Compounds for use as inhibitors of alternative oxidase or cytochrome bc1 complex

Abstract

The invention provides compounds for use in inhibiting a microbial alternative oxidase (AOX) and/or cytochrome bc.sub.1 complex. The invention extends to the use of such inhibitors in agrochemicals and in pharmaceuticals, for treating microbial infections, including fungal infections.

Claims

1. A method of treating a fungal infection in a subject, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of the compound of formula I: ##STR00004## wherein R.sub.1 is a group selected from: CHO; CN; C(O)NH.sub.2; C(O)NHCH.sub.3; C(O)CH.sub.3; COOH; and COOCH.sub.3; R.sub.2 is hydrogen, hydroxyl or an alkoxy group with 1 to 3 C atoms; R.sub.3 is a straight chain or branched alkyl or alkylene with 4 to 20 C atoms, that is optionally mono- or polysubstituted by a C.sub.1 to C.sub.4 alkyl group; R.sub.4 is a hydroxyl group; R.sub.5 is a halogen group; and R.sub.6 is H or a C.sub.1 to C.sub.4 alkyl group, wherein the fungal infection is caused by a fungus comprising an alternative oxidase (AOX).

2. The method according to claim 1, wherein R.sub.2 is a hydroxyl group.

3. The method according to claim 1, wherein R.sub.3 is a straight chain or branched alkyl or alkylene with 6 to 15 C atoms, 8 to 12 C atoms or 8 to 10 C atoms, that is optionally mono- or polysubstituted by a C1 to C4 alkyl group.

4. The method according to claim 1, wherein R.sub.3 is a diene having 6 to 15 C atoms that is substituted with at least one methyl group.

5. The method according to claim 1, wherein R.sub.5 is a chlorine, bromine, fluorine or iodine group.

6. The method according claim 1, wherein R.sub.5 is a chlorine group; or wherein R6 is a methyl, ethyl or propyl group.

7. The method according claim 1, wherein: R.sub.1 is selected from CHO; CN; C(O)NH.sub.2; C(O)NHCH.sub.3; C(O)CH.sub.3; COOH; and COOCH.sub.3; R.sub.2 is a hydroxyl group; R.sub.3 is a straight chain or branched alkyl or alkylene with 4 to 20 C atoms, that is optionally mono- or polysubstituted by a C.sub.1 to C.sub.4 alkyl group; R.sub.4 is a hydroxyl group; R.sub.5 is a chlorine atom; and R.sub.6 is H or a C.sub.1 to C.sub.4 alkyl group.

8. The method according to claim 1, wherein: R.sub.1 is selected from CHO; CN; C(O)NH.sub.2; C(O)NHCH.sub.3; C(O)CH.sub.3; COOH; and COOCH.sub.3; R.sub.2 is a hydroxyl group; R.sub.3 is a straight chain or branched alkyl or alkylene with 6 to 15 C atoms, that is optionally mono- or polysubstituted by a C.sub.1 to C.sub.2 alkyl group; R.sub.4 is a hydroxyl group; R.sub.5 is a chlorine atom; and R.sub.6 is H or a C.sub.1 to C.sub.4 alkyl group.

9. The method according to claim 1, wherein: R.sub.1 is selected from CHO; CN; C(O)NH.sub.2; C(O)NHCH.sub.3; C(O)CH.sub.3; COOH; and COOCH.sub.3; R.sub.2 is a hydroxyl group; R.sub.3 is an alkylene chain having 8 to 10 C atoms, and is substituted with at least one methyl group; R.sub.4 is a hydroxyl group; R.sub.5 is a chlorine atom; and R.sub.6 is a methyl group.

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

11. The method of claim 1, wherein the fungus is selected from a group of genera consisting of Aspergillus; Blumeria; Candida; Cryptococcus; Encephalitozoon; Fusarium; Leptosphaeria; Magnaporthe; Phytophthora; Plasmopara; Pneumocystis; Pyricularia; Pythium; Puccinia; Rhizoctonia; Trichophyton; and Ustilago.

12. The method of claim 11, wherein the fungus is selected from a group of species consisting of Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus parasiticus; Aspergillus terreus; Blumeria graminis; Candida albicans; Candida cruzei; Candida glabrata; Candida parapsilosis; Candida tropicalis; Cryptococcus neoformans; Encephalitozoon cuniculi; Fusarium solani; Leptosphaerianodorum; Magnaporthe grisea; Phytophthora capsici; Phytophthora infestans; Plasmopara viticola; Pneumocystis jiroveci; Puccinia coronata; Puccinia graminis; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Trichophytoninterdigitale; Trichophyton rubrum; and Ustilago maydis.

13. The method of claim 1, wherein the fungus is Saccharomyces spp., S. cerevisiae; Candida spp., or C. albicans.

Description

(1) Embodiments of the invention will now be further described, by way of example only, with reference to the following Examples, and to the accompanying diagrammatic drawings, in which:

(2) FIG. 1 is a schematic drawing showing the mitochondrial respiratory chain, in which I-IV: Respiratory chain complexes; V: ATP synthase; Q: Ubiquinone; N.sub.ext/int: NADH dehydrogenases; Stb: Strobilurin site of action; SHAM: salicylhydroxamic acid site of action; CN/CO: Cyanide/Carbon monoxide inhibition site; AO: Alternative oxidase; IMS/IM/M: Inter-membrane space/inner membrane/matrix of mitochondria;

(3) FIG. 2 shows a crystal structure of an alternative oxidase (AOX) protein from Trypanosoma brucei in the presence of a stoichiometric inhibitor;

(4) FIG. 3 is a schematic figure showing the hydrophobic pocket and hydrogen-bonding of the inhibitor to the di-iron site of the alternative oxidase (AOX) using Sauromatum numbering;

(5) FIGS. 4A-C are a sequence alignment of various alternative oxidases (AOX) of various species showing a consensus sequence. AXIB-ARATH is the AOX enzyme from Arabidposis thaliana (SEQ ID No:2), AOX1_SOYBN is the AOX enzyme from soybean (SEQ ID No:3), AOX1_TOBAC is the AOX enzyme from tobacco (SEQ ID No:4), AOX1A_ORYS is the AOX enzyme from Oryza sativa-rice (SEQ ID No:5), AOX1_SAUGU is the AOX enzyme from Sauromatum guttatum (SEQ ID No:6), AOX_CATRO is the AOX enzyme from Catharanthus roseus (SEQ ID No:7), AOX1_MANIN is the AOX enzyme from Mangifera indica (SEQ ID No:8), AOX_ZEAMA is the AOX enzyme from Maize (SEQ ID No:9), AOX1_CHLRE is the AOX enzyme from Chlamydomonas reinhardtii (SEQ ID No:10), AOX_NEUCR is the AOX enzyme from Neuropsora crassa (SEQ ID No:11), AOX_HANAN is the AOX enzyme from Hansenula anomola (SEQ ID No:12), AOX_TRYBB is the AOX enzyme from Trypanosoma brucei (SEQ ID No:13), and AOX_CHLSP is the AOX enzyme from Chlamydomonas species (SEQ ID No:14);

(6) FIG. 5 represents the chemical formula of Ascofuranone (left-hand side) and various embodiments of the AOX inhibitor according to the invention;

(7) FIG. 6 represents the chemical formula of Colletochlorin B;

(8) FIG. 7 shows the chemical synthesis of Colletochlorin B;

(9) FIG. 8 is a graph showing IC.sub.50 values for Colletochlorin B; and

(10) FIG. 9 is a graph showing the effects of Colletochlorin B on cytochrome bc.sub.1 activity.

EXAMPLES

(11) Materials & Methods

(12) The alternative oxidase (AOX) protein was purified and crystallized according to the techniques outlined in Kido, Y. et al (2010) Biochim. Biophys. Acta 1797, 443-450, and in Kido, Y. et al (2010) Acta. Crystallogr. Sect. F Struct. Biol, Cryst, Commun., 66, 275-278. The crystal structure of the protein was obtained both in the presence and absence of an inhibitor using the vapour hanging-drop technique, as outlined in the papers given above. The inhibitor-binding site was identified from the crystal structure. Analysis of the residues surrounding the pocket revealed that L177, E178, L267, A271 and Y275 shown in FIG. 3 are 100% conserved across all fungal, plant and trypanosomatid species.

Example 1—Characterisation of the Alternative Oxidase (AOX) Binding Pocket

(13) A major breakthrough in this study was the determination of the first ever crystal structure of an alternative oxidase (AOX) protein both in the presence and absence of a stoichiometric inhibitor, as illustrated in FIG. 2. The inventors found that the pocket does not change, i.e. it is substantially the same in the presence or absence of the inhibitor. Knowledge of the crystal structure of AOX in the presence of an inhibitor put the inventors in a very powerful position to undertake some rational fungicidal molecular design, which, as discussed below, has resulted in the production of a library of AOX inhibitor compounds that have the capacity to act as phytopathogenic fungicides specifically targeted at the AOX.

(14) Accordingly, once the inventors had generated the crystal structure of AOX shown in FIG. 2, they went on to characterise the AOX quinone-binding pocket in detail using site-directed mutagenesis. Referring to FIG. 3, there is shown the fully characterised quinone-binding site or pocket of the alternative oxidase enzyme (AOX) of the plant, Sauromatum guttatum (Voodoo Lily). The Figure also shows a representative inhibitor (Colletochlorin B) positioned inside the pocket. The six-membered ring of the inhibitor tightly interacts with the hydrophobic residues of the pocket, and the isoprenyl tail of the inhibitor interacts with Arg173, Glu270 and Ser274 residues of the pocket. FIG. 3 also shows that the inhibitor binding pocket (R173, L177, E178, L267, E270, A271, S274 & Y275) is located near the membrane surface, and is within 4 Å of the active-site of the protein. It should be noted that the numbering on FIG. 3 refers to the plant (i.e. Sauromatum guttatum) AOX protein, as all AOXs tend to be compared with this protein. The head group aldehyde oxygen is hydrogen-bonded by Glu178 and Tyr275. Although not wishing to be bound by theory, these hydrogen bonds are believed to be important for the potent inhibitory activity of these compounds.

(15) As discussed in the Examples below, the inventors have confirmed that the quinone-binding site of the AOX shown in FIGS. 4A-C is a promising target in the treatment of fungal pathogens. The inventors have prepared a sequence alignment of a number of AOX enzymes, which is shown in FIGS. 4A-C, and it can be seen that the architecture of the AOX binding-site is highly conserved across all AOXs, irrespective of the species from which they are derived. The boxed residues in FIGS. 4A-C represent AOX residues which are involved in inhibitor binding. The Arg173, Glu270 and Ser274 residues shown in FIG. 3 are less conserved. However, as these residues are only involved in the binding of the tail, variation is believed to be less significant with respect to inhibitor sensitivity.

(16) Thus, the detailed knowledge of the nature of the binding site of the AOX of S. guttatum is important, as it has revealed that there is a common architecture that can be applied to quinol-binding sites in general, and hence provides further insight into the mechanism of binding. More importantly, this information has assisted in the rational design of phytopathogenic fungicides and human parasites that are specifically targeted to the alternative oxidase.

(17) Based on this information, the inventors set out to design and synthesize a new library of AOX inhibitors, and also to gain further detailed structural knowledge of the nature of the protein-ligand interaction and kinetics. They also tested the extent to which structurally modified inhibitors targeted at the AOX could also inhibit the fungal Qo site, thereby providing a new generation of dual-mode fungicides.

Example 2—Design and Synthesis of AOX Inhibitors

(18) The inventors have designed and synthesised a number of AOX inhibitors based on the compound, ascofuranone, the chemical structures of which are illustrated in FIG. 5. Ascofuranone has a complex synthetic route, and has a reactive aldehyde group (—CHO). Several ascofuranone derivatives were synthesised, namely Colletochlorin B (labelled structure “2” in FIG. 5, where R is CHO), compound “3” shown in FIG. 5, where R is CH.sub.2OH, and 4a-4h, where R is as shown in FIG. 5.

(19) The inhibitory effects of some of these compounds were assessed, and the results are summarised in Table 1.

(20) TABLE-US-00002 TABLE 1 Inhibitory effects on recombinant AOX protein Inhibitor IC50 Ascofuranone  58 pM Colletochlorin B 165 pM Octyl Gallate 105 nM Salicylhydroxamic acid (SHAM)  7 μM

(21) Table 1 summarises the concentration of inhibitor required to reduce the respiration of purified recombinant AOX protein by 50%. Respiration was measured as the rate of oxygen consumption in the presence of 1 mM NADH as substrate and the numbers represent the final I.sub.50 concentration of the inhibitor.

(22) Of the derivatives that were synthesized, Colletochlorin B was one of the most promising candidates, because it has an IC.sub.50 value of approx 165 pM (IC.sub.50 for Ascofuranone 58 pM) when tested upon recombinant AOX proteins. This inhibitor was specific for membrane-bound and purified AOX, and did not appear to inhibit other quinol oxidases. Furthermore, Colletochlorin B can also be synthesized by a simple two-step process, which is a significant advantage over ascofuranone.

Example 3—Synthesis of Colletochlorin B

(23) The chemical structure of Colletochlorin B is shown in FIG. 6, and the method used for its synthesis is shown in FIG. 7.

(24) Step 1: Compound (1) to Compound (2)

(25) Orcinol (5 g, 40 mmol) and Zn(CN).sub.2 (7.1 g, 60 mmol) were placed into a 3 necked flask with mechanical stirrer under N.sub.2. 50 ml of Ether was added, and the reaction was saturated with HCl gas. After 2 hours, the Ether was decanted off and 50 mls of water added to the reaction mixture. This was heated to 100° C. where the product crashed out of solution. The crude product was collected via buchner filtration, and recrystallised from water to yield the aldehyde (4.6 g) in 76% yield.

(26) Step 2: Compound (2) to Compound (3)

(27) Orcinol Aldehyde (527 mg, 3.5 mmol) was put under N.sub.2 and dissolved in anhydrous ether (60 ml) on an ice bath. SO.sub.2Cl.sub.2 (1.35 ml, 4.7 mmol) was diluted in ether (15 ml) and then added dropwise over 15 minutes. The reaction was left to stir overnight, and quenched with the addition of water. The Ether layer was washed with 0.1M NaHCO.sub.3 and water, then dried over MgSO.sub.4 and concentrated under vacuum. The crude solid was then purified via flash chromatography (Toluene: Ethyl acetate 2:1.fwdarw.1:1) to obtain the product (459 mg) in 75% yield.

(28) Step 3: Compound (3) to Compound (4)

(29) 3-chloro-4,6-dihydroxy-2-methyl-benzaldehyde (150 mg, 0.8 mmol) was dissolved in 10% KOH (0.9 ml, 0.8 mmol) yielding a deep red solution. The reaction was placed on an ice bath, and geranyl bromide (0.39 ml, 1.6 mmol) was added. The reaction was stirred vigorously overnight, and extracted with ether. The organic layer was washed with NaHCO.sub.3 and brine, before being concentrated under vacuum. The resultant oil was purified via flash chromatography (petrol ether 40-60: ether 10:1.fwdarw.3:1) to obtain pure Colletochlorin B (52 mg) in 20% yield.

Example 4—Characterisation of Colletochlorin B

(30) The inventors have confirmed that whilst the hydroxyl groups and the chlorine and methyl substituents on the benzene ring of the inhibitor are believed to be important for high potency, the furanone moiety is redundant as long as a hydrophobic side chain, such as the geranyl group, is retained, as in Colletochlorin B (2).

(31) However, the aldehyde group present in ascofuranone (1) and Colletochlorin (2) is believed to represent a problem for anti-parasitic design for several reasons. Besides their ability to function as hydrogen bond acceptor and to undergo dipole-dipole interaction with AOX, aldehyde groups are chemically reactive enough to undergo reversible covalent modifications and would be generally unsuited to standard pharmaceutical formulations. Furthermore, aldehydes are prone to metabolic oxidation to the respective carboxylic acid with the concomitant non-specific binding to basic transport proteins. Therefore, the inventors set out to remove this aldehyde group using the reducing agent NaBH.sub.4, as shown in FIG. 5. Synthesis and analysis of an alcohol-derivative (3) revealed that its inhibitory properties are retained, which is why the various aldehyde bioisosteres, which are represented as compounds 4a-4h in FIG. 5, were produced.

Example 5—Site-Directed Mutagenesis Studies

(32) The inventors have generated mutants of E123 and Y220, and have demonstrated that they are important for enzyme activity and inhibitor-binding.

(33) Site-Directed Mutagenesis and Plasmid Construction

(34) Construction of pREP1-AOX, pREP1-E123A and pREP1-Y220F (used to express wild type AOX and the E123A and Y220F mutants in S. pombe) has been described previously [M. S. Albury, C. Affourtit, P. G. Crichton, A. L. Moore, Structure of the plant alternative oxidase—Site-directed mutagenesis provides new information on the active site and membrane topology, J. Biol. Chem. 277 (2002) 1190-1194: M. S. Albury, P. Dudley, F. Z. Watts, A. L. Moore, Targeting the plant alternative oxidase protein to Schizosaccharomyces pombe mitochondria confers cyanide-insensitive respiration, J. Biol. Chem. 271 (1996) 17062-17066.]. Mutagenesis of AOX was performed using the Quick Change mutagenesis kit (Stratagene) according to manufacturer's instructions, with plasmid pSLM-AOR [M. S. Albury, C. Affourtit, P. G. Crichton, A. L. Moore, Structure of the plant alternative oxidase—Site-directed mutagenesis provides new information on the active site and membrane topology, J. Biol. Chem. 277 (2002) 1190-1194]. Each full length mutant AOX was excised on a BspHI-BamHI fragment and ligated to the yeast expression vector pREP1/N (a modified version of pREP1 [K. Maundrell, Nmt1 of fission yeast—a highly transcribed gene completely repressed by thiamine, J. Biol. Chem. 265 (1990) 10857-10864.] in which the NdeI site was replaced with NcoI) which had been digested with NcoI and BamHI, yielding pREP1-E123A and pREP1-Y220F.

(35) Results

(36) TABLE-US-00003 TABLE 2 Activity (nmol % Inhibition oxygen/min/mg (compared Condition protein to wt) pREP1-AOX—wt 55 0 pREP1-E123A—E123 mutant 2 96 pREP1-Y220F—Y220 mutant 0 100

(37) Activity was measured as oxygen consumed/min/mg protein using NADH as substrate when isolated yeast mitochondria (Schizosaccharomyces pombe) containing the wild-type and mutant form of the AOX. Note that the inhibitor does not bind to the mutant forms of the oxidase.

Example 6—Effect of Colletochlorin B on Cytochrome bc.SUB.1 .Respiratory Activity

(38) Colletochlorin B and its derivatives have been demonstrated in Examples 1-5 to be a specific inhibitor of the alternative oxidase (AOX) in plants and fungi. Following on from this work, the inventors set out to test whether or not these compounds also have any effect on the respiratory activity of cytochrome bc.sub.1 complex. Mitochondria from two sources (rat liver and potato) were titrated with Colletochlorin B (CB), as indicated in typical data summarised in FIG. 9. Both mitochondrial sources did not contain any alternative oxidase (AOX), and so the respiratory activity measured must have been from the cytochrome bc.sub.1 complex.

(39) Respiratory activity was measured in a medium containing 0.3M mannitol, 10 mM KCl, 5 mM MgCl.sub.2, 1 mM potassium phosphate and 10 mM MOPS (3-(N-morpholino)propanesulfonic acid) buffer pH7.4. Either 0.9 mg of rat liver mitochondria respiring on 5 mM succinate or 0.3 mg potato mitochondria respiring on 1 mM NADH were used. Respiration was measured using a Rank oxygen electrode of 0.4 ml volume at 25° C. in the presence of 1 μM CCCP (Carbonyl cyanide m-chloromethoxy phenylhydrazone). The results were compared with ascochlorin (a known bc.sub.1 inhibitor) and azoxystrobin (a commercial fungicide targeted at the bc.sub.1 complex).

(40) Results

(41) TABLE-US-00004 TABLE 3 IC.sub.50 (Rat liver mitochondria) Compound IC.sub.50/nM Ascochlorin 187 Colletochlorin B 515 Azoxystrobin 525

(42) TABLE-US-00005 TABLE 4 IC.sub.50 (Potato mitochondria) Compound IC.sub.50/μM Ascochlorin 0.5 Colletochlorin B 0.75 Azoxystrobin 1.4 Ascofuranone 30

(43) Tables 3 and 4 summarise the concentration of inhibitor that was required to reduce the respiration of cytochrome bc.sub.1 complex (in the absence of AOX) by 50%. Respiration was measured as the rate of oxygen consumption in the presence of 1 mM NADH or 5 mM succinate as substrate, and the numbers represent the final IC.sub.50 concentration of the inhibitor tested. The lower the IC.sub.50 value the better, since it means that a lower amount of the compound is needed to halve the respiratory activity.

(44) Of the inhibitors that were synthesized, Colletochlorin B was a promising candidate, because it has a lower IC.sub.50 value (approx 515 nM) than Azoxystrobin (approx 525 nM) when tested on rat liver mitochondria. When tested on potato mitochondria, the IC.sub.50 value of Colletochlorin B was only 0.75 μM, which was half of the IC.sub.50 of Azoxystrobin (approx 1.4 μM), and significantly less than the IC.sub.50 of Ascofuranone (approx 30 μM).

CONCLUSIONS

(45) Careful titration using mitochondria in which the alternative oxidase is absent has surprisingly revealed that the compound is a specific inhibitor of the cytochrome bc.sub.1 complex in addition to inhibiting AOX. The data suggest that the compound inhibits the cytochrome bc.sub.1 complex at both the Qo and Qi binding-sites of this complex thereby making it a very potent inhibitor of respiration even in the absence of the alternative oxidase. The implications of such a finding suggest that derivatives of this compound would be very specific and potent dual function fungicide, as not only do they inhibit the alternative oxidase (AOX), but also the cytochrome bc.sub.1 complex.

(46) It will be appreciated that commercially available fungicides, such as azoxystrobin, against which Colletochlorin B has been tested herein, inhibit only one site (qo) within the bc1 complex. Accordingly, since Colletochlorin B inhibits the cytochrome bc.sub.1 complex at both the Qo and Qi binding-sites, and also the AOX, this compound and its derivatives can act as a highly potent and robust inhibitor.