CN111138421A

CN111138421A - Antifungal water-soluble compound and preparation method and application thereof

Info

Publication number: CN111138421A
Application number: CN201911363413.5A
Authority: CN
Inventors: 刘伟; 刘国强
Original assignee: Shanghai Innofucheng Biotechnology Co Ltd
Current assignee: Shanghai Innofucheng Biotechnology Co Ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-05-12
Also published as: CN112521378A; WO2021129723A1

Abstract

The invention provides an antifungal water-soluble compound, and a preparation method and application thereof. The structural formula is shown as formula I. The compound has good antifungal effect and water solubility, and can be used for preparing an antifungal liquid preparation for treating and preventing clinical fungal infection, and the optical isomer with the SRSS configuration obtained by chiral synthesis is the optimal configuration of antifungal activity.

Description

Antifungal water-soluble compound and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an antifungal water-soluble compound, and a preparation method and application thereof.

Background

Itraconazole (Itraconazole) is a new generation of triazole high-efficiency broad-spectrum antifungal drug, and can be combined with fungal cytochrome P450 isoenzyme to inhibit the synthesis of ergosterol. The product can be used for treating infection of dermatophytosis, Candida, Cryptococcus neoformans, Pityrosporum, Aspergillus, Histoplasma, Paracoccidiosis Brazilian, Trichosporon schenckii, Eumycota, Cladosporium, and Blastomyces dermatitidis.

Itraconazole is insoluble in water and difficult to prepare into a liquid preparation, so that the commercially available itraconazole injection needs to be prepared into the injection by adopting cyclodextrin inclusion and other technologies and a large amount of auxiliary materials, and cyclodextrin compounds have side effects such as hemolysis and the like and have larger potential safety hazards, thereby increasing the risk of clinical use.

Disclosure of Invention

The invention aims to provide a compound, which has a structural formula shown in a formula I:

the compound of the formula I is shown in the specification,

in the above formula I, R₁Can be amino or carboxyl; r₂、R₃Can be independently selected from H, C1-C5 alkyl, halogen or amino substituted C1-C5 alkyl, benzene ring, halogen or amino or hydroxyl substituted benzene ring, or R₂And R₃Cycloalkyl constituting C3-C6, halogen or amino or hydroxy substituted C3-C6 cycloalkyl;

y may be (-CH)₂) n- (i.e. n contiguous methylene groups) where n is an integer from 0to 18, or n (where n is greater than or equal to 2) contiguous methylene groups may have a pair of unsaturated double bonds.

The compound provided by the invention is a racemate of the compound shown in the formula I, or optical isomers of the compound shown in the formula I in various configurations, and specifically can be optical isomers of SRSS configurations shown in the specification;

R₁can be amino or carboxyl; r₂、R₃Can be independently selected from H, C1-C5 alkyl, halogen or amino substituted C1-C5 alkyl, benzene ring, halogen or amino or hydroxyl substituted benzene ring, or R₂And R₃Cycloalkyl constituting C3-C6, halogen or amino or hydroxy substituted C3-C6 cycloalkyl;

y may be (-CH)₂) n-wherein n is an integer of 0to 18, or n (when n is 2 or more) may have a pair of unsaturated double bonds in the methylene groups connected.

In the formula I, R₁When the carboxyl is adopted, the compound provided by the invention also comprises any one of sodium salt, potassium salt, magnesium salt, tromethamine salt, ethanolamine salt and diethylamine salt of the compound shown in the formula I.

In the formula I, R₁When amino, the compounds provided by the invention also compriseAnd (3) medicinal organic acid salts or inorganic acid salts such as hydrochloride, sulfate, phosphate, methanesulfonate, toluenesulfonate and the like of the compound shown in the formula I.

The compound provided by the invention can be one of the following compounds:

n＝16，R₁＝-NH₂，R₂＝H，R₃＝H；

the compound shown in the formula I is prepared by a method comprising the following steps:

carrying out esterification reaction on a compound shown as a formula II and a compound shown as a formula III to obtain a compound shown as a formula I;

in the compound represented by the formula III, R₁、R₂、R₃And Y is as defined for R in formula I₁、R₂、R₃And the definition of Y is given in the following,

in the above method, the molar ratio of the compound represented by formula II to the compound represented by formula III may be: 3-1: 1;

the esterification reaction is carried out under the catalysis of carbodiimide; the carbodiimide may specifically be EDCI;

the esterification reaction is carried out under an alkaline condition, and the esterification reaction is carried out in an organic solvent, wherein the organic solvent can be dichloromethane;

the temperature of the esterification reaction can be 30-60 ℃, and specifically can be 45 ℃;

the esterification reaction time can be 6-15h, and specifically can be 12 h.

In the above process, the compound of formula II can be prepared by the reaction scheme shown in FIG. 1.

The specific operation is as follows:

compound 1(200g,0.75mol), Compound 2(131g, 0.83mol) was dissolved in dimethyl sulfoxide (2000mL), followed by addition of potassium carbonate (312g, 2.26mol), and after the addition was complete, the mixture was warmed to 130 ℃ and stirred overnight. TLC (thin layer chromatography) on the next day detects that the raw materials disappear, the reaction solution is poured into 6000mL of ice water, stirred for 30 minutes, filtered to collect a filter cake, and dried under reduced pressure to obtain 220g of a compound 3;

adding compound 3(50g, 0.160mol), methanol (500mL), acetic acid (125mL) and palladium-carbon (10g) into a 2000mL hydrogenation bottle respectively, carrying out hydrogenation reaction at room temperature under the pressure of 50Psi overnight, monitoring the disappearance of raw materials by TLC, adding 300mL of water and 100mL of concentrated hydrochloric acid into a reaction solution, stirring for 30 minutes, filtering and removing the palladium-carbon through kieselguhr, concentrating the filtrate under reduced pressure to remove the methanol, adjusting the pH of the residue to 13 with sodium hydroxide solution at the cooling temperature of an ice water bath, filtering and collecting a filter cake, and drying under reduced pressure to obtain 40g of compound 4;

adding compound 4(160g, 0.565mol), sodium bicarbonate (95g, 1.131mol), tetrahydrofuran (1600mL) and water (1500mL) into a 5000mL three-necked flask, cooling to0 ℃, slowly dropwise adding a tetrahydrofuran solution of compound 5(126g, 0.622mol) into the reaction solution, and stirring for 2 hours at 0 ℃ after dropwise adding; TLC monitors the disappearance of the raw material, 1500mL of water is added into the reaction solution, the mixture is stirred for 30 minutes, and filter cake is collected by filtration and dried under reduced pressure to obtain 240g of compound 6;

adding compound 6(270g, 0.603mol) hydrazine monohydrate (177g, 3.01mol) and dioxane (2000mL) into a 5000mL three-necked flask, heating the reaction solution to 100 ℃, reacting for 1 hour, and monitoring the disappearance of the raw materials by TLC; adding 2000mL of ice water into the reaction solution, cooling to room temperature, filtering, collecting a filter cake, and drying under reduced pressure to obtain 170g of a compound 7;

DMF (1000mL), Compound 7(100g, 0.293mol) and formamidine acetate (122g, 1.173mol) were added to a 2000mL three-necked flask, and after stirring at room temperature for 1 hour, 200mL of acetic acid was added to the reaction mixture, which was then warmed to 80 ℃ and reacted overnight; the TLC liquid quality monitoring of the next day shows that the raw materials disappear, the reaction solution is cooled to0 ℃, and the filter cake is collected by filtration and dried under reduced pressure to obtain 80g of compound 8;

a500 mL single vial was charged with Compound 8(10g, 28.5mmol), Compound 9(6g, 5mmol), potassium carbonate (12g, 85.5mmol) and DMF (100mL), and then warmed to 80 ℃ for reaction overnight. LCMS monitors the disappearance of the raw materials, the reaction solution is led into ice water (300mL), filter cake is collected by filtration, and 13g of compound 10 is obtained by decompression and drying;

compound 10(13g, 32mmol) in a 500mL three-necked flask was dissolved in methanol (200mL), and sodium borohydride (3g, 64mmol) was added in portions at 0 ℃ and stirred for 2 hours after the addition was completed, and the disappearance of the starting material was monitored. Removing half of the solvent under reduced pressure, pouring the reaction solution into 300mL of water, extracting with dichloromethane (100 mL. multidot.3), drying and concentrating to obtain 7g of compound 11;

compound 11(12g, 29.2mmol) was dissolved in hydrobromic acid (120mL), stirred at 120 degrees overnight and TLC monitored for disappearance of starting material. The solvent was removed under reduced pressure. Adding 200mL of water into the residue, stirring for 30 minutes, filtering, and drying a filter cake to obtain 15g of a compound 12 which is directly used in the next step;

a100 mL single-neck flask was charged with Compound 12(5g, 12.2mmol), Compound 13(5.5g, 13.4mmol), Potassium hydroxide (3.4g, 61mmol) and DMF (50mL), heated at 80 ℃ and stirred overnight to monitor disappearance of starting material; the reaction mixture was poured into 150mL of ice water, extracted with dichloromethane (50 mL. times.4), dried and concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol: 50; 1) to obtain 3g of compound 14 racemate (i.e., the compound represented by formula II).

The method can further comprise separating the obtained racemate to obtain an isomer with a single configuration, namely an SRRS configuration, an SRSR configuration, an RSRS configuration, an RSSR configuration, an SRRR configuration, an SRSS configuration, an RSRR configuration or an RSSS configuration;

and (3) carrying out esterification reaction on the compound with the single configuration and the compound shown in the formula III to obtain an esterification reaction product with the corresponding configuration, namely the compound shown in the formula I with the single configuration.

The application of the compound shown in the formula I in the preparation of antifungal medicines or medicines for preventing and/or treating diseases caused by fungi also belongs to the protection scope of the invention.

In the application, the fungus may be: fungi of the genera candida, cryptococcus, aspergillus;

the method specifically comprises the following steps: candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans or Aspergillus fumigatus;

more specifically, the following strains can be mentioned: candida albicans standard strain ATCC SC5314, clinical strain CMCCF (F) C of Candida albicans._1tCandida parapsilosis Standard strain ATCC22019 and Candida parapsilosis clinical strain CMCCC (F) C._4iCandida glabrata Standard strain ATCC2001, Candida glabrata clinical strain CMCCC (F) Y._10dCandida tropicalis standard strain ATCC-MYA-750 and Candida tropicalis clinical strain CMCCC (F) Y._2hC, Candida krusei standard strain ATCC6258, and Candida krusei clinical strain CMCCC (F)._6aCryptococcus neoformans Standard strain ATCC4906, Cryptococcus neoformans clinical strain CMCCC (F) D2-12, Aspergillus fumigatus Standard strain ATCCYA 3626 or Aspergillus fumigatus clinical strain CMCCC (F).

The application of the compound shown in the formula II in the preparation of antifungal medicines also belongs to the protection scope of the invention.

the method specifically comprises the following steps: candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans or Aspergillus fumigatus,

more specifically, the following strains can be mentioned: candida albicans standard strain ATCC SC5314, clinical strain CMCCF (F) C of Candida albicans._1tCandida parapsilosis Standard strain ATCC22019 and Candida parapsilosis clinical strain CMCCC (F) C._4iCandida glabrata Standard strain ATCC2001, Candida glabrata clinical strain CMCCC (F) Y._10dCandida tropicalis standard strain ATCC-MYA-750 and Candida tropicalis clinical strain CMCCC(F)Y._2hC, Candida krusei standard strain ATCC6258, and Candida krusei clinical strain CMCCC (F)._6aCryptococcus neoformans Standard strain ATCC4906, Cryptococcus neoformans clinical strain CMCCC (F) D2-12, Aspergillus fumigatus Standard strain ATCCYA 3626 or Aspergillus fumigatus clinical strain CMCCC (F).

The compound shown in the formula II can be a raceme of the compound shown in the formula II or a single configuration compound of the compound shown in the formula II, and more specifically can be an SRSS configuration compound of the compound shown in the formula II.

The compound shown in the formula II can also be any one of sodium salt, potassium salt, magnesium salt, tromethamine salt, ethanolamine salt and diethylamine salt of the compound shown in the formula II.

The invention also provides a liquid preparation for resisting fungi.

The antifungal liquid preparation contains any one of the compound shown in the formula I, the raceme of the compound shown in the formula I, optical isomers of the compound shown in the formula I with various configurations, and salts of the compound shown in the formula I (including the raceme and the optical isomers with various configurations); or any one of the compound shown in the formula II, raceme of the compound shown in the formula II, optical isomers of the compound shown in the formula II with various configurations, sodium salt, potassium salt, magnesium salt, tromethamine salt, ethanolamine salt and diethylamine salt of the compound shown in the formula II (including raceme and optical isomers with various configurations);

more specifically, the antifungal liquid preparation contains any one of an optical isomer of the SRSS configuration of the compound shown in the formula I or a salt of the optical isomer of the SRSS configuration of the compound shown in the formula I, or any one of a sodium salt, a potassium salt, a magnesium salt, a tromethamine salt, an ethanolamine salt and a diethylamine salt of the SRSS configuration of the compound shown in the formula II or the optical isomer of the SRSS configuration of the compound shown in the formula II.

The invention provides a compound shown in formula 1, which has good antifungal effect and water solubility, and can be used for preparing an antifungal liquid preparation for treating and preventing clinical fungal infection. The compound has good antifungal effect and good water solubility, can keep relative stability in a solution, and can be prepared into a liquid preparation for clinical use.

Drawings

FIG. 1 is a reaction scheme for preparing a compound represented by formula II in the present invention.

FIG. 2 shows LCMS spectrum of SZY1402-14A-1(SRRS configuration).

FIG. 3 is an NMR spectrum of SZY1402-14A-1(SRRS configuration).

FIG. 4 is a LCMS spectrum of SZY1402-14A-2(SRSR configuration).

FIG. 5 is an NMR spectrum of SZY1402-14A-2(SRSR configuration).

FIG. 6 is a LCMS spectrum of SZY1402-14B-1(RSRS configuration).

FIG. 7 is an NMR spectrum of SZY1402-14B-1(RSRS configuration).

FIG. 8 is a LCMS spectrum of SZY1402-14B-2(RSSR configuration).

FIG. 9 is an NMR spectrum of SZY1402-14B-2(RSSR configuration).

FIG. 10 shows the LCMS spectrum of SZY1402-14C-1(SRRR configuration).

FIG. 11 is an NMR spectrum of SZY1402-14C-1(SRRR configuration).

FIG. 12 is a LCMS spectrum of SZY1402-14C-2(SRSS configuration).

FIG. 13 is an NMR spectrum of SZY1402-14C-2(SRSS configuration).

FIG. 14 is an LCMS spectrum of SZY1402-14D-1(RSRR configuration).

FIG. 15 is an NMR spectrum of SZY1402-14D-1(RSRR configuration).

FIG. 16 shows the LCMS spectrum of SZY1402-14D-2(RSSS configuration).

FIG. 17 is an NMR spectrum of SZY1402-14D-2(RSSS configuration).

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.

The compound shown in the formula II in the invention is prepared by a reaction scheme shown in a figure 1.

The specific operation is as follows:

Chiral separation to obtain 8 single chiral isomers (SRRS, SRSR, RSRS, RSSR, SRRR, SRSS, RSRR and RSSS).

FIGS. 2-17 are structural representations of 8 single-configuration compounds of formula II, respectively. The antibacterial activity of the 8 single-configuration compounds of formula II is shown in the table below:

as can be seen from the data in the table, the antibacterial activity of SZY1402-14C-2(SRSS configuration) is better than that of the compounds of other configurations.

In a 100mL single-neck flask was added compound 14 (single configuration) (3g, 4.2mmol), compound 15 (0.3g-1.5g,5.4mmol), 4-dimethylaminopyridine (0.5g, 0.4mmol), EDCI (1.0g, 5.4mmol) and dichloromethane (30mL), stirred at room temperature overnight, TLC monitored for disappearance of starting material, washed twice with saturated saline, dried and concentrated. The crude product is purified by high-efficiency preparation liquid phase to obtain the compound with the general formula.

Among them, compound 15 may be specifically: succinic anhydride, malonic acid, phenylalanine or

Examples 1,

Synthetic preparation of 16A-SRSS

The following is a chiral synthetic route:

compound 1(200g,1.69mol,194mL,1.00eq), imidazole (115g,1.69mol,1.00 eq) were dissolved in 2LDMF, to which TBDPSCl (418g,1.52mol,391 mL,0.90eq) was added at 0-10 ℃. Then the reaction is stirred for 3 hours at the temperature of 20-30 ℃. TLC (PE: EA ═ 20:1, Rf ═ 0.60) monitoring indicated completion of the reaction. The reaction was added to 3L of water, followed by extraction with MTBE (1.00L × 2), and the organic phases were combined, washed twice with saturated brine (1.00L × 2), dried over anhydrous sodium sulfate, filtered, and rotary evaporated to give the compound 2 as a yellow oil, 830 g. 1H NMR (400MHz CDCl 3): δ 7.66-7.70(m,4H),7.27-7.43 (m,6H),6.85-6.95(m,2H),4.27-4.28(m,1H),4.00-4.04(m,2H),1.37-1.39 (m,3H),1.14-1.18(m,3H),1.08(s,9H)

Compound 2(260g,729mmol,1.00eq) was dissolved in 1.00L DCM, cooled to-70-60 ℃ and DIBAL-H (1M,1.17L,1.60eq) was added dropwise. The mixture is stirred and reacted for 2 hours at the temperature of-70 to-60 ℃. TLC (PE: EA ═ 10:1, Rf ═ 0.45) showed the reaction was complete. The reaction was quenched with 2.00L of aqueous NH4Cl solution and then filtered. The filtrate was extracted with MTBE (1.50L × 3), the organic phases were combined, dried over Na2SO4 and rotary evaporated under reduced pressure to give 209g of compound 3, compound 3 being a yellow oil. 1H NMR (400MHz CDCl 3): δ 9.64(s,1H),7.65-7.67(m,4H),7.38-7.40(m,6H),4.08-4.10(m,1H), 1.21-1.23(m,1H),1.04-1.11(m,3H)

Compound 3(310g,992mmol,1.00eq) and acetic acid (59.6g,992mmol,56.7mL, 1.00eq) were dissolved in 2.00L of methanol, and then formylhydrazine (59.6g,992mmol,1.00eq) was added thereto, followed by reaction with stirring at 20 to 30 ℃ for 1 hour.

TLC (PE: EA ═ 5:1, Rf ═ 0.28) showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove the solvent, 2.00L of MTBE was added thereto, followed by washing with 500ml of an aqueous Na2CO3 solution, drying over anhydrous sodium sulfate and rotary evaporation under reduced pressure to obtain a product, which was purified by column chromatography (PE: EA 20/1to 5/1). Compound 4 was obtained as a pale yellow solid (325g,917 mmol, 46.2% yield, SFC showed 100% ee). 1H NMR (400MHz CDCl 3): δ 9.13(s,1H), 8.42-8.45(m,1H),7.64-7.68(m,4H),7.39-7.43(m,6H),7.04(s,1H), 4.41-4.47(m,1H),1.29-1.32(m,3H),1.11(s,9H)

Compound 4(160g,451mmol,1.00eq) was dissolved in 1.00L THF, to which MeMgBr (3M,903mL,6.00eq) was added at 0 ℃ and then stirred at 20-30 ℃ for 2 h. TLC (PE: EA ═ 2:1, Rf ═ 0.33) showed most of the starting material reacted. The reaction was quenched with 1.50L of water and then extracted with MTBE (1.50L × 3). The organic phase was washed with 500ml brine and then rotary evaporated under reduced pressure to give the product. The product was purified by column chromatography PE EA 10/1to 2/1). Compound 5 was obtained as a yellow oil (100g,270mmol, 29.9% yield). 1H NMR (400MHz CDCl 3): δ 7.93-8.14(m,1H),7.68-7.72(m,4H), 7.27-7.44(m,6H),6.50-6.71(m,1H),7.27-4.73(m,1H),2.60-2.93(m, 1H), 1.04-1.10(m,15H)

Compound 7(130g,483mmol,1.00eq) was dissolved in 1.00L of DMF, and benzyl chloroformate (90.7g,579mmol,72.6mL,1.20eq) was added thereto at 0 ℃ and the reaction mixture was stirred at 20-30 ℃ for 2 h. TLC (PE: EA ═ 1:2, Rf ═ 0.38) showed the reaction was complete. 1.50L of water was added to the reaction solution, and the mixture was filtered to obtain a cake. The filter cake was washed with 1.5L aqueous NaHCO3 solution and filtered to give a brown filter cake, compound 8, amounting to 145 g. 1H NMR (400MHz MeOD): δ 7.30-7.33(m,2H),6.96-6.98 (m,4H),6.825-6.84(m,2H),6.59-6.61(m,4H),6.39-6.41(m,1H),3.23-3.25 (m,4H),3.10-3.12(m,4H)

The compound 5(100g,270mmol,1.00eq) is dissolved in 1.00L dioxane, then Et3N (54.6g,540mmol,75.1mL,2.00eq) and the compound 8(137g,351mmol,1.30eq) are added, and then the mixture is stirred and reacted at 100-110 ℃ for 12 h. The reaction was cooled to 20-30 ℃, 1.50L of water was added thereto, and then extracted three times with DCM (1.50L × 3). The organic phases were combined and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (PE: EA ═ 2/1to 1/1), TLC (plate1, PE: EA ═ 2:1, Rf ═ 0.40) to give compound 6(17.0g) as a white solid.

1H NMR(400MHz CDCl3)：δ7.71-7.72(m,4H),7.50-7.54(m,1H),7.37-7.40 (m,8H),6.37-7.00(m,6H),4.43-4.45(m,1H),4.19-4.20(m,1H),3.35-3.37 (m,4H),3.22-3.23(m,4H),1.35-1.37(m,3H),0.97-1.08(m,12H)。

Compound 6(17.0g,26.2mmol,1.00eq) was dissolved in 100ml THF, TBAF.3H2O (16.6g,52.5mmol,2.00eq) was added, and the reaction was stirred at 50 ℃ for 12 h. TLC (PE: EA ═ 1:2, Rf ═ 0.32) showed the reaction was complete. The reaction was quenched with 50mL water, extracted three times with EA (100mL x 3), dried over Na2SO4 and concentrated to give the crude product. The crude product was purified by column chromatography (PE: EA: 3/1to 1/2) and washed with EA at 20-30 ℃ for 15 min. Filtration afforded compound YLYY-20190214(6.60g,16.1mmol, 61.4% yield). 1H NMR (400MHz DMSO): Δ 8.88(s,1H),8.30(s,1H),7.48-7.50 (m,2H),7.08-7.11(m,2H),6.84-6.86(m,2H),6.66-6.69(m,2H),4.72(s, 1H),4.00-4.04(m,1H),3.79-3.82(m,1H),3.30-3.34(m,4H),3.09-3.11 (m,4H),1.23-1.25(m,3H),1.10-1.12(m, 3H).

Compound 2b (100g,529mmol,1.00eq) was dissolved in 1000ml acetonitrile, then NBS (94.2g,529mmol,1.00eq) was added thereto by controlling the temperature at 20 ℃ and the reaction was stirred at 20-25 ℃ for 1 hour. Then, p-toluenesulfonic acid (182g,1.06mol,2.00eq) was added to the reaction solution. Then the temperature is increased to 90 ℃ and the reaction is stirred for 5 h. New spots were generated by TLC detection (PE: EA ═ 1: 0). The reaction was diluted with 1L aqueous NaHCO3 and extracted twice with EA (2000mL 2). The organic phase was washed with 500ml of water, then dried over Na2SO4(20.0g) and rotary evaporated to give the product. The product was purified by column chromatography (PE: EA: 1/0-1/1). 80g of compound 2a was obtained as a yellow oil.

1H NMR:(400MHz CDCl3)：δ7.53-7.55(m,1H),7.42-7.45(m,1H),7.33-7.35 (m,1H),4.48(s,2H).

Compound 1(100g,1.08mol,84.8mL,1.00eq) was dissolved in acetonitrile (790g,13.6mol, 1.00L,12.6eq) and bf3.et2o (1.15g,8.10mmol,1.00mL, 7.50e-3eq) was added thereto at 0 ℃. After stirring and reacting at 0 ℃ for 1 hour, the temperature is raised to 40 ℃, and stirring is continued for 4 hours. TLC (PE: EA ═ 1:2) detection showed that new spots were produced. The reaction mixture was concentrated under reduced pressure to remove the solvent, and then used as it was in the next reaction. 171g of crude compound 2 was obtained as a yellow oil. 1H NMR (400MHz CDCl 3): δ 4.28(m,1H), 4.06-4.10(m,1H),3.83-3.86(m,1H),2.52-3.56(m,1H),2.44-3.52 (m,1H),1.41(s,3H),1.33(s,3H)

A mixture of compound 2a (72.0g,269mmol,1.00eq), compound 2(63.1g,419mmol,1.56 eq) and p-toluenesulfonic acid (92.6g,537mmol,2.00eq) was dissolved in 1300mL of toluene and 610mL of butanol, and the reaction was stirred at 130 ℃ for 36 h. TLC (PE: EA ═ 1:0) showed new spots formed. The reaction was cooled to 25 ℃, then poured into 1000mL of 10% aqueous NaHCO3, and extracted with EA (1500mL × 3). The combined organic phases were rotary evaporated under reduced pressure and purified by column chromatography (PE: EA: 1/0to0/1) to give compound 3 as a yellow oil, 54 g. 1H NMR for Compound 3 (400MHzCDCl 3): δ 7.52-7.60(m,1H),7.34-7.36 (m,1H),7.20-7.21(m,1H),4.58(m,1H),4.19-4.20(m,2H),4.02-4.08(m, 2H),3.62-3.74(m,2H)

1H NMR for Compound 2a (400MHz CDCl 3): δ 7.55-7.57(m,1H),7.48(s,1H), 7.35-7.38(m,1H),4.50(s,2H)

Compound 3(49.0g,136mmol,1.00eq) was dissolved in 500mL DMF, then PhCOONa (39.2g,272mmol,81.6mL,2eq) was added, the reaction solution was warmed to 160 ℃ and 165 ℃ and stirred for 5 h. TLC (PE: EA ═ 10:1) monitoring showed that new spots were produced. 2000mL of water was added to the reaction mixture, followed by extraction with ethyl acetate EA (600 mL. times.2), and the organic phases were combined, washed with saturated brine (200 mL. times.1), dried over anhydrous sodium sulfate (10.0g), and rotary evaporated under reduced pressure to give 27g of Compound 4, which was a yellow oil, Compound 4.

Compound 4(59.0g,132mmol,1.00eq) was dissolved in 50ml of methanol and the reaction stirred at 25 ℃ for 1 h. A large amount of solid precipitates from the solution. Cooling to 20 deg.C, and filtering to obtain white solid. HPLC purity 86.8%. The white solid was dissolved with MTBE (177mL × 1) at 60 ℃, MeOH (590mL × 1) was then added, the compound was stirred at 60 ℃ for 0.5h, and then filtered to give a white solid which was purified by column chromatography (PE: EA ═ 1/0to0/1) to give compound 5(20.0g,43.8mmol, 33.1% yield, 97.7% purity, SFC showed 98.5% ee). 1H NMR (400MHz CDCl 3): δ 8.08-8.10(m,2H),7.60-7.66(m,2H),7.44-7.60(m, 2H),7.27-7.29(m,2H),4.56-4.84(m,2H),4.44(m,1H),4.11-4.13(m, 1H), 4.04-4.05(m,1H),3.86-3.98(m,1H),3.50-3.51(m, 1H).

Compound 5(20.0g,43.8mmol,1.00eq) was dissolved in 200ml DMF and after

temperature control

1,2, 4-triazole sodium salt (12.1g,131mmol,3.00eq) was added at 20-25 ℃ and the mixture was stirred at 110 ℃ for 12 h. TLC (PE: EA ═ 1:1) monitored the formation of new spots and the reaction was concentrated under reduced pressure to give crude product which was washed once with 100mL water and then extracted twice with ethyl acetate (50.0mL × 2). The organic phases were combined and concentrated under reduced pressure to give the product. Column chromatography purification (PE: EA ═ 1/0to0/1) gave compound 6(7.00g), a white solid, 1H NMR (400MHz CDCl 3): δ 8.16(s,1H),7.96(s,1H),7.57-7.59(m,1H), 7.48(m,1H),7.25-7.28(m,1H),4.78(s,2H),4.16-4.18(m,1H),3.84-4.16 (m,1H),3.68-3.71(m,2H),3.26-3.28(m, 1H).

Compound 6(3.00g,9.09mmol,1eq) was dissolved in 30mL pyridine, followed by addition of MsCl (2.15g,18.8mmol,1.45mL,2.07eq) at 0 ℃. The mixed solution is stirred and reacted for 3 hours at the temperature of 20-25 ℃. TLC (PE: EA ═ 1:2) monitoring showed new spots formed. The reaction was washed once with water (400mL x 1), then extracted twice with DCM (200mL x 2), the organic phases were combined and the organic phase was washed twice with water (50.0mL x 2). The organic phases were combined and concentrated under reduced pressure to give the product. The product was purified by column chromatography (PE: EA: 1/0to0/1) and LCMS showed a molecular structure. The product was slurried with PE EA ═ 5:1(100mL) and filtered to give SZY1402-14-peak2(4.50g, 99.9% purity, 100% ee) as a white solid. 1H NMR (400MHzCDCl 3): δ 8.22 (s,1H),7.92(s,1H),7.48-7.53(m,2H),7.25(m,1H),4.76-4.85(m, 2H), 4.33(m,1H),3.98-4.02(m,1H),3.87-3.92(m,2H),3.75-3.87(m,1H), 3.09(s,3H)

A100 mL single neck flask was charged with compound 12a (5g, 12.2mmol), compound 13a (5.5g, 13.4mmol), potassium hydroxide (3.4g, 61mmol) and DMF (50mL), stirred at 80 ℃ overnight and monitored for disappearance of starting material. The reaction solution was poured into 150mL of ice water, extracted with dichloromethane (50mL × 4), dried and concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol: 50; 1) to obtain 3g of compound 14 SRSS.

1H NMR(400MHz CDCl3)：δ8.21(s,1H),7.90(s,1H),7.65(s,1H),7.59-7.57 (d,J＝8.4Hz,1H),7.48-7.47(d,J＝2.0Hz,1H),7.43-7.40(m,2H),7.27-7.24(m, 1H),7.05-7.03(d,J＝8.4Hz,2H),6.95-6.93(d,J＝8.8Hz,2H),6.82-6.81(d, J＝2.4Hz,2H),4.86-4.75(dd,J1＝32.4Hz,J2＝14.8Hz,2H),4.38-4.34(m,1H), 4.27-4.22(m,1H),4.03-3.97(m,1H),3.94-3.92(m,1H),3.84-3.78(m,2H), 3.51-3.47(m,2H),3.39-3.36(m,4H),3.25-3.22(m,4H),3.06-3.04(d,J＝8.8Hz, 1H),1.48-1.46(d,J＝7.2Hz,3H),1.26-1.24(d,J＝6.4Hz).MS：[M+H]+＝722.1

n＝1、R₂H, R3 ═ H, R1 ═ carboxylic acid and sodium salt (16A-SRSS)

Dichloromethane (300ml) was added to a 500ml three-necked flask, followed by the addition of 14-SRSS/SZY1402-14C2(7.5g), succinic anhydride (4.7g), N-lutidine (1.91g) and triethylamine (3.16g) in that order, followed by stirring for 10min, stirring until homogeneous, and heating to 45 ℃ for reaction for 6 h. And controlling the TLC to complete the reaction. Cooling the reaction solution to room temperature, washing with water for 2 times, separating liquid, washing the organic phase with 0.5M hydrochloric acid and water, and concentrating under reduced pressure to obtain oil. And dropwise adding 50ml of methanol into the oily substance under stirring, carrying out suction filtration, leaching a filter cake with a small amount of methanol to obtain a white solid, and carrying out forced air drying at 40 ℃ to obtain 5.0g of the compound 15A-SRSS.

To an ethanol solution (50mL) of 15A-SRSS (5.0g), an aqueous sodium hydrogencarbonate solution (1.0g/10mL) was added dropwise, the mixture was stirred overnight at room temperature, and the mixture was spin-dried to obtain 16A-SRSS (5.0 g).

1H NMR(400MHz CDCl3)：δ8.22(s,1H),7.92(s,1H),7.68(s,1H),7.61-7.59 (d,J＝8.4Hz,1H),7.48-7.47(d,J＝2.0Hz,1H),7.42-7.39(m,2H),7.28-7.24(m, 1H),7.05-7.03(d,J＝8.4Hz,2H),6.95-6.93(d,J＝8.8Hz,2H),6.82-6.81(d, J＝2.4Hz,2H),4.86-4.75(dd,J1＝32.4Hz,J2＝14.8Hz,2H),4.38-4.34(m,2H), 4.27-4.22(m,1H),4.03-3.97(m,1H),3.94-3.92(m,1H),3.84-3.78(m,2H), 3.51-3.47(m,1H),3.39-3.36(m,4H),3.25-3.22(m,4H),3.06-3.04(d,J＝8.8Hz, 1H),2.65-2.62(m,2H),2.58-2.53(m,2H),1.48-1.46(d,J＝7.2Hz,3H),1.26-1.24 (d,J＝6.4Hz).MS：[M+H]+＝822.6

Preparation of malonic esters and sodium salts of examples 2, 14-SRSS

Malonic acid (2.0g), DMAP (0.2g) and EDCI (3.0g) were added to a solution of 14-SRSS (5.0g) in dichloromethane (50mL) in this order, the reaction was stirred at room temperature for 6-8h, and after completion of the TLC detection, the reaction was washed with water and dilute hydrochloric acid in this order 2 times with 50mL each time. The organic phase is dried by anhydrous sodium sulfate, and 4.5g of 15B-SRSS is obtained by evaporating the solvent and carrying out column chromatography separation.

To an ethanol solution (50mL) of 15B-SRSS (4.5g), an aqueous sodium hydrogencarbonate solution (1.0g/10mL) was added dropwise, the mixture was stirred overnight at room temperature, and the mixture was spin-dried to obtain 16B-SRSS (4.5 g).

1H NMR(400MHz CDCl3)：δ8.19(s,1H),7.90(s,1H),7.67(s,1H),7.58-7.56 (d,J＝8.4Hz,1H),7.45-7.43(d,J＝2.0Hz,1H),7.41-7.38(m,2H),7.27-7.24(m, 1H),7.05-7.03(d,J＝8.4Hz,2H),6.95-6.93(d,J＝8.8Hz,2H),6.82-6.81(d, J＝2.4Hz,2H),4.86-4.75(dd,J1＝32.4Hz,J2＝14.8Hz,2H),4.38-4.34(m,1H), 4.27-4.22(m,1H),4.03-3.97(m,1H),3.94-3.92(m,1H),3.84-3.78(m,2H), 3.48-3.45(m,1H),3.39-3.36(m,4H),3.25-3.22(m,4H),3.18(s,2H),3.06-3.04 (d,J＝8.8Hz,1H),1.50-1.48(d,J＝7.2Hz,3H),1.25-1.23(d,J＝6.4Hz).MS： [M+H]+＝808.7

Example 3: n-16, R₁＝-NH₂，R₂＝H，R₃＝H；

Boc-amino 18-carbonic acid (2.1g), DMAP (0.2g) and EDCI (3.0g) were added to a solution of 14-SRSS (5.0g) in dichloromethane (50mL) in this order, the reaction was stirred at room temperature for 6-8h, and after completion of the TLC detection, the reaction was washed with water and dilute hydrochloric acid in this order 2 times in 50mL portions. The organic phase was dried over anhydrous sodium sulfate, evaporated and separated by column chromatography to give 4.5g of 15D-SRSS.

Adding 15D-SRSS (4.5g) into 50mL ethyl acetate hydrochloride solution (2mol/L), reacting at room temperature and stirring overnight, separating out solid, and performing suction filtration to obtain the product 16D-SRSS (4.2 g).

1H NMR(400MHz CDCl3)：δ8.24(s,1H),7.88(s,1H),7.64(s,1H),7.59-7.57 (d,J＝8.4Hz,1H),7.48-7.47(d,J＝2.0Hz,1H),7.43-7.40(m,2H),7.27-7.24(m, 1H),7.04-7.02(d,J＝8.4Hz,2H),6.95-6.93(d,J＝8.8Hz,2H),6.82-6.81(d, J＝2.4Hz,2H),4.86-4.75(dd,J1＝32.4Hz,J2＝14.8Hz,2H),4.38-4.34(m,1H), 4.27-4.22(m,2H),4.03-3.97(m,1H),3.94-3.92(m,1H),3.84-3.78(m,2H), 3.51-3.47(m,1H),3.39-3.36(m,4H),3.25-3.22(m,4H),3.06-3.04(d,J＝8.8Hz, 1H),2.65-2.60(m,2H),2.28-2.24(m,2H),1.68-1.55(m,4H),1.48-1.46(d, J＝7.2Hz,3H),1.35-1.28(m,26H)1.26-1.24(d,J＝6.4Hz).MS：[M+H]+＝1004.1

Example 4, n ═ 1, R₁＝-NH₂，R₂Is a benzene ring, R₃＝H

Boc-aminophenylpropionic acid (1.8g), DMAP (0.2g) and EDCI (3.0g) were added to a dichloromethane solution (50mL) of 14-SRSS (5.0g) in this order, the reaction was stirred at room temperature for 6 to 8 hours, and after completion of the TLC detection, the reaction was washed with water and dilute hydrochloric acid in this order 2 times, 50mL each time. The organic phase was dried over anhydrous sodium sulfate, evaporated and separated by column chromatography to give 15E-SRSS 5.5 g.

Adding 15E-SRSS (5.5g) into 60mL of ethyl acetate hydrochloride solution (2mol/L), reacting at room temperature and stirring overnight, separating out a solid, and performing suction filtration to obtain a product 16E-SRSS (4.8 g).

1H NMR(400MHz CDCl3)：δ8.21(s,1H),7.90(s,1H),7.65(s,1H),7.59-7.57 (d,J＝8.4Hz,1H),7.48-7.47(d,J＝2.0Hz,1H),7.43-7.40(m,2H),7.27-7.24(m, 2H),7.10-7.06(m,4H),7.05-7.03(d,J＝8.4Hz,2H),6.95-6.93(d,J＝8.8Hz,2H), 6.82-6.81(d,J＝2.4Hz,2H),4.86-4.75(dd,J1＝32.4Hz,J2＝14.8Hz,2H),4.52-4.49 (m,1H),4.38-4.34(m,1H),4.27-4.22(m,2H),4.03-3.97(m,1H),3.94-3.92(m, 1H),3.84-3.78(m,2H),3.51-3.47(m,1H),3.39-3.36(m,4H),3.25-3.22(m,4H), 3.06-3.04(d,J＝8.8Hz,1H),2.90-2.78(m,2H),1.48-1.46(d,J＝7.2Hz,3H), 1.26-1.24(d,J＝6.4Hz).MS：[M+H]+＝869.8

Example 5 preparation of 8 different optical isomers of 16A

8 single chiral isomers (SRRS, SRSR, RSRS, RSSR, SRRR, SRSS, RSRR, RSSS) were separated by SFC (supercritical fluid chromatography).

In a 100mL single neck flask was added compound 14(SRSS) (3g, 4.2mmol), succinic anhydride (541mg, 5.4mmol), 4-dimethylaminopyridine (0.5g, 0.4mmol), EDCI (1.0g, 5.4mmol) and dichloromethane (30mL), stirred overnight at room temperature, TLC monitored for disappearance of starting material, washed twice with saturated saline, dried and concentrated.

To an ethanol solution (50mL) of 15A-SRSS (5.0g), an aqueous sodium bicarbonate solution (1.0g/10mL) was added dropwise, the mixture was stirred overnight at room temperature, and the sodium salt of 16A-SRSS was obtained by spin-drying.

Using different optical isomers of Compound 14, repeating the above two steps can yield 7 additional optical isomers of 16A-SRSR, 16A-RSRS, 16A-RSSR, 16A-SRRR, 16A-SRSS, 16A-RSRR, 16A-RSSS, respectively.

Examples of the drug effect study: in vitro antifungal primary screening test for 8 new drug compounds

The purpose is as follows: and 8 new drug compounds are tested for the existence of antifungal effect on 7 common pathogenic fungi and 14 common pathogenic fungi in vitro.

Two materials and methods:

1. sample preparation:

1.1 test samples:

16A-SRRS corresponding to SZY1402-14A-1, purity: > 98%, total amount: 100 mg.

16A-SRSR corresponding to SZY1402-14A-2, purity: > 98%, total amount: 100 mg.

16A-RSRS corresponding to SZY1402-14B-1, purity: > 98%, total amount: 100 mg.

16A-RSSR corresponding to SZY1402-14B-2, purity: > 98%, total amount: 100 mg.

16A-SRRR corresponding to SZY1402-14C-1, purity: > 98%, total amount: 100 mg.

16A-SRSS corresponding to SZY1402-14C-2, purity: > 98%, total amount: 100 mg.

16A-RSRR corresponding to SZY1402-14D-1, purity: > 98%, total amount: 100 mg.

Purity of 16A-RSSS corresponding to SZY 1402-14D-2: > 98%, total amount: 100 mg.

Note: the above compounds were all 8 chiral isomers of compound 14 from the synthetic general formula obtained in example 6.

1.2 Positive control:

itraconazole batch No.: PSCZ-180302, purity: 98.85%, total amount: 100 mg.

Posaconazole batch No.: BSP-20180317, purity: 99.90%, total amount: 100 mg.

Fluconazole batch number: lot # D1615050, purity: 98%, total amount: 100 mg.

Experimental bacteria: all the experimental strains are standard strains and clinical strains provided by a fungus branch center of a pathogenic microorganism (virus) strain preservation center of Chinese academy of medicine sciences, and comprise 7 common superficial pathogenic fungi of 14 strains, which are as follows:

candida species: candida albicans standard strain ATCCSC5314

Clinical strain CMCCF (F) C._1t

Candida parapsilosis standard strain ATCC22019

Clinical strain CMCCC (F) C._4i

Candida glabrata standard strain ATCC2001

Clinical strain CMCCC (F) Y._10d

Candida tropicalis standard strain ATCC-MYA-750

Clinical strain CMCCC (F) Y._2h

Candida krusei standard strain ATCC6258

Clinical strain CMCCC (F) C._6a

Cryptococcus genus: cryptococcus neoformans standard strain ATCC4906

Clinical strain CMCCC (F) D._2-12

The genus Aspergillus: aspergillus fumigatus standard strain ATCC-MYA-3626

Clinical strain CMCCC (F) A._1d

3. The experimental method comprises the following steps: liquid microdilution, performed with particular reference to CLSI-M-27A4 and M38-A4, USA.

3.1 detection content: minimum Inhibitory Concentration (MIC) (mg/L)

3.2 technical requirements: repeating the experiment twice before and after, taking geometric mean value of three 6 data each time

3.3 Experimental procedures:

3.3.1.1 preparation of drug sensitive plate for test sample (micro liquid-based dilution method)

The tested sample is a 16A compound corresponding to SZY1402-14A-1, SZY1402-14A-2, SZY1402-14B-1, SZY1402-14B-2, SZY1402-14C-1, SZY1402-14C-2, SZY1402-14D-1 and SZY 1402-14D-2. Formulated with DMSO to 1600 μ g/ml according to CLSI guidelines. And subpackaging the small tubes and freezing the small tubes in a refrigerator of 80 ℃ below zero, wherein the storage life is not more than 6 months. When preparing the drug sensitive plate, the drug sensitive plate is taken out from a refrigerator with the temperature of-80 ℃ for dissolution and then diluted by DMSO step by step in multiple proportion for 10 concentrations. Then diluted 50-fold each to twice as much as the work by RPMI1640 liquid medium. When the residual mother liquor is discarded, the mother liquor is not returned to minus 80 ℃. The drug sensitive plate is stored at-80 ℃ after being prepared, and is taken out for dissolving use when the bacteria liquid is added, and the storage life of the drug sensitive plate at-80 ℃ is not more than 3 months.

3.3.1.2 Final drug working concentrations of the drug media the following compounds are all the corresponding 16A compounds

SZY 1402-14A-1: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14A-2: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14B-1: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14B-2: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14C-1: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14C-2: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14D-1: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

SZY 1402-14D-2: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

3.3.1.3 preparing bacterial liquid:

activating the experimental bacteria, and culturing the yeast on Sabouraud weak agar medium (SDA) at 30 deg.C for 48 hr; culturing filamentous fungi in Potato Dextrose Agar (PDA) culture medium at 26 deg.C for 7-10 days; sterile normal saline is used for preparing bacterial suspension, and a blood counting chamber is used for counting and adjusting the concentration. Final required working concentration of yeast: 0.5 to 2.5 x 10³cf μ/ml, final desired working concentration of filamentous fungi: 0.4 to 5X 10⁴cfμ/ml。

3.3.1.4 inoculation bacterial quantity:

micro-liquid based dilution method: the prepared bacterial liquid is calculated according to the concentration of each bacterial suspension, then an appropriate amount of bacterial liquid is taken and added into 10ml of RPMI1640 liquid culture medium, and 100 mu l of the bacterial liquid is added into a 96-hole culture plate which is placed at room temperature for 2 hours and is coated with the medicine. Adding the bacteria solution into the 1 st to 11 th holes, setting a blank culture medium control, and placing the blank culture medium control in an incubator for incubation. The preparation method of the bacterial liquid for repeated experiments is completely the same as the above.

3.3.1.5 culture conditions: and (3) candida: 24h at 35 DEG C

Cryptococcus: 72h at 35 DEG C

Aspergillus fumigatus: 35 ℃ for 48h

3.3.1.6 method for judging result:

minimum Inhibitory Concentration (MIC): and C, the candida and cryptococcus, the blank control growth is taken as a reference, and the minimum concentration when the bacteria growth amount in the drug-based hole is less than or equal to 50 percent is the MIC value of the drug, and is expressed by mu g/ml. And Aspergillus, taking blank control growth as a reference, and taking the minimum concentration when the bacterial growth amount in the drug-based hole is less than or equal to 100 percent as the MIC value of the drug, wherein the minimum concentration is expressed by mu g/ml. Each test was repeated 2 times in exactly the same way with 3 parallel wells as specified. So as to ensure the accuracy and the authenticity of the experiment.

3.3.2.1 Positive control sample drug sensitive plate preparation (micro liquid base dilution method)

The positive control drugs itraconazole and posaconazole were formulated with DMSO to 1600 μ g/ml according to CLSI guidelines. And subpackaging the small tubes and freezing the small tubes in a refrigerator of 80 ℃ below zero, wherein the storage life is not more than 6 months. The positive control drug fluconazole was formulated with DMSO to 6400 μ g/ml according to CLSI guidelines. And subpackaging the small tubes and freezing and storing the small tubes in a refrigerator at the temperature of 80 ℃ below zero. When preparing the drug sensitive plate, taking out one tube from a-80 ℃ refrigerator respectively, dissolving, and diluting by DMSO step by time to obtain 10 concentrations. Then diluted 50-fold each to twice the working concentration by RPMI1640 liquid medium. When the residual mother liquor is discarded, the mother liquor is not returned to minus 80 ℃. And (3) after the drug sensitive plate is prepared, storing at-80 ℃, taking out the drug sensitive plate for dissolving use when adding a bacterial solution, wherein the storage life of the-80-degree drug sensitive plate is not more than 3 months.

3.3.2.2 drug media Final drug working concentration:

itraconazole: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

Posaconazole: 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.06. mu.g/ml, 0.03. mu.g/ml, growth control, blank control.

And (3) fluconazole: 64. mu.g/ml, 32. mu.g/ml, 16. mu.g/ml, 8. mu.g/ml, 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, growth control, blank control.

3.3.2.3 preparing bacterial liquid: the method is completely the same as the experimental medicine method.

3.3.2.4 inoculation bacterial quantity: the method is completely the same as the experimental medicine method.

3.3.2.5 culture conditions: the method is completely the same as the experimental medicine method.

3.3.2.6 method for judging result:

4. The experimental method comprises the following steps: micro-liquid based dilution method: with particular reference to us CLSI-M-27a4 and M38-a 4.

5 results of the experiment

5.1 liquid microdilution method results: the results of 2 tests of MIC determination of test drugs and positive control drugs on 7 standard strains and clinical strains of 14 common pathogenic fungi show that: 8 samples of the tested drugs have better inhibition effect on 7 common pathogenic fungi of 14 strains in two test results, and the test results are summarized in table 1. The MIC determination results of positive control drugs itraconazole, fluconazole and posaconazole are summarized in table 2.

TABLE 1 MIC assay results (micro-liquid dilution method) of test drugs on 7 common 14 common pathogenic fungi of common superficial fungi

Note: the 16A compounds with corresponding 16A corresponding configurations of SZY1402-14A-1, SZY1402-14A-2, SZY1402-14B-1, SZY1402-14B-2, SZY1402-14C-1, SZY1402-14C-2, SZY1402-14D-1 and SZY1402-14D-2 in the tables

TABLE 2 MIC assay results (micro-liquid dilution method) of positive control drugs for 7 common 14 common pathogenic fungi of common superficial fungi

The experimental study is carried out by carrying out in-vitro MIC (minimal inhibitory concentration) measurement on 8 novel drug compounds on 7 common pathogenic fungi of 14 strains, and the results of 2 times of experiments are shown as follows: the 8 new drug compounds have better bacteriostatic action on 12 tested yeast type fungi (2 strains of candida albicans, 2 strains of candida krusei, 2 strains of candida parapsilosis, 2 strains of candida glabrata, 2 strains of candida tropicalis and 2 strains of cryptococcus), wherein the 16A compound (namely 16A-SRSS configuration) corresponding to SZY1402-14C2 has optimal antibacterial activity.

Example 7, stability experiment:

test compounds:

the stability of the aqueous solution of the above compound was observed for 0to 5 days, and the experimental results were as follows:

since compound 1 is insoluble in water, no stability experiments were performed.

8. Solubility test:

test compounds:

itraconazole

Solvent: water; pH: 7, solubility data at room temperature (24 ℃) (g/100 ml):

compound (I)	1	2	3	4
					Solubility in water	0.05	0.15	3.5	4.8

Compounds 16A-SRSS, 16B-SRSS, 16D-SRSS, 16E-SRSS in vivo antifungal (Candida albicans) test positive controls: itraconazole injection, specification 25ml 0.25g, manufactured by glazosmithklinmemanaging s.p.a, italy, product lot number: 14KQ 163;

materials and methods

Test animals: the ICR mice (cleaning grade) are provided by the Yangzhou university comparison medical center (animal qualification number SCXK (Su 2012-; each group comprises 10 male and female halves, and a test group, a positive control group, an infection control group and a blank control group are simultaneously established.

Infecting strains: candida albicans ATCC5314, provided by the China medical fungi Collection.

The infection route is as follows: the tail vein of the mice was challenged once.

Determination of the amount of infectious bacteria: transferring Candida albicans ATCC5314 with virulence restoring effect in animal body via Saburg agar culture mediumIncubating at 30 deg.C for 48 hr, washing with physiological saline, counting by a blood ball counter, and adjusting concentration to 2.0 × 10⁷cfu/ml、1.0×10⁷cfu/ml、2.0×10⁶cfu/ml、1.0×10⁶cfu/ml、2.0×10⁵cfu/ml. As the amount of the bacteria at the time of tail vein infection challenge, the injection amount per mouse was 0.5 ml/mouse, the number of death days of the animal was measured, and the amount of 100% death minimum infectious bacteria (100% MLD) in the mouse in 7 days was determined. Measured at 0.5X 10⁷Average survival days 7.2 ± 2.216(n ═ 10) days. The dose was used as a tail vein one-time challenge dose for SZY-1402 test on Candida albicans ATCC5314 systemic infection protection.

Test drug 16A (B/D/E) SRSS protection test against Candida albicans systemic infection.

Administration dose: positive control drug itraconazole injection 5mg/kg is used as group I, and four groups of test drugs are administered according to equimolar ratio and are respectively used as experiment A, B, D, E groups

Infection control and blank control were established simultaneously for both the pre-test and the full test, and 0.9% NaCl solution was administered intravenously once each.

The administration route and the method are as follows:

the drug is administered within 2 hours after the challenge of the tail vein bacteria of the mice, the administration dose is calculated according to the average body weight of about 20.0g of each mouse, the drug is dissolved in 0.5ml of 0.9 percent NaCl solution, and the tail vein is administered once; the infection control group and the blank control group were given 0.9% Nacl solution as a placebo.

The observation method comprises the following steps:

mice that died within 24h after dosing were not included in the observations. When all the infection controls died, the number of the surviving animals in each group was counted, and data statistics were performed.

And (3) test results:

the results of the test for the protection of mouse candida albicans ATCC5314 systemic infection model by itraconazole for injection of the new drug and the positive control drug are shown in the following table.

Claims

1. A compound having a structural formula shown in formula I:

in the formula I, R₁Is amino or carboxyl; r₂、R₃Independently selected from H, C1-C5 alkyl, halogen or amino substituted C1-C5 alkyl, benzene ring, halogen or amino or hydroxyl substituted benzene ring, or R₂And R₃Cycloalkyl constituting C3-C6, halogen or amino or hydroxy substituted C3-C6 cycloalkyl;

y is (-CH)₂) n-where n is an integer of 0to 18, or n connected methylene groups may have a pair of unsaturated double bonds.

2. The compound of claim 1, wherein: the compound is a racemate of the compound shown in the formula I, or optical isomers of the compound shown in the formula I in various configurations.

3. The compound of claim 2, wherein: the compound is an optical isomer with SRSS configuration shown in the specification;

R₁is amino or carboxyl; r₂、R₃Independently selected from H, C1-C5 alkyl, halogen or amino substituted C1-C5 alkyl, benzene ring, halogen or amino or hydroxyl substituted benzene ring, or R₂And R₃Cycloalkyl constituting C3-C6, halogen or amino or hydroxy substituted C3-C6 cycloalkyl;

y is (-CH)₂) n-, wherein n is an integer of 0to 18, or n phasesThere may be a pair of unsaturated double bonds in the contiguous methylene group.

4. A compound according to any one of claims 1-3, characterized in that: in the compound, R₁When the carboxyl is the carboxyl, the compound also comprises any one of sodium salt, potassium salt, magnesium salt, tromethamine salt, ethanolamine salt and diethylamine salt of the compound shown in the formula I;

R₁when the compound is amino, the compound also comprises a medicinal organic acid salt or inorganic acid salt of the compound shown in the formula I, and the medicinal organic acid salt or inorganic acid salt can be hydrochloride, sulfate, phosphate, methanesulfonate and toluenesulfonate.

5. A process for the preparation of a compound of formula I as claimed in any one of claims 1to 4, comprising:

in the compound represented by the formula III, R₁、R₂、R₃And Y is as defined for R in formula I in claim 1₁、R₂、R₃And definition of Y.

6. The method of claim 5, wherein: the molar ratio of the compound shown in the formula II to the compound shown in the formula III is as follows: 3-1: 1;

the reaction is carried out under the catalysis of carbodiimide;

the temperature of the esterification reaction is 30-60 ℃;

the esterification reaction time is 6-15 h.

A compound of SRSS configuration having the formula:

。

8. the use of a compound of the formula I as claimed in any of claims 1to 4 for the preparation of an antifungal agent or for the preparation of a medicament for the prophylaxis and/or treatment of diseases which are caused by fungi, or

Use of a compound according to claim 7 for the preparation of an antifungal medicament or for the preparation of a medicament for the prophylaxis and/or treatment of diseases caused by fungi.

9. Use according to claim 8, characterized in that: in the application, the fungi are as follows: fungi of the genera candida, cryptococcus or aspergillus;

the method specifically comprises the following steps: candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, or Aspergillus fumigatus.

10. A liquid preparation for antifungal use is provided,

the liquid preparation does not contain cyclodextrin auxiliary materials;

any one of a compound shown in formula I in claim 1, a racemate of the compound shown in formula I, optical isomers of the compound shown in formula I with various configurations, and salts of the compound shown in formula I;

or

A compound having the SRSS configuration according to claim 7, which is any one of a sodium salt, a potassium salt, a magnesium salt, tromethamine salt, ethanolamine salt and diethylamine salt thereof;

specifically, the liquid preparation is an injection and/or a liquid oral preparation.