Pyrazolo [1, 5-a] pyrimidine-3-formyl hydrazine compound as well as synthesis method and application thereof

Abstract

The invention relates to a compound capable of inhibiting the growth activity of Candida albicans, in particular to a pyrazolo [1, 5-a] pyrimidine-3-formyl hydrazine compound as well as a synthesis method and application of the pyrazolo [1, 5-a] pyrimidine-3-formyl hydrazine compound. The pyrazolo [1, 5-a] pyrimidine-3-formylhydrazine compound disclosed by the invention can be used for preparing a candida albicans antibacterial agent, and the structural formula of the pyrazolo [1, 5-a] pyrimidine-3-formylhydrazine compound is # imgabs0 #, in which R is a hydrogen atom, a halogen atom, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tertiary butyl group, a methoxy group, a trifluoromethoxy group and a trifluoromethyl group. The in-vitro inhibition activity of 31 synthesized target compounds on 6 test bacteria is determined by adopting a microdilution method, and all the compounds have different degrees of inhibition effects on the test bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of compounds having the activity of inhibiting the growth of Candida albicans, and particularly relates to pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds, their synthesis methods and applications. Background Art

[0002] In healthy or immunocompromised individuals, single-celled or multicellular eukaryotic microorganisms that cause severe infections are called fungi. They can not only infect a variety of animal and plant hosts, but also survive in different environments, thus causing allergic syndromes, superficial infections and various life-threatening diseases.

[0003] Currently, fungal infections have become a serious global public health problem, posing a great threat to human health and economic development. However, there are still many unresolved issues regarding the mechanism of fungal infections and the treatment of related diseases.

[0004] According to the location and severity of human fungal infections, they are classified into invasive fungal infections and superficial fungal infections. It is estimated that superficial fungal infections have affected the health of approximately 2 billion people globally, and most invasive fungal infections are more lethal than tuberculosis or malaria. In 2022, the World Health Organization released a report titled - 《WHO fungal priority pathogens list to guide research, development and public health action》. It listed a priority fungal list, among which Candida albicans (C. albicans) is considered a highly infectious pathogen, with a mortality rate of 20 - 50% in the infected population. Candida albicans is the most common in the genus Candida and usually inhabits the urogenital organs, gastrointestinal tract and skin as a commensal bacterium. Approximately 50% - 90% of human candidiasis is caused by Candida albicans, among which vulvovaginal candidiasis has a wide range of effects, and about 75% of women worldwide are deeply troubled by it.

[0005] Currently, the commonly used anti-Candida albicans drugs in clinics are azoles and allylamines, but they cannot eradicate fungal diseases. In addition, Candida albicans has developed high drug resistance to these two types of antifungal drugs, which makes the treatment of fungal diseases more challenging.

[0006] Therefore, the research and development of antifungal drugs with novel structures and better curative effects have important theoretical significance and practical value for the treatment of fungal infection diseases and the solution of fungal drug resistance problems. Summary of the Invention

[0007] The present invention provides a class of pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds, their synthesis methods and applications to solve the problem that the commonly used existing anti-Candida albicans drugs cannot effectively eradicate fungal diseases.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds contain highly active pharmacophores pyrazolo[1,5-a]pyrimidine and carbohydrazide, and the structural formula is:

[0010]

[0011] Among them, R is a hydrogen atom, a halogen atom, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a methoxy group, a trifluoromethoxy group, or a trifluoromethyl group.

[0012] The synthesis route of the above-mentioned pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds is as follows:

[0013]

[0014] The above-mentioned pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds can be used to prepare Candida albicans antibacterial agents.

[0015] Compared with the prior art, the present invention has the following advantages and effects:

[0016] 1) The compounds of the present invention have a simple structure and a small molecular weight, and can inhibit the growth activity of Candida albicans.

[0017] 2) The raw materials of the compounds of the present invention are cheap and easily available during the preparation process, the steps are short, and the substrate scope of application is wide.

[0018] 3) The reaction conditions in the synthesis process of the present invention are mild, the operation is easy, the post-treatment is simple, and it is efficient and concise.

[0019] 4) The antibacterial activity experiments in vitro of the compounds of the present invention show that they have inhibitory activities to varying degrees against all tested bacteria. Among them, the antibacterial activities of compounds A1, A2, A4, A5, A6, A8, and A27 are significantly higher than those of the positive drug. In particular, for compounds A2, A6, and A27, the MIC 80 values for inhibiting the drug-resistant strain C. albicans 5122 are all 16 μg / mL; in addition, the MIC 80 values of A2 for inhibiting the drug-resistant strains C. albicans 5272 and C. albicans 4935 are also as low as 16 μg / mL; their inhibitory activities are more than 8 times that of fluconazole (MIC 80 > 128 μg / mL). Detailed Embodiments

[0020] The present invention will be described in detail below in conjunction with embodiments:

[0021] The structural formula of the pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compound of the present invention is:

[0022]

[0023] Among them, R is a hydrogen atom, a halogen atom, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a methoxy group, a trifluoromethoxy group, or a trifluoromethyl group.

[0024] The chemical structures and physical properties of the target compounds are shown in Table 1:

[0025] Table 1 Structures and Physical Properties of Pyrazolo[1,5-a]pyrimidine-3-carbohydrazide Compounds

[0026]

[0027]

[0028]

[0029]

[0030] The synthetic steps for preparing pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds established in the present invention are as follows:

[0031] Add 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF to a 50 mL round-bottom flask, and stir well until it is completely dissolved. Under vigorous stirring, add 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol), and substituted hydrazine (2 mmol) in sequence. After reacting at room temperature for a period of time, 31 pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds (A1 - A31) can be obtained.

[0032]

[0033] The specific operating steps for synthesizing pyrazolo[1,5-a]pyrimidine-3-carbohydrazide with different substituents attached to the benzene ring are as follows:

[0034] 1. Synthesis of Compound A1 - N'-phenyl-3-pyrazolopyrimidinecarbohydrazide

[0035] Add 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF to a 50 mL round-bottom flask, and stir well until it is completely dissolved. Under vigorous stirring, add 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 216.3 mg of phenylhydrazine (2 mmol) in sequence. After detecting the completion of the reaction by TLC, pour the resulting reaction solution into 150 mL of saturated sodium bicarbonate aqueous solution, extract it 3 times with 50 mL of ethyl acetate, and combine the organic phases; then wash the organic phase with 50 mL of saturated brine, repeat 3 times; dry with anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure to obtain the crude product, and purify it by column chromatography to obtain the target compound A1, with a yield of 43%.

[0036] 2. Synthesis of Compound A2 - N'-(4-fluorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide

[0037] Add 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF to a 50 mL round-bottom flask, and stir well until it is completely dissolved. Under vigorous stirring, add 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 252.3 mg of 4-fluorophenylhydrazine (2 mmol) in sequence. After detecting the completion of the reaction by TLC, pour the resulting mixture into 150 mL of saturated sodium bicarbonate aqueous solution, extract it 3 times with 50 mL of ethyl acetate, combine the organic phases, and then wash the organic phase with 50 mL of saturated brine, repeat 3 times. Dry with anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure to obtain the crude product, and purify it by column chromatography to obtain the target compound A2, with a yield of 52%.

[0038] 3. Synthesis of Compound A4 - N'-(3-fluorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide

[0039] 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF were added to a 50 mL round-bottom flask and stirred well until completely dissolved. Under vigorous stirring, 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 252.3 mg of 3-fluorophenylhydrazine (2 mmol) were added successively. After the reaction was detected to be complete by TLC, the resulting mixture was poured into 150 mL of saturated aqueous sodium bicarbonate solution, extracted three times with 50 mL of ethyl acetate, the organic phases were combined, and then the organic phase was washed with 50 mL of saturated brine three times. Dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure to obtain the crude product, which was purified by column chromatography to obtain the target compound A4 with a yield of 58%.

[0040] 4. Synthesis of Compound A5 - N'-(4-chlorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide

[0041] 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF were added to a 50 mL round-bottom flask and stirred well until completely dissolved. Under vigorous stirring, 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 285.2 mg of 2-chlorophenylhydrazine (2 mmol) were added successively. After the reaction was detected to be complete by TLC, the resulting mixture was poured into 150 mL of saturated aqueous sodium bicarbonate solution, extracted three times with 50 mL of ethyl acetate, the organic phases were combined, and then the organic phase was washed with 50 mL of saturated brine three times. Dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure to obtain the crude product, which was purified by column chromatography to obtain the target compound A5 with a yield of 53%.

[0042] 5. Synthesis of Compound A6 - N'-(3-chlorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide

[0043] Add 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF to a 50 mL round-bottom flask, and stir well until it is completely dissolved. Under vigorous stirring, add 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 285.2 mg of 3-chlorophenylhydrazine (2 mmol) in sequence. After detecting the completion of the reaction by TLC, pour the obtained mixture into 150 mL of saturated sodium bicarbonate aqueous solution, extract with 50 mL of ethyl acetate for 3 times, combine the organic phases, and then wash the organic phases with 50 mL of saturated brine for 3 times. Dry over anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure to obtain the crude product, and purify by column chromatography to obtain the target compound A6 with a yield of 44%.

[0044] 6. Synthesis of Compound A8 - N'-(3-bromophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide

[0045] Add 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF to a 50 mL round-bottom flask, and stir well until it is completely dissolved. Under vigorous stirring, add 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 374.1 mg of 3-bromophenylhydrazine (2 mmol) in sequence. After detecting the completion of the reaction by TLC, pour the obtained mixture into 150 mL of saturated sodium bicarbonate aqueous solution, extract with 50 mL of ethyl acetate for 3 times, combine the organic phases, and then wash the organic phases with 50 mL of saturated brine for 3 times. Dry over anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure to obtain the crude product, and purify by column chromatography to obtain the target compound A8 with a yield of 60%.

[0046] 7. Synthesis of Compound A27 - N'-(3-methoxyphenyl)-3-pyrazolopyrimidinecarboxylic hydrazide

[0047] Add 326.1 mg of pyrazolopyrimidine-3-carboxylic acid (2 mmol) and 10 mL of DMF to a 50 mL round-bottom flask, and stir well until it is completely dissolved. Under vigorous stirring, add 383.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 mmol), 216.2 mg of 1-hydroxybenzotriazole (HOBt, 1.6 mmol) and 276.3 mg of 3-methoxyphenylhydrazine (2 mmol) in sequence. After detecting the reaction completion by TLC, pour the obtained mixture into 150 mL of saturated sodium bicarbonate aqueous solution, extract it 3 times with 50 mL of ethyl acetate, combine the organic phases, and then wash the organic phases with 50 mL of saturated brine for 3 times. Dry over anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure to obtain the crude product, and purify it by column chromatography to obtain the target compound A27 with a yield of 73%.

[0048] Spectral data of some compounds of the present invention

[0049] The present invention uses spectroscopic techniques such as MS, 1 1H NMR and 13 13C NMR to characterize the structures of all target compounds. The following are the nuclear magnetic resonance spectral data of 7 representative compounds.

[0050] N'-phenyl-3-pyrazolopyrimidinecarboxylic hydrazide (A1): 1 1H NMR (500 MHz, DMSO-d 6 ) δ 9.55 (d, J = 2.8 Hz, 1H), 9.35 (dd, J = 7.0, 1.7 Hz, 1H), 8.86 (dd, J = 4.2, 1.6 Hz, 1H), 8.67 (s, 1H), 8.03 (d, J = 2.7 Hz, 1H), 7.31 (dd, J = 7.0, 4.2 Hz, 1H), 7.16 (t, J = 7.7 Hz, 2H), 6.81 (d, J = 8.0 Hz, 2H), 6.73 (t, J = 7.3 Hz, 1H). 13 13C NMR (125 MHz, DMSO) δ 161.96, 153.19, 149.82, 146.33, 146.04, 138.05, 129.26, 119.15, 112.64, 110.58, 104.23. HRMS (ESI) m / z [M+H] + Calcd for C 13 H 12 N 5 O: 254.10364, found 254.10305.

[0051] N'-(4-fluorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide (A2):1 H NMR (500 MHz, DMSO-d 6 ) δ 9.61 (s, 1H), 9.35 (dd, J = 7.1, 1.7 Hz, 1H), 9.03 - 8.81 (m, 1H), 8.67 (d, J = 1.6 Hz, 1H), 7.91 (s, 1H), 7.32 (ddd, J = 6.4, 4.3, 1.5 Hz, 1H), 7.18 (dt, J = 8.8, 5.6 Hz, 1H), 6.88 (t, J = 6.0 Hz, 2H). 13 C NMR (125 MHz, DMSO) δ 161.97, 156.37 (d, J = 233.4 Hz), 153.18, 146.40 (d, J = 1.7 Hz), 146.32, 146.06, 138.05, 115.68 (d, J = 22.3 Hz), 113.85 (d, J = 7.7 Hz), 110.58, 104.20. HRMS (ESI) m / z [M + H] + Calcd for C 13 H 11 FN 5 O: 272.09421, found 272.09358.

[0052] N'-(3-Fluorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide (A4): 1 H NMR (500 MHz, DMSO-d 6 ) δ 9.61 (d, J = 2.5 Hz, 1H), 9.35 (d, J = 7.0 Hz, 1H), 8.86 (d, J = 3.9 Hz, 1H), 8.68 (s, 1H), 8.32 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 7.2, 4.3 Hz, 1H), 7.17 (q, J = 7.7 Hz, 1H), 6.64 (dd, J = 8.1, 2.1 Hz, 1H), 6.60 - 6.43 (m, 2H). 13 C NMR (125 MHz, DMSO) δ 163.68 (d, J = 240.6 Hz), 161.93, 153.20, 152.15 (d, J = 10.4 Hz), 146.35, 146.13, 138.03, 130.86 (d, J = 10.0 Hz), 110.60, 108.65 (d, J = 2.2 Hz), 105.15 (d, J = 21.4 Hz), 104.09, 99.16 (d, J = 25.6 Hz). HRMS (ESI) m / z [M + H] + Calcd for C 13 H 11 FN5 O: 272.09421, found 272.09376.

[0053] N'-(4-chlorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide (A5): 1 H NMR (500 MHz, DMSO-d6) δ 9.60 (s, 1H), 9.35 (dd, J = 7.0, 1.6 Hz, 1H), 8.86 (dd, J = 4.1, 1.7 Hz, 1H), 8.67 (s, 1H), 8.22 (s, 1H), 7.32 (dd, J = 7.1, 4.3 Hz, 1H), 7.19 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H). 13 C NMR (125 MHz, DMSO) δ 161.94, 153.20, 148.86, 146.34, 146.08, 138.04, 129.02, 122.39, 114.15, 110.60, 104.09. HRMS (ESI) m / z [M+H] + Calcd for C 13 H 11 ClN 5 O: 288.06466, found 288.06396.

[0054] N'-(3-chlorophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide (A6): 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.65 (d, J = 2.3 Hz, 1H), 9.37 (dd, J = 7.0, 1.7 Hz, 1H), 8.88 (dd, J = 4.2, 1.7 Hz, 1H), 8.70 (s, 1H), 8.36 (d, J = 2.3 Hz, 1H), 7.33 (dd, J = 7.0, 4.2 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.93 - 6.57 (m, 3H). 13 C NMR (100 MHz, DMSO) δ 161.95, 153.20, 151.53, 146.36, 146.13, 138.02, 134.00, 130.90, 118.52, 111.92, 111.31, 110.61, 104.04. HRMS (ESI) m / z [M+H] + Calcd for C 13 H 11 ClN 5 O: 288.06466, found 288.06420.

[0055] N'-(3-Bromophenyl)-3-pyrazolopyrimidinecarboxylic hydrazide (A8): 1 H NMR (500 MHz, DMSO-d 6 ) δ 9.62 (d, J = 2.4 Hz, 1H), 9.36 (dd, J = 7.0, 1.7 Hz, 1H), 8.87 (dd, J = 4.3, 1.6 Hz, 1H), 8.68 (s, 1H), 8.30 (d, J = 2.4 Hz, 1H), 7.32 (dd, J = 7.0, 4.2 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.80 (dd, J = 8.3, 2.2 Hz, 1H). 13 C NMR (125 MHz, DMSO) δ 161.92, 153.24, 151.67, 146.35, 146.14, 138.05, 131.26, 122.57, 121.42, 114.78, 111.67, 110.64, 104.04. HRMS (ESI) m / z [M + H] + Calcd for C 13 H 11 BrN 5 O: 332.01415, found 332.01343.

[0056] N'-(3-Methoxyphenyl)-3-pyrazolopyrimidinecarboxylic hydrazide (A27): 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.54 (d, J = 1.7 Hz, 1H), 9.35 (dd, J = 7.1, 1.7 Hz, 1H), 8.86 (dd, J = 4.2, 1.7 Hz, 1H), 8.67 (s, 1H), 8.04 (d, J = 2.3 Hz, 1H), 7.31 (dd, J = 7.1, 4.2 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 6.55 - 6.27 (m, 3H), 3.67 (s, 3H). 13 C NMR (100 MHz, DMSO) δ 161.92, 160.64, 153.18, 151.33, 146.31, 146.06, 138.04, 130.12, 110.57, 105.42, 104.46, 104.24, 98.60, 55.23. HRMS (ESI) m / z [M + H] + Calcd for C 14 H 14 N 5 O2 : 284.11420, found 284.11354。

[0057] Antibacterial activity of the pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds of the present invention:

[0058] 1. Test bacteria

[0059] Standard Candida albicans ATCC 10231

[0060] Clinically isolated strain C.albicans SC5314

[0061] Clinically drug-resistant strain C.albicans 4935

[0062] Clinically drug-resistant strain C.albicans 5122

[0063] Clinically drug-resistant strain C.albicans 5172

[0064] Clinically drug-resistant strain C.albicans 5272

[0065] 2. Test method

[0066] First, place the RPMI 1640 medium (containing 800 mg / L of sodium bicarbonate), 3-morpholinopropanesulfonic acid (MOPS) and 96-well plates disinfected with medical alcohol in a laminar flow hood and irradiate them with ultraviolet light to dry them. Then, sterilize several 50 mL conical flasks, 10 μL, 200 μL, 300 μL, 5 mL pipette tips in an autoclave. Then, add 0.85 mL of MOPS buffer solution (concentration of 1 g / mL) to 50 mL of RPMI 1640 medium, adjust the medium to acidic, and prepare the test compound into a test solution of 6.4 mg / mL. Subsequently, add 40 μL of the drug solution to be tested to 960 μL of the prepared RPMI 1640 medium to obtain a test solution of 256 μg / mL at this time. When preparing the bacterial suspension, pick an appropriate amount of Candida albicans from the solid medium and mix it evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0067] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively using the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0068] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0069] Where the minimum concentration with an inhibition rate > 80% is the MIC 80 value of the compound.

[0070] 3. Example of preliminary screening of the antibacterial activity of target compounds

[0071] In vitro antibacterial activity of compound A1 against Candida albicans ATCC 10231

[0072] First, the RPMI 1640 medium (containing 800 mg / L of sodium bicarbonate), 3-morpholinopropanesulfonic acid (MOPS), and the 96-well plate sterilized with medical alcohol were placed in the laminar flow hood and irradiated with ultraviolet light to dry. Then, several 50 mL conical flasks and pipette tips of 10 μL, 200 μL, 300 μL, and 5 mL were placed in the autoclave for sterilization. Then, 0.85 mL of MOPS buffer solution (concentration 1 g / mL) was added to 50 mL of RPMI 1640 medium to adjust the medium to acidic, and the test compound A1 was prepared into a test solution of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium to obtain a test solution of 256 μg / mL at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0073] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0074] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0075] Where the minimum concentration with an inhibition rate > 80% is the MIC 80 value of the compound.

[0076] The MIC 80 value of compound A1 against Candida albicans ATCC 10231 is 16 μg / mL. The in vitro antibacterial activity of compound A2 against the drug-resistant strain C. albicans 5272

[0077] As described for compound A1, the test solution of compound A2 was prepared at a concentration of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and a test solution with a concentration of 256 μg / mL was obtained at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0078] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0079] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A对照组 -A 空白组 ) × 100%

[0080] Among them, the minimum concentration with an inhibition rate > 80% is the MIC of this compound 80 value. All of the above experiments were set up with three control groups

[0081] The MIC value of compound A2 against the drug-resistant strain C. albicans 5272 80 is 16 μg / mL

[0082] The in vitro antibacterial activity of compound A2 against the drug-resistant strain C. albicans 5122

[0083] As described for compound A1, the test compound A2 was prepared into a test solution of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and at this time, a test solution of 256 μg / mL was obtained. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard

[0084] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as the negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as the positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data were recorded and processed. All of the above experiments were set up with three control groups

[0085] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0086] Among them, the minimum concentration with an inhibition rate > 80% is the MIC 80 value

[0087] The MIC value of compound A2 against the drug-resistant strain C. albicans 5122 80 is 16 μg / mL

[0088] The in vitro antibacterial activity of compound A2 against the drug-resistant strain C. albicans 4935

[0089] As described for compound A1, the test compound A2 was formulated into a test solution at a concentration of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and a test solution with a concentration of 256 μg / mL was obtained at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0090] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0091] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0092] Among them, the minimum concentration with an inhibition rate > 80% is the MIC 80 value of this compound.

[0093] The MIC 80 value of compound A2 against the drug-resistant strain C. albicans 4935 is 16 μg / mL.

[0094] In vitro antibacterial activity of compound A4 against the drug-resistant strain C. albicans 5122

[0095] As described for compound A1, the test compound A4 was formulated into a test solution at a concentration of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and a test solution with a concentration of 256 μg / mL was obtained at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0096] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0097] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0098] Where the minimum concentration with an inhibition rate > 80% is the MIC 80 value of the compound.

[0099] The MIC 80 value of compound A4 against the drug-resistant strain C. albicans 5122 is 16 μg / mL.

[0100] In vitro antibacterial activity of compound A5 against the drug-resistant strain C. albicans 5122

[0101] As described for compound A1. The test solution of compound A5 was prepared at a concentration of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and a test solution with a concentration of 256 μg / mL was obtained at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0102] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0103] Inhibition rate calculation formula: Inhibition rate % = (A 对照组-A 实验组 ) / (A 对照组 -A 空白组 )×100%

[0104] Among them, the minimum concentration with an inhibition rate > 80% is the MIC of this compound 80 value.

[0105] The MIC value of compound A5 against the drug-resistant strain C. albicans 5122 80 is 32 μg / mL. The in vitro antibacterial activity of compound A6 against the drug-resistant strain C. albicans 5272

[0106] As described for compound A1, the test compound A6 was prepared into a test solution of 6.4 mg / mL. Subsequently, 40 μL of the drug solution to be tested was added to 960 μL of the prepared RPMI 1640 medium, and at this time, a test solution of 256 μg / mL was obtained. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0107] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. The above experiments were all set up with three control groups.

[0108] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 -A 实验组 ) / (A 对照组 -A 空白组 )×100%

[0109] Among them, the minimum concentration with an inhibition rate > 80% is the MIC 80 value.

[0110] The MIC value of compound A6 against the drug-resistant strain C. albicans 5122 80 is 32 μg / mL. The in vitro antibacterial activity of compound A8 against the drug-resistant strain C. albicans 5272

[0111] As described for compound A1, the test compound A8 was formulated into a test solution at a concentration of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and a test solution at a concentration of 256 μg / mL was obtained at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0112] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as a negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as a positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data were recorded and processed. All the above experiments were set up with three control groups.

[0113] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0114] Where the minimum concentration with an inhibition rate > 80% is the MIC 80 value of this compound.

[0115] The MIC 80 value of compound A8 against the drug-resistant strain C.albicans 5272 was 32 μg / mL.

[0116] In vitro antibacterial activity of compound A27 against the drug-resistant strain C.albicans 5122

[0117] As described for compound A1, the test compound A27 was formulated into a test solution at a concentration of 6.4 mg / mL. Subsequently, 40 μL of the test drug solution was added to 960 μL of the prepared RPMI 1640 medium, and a test solution at a concentration of 256 μg / mL was obtained at this time. When preparing the bacterial suspension, an appropriate amount of Candida albicans was picked from the solid medium and mixed evenly in the liquid medium to make the turbidity the same as that of the McFarland turbidity standard.

[0118] During the experiment, 200 μL of the prepared RPMI 1640 medium was added to each well in the first column of the 96-well plate as the negative control; 100 μL of the bacterial suspension was added to each well in the second to eleventh columns, and 200 μL of the bacterial suspension was added to each well in the twelfth column as the positive control. The concentrations of the test compounds in the second to eleventh columns were made to be 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL respectively by the serial dilution method. After adding the drugs, the mixture was cultured in a sterile incubator at 28 °C for about 24 h. When the absorbance of the blank group at a wavelength of 620 nm was 0.6, the data was recorded and processed. All the above experiments were set up with three control groups.

[0119] Inhibition rate calculation formula: Inhibition rate % = (A 对照组 - A 实验组 ) / (A 对照组 - A 空白组 ) × 100%

[0120] Where the minimum concentration with an inhibition rate > 80% is the MIC 80 value of the compound. All the above experiments were set up with three control groups.

[0121] The MIC 80 value of compound A27 against the drug-resistant strain C. albicans 5122 was 16 μg / mL.

[0122] The in vitro inhibitory activities of 31 synthesized target compounds against 6 test bacteria were determined by the microdilution method, and the preliminary antifungal activity test results are shown in Table 2:

[0123] Table 2 Antifungal activity test results of some compounds in series A (MIC 80 , μg / mL)

[0124]

[0125]

[0126] In summary, in the present invention, standard Candida albicans ATCC 10231, clinical isolate C. albicans SC5314, clinically drug-resistant strains C. albicans 4935, C. albicans 5122, C. albicans 5172, C. albicans 5272, etc. were used as test bacteria (6 strains), and the in vitro inhibitory activities of 31 synthesized target compounds against them were determined by the microdilution method. It was found that all compounds had inhibitory activities to varying degrees against the test bacteria. Among them, compounds A1, A2, A4, A5, A6, A8, and A27 had better activities, and their antibacterial activities against the drug-resistant strains C. albicans 5272 and C. albicans 5122 were significantly higher than those of the positive control drug, fluconazole.

[0127] These results fully demonstrate that the test samples, pyrazolo[1,5-a]pyrimidine-3-carbohydrazide compounds, have antibacterial effects.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. A pyrazolo[1,5-a]pyrimidine-3-carboxylic acid hydrazide compound, characterized in that: The structural formula is: Wherein, R is a hydrogen atom, a halogen atom, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a methoxy group, a trifluoromethoxy group, or a trifluoromethyl group.

2. The method for synthesizing the pyrazolo[1,5-a]pyrimidine-3-carboxylic acid hydrazide compound according to claim 1, characterized in that: The synthetic route is:

3. Use of the pyrazolo[1,5-a]pyrimidine-3-carboxylic acid hydrazide compound according to claim 1 in the preparation of an antibacterial agent for Candida albicans.

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