm infection, the compositions will be administered in oral dosage form in amounts ranging from about 250 micrograms up to about 500 mg or more at least once a day, preferably, up to four times a day, within the dosage range used for therapeutic treatment. The present compounds are preferably administered orally, but may be administered parenterally, topically, in suppository or other form as described hereinabove.

Some of the compounds according to the present invention, because of their low toxicity to host cells, may advantageously be employed prophylactically to prevent a hookworm disease by inhibiting AceMIF in a patient or subject or to prevent the occurrence of clinical symptoms associated with hookworm infection, as described herein. Thus, the present invention also encompasses methods for the prophylactic treatment (preventing, reducing the likelihood or delaying the onset) of hookworm infections, and in particular symptoms, secondary disease states and conditions which occur secondary to hookworm infections. This prophylactic method comprises administering to a patient in need of such treatment or who is at risk for the development of hookworm disease an amount of a compound according to the present invention effective for alleviating, preventing or delaying the onset of the infection. In the prophylactic treatment according to the present invention, it is preferred that the compound utilized should be as low in toxicity and preferably non-toxic to the patient. It is particularly preferred in this aspect of the present invention that the compound which is used should be maximally effective against hookworm and should exhibit a minimum of toxicity to the patient, In the case of compounds of the present invention for the prophylactic treatment of hookworm infections, these compounds may be administered within the same dosage range for therapeutic treatment (as described hereinabove, as a prophylactic agent to prevent the proliferation of the infection or alternatively, to prolong the onset of or reduce the likelihood of a patient contracting a hookworm infection which manifests itself in clinical symptoms.

In addition, compounds according to the present invention may be administered alone or in combination with other agents, including other compounds of the present invention. Certain compounds according to the present invention may be effective for enhancing the biological activity of certain agents according to the present invention by reducing the metabolism, catabolism or inactivation of other compounds and as such, may be

co-administered for this intended effect.

As indicated, compounds according to the present invention may be administered alone or in combination with other anti-hookworm agents for the treatment of a hookworm infection or a symptom, secondary effect or condition which occurs as a consequence of a hookworm infection, especially compounds which are otherwise disclosed as being useful for the treatment of hookworm and related symptoms, disease states and conditions, such as benzimidazole, especially albendazole and mebendazole, as well as levamisole and pyrantel pamoate, or mixtures of these agents, Treatments for anemia as a secondary disease state or condition of hookworm disease may be treated with ferrous sulfate, among other iron- containing agents, as well as folic acid and vitamin B12 and therapies which include these agents coadministered with compounds according to the present invention represent alternative embodiments of the present invention.

The compounds disclosed above may be used in combination with the present compounds for their additive activity or treatment profile against hookworm and/or one or more symptoms, disease states and/or conditions, and in certain instances, for their synergistic effects in combination with compounds of the present invention, Certain compounds according to the present invention may be effective for enhancing the biological activity of certain agents according to the present invention by reducing the metabolism or inactivation of other compounds and as such, are co-administered for this intended effect.

The present invention is now described, purely by way of illustration, in the following examples. It will be understood by one of ordinary skill in the art that these examples are in no way limiting and that variations of detail can be made without departing from the spirit and scope of the present invention.

Chemistry

The novel compounds of the instant invention are generally prepared using chemical synthetic approaches which are well known in the art. Many of the compounds according to the present invention are well-known characterized compounds exhibiting different uses than that of the present invention. One of ordinary skill will readily be able to synthesize compounds according to the present invention using the generally known synthetic methods for synthesizing the known compounds and by analogy without engaging in undue experimentation.

Experimental Procedures

Materials

AceMIF was purified as described previously (Cho, et al., 2007), HPP, L-3,4- dihydroxyphenylalanine methyl ester hydrochloride (Methyl L-DOPA hydrochloride), and sodium (meta) periodate were purchased from Sigma (St. Louis, MO). Chemical analogues were obtained from Sigma, Amb inter (Paris, France) and the NCI/DTP Open Chemical Repository (http://dtp.cancer.gov).

AceMIF immunization

For systemic immunization, groups of five male LVG hamsters (Charles River) were immunized subcutaneously with 100 μg of recombinant AceMIF protein using the adjuvant aluminum hydroxide (alum, Alohydrogel®, HCI Biosector). Control animals were immunized with alum alone, The animals were be boosted subcutaneously twice at 3 week intervals with the same dose. One week following the 3rd immunization, hamsters were infected with 100 A. ceyl nic m L₃ by oral gavage. For each of the vaccination groups studied, hamsters were weighed every other day, and serum hemoglobin was measured using a commercially available kit (Sigma). Animals were followed for 40 days post-infection, and recombinant AceMIF protein-immunized animals were compared to adjuvant unimmunized and uninfected controls. For these studies, statistical analyses were carried out using the StatView, version 4,51, statistical software package (Abacus Concepts, Inc.). Pairwise comparisons were performed using Student's t test. For multiple group comparisons, analysis of variance was performed followed by Fisher's protected least significant difference as a posttest, P values of <0,05 were considered significant.

High-throughput screening for AceMIF inhibitors

AceMIF was screened against a small chemical library at the Center for Chemical Genomics at Yale University. The Gen-Plus chemical library was obtained from MicroSource Discovery Systems Inc. (GaylordsviUe, CT) and contains 960 bioactive compounds including marketed pharmaceuticals.

Measurement of Kj

The MIF keto-enol hydroxyphenylpyruvate (HPP) isomerase activity was used to obtain K, values for each inhibitor identified by HTS.

PBMC migration inhibition assay

A preliminary peripheral blood mononuclear cell (PBMC) migration assay was performed as previously described (Cho, et al., 2007) with one concentration of small molecule compounds (10 μΜ) and one concentration of AceMIF (8 nM). Cell migration was assessed using the QCM chemotaxis 96-well assay (Chemicon, Billerica, MA). Fluorescence of migrated cells was measured using the 480/520 nm filter set of a Tecan Infinite M200 plate reader (Durham, NC). Results are graphically represented in relative fluorescence units (RFU) of experimental wells after subtraction of negative (no AceMIF) and background controls (no cells). A more comprehensive dose-response migration assay of PBMC chemotaxis was used to determine the IC50 of compound 2 (Figure 7).

MI F receptor CD74 capture assay

Due to the sensitivity of the capture assay to DMSO, 10 mM of each compound in 100% DMSO was diluted to a final DMSO concentration of less than 0.04% DMSO before assaying. To elucidate CD74 inhibition by the compounds, a 96-well Immunoplate (NUNC, Rochester, NY) was coated with soluble CD74 ectodomain (sCD74 ^3~232) as described (Leng, et al., 2003). The inhibitors were pre-incubated with biotinylated AceMIF (2 ng/μΐ) prepared using the Biotin Labeling Kit (Roche Molecular Biochemicals, Nutley, NJ) for 1 hr at room temperature in the dark. Immobilized sCD74 ^" was mixed and incubated with the pre- incubated inhibitor-biotinylated AceMIF at 4°C overnight. The plate was washed with 250 μΐ/well Tween-20, Tris-buffered saline (Thermo Scientific, Pittsburgh, PA). The biotinylated AceMIF was detected by streptavidin-alkaline phosphatase (R&D Systems, Minneapolis, MN) followed by color development with 60 μΐ/well of p-nitrophenylphosphate (Sigma, St. Louis, MO) observed at 405 nm.

Toxicity against A, ceylanicum ex vivo

A. ceylanicum worms were removed from the small intestine of infected Syrian hamsters (Bungiro, et al., 2003) and cultured in hookworm culture medium (HCM) consisting of RPMI 1640, 50% FCS, 20U/20μg/mL penicillin/streptomycin, 10 μ^ιηΐ Fungizone as described (Cappello, et al., 2006). The susceptibility of A, ceylanicum to AceMIF-specific inhibitors was evaluated by culturing wells of adult worms (10 worms/well, 2 wells/ treatment) in the presence of 100 μΜ concentration of each compound. Control wells contained equivalent volumes of DMSO and/or albendazole

(Sigma), the cui'rent standard for treating hookworm infections. At 24 hour intervals the wells were evaluated by light microscopy to determine the percent live/dead worms.

Struct ure-a ctivity relations h ips The structural similarity search was performed in the PubChem small molecule database of the National Center for Biotechnology Information (NCBI) with Tanimoto coefficients (structural similarity index) of 80 and 85, Seven structural analogues (compounds 7 to 13) of compound 2 were available for further studies (Figure 6, Table 1).

Flux experiment

Target specifipity of the furosemide analogues were tested against the Na- -Cl cotransporter N CC1 in a flux experiment as previously described (Darman and Forbush, 2002). In this study, we used low chloride hypotonic medium to preactivate NKCCl: for 40 min, then 20 mM CI- medium (135 mM NaCl, 5 mM +, 124 mM gluconate, 1 mM Mg+, 1 mM Ca+) for a 10 min preincubation with the testing compounds, and finally a 1.1 min flux in the regular flux medium, consisting of 135 mM NaCl, 5 mM RbCl, 1 mM CaC12/MgC12, 1 mM Na2HP04 Na2S04, ~1 μα/ml 86Rb, 10-4 M ouabain, and 15 mM NaHEPES, pH 7,4, without inhibitors and at two concentrations (10 μΜ and 100 nM) of bumetanide, furosemide, and analogues,

X-ray Crystallography

The carboxyl terminal residues Thrl 17 and Metl 18 were truncated (AC2 AceMIF) to overcome a crystal -packing artifact that hinders co-crystallization of the selected inhibitors. AC2AceMIF (32.5 mg/ml) was crystallized in complex with 2.5 mM of compound 2 or 9 in 0.1 M imidazole, pH 8.0, 0, 16-0.24 M zinc acetate, and 15-25% PEG3000. Crystals were grown to 400 μπι at 18°C within two weeks.

Accession Number

The coordinates for the structure of AceMIF and furosemide complex is 3RF4 and of the AceMIF and compound 9 complex is 3RF5, and have been deposited in the Protein Data Bank

Further Experimental Procedures (Second Reference Set) High-throughput screening for AceMIF inhibitors L-dopachrome was used as a substrate. MIF was pre-diluted in 20 mM Tris HC1, pH7.4 and 20 mM NaCl, mixed in 10 mM potassium phosphate, pH 6.0 to a final

concentration of 2.5 μΜ, and distributed to a 96 well microplate. Several wells received either substrate alone or protein alone as enzyme inhibition (positive) and no-enzyme inhibition (negative) controls, respectively. The microplate was placed in a liquid handling TECAN Aquarius which dispensed 10 μΐ, volumes per well into 384-well assay plates.

Compounds in the chemical libraries were transferred to the assay plates by a 384-pin tool (to a final concentration of approximately 10 μΜ in 20 nL). Both positive and negative controls received DMSO-vehicle, but no compound. The assay plate was incubated at 4°C for 15 minutes. A L-dopachrome stock solution was prepared immediately before starting the inhibition assays by mixing equal volumes of 10 mM methyl L-DOPA hydrochloride and 20 mM sodium periodate at room temperature for 5 minutes, and then storing on ice. Ten ΐ of 1 mM dopachrome was added into each well for a final assay concentration of 0.5 mM, The 384-well plates were centrifuged briefly at low speed, incubated for 30 minutes at room temperature, and the absorbance was measured by an Envision plate reader (PerkinElmer) at 480 nm.

The reliability of the assay was evaluated by calculating a Z-factor (Ζ') (Eq SI , below), which is statistical parameter to assess whether the response of an assay is robust for high throughput screening,

Z'=l - (3sigma(+) + 3sigma(-))/Absolute[mu(+) - mu(-)] (SI) Measurement of Ki

HPP was dissolved in 0.5 M acetate buffer, pH 6.0 to the concentration of 50 mM and incubated overnight and allowed to equilibrate into the enol to keto forms at room temperature. Fifty nM MIF was premixed in 424 mM boric acid and transferred to a 96 well UV transparent bottom half-area plate (Corning, NY). Inhibitors were dissolved in DMSO to 50 mM and added into each well and incubated for more than 10 minutes until the assay was started by addition of HPP at various concentrations, MIF activity was monitored at 306 nm for formation of the borate-enol complex using the plate reader Infinite M200

(TECAN, Durham, NC) for 90 seconds. Calculation of initial velocities and nonlinear regression were repeated three times and combined for analysis using the program Prism4 (GRAPHPAD, La Jolla, CA). Toxicity against Λ, ceylanicum ex vivo

Worm viability was monitored by light microscopy at 24h time points, and the data are represented as the percentage of surviving worms relative to controls (mean of duplicate wells). Data are presented as a Kaplan-Meier survival plot. Statistical analyses were performed using the logrank test to compare survival curves of AceMIF inhibitor-treated and control worms. P values of <0.05 were considered significant.

X-ray Crystallography

Single crystals were flash frozen in the mother liquor with 20% glycerol for data collection. Diffraction data were collected at X29 beam line of National Synchrotron Light Source ( SLS) in the Brookhaven National Laboratory (BNL), Upton, NY or at the Yale University School of Medicine (see Table 2, Figure 1 1 for details of data collection and structural refinement). The data were reduced using MOSFLM and scaled with SCALA in the CCP4 program package [6]. Molecular replacement was performed using PHASER and rigid body refinement was done by Refmac5 [7], Crystallographic molecular dynamics and thermal factor refinement were done with CNS [8], and routine structure refinement was done by Refmac5 with manual intervention of protein and the inhibitor model using COOT [9].

The inhibitor model and its hetero atom library were generated by the PRODRG2 server to produce the PDB format [10]. The electron density of compound 2 and the refined protein structure were visualized using PyMol [1 1]. Hydrogen bonds were searched by HBPlus integrated in SPOCK [12]. Hydrophobic interactions were analyzed by LigPlot [13]. The protein surface was generated by Chimera [14] and the electrostatic potential was calculated by APBS [15] integrated in PyMolXl lHybrid(l .0). The two inhibited protein structures were superposed using the program THESEUS [16] to quantitate conformational changes upon different inhibitor binding and also to visualize positional difference of the bound inhibitors.

Meta-analysis of the GenPlus library To characterize the GenPlus library (MicroSource Discovery Systems, Inc.,

Gaylordsville, CT) compounds in greater detail, we performed a meta-analysis of results from fifteen unrelated high-throughput screens performed at the Center for Chemical Genomics at Yale University and compared the identified compounds to those of the AceMIF screen. Hexachlorophene was common in six different assays including for MIF and was excluded from further studies with AceMIF, Compounds 1, 2, 4, 5, and 6 were common in another screen for antioxidants. Compound 3 was common in two screens, a screen for anti-oxidants and a biochemical screen for molecules that interrupt transmembrane homodimers. These results indicate a high percentage of compounds in the GenPlus library can be re-positioned for other indications if the target is involved in disease, as long as the drug does not result in adverse effects. The GenPlus library contains 20 compounds with known anthelmintic activities. However, only two of these compounds were identified as specific inhibitors of AceMIF enzymatic activity.

Results

AceMIF immunization

In order to test whether neutralization of AceMIF would impact hookworm-induced pathogenesis, Syrian hamsters were immunized with rAceMIF and followed for changes in body weight and blood hemoglobin levels after hookworm infection. Hamsters were immunized subcutaneously with 100 μg of rAceMIF in alum adjuvant, while controls received alum only. After boosting (50 μg of rAceMIF 2X), animals were challenged with 100 A, ceylanicum infective larvae and monitored for 40 days. As shown in Figure 1 A, immunization of hamsters with rAceMIF was associated with partial protection from hookworm-associated growth delay, with significantly higher body weights noted at days 26- 40 post infection (p<0.03), compared to controls. Immunized animals also exhibited less severe anemia following challenge infection, with higher blood hemoglobin levels noted from days 23-33 post-infection (Figure IB, p<0.05). The demonstration that vaccination confers partial protection from clinical symptoms of hookworm-associated disease (weight loss and anemia) suggests that AceMIF, which is secreted by adult s, ceylanicum, is an important virulence factor that plays a role in pathogenesis. It also suggests that AceMIF is accessible by antibodies in the bloodstream of infected animals and therefore could be targeted using small molecule inhibitors. In previous studies, inhibitors targeting the catalytic site of human MIF were therapeutic in vivo, further providing a rationale for inhibiting this site of AceMIF (Crichlow, et al., 2007; (Dagia, et al., 2009; Leng, et al„ 201 1)

Inhibitors from a chemical library

A HTS assay of i-dopachrome tautomerase activity (Z' (Eq. SI) value >0.8) was developed for AceMIF using an automatic liquid handler and plate reader to screen a small chemical library composed of FDA-approved drugs and other bioactive compounds

(GenPlus, MicroSource) (Zhang, et al., 1999). The rationale for developing this assay was based on previous studies that identified small molecules that bind the MIF active site and have proven to have therapeutic effect in animal models of disease (Crichlow, et al, 2007; (Dagia, et al., 2009; Leng, et al., 201 1). Eleven compounds that inhibited AceMIF - dopachrome tautomerase activity by more than 60 percent relative to controls in the HTS assay were selected and purchased for laboratory studies. Five of those that also inhibited human MIF or were false positives were removed from further characterization. Cross- reactivity of the six remaining AceMIF inhibitors (Table 1 , Figure 6) was studied using the HPP tautomerase assay against human MIF and the Kj's were 3-104 fold greater than AceMIF (Table 2, Figure 7). Human MIF-mediated PBMC cell migration was not significantly inhibited by any compound. Interaction of human MIF with sCD74 was also not inhibited by any of the compounds.

Among these six compounds were: pyrantel pamoate (compound 4) and

hexylresorcinol (compound 6), two compounds with anthelminthic properties, sodium meclofenamate (compound 1), a non-steroidal anti-inflammatory drug (NSAID), furosemide (compound 2), a diuretic used to treat hypertension and heart failure, hydroxyzine pamoate (compound 3), an anti-anxiety medicine and cobalamine (compound 5), We also performed a meta-analysis of 15 unrelated screens performed at the Yale Center for Proteomics and Genomics in order to characterize the specificity of the GenPlus library, Hexachlorophene was common in six different assays including for MIF and was excluded from further studies with AceMIF. Of the compounds, 1, 2, 4, 5, and 6 were common in another screen for antioxidants. Compound 3 was common in two screens (Further Experimental Procedures).

Kinetic properties of the inhibitors For extensive kinetic studies, HPP was used as a substrate instead of X-dopachrome, which is stable for only 20-30 minutes at ambient temperatures. Initial velocities of AceMIF tautomerase activity with three different inhibitor concentrations and HPP concentrations ranging from 0.01 to 2 raM were measured and analyzed to determine the inhibition type using Lineweaver-Burk plot analysis. In two recent studies inhibitors were found to bind to an allosteric site adjacent to the active site and function as non-competitive inhibitors (Cho, et al., 2010; McLean, et al., 2010), Representative graphs of a competitive, non-competitive, and mixed inhibition are shown in Figure 2A, B, and C, respectively, A complete summary of Ki's and inhibition types of all the inhibitors is listed in Table 1 (Figure 6), Compounds 1, 2 and 4 are competitive inhibitors with Kj's in the sub- to micromolar range. Compounds 3 ( _j 5.71 μΜ) and 6 (Kj 136.6 μΜ) are both mixed inhibitors with varied Kj's. Compound 5 was determined to be a non-competitive inhibitor of AceMIF tautomerase activity and had a j of 124.4 micromolar.

Inhibition of AceMIF-induced chemotaxis

Chemotaxis assays with human peripheral blood mononuclear cells (PBMCs) were performed at a single concentration of AceMIF with an excess of each compound (Figure 2D, Table 2, Figure 1 1). Interestingly, the anti-chemotactic activity of inhibitor compounds did not correlate with the potency of enzymatic inhibition. For example, compound 5 (Kj of 124.4 μΜ) was a more potent anti-chemotactic agent than compounds 2, 3, and 4, which had Kj's from 0.56 to 8.16 μΜ, It is clear from Figure 2D that the type of inhibition also bears no relevance to the potency of anti-chemotactic activity because competitive, non-competitive, and mixed inhibitors (compounds 1, 2, 4, 5, and 6) are all potent inhibitors of AceMIF- mediated monocyte chemotaxis. For compound 2, a dose-dependent inhibition assay was performed to estimate an IC₅o value, which was approximately 1 μΜ (Figure 7).

Inhibition of AceMIF binding to the human CD74 receptor

AceMIF interacts with the human MIF receptor CD74, presumably to modulate and potentially evade the host immune response (Cho, et al., 2007). To determine whether any of the inhibitors block the binding of AceMIF to CD74, we employed the same method used previously to detect interactions with the immobilized soluble extracellular domain (residues 73-232) of CD74 (sCD74). Compounds 2, 4, and 6 specifically interfered with AceMIF interactions with sCD74 at sub-micromolar IC₅₀s (Table 1, Figure 6). A representative dose- response graph of compound 2 is shown in Figure 2E.

Worm killing assays

The antihelminthic activity of each of the inhibitors was tested against A. ceylanicum in an ex vivo worm-killing assay (Cappello, et al., 2006). The rationale for these experiments was based on the presence of AceMIF in homogenates of L₃ larvae and adult . ceylanicum (Cho, et al., 2007), and that AceMIF may serve a worm-specific function(s) in the absence of host immune effectors, similar to that of human MIF (Mitchell, et al., 2002). These experiments were also important in evaluating the nematicidal potential of each compound for future in vivo studies, Survival curves of AceMIF inhibitor-treated worms were significantly different from control worms treated with DMSO alone (Table 1 , Figure 6 and Figure 2F; compound 2, p=0.0007; compound 3, p=0,0005; compound 4, p=0.0023).

However, survival curves of these compounds were not significantly different from each other. Compounds 2, 3, and 4 inhibited survival with 40-50% worm-killing activity. All other compounds, including compound 1, the most potent enzymatic inhibitor, had worm killing activity of 0-15%, indicating that the blocking AceMIF active site is insufficient to kill hookworms ex vivo,

Structure-Activity Relationships of Compound 2

Compound 2 exhibited the best combination of inhibitory activities - sub-micromolar for both the , for enzyme activity (binding to the AceMIF active site) and IC₅₀ for CD74 receptor binding, IC50 of 1 μΜ in monocyte chemotaxis experiments, and the highest potency in the worm-killing assay. Therefore, compound 2 was selected for further study. To investigate the contribution of each functional group of compound 2 in its inhibitory effect, structurally similar molecules were selected from a search of PubChem (website at pubchem.ncbi.nlm.nih.gov ) and assayed for comparison, Chemical structures and kinetic parameters of these structural analogues are shown in Table 1 (Figure 6). Compound 9 (2- (furan-2-ylmethylamino)benzoic acid) and compound 2 have furfurylamine at the Rl position and have relatively low ¾ values compared to compound 7 (4-chloro-5-(furan-2- ylmethylsulfamoyl)-2-piperidin-l-ylbenzoic acid), which has piperidine at the same position, and compound 8 (4-chloiO-N-(furan-2-ylmethyl)-3-[methyl(phenyl)sulfamoyl]benzamide)₎ which has no Rl group. This implies that the furfurylamine group plays an important role in interacting with AceMIF, and its affinity is significantly reduced when its position is changed to R2 as in compound 7.

Seven structural analogues of compound 2 were tested in the CD74 capture assay, PBMC migration assay, and ex vivo worm survival assay to determine their activities relative to compound 2 (Table 1 , Figure 6). Compound 8 significantly inhibited the interaction of AceMIF with CD74 (IC₅₀ of 3.3 μΜ) while compounds 7 and 9 inhibited only 38% and 15% of AceMIF-CD74 binding, respectively. In the PBMC migration assay, compounds 8 and 9 were similarly active in inhibiting AceMIF-mediated monocyte migration relative to compound 2 while compound 7, which is missing a furfurylamine group, was only half as active as the parent compound. Interestingly, none of the structural analogues of compound 2 possessed worm-killing activity. Compound 10 has two methyl groups at Rl position instead of a furfuryl group and two methyl ethers at R2 and R3 positions.

The structural differences in the fuiOsemide analogues caused significant changes in their ability to inhibit AceMIF tautomerase activity. For example, the only chemical difference between compounds 9 and 1 1 is the replacement of a methylene group with a carbonyl oxygen next to the anthranilate nitrogen to form a peptide bond (of compound 1 1) resulting in a 4-fold and 9-fold higher Kj, respectively, compared to compound 2 (Kj 0.56 μΜ). Compound 12 is structurally similar to compound 1 1, but has an extra methyl group attached to a ring carbon of the furan group. This difference results in a fourteen fold higher Ki value compared to that of compound 2. Interestingly, Kj is increased 102 fold relative to compound 2. Replacement of the secondary amine linker with a methylated tertiary amine in compound 13 increases the Kj 102 fold relative to compound 2. The crystal structure of compound 2 indicates this linker is buried in the active site and replacing the amine proton with a methyl group would result in a steric clash, destabilizing the interaction between inhibitor and AceMIF. Taken together, these results show that structural alterations of furosemide (compound 2) significantly impact the nature of their interaction with the AceMIF protein (and resulting inhibitory activities) and furosemide provides an excellent pharmacophore for novel anthelmintics. NKCCl flux assay

The diuretic activity of furosemide in humans makes it unsuitable as an anti- anthelminthic therapeutic for human use. To determine whether any of the furosemide analogues inhibited Na-K-Cl symporter (NKCCl), a flux assay measuring the influx of ⁸⁶Rb as a surrogate marker of K⁺ in the presence of furosemide and its structural analogues was measured (Darman and Forbush, 2002). Bumetanide was used as a positive control of inhibition. The assay was performed at 10 μΜ and 100 nM concentrations of each compound. Only bumetanide and furosemide inhibited the Rb influx while the furosemide analogues did not (Figure 3).

X-ray Crystallography

Attempts at co-crystallizing full-length AceMIF with the selected compounds in the original condition or in -500 commercial conditions were unsuccessful presumably due to the crystal packing artifact in the apo crystal structure (Figure 8). Various C-terminal truncations of AceMIF were tested for co-crystallization. Deletion of two C-terminal residues Thrl 17 and Metl 18 (AC2AceMIF) was the only truncation that resulted in co-crystals, diffracting at 1.8A in space group P3 |21. The crystal structure was solved by molecular replacement and refined to R and Rf_rcc of 17% and 20%, respectively. Electron density generated without a model of compound 2 shows the inhibitor in the active site in Figure 4A, The carboxylic acid oxygen (04) of the anthranilate pharmacophore hydrogen bonds with the side chain hydroxyl group of Ser63, the backbone nitrogen atom of Ile64, and the side chain nitrogen atom of Lys32 (Figure 4B), The second carboxyl oxygen (05) forms a hydrogen bond with the nitrogen atom of Pro 1, completing a tight hydrogen-bonding network with the hydrophilic residues in the active site. The sulfur atom of Met2 also hydrogen bonds with the nitrogen of the furfurylamine group of compound 2, No water-mediated hydrogen bonding interaction is visible in the electron density. Hydrophobic interactions exist between the furfuryl group and the residues consisting of one half of the active site (Figure 4A-B). The sulfonamide group and chloride atom are exposed to the solvent (Figure 4C-D). In contrast to the fully occupied active site by the furfuryl and carboxyl groups, there is an unoccupied site (Figure 4C-D) adjacent to the chloride atom where chemical modifications can be made to improve the affinity of this compound for AceMIF. This unoccupied site begins with a hydrophobic groove (Vail 06, Phel08, and Vail 13) and ends with hydrophilic residues (Asnl09 and Thrl 12).

Compound 9 was also co-crystallized with AC2AceMIF and its X-ray diffraction data were collected at 2.1 A (Figure 5 and Table 2, Figure 1 1). Compound 9 is a structural analogue of compound 2 and lacks the solvent exposed sulfonamide group and chloride atom. It has a 4-fold higher j than that of compound 2. Crystal structures of AceMIF in complex with compounds 2 and 9 are superimposed using a maximum likelihood method (Theobald and Wuttke, 2006) with only protein Ca atoms at the root mean square deviation (r.m.s.d.) of 0.11 A and show an almost identical fit of the compounds in the active site (Figure 5C). The loss of the sulfonamide group and chloride atom from Compound 2 results in a non-diuretic compound that retains its inhibition against AceMIF catalytic and migration activity.

Discussion

A previous report showed that AceMIF is secreted by adult hookworms and competes for binding to the human MIF receptor in vitro (Cho, et al., 2007). Evidence also exists that protozoan (Leishmania major (Kamir, et al., 2008)) and helminth (Brugia malay (Prieto- Lafuente, et al., 2009)) parasite MIFs modulate host immune responses, presumably through interaction with mammalian receptors on immune cells. Moreover, the development of specific small molecule inhibitors synthesized to neutralize mammalian MIF has shown significant promise in their ability to ameliorate disease pathogenesis (Al-Abed, et al., 2005; Crichlow, et al., 2007; Dabideen, et al., 2007). The AceMIF vaccination experiment in this study shows that immunoneutralization provides partial protection to the host. The partial protection could potentially be explained by the presence of multiple MIF isoforms in A. ceylanicum (unpublished observation), which has also been demonstrated for other nematodes (Vermeire et al, 2008), It is possible that targeting both hookworm MIF proteins through vaccination or small molecule chemotherapy might further improve protection. The human MIF inhibitor, ISO-1, does not inhibit AceMIF catalytic nor macrophage migration activity, indicating the active sites were sufficiently different that a specific AceMIF small molecule inhibitor could be designed or discovered (Cho, et al., 2007). Taken together, these studies support the rationale that targeting of AceMIF represents a viable strategy to reduce hookworm pathogenesis and disease.

High throughput screening and drug repositioning

Among almost 1 ,000 FDA-approved drugs or other bioactive compounds for the HTS screen, compounds that showed >60% inhibition of the Z-dopachrome-methyl ester tautomerase activity of AceMIF in the initial screen were subjected to a more comprehensive analysis to characterize the j, which varied from sub-micromolar to hundreds of micromolar. The six AceMIF-specific inhibitors identified in the HTS screen were characterized in other AceMIF assays. For example, plate-based chemotaxis and competition ELISA experiments to probe the effect of inhibitor compounds on AceMIF-mediated cell migration and CD74 receptor binding were studied. Finally, all six compounds and selected analogues were tested to determine toxicity against A. ceylanic m in an ex vivo assay

(Cappello, et al., 2006). Only compounds 2, 3, and 4 (or their metabolites) demonstrated any worm-killing activity, which was clearly independent of any human target. Interestingly, compound 4 (pyrantel pamoate) is a neuromuscular depolarizing agent known to cause paralysis in helminths resulting in parasite expulsion through a mechanism involving inhibition of parasite choHnesterase. Identification of pyrantel pamoate as a specific inhibitor of Ace IF suggests this compound may have multiple targets within parasitic nematodes that are responsible for its anthelminthic properties, Among the six compounds that were identified, only compound 1 (sodium meclofenamate), an anti -inflammatory therapeutic that inhibits cyclooxygenase pathway (Schober, et al., 2002) that might be re-positioned as anthelminthic. The trough and peak levels of the this drug, 0.2 μg/ml and 15

(0.6 μΜ and 45 μ ), respectively, at an oral dose of 100 mg (Conroy, et al., 1991). The trough level is higher than the K, for catalytic activity, and 10 μΜ of sodium meclofenamate would inhibit nearly 100% of macrophage migration (Figure ID, Table 1, Figure 6). The adverse effect of sodium meclofenamate includes impeding the immune system, which may prevent an appropriate response to hookworm infection. It is therefore very important to monitor the efficacy and adverse effects of compound 1 in the Syrian hamster model of hookworm disease and any future clinical trials in humans.

Kj and IC₅0 for receptor binding, macrophage migration, and hookworm survival

There was no strict interdependence among the j of enzymatic activity and IC50 values for receptor binding and chemotaxis inhibition. This paradox does not only involve the inhibitors in this study but also cellular mutagenesis studies (Lubetsky, et al., 2002; Swope, et al., 1998) and mice knock-in of a tautomerase-null, Prol-to-Gly MIF protein (P1G-MIF) replacing the endogenous iw/gene (Fingerle-Rowson, et al., 2009) that show a large decrease in catalytic activity is accompanied by a smaller decrease in biological activity. This suggests that protein-protein interactions are critical for the immunological function of MIF rather than the catalytic activity of MIF (Fingerle-Rowson, et al., 2009). Taken together, these observations imply MIF-receptor binding occurs at or near the active site and that some small molecule active site inhibitors have chemical moieties that interfere with the MIF-receptor interactions. In this regard, structural studies indicate that compound 2, which has a sulfonamide group and chloride atom that protrude from the active site and make no interaction with AceMIF, inhibits that interaction between AceMIF and human MIF receptor. These two groups are removed in compound 9. X-ray crystallography indicates the interactions within the active site are retained but this compound does not inhibit AceMIF- human MIF receptor interactions.

The ex vivo worm survival assay (at the single concentration of 100 μΜ) to test the toxicity of inhibitors to the worm showed no clear relationship between inhibition of AceMIF enzymatic activity and A. ceylanicum-killmg. Again, this implies that the inhibition of the catalytic activity of AceMIF does not affect the pathology induced by the protein during hookworm infection. These incongruent results may in part be explained by the

multifunctional nature of MIF proteins: (1) an active site that catalyzes a chemical reaction and (2) a binding site near the active site that interacts with the MIF receptor. By selecting for inhibitors of tautomerase activity in the HTS, we have identified compounds effective in inhibiting the catalytic activity including a few compounds that also disrupt MIF-receptor interactions,

Pharmacophore identification and erystallographic analysis

Compound 2 (furosemide) exhibited sub-micromolar K, (0.56 μΜ) and IC₅o (0.33 μΜ) values for the enzymatic and receptor binding inhibition activities, respectively, and inhibition of AceMIF-mediated monocyte chemotaxis (IC₅o of 1 μΜ). In addition, it was also toxic to adult worms (50% killing relative to controls). Furosemide is a diuretic that that blocks the Na-K-Cl cotransporter (Kirkendall and Stein, 1968; Puschett, 1981). It has an anthranilate scaffold, as does meclofenamate, and can be represented by a benzene scaffold for a broader structural analogue search. A furosemide-based search of commercially available reagents with decreased Tanimoto coefficients yielded seven structurally related compounds (compounds 7 to 13) to test the significance of each R-group (see Table 1, Figure 6) in AceMIF assays and in inhibiting the Na-K-Cl symporter. Kinetic assays revealed higher Ki values for all seven analogues, ranging from 5-247 μΜ depending on the R-groups.

Compounds 8 and 9 possessed similar activity to compound 2 in their ability to inhibit AceMIF-mediated PBMC migration (82% inhibition for both). Compound 8 also acts as an antagonist in solution with AceMIF, preventing its binding to CD74. However, none of the compound 2 analogues showed hookworm-killing activity, nor did any of the seven analogues inhibit the Na-K-Cl symporter. Crystal lo graphic analysis of compounds 2 and 9 complexed to AceMIF was performed to more fully understand the interaction of these compounds with AceMIF and to form the basis of future structure-based drug design studies. A superposition of the two structures is shown in Figure 5C and reveals an excellent overlay of the two compounds in the active site. The solvent accessible sulfonamide and chloride R-groups have no

interactions with the protein for compound 2 and result in a marginal difference in Kj for compound 9, which does not contain either of these R-groups.

We also compared the crystal structures of AceMIF-compound 2 to human MIF-ISO- 1 and to carbonic anhydrase-compound 2 to reveal the basis of these interactions (Figure 9). The specificity of the ISO-1 inhibitor for human MIF relative to AceMIF is based on different residues for one side (half) of the active site. In the carbonic anhydrase-compound 2 (PDB ID 1Z9Y) crystal structure (Puschett, 1981) the sulfonamide and the chloride make important binding interactions with carbonic anhydrase (Figure 10), whereas in the AceMIF co-complex these two R-groups point out of the active site into the solvent and make no interactions with the protein (Figure 4).

In summary, the AceMIF vaccination study provides the "proof-of-concept" for targeting a helminth immunomodulator with small molecule compounds. The high throughput screen identified six bioactive molecules. One AceMIF-speciflc inhibitor, sodium meclofenamate, could be repositioned an as hookworm therapeutic with the caveat that its adverse effects do not interfere with the immune response during treatment in animal model studies. Functional and structural studies of furosemide led to a pharmacophore and SAR studies. The differential effects of the compounds identified from the screen and the furosemide analogues will be useful in probing the relative significance of AceMIF-induced activities in the pathology of hookworm infection in vivo. This information will be useful for future structure-based drug design.

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Claims

Claims:

1. A method for inhibiting A. ceylanicum macrophage migration inhibitory factor (AceMIF) and/or treating a hookworm infection or a symptom or secondary disease state or condition of a hookworm infection in a patient in need, the method comprising administering to said patient an effective amount of at least one compound according to the chemical structure:

Where RM is H or a Ci-C₂o optionally substituted alkyl group;

RMI is H or a C C₃ alkyl group which is optionally substituted with one or two hydroxyl groups;

R 2 and RM₃ are each independently a H, a halogen (F, CI, Br, I, preferably F or CI), nitro, cyano, hydroxyl, -(CH₂)irC0₂H, (C 1 -C12) alkyl (preferably Ci-C₆ alkyl) which is optionally substituted, -(CH₂)„OC(0)(C₁-Cio) alkyl ester (oxyacylester) which is optionally substituted, -(CH2)„C(0)0-(Ci-C|o) alkyl ester (carboxy ester) which is optionally substituted,

-(CH2)_nC(0)-(Ci-C[o) alkyl (acyl group) which is optionally substituted, or a -(CI-I₂)n-0-(C i- C 10) alkyl (alkoxy) which is optionally substituted;

is H, halogen, nitro, cyano, hydroxyl, a (C 1 -C 12) hydro carbyl group which is optionally substituted, -(CH2)_nOC(O)(C Ci₀) alkyl ester (oxyacylester) which is optionally substituted, -(CH2)_nC(0)0-(Ci"Cio) alkyl ester (carboxy ester) which is optionally substituted,

-(CH2)_nC(0)-(Ci-C|o) alkyl (acyl group) which is optionally substituted, or a -(CH₂)_n-0-(Ci- Cjo) alkyl (alkoxy) which is optionally substituted;

n is 0, 1 ,2,3, 4 or 5, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof, or at least one compound according to the chemical structure:

Where R ' is OR', NR"R"' or a Ci-C₂₀ alkyl group which is optionally substituted or an aryl group which is optionally substituted;

R' is H, a C C₂o alkyl group which is optionally substituted or an aryl group which is optionally substituted;

R" is H or a Ci-C₆ alkyl group which is optionally substituted with a hydroxyl group or together with R'" forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

R'" is H, a C[-C₂o alkyl which is optionally substituted, an optionally substituted phenyl group, or a 5- or 6-membered heterocyclic group which is optionally substituted with one or two C 1-C3 alkyl groups, or together with R" forms a 5-or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

RFI is H, a Q-Q alkyl group which is optionally substituted with a 5-or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group, or a NR^{F 1}R^{F L A} group;

R^{F 1} is H or a Cj-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^{F L A} forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

R^{F L A} is H or a Ct-C₂₀ alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a C(0)R^{F I N} group where R^{F 1 N} is an optionally substituted Q-Ce alkyl group, an amine group which is optionally substituted with one or two C|-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted (preferably optionally substituted with one or two C 1-C3 alkyl groups) or together with R^{F 1} forms a 5- or 6- membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

RF2 is H, C\-C alkyl which is optionally substituted, 0-(Ci-C₆) alkyl which is optionally substituted, 0-C(0)-(C_rC₆)alkyl which is optionally substituted, -C(0)-0-(Ci-C₆)alkyl which is optionally substituted, -C(0)-(C|-C₆)alkyl which is optionally substituted, a -C(0)NR^F2R^F2A group, a NR^F2RF ^A group or a -S(0)(0)-R^F2S (sulfonyl) group;

R^F2 is H or a Ci-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^1*2" forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

R^F2A is H or a C C₂o alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F2N group, or R^1'2" together with R^F2 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1 -C3 alkyl group;

R'^"2N is an optionally substituted C C₆ alkyl group, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted;

R^F2S is a Ci-C₆ optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted 5- or 6-membered heterocyclic group or a NR^F2SNR^F2SNil group;

R^F2SN is H, or a C1-C3 alky group which is optionally substituted with one or two hydroxyl groups or together with R^F2SNA forms a 5- or 6-membered optionally substituted heterocyclic group;

R^F2SNA is H, an optionally substituted Ci-C₆ alkyl group, an optionally substituted phenyl group or an optionally substituted 5- or 6-membered heterocyclic group, or R^F2SNA together with R^F2SN, forms a 5- or 6-membered optionally substituted heterocyclic group;

RF3 is H, a halogen, a Ci-C₆ alkyl which is optionally substituted, 0-(C|-C₆)alkyl which is optionally substituted, 0-C(0)-(C|-C₆)alkyl which is optionally substituted, -C(0)-0-(C[- C₆)alkyl which is optionally substituted, -C(0)-(Ci-C₆)alkyl which is optionally substituted, a -C(0)NR^F3R^F3IL group or a NR^F3RF^3A group;

R^F3 is H or a Q-Ce alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F3A forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1 -C3 alkyl group; R^F3A is H or a Ci-C₂₀ alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F3 group, or R^F3A together with R^{1 3} forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

R^F3 is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two Ci-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted, with the proviso that R_Fi, RF₂ and RF3 are not all simultaneously H, or

a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof; pound according to the chemical structure:

O

O O

or a pharmaceutically acceptable salt, solvate or polymorph thereof; or a compound mixture according to the chemical structure:

or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof.

2. The method according to claim 1 which is directed to inhibiting AceMIF in a patient.

3. The method according to claim 1 which is directed to treating a hookworm infection.

4. The method according to claim 1 which is directed to treating a symptom or a secondary disease state or condition of a hookworm infection.

5. The method according to claim 4 wherein said symptom or a secondary disease state or condition of a hookworm infection is gastrointestinal discomfort, nausea, abdominal pain, diarrhea, blood loss leading to anemia, iron deficieincy, protein loss and/or

malnutrition, capricious appetite, cough and pneumonitis, intestinal blood loss, dysentery, hemorrhage, edema, iron deficiency anemia, thready pulse, coldness of the skin, pallor of the mucous membranes, fatigue and weakness, erythrocyte rupture, hemoglobin degradation, facial and peripheral edema; eosinophilia, pica, growth retardation, intellectual and co nitive impairments or adverse maternal-fetal outcomes,

6. The method according to any of claims 1-5 wherein said compound is

Where Rp> is OR', NR"R"' or a Q-C20 alkyl group which is optionally substituted or an group which is optionally substituted;

R' is H, a Q-C20 alkyl group which is optionally substituted or an aryl group which is optionally substituted; R" is H or a Ci-C₆ alkyl group which is optionally substituted with a hydroxyl group or together with R'" forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C]-C₃ alkyl group;

R"' is H, a Ci-C₂o alkyl which is optionally substituted, an optionally substituted phenyl group, or a 5- or 6-membered heterocyclic group which is optionally substituted with one or two C 1-C3 alkyl groups, or together with R" forms a 5-or 6-membered heterocyclic group which is optionally substituted with a C|-C₃ alkyl group;

RFI is H, a Ci-C₆ alkyl group which is optionally substituted with a 5-or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group, or a

group;

R^{F 1} is H or a Ci-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^{FL A} forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C[-C₃ alkyl group;

R^{F ! A} is H or a C 1 -C20 alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a C(0)R^{F 1 N} group where R^{F I N} is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two Ci-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted (preferably optionally substituted with one or two C1 -C3 alkyl groups) or together with R^{F I} forms a 5- or 6- membered heterocyclic group which is optionally substituted with a C[-C₃ alkyl group;

RF2 is H, Ci-C<3 alkyl which is optionally substituted, 0-(C|-C₆)alkyl which is optionally substituted, 0-C(O)-(C₁-C_{ )alkyl which is optionally substituted, -C(0)-0-(C[-C₆)alkyl which is optionally substituted, -C(0)-(C₁-C₆)alkyl which is optionally substituted, a -C(0)NR^F2R^F2A group, a NR^F2RF^2A group or a -S(0)(0)-R^F2S (sulfonyl) group;

R^F2 is H or a Ci-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F2A forms a 5- or 6-membered heterocyclic group which is optionally substituted with a Ci-C alkyl group;

R^{F A} is H or a Ci-C₂o alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F2N group, or R^F2FL together with R^F2 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C|-C₃ alkyl group;

R^F2N is an optionally substituted Ci-C₆ alkyl group, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted; R is a Ci-C₆ optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted 5- or 6-membered heterocyclic group or a NR ^' R group;

R^F2SN is H, or a C|-C₃ alky group which is optionally substituted with one or two hydroxyl groups or together with R^F2SNA forms a 5- or 6-membered optionally substituted heterocyclic group;

R^F2SNA is H, an optionally substituted Ci-C₆ alkyl group, an optionally substituted phenyl group or an optionally substituted 5- or 6-membered heterocyclic group, or R^F2SNA together with R^F2SN, forms a 5- or 6-membered optionally substituted heterocyclic group;

R_F3 is H, a halogen, a C C₆ alkyl which is optionally substituted, 0-(Ci-C₆)alkyl which is optionally substituted, 0-C(0)-(Ci-C₆)alkyl which is optionally substituted, -C(0)-0-(Ci- C₆)alkyl which is optionally substituted, -C(0)-(C]-C₆)alkyl which is optionally substituted, a -C(0)NR^F3R^F3FL group or a NR^F3RF^3A group;

R^F3 is H or a C i-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F3 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

R^F3A is H or a C ]-C2o alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F3N group, or R^F3A together with R^F3 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C₁-C3 alkyl group;

R^F3N is an optionally substituted C|-C₆ alkyl group, an amine group which is optionally substituted with one or two Q-Q alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted, with the proviso that R_FI , R_F2 and R_F3 are not all simultaneously H, or

a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof.

7, The method according to claim 6 wherein R_F' is H, OR' where R' is a C₁ -C3 alkyl group or a -NH-2-furanyl group.

8. The method according to claim 7 wherein R' is a methyl group.

9. The method according to claim 6 wherein R_FI is a -NH-CH₂-2-furanyl group, a N- piperidine group, a N-morpholine group, a ~NHC(0)-2-furanyl group which is optionally substituted with one or two methyl groups, or a -NHC(0)NH-(Ci-C₃) alkyl group, preferably an isopropyl group.

10. The method according to any of claims 6-9 wherein Rp₂ is H,

more preferably -OCH3, or a sulfonamide group -S(0)(0)NH₂ (sulfonamide) group which is optionally substituted on the nitrogen with a C1-C3 alkyl group and/or an optionally substituted heteroaryl or an optionally substituted phenyl group (the nitrogen of the sulfonamide preferably contains a methyl group).

1 1. The method according to claim 10 wherein said optionally substituted heteroaryl group is a -CH₂-2-furanyl group.

12. The method according to any of claims 6-9 where Rn is H, -0(C|-C₆)alkyl or a halogen.

13. The method according to claim 12 where RF₃ is H, -OCH3, CI or F.

14. The method according to claim 12 where RF₃ is H, -OCH3 or CI.

15. The method according to claim 6 wherein R is H, OR' where R' is a C 1 -C3 alkyl group or a -NH-2-furanyl group, R^ is a ~NH-CH₂-2-furanyl group, a N-piperidine group, a N-morpholine group, a -NHC(0)-2-furanyl group which is optionally substituted with one methyl group or a ~NHC(0)NH-(C,-C₃) alkyl group, R_F2 is H, -0(C_rC₆)alkyl or a sulfonamide group -S(0)(0)NH₂ (sulfonamide) group which is optionally substituted on the nitrogen with a C1-C3 alkyl group and/or an optionally substituted heteroaryl or an optionally substituted phenyl group and RF3 is H, -0(Ci-C₆)alkyl or a halogen.

16. The method according to any of claims 1-5 wherein said compound is Where RM is H or a Ci-C₂₀ optionally substituted alkyl group;

R-M t is H or a C_rC₃ alkyl group which is optionally substituted with one or two hydroxyl groups;

RM2 and RM3 are each independently a H, a halogen, nitro, cyano, hydroxyl, -(CH₂)_N-C02H, (C1-C12) alkyl which is optionally substituted, -(CH₂)„OC(O)(C₁-Ci₀) alkyl ester

(oxyacylester) which is optionally substituted,

-(CH2)_nC(0)0-(Ci-Cio) alkyl ester (carboxy ester) which is optionally substituted,

-(CH )„C(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted or a -(CH₂)_irO-(Ci- C10) alkyl (alkoxy) which is optionally substituted;

RM4 is H_} halogen, nitro, cyano, hydroxyl, a (C 1 -C 12) hydrocarbyl group which is optionally substituted, -(CH₂)_nOC(0)(C[-C|o) alkyl ester (oxyacylester) which is optionally substituted, -(CH₂)„C(0)0-(Ci-C]o) alkyl ester (carboxy ester) which is optionally substituted,

-(CH₂)_nC(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted, or a -(CH₂)_n-0-(C_r C₁₀) alkyl (alkoxy) which is optionally substituted;

n is 0, 1 ,2,3, 4 or 5,

or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof.

17. The method according to claim 16 wherein R_M is H,

18. The method according to claim 16 or 17 wherein R I is H,

1 . The method according to any of claims 16-18 wherein R_M2 and R ₃ are both halogens.

20. The method according to any of claims 16-19 wherein R_M4 is C|-C₃ aikyl group.

21. The method according to any of claims 16-19 wherein ₂ and RM₃ are both CI and RM4 is a methyl group.

22. The method according to any of claims 1-5 wherein said compound is

or a pharmaceutically acceptable salt, solvate or polymorph thereof.

23. A method according to any of claims 1-5 wherein said compound is a compound mixture acc

or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof.

24, A pharmaceutical composition comprising an effective amount of a compound according to the structure:

Where RM is H or a Ci-C₂o optionally substituted alkyl group;

RMI is H or a C1-C3 alkyl group which is optionally substituted with one or two hydroxyl groups;

R 2 and RM₃ are each independently a H, a halogen (F, CI, Br, I, preferably F or CI), nitro, cyano, hydroxyl, -(CH₂)_n-CC>2H, (C1-C12) alkyl (preferably Ci-C₆ alkyl) which is optionally substituted, -(CH2)_nOC(0)(C|-Cio) alkyl ester (oxyacylester) which is optionally substituted, -(CH₂)_nC(0)0-(Ci-Cio) alkyl ester (carboxy ester) which is optionally substituted,

-(CH2)nC(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted, or a -(CH2) -0-(Ci~ Cio) alkyl (alkoxy) which is optionally substituted;

RM4 is H, halogen, nitro, cyano, hydroxyl, a (Ci-C[2) hydrocarbyl group which is optionally substituted, -(CH2)_nOC(O)(C]-C_l0) alkyl ester (oxyacylester) which is optionally substituted, -(CH2)_nC(0)0-(C[-Cjo) alkyl ester (carboxy ester) which is optionally substituted,

-(CH₂)_nC(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted, or a ~(CH2)_n-0-(Ci- Cio) alkyl (alkoxy) which is optionally substituted;

n is 0, 1,2,3, 4 or 5,

or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof, or at least one compound according to the chemical structure:

Where Rp> is OR', NR"R'" or a C[-C₂o alkyl group which is optionally substituted or an aryl group which is optionally substituted;

R' is H, a C i-C₂o alkyl group which is optionally substituted or an aryl group which is optionally substituted;

R" is H or a C i-C₆ alkyl group which is optionally substituted with a hydroxyl group or together with R'" forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

R'" is H, a C i -C₂o alkyl which is optionally substituted, an optionally substituted phenyl group, or a 5- or 6-membered heterocyclic group which is optionally substituted with one or two Q-C3 alkyl groups, or together with R" forms a 5-or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

Rpi is H, a C i -Cfi alkyl group which is optionally substituted with a 5-or 6-membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group, or a NR^FIR^FI group;

R^F1 is H or a C i-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F forms a 5- or 6-membered heterocyclic group which is optionally substituted with a CrC3 alkyl group;

R^{Fl a} is H or a C] -C2o alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a C(0)R^FIN group where R^{F! N} is an optionally substituted Cj-Ce alkyl group, an amine group which is optionally substituted with one or two C_\-C& alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted (preferably optionally substituted with one or two Q-C3 alkyl groups) or together with R^F1 forms a 5- or 6- membered heterocyclic group which is optionally substituted with a C1-C3 alkyl group;

RF2 is H, Cj-C₆ alkyl which is optionally substituted, 0-(Ci-C₆)alkyl which is optionally substituted, 0-C(0)-(Ci-C₆)alkyl which is optionally substituted, -C(0)~0-(C|-C₆)alkyl which is optionally substituted, -C(0)-(Ci-C₆)alkyl which is optionally substituted, a -C(0)NR^R2R^F2A group, a NR^F2RF ^A group or a -S(0)(0)-R^F2S (sulfonyl) group;

R^F2 is H or a C | -C₆ alkyl group which is optionally substituted with one or two hydroxy 1 groups or together with R^1'28 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C_rC₃ alkyl group;

R^{F A} is H or a C1-C20 alkyl which is optionally substituted, an optionally substituted phenyl group, a 5 - or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F2N group, or R^F2A together with R^F2 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

R^{F N} is an optionally substituted Ci-C₆ alkyl group, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted;

R^F2S is a C]-C₆ optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted 5- or 6-membered heterocyclic group or a NR^F2SNR^F2SNA group;

R^F2SN is H, or a C1-C3 alky group which is optionally substituted with one or two hydroxy 1 groups or together with R^F2SNa forms a 5- or 6-membered optionally substituted heterocyclic group;

R^F2SNA is H, an optionally substituted C]-C₆ alkyl group, an optionally substituted phenyl group or an optionally substituted 5- or 6-membered heterocyclic group, or R^F2SNN together with R , forms a 5- or 6-membered optionally substituted heterocyclic group;

RF3 is H, a halogen, a Ci-C₆ alkyl which is optionally substituted, 0-(C₁-C₆)alkyl which is optionally substituted, 0-C(0)-(Ci-C₆)alkyl which is optionally substituted, -C(0)-0-(C | - C₆)alkyl which is optionally substituted, -C(0)-(Ci-C₆)alkyl which is optionally substituted, a -C(0)NR^F3R^F3A group or a NR^F3RF^3A group;

R^F3 is H or a C ₍-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F3A forms a 5- or 6-membered heterocyclic group which is optionally substituted with a Ci-C₃ alkyl group;

R^F3A is H or a C i-C₂o alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F3N group, or R^F3N together with R^F3 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group; R^F3N is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two C_rC₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted, with the proviso that Rn, Rp₂ and FB are not all simultaneously H, or

a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof; or a compound according to the chemical structure:

O

O O

or a pharmaceutically acceptable salt, solvate or polymorph thereof; or a compound mixture according to the chemical structure:

or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof in combination with a pharmaceutically acceptable carrier, additive or excipient and further optionally in combination with at least one additional bioactive agent selected from the group consisting of benzimidazole, levamisole, pyrantel pamoate, ferrous sulfate, folic acid, vitamin B12 or mixtures of these agents.

25. The composition according to claim 24 wherein said benzimidazole is

albendazole or mebendazole.

26. The composition according to claim 24 which is directed to inhibiting AceMIF in a patient.

27. The composition according to claim 24 which is directed to treating a hookworm infection.

28. The composition according to claim 24 which is directed to treating a symptom or a secondary disease state or condition of a hookworm infection.

29. The composition according to claim 28 wherein said symptom or a secondary disease state or condition of a hookworm infection is gastrointestinal discomfort, nausea, abdominal pain, diarrhea, blood loss leading to anemia, iron deficieincy, protein loss and/or malnutrition, capricious appetite, cough and pneumonitis, intestinal blood loss, dysentery, hemorrhage, edema, iron deficiency anemia, thready pulse, coldness of the skin, pallor of the mucous membranes, fatigue and weakness, erythrocyte rupture, hemoglobin degradation, facial and peripheral edema; eosinophilia, pica, growth retardation, intellectual and cognitive impairments or adverse maternal-fetal outcomes,

30. The composition according to any of claims 24-29 wherein said compound is

Where Rp> is OR', NR"R"' or a C|-C₂o alkyl group which is optionally substituted or an aryl group which is optionally substituted; R' is H, a C|-C₂o alkyl group which is optionally substituted or an aryl group which is optionally substituted;

R" is H or a C)-C₆ alkyl group which is optionally substituted with a hydroxyl group or together with R'" forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

R'" is H, a C 1 -C20 alkyl which is optionally substituted, an optionally substituted phenyl group, or a 5- or 6-membered heterocyclic group which is optionally substituted with one or two C 1 -C3 alkyl groups, or together with R" forms a 5-or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

Rpi is H, a C_\-C₆ alkyl group which is optionally substituted with a 5-or 6-membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group, or a NR^F1R^{Fl a} group;

R^F1 is H or a Ci-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^{Fl a} forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C|-C₃ alkyl group;

R^Fla is H or a C 1 -C20 alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a C(0)R^F1N group where R^F,N is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two C|-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted (preferably optionally substituted with one or two C 1-C3 alkyl groups) or together with R^F1 forms a 5- or 6- membered heterocyclic group which is optionally substituted with a C 1-C3 alkyl group;

RF2 is H, C|-C₆ alkyl which is optionally substituted, 0-(Ci-C₆)alkyl which is optionally substituted, 0-C(0)-(Ci-C₆)alkyl which is optionally substituted, -C(0)-0-(Ci-C₆)alkyl which is optionally substituted, -C(0)-(Ci-C₆)alkyl which is optionally substituted, a -C(0)NR^F2R^F2a group, a NR^F2RF^2fi group or a -S(0)(0)-R^F2S (sulfonyl) group;

R^F2 is H or a Ci-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F2a forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1 -C3 alkyl group;

R^F2a is H or a C 1 -C20 alkyl which is optionally substituted, an optionally substituted phenyl group, a 5 - or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F2N group, or R^F2a together with R^F2 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C 1 -C3 alkyl group; R^F2N is an optionally substituted Ci-C₆ alkyl group, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted;

R^F2S is a Ci"C6 optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted 5- or 6-membered heterocyclic group or a NR^F2SNR^F2SNa group;

R^F2SN is H, or a C C₃ alky group which is optionally substituted with one or two hydroxyl groups or together with R^F2SNa forms a 5- or 6-membered optionally substituted heterocyclic group;

R^!'2SNA is H, an optionally substituted Ci-C₆ alkyl group, an optionally substituted phenyl group or an optionally substituted 5- or 6-membered heterocyclic group, or R^l'2SNa together with R^F2SN, forms a 5- or 6-membered optionally substituted heterocyclic group;

Rp₃ is H, a halogen, a Ci-C₆ alkyl which is optionally substituted, 0-(Ci-C₆)alkyl which is optionally substituted, 0-C(0)-(Ci-C₆)alkyl which is optionally substituted, -C(0)-0-(Cj- C₆)alkyl which is optionally substituted, -C(0)-(Ci-C₆)alkyl which is optionally substituted, a -C(0)NR^F3R^F3A group or a NR^F3RF^3h group;

R ³ is H or a Ci-C₆ alkyl group which is optionally substituted with one or two hydroxyl groups or together with R^F3A forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C[-C₃ alkyl group;

R^F3A is H or a C 1-C20 alkyl which is optionally substituted, an optionally substituted phenyl group, a 5- or 6-membered heterocyclic group which is optionally substituted, a -C(0)R^F3N group, or R^F3A together with R^1'3 forms a 5- or 6-membered heterocyclic group which is optionally substituted with a C|-C₃ alkyl group;

R^F3N is an optionally substituted Ci-C₆ alkyl group, an amine group which is optionally substituted with one or two Ci-C₆ alkyl groups which themselves may be optionally substituted with one or two hydroxyl groups, a phenyl group which is optionally substituted, or a heterocyclic group which is optionally substituted, with the proviso that RR, RF₂ and RF₃ are not all simultaneously H, or

a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof.

31. The composition according to claim 30 wherein Rp is H, OR' where R' is a C 1 -C3 alkyl group or a -NH-2-furanyl group.

32, The composition according to claim 31 wherein R' is a methyl group.

33. The composition according to claim 30 wherein Rpi is a -NH-CH₂-2-furanyl group, a N-piperidine group, a N-morpholine group, a -NHC(0)-2-furanyl group which is optionally substituted with one or two methyl groups, or a -NHC(0)NH-(Ci-C3) alkyl group, preferably an isopropyl group.

34. The composition according to any of claims 30-33 wherein Rp₂ is H, -0(C_!- C₆)alkyl, more preferably -OCH3, or a sulfonamide group -S(0)(0)NH₂ (sulfonamide) group which is optionally substituted on the nitrogen with a C1-C3 alkyl group and/or an optionally substituted heteroaryl or an optionally substituted phenyl group (the nitrogen of the sulfonamide preferably contains a methyl group).

35. The composition according to claim 34 wherein said optionally substituted heteroaryl group is a -C¾-2-furanyl group.

36. The composition according to any of claims 30-33 where Rp₃ is H, -0(C[- C₆)alkyl or a halogen.

37. The composition according to claim 36 where Rp₃ is H, -OCH₃, CI or F.

38. The composition according to claim 36 where Rp₃ is H, -OCH₃ or CI.

39. The composition according to claim 30 wherein R is H, OR' where R' is a Ci- C₃ alkyl group or a -NH-2-furanyl group, R_Fi is a -NH-CH₂-2-furanyl group, a N-piperidine group, a N-morpholine group, a -NHC(0)-2-furanyl group which is optionally substituted with one methyl group or a -NHC(0)NH-(C C₃) alkyl group, R_F2 is H, -0(C_rC₆)alkyl or a sulfonamide group -S(0)(0)NH₂ (sulfonamide) group which is optionally substituted on the nitrogen with a Ci-C₃ alkyl group and/or an optionally substituted heteroaryl or an optionally substituted phenyl group and Rp is H, -0(Ci-C₆)alkyl or a halogen.

40. The composition according to any of claims 24-29 wherein said compound is Where RM is H or a C|-C₂o optionally substituted alkyl group;

R I is H or a C1-C3 alkyl group which is optionally substituted with one or two hydroxyl groups;

R 2 and RM₃ are each independently a H, a halogen, nitro, cyano, hydroxyl, -(CH₂)_TL-C0₂H, (C1-C12) alkyl which is optionally substituted, -(CH₂)_nOC(0)(Ci-Cio) alkyl ester

(oxyacylester) which is optionally substituted,

-(CH2)_nC(0)0-(Ci-Cio) alkyl ester (carboxy ester) which is optionally substituted,

-(CH₂)_nC(0)-(Ci-Cio) alkyl (acyl group) which is optionally substituted or a -(CH₂)_N-0-(Ci- Cio) alkyl (alkoxy) which is optionally substituted;

R 4 is H, halogen, nitro, cyano, hydroxyl, a (C1-C12) hydrocarbyl group which is optionally substituted, -(CH2)„OC(0)(Ci-Cio) alkyl ester (oxyacylester) which is optionally substituted, -(CH2)nC(0)0-(Ci-Cio) alkyl ester (carboxy ester) which is optionally substituted,

-(CH₂)_nC(0)-(Ci-C₁o) alkyl (acyl group) which is optionally substituted, or a -(CH2)_N-0-(Ci- C10) alkyl (alkoxy) which is optionally substituted;

n is 0, 1 ,2,3 , 4 or 5,

or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof.

41 . The composition according to claim 40 wherein RM is H.

42. The composition according to claim 40 or 41 wherein R_{M J} is H.

43. The composition according to any of claims 40-42 wherein R_M2 and RM3 are both halogens.

44. The composition according to any of claims 40-43 wherein R ₄ is CpC3 alkyl group,

45. The composition according to any of claims 40-43 wherein RM₂ and R are both CI and M4 is a methyl group.

46. The composition according to any of claims 24-29 wherein said compound is

or a pharmaceutically acceptable salt, solvate or polymorph thereof.

47. A composition according to any of claims 24-29 wherein said compound is a compound mixture according to the chemical structure:

or a pharmaceutically acceptable salt, enantiomer, solvate or polymorph thereof.

48. Use of a composition according to any claims 24-47 in the manufacture of a medicament for the inhibition of AceMIF in a patient in need or for the treatment of a hookworm infection or a symptom, secondary disease state or condition of a hookworm infection.

49. Use according to claim 48 wherein said composition comprises at least one additional bioactive agent selected from the group consisting of benzimidazole, levamisole, pyrantel pamoate, ferrous sulfate, folic acid, vitamin B 12 or mixtures of these agents.

50. Use according to claim 49 wherein said benzimidazole is albendazole or mebendazole,

51. Use according to any of claims 48-50 which is directed to inhibiting AceMIF in a patient.

52. Use according to any of claims 48-50 which is directed to treating a hookworm infection or a symptom, secondary disease state or condition of a hookworm infection.

53. Use according to claim 52 which is directed to treating a symptom or a secondary disease state or condition of a hookworm infection.

54. Use according to claim 53 wherein said symptom or a secondary disease state or condition of a hookworm infection is gastrointestinal discomfort, nausea, abdominal pain, diarrhea, blood loss leading to anemia, iron deficieincy, protein loss and/or malnutrition, capricious appetite, cough and pneumonitis, intestinal blood loss, dysentery, hemorrhage, edema, iron deficiency anemia, thready pulse, coldness of the skin, pallor of the mucous membranes, fatigue and wealcness, erythrocyte rupture, hemoglobin degradation, facial and peripheral edema; eosinophilia, pica, growth retardation, intellectual and cognitive impairments or adverse maternal-fetal outcomes,

55. Use of a composition according to any of claims 24-47 in the manufacture of a medicament for the treatment of a parasitic infection in a patient or subject,

56. Use according to claim 55 wherein said parasitic infection is a protozoa, a cestode, a trematode or a nematode.

57. Use according to claim 56 wherein said protozoa is Entamoeba histolytica, Trypanosome spp,, and Giardia spp,

58. Use according to claim 56 wherein said cestode is T. solium, T. saginata,

Diphyllobothrium spp., Hymenolepsis spp,, or Echinococcus spp,, and said trematode is Paragonimus westermani (lung fluke), Clonorchis sinensis or Fasciola hepatica (liver fluke).

59. Use according to claim 56 wherein said nematode is an intestinal nematode or a tissue nematode.

60. Use according to claim 59 wherein said intestinal nematode is Trichuris trichiura (thread worm/whip worm), Enterobius vermicularis (pinworm), Ascaris lumbricoides, or Strongyloides stercoralis (Cochin-China diarrhea)

61. Use according to claim 59 wherein said tissue nematode is Trichinella spirala, Trichinella nativa, Toxocaria canis, Filaria spp., Wucheria spp. or Onchocera volvulus.

62. A compound as set forth in any of claims 24-47 hereof.

63. A method of generating an immunogenic response to AceMIF in a patient or subject comprising administering to said patient or subject an immunogenic effective amount of AceMIF in a pharmaceutically acceptable carrier, additive or excipient.

64. The method according to claim 63 wherein said AceMIF is recombinant AceMIF.

65. The method according to claim 63 or 64 wherein said immunogenic response is prophylactic for (prevents) a hookworm infection in said patient or subject.

66. The method according to any of claims 63-65 wherein said patient or subject is a domesticated or agricultural animal.