Antifungal compounds

Abstract

The invention relates to compounds for use in the treatment or prevention of a fungal infection in a subject.

Description

[0001] ANTIFUNGAL COMPOUNDS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the treatment of fungal infections on animals and on plants.

[0004] BACKGROUND OF THE INVENTION

[0005] Pathogenic fungi pose a profound threat to human health. It is estimated that, worldwide, over one billion people suffer from a fungal infection. Superficial infections such as dermal infections, vaginal yeast infections and oral thrush are very common, for example, vaginal candidiasis alone affects 75 percent of women at some point in their lives. These kinds of local infections cause great discomfort and distress to the patient. However, when the pathogen reaches the bloodstream and deeper tissues, this can result in an invasive infection associated with severe disease and potentially death. Once an infection becomes invasive, the prospect of survival for the patient rarely exceeds 50%. This results in as much as 1.7 million deaths per year. The most frequently isolated species in fungal infections are Candida, Aspergillus, Cryptococcus and Pneumocystis.

[0006] Candida species are the most common cause of fungal invasive infections and the fourth- and sixth-most common cause of bloodstream-associated infections in the United States and Europe, respectively. Several Candida species are opportunistic commensals, becoming pathogenic when the host immune system is compromised. Candida albicans is isolated most commonly among all Candida species and was recently classified by the World Health Organization as a critical pathogen that is a concern to public health and requires immediate attention.

[0007] Non-albicans Candida species, such as C. glabrata and C. auris are vastly increasing in occurrence. These latter species pose a serious threat to general health since both are inherently (multidrug-) resistant to some of the limited available antifungal drugs. C. auris was first described in 2009 and has been classified by the Centres for Disease Control and Prevention as one of the most urgent threats of current medicine. Later it was also classified by the World Health Organization as a critical pathogen. Apart from its multidrug-resistant character, it also spreads easily between patients leading to hospital outbreaks. The limited availability of antifungal therapies and the increasing onset of resistance against these drugs hamper the efficient eradication of infections and treatment of patients.

[0008] Alarmingly, fungal infections are on the rise due to the increase in susceptible patients. Ironically, this is mostly due to the progress made in medicine. The increased use of broad-spectrum antibiotics, immunosuppressive drugs used to treat cancer patients and organ recipients, and the increasing numbers of people with a comprised immune system (AIDS, diabetes, SCID,...) lead to a growing population of susceptible patients.

[0009] Additionally, the routine usage of medical devices such as catheters, pacemakers and prosthetic joints increases the risk of introducing the pathogen directly into the body as Candida cells attach to these abiotic surfaces. Furthermore, the prophylactic and long-term use of antifungal drugs have promoted the manifestation of (multi)drug- resistant fungi.

[0010] A major problem concerning fungal infections is the limited availability of antifungal therapies. There are only three major classes of antifungal drugs that can be used to treat systemic infections: azoles, polyenes and echinocandins. Alarmingly, 90 percent of C. auris isolates are resistant to at least one class and 30 percent is resistant to at least two antifungal drug classes. The limited amount of drugs in combination with the rising resistance highlights the urgent need for new antifungal drugs.

[0011] Very few new antifungal drugs have reached the clinical phase over the last decades of which near to none represent drugs with a novel mode of action. This indicates a need for alternative drug targets. A main hurdle when exploring metabolic pathways as potential drug targets is the high level of conservation between the fungal actors and the human ones, as both are eukaryotes. Drugs targeting conserved processes generally exhibit significant side effects.

[0012] Other frequent problems surrounding antifungal drug development are: the lack of cell permeability and cellular bioavailability of the inhibiting compound. Most often, potent inhibitors are too polar and hydrophilic to pass the cell membrane. Furthermore, some compounds work fungistatic rather than fungicidal or give significant off-target effects.

[0013] Furthermore, there is a lack of discovery programs devoted to the development of new therapeutics as pharmaceutical companies expect limited financial return. SUMMARY OF THE INVENTION

[0014] The present invention relates to a group of compounds that have antifungal activity against Candida species.

[0015] The group of compounds surpass the primary challenges associated with antifungal drug development.

[0016] Firstly, they exhibit minimal to no toxicity toward mammalian cell lines. Host toxicity is a big problem in antifungal drug development due to the conservation of core processes and pathways between fungi and humans as both are eukaryotes. In many cases, the targets of antifungal drugs are not sufficiently selective, leading to undesirable side effects in humans. However, these compounds demonstrate improved selectivity and reduced toxicity toward host cells.

[0017] Secondly, they can effectively penetrate the fungal cell wall and membrane. Wholecell bioactivity is crucial, as the ability to reach the target site within the fungal cells is often a limiting factor for the efficacy of antifungal compounds.

[0018] Thirdly, some of these compounds have demonstrated efficacy against a range of fungal species including Candida auris, Candida giabrata, Candida krusei and Candida tropica lis.

[0019] A further aspect of the invention is the identification of antifungal compounds. In a first step compounds are screened for inhibition of riboflavin synthesis. In a second step such compounds are tested for their antifungal activity.

[0020] The alleged lack of cytotoxicity can be confirmed by testing toxicity on human cells.

[0021] The invention is further summarised in the following statements:

[0022] 1. A composition comprising a compound selected from a group of compounds consisting of (6,7-dimethoxyquinolin-3-yl)-(2-hydroxyphenyl)methanone (1J), N- [3-( 1,2,3, 4-tetrahydrona phthalen- 1-yloxy) propyl] tetrazolof 1,5- b]pyridazin-6-amine (ID), N-(l,3-dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl) benzamide (1A), N-(l,3-dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl)-4- fluorobenzamide (4A), N-[(2-methyl-lH-indol-3-yl)-pyridin-2-ylmethyl]pyridin-2-amine (II), and 3-[[5-[(4-chlorophenoxy)methyl]-l,3,4-oxadiazol-2-yl]sulfanyl]pyrazine-2- carbonitrile (IF), for use in the treatment or prevention of a fungal infection in a subject.

[0023] 2. The composition for use according to statement 1, wherein the compound is IJ or ID

[0024] 3. The composition for use according to statement 1 or 2, wherein said composition further comprises an antibacterial agent.

[0025] 4. The composition for use according to any one of statements 1 to 3, wherein the subject is a human.

[0026] 5. The composition for use according to any one of statements 1 to 4, wherein the fungal infection is a Candida sp infection.

[0027] 6. The composition for use according to any one of statements 1 to 5, wherein the fungal infection is an infection of Candida auris, Candida giabrata, Candida krusei or Candida tropica lis.

[0028] 7. The composition for use according to any one of statements 1 to 6, wherein the fungal infection is of a fluconazole and / or echinocandin-resistant fungus.

[0029] 8. A compound selected from the group consisting of 1J, II, IF and ID for the treatment of agricultural fungal infections.

[0030] 9. A compound selected from the group consisting of 1A, 4A, 1J, IF and ID, for use as a medicament.

[0031] FIGURES

[0032] Figure 1A. The MICso value based on the dose-response curve.

[0033] Figure IB. Cidality assay for compounds 4A, IF, II .

[0034] Figure 2A. Potency of IF and its commercial analogues was tested against C. albicans SC5314.

[0035] Figure 2B. Potency of II and its commercial analogues was tested against C. albicans SC5314.

[0036] Figure 2C. MICso values based on the dose-response curves for II and its analogues Figure 3A. The potency of the compounds against several species was investigated via a dose-response and cidality assay.

[0037] Figure 3B. The cidality of compounds that seemed fungicidal after incubation with cells was investigated via a cidality assay.

[0038] Figure 4. Susceptibility of several yeast and bacterial species to 50 pM compounds. Figure 5. Cytotoxicity of compounds (A) and concentration (B) against HeLa cells. DETAILED DESCRIPTION

[0039] The present invention relates to a group of compounds that can be used for antifungal therapy to treat fungal infections in humans.

[0040] According to an embodiment, one or more compounds may also be used in combination with antibacterial agents.

[0041] The group of compounds of the present invention may be used for medical purposes, particularly against Candida sp.

[0042] In another embodiment, the group of compounds of the present invention may be used as a medicament.

[0043] In another embodiment, a composition comprising one or more compounds may be used.

[0044] According to another embodiment, one or more compounds may also be used for agricultural purposes.

[0045] According to another embodiments one or more compounds may be used to treat agricultural fungal infections. In certain embodiments, one or more compounds may be used for topical applications to treat fungal infections in plants and / or animals such as cattle.

[0046] A high-throughput screening (HTS) was performed on a drug library wherein druglike structural diversity set provided by Enamine was used to identify hits and validate them for their antifungal potential.

[0047] The HTS was performed to identify compounds with antifungal activity against Candida species, such as C. albicans.

[0048] This drug library consisted of 20.000 compounds selected on their physicochemical properties to increase membrane permeability, solubility and enhanced bioavailability. In the HTS, 50 pM of compound was tested for growth inhibition of C. albicans SC5314 via an absorbance and fluorescence read-out.

[0049] The group of compounds of the present invention are a result of the HTS.

[0050] Herein riboflavin biosynthesis is a target for its mode of action, preferably against Candida species.

[0051] Riboflavin is essential for all living cells. In humans, the pathway for riboflavin synthesis is absent. As a result, humans generally get their riboflavin or vitamin B2 riboflavin intake through food consumption. Unlike humans, fungi like C. albicans synthesise riboflavin themselves. Therefore, by targeting this essential pathway, host toxicity is limited.

[0052] Compounds of the present invention may be selected from the following group of compounds that show antifungal activity (z numbers are PubChem substance numbers).

[0053]

[0054] Antifungal activity of the compounds was tested using broth dilution assays performed in in RPMI-MOPS containing 2% glucose to mimic the human plasma and growth was measure via absorbance at 600nm after 24hours at 37oC unless stated otherwise.

[0055] The antifungal activity of one or more compounds was investigated against C. albicans SC5314 via a dose-response assay. EXAMPLES

[0056] Example 1 Potency of the compounds

[0057] The antifungal activity of the hit compounds against C. albicans SC5314 was investigated via a dose-response assay. 1A, 4A, ID, IF, II and 11 inhibited growth of C. albicans. Compound 11 showed a rather surprising dose-response relationship, since the relative growth at 128 and 64 pg / mL compound was higher compared to 32 and 16 pg / mL IF. This was observed throughout all independently performed assays and is not due to absorbance of the compound.

[0058] MICso was calculated based on the dose-response curve as shown in Figure 1A. The MIC50 value is defined as the lowest compound concentration that corresponds with minimal 50% growth reduction, thus the lower the MIC50 value, the more potent the compound. In order to elucidate whether the compound might act fungicidal or fungistatic, a cidality assay was performed. C. albicans cells preincubated for 24 hours in the presence of compound were spotted on YPD agar plates, but only when the compound seemed fungicidal during the broth dilution assay (Figure IB). Cells preincubated with compound IF and 4A showed lack of growth for compound concentrations higher than the respective MIC50 value, indicating that the cell has fungicidal activity. Growth of cells preincubated with compound II was observed for all tested compound concentrations, supporting a fungistatic activity of the compound.

[0059] Compounds were evaluated for their potency against C. albicans SC5314. Growth in the presence of 50 pM (~ 16 pg / mL) of each analogue was assessed and the results are shown in Figure 2A for the analogues of IF and in Figure 2B for the analogues of II. 2F reduced the growth of C. albicans, but to a lesser extent than IF. All analogues of I showed more than 50% growth inhibition, but only 21 and 31 exhibited similar growth reduction as II. Therefore, a dose-response assay was performed for these two analogues. The MIC50 was calculated and 21 was identified as the most potent compound among the I series (Figure 2C). However, II was found to be more promising than 21, because 21 was not as soluble as II in the medium and was thus expected to create more variation between results. The spectrum of the compound's antifungal activity was studied by testing the potency of the compound against several species in a dose-response and cidality assay. The susceptibility of strains of emerging pathogens C. auris and C. glabrata and of a vaginal isolate of C. albicans were studied, as well as the activity of the compounds against S. cerevisiae S288C (Figure 3A). Compound 4A was observed to be active against all Candida species except towards C. auris B8441. Compound ID only showed inhibition of growth of C. albicans SC5314 and 149 among the Candida species. IF reduced growth of all tested Candida species, but to a different extent. Compound II was observed to be active toward both C. albicans species and showed some activity towards C. auris B11220. Finally, compound 11 reduced growth of both C. albicans species and C. auris B8441.

[0060] N-(l,3-dimethyl-2,4,7-trioxo-lH,2H,3H,4H,7H-pyrano[2,3-d]pyrimidin-6-y) benzamide lacking the fluor group of compound 4A also showed significant activity against Candida albicans but less active than compound 4A.

[0061] The cidality of compounds that seemed fungicidal after incubation with cells was investigated via a cidality assay (Figure 3B). C. albicans 149 cells pre-treated with 4A, ID and II showed growth, but no growth was observed for some concentrations after pre-treatment with IF and 11. IF was fungicidal towards all tested species, but the concentration of IF for which cells showed no growth variated between repeats. Variation in growth between repeats for the same concentration of IF was observed for the C. auris B11220 cells pre-treated with IF.

[0062] Insusceptibility of S. cerevisiae to a compound would be advantageous in combination treatment with probiotics for bacteria. Therefore, we performed a broth dilution assay with S. cerevisiae as well. Compounds ID and 11 showed minimal growth reduction of S. cerevisiae, while 4A, II and IF exhibited growth inhibition, with IF emerging as the most potent (Figure 3A).

[0063] The activity spectrum of the compounds was studied and compounds were screened for susceptibility towards haploid organisms, as susceptible haploid species would be advantageous in a next step, the bioactivity of the compound was assessed via growth of several species in the presence of 50 pM (~ 16 pg / mL) compound. The panel of organisms included strains of wild-type Candida species, but also the pathogenic bacteria Staphylococcus aureus and several haploidic species, such as E. coll, S. cerevisiae and Pichia yeasts. The results are shown in Figure 4. All selected compounds showed activity towards both C. albicans strains of which one is the clinical isolate 3153A and towards C. albicans' closest related species, C. dubliniensis. Moreover, compound 4A also showed activity towards C. parapsiliosis, C. glabrata and P. pastoris GS200, and minimal to no activity towards the other species. Compound ID showed growth inhibition of P. pastoris GS200 and minimal activity towards C. tropicalis and S. aureus. Compound IF was observed to reduce growth of all Candida species and showed some activity towards S. cerevisiae and P. pastoris GS115, but minimal to no activity towards the other species. P. guilliermondii and P. pastoris GS115 were both susceptible to 50 pM of compound II. Compound 11 reduced growth of C. tropicalis, S. cerevisiae, all three Pichia strains and S. aureus.

[0064] Example 2. Activity of compound against fluconazole and echinocandin- resistant strains

[0065] The group of compounds of the present invention may be used to develop drugs with a mode of action different from the mechanisms of azoles, echinocandins and polyenes.

[0066] The compounds were investigated to exhibit bioactivity against C. albicans C147, a fluconazole resistant (FCZR) strain and against C. albicans C343, an echinocandin resistant (ECNR) strain via a dose-response assay. Compounds 4A, IF and II showed activity against both resistant strains and IF showed fungicidal towards both strains. Compound ID was observed to be inactive against FCZR, and showed activity to ECNR. The FCZR strain showed insusceptibility to compound 11, but growth of the ECNR strain was reduced to 50% in the presence of 16 pg / mL 11 and. 11 showed fungicidal towards ECNR.

[0067] Example 3. Cytotoxicity of compounds against HeLa cells

[0068] Antifungals are supposed to selectively eradicate fungal pathogens with minimal cytotoxicity to the host. The compounds of the present invention were screened for toxicity against the human HeLa cell line via an LDH cytotoxicity assay.

[0069] The results are shown in Figure 5. Compound 4A showed minimal toxicity towards the HeLa cells compared to the toxicity imposed by the internal standard. Compounds ID, II and 11 showed less than 6% cytotoxicity in comparison to the standard. Compound IF was observed to impose approximately 31% toxicity at 350 pM (~ 112 pg / mL) compound. As the minimal inhibitory concentration to reduce 50% growth of C. albicans SC5314 was determined as 16 pg / mL, the toxicity of compound concentrations approximately equal to 4*MIC50 (200 pM) and 2*MIC50 (100 pM) was investigated as well (Figure 5B). Lower concentrations of IF resulted in lower toxicity against the HeLa cells with 12% and 3.5% toxicity for 200 pM and 100 pM compounds, respectively, compared to the toxicity imposed by the standard.

[0070] Example 4. Proposed mode of action of compound ID

[0071] Compound ID revealed unsusceptible to a FCZR stain, which indicates that either ID works as an azole or the compound can be efflux out by the upregulated efflux pumps. Interestingly, the structure of the compound lacks the antimicrobial azole pharmacophore. Under consideration that ID would indeed interfere with the ergosterol synthesis, the activity of ID towards S. aureus, which lacks ergosterol in the cell wall, was rather unexpected. However, some azoles perform antibacterial activity towards S. aureus by inducing oxidative stress upon binding to flavohemoglobin. In C. albicans, imidazoles inhibit the NO detoxification mechanism of flavohemoglobin. Surprisingly, this might be linked to a previously obtained observation in which the antifungal activity of ID was lost in the presence of external RF. RF is the precursor of FAD which is a cofactor of flavohemoglobin. In analogy with imidazole, ID possibly can coordinate with the iron heme of flavohemoglobin, resulting in uncompetitive inhibition with NO and thereby blocking the detoxification activity of flavohemoglobin. Excess of RF or FAD might recover the NO detoxification activity of flavohemoglobin to abolish the oxidative stress produced by the compound. The question remains whether excess RF or FAD can indeed restore the activity of flavohemoglobin. Therefore, we suggest performing a small pilot experiment in which the antifungal activity of imidazole against S. aureus and C. albicans in presence and absence of external RF is determined and we also need to investigate that ID targets flavohemoglobin.

Claims

Claims1. A composition comprising a compound or a salt thereof selected from a group of compounds consisting of : N-(l,3-dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl)-4-fluorobenzamide (4A), (6,7-dimethoxyquinolin-3-yl)-(2-hydroxyphenyl)methanone (1J),N -[3-( 1,2,3, 4-tetrahydrona phtha len-l-yloxy) propy l]tetrazolo[l, 5-b]pyridazin- 6-amine (ID), 3-[[5-[(4-chlorophenoxy)methyl]-l,3,4-oxadiazol-2-yl]sulfanyl]pyrazine-2- carbonitrile (IF), N-[(2-methyl-lH-indol-3-yl)-pyridin-2-ylmethyl]pyridin-2-amine (II), andN-(l,3-dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl)benzamide (1A), for use in the treatment or prevention of a fungal infection in a subject.

2. The composition for use according to claim 1, wherein the compound is N-(l,3- dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl)-4-fluorobenzamide (4A), or(6,7-dimethoxyquinolin-3-yl)-(2-hydroxyphenyl)methanone (1J).

3. The composition for use according to claim 1 or 2, wherein said composition further comprises an antibacterial agent.

4. The composition for use according to any one of claims 1 to 3, wherein the subject is a human.

5. The composition for use according to any one of claims 1 to 4, wherein the fungal infection is a Candida sp. infection.

6. The composition for use according to any one of claims 1 to 5, wherein the fungal infection is an infection of Candida auris, Candida giabrata, Candida krusei or Candida tropicalis.

7. The composition for use according to any one of claims 1 to 6, wherein the fungal infection is of a fluconazole and / or echinocandin-resistant fungus.

8. A compound or salt thereof selected from the group consisting of: N-(l,3-dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl)-4-fluorobenzamide (4A), (6,7-dimethoxyquinolin-3-yl)-(2-hydroxyphenyl)methanone (1J),N -[3-( 1,2,3, 4-tetrahydrona phtha len-l-yloxy) propy l]tetrazolo[l, 5-b]pyridazin- 6-amine (ID), 3-[[5-[(4-chlorophenoxy)methyl]-l,3,4-oxadiazol-2-yl]sulfanyl]pyrazine-2- carbonitrile (IF), N-[(2-methyl-lH-indol-3-yl)-pyridin-2-ylmethyl]pyridin-2-amine (II), andN-(l,3-dimethyl-2,4,7-trioxopyrano[2,3-d]pyrimidin-6-yl)benzamide (1A), for use as a medicament.

9. The compound for use as a medicament, according to claim 1, wherein the compound is N-(l, 3-d imethy 1-2,4, 7-trioxopyrano[2, 3-d] pyrimidin-6-y l)-4- fluorobenzamide (4A).

10. A compound or a salt thereof selected from the group consisting of 11, II, IF and ID for the treatment of agricultural fungal infections.

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