WO2022048533A1 - Five-membered heteroaryl compound and use thereof - Google Patents

Abstract

Provided are a series of five-membered heteroaryl compounds and the use thereof, and specifically disclosed are a compound as represented by formula (I) and a pharmaceutically acceptable salt thereof, wherein the compound can be used to prepare a drug for treating infections related to deep fungal infections.

Description

Five-membered heteroaryl compounds and their applications

The present invention claims the following priority:

CN202010918094.6, application date: September 3, 2020;

CN202011288100.0, application date: November 17, 2020.

technical field

The present invention relates to a series of five-membered heteroaryl compounds and applications thereof, in particular to compounds represented by formula (I) and pharmaceutically acceptable salts thereof.

Background technique

Invasive fungal diseases (IFDs) are the most lethal type of fungal infections, and the morbidity and mortality are on the rise. The fungal cell wall is mainly composed of glucan, chitin and mannoprotein, and glycosylphosphatidylinositol egg-anchored protein (GPI-AP) is anchored on the cell membrane and cell wall, and mediates the process of mannoprotein and glucan. Cross-linking plays an important role in fungal cell wall synthesis, adhesion and morphological transformation. Among them, Gwt1 is a key acetylase in GPI synthesis and plays an important role in the formation of GPI precursors. Inhibition of Gwt1 activity leads to the inhibition of GPI-AP synthesis, and the fungal surface mannoprotein cannot be cross-linked to the cell wall, thereby destroying its ability to adhere to the host surface and cell wall integrity, and exert antifungal effect.

SUMMARY OF THE INVENTION

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

in,

Ar is selected from

T ₁ and T ₂ are selected from CH and N;

L ₁ is selected from -O-, -CH ₂ O- and -OCH ₂ -;

R ₁ is selected from H, F, Cl, Br, I, OH and NH ₂ ;

which does not include molecules

In some aspects of the invention, the Ar is selected from

In some aspects of the invention, the structural unit

selected from

Other variables are as defined in the present invention.

There are also some solutions of the present invention that are formed by any combination of the above variables.

The present invention also provides the following compounds or their pharmaceutically acceptable salts,

The present invention also provides the application of the compound or a pharmaceutically acceptable salt thereof in preparing a medicament for treating deep fungal infection. The present invention also provides following synthetic method:

Synthesis method 1:

Synthesis method 2:

Synthesis method 3:

Synthetic method 4:

Synthesis method 5:

technical effect

The compounds of the present invention have better binding effect on Gwt1 protein and can inhibit GPI-anchored protein. The compounds of the present invention have good inhibitory activity against Candida, Cryptococcus and Aspergillus, and have good PK properties and antibacterial effects in vivo.

Description of drawings

Figure 1. Binding pattern map of Compound 1 and Gwt1 protein.

Figure 2. Binding pattern map of Compound 2 and Gwt1 protein.

Figure 3. Binding pattern map of compound 3 and Gwt1 protein.

Figure 4. Binding pattern map of compound 4 and Gwt1 protein.

Figure 5. Binding pattern map of compound 5 and Gwt1 protein.

Related Definitions

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear unless specifically defined, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to this within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

Unless otherwise indicated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.

Unless otherwise specified, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

Use solid wedge keys unless otherwise specified

and wedge-dotted keys

Indicate the absolute configuration of a stereocenter, using a straight solid key

and straight dashed keys

Indicate the relative configuration of the stereocenter, with a wavy line

Represents a solid wedge key

or wedge-dotted key

or with wavy lines

Represents a straight solid key

and straight dashed keys

Unless otherwise specified, the term "tautomer" or "tautomeric form" refers to isomers of different functional groups that are in dynamic equilibrium and are rapidly interconverted at room temperature. A chemical equilibrium of tautomers can be achieved if tautomers are possible (eg, in solution). For example, proton tautomers (also called prototropic tautomers) include interconversions by migration of protons, such as keto-enol isomerization and imine-ene Amine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in one enantiomer" refer to one of the isomers or pairs The enantiomer content is less than 100%, and the isomer or enantiomer content is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between two isomers or relative percentages of two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80% .

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). For another example, deuterated drugs can be formed by replacing hydrogen with deuterium, and the bonds formed by deuterium and carbon are stronger than those formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

The terms "optional" or "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. .

The term "substituted" means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable of. When the substituent is oxygen (ie =O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically achievable basis.

When any variable (eg, R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may optionally be substituted with up to two Rs, with independent options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of a linking group is 0, such as -(CRR) ₀ -, it means that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups connected to it are directly connected, for example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When the listed linking group does not indicate its direction of attachment, the direction of attachment is arbitrary, for example,

The linking group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right.

It is also possible to connect ring A and ring B in the opposite direction to the reading order from left to right.

Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, when a group has one or more attachable sites, any one or more sites in the group can be linked to other groups by chemical bonds. When the connection method of the chemical bond is not located, and there is an H atom at the linkable site, when the chemical bond is connected, the number of H atoms at the site will be correspondingly reduced with the number of chemical bonds connected to the corresponding valence. the group. The chemical bond connecting the site to other groups can be represented by straight solid line bonds

straight dotted key

or wavy lines

Express. For example, a straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in this group;

The straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;

The wavy line in the phenyl group indicates that it is connected to other groups through the 1 and 2 carbon atoms in the phenyl group;

Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least

These 4 connection methods, even if the H atom is drawn on -N-, but

still includes

For the group in this connection mode, when one chemical bond is connected, the H at the site will be correspondingly reduced by one to become the corresponding monovalent piperidinyl group.

Unless otherwise specified, _Cn-n+m or _Cn - _Cn+m includes any particular instance of n to n+ _m carbons, eg _C1-12 includes _C1 , _C2 , C3, C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , and C ₁₂ , also including any range from n to n+ _m , eg C _1-12 includes C _1-3 , C _1-6 , C _1-9 , C _3-6 , C _3-9 , C _3-12 , C _6-9 , C _6-12 , and C _9-12 , etc.; in the same way, n yuan to n +m-membered means that the number of atoms in the ring is from n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring , 10-membered ring, 11-membered ring, and 12-membered ring, also including any one range from n to n+m, for example, 3-12-membered ring includes 3-6 membered ring, 3-9 membered ring, 5-6 membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, a nucleophilic substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, and the like.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: formyl; acyl groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.

The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffractometry (SXRD), the cultivated single crystal is collected by Bruker D8venture diffractometer, the light source is CuKα radiation, and the scanning method is as follows:

After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.

Compounds are named according to conventional nomenclature in the art or are used

Software naming, commercially available compounds use supplier catalog names.

detailed description

The present invention will be described in detail by the following examples, but it does not mean any unfavorable limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. For those skilled in the art, various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention. will be obvious.

Calculation example 1

The molecular docking process was performed by using Maestro (

Glide SP ^[1] and default options in version 2017-2). The three-dimensional model of Gwt1 was obtained through the I-TASSER website. To prepare the protein, hydrogen atoms were added using the Protein Preparation Wizard module of Maestro ^[2] and the OPLS3 force field was used. For the preparation of ligands, the three-dimensional structure of the molecule was generated using LigPrep, and energy minimization was performed ^[3] , and the Macromodel module was used to search the conformation of small molecules. Taking the three amino acid residues Trp159, Asp457 and Tyr470 in Gwt1 as the centroid, the edge length was generated as

The cube docking grid. The interaction types between protein receptors and ligands were analyzed, and the interaction types between protein receptors and ligands were analyzed, and then reasonable docking conformations were selected and saved according to the calculated docking score and globalStrain value.

[1]Glide,

LLC, New York, NY, 2017.

[2] Maestro,

LLC, New York, NY, 2017.

[3]LigPrep,

LLC, New York, NY, 2017.

Conclusion: The compound of the present invention has good binding with Gwt1 protein.

Example 1: WX001

synthetic route:

Step 1: Synthesis of compound WX001-2

To compound WX001-1 (CAS: 936342-55-1, see patent for synthesis: US20090082403) (2 g, 8.56 mmol) in tetrahydrofuran (20 mL) solution, add potassium carbonate (3.55 g, 25.67 mmol), methanol (10 mL) and Bis(triphenylphosphine)palladium dichloride (1.2 g, 1.71 mmol) was stirred at 80° C. and 50 Psi for 12 hours under a carbon monoxide atmosphere. After cooling the reaction solution, it was filtered and concentrated under reduced pressure. The crude product was isolated and purified by column chromatography (petroleum ether:ethyl acetate v/v=100:0 to 10:1). Compound WX001-2 was obtained. ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.19-8.18 (m, 1H), 7.61-7.59 (m, 1H), 7.44 (d, J=8.0 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 6.91-6.88(m, 1H), 6.81(d, J=8.4Hz, 1H), 5.37(s, 2H), 3.70(s, 3H) 3.65(s, 2H).

Step 2: Synthesis of compound WX001-3

Compound WX001-2 (1 g, 3.89 mmol) and tetrahydrofuran (10 mL) were added to a dry reaction flask, lithium hydroxide monohydrate (489 mg, 11.66 mmol) and water (5 mL) were added, and the mixture was stirred at 25° C. for 12 hours. The reaction solution was concentrated under reduced pressure, added to water (20 mL), adjusted to pH～4 with dilute hydrochloric acid (2M), extracted with ethyl acetate (20 mL×3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the compound WX001-3. ¹ H NMR (400MHz, DMSO-d ₆ )δ=12.32(s, 1H), 8.18-8.16(m, 1H), 7.74-7.69(m, 1H), 7.38(d, J=8.0Hz, 2H), 7.25 (d, J=8.0Hz, 2H), 7.00-6.97 (m, 1H), 6.86 (d, J=8.4Hz, 1H), 5.32 (s, 2H), 3.57 (s, 2H).

Step 3: Synthesis of Compound WX001

Compound WX001-3 (240 mg, 985.84 μmol) and dichloromethane (5 mL) were added to a dry bottle, DCC (102 mg, 492.92 μmol) was added at 0°C, stirred for 2 hours, filtered, the solution was concentrated under reduced pressure, and compound 4 ( CAS: 68640-74-4, see patent for synthesis: EP2065377) (150 mg, 985.84 μmol) and pyridine (5 mL), the resulting reaction solution was stirred at 90° C. for 4 hours. After the reaction solution was cooled, it was directly concentrated under reduced pressure. The crude product was subjected to preparative HPLC (neutral system) (chromatographic column: Phenomenex Gemini-NX C18 75 mm×30 mm×3 μm; mobile phase: [H ₂ O(10 mM NH ₄ HCO ₃ )-ACN]; B(ACN)%: 35% -55%, 8min) separation and purification. Compound WX001 was obtained. MS m/z (ESI): 360.1 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.18-8.16 (m, 3H), 7.74-7.70 (m, 1H), 7.46-7.39 (m, 4H), 7.00-6.97 (m, 1H), 6.86 (d, J=8.4Hz, 3H), 6.74-6.71 (m, 1H), 5.34 (s, 2H), 4.45 (s, 2H).

Example 2: WX002

synthetic route:

Step 1: Synthesis of compound WX002

In a dry vial compound WX002-1 (CAS: 53511-62-9, see patent for synthesis: EP2065377) (50 mg, 308.35 μmol) was dissolved in DMF (3 mL) and compound WX001-1 (86 mg, 370.02 μmol) was added With K ₂ CO ₃ (85 mg, 616.71 μmol) and NaI (55 mg, 370.02 μmol), the reaction was stirred at 60° C. for 1 hour. Direct filtration, the crude product was subjected to preparative HPLC (TFA system) (chromatographic column: Phenomenex Luna C18 100mm×40mm×5μm; mobile phase: [H ₂ O(0.1%TFA)-ACN]; B(ACN)%: 15%-48 %, 8min) separation and purification. Compound WX002 was obtained. MS m/z (ESI): 360.1 [M+1] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.56 (d, J=7.6 Hz, 1H), 8.21-8.15 (m, 2H), 8.00-7.70(m, 3H), 7.49-7.44(m, 4H), 6.98(s, 2H), 6.86(m, J=8.4Hz, 1H), 6.06(s, 2H), 5.35(s, 2H) .

biological test

Test Example 1. Inhibitory Concentration Test

1. Experimental purpose

The minimum inhibitory concentration (MIC) and minimum effective concentration (MEC) of the test drug against fungi.

2. Experimental strains and test medium

Experimental strains: Candida parapsilosis ATCC 22019; Candida albicans ATCC MYA-2876; Candida albicans WX-CA009; Candida glabrata ATCC15126; Candida tropicalis ATCC 750;

Cryptococcus neoformans H99ATCC 208821;

Aspergillus fumigatus ATCC-MYA-4609; Aspergillus flavusATCC MYA-1004

Test medium: RPMI1640 (containing 0.165M MOPS, pH 7.0)

3. Experimental Procedure

3.1. Preparation of compound masters

On the day of the experiment, the compound in the vial was dissolved in 100% DMSO to a stock concentration of 6.24 mg/mL. Then diluted 10-fold with DMSO to 0.624 mg/mL, and set aside.

Compound solutions (0.624 mg/mL) were serially diluted 2-fold with DMSO on a 96-microwell plate (V-bottom) to sequentially obtain 100× working solutions (well 1 to well 11). 624, 312, 156, 78, 39, 20, 10, 5, 25, 12.5, 6.25 μg/mL. 100% DMSO served as a positive control (well 12). This is the compound master.

3.2. Preparation of the inoculum

Candida parapsilosis ATCC 22019 and Candida albicans ATCC MYA-2876; Candida albicans WX-CA009; Candida glabrata ATCC15126; Candida tropicalis ATCC 750 were streaked on SDA plates and placed in a 35±2℃ incubator for aerobic culture. 24h.

The -80 ℃ cryopreserved bacteria Cryptococcus neoformans H99 ATCC 208821 was streaked on the SDA plate and placed in a 35 ± 2 ℃ incubator for aerobic culture for 48h.

The -80 ℃ frozen bacteria Aspergillus fumigatus ATCC-MYA-4609; Aspergillus flavus ATCC MYA-1004 were streaked on SDA plates. Place in 30±2℃ anaerobic incubator for 6 days.

On the day of the experiment, for the strains Candida parapsilosis ATCC 22019, Candida albicans ATCC MYA-2876; Candida albicans WX-CA009; Candida glabrata ATCC15126; In physiological saline, the turbidity of the bacterial suspension was adjusted to OD600=0.2 with a turbidimeter, and the bacterial suspension contained ~3.0×10 ⁶ CFU/mL. The turbidity-adjusted bacterial suspension was then diluted with test medium to a concentration of ~3.0×10 ³ CFU/mL. This is the inoculum.

For strain Aspergillus fumigatus ATCC MYA-4609; Aspergillus flavus ATCC MYA-1004, after removing the plate, add 3 mL of 0.9% saline containing 0.1% Tween20 to the plate and gently collect the spores, count with a hemocytometer and adjust the spore suspension to ~ ⁵ x 106 spores/mL. The spore suspension was then diluted to 0.8-1 x ¹⁰⁵ spores/mL with test medium.

3.3. MIC and MEC detection

Transfer 2 μL of 100× working solution from the compound master plate (prepared in 3.1) to a round-bottom 96-well plate (containing 98 μL of test medium), and then add 100 μL of bacterial inoculum (prepared in 3.2) to each well to obtain an MIC test plate . Therefore, the final test concentrations of the compounds were 6.24, 3.12, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, 0.25, 0.0125, 0.006 μg/mL. 1% DMSO was used as growth control.

For the strains Candida parapsilosis ATCC 22019; Candida albicans ATCC MYA-2876; Candida albicans WX-CA009; Candida glabrata ATCC15126;

For the mold strains Aspergillus fumigatus ATCC-MYA-4609 and Aspergillus flavusATCC MYA-1004, all test plates were incubated aerobically at 35±2°C for 48h.

For Cryptococcus neoformans H99 ATCC 208821, all test plates were incubated aerobically at 35±2°C for 72h.

3.4. Read MIC and MEC

After incubation, the MIC (μg/mL) and MEC (μg/mL) of the test compounds against yeast and mold were judged by visual observation or microscopic observation of the test plate according to the criteria in the table below.

MIC/MEC judgment criteria for the compounds to be tested against fungi

4. Experimental results

Note: The experimental results are the results of 3 independent experiments, the unit is μg/mL

Conclusion: The compounds of the present invention have good inhibitory activity against Candida, Cryptococcus and Aspergillus.

Test Example 2: Pharmacokinetic Evaluation Experiment in Mice

Experimental purpose: Taking female CD-1 mice as the test animals, the plasma drug concentrations of the test compounds at different times after intraperitoneal injection were determined by LC/MS/MS method. To study the pharmacokinetic behavior of the test compound in mice, and to evaluate its pharmacokinetic characteristics.

Drug preparation: Weigh an appropriate amount of sample to prepare a clear solution. Vehicle: 10% DMSO/10% Solutol/80% _H2O , pH ~5.

Dosing regimen: Take 2 healthy female CD-1 mice, purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd., with normal diet. Intraperitoneal injection. Operation steps: 2h before administration, 1-aminobenzotriazole (ABT) (50mg/kg, 5mg/mL in normal saline) was administered orally, after administration to animals, at 0.083, 0.25, 0.5, 1, 2, 4 About 30 μL of blood was collected at 8 hours, 8 hours and 24 hours respectively, and placed in a commercial anticoagulation tube pre-added with EDTA-K2. The tubes were centrifuged for 10 minutes to separate the plasma and stored at -60°C. The content of target compounds in plasma samples was determined by LC/MS/MS method.

Test Example 3: Mouse Candidemia Pharmacodynamic Model

Experimental animals: female CD-1 mice, 7 weeks old, 27-29 g, n=5;

Microbial pathogen: Candida albicans ATCC MYA-2876;

Inoculation level and route of inoculation: 2.0～4.0E+05CFU/mouse, infection by tail vein injection;

Treatment: The treatment started 1 h after infection, firstly, 1-aminobenzotriazole (ABT) was orally administered, and 2 h later, the test compound was injected intraperitoneally, once a day, for a total of 7 days, and the administration volume was 10 mL/kg.

Observation indicators: Changes in body weight and death of mice in each group within 7 days after infection.

Conclusion: CD-1 mice were injected with a certain dose of Candida albicans ATCC MYA-2876 via tail vein, the mortality of animals reached 100% within 7 days, resulting in severe mouse candidemia. In this model, after the animals were treated with 50mg/kg ABT orally, the test compound WX001 at a low dose of 26mg/kg could completely resist the death of mice caused by Candida albicans ATCC MYA-2876 candidemia.

Claims (4)

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

   

   in,

   Ar is selected from

   

   T ₁ and T ₂ are selected from CH and N;

   L ₁ is selected from -O-, -CH ₂ O- and -OCH ₂ -;

   R ₁ is selected from H, F, Cl, Br, I, OH and NH ₂ ;

   and formula (I) does not include a molecule

   
2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural unit

   

   selected from

   
3. The following compounds or their pharmaceutically acceptable salts,

   

   
4. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating deep fungal infections.

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