An aminophenine analogue, and a preparation method and application thereof

Abstract

The present application discloses an antifungal phenylhydantoin analogue, which is selected from one of the following structures: the antifungal phenylhydantoin analogue is obtained by extraction and separation from fermentation of H. carpetanum. The antifungal phenylhydantoin analogue can be used to prepare an antifungal medicine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an analogue of phenazine and its preparation method and application. Background Technology

[0002] Triterpenes are a large subclass of terpenoids, consisting of a basic carbon skeleton of 30 carbon atoms. Most can be considered as six isoprene monomers linked together, existing in a free state or as glycosides. Most glycosides are water-soluble and are also called triterpenoid saponins. Based on the type of basic carbon skeleton in their molecules, triterpenoids can be classified into acyclic triterpenoids, monocyclic triterpenoids, bicyclic and tricyclic triterpenoids, and tetracyclic and pentacyclic triterpenoids. Triterpenoids are widely distributed in nature and possess important biological activities, such as hemolysis, anticancer, anti-inflammatory, antibacterial, molluscicidal, and antifertility activities. Most triterpenoids are tetracyclic and pentacyclic triterpenoids. Tetracyclic triterpenes are classified into: lanostane, dammarane, triucallane, cycloartane, and cucurbitane; pentacyclic triterpenes are classified into: oleanane, lupane, ursane, friedelane, amniodane, isoamniodane, hopane, and isoparane.

[0003] Fungal infections are becoming a threat to individuals, affecting nearly 100 million people worldwide [8. AM Rauseo, A. Coller-Reilly, L. Larson, A. Spec, Hope on the Horizon: Novel Fungal Treatments in Development, Open Forum Infectious Diseases 2020, 7.]. Fungal infections can be specifically divided into two categories: superficial fungal infections and invasive fungal infections [9. D.D. Wenning, M.J. Bromley, How to bolster the antifungal pipeline, 2015, 347, 1414-1416.]. Conventional antifungal drugs mainly fall into four categories: polyenes, azoles, echinocandins, and flucytosine [10. T.J. Gintjee, M.A. Donnelley, G.R. Thompson, Aspiring Antifungals: Review of Current Antifungal Pipeline Developments, 2020, 6, 28.]. While conventional antifungal drug treatment is effective in the treatment of fungal infections, some treatments have long treatment cycles and are prone to recurrence, such as onychomycosis [11. J. Mohr, M. Johnson, T. Cooper, J. S. Lewis, L. Ostrosky-Zeichner, Current Options in Antifungal Pharmacotherapy, 2008, 28, 614-645.].While these drugs treat diseases, they also reveal some drawbacks, such as drug resistance, pharmacokinetic changes, toxicity, reduced bioavailability, and drug interactions [12. TF Patterson, GRT Hompson, III, DWD Benning, JAFishman, S. Hadley, R. Herbrecht, DPK Ontoyiannis, KA Marr, VA Morrison, MHN Guyen, BHSegal, WJS Teinbach, DA Stevens, TJ Walsh, JR Wingard, J.-AH Young, JEB Nennett, Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America, Clinical Infectious Diseases 2016, 63, e1-e60.]. Furthermore, some adverse events have occurred during the treatment of fungal infections [13. A. Khurana, K. Sardana, A. Chowdhary, Antifungal resistance in dermatophytes: Recent trends and therapeutic implications, Fungal Genetics and Biology 2019, 132, 103255.].In recent years, antifungal resistance has received increasing attention in clinical practice [14. J.A. Hendrickson, C. Hu, S.A. Laitken, N. Beyda, Antifungal Resistance: a Concerning Trend for the Present and Future, Current Infectious Disease Reports 2019, 21, 47.], and resistance to echinocandins has also emerged in major European regions [15. B.D. Alexander, M.D. Johnson, C.D. Feiffer, C. Jiménez-Ortigosa, J. Catania, R. Booker, M. Castanheira, S.A. Messer, D.S. Erlin, M.A. Faller, Increasing Echinocandin Resistance in Candida glabrata: Clinical Failure Correlates With Presence of FKS Mutations and Elevated Minimum Inhibitory Concentrations, Clinical Infectious Disease Reports 2019, 21, 47.]. [Diseases 2013, 56, 1724-1732.] and acquired resistance in azole-resistant strains [16. J.M.F.E., A. Chowdhary, J.L.R. Hodes, M.C.Fisher, P.E. Verweij, Clinical implications of globally emerging azole resistance in Aspergillus fumigatus, 2016, 371, 20150460.]. Antifungal resistance not only presents greater challenges for researchers and pharmaceutical companies in developing new drugs, but also complicates the treatment of patients for physicians. The development of novel antifungal drugs and new treatment strategies is urgently needed.However, in recent years, there has been very little progress in the development of new antifungal drugs [17. L. Scorzoni, ESAC de Paula, CM Marcos, PAAsato, WC de Melo, HC de Oliveira, CB Costa-Orlandi, MJ Mendes-Giannini, AM Fusco-Almeida, Antifungal Therapy: New Advances in the Understanding and Treatment of Mycosis, Frontiers in Microbiology 2017, 8, 36.]. The U.S. Food and Drug Administration (FDA) has only approved two new drugs: Oteseconazole (April 2022) [18. SM Hoy, Oteseconazole: First Approval, Drugs 2022, 82, 1017-1023.] and Ibrexafungerp (June 2021) [19. KN Barnes, AMYancey, AB Forinash, Ibrexafungerp in the Treatment of Vulvovaginal Candidiasis,2023,57,99-106.].

[0004] Ibrexafungerp is one of the semi-synthetic derivatives of the natural triterpenoid glycoside compound enfumafungin. Enfumafungin has very good antifungal activity. Subsequently, in order to improve the oral efficacy and pharmacokinetic properties of enfumafungin, Merck and Scynexis prepared a series of semi-synthetic enfumafungin derivatives [20. RF Hector D.E. Bierer, New β-glucan inhibitors as antifungal drugs, Expert Opinion on Therapeutic Patents 2011, 21, 1597-1610.]. Compared to enfumafungin, ibrexafungerp (formerly known as SCY-078 or MK-3118) was finally successfully developed by replacing the acetoxy group at C-2 with a pyridine triazole group, the glycoside group at C-3 with an amino ether, and the hemiacetal group at C-25 with an ether [21. MAPfaller, SAMesser, MRMotyl, RNJones, M.Castanheira, Activity of MK-3118, a new oral glucan synthase inhibitor, tested against Candida spp. by two international methods (CLSI and EUCAST), The Journal of Antimicrobial Chemotherapy 2013, 68, 858-863. 22. S.Wring, K.Borroto-Esoda, E.Solon, D.Angulo, SCY-078, a Novel Fungicidal Agent, Demonstrates Distribution to Tissues Associated with Fungal Infections during Mass Balance Studies with Intravenous and Oral[(14)C]SCY-078 in Albino and Pigmented Rats, Antimicrob Agents Chemother 2019, 63.]. Ibrexafungerp( 29) Approved by the FDA in June 2021 as the only non-azole drug for the treatment of vulvovaginal candidiasis, which occurs at least once in the lifetime of approximately 75% of women and is primarily caused by Candida albicans infection [23. MC Masone, Ibrexafungerp to treat acute vulvovaginal candidiasis, Nature Reviews Urology 2021, 18, 638-638.]. Ibrexafungerp is an orally active β-(1,3)-glucan synthesis inhibitor and is the first novel antifungal drug approved in the past 20 years. Although ibrexafungerp differs structurally from echinocandins, its mechanism of action is similar to that of echinocandins. It disrupts fungal cell wall formation by non-competitively inhibiting β-(1,3)-D-glucan synthase (GS) [24. S. Jallow N. Govender, Ibrexafungerp: A First-in-Class Oral Triterpenoid Glucan Synthase Inhibitor, Journal of Fungi 2021, 7, 163.]. However, ibrexafungerp and echinocandins do not bind to the same enzyme but rather partially overlap, resulting in very limited cross-resistance between resistant strains [25. C. Jiménez-Ortigosa, W. B. Berez, D. Angulo, K. Borroto-Esoda, D. S. Demerlin, De Novo Acquisition of Resistance to SCY-078 in Candida glabrata Involves FKS Mutations That bothOverlap and Are Distinct from Those Conferring Echinocandin Resistance, 2017, 61, 10. 1128 / aac.00833-00817.].

[0005] Enfumafungin is a natural triterpenoid glycoside compound, first isolated from *Hormonema carpetanum* ATCC 74360. To search for new enfumafungin analogs and explore the biosynthesis of enfumafungin, *H. carpetanum* ACTT 74360 was re-examined in hopes of discovering new analogs. Summary of the Invention

[0006] The first objective of this invention is to provide an analogue of benzophenone.

[0007] A second objective of this invention is to provide a method for preparing the aforementioned phenamine analogue.

[0008] A third object of the present invention is to provide the use of the aforementioned fenofosine analogue in the preparation of antifungal drugs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides an analogue of phenazine, the structure of which is selected from one of the following structures:

[0011]

[0012] The amiphene analogue was extracted and isolated from the fermentation product of the fungus H. carpetanum ATCC 74360.

[0013] A second aspect of the present invention provides a method for extracting the aforementioned phenamine analogue, comprising the following steps:

[0014] The Hormonema carpetanum strain was inoculated into bottles containing seed culture medium and cultured for 3-4 days. The seed culture was then transferred to fermentation medium and cultured for another time to obtain the fermentation broth.

[0015] The above fermentation broth was extracted with an equal volume of ethyl acetate until the extract was basically colorless, and then concentrated under reduced pressure to obtain a crude extract.

[0016] The crude extract was dissolved in an equal volume of methanol, mixed with MCI packing material, and subjected to MCI column chromatography with methanol-water gradient elution to obtain 18 fractions Fr.1-Fr.18.

[0017] Fr.13 was purified by Sephadex LH-20 gel column chromatography and RP-HPLC to obtain enfumafungin B (13.1 mg, tR 14.9 min);

[0018] Fr.14 was purified by Sephadex LH-20 gel column chromatography and RP-HPLC to obtain enfumafungin C (6 mg, tR 13.3 min).

[0019] The conditions for culturing for 3-4 days are: culturing at 25℃ and 220 rpm for 3-4 days.

[0020] The conditions for continued cultivation were: fermentation broth obtained by culturing at 25°C for 28 days.

[0021] The seed culture medium contains 40 g / L maltose, 10 g / L malt extract, and 2.1 g / L peptone per 100 ml.

[0022] The fermentation medium contains 50 g / L rice, 1 g / L malt extract, and 0.5 g / L potassium dihydrogen phosphate per 60 liters.

[0023] The MCI column refers to a column with an inner diameter of 3.8cm and a height of 46cm.

[0024] The conditions for the methanol-water gradient elution were: MeOH / H2O 30%-100%, 25.0 mL / min.

[0025] The 18 components Fr.1-Fr.18 were obtained by detection using an HSGF254 thin-layer chromatography plate with 10% vanillin sulfate for color development.

[0026] The Sephadex LH-20 gel column chromatography conditions were: CH2Cl2 / MeOH, 2:1.

[0027] The RP-HPLC conditions were: 70% MeCN, 2.0 mL / min.

[0028] A third aspect of the present invention provides the use of the aforementioned fenofosine analogue in the preparation of an antifungal medicament.

[0029] The fungi were selected from Candida albicans SC5314, Candida tropicalalis 8915, Candida glabrata 537, Candida krusei 4996, Candida auris 919, Candida auris 918, Candida parapsilosis 22019, and Cryptococcus neoformans 32609.

[0030] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0031] The fenofibrate analogue provided by this invention is an active secondary metabolite discovered in microorganisms and possesses antifungal activity. It exhibits antifungal activity similar to fenofibrate against Candida albicans, Candida tropicalis, Candida glabrata, Candida malariae, Cryptococcus neoformans, and other fungi. Detailed Implementation

[0032] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] Preparation of enfumafungin B and enfumafungin C

[0035] The *Hormonema carpetanum* ATCC 74360 strain (purchased) was inoculated into ten 250 mL Erlenmeyer flasks, each containing 100 mL of seed culture medium (40 g / L maltose, 10 g / L malt extract, 2.1 g / L peptone). The culture was incubated at 25°C and 220 rpm for 3–4 days. The seed culture was then transferred to 60 L of fermentation medium (50 g / L rice, 1 g / L malt extract, 0.5 g / L potassium dihydrogen phosphate) and incubated at 25°C for 28 days to obtain the fermentation broth. The fermentation broth was extracted with an equal volume of ethyl acetate, 4–5 times, until the extract was essentially colorless. The extract was then concentrated under reduced pressure to obtain 40 g of crude extract. The crude extract was dissolved in 10 mL of methanol, mixed with 40 g of MCI packing material, and subjected to MCI column chromatography (column inner diameter 3.8 cm, column height 46 cm) with methanol-water gradient elution (MeOH / H2O 30%-100%, 25.0 mL / min). The fractions were collected according to polarity and detected using an HSGF254 thin-layer chromatography plate with 10% vanillin sulfate as a colorimetric indicator, yielding 18 fractions (Fr.1-Fr.18). Fr.13 was subjected to Sephadex LH-20 (CH2Cl2 / MeOH, 2:1) gel column chromatography and further purified by RP-HPLC (70% MeCN, 2.0 mL / min) to obtain enfumafungin B (13.1 mg, tR 14.9 min). Fr.14 was subjected to Sephadex LH-20 (CH2Cl2 / MeOH, 2:1) gel column chromatography and further purified by RP-HPLC (70% MeCN, 2.0 mL / min) to obtain enfumafungin C (6 mg, tR 13.3 min).

[0036] Structural identification of enfumafungin B and enfumafungin C:

[0037] This invention isolates two novel compounds from the secondary metabolites of *H. carpetanum* ATCC 74360, named enfumafungin B and enfumafungin C. The physicochemical data of these two compounds are as follows:1 H and 13 The C NMR data (Table 1-2) are as follows:

[0038] enfumafungin B: amorphous powder; R f 0.3 and 0.4 (EtOAc / MeOH / H2O 90:10:1); Optical rotation [α] 24.1 D -33 (c 0.4, CH2Cl2); Infrared absorption data IR(film)ν max 33379,2960,2872,1701,1385,1160,1073,1029cm -1 ; 1 H and 13 C10 NMR data are shown in Table 1; High-resolution mass spectrometry (HRESIMS) m / z: 665.3907 [MH]-(calcd for C10) 36 H 57 O 11 ,665.3901),711.3958[M+COOH]-(calcd for C 37 H 59 O 13 ,711.3956),1331.7850[2M-H]-(calcd for C 72 H 115 O 22 ,1331.7880).

[0039]

[0040] enfumafungin C: amorphous powder; R f 0.4 and 0.5 (EtOAc / MeOH / H2O 90:10:1); Optical rotation [α] 24.1 D -21 (c 0.2, CH2Cl2); Infrared absorption data IR(film)ν max 3381,2929,1732,1452,1373,1262,1069,1029cm -1 ; 1 H and 13 C10 NMR data are shown in Table 2; High-resolution mass spectrometry (HRESIMS) m / z: 708.4333 [M+NH4]+ (calcd for C10 NMR). 38 H 62 NO 11,708.4323),1398.8376[2M+NH4]+(calcd for C 76 H 120 NO 22 ,1398.8302),511.3415[M-glucose+H]+(calcd for C 32 H 47 O5,511.3423).

[0041]

[0042] Table 1. Compound enfumafungin B 1 H and 13 C NMR data a

[0043]

[0044]

[0045] a In CD3OD, 600MHz for 1 H and 150MHz for 13 C NMR.

[0046] Table 2. Compound enfumafungin C 1 H and 13 C NMR data a

[0047]

[0048]

[0049] a In CD3OD, 600MHz for 1 H and 150MHz for 13 C NMR.

[0050] Example 2

[0051] The antifungal activity of enfumafungin B and enfumafungin C of the present invention.

[0052] (I) Experimental Methods

[0053] 1. Selection of fungal strains

[0054] Clinical fungi were obtained from the fungal collections of Shanghai Changzheng Hospital (China) and our laboratory. The following eight clinical fungi were selected for antifungal activity against enfumafungin B and enfumafungin C: *Candida albicans* SC5314, *Candida tropicalis* 8915, *Candida glabrata* 537, *Candida krusei* 4996, *Candida auris* (Candida auris 919, Candida auris 918), *Candida parapsilosis* 22019, *Cryptoccus neoformans* 32609, and two fluconazole-resistant fungi: *Candida albicans* 10231 and *Cryptoccus neoformans* H99.

[0055] 2. Antifungal activity detection

[0056] Antifungal activity was determined using a broth microdilution method adjusted according to CLSIM38-A2 and M27-A3 standards. The fungal suspension was applied to RPMI 1640 medium at a concentration of 10... 3 96-well plates with CFU / mL were used. Fluconazole and enfumafungin were selected as positive controls. The test compound, fluconazole, and enfumafungin were serially diluted to a final concentration range of 64 to 0.125 μg / mL. To evaluate efficacy, fluconazole was added to the fungal suspension at a concentration of 8 μg / mL before application to the 96-well plates, while the compound was serially diluted. The 96-well plates were incubated at 30°C for 48 hours. The optical density was then measured at 630 nm using a microplate reader, and background optical density was removed. The minimum inhibitory concentration (MIC80) was the concentration that inhibited the growth of 80% of the test strains.

[0057] 3. Statistical Analysis

[0058] Statistical analysis was performed using GraphPad Prism 8.0. Data were analyzed using one-way ANOVA, followed by Tukey's test to compare the control and blank groups. A p-value <0.05 was considered statistically significant.

[0059] (II) Experimental Results

[0060] The antifungal activities (MIC80 value, μg / mL) of enfumafungin B, enfumafungin C, fluconazole, and enfumafungin are shown in Table 3.

[0061] Table 3

[0062]

[0063] The results in Table 3 show that the compound enfumafungin B of the present invention has antifungal activity against Candida glabrata and Candida malformans; enfumafungin C has antifungal activity similar to that of fentanyl against Candida albicans, Candida tropicalis, Candida glabrata, Candida malformans, Cryptococcus neoformans, etc.; among which enfumafungin C has better activity than enfumafungin B.

[0064] Enfumafungin B and C are structural analogs of fenofungin, while Ibrexafungerp is a semi-synthetic derivative obtained based on the structure of natural fenofungin. To improve the oral efficacy and pharmacokinetic properties of this class of antifungal drugs, researchers are continuously working on the synthesis of second-generation fenofungin-based antifungal drugs [S. Chu, L. Long, TSM McCormick, K. Borroto-Esoda, S. Barat, MAGhannoum, A Second-Generation Fungerp Analog, SCY-247, Shows Potent In Vivo Activity in a Murine Model of Hematogenously Disseminated Candida albicans, Antimicrob Agents Chemother 2021, 65, e01988-20.]. The two novel fenofungin analogs provided in this invention will offer new natural raw materials for the structural modification of second-generation fenofungin-based antifungal drugs. Through structural modification and analog synthesis, they will help obtain new semi-synthetic derivatives with improved oral efficacy and pharmacokinetic properties.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A methamphetamine analogue, characterized in that, The structure is selected from: 。 2. A method for extracting an analogue of phenazine as described in claim 1, characterized in that, Includes the following steps: Will The strain was inoculated into bottles, each containing seed culture medium, and cultured for 3-4 days; the seed culture was then transferred to fermentation medium and cultured for further culturing to obtain fermentation broth. The above fermentation broth was extracted with an equal volume of ethyl acetate until the extract was basically colorless, and then concentrated under reduced pressure to obtain a crude extract. The crude extract was dissolved in an equal volume of methanol, mixed with MCI packing material, and subjected to MCI column chromatography with methanol-water gradient elution to obtain 18 fractions Fr.1-Fr.

18. Fr.13 was purified by Sephadex LH-20 gel column chromatography and then by RP-HPLC to obtain enfumafungin B; Fr.14 was purified by Sephadex LH-20 gel column chromatography and then purified by RP-HPLC to obtain enfumafungin C; The conditions for culturing for 3-4 days are: culturing at 25℃ and 220 rpm for 3-4 days; The conditions for continued cultivation were: fermentation broth obtained by culturing at 25°C for 28 days; The seed culture medium contains 40 g / L maltose, 10 g / L malt extract, and 2.1 g / L peptone per 100 ml. The fermentation medium contains 50 g / L rice, 1 g / L malt extract, and 0.5 g / L potassium dihydrogen phosphate per 60 liters; The MCI column refers to a column with an inner diameter of 3.8 cm and a height of 46 cm; The conditions for the methanol-water gradient elution were: MeOH / H2O 30%-100%, 25.0 mL / min.

3. The method for extracting phenazine analogues according to claim 1, characterized in that, The 18 components Fr.1-Fr.18 were obtained by detection using an HSGF254 thin-layer chromatography plate with 10% vanillin sulfate for color development.

4. The method for extracting phenazine analogues according to claim 1, characterized in that, The conditions for the Sephadex LH-20 gel column chromatography were: CH2Cl2 / MeOH, 2:1; The RP-HPLC conditions were: 70% MeCN, 2.0 mL / min.

5. The use of the phenazine analogue of claim 1 in the preparation of an antifungal medicament, characterized in that, The fungi are selected from Candida albicans, Candida tropicalis, Candida glabrata, Candida malformans, and Cryptococcus neoformans.

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