AU2021104338A4 - Antifungal compounds and their applications - Google Patents

Abstract

The present invention relates to the technical field of antifungal compounds, and specifically to an antifungal compound and its application, the antifungal compound being specifically Methyl-5-({5-nitro-1,3-thiazol-2-yl} sulfonyl)-1,3,4-thiadiazol-2-ylcarbamic acid tert-butyl ester. The antifungal compounds provided by the present invention have been tested and verified to have strong inhibitory activity against Candida albicans, Cryptococcus neoformans, Candida glabrata and Trichophyton rubrum, its activity is even better than that of common antifungal drugs (fluconazole). It showed some inhibitory activity in vitro against Candida parapsilosis, Aspergillusfumigatus and Microsporum gypsum fungi.

Description

Antifungal compounds and their applications

TECHNICAL FIELD

The present invention relates to the technical field of antifungal compounds, and

specifically to an antifungal compound and its application.

BACKGROUND

Fungal infection is one of the common causes of disease in hot zone

environments and summer operations. The incidence of superficial fungal disease

among operators performing special tasks (ships, rainforests, islands, flood rescue)

remains high, and it is easy to be transmitted among operators which is a major

feature of dermatophytosis. In particular, the fatigue and reduced resistance of the

body make the prevalence of skin fungal diseases increase sharply. Therefore, fungal

skin disease not only affects the health of patients, but also seriously reduces the

ability of workers working in hot and humid environments. Antifungal drug research

has made great progress in recent years, but their toxic side effects and the emergence

of drug-resistant strains pose challenges for clinical antifungal treatment. Coupled

with the abuse of broad-spectrum anti-infective drugs and the high doses of

antineoplastic drugs and immunosuppressive agents, the incidence of fungal diseases

is increasing. Therefore, screening for inhibitory compounds that can target novel

targets of fungi is important for the prevention and treatment of skin fungal diseases.

Carbon dioxide (C02) is not only an end product of cellular respiratory

metabolism, but also an important signaling molecule that is involved in the

regulation of multiple biological processes. For example, C02 is the main signaling molecule for blood-sucking female mosquitoes to find suitable targets, and highC02 concentrations promote maturation and swimming of animal germ cells, even affecting the lifespan of small animals (e.g., nematodes). Many fungi (e.g. Candida albicans) are both important pathogenic fungi in humans and common commensal organisms in healthy people. C02, as an important signaling molecule for microbial life activities, is about 0.03% in normal air and about 4.5% to 30% in animals, etc. It is involved in the regulation of various biological processes. TheC02 concentration in the human body is much higher than theC02 concentration in the air, and studies have shown that C02 plays an important regulatory role in the morphological transformation of the pathogenic fungus Candida albicans and in the process of infecting the host. The high concentration of C02 promotes mycelial growth and opaque formation, thus promoting the colonization of Candida albicans in the body.

This response is the result of the interaction of the bacterium with its host and its

long-term adaptive evolution. Thus, C02 plays a very important role in biological

evolution and cellular life activities.

Carbonic anhydrase (CA) is a Zn 2 + containing metalloenzyme that effectively

catalyzes the reversible hydration reactionof C02and HC03-. Based on their genetic

differences, CAs are classified into five families a, P, y, 6, and . Among them, a-CA

is the most intensively studied, including 16 isoforms, present in mammals (human

and mouse), plants, prokaryotes and fungi. It plays an important role in biological

processes such as respiration, photosynthesis, C02 transport, maintenance of

acid-base balance, and ion transport. In recent years, the presence of p-CA has begun to be reported from plants, prokaryotes and fungi, not present in mammals, and the active center of p-CA differs from that of human-derived a-CA, making it an ideal target for antifungal drugs. The current study found that p-CA not only plays an important role in theC02 sensing system of pathogenic fungi, but also participates in fungal sexual reproduction and pathogenesis, it mainly acts on the cAMP signaling pathway to activate PKA, while playing a role in infection, virulence transfer and reproduction, a pathway that is now well established. Therefore, p-CA is an ideal antifungal drug target. It has also been reported that a series of sulfonamide precursors were synthetically screened against p-CA (CAN and NCE), the causative agents of deep fungal infections such as Candida albicans and Cryptococcus neoformans. And the preliminary exploration of its mechanism of action, the class of compounds is mainly modified in the structure of the currently existing sulfonamides, and its innovation in drug structure needs to be further enhanced. Our researchers discovered for the first time that Flo8, which contains a LisH structural domain conserved in eukaryotes, is necessary for bothC02-induced mycelial growth and opaque formation.

It not only regulates the C2-induced "yeast-mycelium morphology" transition of

Candida albicans, but also controls the transition between "white gungus and opaque".

The sensitivity of Candida albicans cells in sensing C02 was enhanced by

overexpression of the Flo8 gene, and 3-CA played an important role in this process.

Thus, both the study of existing drug-derived compounds acting on p-CA and the

study of important genes and proteins in pathways related to p-CA indicate the

specificity and importance of p-CA in the life system of fungi, it is the basis for being an ideal drug target and deserves further in-depth study. Therefore, conducting screening studies for inhibitors targeting p-CA is important for the research of new generation of drugs against fungal diseases.

SUMMARY

Based on the above, the present invention provides an antifungal compound and

its application.

To solve the technical problems described above, the present invention provides

the following technical solutions:

One of the technical solutions, an antifungal compound, specifically tert-butyl

methyl-5-({5-nitro-1,3-thiazol-2-yl} sulfonyl)-1,3,4-thiadiazol-2-ylcarbamate, with

the chemical structural formula: 0

HN

S N fi--N

//\ SS

Technical solution No. 2, application of the antifungal compounds above in the

preparation of antifungal drugs.

Further, the antifungal compounds above are used in the preparation of

anti-superficial fungal disease drugs.

Further, the fungi mentioned above include Candida albicans, Candida

parapsilosis, Candida glabrata, Cryptococcus neoformans, Microsporum gypsum,

Trichophyton rubrum, and Aspergillusfumigatus.

Further, the antifungal compounds described above are used in the preparation of

antifungal drugs targeting p-carbonic anhydrase.

Compared with the prior art, the present invention has the following beneficial

effects:

The antifungal compounds provided by the present invention have been tested

and verified to have strong inhibitory activity against Candida albicans, Cryptococcus

neoformans, Candida glabrata and Trichophyton rubrum. Its activity was even better

than that of common antifungal drugs (fluconazole), showing some inhibitory activity

in vitro against Candida parapsilosis, Aspergillus fumigatus and Microsporum

gypsumfungus.

Analysis of the interaction of the antifungal compounds provided by the present

invention with p-CA revealed that the total energy of its interaction with p-CA

amounted to -27.94 Kcal/mol. The high affinity for interaction demonstrates that the

antifungal compounds provided by the present invention can specifically bind to p-CA

and have the effect of targeting p-CA antifungal drugs.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention are now described in

detail, and this detailed description should not be considered a limitation of the

invention, but should be understood as a more detailed description of certain aspects,

features and embodiments of the invention.

It is to be understood that the terms described in the present invention are intended to describe particular embodiments only and are not intended to limit the invention. Furthermore, for the range of values in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within a stated range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the scope.

Unless otherwise indicated, all technical and scientific terms used herein have

the same meaning as commonly understood by those of ordinary skill in the art

described in the present invention. Although the present invention describes only

preferred methods and materials, any methods and materials similar or equivalent to

those described herein may also be used in the implementation or testing of the

present invention. All literature referred to in this specification is incorporated by

reference for the purpose of disclosing and describing the methods and/or materials

associated with the literature. In the event of conflict with any incorporated literature,

the contents of this specification shall prevail.

Without departing from the scope or spirit of the present invention, various

improvements and variations may be made to specific embodiments of the

specification of the present invention. It will be obvious to those skilled in the art.

Other embodiments obtained from the specification of the present invention will be

apparent to the skilled person. The specification and embodiments of the invention are exemplary only.

The terms "contains", "includes", "has", " comprises", etc., as used in this

document are open-ended terms, that is meaning including but not limited to.

Embodiment 1

An antifungal compound (T21), chemically named

methyl-5-({5-nitro-1,3-thiazol-2-yl} sulfonyl)-1,3,4-thiadiazol-2-ylcarbamic acid

tert-butyl ester, with the structural formula: 0

HN S NN N //\ SS

Embodiment 2 Validation of effect

I. Test materials

1. The test strains are shown in Table 1 (the test strains were provided by the

fungal strain library of the New Drug Research Center, School of Pharmacy, Second

Military Medical University).

Table 1 Species Strain number Candida albicans Y0109

Candida albicans SC5314

Candidaparapsilosis ATCC 22019 Candidaglabrata 537 cryptococcus neoformans 32609

Microsporum gypseum Cmcc fmza

Trichophyton rubrum Cmccftla

Aspergillusfumigatus 07544

2. Fungal culture medium

(1) RPMI 1640 culture solution: RPMI 1640 (Gibco BRL, Invitrogen) 10g,

NaHCO3 2.0g, morpholinepropanesulfonic acid (MOPS, Sigma) 34.5 g (0.165 M),

add 900 ml of triturated water Dissolve, adjust pH to 7.0 (25°C) with 1 M NaOH,

volume to 1000 ml, filter and remove bacteria, store at 4°C.

(2) YEPD culture solution: yeast extract 10 g, peptone 20 g, glucose 20 g,

dissolve with 900 ml of triple distilled water, add 2 mg/ml chloramphenicol aqueous

solution 50 ml, fix the volume to 1000 ml, autoclave and store at 40 C.

(3) Sabouraud dextrose agar medium (SDA): lOg of peptone, 40g of glucose,

18g of agar, dissolved in 900 ml of triturated water, and 50 ml of 2mg/ml of

chloramphenicol aqueous solution was added. Adjust pH to 7.0, volume to 1,000 ml,

115°C, autoclave and store at 40 C.

(4) Potato dextrose agar medium (PDA medium): peeled potato 200 g, glucose

g, agar 20 g. Add 900 ml of triple distilled water, dissolve, fix the volume to 1000

ml, autoclave and store at 40 C.

3. Anti-fungal compounds

(1) Drug to be tested: methyl-5-({5-nitro-1,3-thiazol-2-yl}

sulfonyl)-1,3,4-thiadiazol-2-ylcarbamic acid tert-butyl ester, molecular formula

C7H5N504S3, molecular weight 319.35, chemical formula:

0

HN S N N\ SS

(2) Control drug: carbonic anhydrase inhibitor-acetazolamide (AAZ), molecular

formula:

0

N N

HN 0

S/

/ NH 2 0

(3) Positive control drugs: fluconazole (FCZ, purchased from Pfizer

Pharmaceutical Co., Ltd.), itraconazole (ICZ, purchased from Sigma), voriconazole

(VCZ, purchased from Sigma), ketoconazole (KCZ, purchased from Sigma),

terbinafine (TBF, purchased from Shanghai Institute of Pharmaceutical Industry).

II. Test Methods

The minimal inhibitory concentration of the compounds to be screened against

Pseudomonas and filamentous fungi were determined using the Broth Microdilution

method as recommended by CLSI-M27A3 and M38A2 of the American Clinical and

Laboratory Standards Institute (CLSI). Screening for antifungal activity with the

following experimental methods:

1. Preparation of the compound to be tested: the compound of embodiment 1

(T21) was dissolved with DMSO and prepared as 6.4 mg/ml of mother liquor and

stored at -80°C.

2. Positive control drug concentration preparation:

(1) Fluconazole 2mg/ml injection for storage; Itraconazole, voriconazole,

ketoconazole and terbinafine dissolved in DMSO to prepare 6.4mg/ml mother liquor,

stored at -80°C.

(2) Fluconazole mother liquor was formulated at a concentration of 1280[tg/ml,

and itraconazole, voriconazole, ketoconazole, and terbinafine mother liquor was

formulated at a concentration of 6.4 mg/ml. The mother liquor was added to RPMI

1640 medium before the preparation of the drug-sensitive plates, shaken and mixed

thoroughly, and diluted to an intermediate concentration of 640[tg/ml.

All the above antifungal drug mother liquor is stored at -80°C.

3. Minimal Inhibitory Concentration (MIC) assay plate preparation

(1) Before preparation of the drug-sensitive plate, 50 1 of the drug to be tested,

the control drug and the positive drug mother solution at a concentration of 6.4 mg/ml

were added to a 1 ml deep-well plate containing 450 1 of RPMI 1640 culture

medium and mixed. 160[ 1of fluconazole mother liquor was taken and added to a 1 ml

deep well plate containing 340[ 1of RPMI 1640 medium and mixed to obtain a

concentration of 640 g/ml of each drug test solution. 10 levels of multiplicative

dilution to 10 concentrations of 640-1.25 g/ml test solution.

(2) Take 20 1 of test solution of each drug at 10 concentrations of 640-1.25

[g/ml and dispense them in each row of wells 2 to 11 of a 96-well plate to make a

drug-sensitive plate. If the plates are not used immediately, they are sealed with film

and stored at -80°C.

4. Preparation of fungal suspensions

A. Saccharomyces suspension preparation

(1) Activate the test strains on SDA dishes 2 times to ensure their viability.

(2) The activated strains were inoculated in line in SDA dishes and incubated at

°C for 24h.

(3) Pick 5 colonies >1mm in diameter, dissolve them in sterilized triple-distilled

water and shake them on a shaker for 15sec to prepare a bacterial suspension.

(4) Adjusting the concentration of the bacterial suspension to 1x 106 to 5 x 106

(equivalent to a turbidity of 0.5) CFU/ml with RPMI 1640 culture solution by using a

hemocytometer plate.

Calculation formula: Bacterial fluid concentration = number of cells in 5 medium

squares x 5 x 104

(5) The prepared bacterial suspension was diluted 1000 times with RPMI 1640

medium to a concentration between 1x103-5x103 CFU/ml.

B. Filamentous fungal suspension preparation

(1) Activation of the test strains on PDA dishes for 7 d to induce conidia as well

as sporangiospore formation.

(2) Prepare a bacterial suspension by adding 1 ml of 0.85% saline containing

0.01 ml of Tween 20 (1 drop) to the colony incubated for 7 d.

(3) After the suspension is left for 3-5 min, the large particles sink to the bottom

and the upper layer of homogeneous liquid (containing ascospores or conidia and

mycelial fragments) is taken.

(4) Adjust the concentration of the bacterial suspension to 2x1O6~1x10' CFU/ml

with RPMI 1640 culture medium using a hemocytometer plate.

Calculation formula: Bacterial liquid concentration = number of spores in 5

medium squares x 5 x 104.

(5) The above prepared bacterial suspension was diluted 1000 times with RPMI

1640 medium to a concentration between 2x10 3 ~1x104 CFU/ml.

5. Inoculation

(1) Take the drug-sensitive plate and add 200[ 1of RPMI 1640 culture solution to

each well of row 1 as a blank control. Add 200[ 1of the bacterial suspension to be

tested in well 12 as a negative control; add 180[ 1of bacterial suspension to each row

of wells 2~11 of the drug sensitive plate, and mix thoroughly. The final drug

concentration of each well was made 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125[g/ml,

respectively, and the DMSO content in each well was less than 1%; well 12 contained

no drug and was used as a negative control.

(2) Drug sensitivity plate H row for fluconazole and quality control strain

CandidaparapsilosisATCC22019.

6. Cultivation

(1) Candida albicans, Candida glabrata and Candida parapsilosis were

incubated at 35°C for 24 hours and then the results were observed.

(2) Cryptococcus neoformans was incubated at 35°C for 72 hours and then

observe the results.

(3) Trichophyton rubrum was incubated at 30°C for 4-7 days and then observe

the results.

(4) The microsporum grysumum was incubated at 30°C for 4-7 days and then

observe the results.

(5) Aspergillusfumigatus was incubated at 35 °C for 24 hours and then observed

the results.

7. Interpretation of results

(1) Absorbance determination method: The absorbance value of the 96-well plate

was detected by the enzyme standardization instrument, and the absorbance value

decreased by 80% compared with the negative control hole as the minimal inhibitory

concentration (MIC) of the drug.

(2) Determination of visual results: When reading MIC values, the fungal growth

in each tested hole should be compared with the growth control hole with the aid of a

reading microscope. A score of 4 indicates no growth inhibition; a score of 3 indicates

a slight reduction in growth or 75% of the growth control; a score of 2 indicates a

significant reduction in growth or 50% of the growth control. A score of1 indicates:

slight growth or 25% of the growth control; a score of 0 indicates: clarified by visual

observation or no growth was seen.

Where the yeast determination: the MIC value of the compound to be tested was

determined to be the minimal drug concentration (reading of 2) equivalent to when the growth control had a 50% or greater reduction in growth.

Filamentous fungal determination: the MIC value of the compound to be tested

was determined to be the minimal drug concentration (reading of 2) equivalent to

when the growth control had 50% or more growth reduction. The voriconazole MIC

value was determined to be the minimum drug concentration at which there was no

visible growth (reading of 0); the fluconazole, itraconazole, ketoconazole, and

terbinafine MIC values were determined to be the minimum drug concentration

equivalent to a growth control with 50 % or more growth reduction (reading of 2).

To determine the results, the MIC was first determined by absorbance

measurement, and the reliability of the results was confirmed by visual result

determination.

8. Quality control

The purpose of row H of each drug-sensitive plate for quality control strains and

quality control compounds is to monitor the accuracy, precision, reagents used, test

conditions, and instrumentation of the drug-sensitivity test. Ensures the testers'

operation and the accuracy and objectivity of the results. The quality control

compound was fluconazole and the quality control strain was Candida parapsilosis

ATCC22019. The MIC reference values of Candidaparapsilosis ATCC22019 are

shown in Table 2, and the test operation was considered accurate and reliable only

when its MIC values were bounded by the above range. At the same time, the test

strain grows well, then the test can be considered successful and the results are

acceptable.

Table 2

MIC (pg/mL) ranges for microdilution tests

Antifungal 24-h % within 48-h %within Organism Agent Range Mode Range Range Mode Range Candida Amphotericin B 0.25-2.0 0.5 97 0.5-4.0 2.0 92 parapsilosis 5FC 0.06-0.25 0.12 99 0.12-05 025 98 ATCC*22019 Fluconazole 05-4.0 2.0 98 LO-4.0 2.0 99 Itraconazole 0.12-05 0.25 96 0.12-0.5 0.25 98 Ketoconazole 0.03-0.25 0.06/0.12 98 0.06-0.5 0.12 98 Voricanazole 0.016-0.12 0.06 100 0.03-0.25 0.06 100 Ravuconazole 0.016-0.12 0.06 96 0.03-0.25 0.06 98 Posaconazole 0.06-0.25 0.12 97 0.06-0.25 0.12 99

III. Results

According to the quality control, each drug-sensitive plate in this embodiment is

provided with a quality control strain and quality control compound, and the quality

control compound fluconazole MIC fluctuates within no more than 1 drug gradient

concentration during the experiment. All were within the MIC standard range of

quality control strains published by CLSI-M27A3 and CLSI-M38A2, indicating that

the experimental results data were reliable.

The minimal inhibitory concentration (MIC) values of T21 compounds against

eight common pathogenic fungi were measured by the CLSI-recommended

micro-liquid-based dilution method, and the experimental data are shown in Table 3.

T21 showed strong inhibitory activity against Y0109, SC53148, cryptococcus

neoformans 32609, Candida glabrata537 and Trichophyton rubrum Cmccftla, and its

activity was even better than that of common antifungal drugs (FCZ). It exhibited

some inhibitory activity in vitro against the Candidaparapsilosis22019, Aspergillus

fumigatus 07544 and Microsporum gypseum Cmccfmz. The results above indicate

that T21 has good fungal inhibitory activity.

Table 3 In vitro activity of series compounds against common pathogenic fungi (MIC,

pg/ml)

Candida cryptoc Aspergi Trichoph Microspo occus Candida 1/us fu tonru vcu rum compo SC531 parapsilo adia cop Y0109 neoform glabrata fumigat ' gypseum und 4C3 paasio an baafmgt rubrum Cmcm ans 537 usCmcz 22019 Cmccftla 32609 07544 a T-21 <0.125 <0.125 32 <0.125 <0.125 8 <0.125 8 ICZ 1 1 1 1 1 1 <0.125 0.25 TBF 64 >64 2 0.5 4 0.25 <0.125 <0.125 KCZ <0.125 <0.125 0.25 0.5 0.25 4 <0.125 1 VCZ <0.125 <0.125 <0.125 <0.125 0.25 0.25 <0.125 <0.125 FCZ <0.125 0.25 2 2 1 >64 1 32

Note: Fluconazole(FCZ), Itraconazole (ICZ), Voriconazole (VCZ), Ketoconazole

(KCZ), Terbinafine (TBF)

The crystal structure analysis of p-CA revealed that the drug action target

cavities were small and shallow, probably relying mainly on the chelation of Zn2

. The compound contains acid groups or neutral groups containing lone electron pairs,

which facilitate the interaction with Zn2. The analysis of the interaction of T21 with

p-CA is shown in Table 4, which found that the total energy of its interaction with

p-CA amounted to -27.94Kcal/mol with high interaction affinity. While the total

energy of interaction between AAZ and p-CA reached -25.38Kcal/mol, the T21

affinity activity was better than that of the positive control drug AAZ. The above

results suggest that T21 can bind specifically to p-CA and has the effect of targeting

p-CA antifungal drugs.

Table 4 Comparison of T21 and acetoazolamide (AAZ) interactions with p-CA

Target Compound Number Hydrophobic Van der Static Total Points of interaction Waals energy Energy hydrogen residues energy KJ/mol KJ/mol bonds KJ/mol

AAZ 4 2 -16.08 -9.30 -25.38 P-CA T21 3 4 -22.78 -4.41 -27.19

The foregoing is only a preferred embodiment of the invention and is not

intended to limit the invention. Any modifications, equivalent substitutions and

improvements made within the spirit and principles of the invention shall be included

in the scope of protection of the invention.

Claims (5)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An antifungal compound characterized, specifically, as tert-butyl

methyl-5-({5-nitro-1,3-thiazol-2-yl} sulfonyl)-1,3,4-thiadiazol-2-ylcarbamate, with

the chemical structural formula: 0

HN

S NN

SS

2. An application of the antifungal compound according to claim 1 in the

preparation of an antifungal drug.

3. Application of the antifungal compound according to claim 2 in the

preparation of an anti-superficial fungal disease drug.

4. The application according to claim 2 or 3, characterized in that the fungi

comprise Candida albicans, Candida parapsilosis, Candida glabrata, Cryptococcus

neoformans, Microsporum gypsum, Trichophyton rubrum and Aspergillusfumigatus.

5. Application of the antifungal compound according to claim 2 in the

preparation of antifungal drugs targeting p-carbonic anhydrase.