CN112521378A

CN112521378A - Antifungal water-soluble prodrug and preparation method thereof

Info

Publication number: CN112521378A
Application number: CN202011443213.3A
Authority: CN
Inventors: 刘伟; 刘国强; 王延东
Original assignee: Shanghai Innofucheng Biotechnology Co ltd
Current assignee: Shanghai Innofucheng Biotechnology Co ltd
Priority date: 2019-12-26
Filing date: 2020-12-08
Publication date: 2021-03-19
Also published as: CN111138421A; WO2021129723A1

Abstract

The invention provides an antifungal water-soluble prodrug and a preparation method thereof. The structural formula is shown as formula I: in formula I, R is when n is 0₁Is methyl, R₂Is H; when n is an integer of 1 to 4, R₁Is H, -CH₃or-C₂H₅，R₂Is H or NH₂. The compounds of examples 1-6 of the invention have better solubility, the solubility at pH5-6 can meet the requirement of preparing injection, the solution prepared by the compounds 1-6 is more stable at 2-8 ℃ than room temperature at pH5-6, the prepared solution can be partially converted into hydroxyitraconazole in plasma by enzyme incubation, which shows that the compounds 1-6 can become prodrugs of the compounds shown in formula 1, and the solution prepared by the compounds 1-6 has no irritation and hemolysis, and is suitable for preparing injection.

Description

Antifungal water-soluble prodrug and preparation method thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an antifungal water-soluble prodrug and a preparation method 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, chromocor, Cladosporium, and Blastomyces dermatitidis.

The compound represented by formula 1 is one of metabolites of itraconazole, has antifungal activity similar to that of itraconazole (itraconazole and hydroxyitraconazole have in vitro antibacterial activity comparison; journal of mycology, china, 2013-01-009), and has inhibitory activity against various fungi (see PCT, WO 00/66100,2R,4S-hydroxyitraconazole analyzers).

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, so that the risk of clinical use is increased. It is therefore important to find an antifungal compound with suitable solubility, good solution stability, and no irritation.

Disclosure of Invention

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

in the formula I, n can be an integer of 0-4;

when n is 0, R₁Is methyl, R₂Is H;

when n is an integer of 1 to 4 (specifically 1,2, 3 or 4), R₁Is H, -CH₃or-C₂H₅，R₂Is H or NH₂。

In particular, the compounds of formula I have the following representative forms:

the pharmaceutically acceptable salts of the compounds represented by the formula I and the optical isomers thereof also belong to the protection scope of the invention.

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 formula III, n can be an integer of 0-4;

when n is 0, R₁Is methyl, R₂Is H;

In the above method, the molar ratio of the compound represented by the formula II to the compound represented by the formula III may be: 1-2: 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 20-60 ℃, and specifically can be 45 ℃;

the esterification reaction time can be 10-15h, and specifically can be 12 h.

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.

The invention also provides a liquid preparation for resisting fungi, which takes the compound shown in the formula I or the pharmaceutically acceptable salt or the optical isomer thereof as an active ingredient.

The liquid preparation can be injection; the liquid preparation takes water as a solvent and does not contain a cosolvent, and specifically, the liquid preparation can contain no cyclodextrin.

The compounds of examples 1-6 are obtained by screening, the compounds of examples 1-6 have better solubility, the solubility at the pH of 5-6 can meet the requirement of preparing injection, the solution prepared by the compounds 1-6 is more stable at the temperature of 2-8 ℃ at the pH of 5-6 than room temperature, the prepared solution can be partially converted into itraconazole in plasma by enzyme incubation, the compounds 1-6 can become prodrugs of the compounds shown in the formula I, and the solution prepared by the compounds 1-6 has no irritation and hemolysis and is suitable for preparing injection.

Drawings

FIG. 1 is a scheme showing the synthesis scheme for the preparation of Compound 1 in example 1 of the present invention.

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.

Example 1 Synthesis of Compound 1

Compound 1 was synthesized according to the synthetic scheme shown in fig. 1, with the following specific steps:

1.1, synthesis of SZY 1402-3:

adding a starting material 1(2kg), a starting material 2(1.3kg) and dimethyl sulfoxide (20L) into a 50L reaction kettle in sequence, stirring uniformly, adding potassium carbonate (3.5kg), heating to 130 ℃, and stirring for reaction for 20 hours. And (3) monitoring by HPLC (high performance liquid chromatography) until the raw materials disappear, cooling the temperature of the reaction solution to room temperature, pouring the reaction solution into 20L of ice water, stirring for 30 minutes, carrying out suction filtration, leaching a filter cake with water until the pH of the filtrate is neutral, and drying the filter cake to obtain 2.15kg of a compound SZY 1402-3.

1.2, synthesis of SZY 1402-4:

A50L reaction kettle is sequentially added with a compound SZY1402-3(1.5kg), methanol (14L), acetic acid (4.5L) and wet palladium carbon (150g), stirred uniformly and subjected to hydrogenation reaction for 20 hours at room temperature under normal pressure. HPLC (high performance liquid chromatography) monitoring until the raw materials disappear, adding 9.0L of water and 1.5L of concentrated hydrochloric acid into the reaction solution, stirring for 30 minutes, filtering by using kieselguhr to remove palladium carbon, concentrating the filtrate under reduced pressure to remove methanol, adjusting the pH of the concentrated solution to 13 by using a sodium hydroxide solution at the cooling temperature of an ice water bath, performing suction filtration, leaching a filter cake by using water until the pH of the filtrate is neutral, and drying the filter cake to obtain 1.3kg of a compound SZY 1402-4.

1.3, synthesis of SZY 1402-7:

the compound SZY1402-4(1.3kg), potassium carbonate (1.27kg) and dioxane (20L) are sequentially added into a 50L reaction kettle, the mixture is uniformly stirred, the temperature is reduced to 5-10 ℃, dioxane solution of the compound 5(943g) is slowly dripped into the reaction liquid, and the reaction is stirred for 2 hours at 20 ℃ after the dripping is finished. HPLC monitored until disappearance of starting material.

Hydrazine monohydrate (700ml) and dioxane (5L) were added to the reaction mixture, the reaction mixture was heated to 80 ℃ for 2 hours, and HPLC monitored until the starting material disappeared. And cooling the reaction solution to room temperature, adding 20L of water into the reaction solution, stirring for 30 minutes, carrying out suction filtration, leaching a filter cake by using water until the pH of the filtrate is neutral, and carrying out forced air drying at 40 ℃ to obtain 1.45kg of a compound SZY 1402-7.

1.4, synthesis of SZY 1402-8:

DMF (13L), compound SZY1402-7(1.3kg) and formamidine acetate (603g) were added to a 20L three-necked flask in this order, stirred at room temperature for 1 hour, and 2.65L of glacial acetic acid was added to the reaction solution, followed by heating to 70 ℃ for reaction for 2 hours. HPLC monitored until disappearance of starting material. Cooling the reaction solution to room temperature, carrying out suction filtration, leaching the filter cake with absolute ethyl alcohol, pumping till the filter cake is nearly dry, pulping the filter cake with absolute ethyl alcohol for 1h, carrying out suction filtration, and carrying out forced air drying on the filter cake at 40 ℃ to obtain 1.2kg of a compound SZY 1402-8.

1.5, synthesis of SZY 1402-10:

the compound SZY1402-8(100g), 3-chloro-2-butanone (60g), potassium carbonate (120g) and DMF (1L) are added into a 3L flask respectively, stirred uniformly and then heated to 80 ℃ for reaction for 3 h. HPLC (high performance liquid chromatography) is used for monitoring disappearance of raw materials, the reaction solution is introduced into ice water (3L), the filtration is carried out, a filter cake is collected, water is leached until the filtrate is neutral, and the filter cake is dried by air blowing to obtain 130g of a compound SZY 1402-10.

1.6, synthesis of SZY 1402-11:

compound SZY1402-10(130g) in a 5L three-necked flask was dissolved in methanol (2L), and sodium borohydride (30g) was added thereto in portions at 0 ℃ and stirred for 2 hours after the addition was completed, followed by monitoring the disappearance of the starting material. Half of the solvent was removed under reduced pressure, then the reaction solution was poured into 3L of water, extracted with dichloromethane (1.0L. times.3), dried and concentrated to give 70g of compound SZY1402-11.

1.7, synthesis of SZY 1402-12:

dichloromethane (800mL) and compound SZY1402-11(40.0g) are added into a 2.0L three-necked flask, the temperature is reduced to-30 ℃, and BBr is slowly added dropwise₃(39.5ml), after dropping, the temperature is raised to 25-30 ℃ and the reaction is stirred for 20 h. HPLC monitored the disappearance of starting material. Slowly adding 88% ethanol water solution 150ml into the reaction solution, adding 500ml water, stirring for 30min, separating, discarding organic phase, and collecting water phase with saturated NaHCO₃The pH was adjusted to 7-8, suction filtered and the filter cake dried to give 21g of compound SZY 1402-12.

1.8, synthesis of SZY 1402-14:

DMF (135ml) was charged into a 250ml three-necked flask, followed by addition of the compound SZY1402-12(4.5g) and the compound cis-methanesulfonic acid- [2- (2, 4-dichlorophenyl) -2- (1H-1,2, 4-triazol-1-ylmethyl) -1, 3-dioxolan-4-yl]Methyl ester (SZY1402-13) (5.39g) and granular potassium hydroxide (1.54g) were added to methyl isobutyl ketone (9ml), and the mixture was stirred well, N₂Replacing gas for 3 times, heating to 60 ℃ and reacting for 4 hours. Control to reaction in TLCAnd (6) finishing. The reaction solution is cooled to room temperature, filtered, the filter cake is leached by a small amount of DMF, and 500ml of water is slowly dropped into the filtrate under stirring. After dropping, stirring for 30 min. Suction filtration is carried out, the filter cake is dispersed once again by 500ml of water, white solid is obtained by suction filtration, and 6.0g of compound SZY1402-14 is obtained by air blast drying at 40 ℃.

1.9 Synthesis of Compound 01-1:

dichloromethane (200ml) was placed in a 500ml three-necked flask, and the compound SZY1402-14(10g) was added thereto and dissolved with stirring, and Boc-N-sarcosine (5.3g), EDCI (5.3g) and DMAP (5.1g) were successively added thereto and reacted with stirring at room temperature for 6 hours. And controlling the TLC to complete the reaction. Reaction solution is saturated NH₄The mixture was washed with Cl and saturated brine, separated, and concentrated under reduced pressure to give an oil, which was purified by preparative liquid phase to give 10.0g of a white foamy solid compound 01-1.

1.10, Synthesis of Compound 1:

ethyl acetate (100ml) is added into a 500ml three-neck flask, compound 01-1(10g) is added and stirred to dissolve, the solution is a clear solution, 2N HCl/EA (100ml) is dropwise added under stirring, white turbidity is generated in the solution along with the addition of HCl/EA, the reaction is stirred at room temperature for 2 hours, and the TLC point plate reaction is finished. And (3) carrying out suction filtration to obtain white solid hydrochloride, leaching a filter cake by EA, washing HCl by EA slurry, carrying out suction filtration, and carrying out reduced pressure spin drying on the filter cake to obtain 10g of a white powdery solid compound 1.

1HNMR CH₃OHδ:10.07(s,1H),8.91(s,1H),8.20(s,1H),7.85-7.82((d,J＝9.2Hz,3H),7.63-7.59(m,3H),7.46-7.44(d,J＝9.2Hz,1H),7.31-7.29(d,J＝8.8Hz,2H),7.16-7.13(d,J＝8.8Hz,2H),5.38-5.35(m,1H)，5.20-5.10(m,2H)，4.51-4.46(m,2H)，4.23-4.19(m,1H)，4.05-3.85(m,13H)，2.74(s,3H)，2.04(s,1H)，1.49-1.47(d,J＝7.2Hz,3H),1.41-1.40(d,J＝6.4Hz,3H).

LC-MS:m/z＝793.7(M+1).

Example 2 Synthesis of Compound 2

Dichloromethane (20ml) was placed in a 50ml three-necked flask, and the compound SZY1402-14(1g) was added thereto and dissolved with stirring, and Boc-3-aminopropionic acid (0.5g), EDCI (0.54g) and DMAP (0.5g) were successively added thereto and reacted with stirring at room temperature for 6 hours. And controlling the TLC to complete the reaction. Reaction solution is saturated NH₄The mixture was washed with Cl and saturated brine, separated, and concentrated under reduced pressure to give an oil, which was purified by preparative liquid phase to give 0.9g of a white foamy solid compound 02-1.

Ethyl acetate (20ml) was added to a 50ml three-necked flask, compound 02-1(0.9g) was added and dissolved with stirring to obtain a clear solution, 2N HCl/EA (10ml) was added dropwise with stirring, a white turbidity was generated in the solution with the addition of HCl/EA, the reaction was stirred at room temperature for 2 hours, and the TLC spot plate reaction was completed. And (3) carrying out suction filtration to obtain a white solid, seriously wetting, leaching a filter cake with EA, and then carrying out reduced pressure spin drying to obtain a powdery solid compound 2(0.5 g).

1H NMR(400MHz DMSO)：δ8.21(s,1H),7.95(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),3.72-3.68(m,2H),3.51-3.47(m,2H),3.39-3.36(m,4H),3.25-3.22(m,4H),3.06-3.00(m,3H),1.48-1.46(d,J＝7.2Hz,3H),1.26-1.24(d,J＝6.4Hz).MS：[M+H]+＝793.7

Example 3 Synthesis of Compound 3

Dichloromethane (20ml) was placed in a 50ml three-necked flask, and the compound SZY1402-14(1g) was added thereto and dissolved with stirring, and N-methyl-Boc-2, 4-diaminobutyric acid (1.2g), EDCI (0.54g) and DMAP (0.5g) were successively added thereto and reacted with stirring at room temperature for 6 hours. And controlling the TLC to complete the reaction. Reaction solution is saturated NH₄The mixture was washed with Cl and saturated brine, separated, and concentrated under reduced pressure to give an oil, which was purified by preparative liquid phase to give 1.8g of a white foamy solid compound 03-1.

Ethyl acetate (20ml) was added to a 50ml three-necked flask, compound 03-1(1.8g) was added and dissolved with stirring to obtain a clear solution, 2N HCl/EA (10ml) was added dropwise with stirring, a white turbidity was generated in the solution with the addition of HCl/EA, the reaction was stirred at room temperature for 2 hours, and the TLC spot plate reaction was completed. And (3) carrying out suction filtration to obtain a white solid, seriously wetting, leaching a filter cake with EA, and then carrying out reduced pressure spin drying to obtain a powdery solid compound 3(1 g).

1H NMR(400MHz DMSO)：δ8.19(s,1H),7.88(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),7.08-7.03(m,2H),6.95-6.93(d,J＝8.8Hz,2H),6.82-6.81(d,J＝2.4Hz,2H),4.86-4.75(m,2H),4.40-4.39(m,1H),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),2.66-2.62(m,4H),1.48-1.46(d,J＝7.2Hz,3H),1.26-1.24(d,J＝6.4Hz,1H).MS：[M+H]+＝822.7

Example 4 Synthesis of Compound 4

A500 ml three-necked flask was charged with methylene chloride (200ml), and Compound 14(10g) was added thereto and dissolved with stirring, followed by the addition of Boc-N-methylaminopropionic acid (5.63g), EDCI (5.3g) and DMAP (5.1g)The reaction was stirred at room temperature for 6 h. And controlling the TLC to complete the reaction. Reaction solution is saturated NH₄The mixture was washed with Cl and saturated brine, separated, and concentrated under reduced pressure to give an oil, which was purified by preparative liquid phase to give 10.0g of a white foamy solid 04-1.

Ethyl acetate (100ml) is added into a 500ml three-neck flask, compound 04-1(10g) is added and stirred to be dissolved, the solution is clear, 2N HCl/EA (100ml) is dropwise added under stirring, white turbidity is generated in the solution along with the addition of the HCl/EA, the reaction is stirred at room temperature for 2 hours, and the TLC point plate reaction is finished. And (3) carrying out suction filtration to obtain white solid hydrochloride, leaching a filter cake by EA, washing HCl by EA slurry, carrying out suction filtration, and carrying out reduced pressure spin drying on the filter cake to obtain 10g of white powdery solid compound 4.

1HNMR CH₃OHδ:10.07(s,1H),8.91(s,1H),8.20(s,1H),7.85-7.82((d,J＝9.2Hz,2H),7.76-7.74(d,J＝8.4Hz 1H),7.63-7.59(m,3H),7.46-7.44(d,J＝9.2Hz,1H),7.31-7.29(d,J＝8.8Hz,2H),7.16-7.13(d,J＝8.8Hz,2H),5.38-5.35(m,1H)，5.20-5.10(m,2H)，4.51-4.46(m,2H)，4.23-4.19(m,1H)，4.05(m,3H)，3.9(m,4H)，3.85(m,8H)，2.74(s,3H)，2.04(s,1H)，1.49-1.47(d,J＝7.2Hz,3H),1.41-1.40(d,J＝6.4Hz,3H).

LC-MS:m/z＝807.7(M+1).

Example 5 Synthesis of Compound 5

Dichloromethane (200ml) was placed in a 500ml three-necked flask, and the compound SZY1402-14(10g) was added thereto and dissolved with stirring, and Boc-aminobutyric acid (5.63g), EDCI (5.3g) and DMAP (5.1g) were successively added thereto and reacted with stirring at room temperature for 6 hours. And controlling the TLC to complete the reaction. Reaction solution is saturated NH₄The solution was washed with Cl and saturated brine, separated, and concentrated under reduced pressure to give an oil, which was purified by preparative liquid phase to give 10.0g of a white foamy solid 05-1.

Ethyl acetate (100ml) is added into a 500ml three-neck flask, compound 05-1(10g) is added and stirred to dissolve, the solution is a clear solution, 2N HCl/EA (100ml) is dropwise added under stirring, white turbidity is generated in the solution along with the addition of HCl/EA, the reaction is stirred at room temperature for 2 hours, and the TLC point plate reaction is finished. And (3) carrying out suction filtration to obtain white solid hydrochloride, leaching a filter cake by EA, washing HCl by EA slurry, carrying out suction filtration, and carrying out reduced pressure spin drying on the filter cake to obtain 10g of white powdery solid compound 5.

1HNMR CH₃OHδ:8.91(s,1H),8.20(s,1H),7.85-7.82((d,J＝9.2Hz,2H),7.76-7.74(d,J＝8.4Hz 1H),7.63-7.59(m,3H),7.46-7.44(d,J＝9.2Hz,1H),7.31-7.29(d,J＝8.8Hz,2H),7.16-7.13(d,J＝8.8Hz,2H),5.38-5.35(m,1H)，5.20-5.10(m,2H)，4.51-4.46(m,2H)，4.23-4.19(m,1H)，4.05(m,3H)，3.9(m,4H)，3.85(m,8H)，2.74(s,3H)，2.04(s,1H)，1.49-1.47(d,J＝7.2Hz,3H),1.41-1.40(d,J＝6.4Hz,3H).

LC-MS:m/z＝807.7(M+1).

Example 6 Synthesis of Compound 6

Dichloromethane (200ml) was placed in a 500ml three-necked flask, and the compound SZY1402-14(10g) was added thereto and dissolved with stirring, and Boc-N-methylaminobutyric acid (6.02g), EDCI (5.3g) and DMAP (5.1g) were successively added thereto and reacted with stirring at room temperature for 6 hours. And controlling the TLC to complete the reaction. Reaction solution is saturated NH₄The mixture was washed with Cl and saturated brine, separated, and concentrated under reduced pressure to give an oil, which was purified by preparative liquid phase to give 10.0g of a white foamy solid 06-1.

Ethyl acetate (100ml) is added into a 500ml three-neck flask, a compound 06-1(10g) is added and stirred to be dissolved, the solution is a clear solution, 2N HCl/EA (100ml) is dropwise added under stirring, white turbidity is generated in the solution along with the addition of the HCl/EA, the reaction is stirred at room temperature for 2 hours, and the TLC point plate reaction is finished. And (3) carrying out suction filtration to obtain white solid hydrochloride, leaching a filter cake by EA, washing HCl by EA slurry, carrying out suction filtration, and carrying out reduced pressure spin drying on the filter cake to obtain 10g of white powdery solid compound 6.

1HNMR CH₃OHδ:10.07(s,1H),8.91(s,1H),8.20(s,1H),7.85-7.82((d,J＝9.2Hz,2H),7.76-7.74(d,J＝8.4Hz 1H),7.63-7.59(m,3H),7.46-7.44(d,J＝9.2Hz,1H),7.31-7.29(d,J＝8.8Hz,2H),7.16-7.13(d,J＝8.8Hz,2H),5.38-5.35(m,1H)，5.20-5.10(m,2H)，4.51-4.46(m,2H)，4.23-4.19(m,1H)，4.05(m,5H)，3.9(m,4H)，3.85(m,8H)，2.74(s,3H)，2.04(s,1H)，1.49-1.47(d,J＝7.2Hz,3H),1.41-1.40(d,J＝6.4Hz,3H).

LC-MS:m/z＝821.7(M+1).

Example 7 solubility test

Solubility of the compounds of examples 1-6 in water (pH 5-6), methanol, ethyl acetate, dichloromethane (table 1)

Table 1 solubility of the compounds of examples 1-6 in various solvents

Example 8 testing of the stability of the example Compounds

Chromatographic conditions

A chromatographic column: waters Xbridge Shield RP C18 (4.6X 150mm 5 μm)

Detection wavelength: 254nm

Flow rate: 1ml/min

Sample introduction amount: 10 μ L

Column temperature: 30 deg.C

Gradient: a: 0.05% phosphoric acid aqueous solution B: acetonitrile

TABLE 2 elution scheme

Time/min	A/％	B/％
			0	90	10
20	10	90
			25	10	90
26	90	10
			35	90	10

Sample preparation: the sample was dissolved in purified water and diluted to 0.5mg/mL

1. The stability of the aqueous solution of the compound of example at each time point was evaluated at 2 to 8 ℃ and pH5 to 6, and the results were as follows (purity of main peak by HPLC,%):

table 3 stability of aqueous solutions of the compounds of the examples at various time points (2-8 ℃, pH 5-6)

2. The stability of the aqueous solution of the compound of example at each time point was measured at room temperature (25 ℃) and pH5 to 6, and the results were as follows (purity of main peak by HPLC,%):

table 4 stability of aqueous solutions of the compounds of the examples at various time points (room temperature (25 ℃), pH 5-6)

When the pH value is 5-6, the stability of the aqueous solution at 2-8 ℃ is better than that at room temperature, and the clinical requirement of preparing an injection preparation can be met.

Example 9 plasma stability test of Compounds

Chromatographic conditions

A chromatographic column: waters Xbridge Shield RP C18 (4.6X 150mm 5 μm)

Detection wavelength: 254nm

Flow rate: 1ml/min

Sample introduction amount: 10 μ L

Column temperature: 30 deg.C

Gradient: a: 0.05% phosphoric acid aqueous solution B: acetonitrile

TABLE 5 elution scheme

Time/min	A/％	B/％
			0	90	10
20	10	90
			25	10	90
26	90	10
			35	90	10

The compounds of the examples were added to fresh human whole blood collected at 37 ℃ at initial incubation concentrations of 10ug/ml, and the concentrations of compounds of formula 1 in plasma were measured after 0, 0.083, 0.25, 0.5, 1,2, 4h, respectively, with the following results (ng/ml):

TABLE 6 concentration of the Compound of formula 1 in plasma

Examples 1-6 the compounds were all able to be partially metabolized to compounds of formula 1 in whole blood incubations, and compounds 1-6 could be prodrugs of formula 1.

Example 10 solution irritation test of example Compounds

Injection intramuscular stimulation test of the Compounds of the examples

The experiment uses 6 New Zealand rabbits, adopts the self-contrast method of the left and right muscle quadriceps muscle of the same body, and each four muscle of each side is injected with 1.0mL per one intramuscular once a day for 3 days continuously, each administration group is administered with the solution prepared by the compound of the embodiment and the water for injection with the corresponding concentration, the concentration is 1.88mg/mL, and the right side is administered with the sodium chloride injection with the equal volume of 0.9% (0.9g/100mL) as the control. Performing cesarean examination 48 hours after the last administration of the drug, and observing the texture of deep muscle tissues of bilateral injection parts of the rabbits by naked eyes, wherein the texture is rich in elasticity and luster, and no irritation reaction symptoms such as red swelling, congestion and the like are generated; pathological histological examination shows that no remarkable stimulating reaction such as degeneration or necrosis and the like is found in deep muscle tissues of bilateral injection parts of the rabbits. The intramuscular irritation test of the injections prepared in examples 1 to 6 was satisfactory. The specific experimental methods and experimental results are as follows:

preparing a test substance:

1 bottle of the injection (4mL:94 mg/bottle) prepared in examples 1 to 6 was aspirated to 1.0mL, and then diluted to 12.5mL with 0.9% sodium chloride injection to give a solution having a concentration of 1.88 mg/mL.

Animal weighing: before dosing and 48 hours and 14 days after the last dose were weighed once each.

The administration method comprises the following steps:

taking a healthy New Zealand white rabbit, and respectively injecting a test substance into the left four thighs of the rabbit and injecting an equal volume of 0.9% sodium chloride injection into the right four thighs of the rabbit by an aseptic method to serve as a control.

And (3) test observation:

the animals and the site of intramuscular injection were observed and recorded before daily administration, and 48 hours after the last administration, the test animals were taken, sacrificed by exsanguination, the quadriceps muscle was exposed by dissection, cut longitudinally, and the site of injection was observed and recorded visually, and the muscle at the site of injection was examined pathologically and histologically.

And (4) judging a result:

and carrying out comprehensive judgment according to the examination results of visual observation and histopathology.

And (3) test results: .

EXAMPLES solutions formulated with the compounds were responsive to rabbit muscle stimulation (visual observation)

The visual observation time of the rabbits is 48 hours after the last administration

Table 8 compound injection of the example responds to rabbit muscle stimulation (pathological observation)

The results of visual observation and pathological report at 48 hours after the last administration show that the compound solution of the example has no obvious irritative change such as degeneration, necrosis and the like on the muscle of the injection site of the New Zealand rabbit.

Examples 1-6 compound solutions (7.5 and 22.5mg/kg · bw) were negative to the guinea pig systemic active hypersensitivity test; solutions of the compounds of examples 1-6 (1.88mg/mL) were non-irritating to rabbit auricle blood vessels and surrounding tissue; examples 1-6 solutions of the compounds (1.88mg/mL) were non-irritating to the muscle tissue at the site of rabbit injection; example Compounds 1-6 solutions (1.88mg/mL) were negative to rabbit in vitro hemolytic tests.

Experiments show that the compounds 1 to 6 in the examples have proper solubility, meet the requirements on solution stability, have no irritation, allergy or hemolysis, and are suitable for preparing liquid formulations such as injection and the like.

Example 11, in vivo antifungal experiment with the compound of example:

positive control drug: itraconazole injection, specification 25ml 0.25g, manufactured by GlazoSmithkline manufacturing s.p.a, italy, product lot number: 14KQ 163;

CR mice (cleaning grade) provided by Yangzhou university comparison medical center are 6 weeks old and 18.0-20.0 g in weight, and are randomly grouped; each group comprises 10 male and female halves, and experimental group, positive control group, and infection 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.

Subculturing Candida albicans ATCC5314 with sandcastle agar culture medium, incubating at 30 deg.C for 48 hr, washing with physiological saline, counting with blood ball counter plate, 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) bacteria 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 the tail vein one-time challenge dose for the systemic infection protection test of Candida albicans ATCC5314 with the example compounds.

Examples protection test of compounds against candida albicans systemic infection:

administration dose: the positive control drug, namely itraconazole injection is 5mg/kg, the test drugs in the six groups of examples are administrated according to equimolar amount, the test drugs are respectively used for 1-6 groups, infection control is simultaneously established in the pre-test and the formal test, and 0.9% NaCl solution is intravenously administrated at one time.

Administered within 2 hours after challenge with caudal vein bacteria of mice at a dose of about 20.0g per average mouse weight dissolved in 0.5ml of 0.9% NaCl solution and administered in tail vein at once; the infection control group was given 0.9% NaCl solution as a placebo.

When all the infection controls died, the number of the surviving animals in each group was counted, and data statistics were performed.

The results of the protection test of the compound of examples and the positive control itraconazole injection on the mouse candida albicans ATCC5314 systemic infection model are shown in the following table.

Compounds 1-6 are capable of being converted to itraconazole in plasma in part by enzymatic incubation and have similar antifungal activity to the compounds of formula I, and example 11 demonstrates the inhibitory effect of compounds 1-6 on mouse Candida albicans ATCC 5314.

Claims

1. A compound having the formula I:

in the formula I, n is an integer of 0-4;

when n is 0, R₁Is methyl, R₂Is H;

when n is an integer of 1 to 4, R₁Is H, -CH₃or-C₂H₅，R₂Is H or NH₂。

2. The compound of claim 1, wherein: the compounds of formula I have the following representative forms:

3. a pharmaceutically acceptable salt of the compound of claim 1 or 2 or an optical isomer thereof.

4. A process for the preparation of a compound of formula i as claimed in claim 1, comprising the steps of:

in the formula III, n is an integer of 0-4;

when n is 0, R₁Is methyl, R₂Is H;

5. Use of a compound according to any one of claims 1 or 2 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.

6. Use according to claim 5, characterized in that: the fungi are: fungi of the genera Candida, Cryptococcus, Aspergillus.

7. A liquid formulation for antifungal comprising the compound represented by the formula i of claim 1 or 2 or a pharmaceutically acceptable salt or an optical isomer thereof as an active ingredient.

8. The liquid formulation of claim 7, wherein: the liquid preparation takes water as a solvent and does not contain a cosolvent.

9. The liquid formulation of claim 7 or 8, wherein: the liquid preparation is an injection.