Osidiofundin analogues, methods for their preparation and use

Abstract

The present invention relates to methods for synthesizing osidiofundin analogs using mutants of osidiofundin containing aspartic acid (ASP7) or glutamic acid (GLU7) at position 7 instead of the previously characterized asparagine (ASN7). Selected semisynthetic analogs produced using the ASP7 and GLU7 mutants exhibited improved antifungal activity compared to the ASN7 osidiofundin mutant. The ASP7 and GLU7 mutants of osidiofundin and compositions thereof can be used to treat fungal infections, parasitic infections, and cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 443,060, filed February 3, 2023, the disclosure of which is incorporated by reference in its entirety, including figures, tables, and amino acid or nucleic acid sequences.

[0002] Sequence Listing Statement The Sequence Listing of the present application is labeled "Seq-List.xml", was created on February 1, 2024, and is 32,956 bytes. The entire contents of the Sequence Listing are incorporated herein by reference in their entirety.

[0003] Statement Regarding Federally Sponsored Research This invention was made with government support under 2R42AI131792-02A1 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0004] Osidiofundin is a soil bacterium Burkholderia contaminans Osidiofundin is an antifungal agent produced by MS14 (Reference 1). It is a nonribosomally produced cyclic glycopeptide consisting of eight amino acids (Figure 1).

[0005] The proposed mechanism of action of osidiofundin is the inhibition of the physiological activity of actin. C. glabrata and C. auris (Reference 2) Based on these results, osidiofundin is being developed as a treatment for vulvovaginal (demonstrated in mouse models (References 2, 3)), oral, and cutaneous fungal infections. Osidiofundin has been shown to be effective in treating systemic fungal infections, likely due to its strong binding to serum proteins in animal model systems (Reference 3). in vivo The treatment was not effective.

[0006] Studies have shown that osidiofundin also exhibits chemotherapeutic activity against multiple cancer cell lines. Its anticancer properties are more potent than its antifungal activity (4), with minimal toxicity. Osidiofundin has also been shown to be effective against gastrointestinal parasites. Cryptosporidium parvum (Reference 5) in vitro Therefore, there is a need to produce analogs of osidiofundin to provide novel variants optimized for the treatment of systemic fungal infections, as anticancer agents, or as antiparasitic agents. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides methods for producing novel osidiofundin analogs derived from osidiofundin variants, preferably the osidiofundin aspartic acid 7 variants (OCF-I and OCF-J) and glutamic acid 7 variants (OCF-M and OCF-N). The OCF-I and OCF-J variants have an aspartic acid at position 7 (ASP7), while the OCF-M and OCF-N variants have a glutamic acid at position 7 (GLU7), replacing the asparagine (ASN7) found in the previously isolated and characterized osidiofundin variants (OCF-A and OCF-B). In certain embodiments, osidiofundin variants can be modified to produce semisynthetic analogs by selective modification of the carboxylic acid functionality at position 7 via carbodiimide-mediated amidation.

[0008] In certain embodiments, the various OCF-I, OCF-J, OCF-M, and OCF-N analogs described have antifungal activity. In certain embodiments, osidiofundin variants derived from OCF-I, OCF-J, OCF-M, and OCF-N exhibit improved antifungal activity compared to ASP7 and GLU7 variants (e.g., OCF-I, OCF-J, OCF-M, and OCF-N). Accordingly, certain embodiments of the present invention provide methods for controlling fungal and parasitic infections by administering to a subject in need thereof OCF-I, OCF-J, OCF-M, and OCF-N analogs described herein.

[0009] In certain embodiments, the ASP7 or GLU7 residues of OCF-I, OCF-J, OCF-M, and OCF-N analogs can be modified to include a linker attached to a monoclonal antibody, allowing for a targeting-based delivery system to specific cancer cells. In certain embodiments, the engineered linkage at the ASP7 or GLU7 position can include various additional optional functional groups, such as, for example, an amine, aldehyde, alkyne, or alcohol.

[0010] This patent or application contains at least one drawing in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0011]

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[0012] As used herein, the singular forms "a," "an," and "the" are to be construed as including the plural forms unless the context clearly dictates otherwise. When "including," "includes," "having," "has," "with," or variations thereof are used in the description and / or claims, these terms are to be interpreted inclusively in the same manner as "comprising." Transitional words and their grammatical variations, such as "comprising," "comprises," "consisting essentially of," "consists essentially of," and "consisting," can be used interchangeably.

[0013] The phrase "consisting essentially of" indicates that the described embodiments include embodiments that include the specified materials or steps and that do not materially affect the basic and novel characteristics of the described embodiments.

[0014] The term "about" refers to a range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on the limitations of the method of measurement or determination, i.e., the measurement system. When the term "about" is used in reference to the length of a polynucleotide, these polynucleotides contain the stated number of bases or base pairs with a 0-10% variation (X ± 10%) around that value. When the term "about" or "approximately" is used in reference to the content of a component in a composition, these compositions contain the stated amount of the component with a 0-10% variation (error margin) (X ± 10%) around the stated value.

[0015] Ranges are described herein in abbreviated notation. This avoids the need to describe each and every value within the range in detail. Any appropriate value within the range can be selected as the upper, lower, or end value of the range, as needed. For example, a range of 0.1 to 1.0 includes the end values ​​0.1 and 1.0, as well as intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, and 0.7 to 1.0. Values ​​within a range with at least two significant digits are contemplated. For example, a range of 5 to 10 includes all values ​​between 5.0 and 10.0, including the end values, and all values ​​between 5.00 and 10.00.

[0016] When ranges are used herein, such as, for example, dosage ranges, combinations and subcombinations of ranges (e.g., subranges within the disclosed ranges) are intended to explicitly include specific embodiments within the ranges.

[0017] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or State government, or a corresponding agency in a country other than the United States, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia, and approved for use in animals, and particularly humans.

[0018] "Pharmaceutically acceptable salts" refers to salts of the osidiofundin analogs of the present invention that are pharmaceutically acceptable and possess the desired pharmacological activity of the parent osidiofundin analog. Particularly, such non-toxic salts may be inorganic or organic acid addition salts and base addition salts.

[0019] The term "pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier used to administer the osidiofundin analog of the present invention. The term "pharmaceutically acceptable vehicle" refers to a non-toxic, biologically tolerable, or otherwise biologically suitable substance for administration to a subject, such as an inert substance added to a pharmacological composition or used to facilitate drug administration and compatible therewith. Examples of vehicles include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, etc.

[0020] A "subject" includes a human or non-human animal, particularly a mammal such as a cow, pig, dog, rodent, or cat.

[0021] "Treatment" or "therapy" of an infectious disease, in one embodiment, refers to ameliorating the infection or cancer (i.e., preventing or reducing the onset of the disease or the progression of at least one of its clinical symptoms). In other embodiments, "treatment" or "therapy" refers to improving at least one physical parameter, which may not be discernible to the subject. In yet other embodiments, "treatment" or "therapy" refers to modulating the infection physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. In yet other embodiments, "treatment" or "therapy" refers to delaying the onset of the infection.

[0022] As used herein, the terms "reduce," "inhibit," "block," "prevent," "alleviate," or "alleviate" with respect to an osidiofundin analog mean that the osidiofundin analog reduces the incidence, severity, size, volume, or associated symptoms of an infection or cancer by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% or more compared to the infection or cancer that would normally be present in the absence of application of the osidiofundin analog or a composition comprising the osidiofundin analog.

[0023] In certain embodiments, the methods of the present invention involve administering a therapeutically effective amount of an osidiofundin analog to a subject suffering from or diagnosed with an infectious disease or cancer. A "therapeutically effective amount" refers to an amount or dose sufficient to generally produce the desired therapeutic or prophylactic effect in a patient in need of treatment for the specified infectious disease or cancer.

[0024] The effective amount or dosage of the osidiofundin analog of the present invention can be determined by conventional methods such as modeling, dose escalation studies, or clinical trials, taking into account commonly considered factors, such as the method or route of administration, the pharmacokinetics of the osidiofundin analog, the severity and course of the infection, previous or ongoing treatments, the subject's health status and response to the drug, and the judgment of the treating physician. Exemplary dosages include about 0.001 to about 200 mg of osidiofundin analog per kg of subject body weight per day, preferably about 0.05 to 100 mg / kg / day, or about 1 to 35 mg / kg / day, and more preferably about 1, 5, 10, or 20 mg / kg / day, administered as a single dose or in divided doses (e.g., bid, tid, or qid). An exemplary range of suitable dosage for a 70 kg human is about 0.05 to about 7 g / day, preferably about 0.07 to about 2.45 g / day, and more preferably about 0.07, 0.35, 0.7, or 1.4 g / day.

[0025] The structures of natural analogues of osidiofundin are shown by formula (IX) and formula (X). [ka] In the formula, R 1 is H or OH, and R 2 are NH2 and OH. [ka] In the formula, R 1 is H or OH, and R 2 are NH2 and OH.

[0026] As seen in formulas (IX) and (X), natural analogs of osidiofundin are composed of eight amino acids (SEQ ID NOS: 1-14) and C18 aliphatic amino acids (NAA), including xylose sugar and 2,4-diaminobutyric acid (DABA). (NAA is 3-amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid, which is present at two positions in SEQ ID NOS: 1-4 and 9-14.) The structural analogs of formulas (IX) and (X) differ in the addition of oxygen to asparagine 1 (Asn1) to form β-hydroxyasparagine 1 (BHN1) and in the residue at position 7. In formula (IX), osidiofundin analogs OCF-A (SEQ ID NOS: 1) and OCF-B (SEQ ID NOS: 2) have an asparagine residue at position 7. OCF-A (1200.59 Da) has an asparagine residue at position 11, and OCF-B (1216.59 Da) has a β-hydroxyasparagine residue at position 11. OCF-I (SEQ ID NO: 9) and OCF-J (SEQ ID NO: 10) each have an aspartic acid residue at position 7 and a hydrogen or hydroxy group at position 11. In formula (X), OCF-K (SEQ ID NO: 11) and OCF-L (SEQ ID NO: 12) have a glutamine residue at position 7. OCF-K has an asparagine residue at position 1, and OCF-L (1230.61 Da) has a β-hydroxyasparagine residue at position 11. OCF-M (SEQ ID NO: 3) and OCF-N (SEQ ID NO: 14) have a glutamic acid residue at position 7. OCF-M has an asparagine residue at position 1, and OCF-N (1231.59 Da) has a β-hydroxyasparagine residue at position 1. HRMS analysis was performed to confirm the identity of the osidiofundin analogs (Table 4). The aspartic acid 7 variants (OCF-I and OCF-J) and glutamic acid variants (OCF-M and OCF-N) of osidiofundin were isolated using reversed-phase high-performance liquid chromatography (RP-HPLC), and they were resolved from the previously identified aspartic acid 7 variants (OCF-A and OCF-B) (Table 1). OCF-K and OCF-L coeluted with OCF-A and OCF-B, while OCF-M and OCF-N coeluted with OCF-I and OCF-J by RP-HPLC.Nuclear magnetic resonance (NMR) analysis confirmed that the mutants contained either aspartic acid or glutamic acid at residue 7.

[0027] Certain embodiments of the present invention provide osidiofundin analogs and methods for making osidiofundin analogs.

[0028] The OCF-I, OCF-J, OCF-M, and OCF-N variants offer novel sites for analog synthesis because the terminal side chain of ASP7 or GLU7 is the only carboxylic acid in the compound. The single carboxylic acid in the molecule provides a selective region for natural product modification using various synthetic chemistry methods. In certain embodiments, condensation with various amines via carbodiimides, such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC)), has been carried out to form an amide bond between the carboxylic acid at the 7-position (Figure 5A, Table 5). All semisynthetic analogs were confirmed by mass spectrometry and NMR analysis.

[0029] In certain embodiments, carbodiimide-mediated condensation is a simple, selective, one-step reaction that can be used for high-throughput semisynthetic analog production. This carbodiimide coupling reaction has been carried out with a variety of primary and secondary amines on the carboxylic acid side chain of ASP7 or GLU7. In certain embodiments, the amine is ammonia, methylamine, dimethylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, nonylamine, decylamine, undecylamine, dodecylamine, 3-butyn-1-amine, 3-amino-1-propanol, 3-aminopropyl-dihydrogenphosphate, amines attached to prodrug functional groups, such as phosphate groups, methyl succinate, or benzyl succinate groups (e.g., 3-amino-1-propanol), or primary or secondary amines bearing various alkyl and / or aryl groups. In certain embodiments, 1-hydroxybenzotriazole (HOBt), 4-(dimethylamino)pyridine (DMAP), N-hydroxysulfosuccinimide (sulfo-NHS), bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide (HATU), or any combination thereof, is added to the condensation reaction. In certain embodiments, the condensation reaction is carried out in N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), methyl tert-butyl ether (TBME), cyclopentyl methyl ether (CPME), dichloromethane, dimethyl carbonate (DMC), ethyl acetate (EtOAc), or isopropyl alcohol (IPA). In certain embodiments, for example, a 10x molar excess of the desired amine, EDC, and 1-hydroxybenzotriazole (HOBt) relative to the osidiofundin analog is used in DMF or DMSO at room temperature (e.g., about 18°C ​​to about 25°C) for about 16, 18, 20, 22, 24, 26, 28, 30, 36, or 48 hours or more, preferably protected from light.In certain embodiments, for some of the amines used in the reaction, particularly the larger amines, longer incubation times and / or incubation at about 35° C. have been shown to improve efficiency.

[0030] In certain embodiments, a cross-linking catalyst such as, for example, N-hydroxysuccinimide can activate the carboxyl group, and the activated osidiofulundin intermediate can be used to produce additional analogs in high-throughput combinatorial chemistry, allowing for the rapid production of large libraries of analogs.

[0031] In certain embodiments, to utilize ASP7 (OCF-I and OCF-J) or GLU7 (OCF-M and OCF-N) osidiofudin variants containing carboxylic acid modification sites, additional chemistries can be utilized, such as multicomponent reactions such as the Ugi reaction or the Passarini reaction. In certain embodiments, the Ugi reaction can include, for example, reacting OCF-I or OCF-J with a primary or secondary amine (e.g., an amine disclosed above), methyl isocyanide, and an aldehyde to yield the corresponding Ugi condensation product.

[0032] The reactants and products of OCF-I and OCF-J follow the scheme below. [ka]

[0033] The reactants and products of OCF-M and OCF-N follow the scheme below. [ka]

[0034] In certain embodiments, the Passarini reaction can include, for example, reacting OCF-I or OCF-J with methyl isocyanide and an aldehyde or ketone to produce the corresponding Passarini condensation product.

[0035] The reactants and products of OCF-I and OCF-J follow the scheme below. [ka]

[0036] The reactants and products of OCF-M and OCF-N follow the scheme below. [ka]

[0037] In certain embodiments, the osidiofundin analog is prepared using the condensation of a carbodiimide with an amine and can be represented by formula (I), formula (II), formula (III), or formula (IV). [ka] In the formula, R 1 is H and R 2 is H or CH3, and R 3 are H, CH3, CH2CH3, CH2CH2CH3, (CH2) 10 CH3 or (CH2) 11 In a preferred embodiment, R 1 is H or OH, and R 2 is H and R 3 are H, CH3, CH2CH3, CH2CH2CH3, (CH2) 10 CH3 or (CH2) 11 CH3; or R 1 is H or OH, and R 2 is CH3 and R 3 is CH3.

[0038] [ka] In the formula, R 1 is H or OH, and R 2 is H or CH3, and R 3 are H, CH3, CH2CH3, CH2CH2CH3, (CH2) 10CH3 or (CH2) 11 In a preferred embodiment, R 1 is H or OH, and R 2 is H and R 3 is H, CH 3、 CH2CH3, CH2CH2CH3, (CH2) 10 CH3 or (CH2) 11 CH3; or R 1 is H or OH, and R 2 is CH3 and R 3 is CH3.

[0039] [ka] In the formula, R 1 is H or OH, and Z is an ester, thioester, acid anhydride, acid chloride, amide, or carboxylate ion.

[0040] [ka] In the formula, R 1 is H or OH, and Z is an ester, thioester, acid anhydride, acid chloride, amide, or carboxylate ion.

[0041] In certain embodiments, the carbodiimide (e.g., EDC)-mediated condensation of OCF-I and OCF-J with ammonia converts ASP7 to ASN7, yielding OCF-A and OCF-B variants (designated OCF-S1 and OCF-S2) (Figure 5A, Table 5). Electrospray ionization mass spectrometry (ESI-MS) data from the ammonia reaction confirmed the OCF-A and OCF-B variants, but also produced additional products (OCF-S15 and OCF-S16) with masses of 1183.56 Da and 1998.57 Da. The masses of these variants in the reaction correspond to OCF-S15 and OCF-S16, which are undesired dehydration byproducts of OCF-I and OCF-J. This byproduct is not observed in OCF-M and OCF-N (GLU7 variants) by mass spectrometry due to its low abundance.

[0042] In certain embodiments, the condensation reaction can employ an additional amine, preferably an amine attached to a prodrug functional group. In preferred embodiments, the prodrug functional group comprises methyl succinate, benzyl succinate, an ether, a phosphate group, or other known prodrug functional groups, such as those described in "Rautio J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Jarvinen T, Savolainen J. Prodrugs: design and clinical applications. Nat Rev Drug Discov. 2008 Mar;7(3):255-70. doi: 10.1038 / nrd2468. Erratum in: Nat Rev Drug Discov. 2008 Mar;7(3):272," incorporated herein by reference in its entirety.

[0043] In certain embodiments, direct condensation may not be achieved using amines such as, for example, ethanolamine, 3-amino-1-butanol, 4-amino-1-butanol, 3-amino-1-propanol, 3-aminopropyl-dihydrogenphosphate, or higher alkyl chain amino alcohols (e.g., n-amino-n-alkyl-1-ol). Therefore, a portion of the amine, e.g., the alcohol group of the amine, can be protected prior to condensation. In certain embodiments, 3-amino-1-propanol is protected with tert-butyldimethylsilyl (TBS) by adding 1 molar equivalent each of 3-amino-1-propanol, triethylamine, and tert-butyldimethylsilyl chloride (TBS-Cl) in dichloromethane (DCM). In certain embodiments, the reaction mixture is mixed and incubated at room temperature for at least about 4, 5, 6, 7, 8, 9, 10, or more hours. The resulting oily layer after incubation contains the protected 3-amino-1-propanol and is used as the amine in the condensation reaction. In a specific embodiment, 1 molar equivalent of 3-aminopropyl-dihydrogen phosphate, triethylamine, and TBS-Cl are incubated in DCM. The reaction mixture is mixed and an equal volume of 35% acetonitrile (65% double-distilled water) 0.1% trifluoroacetic acid is added, followed by incubation at room temperature for at least about 4, 5, 6, 7, 8, 9, 10, or more hours. The aqueous upper layer contains the protected 3-aminopropyl-dihydrogen phosphate and is then used in the condensation reaction.

[0044] In certain embodiments, a prodrug functional group, such as methyl succinate or benzyl succinate, can be attached to the amine prior to the condensation reaction. In a preferred embodiment, tert-butoxycarbonyl (Boc) 3-amino-1-propanol (Boc-3-amino-1-propanol), allyloxycarbonyl (Alloc) 3-amino-1-propanol (Alloc-3-amino-1-propanol), or benzyloxycarbonyl (Boc) 3-amino-1-propanol (Boc-3-amino-1-propanol) was first attached to the methyl or benzyl succinate ester. In certain embodiments, alternatives to the Boc hydroxy protecting group include, for example, methoxymethyl ether, tetrahydropyranyl ether, allyl ether, benzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, acetate, pivalate, or benzoate. In a preferred embodiment, 3-amino-1-propanol, N,N'-dicyclohexylcarbodiimide (DCC), methyl / benzyl succinate (1 molar equivalent), and 4-dimethylaminopyridine (DMAP) (0.1 molar equivalent) were added to anhydrous dimethylformamide (DMF). In certain embodiments, the reaction was stirred at room temperature for at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, or more hours. In certain embodiments, the amine-linked prodrug functionality can be further purified from the reaction mixture.

[0045] In certain embodiments, an amine such as, for example, 3-amino-1-propanol is used to prepare osidiofundin analogs (according to Formula (V) and Formula (VI)) containing a terminal alcohol group, e.g., OCF-S19 and OCF-S20 (Table 5). In certain embodiments, the terminal alcohol group of the osidiofundin analog links the osidiofundin to a prodrug functional group, e.g., a phosphate group, methyl succinate, ether, or benzoate ester. [ka] In the formula, R 1 is H or OH, and R2 is a prodrug functional group. [ka] In the formula, R 1 is H or OH, and R 2 is a prodrug functional group.

[0046] In certain embodiments, the prodrug functional group acts as a prodrug moiety that can be removed by chemical or enzymatic action. In certain embodiments, the prodrug is activated by an enzyme or chemical in the subject, for example, an enzyme or chemical present in the blood or an enzyme or chemical present in a cell. In certain embodiments, the enzyme is a phosphatase, esterase, or protease. In certain embodiments, the prodrug functional group acts as a prodrug moiety to generate OCF-S19 and OCF-S20 upon chemical or enzymatic removal of the prodrug functional group. Additional prodrug moieties can use linkers to leave a group at the AS7 position using a phosphatase, esterase, or protease.

[0047] In certain embodiments, amines such as, for example, 3-butyn-1-amine can be used to prepare osidiofundin analogs (according to Formula (I) and Formula (II)) containing a terminal alkyne group, e.g., OCF-S23 and OCF-S24 (Table 5). In certain embodiments, the terminal alkyne group of the osidiofundin analog can be used to attach the osidiofundin to a functional group, e.g., an azide functional group, in subsequent click reaction chemistry.

[0048] In certain embodiments, analogs of osidiofundin are according to formula (VII) and formula (VIII). [ka] In the formula, R 1 is H or OH. [ka] In the formula, R 1 is H or OH.

[0049] In certain embodiments, the condensation reaction can utilize additional amines, preferably those that function as or are attached to antibodies. In preferred embodiments, the antibodies are monoclonal antibodies. In certain embodiments, ASP7 residues are modified using the aforementioned condensation reaction to include a linker attached to the monoclonal antibody, allowing for targeted delivery systems to specific cancer cells.

[0050] In certain embodiments, the antibody can target at least one antigen, such as CD19, CD3E, ERBB2 (HER2), EGFR, MS4A1 (CD20), CD22, PDCD1 (PD-1), MSLN (mesothelin), ERBB3 (Her3), a Th17 cytokine, IL17A, or any other antigen currently targeted by antibodies for the treatment of cancer or other diseases, as disclosed by Strohl WR, Current progress in innovative engineered antibodies. Protein Cell. 2018 Jan;9(1):86-120. doi: 10.1007 / s13238-017-0457-8. Epub 2017 Aug 18, which is incorporated herein by reference in its entirety.In certain embodiments, the antibody is selected from the group consisting of muromonab-CD3, abciximab, rituximab, daclizumab, infliximab, palivizumab, trastuzumab, etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefecept, omalizumab, tositumomab-i131, efalizumab , cetuximab, bevacizumab, natalizumab, abatacept, ranibizumab, panitumumab, eculizumab, rilonacept, romiplostim, golimumab, ustikinumab, catumaxomab, certolizumab pegol, canakinumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, belatacept, brentuximab vedotin, afla Bercept, mogamulizumab, pertuzumab, zib-aflavercept, racibacumab, trastuzumab emtansine, obinutuzumab, eftrenonacog alfa, ramucirumab, siltuximab, vedolizumab, efmoroctocog alfa, pembrolizumab, dulaglutide, alemtuzumab, blinatumomab, nivolumab, sectinimab, dinutuximab and / or any combination thereof.

[0051] Salts of Osidiofundin Analogues of the Present Invention In some embodiments, the present invention provides salts of the osidiofundin analogs described herein, such as salts with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid; organic acids such as trifluoroacetic acid (TFA), formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid; or bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, and trialkyl and aryl amines, and substituted ethanolamines.

[0052] Further salts include (1) salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or with acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] Included are acid addition salts formed with organic acids such as oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed by replacing an acidic proton present in the parent osidiofundin analog with a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or salts formed with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and, if the osidiofundin analog contains a basic functional group, salts of non-toxic organic or inorganic acids such as chloride, bromide, tartrate, mesylate, acetate, maleate, oxalate, and the like.

[0053] Certain embodiments provide amorphous forms of the salts of the osidiofundin analogs disclosed herein, which are advantageous for oral, pulmonary, buccal, vaginal, or suppository delivery.

[0054] Route of administration and dosage form In certain embodiments, osidiofundin analogs can be administered intramuscularly, subcutaneously, intrathecally, intravenously, or intraperitoneally by infusion or injection. A solution of osidiofundin analogs can be prepared in water, optionally mixed with a non-toxic surfactant. Under normal storage and use conditions, a preservative can be included to prevent microbial growth.

[0055] Pharmaceutical formulations suitable for injection or infusion include sterile aqueous solutions or dispersions or sterile powders containing osidiofundin analogs. These are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The final dosage form is desirably sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles can be solvents or liquid dispersion vehicles containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, nontoxic glyceryl esters, cyclodextrins, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic agents, such as sugars, buffers, and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin.

[0056] Sterile injectable solutions are prepared by combining the osidiofulvin analog in the required amount in a suitable solvent as described herein, optionally with the addition of various other ingredients as described herein, and preferably followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient containing any additional ingredients present in the previously sterile-filtered solution.

[0057] The compositions of the present invention may also be administered orally in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly with the food of the subject's diet.

[0058] For oral therapeutic administration, osidiofundin analogs may be combined with one or more excipients and used in the form of oral tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations should contain at least 0.1% of the osidiofundin analogs of the present invention. Of course, the percentage of the osidiofundin analogs of the present invention present in these compositions and formulations may vary, conveniently ranging from about 2% to about 60% by weight of a particular unit dosage amount. The amount of osidiofundin analog in a therapeutically useful composition is determined so as to provide an effective dosage level.

[0059] Tablets, troches, pills, capsules, etc. may contain one or more of the following ingredients: binders (such as gum tragacanth, acacia, cornstarch, gelatin, etc.), excipients (such as dicalcium phosphate, etc.), disintegrants (such as cornstarch, potato starch, alginic acid, etc.), lubricants (such as magnesium stearate, etc.), sweeteners (such as sucrose, fructose, lactose, aspartame, etc.), or flavorings (such as peppermint, oil of wintergreen, cherry flavor, etc.).

[0060] When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a vegetable oil or polyethylene glycol.

[0061] Various other materials may be present to coat or otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, and capsules may be coated with gelatin, wax, shellac, sugar, etc. Syrups and elixirs may contain osidiofundin analogs, sucrose or fructose as a sweetener, methylparaben and propylparaben as preservatives, coloring agents, and flavorings such as cherry or orange flavor.

[0062] Of course, any material used in preparing any dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.

[0063] Additionally, the ocidiofundin analogs can be formulated into sustained-release preparations and devices, for example, the ocidiofundin analogs can be formulated into sustained-release capsules, sustained-release tablets, sustained-release pills, and sustained-release ocidiofundin analogs and nanoparticles.

[0064] Pharmaceutical compositions for topical administration of osidiofundin analogs to the epidermis (mucosa or skin surface) can be formulated as ointments, creams, lotions, gels, or transdermal patches. Such transdermal patches can contain penetration enhancers such as linalool, cyclodextrin, carvacrol, thymol, citral, menthol, t-anethole, etc. Ointments and creams can comprise, for example, an aqueous or oily base to which appropriate thickeners, gelling agents, colorants, etc. are added. Lotions and creams can comprise an aqueous or oily base and typically contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners, colorants, etc. Gels preferably comprise an aqueous carrier base and a gelling agent such as cross-linked polyacrylic acid polymers or derivatized polysaccharides (e.g., carboxymethylcellulose).

[0065] Pharmaceutical compositions suitable for topical administration in the mouth (e.g., buccal or sublingual administration) include lozenges containing the composition in a flavored base such as sucrose, acacia, tragacanth, etc.; pastilles containing the composition in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier. Pharmaceutical compositions for topical administration in the mouth can optionally contain a penetration enhancer.

[0066] Useful solid carriers include finely dispersed solids such as talc, clay, microcrystalline cellulose, silica, and alumina. Other solid carriers include nontoxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohol, glycol, or water / alcohol / glycol blends in which the osidiofundin analogs can be dissolved or dispersed at effective levels, optionally with the aid of nontoxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for specific applications. The resulting liquid compositions can be applied from absorbent pads, impregnated into bandages or other dressings, or sprayed onto the affected area using pump-action or aerosol sprayers.

[0067] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials can also be combined with liquid carriers to form spreadable pastes, gels, ointments, soaps, etc., which can be applied directly to the user's skin.

[0068] Examples of useful dermatological compositions that can be used to deliver osidiofundin analogs to the skin are known in the art, see, for example, U.S. Patent No. 4,608,392, U.S. Patent No. 4,992,478, U.S. Patent No. 4,559,157, and U.S. Patent No. 4,820,508, all of which are incorporated herein by reference.

[0069] The concentration of the osidiofundin analogs of the present invention in such formulations can vary widely depending on the nature of the formulation and the intended route of administration. For example, the concentration of the osidiofundin analog in a liquid composition such as a lotion is preferably about 0.1 to 25% by weight, more preferably about 0.5 to 10% by weight. The concentration in a semi-solid or solid composition such as a gel or powder is preferably about 0.1 to 5% by weight, more preferably about 0.5 to 2.5% by weight.

[0070] Pharmaceutical compositions for spinal administration or amniotic infusion may be provided in single dose form in ampoules, prefilled syringes, small volume infusions, or in multidose containers and may contain added preservatives. Compositions for parenteral administration may be suspensions, solutions, or emulsions and may contain excipients such as suspending agents, stabilizers, dispersing agents, etc.

[0071] Pharmaceutical compositions suitable for rectal administration are those in which the osidiofundin analogues of the present invention are combined with solid or semi-solid (e.g., cream or paste) carriers or vehicles. For example, such rectal compositions can be provided as single-dose suppositories. Suitable carriers or vehicles include cocoa butter and other materials commonly used in the art.

[0072] According to one embodiment, pharmaceutical compositions of the present invention suitable for vaginal administration are provided as pessaries, tampons, creams, gels, pastes, foams, or sprays containing the osidiofundin analogs of the present invention in combination with carriers known in the art, including, for example, cyclodextrins. Alternatively, compositions suitable for vaginal administration can be administered in liquid or solid dosage forms.

[0073] Pharmaceutical compositions suitable for intranasal administration are also included in the present invention. Such intranasal compositions contain the ocidiofundin analogs of the present invention in a vehicle and suitable administration device for administering a liquid spray, dispersible powder, or drops. Drops can be formulated with an aqueous or non-aqueous base containing one or more dispersing agents, solubilizing agents, or suspending agents. Liquid sprays are conveniently administered from a pressurized pack containing the ocidiofundin analog, a sprayer, a nebulizer, or other convenient means for administering an aerosol. The pressurized pack contains a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas known to those skilled in the art. The amount of aerosol administered can be controlled by providing a valve that allows for the metered delivery of the ocidiofundin analog.

[0074] The osidiofundin analogs can be combined with an inert powder carrier and inhaled or injected by the subject.

[0075] Pharmaceutical compositions for administration by inhalation or insufflation may be provided in the form of a dry powder composition, for example a powder mix of the osidiofundin analogue and a suitable powder base such as lactose or starch. Such powder composition may be presented in unit dose form, for example, in capsules, cartridges, gelatin packs, blister packs or the like from which the powder may be administered with the aid of an inhalator or insufflator.

[0076] The exact amount (effective dose) of an osidiofundin analog varies from subject to subject. It depends, for example, on the species, age, weight, general or clinical condition of the subject, the severity or pathology of the infection to be treated, the specific drug or vehicle used, the method and schedule of administration, etc. A therapeutically effective dose can be empirically determined by conventional procedures well known to those skilled in the art. See, for example, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, eds., Macmillan Publishing Co., New York." For example, an effective dose can be determined initially by: in vivoThe therapeutic effect of a compound can be estimated through analysis or appropriate animal models. Animal models may also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans. Methods for extrapolating effective dosages in mice, and other animals, to humans are known in the art. See, for example, U.S. Patent No. 4,938,949, incorporated herein by reference. Therapeutic doses can also be selected by analogy with dosages of similar therapeutic agents.

[0077] The particular method and regimen of administration can be selected by the attending clinician taking into account the specifics of the case (e.g., the subject, the disease, the condition involved, whether the treatment is prophylactic), etc. Treatment may involve single or multiple daily administrations of the compound(s) for periods ranging from several days to several months or even years.

[0078] However, in general, a suitable dosage is in the range of about 0.001 to about 100 mg / kg body weight per day, preferably about 0.01 to about 100 mg / kg body weight per day, more preferably about 0.1 to about 50 mg / kg body weight per day, and even more preferably about 1 to about 10 mg / kg body weight per day. For example, a suitable dosage is about 1 mg, 10 mg, or 50 mg / kg body weight per day.

[0079] Osidiofundin analogs can be conveniently administered in unit dosage forms, for example, containing about 0.05 to about 10,000 mg, about 0.5 to about 10,000 mg, about 5 to about 1,000 mg, or about 50 to about 500 mg of active ingredient per unit dosage.

[0080] The osidiofundin analog can be administered to achieve a peak plasma concentration of, for example, about 0.25 to about 200 μM, about 0.5 to about 75 μM, about 1 to about 50 μM, about 2 to about 30 μM, or about 5 to about 25 μM. Examples of desirable plasma concentrations include at least 0.25, 0.5, 1, 5, 10, 25, 50, 75, 100, or 200 μM. For example, plasma levels can be about 1 to about 100 micromolar or about 10 to about 25 micromolar. This can be achieved, for example, by intravenous injection of a 0.05 to 5% solution of the osidiofundin analog, optionally in saline, or by oral administration as a bolus containing about 1 to about 100 mg of the osidiofundin analog. Desired blood concentrations can be maintained by continuous or intermittent infusion.

[0081] The osidiofundin analogs can be included in the compositions within a therapeutically useful and effective concentration range, as determined by standard procedures well known in the medical pharmaceutical arts. For example, a typical composition can include one or more osidiofundin analogs at a concentration ranging from at least about 1 mg / ml, preferably at least about 4 mg / ml, more preferably at least 5 mg / ml, and most preferably at least 6 mg / ml.

[0082] The osidiofundin analogue may conveniently be provided in a single dose or in divided doses administered at appropriate intervals, for example, once daily or as two, three, four or more divided doses daily, which sub-dose itself may be further divided, into a number of discrete, spaced apart administrations, such as, for example, multiple inhalations from an inhaler.

[0083] Optionally, the pharmaceutical composition of the present invention may contain one or more other therapeutic agents, for example, as a combination therapy. The additional therapeutic agent is contained in the composition within a therapeutically useful and effective concentration range, as determined by standard methods well known in the medical pharmaceutical field. The concentration of a particular additional therapeutic agent can be the same range as the typical range used as a monotherapy. Alternatively, if a synergistic effect occurs when combined with the osidiofundin analog of the present invention, its concentration may be set lower than the typical monotherapy concentration.

[0084] Treatment method In certain embodiments, the osidiofundin analogs disclosed herein are, in particular Candida Genus and Saccharomyces Therefore, certain embodiments of the present invention are useful for treating infections, particularly fungal infections, more particularly yeast infections, and even more particularly Candida Provided are methods for administering an osidiofundin analog to a subject in need thereof to treat or prevent an infection. In certain embodiments, the present invention provides a method for administering an osidiofundin analog to a subject in need thereof, e.g., Trichophyton mentagrophytes , Trichophyton rubrum , Rhizopus microspores , Mucor circinelloides , Mucor fragilis , Fusarium solani , Fusarium oxysporum , Aspergillus flavus , Aspergillus fumigatus , Candida albicans , Candida glabrata , Candida krusei , Candida auris , Candida parapsilosis , Candida tropicalis , Saccharomyces cerevisiae or Cryptococcus neoformans Including Candida spp., Saccharomyces spp., Trichophyton spp., Rhizopus spp., Mucor spp., Fusarium spp., Aspergillus spp. or Cryptococcus The present invention provides a method for treating or preventing infections caused by fungi selected from the group consisting of Pseudomonas spp.

[0085] In certain embodiments, the osidiofundin analogs disclosed herein are particularly suitable for example:Cryptosporidium parvum , Entamoeba histolytica , Giardia duodenalis or Trichomonas vaginalis Including spp. Accordingly, certain embodiments of the present invention provide methods of administering an osidiofundin analog to a subject in need thereof to treat or prevent a parasitic infection.

[0086] In certain embodiments, osidiofundin analogs can be used to treat cancer. In certain embodiments, osidiofundin analogs exhibit anticancer activity, including anticancer activity against ovarian cancer, astrocytoma brain cancer (SW1088), and B-cell non-Hodgkin's lymphoma. The produced osidiofundin analogs can be designed to bind to antibodies that target specific tumors or cancer cell lines. In certain embodiments, the antibodies can be designed to deliver enzymes that activate the prodrug osidiofundin analog at the site of the tumor or infectious pathogen.

[0087] Cancers suitable for treatment with the disclosed methods include, but are not limited to, acanthoma, acinic cell carcinoma, acoustic neuroma, acral melanoma, amphoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, differentiated acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamtinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, and leukemia. lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratosarcomatoid rhabdomyosarcoma, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown tumor, Burkitt lymphoma, cancer of unknown primary origin, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary origin, carcinosarcoma, Castleman disease, central nervous system germ cell tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic Leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Dego disease, dermatofibrosarcoma protuberans, dermoid cyst, dysplastic small round cell tumor, diffuse large B-cell lymphoma, dysplastic neuroepithelial tumor, germ cell carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroblastic leukemia, esophageal carcinoma, olfactory neuroblastoma, Ewing's tumor complex, Ewing's sarcoma, extracranial germ cell tumor, extragonadal Germ cell tumors, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal tumor in utero, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, digestive cancer, digestive carcinoid tumor, digestive stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational stroma tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma,Hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioblastoma, inflammatory breast cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, renal cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, lentigo maligna melanoma, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioblastoma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell carcinoma Leukocyte lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplasia, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorders, myxoma, nasal carcinoma, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer Cell lung cancer, ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-grade malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, papillary thyroid carcinoma, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid carcinoma, penile cancer, perihemangioblastoma, pharyngeal carcinoma, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, polyembryomatous tumor, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, chromosome 15 NUT gene-related respiratory cancer, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, Schwannomatosis, sebaceous gland carcinoma, secondary neoplasms, testicular tumor, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumor,Spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, cerebral hemispheric primitive neuroectodermal tumor, superficial epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, peripheral lymphatic duct carcinoma, testicular cancer, testicular tumor, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, renal pelvis and ureteral transitional cell carcinoma, transitional cell carcinoma, ureteral germ cell carcinoma, urethral cancer, genitourinary neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, optic pathway glioblastoma, vulvar cancer, Waldenstrom's megalocytic proteinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

[0088] In some embodiments, the cancer treated according to the present invention is skin cancer, particularly melanoma; rectal; bladder; cervix; head and neck; thyroid; breast; prostate; uterine; colon; adrenal; hepatocellular; lung, particularly lung adenoma or lung squamous cell carcinoma; kidney cancer, particularly renal chromophobe carcinoma, renal clear cell carcinoma, or renal papillary carcinoma; or glioblastoma / glioblastoma.

[0089] The method for treating or preventing an infectious disease or cancer can be practiced in any subject, such as a mammal, including a human. Such a method comprises administering an effective amount of an osidiofundin analog of the present invention to a subject in need of prevention or treatment of the infectious disease or cancer. The osidiofundin analog can be administered in the form of a pharmaceutical composition of the osidiofundin analog.

[0090] Preferably, the osidiofundin analog is administered parenterally or enterally. More preferably, the osidiofundin analog is administered intravaginally, intraperitoneally, subcutaneously, or intravenously. The effective dose of the osidiofundin analog may vary depending on the age and condition of each subject to be treated. However, an appropriate unit dose is usually in the range of about 0.01 to about 100 mg. For example, the unit dose can be in the range of about 0.2 mg to about 50 mg. Such a unit dose can be administered once or more times a day, for example, twice or three times a day.

[0091] In certain embodiments, culture media are also included in the present disclosure. For example, the culture medium comprises or consists of ddH2O supplemented with dipotassium hydrogen phosphate, ammonium chloride, magnesium sulfate heptahydrate, and a carbon source. In preferred embodiments, the carbon source is an amino acid or a derivative thereof, citric acid, cis-aconitate, D-isocitric acid, α-ketoglutarate, succinyl-CoA, succinate, fumarate, maleate, oxaloacetate, pyruvate, or acetyl-CoA. In certain embodiments, the concentration of potassium dihydrogen phosphate is about 1 g / L to about 100 g / L, about 5 g / L to about 50 g / L, or about 9 g / L. The concentration of ammonium chloride is about 0.1 g / L to about 10 g / L, about 0.5 g / L to about 5 g / L, or about 1 g / L. The concentration of magnesium sulfate heptahydrate is about 0.01 g / L to about 10 g / L, about 0.1 g / L to about 1 g / L, or about 0.2 g / L. The concentration of the carbon source is about 0.1 g / L to about 10 g / L, about 0.5 g / L to about 5 g / L, or about 1 g / L.

[0092] Cryptosporidium Stocks etc. B. contaminans MS14 Also provided are methods for producing the disclosed osidiofundin analogs by culturing the cells in the disclosed medium at a temperature of about 20°C to about 30°C, e.g., about 28°C, for about 6 hours to about 10 days or more (e.g., about 6 to 24 hours, or about 4 to 10 days, or about 4, 5, 6, 7, 8, 9, or 10 days).

[0093] Materials and Methods Product isolation The primary culture was prepared in a 2 L culture flask. 1 L of double-distilled water (ddH2O) was supplemented with potassium dihydrogen phosphate (9 g / L), ammonium chloride (1 g / L), heptahydrate magnesium sulfate (0.2 g / L), and L-asparagine (1 g / L). The pH of the culture was adjusted to 7.0-7.2 and autoclaved for 20 minutes. The secondary culture was prepared in a 2.8 L culture flask. 900 mL of ddH2O was supplemented with potassium dihydrogen phosphate (9 g / L), ammonium chloride (1 g / L), heptahydrate magnesium sulfate (0.2 g / L), and L-asparagine (1 g / L). The pH of the culture was adjusted to 7.0-7.2 and autoclaved for 20 minutes. B. contaminans One ml of the frozen glycerol stock was added to the overnight culture and incubated at 28°C with shaking at 150 RPM. The OD600 of the primary culture was measured after 8–15 hours, and when the OD600 reached 0.65–0.9, 100 ml of the primary culture was added to each secondary culture flask. The secondary cultures were incubated at 28°C for 3–7 days without shaking before extraction. Prior to extraction, the secondary cultures were pooled and heated to 55–60°C to heat-kill the bacteria, then cooled to below 40°C.

[0094] The column was prepared by packing at least 2 g of Amberlite XAD1140 resin per liter of culture broth to be extracted. Prior to packing, the resin was soaked in 80% isopropanol and mixed at room temperature for 30 minutes, after which the supernatant was discarded. The resin was then soaked in ddH2O and mixed at room temperature for 15 minutes, after which the supernatant was discarded. This water wash was repeated two more times. After the water wash, the resin was packed into the column. The resin can be used for up to four extractions before disposal. A peristaltic pump was attached to the column using Masterflex tubing, and the pump flow rate was measured. Heat-sterilized culture broth was pumped through the column for a time required for four times the total culture volume to circulate through the column. The resin was then rinsed with at least 75 ml of double-distilled water per gram of resin. The resin was then rinsed with at least 150 ml of 20% isopropanol (IPA) in ddH2O containing 0.1% trifluoroacetic acid (TFA) for at least 30 minutes. The resin was then rinsed twice with at least 150 ml of 80% IPA in ddH2O containing 0.1% TFA for 60 min or more to elute the active compound. The 80% IPA rinses were combined and dried in an oven or rotary evaporator to remove the IPA. The aqueous phase was centrifuged at 13,000 g, and the resulting pellet was suspended in 35% acetonitrile (ACN) containing 0.1% TFA. The resulting material was further purified by reverse-phase high-performance liquid chromatography (RP-HPLC).

[0095] Product purification The product was purified using reversed-phase high-performance liquid chromatography (RP-HPLC) on a SinoChrom ODS-BP 5 μm (10.0 mm x 250 mm, SN: 3018159x) column. The mobile phases were ACN and ddH2O containing 0.1% TFA, respectively. The gradient changed from 90% ddH2O to 20% ddH2O over 30 min. Osidiofudin eluted at approximately 45%–43% ddH2O with 0.1% TFA. The sample could then be further purified to separate the osidiofudin variants. The collected peaks were then run on a Supercell ODS2 5 μm (4.6 mm x 250 mm, SN: E3018660) column, both with ACN containing 0.1% formic acid (FA) and 10 mM triethylamine. The gradient changes from 90% 10 mM triethylamine to 60% 10 mM triethylamine, holds for 10 minutes, and then drops to 20% 10 mM triethylamine. Osidiofundin variants elute during the isocratic hold at 60% 10 mM triethylamine.

[0096] Mass spectrometry (MS) Product characterization is performed using mass spectrometry (MS) on a ThermoFisher Q Exactive Orbitrap mass spectrometer (ESI-MS) or a Bruker Ultraflextreme MALDI-TOF-TOF.

[0097] For ESI-MS, compounds were dissolved in 50% ACN containing 0.1% FA at a concentration of 1 μg / ml or higher. Samples were directly injected at a rate of 1 μL / min. The protonated mass of the compounds was monitored.

[0098] For MALDI-TOF MS, the sample was mixed with alpha-cyano-4-hydroxycinnamic acid matrix (10 mg / ml) at a 1:1 ratio and dried on the MALDI-TOF plate. Samples were analyzed in reflectance mode with low laser power settings (instrument: ultrafleXtreme TOF / TOF (Bruker, Billerica, MA); software: flexControl (version 3.4.135.12) / flexAnalysis (version 3.4.78.0)) (laser: repetition rate - 1000 Hz; shot count - 1000; beam focus - 6; beam attenuation - 70) (spectrometer: polarity - anode voltage polarity; PIE delay - 120 ns; reflector detector voltage - 2.625 kV; lens voltage - 7.8 kV; ion source 1 - 20 kV; ion source 2 - 17.85 kV). Samples were scanned over the 650-3000 Da range. MS data obtained from MALDI-TOF showed high signal intensities for the sodium (+22.98 Da) and potassium (+38.96 Da) adducts. The protonated sample showed lower signal intensities compared to these two adducts. Observation of protonated osidiofundin mutants with ASN1 instead of BHN1 was not always clear due to the weaker signal intensities compared to the adducts.

[0099] nuclear magnetic resonance (NMR) A 3-4 mg sample of osidiofulvin was dissolved in 600 μl of dimethyl sulfoxide (DMSO-d6; Cambridge Isotopes), and NMR data were collected as previously reported (References 6, 7). 1 H resonances were assigned using correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY) experiments according to standard methods (ref. 8). Rotating system Overhauser enhanced spectroscopy (ROESY) and 13C-heteronuclear single quantum correlation (HSQC) experiments are used to resolve ambiguous regions in TOCSY and NOESY spectra. Spectral sweep widths for TOCSY, NOESY, and ROESY are typically 11.35 ppm in both dimensions. HSQC sweep widths are typically 11.35 ppm in the proton dimension and 100 and 150 ppm in the carbon dimension. All two-dimensional (2D) data are collected at 2,048 complex points in the acquisition dimension and 256 complex points in the indirect dimension, except for HSQC, which collects 2,048 complex points in the direct dimension and 128 complex points in the indirect dimension. Data are processed with nmrPipe (Ref. 9) and analyzed with the interactive computer program NMRView (Ref. 10).

[0100] Overlay assay Yeast extract peptone dextrose (YPD) medium plates (20 g / L peptone, 20 g / L dextrose, 10 g / L yeast extract, 15 g / L agar) were overlaid with YPD soft medium agar (20 g / L peptone, 20 g / L dextrose, 10 g / L yeast extract, 7.5 g / L agar). In the state containing (OD 600 (=0.15, then 25-fold dilution) and allow to solidify. Spot the compound in solution onto the plate and allow it to dry. To compare different compounds, spot equal volumes onto the plate. Incubate the plate at 35°C for 24 hours. Then observe the growth inhibition zone on the plate.

[0101] Minimum inhibitory concentration (MIC) measurement MIC assays were performed in 96-well plates in YPD growth medium or YPD growth medium supplemented with human or mouse serum, using a solution of 20% serum and 80% growth medium (serum MIC) according to a modified CLSI M27-A3 method. Plates were incubated at 35°C for 24 hours, and MIC values ​​were determined by examining individual wells for the presence of fungi.

[0102] Production of semisynthetic analogues To prepare analogs of ASP7 mutants, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was used as a condensation reagent to generate various amides at the carboxyl group of ASP7 (Figure 5A). OCF-I and OCF-J were incubated in N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) at a 10-fold molar excess of the target amine, EDC, and 1-hydroxybenzotriazole (HOBt) for at least 16 hours at room temperature, protected from light. Depending on the amine used, particularly larger amines, longer incubation times or incubation at 35°C have been observed to improve efficiency. Under the described conditions, the reaction did not proceed beyond 4 hours of incubation. Primary amines with chain lengths up to 12 carbons (dodecylamine) have been used successfully in this reaction. Two primary amines with terminal alkyne groups, 3-butyn-1-amine and propargylamine, have also been used successfully in this reaction. Furthermore, by using a GLU7 mutant, it is possible to generate an amide at the carboxyl group of GLU7 (Figure 5B). The reaction conditions are the same as for the ASP7 mutant.

[0103] Direct EDC condensation using 3-amino-1-propanol was not achieved. This is likely due to the alcohol group present in the compound. The alcohol group of 3-amino-1-propanol was protected with tert-butyldimethylsilyl (TBS) by adding 1 molar equivalent of 3-amino-1-propanol, triethylamine, and tert-butyldimethylsilyl chloride (TBS-Cl) in dichloromethane (DCM). The reaction mixture was mixed and incubated at room temperature for at least 4 hours. The oily layer formed after incubation contained the protected 3-amino-1-propanol and was used as the amine in the EDC condensation reaction. The generation of phosphate prodrug designs OCF-S21 and OCF-S22 also required TBS protection of 3-aminopropyl-dihydrogenphosphate. 1 molar equivalents of 3-aminopropyl-dihydrogenphosphate, triethylamine, and TBS-Cl were incubated in DCM. The reaction mixture is mixed and an equal volume of 35% acetonitrile / 0.1% trifluoroacetic acid is added, followed by incubation at room temperature for at least 4 hours. The upper aqueous layer contains the protected 3-aminopropyl-dihydrogen phosphate, which is used in the subsequent EDC condensation reaction.

[0104] To prepare the methyl and methyl succinate prodrugs, Boc-3-amino-1-propanol was first coupled with methyl or benzyl succinate. To anhydrous dimethylformamide (DMF) were added 3-amino-1-propanol, N,N'-dicyclohexylcarbodiimide (DCC), 1 molar equivalent of methyl or benzyl succinate, and 0.1 molar equivalent of 4-dimethylaminopyridine (DMAP). The reaction was stirred at room temperature for at least 12 hours. The by-product, dicyclohexylurea, was observed as a white precipitate. The solution was filtered through 2-micron filter paper to remove the by-product. Saturated ammonium chloride solution was added to the reaction mixture, and the reaction mixture was extracted twice with ethyl acetate. The combined ethyl acetate extracts were dried over anhydrous magnesium sulfate. The ethyl acetate was then filtered through 2-micron filter paper and dried on a rotary evaporator. The residue, the product, was placed in a 20% trifluoroacetic acid / 80% dichloromethane solution and left at room temperature for 24 hours. A 1 molar excess of saturated sodium bicarbonate was then added to neutralize the TFA. The reaction solution was extracted twice with ethyl acetate, and the combined ethyl acetate extracts were dried over anhydrous magnesium sulfate and filtered through a 2-micron filter. The ethyl acetate was evaporated to dryness using a rotary evaporator, taken up in ethanol as a stock solution, and stored at -20°C. The final product was confirmed by mass spectrometry. The synthesized EDC condensation reaction product was diluted to 1 ml with 35% ACN / 0.1% TFA, cooled, and loaded onto a Supercell ODS2 5 μm (4.6 mm x 250 mm) column. The resulting peaks were collected and analyzed by MALDI-TOF or ESI-MS to confirm the expected product.

[0105] OCF-S21 / OCF-S22 phosphate prodrug activation OCF-S21 / OCF-S22 B. contaminans MS14Phosphate removal in OCF-S19 / OCF-S20 was performed using calf intestinal alkaline phosphatase (Quick CIP; New England Biolabs). For the overlay assay, 5 μg of OCF-S21 / OCF-S22 (approximately 5000 pmol) was incubated with 0.25 μl of alkaline phosphatase, 0.5 μl of rCutSmart® buffer (1X), and 3.25 μl of ddH2O. The reaction mixture was incubated at 37°C for at least 90 minutes. Phosphate removal to generate OCF-S19 / OCF-S20 was confirmed by MALDI-TOF MS and the overlay assay described above.

[0106] Minimum inhibitory concentration (MIC) assays were performed according to a modified CLSI M27-A3 method using YPD growth medium. To each well, 10 μl of alkaline phosphatase and 2 μl of rCutSmart® Buffer (10X) were added, bringing the final volume per well to 200 μl. A negative control containing 10 μl of alkaline phosphatase and 2 μl of rCutSmart® Buffer (10X) but no OCF-S21 / OCF-S22 showed no inhibition of fungal growth.

[0107] All patents, patent applications, provisional applications, and literature referred to or cited herein are incorporated by reference in their entirety, including all figures and drawings, to the extent not inconsistent with the explicit teachings of this specification.

[0108] The following examples illustrate procedures for carrying out the present invention and should not be construed as limiting. Unless otherwise specified, all percentages are by weight and all solvent mixture proportions are by volume.

[0109] Example 1 - Aspartic Acid 7-Osidiofulvin Osidiofudin was used as detailed in the Methods section. S. cerevisiae DGY6 It is extracted from MS14 culture medium. Osidiofudin is a nonribosomally produced cyclic glycopeptide consisting of eight amino acids (Figure 1). Briefly,in vitro MS14 culture medium was pumped through Amberlite XAD1180 resin to collect osidiofundin and other substances. Osidiofundin was eluted from the resin using 80% isopropanol (IPA) containing 0.1% trifluoroacetic acid (TFA). The 80% IPA 0.1% TFA was dried and placed in a solution of 35% acetonitrile (ACN) 0.1% TFA. This solution was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using ACN with 0.1% TFA and double-distilled water (ddH2O). Osidiofundin eluted as two peaks between 45 and 43% ddH2O 0.1% TFA. These peaks were collected and further purified using a mobile phase of ACN and 10 mM triethylamine (0.1% FA). This method allowed for the separation of multiple osidiofundin mutants (Figure 2). Peaks 2 and 3 on the chromatogram contain OCF variants with asparagine (OCF-A and OCF-B) or glutamine (OCF-K and OCF-L), which NMR structural analysis indicates are located at residue 7. Peaks 4 and 5 contain OCF variants with aspartic acid (OCF-I and OCF-J) or glutamic acid (OCF-M and OCF-N) at residue 7. Table 1: Shows the variants eluting from the separated peaks in Figure 2. [Table 1]

[0110] High-resolution mass spectrometry, which provides accurate predictions of elemental composition, showed that mutants of native osidiofudin contain aspartic acid (ASP), glutamic acid (GLN), and glutamic acid (GLU) instead of aspartic acid (ASN) (Figures 3A-3B, Table 2). NMR data from two samples confirmed the presence of ASP at position 7 (Figures 3A-3B). Samples containing mixtures of OCF-A / OCF-B and OCF-I / OCF-J (Figures 3A-3B) showed the presence of a spin system corresponding to ASP7 in the TOCSY fingerprint region. The fingerprint region is useful for assigning amino acid identity, and H N The spin system with a shift of 8.48 ppm was assigned as ASN7 in a previous study (Reference 7) (Figure 3A). ASN and ASP have similar proton shifts, but ASP lacks a proton shift corresponding to the delta proton of the terminal amide side chain found in the ASN residue. This new spin system does not show the NOESY proton shift from the beta proton to the delta proton of ASN, and the new spin system was used to assign it as ASP (Figure 3B). H of the ASP spin system α and H β From SER8 H N to, and Gly6 H α from the ASP spin system H N These NOEs are at 8.41 and 8.32 ppm H N This is also seen in the ASN7 spin system with GLN7 and GLU7 residues, helping to confirm that the ASP residue is at position 7. HRMS data identified OCF-L and OCF-N with GLN7 and GLU7 residues (Figures 3A-3B, Table 2). TOCSY data revealed that H N A new spin system was identified, assigned as GLN7 at a shift of 8.30. This spin system contains the alpha, beta, epsilon, and gamma protons corresponding to GLN. α and H β From SER8 H N NOE to be observed.

[0111] Table 2. Proton shifts for samples containing mixtures of OCF-A / OCF-B and OCF-I / OCF-J. The newly assigned spin system for ASP7 is shown, as is the newly assigned spin system for GLN7. Chemical shift values ​​correspond to the NMR data shown in Figures 3A-3B. [Table 2-1] [Table 2-2]

[0112] H N The GLY6 spin system at a shift of 7.71 was also assigned (Figure 3A). α From H N The NOE to GLN7 supports the position of GLN7 at position 7. Due to the low concentrations of GLN7 and the GLU7 variant, identification by NMR is difficult due to the low intensity of the proton shifts and overlap with the proton shifts of more variants. The GLU7 spin system is unassigned, but HRMS data and chemical modifications support the presence of the GLU7 variant. TOCSY and NOESY analyses were also used to assign spin systems corresponding to previously identified amino acids in osidiofundin (References 7, 8). The TOCSY data contained a small number of unassigned spin systems, likely corresponding to solvent-borne contaminants, the purification process, or trace amounts of osidiofundin variants. HSQC data were utilized to assign the proton shifts of the NAA2 side chain and the xylose sugar.

[0113] Osidiofudin (from the same batch used for NMR analysis) was subjected to an EDC-mediated condensation reaction with methylamine to produce OCF-S3 and OCF-S4 (Figures 4A-4B, Table 4). TOCSY of the fingerprint region showed the disappearance of the ASP7 spin system (Figure 4A, Table 3). EDC condensation with methylamine was the most efficient reaction, and MS data showed that all OCF-I / OCF-J was converted to OCF-S3 / OCF-S4, which is consistent with the disappearance of the ASP7 spin system. NA new spin system with a π of 8.40 exists, slightly overlapping with another spin system assigned as ASN7 (Figure 4A, Table 3). Both the ASN7 and GLN7 spin systems are visible in TOCSY, indicating that they are unchanged by the EDC condensation reaction. The new spin system is assigned as modified ASP7 with a terminal N-methylamide. The N-methylamide on ASP7 is H α and H β The proton shift in the N-methylamide 7 spin system is similar to that of ASP7. This is because this part of the amino acid is unchanged. α and H β From SER8 H N NOE to, and H of Gly6 α from the ASP spin system H N NOEs to H are observed (Figure 4B). β NOEs from the γ-NH and C6-CH3 terminal methyl groups are observed. The terminal methyl amide has two spin systems: the γ-NH proton shifts are 7.87 and 7.82, and the C6-CH3 proton shifts are both 2.56. HSQC NMR shows the C13 shift of the CH3 on the amide nitrogen is 26.11. TOCSY data also reveals three new spin systems for the terminal amine of DABA5 (Figure 4A). This suggests that DABA5 may be involved in byproduct generation in the EDC condensation reaction, and further optimization of the reaction is required. No significant changes in the spin systems of the other amino acids in the compound were observed. HSQC data were utilized to assign proton shifts for the NAA2 side chain and the xylose sugar.

[0114] Table 3. Proton shifts for OCF-S3 and OCF-S4 samples generated by EDC coupling of OCF-I and OCF-J (Figure 5A). The ASP7 spin system is no longer present. The spin system assigned to N-methylamide ASP7 is present, and ASP7 has been modified by EDC condensation with methylamine. A proton shift of 2.56 ppm is observed, corresponding to the terminal carbon of the side chain. The chemical shift values ​​are consistent with the NMR data shown in Figures 4A-4B. [Table 3-1] [Table 3-2]

[0115] High-resolution mass spectrometry of osidiofudin also reveals a mass corresponding to OCF-K / OCF-L or OCF-M / OCF-N (GLU), which contain glutamine (GLN) at residue 7 (Table 4). During HPLC purification, OCF-K / OCF-L coelutes with OCF-A / OCF-B, while OCF-M / OCF-N coelutes with OCF-I / OCF-J. Glutamine and glutamic acid have one more carbon atom in their side chains compared to asparagine and aspartic acid, respectively. Given the structural similarity between the amino acids at residue 7 in the mutants, the nonribosomal peptide synthetase responsible for adding this residue (OcfD) is likely able to accommodate structurally related amino acids. Nonribosomal peptide synthetases may exhibit nonspecific but preferential binding to certain amino acids (Reference 15). OcfD preferentially binds asparagine but may also bind aspartic acid, glutamine, or glutamic acid. At the time of NMR analysis of the thioesterase ocfN mutants, the existence of ASP7, GLN7, and GLU7 mutants was unknown (Reference 16). OcfN was shown to recognize ASN1 to generate OCF-A, but it is possible that it recognizes ASP7, GLN7, or GLU7 to generate other mutants.

[0116] Table 4: Naturally occurring osidiofundin variants identified by HRMS, with their descriptions and predicted elemental compositions. The descriptions predict the structure of the variants and are supported by chemical modifications, NMR, and HRMS. [Table 4]

[0117] Table 5: Semisynthetic osidiofundin analogs produced using OCF-I and OCF-J variants. Description, theoretical mass, and observed mass by MALDI-TOF or ESI-MS are shown. The theoretical mass corresponds to the predicted protonated mass. Some of the theoretical masses are not observed using MALDI-TOF MS because the sodium and potassium adducts exhibit higher signal intensities than the protonated compounds. Semisynthetic analogs that can also be produced by EDC coupling using OCF-M and OCF-N, which contain glutamic acid at position 7, are not listed. [Table 5-1] [Table 5-2]

[0118] Example 2 - Semisynthetic Analogues of Aspartic Acid 7 EDC condensation is carried out by incubating OCF-I and OCF-J with EDC, hydroxybenzotriazole (HOBt), and a 10 molar excess of the selected amine in dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) (Figure 5A). EDC condensation can also be carried out with the OCF-M and OCF-N variants (Figure 5B). The reaction mixture is incubated and purified by RP-HPLC as described in the Methods section.

[0119] The modification itself may alter the retention time during RP-HPLC. Condensation reactions were successful with several primary amines, including one secondary amine (dimethylamine) (Figure 5A). Benzylamine condensation was unsuccessful, even at high temperatures and long reaction times, likely due to steric hindrance from the benzene ring. Recovery of OCF-A and OCF-B from OCF-I and OCF-J was also possible by EDC-mediated condensation with ammonia. The larger amines used in condensation increase the hydrophobicity of the analogs, eluting at higher acetonitrile concentrations in the HPLC gradient. Mass spectrometry (ESI-MS or MALDI-TOF) confirmed the expected masses of the analogs (Table 5). NMR analysis of the analogs, OCF-S3, and OCF-S4, confirmed their structures as modified forms of ASP7 (Figures 3A-3B and 4A-4B). EDC condensation reactions were performed on OCF-A and OCF-B without the carboxylic acid, but the analogs were formed, confirming the specificity of this chemical reaction.

[0120] The minimum inhibitory concentration (MIC) test was performed according to the CLSI M27-A3 method using three indicator strains: B. contaminans DGY6, B. contaminans ATCC3147, S. cerevisiae ATCC 2001. The MIC values ​​of ASP7 osidiofundin, OCF-I, and OCF-J were 1-2 μg / ml, and more active semisynthetic analogs can be synthesized from OCF-I and OCF-J.

[0121] OCF-S3 and OCF-S4, which used methylamine in the EDC condensation reaction, showed MIC values ​​two-fold lower than those of OCF-I and OCF-J. OCF-S9 and OCF-S10, which used propylamine in the EDC condensation reaction, also showed comparable MICs. Interestingly, EDC condensation using ethylamine showed MIC values ​​of 1–2 μg / ml, which is comparable to those of OCF-I and OCF-J. A linear relationship is expected between the size of the amine used and the observed change in antifungal activity. However, the MICs of OCF-S7 and OCF-S8 (ethylamine) did not show this pattern, with OCF-S9 and OCF-S10 (propylamine) showing a 4–8-fold increase in MIC compared to OCF-S9 and OCF-S10. The larger primary amines dodecylamine (MW = 185.35 g / mol) and undecylamine (MW = 171.32 g / mol) were successfully used to generate the analogs OCF-S11 / OCF-S12 and OCF-S13 / OCF-S14. OCF-S13 and OCF-S14 were C. albicans showed an MIC value of 8 μg / ml against β-glucan, indicating that this modified region is amenable to a large structural modification repertoire that still allows for low micromolar levels of inhibitory activity (Table 6).

[0122] EDC-mediated condensation of OCF-I and OCF-J with ammonia yielded OCF-A and OCF-B mutants (designated OCF-S1 and OCF-S2), which converted ASP7 to ASN7 (Table 6). The reaction mixture of OCF-S1 and OCF-S2 showed a 2-fold improvement in inhibitory activity compared to OCF-I and OCF-J, but did not restore the activity levels reported for the native OCF-A and OCF-B mutants (Table 6). ESI-MS data from the ammonia reaction revealed recovery of the OCF-A and OCF-B mutants, but also produced additional products with masses of 1183.56 Da and 1998.57 Da (OCF-S15 and OCF-S16). The masses of these mutants in the reaction correspond to OCF-S15 and OCF-S16, which are undesired dehydration byproducts of OCF-I and OCF-J. These dehydration by-products had higher MIC values ​​compared to OCF-I and OCF-J, resulting in differences in the MIC values ​​reported for reaction mixtures containing OCF-S1, OCF-S2, OCF-S15, and OCF-S16 (Table 6).

[0123] Table 6. Growth of natural osidiofundin mutants and semi-synthetic osidiofundin analogues in unsupplemented YPD medium C. glabrata DGY6, C. glabrata ATCC3147, S. cerevisiae MIC values ​​against ATCC2001. OCF-I / OCF-J showed higher MIC values ​​than OCF-S1 / OCF-S2, OCF-S3 / OCF-S4, OCF-S5 / OCF-S6, OCF-S9 / OCF-S10, and OCF-S19 / OCF-S20. The MIC values ​​of OCF-I / OCF-J were equal to those of OCF-S7 / OCF-S8. C. albicans The MICs for DGY6 were lower than those for OCF-S11 / OCF-S12, OCF-S13 / OCF-S14, and OCF-S21 / OCF-S22. ND means not determined. The MICs for OCF-S1 and OCF-S2 were determined using a sample containing a mixture of OCF-S1 / OCF-S2 and OCF-S15 / OCF-S16, because they could not be separated chromatographically when ammonia was used as the amine after the EDC reaction with OCF-I and OCF-J. [Table 6]

[0124] EDC condensation of OCF-I and OCF-J variants can also be performed with amines bearing terminal functional groups for downstream synthesis of various analogs. 3-Amino-1-propanol was selected to generate analogs bearing a terminal alcohol group at the ASP7 residue (OCF-S19 and OCF-S20). Condensation was also successful with primary amines as large as dodecylamine, suggesting that the size of the amine does not hinder the reaction. The alcohol group of 3-amino-1-propanol was protected by reaction with tert-butyldimethylsilyl (TBS) chloride, and this protected compound was successfully used in EDC condensation to generate OCF-S19 and OCF-S20. These findings suggest that analogs of 3-amino-1-propanol can also participate in EDC condensation reactions with OCF-I and OCF-J (Figure 6A). The MICs of OCF-S19 / OCF-S20 were tested, revealing a two-fold decrease in MIC compared to OCF-I and OCF-J (Table 6). The analogs exhibit improved activity compared to ASP7 osidiofulvin while maintaining submicromolar activity, making them potential linkers for the synthesis of metabolizable prodrug compounds (Figure 6B).

[0125] MIC assays using human serum can be an indicator of a compound's efficacy in systemic infections, but drugs tested in the presence of serum typically exhibit reduced activity due to plasma-binding proteins. Semisynthetic analogs generated by EDC condensation with primary amines showed 2- to 8-fold increases in MIC values ​​upon serum addition (Table 7). The MIC values ​​of OCF-S19 and OCF-S20 increased 4- to 8-fold upon serum addition (Table 7). These values ​​are comparable to the increases in MIC values ​​of OCF-A and OCF-B in serum.

[0126] Table 7. Osidiofundin mutants and analogs in YPD growth medium supplemented with 20% human serum C. glabrata DGY6, S. cerevisiae ATCC3147, and S. cerevisiaeMIC values ​​against ATCC2001. The numbers in parentheses indicate the fold increase compared to the MIC value in serum-free YPD medium. The MIC values ​​of osidiofundin semi-synthetic analogs increased 2- to 8-fold in serum-supplemented medium. [Table 7]

[0127] OCF-S19 and OCF-S20 are C. albicans OCF-S19 and OCF-S20 were selected as the final metabolites for the prodrug analogs because they exhibit potent antifungal activity in the prodrug analogs. The terminal alcohol group acts as a linker arm to the prodrug functional group, which is metabolized to release the products OCF-S19 and OCF-S20 (Figure 6B). The prodrug functional groups considered include phosphate, methyl succinate, and benzoate. The current lead design is the phosphate prodrug analogs OCF-S21 and OCF-S22, which are metabolized by phosphatase enzymes to generate OCF-S19 and OCF-S20 (Figure 6C). The generation of OCF-S19 and OCF-S20 by phosphate removal from OCF-S21 and OCF-S22 was confirmed by MALDI-MS. Fosfluconazole is an example of a phosphate-based antifungal prodrug that is metabolized to fluconazole by nonselective host alkaline phosphatase (Refs. 17, 18). Fosmanogepix, another phosphate prodrug antifungal agent, recently passed a phase II clinical trial for the treatment of candidemia (Reference 19). The MICs of OCF-S21 and OCF-S22 were 16 μg / ml, 16-fold higher than those of OCF-I and OCF-J. This significant increase in MIC is expected to be due to the addition of a phosphate group. The phosphate group was removed from OCF-S21 and OCF-S22 using nonspecific alkaline phosphatase (Quick CIP; New England Biolabs) to generate OCF-S19 and OCF-S20. This was confirmed by MALDI-TOF MS. C. glabrataThe activity of the prodrugs was determined using an overlay assay with DGY6 (Figure 7). The inhibition zones of OCF-S19 and OCF-S20 (Figure 7, spot A) served as positive controls. OCF-S21 and OCF-S22 (Figure 7, spot D) had smaller inhibition zones compared to OCF-S19 and OCF-S20. When alkaline phosphatase-added OCF-S21 and OCF-S22 were directly spotted onto the plate, no increase in the inhibition zone was observed because the enzyme had not yet removed the phosphate group (Figure 7, spot E). Incubating the enzyme with OCF-S21 and OCF-S22 at 37 °C for 90 min resulted in an inhibition zone size comparable to that of OCF-S19 and OCF-S20 (Figure 7, spot F). The alkaline phosphatase reaction mixture was directly spotted onto the plate. Postincubation did not have any inhibitory effect on fungal growth (Figure 7, spots B and C). Incubation of OCF-S21 and OCF-S22 with the enzyme resulted in a four-fold decrease in MIC values ​​compared to OCF-S21 and OCF-S22 alone (Table 6). The decrease in MIC values ​​indicates that phosphate was removed by the enzyme for antifungal activity. The MIC values ​​of OCF-S21 and OCF-S22 with alkaline phosphatase did not reach the same levels as those of OCF-S19 and OCF-S20, which may be due to limitations of the medium or suboptimal assay conditions. This data supports the premise that alkaline phosphatase present in blood activates prodrugs over time, supporting the systemic formulation of prodrugs for the treatment of systemic infections and cancer.

[0128] Phosphorylated compounds may have enhanced serum activity and pharmacokinetic activity. in vitro S. cerevisiae in vitroThe activity in mouse serum at 1000 kJ / kg / day is important because any prodrugs must first be tested in rodent or other small animal models. The alkaline phosphatase used to activate OCF-S21 and OCF-S22 to OCF-S19 and OCF-S20 was confirmed to be nonspecific, suggesting that dephosphorylation of OCF-S21 and OCF-S22 is facilitated by various nonspecific phosphatases.

[0129] Additional prodrugs were designed by attaching methyl succinate and benzyl succinate moieties to the terminal alcohol groups of OCF-S19 and OCF-S20. To generate the methyl and methyl succinate prodrugs, 3-amino-1-propanol was first attached to methyl or benzyl succinate. tert-Butoxycarbonyl (Boc) 3-amino-1-propanol was then attached to methyl or benzyl succinate using N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), followed by removal of the Boc group. The purified intermediate compounds were then used in EDC condensation to generate prodrugs of osidiofundin. Modification of the aspartic acid 7 or glutamic acid 7 at the 7-position of osidiofundin is the most suitable point for generating novel analogs of the parent molecule. EDC-mediated condensation reactions are an efficient method for producing large libraries of semisynthetic analogs. EDC condensation can also be used to generate a variety of linkers and functional groups, allowing for the generation of a wide range of analogs. Further expansion and screening of the analogs may lead to the discovery of one or more analogs that are active in serum or other biological matrices.

[0130] The discovery of OCF-I, OCF-J, OCF-M, and OCF-N provides favorable targets for the synthesis of analogs, which can be selectively generated by an efficient and convenient single-step reaction without the need for protection and deprotection. Furthermore, modification of the terminal side groups of these residues does not significantly affect their antifungal activity (Table 6).

[0131] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art may suggest various changes or modifications based thereon, which are within the principles and scope of this application and the scope of the appended claims. In addition, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any other element or limitation (single or in any combination) or with any other invention or embodiment thereof disclosed herein, and all such combinations are considered to be within the scope of the present invention, without limitation.

[0132] References 1. Lu, SE et al. Occidiofungin, a unique antifungal glycopeptide produced by a strain of Burkholderia contaminans. Biochemistry 48, 8312-8321 (2009). 2.Ravichandran, A. et al. A novel actin binding drug with in vivo efficacy. Antimicrob. Agents Chemother. 63, 1-13 (2019). 3.Ravichandran, A. et al. Formulation, Pharmacological Evaluation, and Efficacy Studies of Occidiofungin, a Novel Antifungal. Antimicrob. Agents Chemother. 64, (2020). 4.Hing, SL et al. Toxicological Evaluation of Occidiofungin against Mice and Human Cancer Cell Lines. Pharmacol. & Pharm. 05, 1085-1093 (2014). 5.Ma, J. et al. Novel antiparasitic activity of the antifungal lead occidiofungin. Antimicrob. Agents Chemother. 64, (2020). 6.Gu, G., Smith, L., Liu, A. & Lu, S. E. Genetic and biochemical map for the biosynthesis of occidiofungin, an antifungal produced by Burkholderia contaminans strain MS14. Appl. Environ. Microbiol. 77, 6189-6198 (2011). 7.Ravichandran, A., Gu, G., Escano, J., Lu, S. E. & Smith, L. The presence of two cyclase thioesterases expands the conformational freedom of the cyclic peptide occidiofungin. J. Nat. Prod. 76, 150-156 (2013). 8.Wuthrich, K. NMR of Proteins and Nucleic Acids. 336-361 (1986) doi:10.1039 / 9781847555236-00336. 9.F, D. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-293 (1995). 10.Johnsona, B. A. & Blevinsb, R. A. NMRView: A computer program for the visualization and analysis of NMR data*. J. Biomol. NMR 4, (1994). 11.Izore, T. et al. Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity. Nat. Commun. 2021 121 12, 1-14 (2021). 12.Calcott, M. J., Owen, J. G. & Ackerley, D. F. Efficient rational modification of non-ribosomal peptides by adenylation domain substitution. Nat. Commun. 2020 111 11, 1-10 (2020). 13.Tavares, M. et al. Adaptation and Survival of Burkholderia cepacia and B. contaminans During Long-Term Incubation in Saline Solutions Containing Benzalkonium Chloride. Front. Bioeng. Biotechnol. 8, 630 (2020). 14.Sass, A. M. et al. The unexpected discovery of a novel low-oxygen-activated locus for the anoxic persistence of Burkholderia cenocepacia. ISME J. 7, 1568 (2013). 15.Morgan, G. L. et al. Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor. Biochemistry 58, 5249-5254 (2019). 16. Ravichandran, A., Gu, G., Escano, J., Lu, S.-E. & Smith, L. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin. J. Nat. Prod. 76, 150 (2013). 17.Bentley, A. et al. The discovery and process development of a commercial route to the water soluble prodrug, fosfluconazole. Org. Process Res. Dev. 6, (2002). 18. Aoyama, T. et al. Population pharmacokinetics of fluconazole after administration of fosfluconazole and fluconazole in critically ill patients. J. Clin. Pharm. Ther. 37, (2012). 19. Pappas, P. et al. 147. Clinical Safety and Efficacy of Novel Antifungal, Fosmanogepix, in the Treatment of Candidemia: Results from a Phase 2 Proof of Concept Trial. Open Forum Infect. Dis. 7, (2020). [Sequence List Free Text]

[0133] A brief description of the sequence SEQ ID NO: 1: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-β-BHY4-DABA5-Gly6-Asn7-Ser8]. SEQ ID NO: 2: Sequence of the cyclic osidiofundin antibiotic: β-hydroxyAsn1-3amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asn7-Ser8]. SEQ ID NO: 3: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-chloro-BHY4-DABA5-Gly6-Asn7-Ser8]. SEQ ID NO: 4: Sequence of the cyclic osidiofundin antibiotic: β-hydroxyAsn1-3amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-chloro-BHY4-DABA5-Gly6-Asn7-Ser8]. SEQ ID NO: 5: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6,7-trihydroxy-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asn7-Ser8]. SEQ ID NO: 6: Sequence of the cyclic osidiofundin antibiotic. β-HydroxyAsn1-3amino-5,6,7-trihydroxy-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asn7-Ser8]. SEQ ID NO: 7: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6,7-trihydroxy-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asp7-Ser8]. SEQ ID NO: 8: Sequence of the cyclic osidiofundin antibiotic. β-HydroxyAsn1-3amino-5,6,7-trihydroxy-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asp7-Ser8]. SEQ ID NO: 9: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asp7-Ser8]. SEQ ID NO: 10: Sequence of the cyclic osidiofundin antibiotic. β-HydroxyAsn1-3amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Asp7-Ser8]. SEQ ID NO: 11: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Gln7-Ser8]. SEQ ID NO: 12: Sequence of the cyclic osidiofundin antibiotic. β-HydroxyAsn1-3amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Gln7-Ser8]. SEQ ID NO: 13: Sequence of the cyclic osidiofundin antibiotic. Asn1-3 amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Glu7-Ser8]. SEQ ID NO: 14: Sequence of the cyclic osidiofundin antibiotic. β-HydroxyAsn1-3amino-5,6-dihydroxy-7-O-xylose-octadecanoic acid 2-Ser3-BHY4-DABA5-Gly6-Glu7-Ser8].

Claims

1. An analogue of osidiofundin having formula (I), formula (II), formula (III) or formula (IV). 【Chemistry 1】 In the formula, R １ is H or OH, and R ２ is H or CH ３ and R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , (CH ２ ) １０ CH ３ , (CH ２ ) １１ CH ３ , a terminal alkyne group, a terminal alcohol group, a terminal alcohol group bound to a prodrug functional group, a prodrug functional group, or an antibody. 【Chemistry 2】 In the formula, R １ is H or OH, and R ２ is H or CH ３ and R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , (CH ２ ) １０ CH ３ , or (CH ２ ) １１ CH ３ , a terminal alkyne group, a terminal alcohol group, a terminal alcohol group bound to a prodrug functional group, a prodrug functional group, or an antibody. 【Transformation 3】 In the formula, R １ is H or OH, and Z is an ester, thioester, acid anhydride, acid chloride, amide, or carboxylate ion. 【Chemistry 4】 In the formula, R １ is H or OH, and Z is an ester, thioester, acid anhydride, acid chloride, amide, or carboxylate ion.

2. The osidiofundin analog of claim 1, wherein the osidiofundin analog is represented by formula (I) or formula (II). 【Transformation 5】 In the formula, R １ is H or OH, and R ２ is H and R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , (CH ２ ) １０ CH ３ , (CH ２ ) １１ CH ３ or CH ２ CH ２ CCH; or wherein R １ is H or OH, and R ２ is CH ３ and R ３ is CH ３ is. 【Transformation 6】 In the formula, R １ is H or OH, and R ２ is H and R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , (CH ２ ) １０ CH ３ , (CH ２ ) １１ CH ３ or CH ２ CH ２ CCH; or wherein R １ is H or OH, and R ２ is CH ３ and R ３ is CH ３ is.

3. 2. The osidiofundin analog of claim 1, wherein the osidiofundin analog has formula (V) or formula (VI). 【Transformation 7】 In the formula, R １ is H or OH, and R ２ is a prodrug functional group. 【Transformation 8】 In the formula, R １ is H or OH, and R ２ is a prodrug functional group.

4. 4. The osidiofundin analogue of claim 1 or 3, wherein the prodrug functional group is a phosphate group, a methyl succinate, an ether, or a benzoate ester.

5. The osidiofundin analogue according to claim 3, wherein the osidiofundin analogue is represented by formula (VII) or formula (VIII). 【Chemistry 9】 In the formula, R １ is H or OH. 【Chemistry 10】 In the formula, R １ is H or OH.

6. A pharmaceutical composition comprising the osidiofundin analogue of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. A method for treating or preventing a fungal infection, comprising administering to a subject in need thereof an effective amount of an osidiofundin analog of any one of claims 1 to 5, or an effective amount of the pharmaceutical composition of claim 6.

8. 8. The method of claim 7, comprising administering the osidiofundin analog or the pharmaceutical composition intravaginally, intramuscularly, subcutaneously, intrathecally, intravenously, or intraperitoneally.

9. 9. The method according to claim 7 or 8, wherein the fungal infection is caused by Candida spp., Saccharomyces spp., Trichophyton spp., Rhizopus spp., Mucor spp., Fusarium spp., Aspergillus spp. or Cryptococcus spp.

10. The method of claim 9, wherein the Candida spp. is C. albicans, C. glabrata, C. krusei, C. auris, C. parapsilosis or C. tropicalis.

11. A method for treating or preventing a parasitic infection, comprising administering to a subject in need thereof an effective amount of an osidiofundin analogue described in any one of claims 1 to 5, or an effective amount of a pharmaceutical composition described in claim 6.

12. 12. The method of claim 11, comprising administering the osidiofundin analog or the pharmaceutical composition intravaginally, intramuscularly, subcutaneously, intrathecally, intravenously, or intraperitoneally.

13. 13. The method of claim 11 or 12, wherein the parasitic infection is caused by Cryptosporidium spp.

14. A method for treating or preventing cancer, comprising administering to a subject in need thereof an effective amount of an osidiofundin analogue described in any one of claims 1 to 5, or an effective amount of a pharmaceutical composition described in claim 6.

15. 15. The method of claim 14, comprising administering the osidiofundin analog or the pharmaceutical composition intravaginally, intramuscularly, subcutaneously, intrathecally, intravenously, or intraperitoneally.

16. A method for producing an osidiofundin analog of formula (I) or formula (II), comprising condensing an osidiofundin analog of formula (IX) or formula (X), respectively, with an amine via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). 【Chemistry 11】 In the formula, R １ is H or OH, and R ２ is H or CH ３ and R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , (CH ２ ) １０ CH ３ , or (CH ２ ) １１ CH ３ , a terminal alkyne group, a terminal alcohol group, a terminal alcohol group bound to a prodrug functional group, a prodrug functional group, or an antibody. 【Chemistry 12】 In the formula, R １ is H or OH, and R ２ is NH ２ Or OH. 【Chemistry 13】 In the formula, R １ is H or OH, and R ２ is H or CH ３ and R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , (CH ２ ) １０ CH ３ , or (CH ２ ) １１ CH ３ , a terminal alkyne group, a terminal alcohol group, a terminal alcohol group bound to a prodrug functional group, a prodrug functional group, or an antibody. 【Chemistry 14】 In the formula, R １ is H or OH, and R ２ is NH ２ Or OH.

17. 17. The method of claim 16, wherein the amine is ammonia, methylamine, dimethylamine, ethylamine, propylamine, undecylamine, dodecylamine, 3-butyn-1-amine, 3-amino-1-propanol, protected 3-amino-1-propanol, 3-amino-1-propanol bound to methyl succinate, 3-amino-1-propanol bound to benzyl succinate, or 3-amino-1-propanol bound to a phosphate group.

18. 17. The method of claim 16, further comprising adding 1-hydroxybenzotriazole (HOBt) to the diofundin analog of formula III, EDC, and the amine.

19. 17. The method of claim 16, wherein the condensation reaction is carried out in N,N-dimethylformamide or dimethyl sulfoxide.

20. A method for producing an osidiofundin analog of formula (V) or formula (VI), comprising condensing an osidiofundin analog of formula (IX) or formula (X) with an amine containing a prodrug functional group via EDC to produce an osidiofundin having a terminal alcohol arm. 【Chemistry 15】 In the formula, R １ is H or OH, and R ２ is a prodrug functional group. 【Chemistry 16】 In the formula, R １ is H or OH, and R ２ is a prodrug functional group. 【Chemistry 17】 In the formula, R １ is H or OH, and R ２ is NH ２ Or OH. [Chemistry 18] In the formula, R １ is H or OH, and R ２ is NH ２ Or OH.

21. 21. The method of claim 20, wherein the prodrug functional group is a phosphate group, methyl succinate, or benzyl succinate.

22. 21. The method of claim 20, wherein the amine is 3-amino-1-propanol, protected 3-amino-1-propanol, 3-amino-1-propanol coupled to methyl succinate, or 3-amino-1-propanol coupled to benzyl succinate.

23. 21. The method of claim 20, further comprising adding 1-hydroxybenzotriazole (HOBt) to the osidiofundin analog of Formula III, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the amine containing a prodrug functional group.

24. 21. The method of claim 20, wherein the condensation reaction is carried out in N,N-dimethylformamide or dimethyl sulfoxide.

25. 21. The method of claim 16 or 20, wherein formula (IX) is represented by the following formula: 【Chemistry 19】 In the formula, R １ is H or OH, and R ２ is OH.

26. The method according to claim 16 or 20, wherein formula (X) is represented by the following formula: 【Chemistry 20】 In the formula, R １ is H or OH, and R ２ is OH.

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