COMPOUNDS FOR THE TREATMENT OF MALARIA

Abstract

The present invention provides methods of treating malaria by administration of a compound of Formula (I):

##STR00001##

or a pharmaceutically acceptable salt of said compound, to a subject in need thereof, wherein the variables X, R.sup.1, R.sup.3, R.sup.4, R.sup.5, A, B, L, m and n are as defined herein. The invention also provides uses of the compounds of Formula (I), as defined herein, for inhibiting plasmepsin V activity, for treating a Plasmodium infection, and for treating malaria. Also provided are methods of treatment further comprising administration of one or more additional anti-malarial compounds.

Claims

1. A method for treating a Plasmodium infection, or for treating malaria, which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, said compound having the structural Formula (I): ##STR00062## wherein: X is a bond or CH(R.sup.2); R.sup.2 is selected from the group consisting of hydrogen, halo, -C.sub.1-C.sub.6 alkyl, and phenyl, wherein said -C.sub.1-C.sub.6 alkyl and said phenyl are optionally substituted with one to three halo; ring A is phenyl or ##STR00063## is ##STR00064## wherein, R.sup.11 is hydrogen, halo, OH, -C.sub.1-C.sub.6alkyl, optionally substituted with one to three halo, C.sub.3-C.sub.6cycloalkyl, optionally substituted with one to three halo, or NHC(O)O-C.sub.1-C.sub.6alkyl; each occurrence of R.sup.1 is independently selected from halo, CN, OH, -C.sub.1-C.sub.6alkyl, O-C.sub.1-C.sub.6 alkyl, -C.sub.1-C.sub.6haloalkyl, O-C.sub.1-C.sub.6 haloalkyl, and AryA; AryA is a 5- or 6-membered monocyclic aromatic ring with 0, 1, or 2, heteroatoms independently selected from N, O and S; R.sup.3 is -C.sub.1-C.sub.6alkyl, -C.sub.4-C.sub.6cycloalkyl, (CH.sub.2).sub.n-C.sub.4-C.sub.6 heterocycloalkyl, phenyl, or (CH.sub.2).sub.n-cyclopropyl, wherein each of said -C.sub.1-C.sub.6 alkyl, said -C.sub.4-C.sub.6 cycloalkyl, said (CH.sub.2).sub.n-C.sub.4-C.sub.6 heterocycloalkyl, and said (CH.sub.2).sub.n-cyclopropyl are optionally substituted with one or two substituents, independently selected from halo, -OH, and -O-C.sub.1-C.sub.6alkyl, and wherein said phenyl is optionally substituted with one to three substituents, independently selected from OH, halo, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6fluoroalkyl, CN, OCF.sub.3, OCF.sub.2, and S(O).sub.k-C.sub.1-C.sub.6alkyl; R.sup.4 is C.sub.1-C.sub.6alkyl, or phenyl, wherein the phenyl or -C.sub.1-C.sub.6alkyl are optionally substituted with one to three substituents, independently selected from halo, OH, O-C.sub.1-C.sub.3alkyl, -C.sub.1-C.sub.3alkyl and cyclopropyl; n is 0, 1, 2, or 3; and m is 0, 1, 2, 3, 4, 5, or 6.

2. The method of claim 1, wherein in the compound of structural Formula (I), or the pharmaceutically acceptable salt thereof, R.sup.3 and R.sup.4 are independently selected from hydrogen, methyl, isopropyl, -C.sub.1-C.sub.6alkyl, -C.sub.4-C.sub.6 cycloalkyl, (CH.sub.2).sub.n-C.sub.4-C.sub.6 heterocycloalkyl, CH.sub.2-cyclopropyl and phenyl, wherein said phenyl is optionally substituted with one to three halo.

3. The method of claim 1, wherein in the compound of structural Formula (I), or the pharmaceutically acceptable salt thereof, R.sup.3 is methyl, isopropyl, -C.sub.1-C.sub.6alkyl, C.sub.4-C.sub.6 cycloalkyl, (CH.sub.2).sub.n-C.sub.4-C.sub.6 heterocycloalkyl, CH.sub.2-cyclopropyl or phenyl, wherein said phenyl is optionally substituted with one to three halo and R.sup.4 is phenyl, wherein said phenyl is optionally substituted with one to three halo.

4. The method of claim 1, wherein in the compound of structural Formula (I), or the pharmaceutically acceptable salt thereof, X is CH(R.sup.2) and R.sup.2 is phenyl.

5. The method of claim 1, wherein in the compound of structural Formula (I), or the pharmaceutically acceptable salt thereof, ring A in structural Formula (I) is: ##STR00065## wherein R.sup.1 is selected from halo and CF.sub.3.

6. (canceled)

7. (canceled)

8. A method for treating a Plasmodium infection, or for treating malaria, which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, said compound having the structural Formula (IA): ##STR00066## wherein each occurrence of R.sup.8 is independently selected from halo and CF.sub.3; ring B is selected from: ##STR00067## wherein each occurrence of R.sup.9 is independently selected from H, O, halo, and C.sub.1-C.sub.6alkyl; and each occurrence of R.sup.10 independently selected from H, halo, and CF.sub.3, and each occurrence of R.sup.7 is halo; m is 0, 1, 2, 3, 4, 5, or 6; and n is 0, 1, 2, or 3.

9. A method for treating a Plasmodium infection, or for treating malaria, which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, said compound having the structural Formula (IB): ##STR00068## wherein R.sup.2 is selected from the group consisting of hydrogen, halo, -C.sub.1-C.sub.6 alkyl, and phenyl, wherein said -C.sub.1-C.sub.6 alkyl and said phenyl are optionally substituted with one to three halo; R.sup.6 is selected from H, -(C.sub.1-C.sub.6)alkyl and -(C.sub.1-C.sub.6)heteroalkyl; ring B is a C.sub.3-C.sub.7cycloalkyl, a C.sub.3-C.sub.7heterocycloalkyl, or AryB; AryB is: (i) a 5- or 6-membered monocyclic aromatic ring with 0, 1, 2, or 3, heteroatoms independently selected from N, O and S, or (ii) a 9- to 11-membered bicyclic aromatic ring with 0, 1, 2, or 3 heteroatoms independently selected from N, O and S; R.sup.4 is C.sub.1-C.sub.6alkyl, or phenyl, wherein the phenyl or -C.sub.1-C.sub.6alkyl are optionally substituted with one to three substituents, independently selected from halo, OH, O-C.sub.1-C.sub.3alkyl, -C.sub.1-C.sub.3alkyl and cyclopropyl; each occurrence of R.sup.5 is independently halo, OH, O, CN, S(O).sub.ZC.sub.1-C.sub.4 alkyl, C(O)(C.sub.1-C.sub.6alkyl), C(O)O(C.sub.1-C.sub.6alkyl), C(O)N(H)(C.sub.1-C.sub.6alkyl), C(O)N(C.sub.1-C.sub.6alkyl).sub.2, C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.6cycloalkyl, NHC(O)O-C.sub.1-C.sub.6alkyl, or OC.sub.1-C.sub.6alkyl, wherein said S(O).sub.ZC.sub.1-C.sub.4 alkyl, said C(O)(C.sub.1-C.sub.6alkyl), said C(O)O(C.sub.1-C.sub.6alkyl), said C(O)N(H)(C.sub.1-C.sub.6alkyl), said C(O)N(C.sub.1-C.sub.6alkyl), said C.sub.1-C.sub.6alkyl, said C.sub.3-C.sub.6cycloalkyl, said NHC(O)O-C.sub.1-C.sub.6alkyl, and said OC.sub.1-C.sub.6alkyl are optionally substituted with one to three substituents, independently selected from halo, OH, CN, and OC.sub.1-C.sub.6alkyl, and m is 0, 1, 2, 3, 4, 5, or 6.

10. A method for treating a Plasmodium infection, or for treating malaria, which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, said compound having the structural Formula (IC): ##STR00069## wherein each occurrence of R.sup.7 is halo; ring B is a C.sub.3-C.sub.7cycloalkyl, a C.sub.3-C.sub.7heterocycloalkyl, or AryB, AryB is: (i) a 5- or 6-membered monocyclic aromatic ring with 0, 1, 2, or 3, heteroatoms independently selected from N, O and S, or (ii) a 9- to 11-membered bicyclic aromatic ring with 0, 1, 2, or 3 heteroatoms independently selected from N, O and S, R.sup.4 is C.sub.1-C.sub.6alkyl, wherein -C.sub.1-C.sub.6alkyl are optionally substituted with one to three substituents, independently selected from halo, OH, OC.sub.1-C.sub.3alkyl, -C.sub.1-C.sub.3alkyl and cyclopropyl, each occurrence of R.sup.5 is independently halo, OH, O, CN, S(O).sub.ZC.sub.1-C.sub.4 alkyl, C(O)(C.sub.1-C.sub.6alkyl), C(O)O(C.sub.1-C.sub.6alkyl), C(O)N(H)(C.sub.1-C.sub.6alkyl), C(O)N(C.sub.1-C.sub.6alkyl).sub.2, -C.sub.1-C.sub.6alkyl, -C.sub.3-C.sub.6cycloalkyl, NHC(O)O-C.sub.1-C.sub.6alkyl, or OC.sub.1-C.sub.6alkyl, wherein said S(O).sub.ZC.sub.1-C.sub.4 alkyl, said C(O)(C.sub.1-C.sub.6alkyl), said C(O)O(C.sub.1-C.sub.6alkyl), said C(O)N(H)(C.sub.1-C.sub.6alkyl), said C(O)N(C.sub.1-C.sub.6alkyl), said -C.sub.1-C.sub.6alkyl, said C.sub.3-C.sub.6cycloalkyl, said NHC(O)O-C.sub.1-C.sub.6alkyl, and said OC.sub.1-C.sub.6alkyl are optionally substituted with one to three substituents, independently selected from halo, OH, CN, and OC.sub.1-C.sub.6alkyl, m is 0, 1, 2, 3, 4, 5, or 6; and n is 0, 1, 2, or 3.

11. (canceled)

12. The method according to claim 1, wherein the compound has the structure: ##STR00070## ##STR00071## ##STR00072## ##STR00073## or a pharmaceutically acceptable salt thereof.

13. The method according to claim 1, wherein the compound has the structure: ##STR00074## or a pharmaceutically acceptable salt thereof.

14. The method of claim 1, wherein the subject is human.

15. The method of claim 14, wherein the compound or the pharmaceutically acceptable salt thereof is administered orally or via subcutaneous, intramuscular, or intravenous administration.

16. The method of claim 1, further comprising administration of one or more additional anti-malarial agents to the subject.

17. The method of claim 16, wherein the one or more additional anti-malarial agents is selected from the group consisting of: artemether, lumefantrine, dihydroartemisinin, piperaquine, pyronaridine, artesunate, amodiaquine, mefloquine, sulfadoxine, pyrimethamine, lumefantrine, quinine, chloroquine, atovaquone, and proguanil.

18. The method of claim 1, wherein the Plasmodium strain is drug resistant.

19-21. (canceled)

Description

EXAMPLES

[0252] In general, the compounds used in the methods of the invention, i.e. compounds of Formula (I), (IA), (IB) or (IC), may be produced by a variety of processes known to those skilled in the art and by known processes analogous thereto. The invention disclosed herein is exemplified by the following preparations which should not be construed to limit the scope of the disclosure. Alternative mechanistic pathways and analogous structures will be apparent to those skilled in the art. The practitioner is not limited to these methods.

[0253] One skilled in the art will recognize that one route will be optimized depending on the choice of appendage substituents. Additionally, one skilled in the art will recognize that in some cases the order of steps has to be controlled to avoid functional group incompatibility. The prepared compounds may be analyzed for their composition and purity as well as characterized by standard analytical techniques such as, for example, elemental analysis, NMR, mass spectroscopy and IR spectra.

[0254] One skilled in the art will recognize that reagents and solvents actually used may be selected from several reagents and solvents well known in the art to be effective equivalents. Hence, when a specific solvent or reagent is mentioned, it is meant to be an illustrative example of the conditions desirable for that particular reaction scheme and in the preparations and examples described below.

[0255] The invention disclosed herein is exemplified by the following illustrative processes which should not be construed to limit the scope of the disclosure. Alternative mechanistic pathways and analogous structures will be apparent to those skilled in the art.

Method A:

[0256] Method A is a general method for compounds of Formula (I) that relies on the formation of intermediate A8. In this method, a ketone represented by structure Al is condensed with a sulfoxamine such as A2 to provide an imine A3. This imine A3 is subsequently reacted with an appropriate ester A4 under basic conditions to give intermediate A5 according to the procedures of Ellman et al. (Acc. Chem. Res. 35 (11): 984-995 (2002)). Deprotection under acidic conditions to give amino ester A6 and coupling with a protected isothiocyanate (shown here for example using 2,4 dimethoxybenzyl isothiocyanate I-3) affords an iminoimidazolidone A7. Removal of the protecting group under hydrogenolysis conditions gives intermediate A8. Condensation of A8 with alcohols such as A9 provides compounds of type A10 which can be further reacted under acidic conditions to provide the compound of Formula (I).

##STR00039##

Method A

[0257] A modification of this route provided a convergent synthesis as shown here:

##STR00040##

[0258] Compound A6 is condensed with the Boc-protected thioureas 1-4 using a reagent such as a carbodiimide to provide the compounds A10 which are elaborated into compounds of Formula I as in method A.

Method B:

[0259] Method B is a general alternate method for compounds of Formula (I) that relies on using compounds such as B9 (in place of A9) wherein the ring A contains a functional group (such as Cl, Br, I or CN) to provide compounds B10. The functional group (FG) is then converted into the L-phenyl-(R.sup.5).sub.m (or alternative L-ring B-(R.sup.5).sub.m) substitutent and then subsequently deprotected to provide compounds of formula I.

##STR00041##

[0260] Specific compounds useful in the methods of the invention were synthesized using generally the same procedures as described in Khan et al., WO 2013/142396, substituting the appropriate reactants and reagents.

Assay 1

[0261] Summary: A modified version of the assay described in Gamo, F. J., Sanz, L. M., Vidal, J., de Cozar, C., Alvarez, E., Lavandera, J. L., Vanderwall, D. E., Green, D. V., Kumar, V., Hasan, S., Brown, J. R., Peishoff, C. E., Cardon, L. R., Garcia-Bustos, J. F., Nature, 465 (2010) 305-310 (Gamo et al.) was used to assess the activity of compounds against asexual P. falciparum 3D7 parasites. Compounds were pre-dispensed in 384-well plates, RPMI/AlbuMAX growth media was added and P. falciparum inoculated. Plates were incubated for 72 h and then frozen at 80 C. overnight. LDH activity was quantified with the modified cofactor 3-acetylpyridine adenine dinucleotide (APAD) (Sigma Aldrich) by measuring absorbance of the tetrazolium indicator nitro blue tetrazolium (NBT) (Sigma Aldrich) at 650 nm.

[0262] Parasite conditions: An inoculum of synchronous P. falciparum (3D7 strain) parasitized red blood cells (PRBC) at 0.7% parasitaemia and 2% haematocrit in RPMI-1640, 5% AlbuMAX, 2% D-sucrose, 0.3% glutamine and 150 M hypoxanthine was used for the assay.

[0263] Growth inhibition assay: Compound master plates (384-well) were prepared by a 10 pt serial dilution of compounds, from 1 mM to 50.8 nM, in columns 3-12 and 13-22. DMSO was dispensed into columns 1 and 23 of the compound master plate to be used as the positive growth control (100% viability). Columns 2 and 24 of the compound master plate had a stock concentration of 200 M chloroquine solution (0% viability) as negative growth control (final assay concentration of 200 nM). Intermediate compound dilution plates were prepared by dispensing 1l from each well of the compound master plate into 11.5 l of RPMI/AlbuMAX growth media. Duplicate assay plates (384-well) were then prepared by dispensing 0.5 L of compound from the intermediate dilution plates into 9.5 l of RPMI/AlbuMAX growth media. The parasite inoculum (30 L) was dispensed into the assay plates containing compounds using a Multidrop dispenser (Thermo Scientific) such that the final assay volume was 40 L and final compound concentration was 1-0.05 nM (the volume of compound addition can be adjusted to the preferred and agreed screening concentration). The final DMSO concentration was 0.1% (ideally 0.2% to limit toxicity to parasites), but this is dependent on volume of compound DMSO stock solution that can be supplied. Plates were incubated at 37 C. for 72 h in an atmosphere of 5% CO.sub.2, 5% O.sub.2, 95% N.sub.2.

[0264] Evaluation of parasite growth measuring LDH activity: After 72 h of incubation, plates were frozen at 80 C. overnight and then thawed at room temperature for at least 4 h. To evaluate LDH activity, 45 L of freshly made reaction mix (174 mM sodium L-lactate (Sigma Aldrich), 214 M 3-acetyl pyridine adenine dinucleotide (APAD) (Sigma Aldrich), 270 M nitro blue tetrazolium chloride (NBT) (Sigma Aldrich), 4.35 U/mL diaphorase (from Clostridium kluyveri) (Sigma Aldrich), 0.7% Tween 20, 100 mM Tris-HCl pH 7.5) was dispensed using a Multidrop dispenser (Thermo Scientific). Plates were shaken to ensure mixing and absorbance at 650 nm was monitored using a Perkin Elmer Envision plate reader after 30 min of incubation at room temperature. Data were normalized to percent growth inhibition using positive and negative controls, and analysed using TIBCO Spotfire software.

[0265] Counterscreen: A buffered solution of 30 L Bovine LDH (12.5 U/ml) (Sigma Aldrich) was dispensed into compound ready plates. The same protocol then was undertaken for measuring the LDH activity using parasites.

Assay 2

[0266] The assay described in Gamo et al. is as follows:

[0267] P. falciparum strains 3D7 and Dd2 used in this study were obtained from the Malaria Research and Reference Reagent Resource Center (MR4). Parasite strains were cultured using standard procedures as described (Trager, W. & Jensen, J. B. Science 193, 673-675 (1976)). An inoculum of parasitized red blood cells (PRBC) at 0.25% parasitaemia and 2% haematocrit in RPMI-1640, 5% AlbuMAX, 2% D-sucrose, 0.3% glutamine and 150 hypoxanthine was used for the assay.

[0268] Assay plates were prepared by dispensing 0.05 l of compound from master plates at 1 mM in each well. Final assay volume was 25 l and final compound concentration was 2 M. The sixth column was the positive growth control and had 0.05 l of DMSO. The eighteenth column had 0.05 l of a mixture of 50 M chloroquine and 50 M artemisinin stock solutions as negative growth control. The parasite inoculum (25 l) was dispensed into plates containing compounds using a Multidrop Combi dispenser (Thermo Scientific). Plates were shaken for 10 s to ensure mixing and then incubated at 37 C. for 72 hours in an atmosphere of 5% CO.sub.2, 5% O.sub.2, 95% N.sub.2.

Evaluation of Parasite Growth Using Lactate Dehydrogenase (LDH) Activity

[0269] After 72 hours of incubation, plates were frozen at 70 C. overnight and then thawed at room temperature for at least 4 hours. To evaluate LDH activity, 70 l of freshly made reaction mix (143 mM sodium 1-lactate, 143 M 3-acetyl pyridine adenine dinucleotide (APAD), 178.75 M Nitro Blue tetrazolium chloride (NBT), 286 g ml.sup.1 diaphorase (2.83 U ml.sup.'), 0.7% Tween 20, 100 mM Tris-HCl pH 8.0) was dispensed using a Multidrop Combi dispenser. Plates were shaken to ensure mixing, and absorbance at 650 nm was monitored in a plate reader after 10 min of incubation at room temperature. Data were normalized to percent growth inhibition using positive and negative controls and the equation:

[00001]$\mathrm{Percentage}.Math..Math.\mathrm{inhibition}.Math..Math.\mathrm{growth}=\left[1-\left(\frac{A_{\mathrm{well}}-A_{\mathrm{neg}}}{A_{\mathrm{pos}}-A_{\mathrm{neg}}}\right)\right]100$

where A.sub.well is the absorbance measured in the well, and A.sub.pos and A.sub.neg are the average absorbances measured for the positive and negative controls, respectively. This method is a modification of existing ones (Makler et al., Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am. J. Trop. Med. Hyg. 48: 205-210 (1993)) that requires only a single pipetting step after compound incubation and gave a signal to noise ratio of 10 under the conditions chosen. The approach allowed kinetic and end-point readouts and produced a Z quality factor (Zhang et al., J. Biomol. Screen. 4: 67-73 (1999)) higher than 0.7 in validation assays (Supplementary FIG. 2, Gamo et al., Nature 465:305-312 (2010)). Potencies of standard antimalarial agents in this assay were comparable to those determined by the current gold-standard, 96-well, hypoxanthine incorporation assay (Desjardins et al. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16: 710-718 (1979)) (Supplementary Table 3, Gamo et al., 2010, supra).

[0270] At this level of miniaturization, integrity of erythrocytes and LDH activity can be inspected visually, allowing for rapid detection of dispensing errors, interference by colored compounds, or haemolysis, making the method very useful for low technology settings (Supplementary FIG. 3, Gamo et al., 2010, supra). Proliferation of asynchronous parasites was measured after 72 h of incubation in the presence of 2 M compound. We chose a 72 hour incubation time to ensure all parasites traversed at least once through each stage of the cell cycle and to increase the chances of identifying slow acting and delayed death phenotype inhibitors (Goodman et al., The effects of anti-bacterials on the malaria parasite Plasmodium falciparum Mol. Biochem. Parasitol. 152, 181-191 (2007); Ramya et al., A. Inhibitors of nonhousekeeping functions of the apicoplast defy delayed death in Plasmodium falciparum. Antimicrob. Agents Chemother. 51, 307-316 (2007)).

[0271] Given the large number of positives, it was necessary to estimate the concentrations producing 50% inhibition using the LDH assay above and generating dose response curves with fivefold dilution steps down to 3 nM compound in an interplate design, instead of using the hypoxanthine incorporation assay with two-fold dilution intraplate series generally considered the standard method to calculate IC.sub.50 for antimalarials (Fidock et al., Antimalarial drug discovery: Efficacy models for compound screening. Nature Rev. Drug Discov. 3, 509-520 (2004)). The lowest concentration tested was 3 nM. Agreement between the two methods was found to be within the expected limits with standard antimalarials (Supplementary Table 3, Gamo et al., 2010, supra). To eliminate the possibility of retaining inhibitors of the biochemical readout system, one set of the primary hits was assayed against parasite LDH activity under identical screening conditions.

Preparation of Extracts to Evaluate Direct LDH Inhibition by Hit Compounds

[0272] P. falciparum 3D7 strain was grown as described in Assay 1, at 37 C. for 72 hours. The culture was frozen at 80 C. overnight. Cultures were thawed at room temperature for at least 4 hours and the reaction mixture described in Assay 1 was made in order to measure the possible direct inhibition of LDH by the following compounds, assayed as above in Assay 1.

TABLE-US-00001 Compound P. falciparum LDH No. Structure IC.sub.50 Value (M) 1 [00042] 0.0045 2 [00043] 0.0058 3 [00044] 0.0096 4 [00045] 0.0097 5 [00046] 0.0212 6 [00047] 0.0218 7 [00048] 0.0251 8 [00049] 0.0566 9 [00050] 0.0866 10 [00051] 0.1203 11 [00052] 0.1720 12 [00053] 0.2207 13 [00054] 0.2234 14 [00055] 0.2248 15 [00056] 0.2394 16 [00057] 0.2396 17 [00058] 0.3100 18 [00059] 0.3128 20 [00060] 0.3556 [00061]