1, 4-diphenyl semicarbazide compound and application thereof

Abstract

The invention relates to a compound containing amino and ureido and having Candida albicans growth inhibition activity, in particular to a 1, 4-diphenyl semicarbazide compound and a preparation method thereof, the compound is simple in structure, short in synthetic route and efficient; the compound can be applied to preparation of candida albicans antibacterial drugs. The molecular structural formula of the 1, 4-diphenyl semicarbazide compound is shown in the specification, and R1 and R2 are hydrogen atoms, halogen atoms, cyano groups, methyl groups, ethyl groups, propyl groups, methoxyl groups, trifluoromethyl groups and trifluoromethoxyl groups.

Description

Technical Field

[0001] This invention relates to a class of compounds containing amino and urea groups that have inhibitory activity against the growth of Candida albicans, specifically to 1,4-diphenylaminourea compounds and their applications. Background Technology

[0002] In healthy or immunocompromised individuals, single-celled or multicellular eukaryotic microorganisms that cause serious infections are called fungi. Fungal infections can lead to a range of health problems, including skin infections, respiratory illnesses, digestive disorders, and nervous system disorders, posing a serious threat to public health.

[0003] Currently, due to the less-than-ideal pharmacodynamic properties, toxicological parameters, and antibacterial spectrum of antifungal drugs used clinically, long-term and frequent use has led to an increasingly serious problem of fungal resistance. Therefore, fungal resistance to antimicrobial drugs has become a global public health crisis threatening human health, with approximately 1.5 million people dying worldwide each year from invasive fungal infections. Among these, invasive candidiasis has a mortality rate of 30%-40%, disseminated cryptococcosis a mortality rate of 20%-30%, and invasive aspergillosis a similar rate; these infections are more common in immunocompromised patients. Furthermore, superficial fungal infections can occur on the skin and mucous membranes, with an incidence even higher than invasive infections, severely impacting patients' quality of life.

[0004] Therefore, developing novel antifungal drugs is of great theoretical and practical significance for treating fungal infections and addressing the problem of fungal resistance. Summary of the Invention

[0005] In view of this, the present invention provides 1,4-diphenylaminourea compounds and their applications, which have simple compound structures and short and efficient synthetic routes.

[0006] To solve the above problems, the technical solution adopted in this invention is: a 1,4-diphenylaminourea compound with the following molecular structural formula:

[0007]

[0008] Wherein, R1 and R2 are hydrogen atoms, halogen atoms, cyano groups, methyl groups, ethyl groups, propyl groups, methoxy groups, trifluoromethyl groups, and trifluoromethoxy groups.

[0009] The synthetic routes for the above series of compounds are as follows:

[0010] (1) Synthetic routes of A-series compounds

[0011]

[0012] (2) Synthetic routes of B-series compounds

[0013]

[0014] (3) Synthetic routes of C-series compounds

[0015]

[0016] The application of the above-mentioned 1,4-diphenylaminourea compounds in the preparation of antibacterial drugs for Candida albicans.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The compounds of the present invention have simple structures, small molecular weights and high activity.

[0019] (2) The synthesis method of the present invention is short, simple to operate, highly efficient, convenient for post-processing, and its structure has been characterized.

[0020] (3) In vitro antibacterial activity tests of this invention demonstrated that the target compounds exhibited varying degrees of inhibitory activity against all tested bacteria. Among them, nine compounds (A5, B3, C4, C6, C9, C10, C11, C12, and C17) showed superior inhibitory activity against all pathogens compared to fluconazole, with C12 and C17 exhibiting the best activity. Specifically, C12 showed the highest MIC against the drug-resistant strain *C. albicans* 5272. 80 The value was as low as 0.25 μg / mL, and its inhibitory activity was 256 times that of fluconazole (MIC). 80 >128μg / mL). Detailed Implementation

[0021] The specific embodiments of the present invention will be described in detail below with reference to examples, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0022] The general structural formula of the 1,4-diphenylaminourea compounds of this invention is as follows:

[0023]

[0024] Wherein, R1 and R2 are hydrogen atoms, halogen atoms, cyano groups, methyl groups, ethyl groups, propyl groups, methoxy groups, trifluoromethyl groups, and trifluoromethoxy groups.

[0025] The above-mentioned 1,4-diphenylaminourea compounds are used in the preparation of antibacterial drugs for Candida albicans.

[0026] The chemical structures and physical properties of the target compounds are shown in Table 1.

[0027] Table 1. Structure and physical properties of 1,4-diphenylaminourea compounds

[0028]

[0029]

[0030]

[0031]

[0032]

[0033] The synthetic steps of the 1,4-diphenylaminourea compounds of this invention are as follows:

[0034] 1. Synthetic steps of A-series compounds

[0035] Add CDI (5.5 mmol, 0.89 g) and DMF (15 mL) to a 50 mL round-bottom flask and stir thoroughly until completely dissolved. Slowly add 3 mL of a dichloromethane solution containing substituted aniline (5 mmol) to the reaction flask and stir at room temperature for 6–12 h. After the reaction is complete, slowly add phenylhydrazine (4.7 mmol) dropwise to the reaction flask under vigorous stirring, heat to 60 °C, and reflux for 12 h. The target compounds of the A series (A1–A11) are obtained.

[0036] The synthetic route is as follows:

[0037]

[0038] 2. Synthetic steps of B-series compounds

[0039] Add CDI (5.5 mmol, 0.89 g) and DMF (15 mL) to a 50 mL round-bottom flask and stir thoroughly until completely dissolved. Slowly add 3 mL of a dichloromethane solution containing substituted aniline (5 mmol) to the reaction flask and stir at room temperature for 6–12 h. After the reaction is complete, slowly add substituted phenylhydrazine hydrochloride (5 mmol) dropwise to the reaction flask under vigorous stirring and heat to 60 °C, then reflux for 12 h. This yields the B series target compounds (B1–B7).

[0040] The synthetic route is as follows:

[0041]

[0042] 3. Synthetic steps of C-series compounds

[0043] Add CDI (5.5 mmol, 0.89 g) and DMF (15 mL) to a 50 mL round-bottom flask and stir thoroughly until completely dissolved. Slowly add 3 mL of a dichloromethane solution containing substituted aniline (5 mmol) to the reaction flask and stir at room temperature for 6–12 h. After the reaction is complete, slowly add substituted phenylhydrazine hydrochloride (5 mmol) dropwise to the reaction flask under vigorous stirring and heat to 60 °C, then reflux for 12 h. After the second reaction is complete, the C-series target compounds (C1–C30) are obtained.

[0044] The synthetic route is as follows:

[0045]

[0046] The specific steps for synthesizing different types of 1,4-diphenylaminourea compounds are as follows:

[0047] 1. Synthesis of compound A1—1,4-diphenylaminourea

[0048] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of aniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 6 h. After the reaction was complete as detected by TLC, phenylhydrazine (4.7 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with 10 mL of water, and the organic phases were combined. The solution was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound A1 in 84% yield.

[0049] 2. Synthesis of compound A4—1-phenyl-4-m-chlorophenylaminourea

[0050] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of m-chloroaniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 8 h. After the reaction was complete as detected by TLC, phenylhydrazine (4.7 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with 10 mL of water, and the organic phases were combined. The solution was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound A4 in 80% yield.

[0051] 3. Synthesis of compound B1—1-o-fluorophenyl-4-phenylaminourea

[0052] 0.89 g of CDI (5.5 mmol) and 10 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Next, 3 mL of a dichloromethane solution of aniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 6 h. After the reaction was complete as detected by TLC, o-fluorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL of saturated sodium bicarbonate solution and 3 mL of water. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound B1 in 83% yield.

[0053] 4. Synthesis of compound B3—1-p-fluorophenyl-4-phenylaminourea

[0054] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Next, 3 mL of aniline (5 mmol) in dichloromethane was slowly added to the reaction flask, and the mixture was stirred at room temperature for 6 h. After the reaction was complete as detected by TLC, p-fluorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL saturated sodium bicarbonate solution and 3 mL water. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound B3 in 84% yield.

[0055] 5. Synthesis of compound C1—1-p-chlorophenyl-4-o-chlorophenylaminourea

[0056] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of o-chloroaniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 12 h. After the reaction was complete as detected by TLC, p-chlorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL of saturated sodium bicarbonate solution and 3 mL of water. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound C1 in 76% yield.

[0057] 6. Synthesis of compound C4—1-p-chlorophenyl-4-m-chlorophenylaminourea

[0058] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of m-chloroaniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 8 h. After the reaction was complete as detected by TLC, p-chlorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL of saturated sodium bicarbonate solution and 3 mL of water. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound C4 in 82% yield.

[0059] 7. Synthesis of compound C6—1-p-fluorophenyl-4-m-chlorophenylaminourea

[0060] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of p-fluoroaniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 8 h. After the reaction was complete as detected by TLC, m-chlorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL saturated sodium bicarbonate solution and 3 mL water. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound C6 in 77% yield.

[0061] 8. Synthesis of compound C12—1-p-fluorophenyl-4-p-chlorophenylaminourea

[0062] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of p-chloroaniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 8 h. After the reaction was complete as detected by TLC, p-fluorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL of saturated sodium bicarbonate solution and 3 mL of water. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound C12 in 73% yield.

[0063] 9. Synthesis of compound C17—1-p-chlorophenyl-4-p-fluorophenylaminourea

[0064] 0.89 g of CDI (5.5 mmol) and 15 mL of DMF were added sequentially to a 50 mL round-bottom flask and stirred thoroughly until completely dissolved. Then, 3 mL of a dichloromethane solution of p-fluoroaniline (5 mmol) was slowly added to the reaction flask, and the mixture was stirred at room temperature for 6 h. After the reaction was complete as detected by TLC, p-chlorophenylhydrazine (5 mmol) was slowly added dropwise to the reaction flask under vigorous stirring. The mixture was heated to 60 °C and refluxed for 12 h. The reaction solution was then poured into a mortar, 10 mL of dichloromethane was added, and the mixture was ground for 5 min. The mixture was washed three times with a mixture of 10 mL saturated sodium bicarbonate solution and 3 mL water. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target compound C17 in 72% yield.

[0065] I. Spectral data of some compounds

[0066] This invention employs spectral technology ( 1 H NMR, 13 All compounds were structurally characterized by 12C NMR and HRMS. The following are the NMR spectra of nine representative compounds.

[0067] 1,4-Diphenylbiurea (A1): 1H NMR(500MHz,DMSO-d6)δ9.55(d,J=2.8Hz,1H),9.35(dd,J=7.0,1.7Hz,1H),8.86(dd,J=4.2,1.6Hz,1H),8.67(s,1H),8 .03(d,J=2.7Hz,1H),7.31(dd,J=7.0,4.2Hz,1H),7.16(t,J=7.7Hz,2H),6.81(d,J=8.0Hz,2H),6.73(t,J=7.3Hz,1H). 13 C NMR (125MHz, DMSO) δ161.96,153.19,149.82,146.33,146.04,138.05,129.26,119.15,112.64,110.58,104.23.

[0068] 1-Phenylacetyl-4-m-chlorophenylaminourea (A4): 1 H NMR(400MHz,DMSO-d6)δ8.94(s,1H),8.26(s,1H),7.79(s,1H),7.75(s,1H),7.47(d, J=7.8Hz,1H),7.30-7.11(m,3H),6.97(dd,J=7.9,1.2Hz,1H),6.77(d,J=8.9Hz,3H); 13 C NMR (100MHz, DMSO-d6) δ149.73,141.93,133.32,130.52,129.25,121.76,119.58,117.64,113.09.

[0069] 1-p-Fluorophenyl-4-phenylaminourea (B3): 1 H NMR (400MHz, DMSO-d6) δ8.70(s,1H),8.19(s,1H),7.72(s,1H),7.53(d,J=8.0Hz,2H),7.22 (t,J=7.8Hz,2H),7.03(t,J=8.8Hz,2H),6.93(t,J=7.4Hz,1H),6.77(dd,J=9.0,4.5Hz,2H); 13 C NMR (100MHz, DMSO-d6) δ157.72, 156.20 (d, J = 161.9Hz), 146.41, 140.18, 128.94, 122.23, 119.24, 115.69 (d, J = 22.3Hz), 114.17 (d, J = 7.7Hz).

[0070] 1-p-Chlorophenyl-4-m-chlorophenylaminourea (C4): 1 H NMR(400MHz,DMSO-d6)δ8.98(s,1H),8.37(s,1H),7.92(s,1H),7.77(s,1H),7.46(d, J=8.3Hz,1H),7.29–7.18(m,3H),6.98(dd,J=8.0,1.2Hz,1H),6.78(d,J=8.9Hz,2H); 13 C NMR(100MHz,DMSO-d6)δ156.82,148.79,141.85,133.33,130.54,129.02,122.78,121.84,118.63,117.66,114.52.HRMS(ESI)m / z[M+H]+Calcd for C 13 H 11 Cl2N3O:296.0352.found296.0352.

[0071] 1-p-Fluorophenyl-4-m-chlorophenylaminourea (C6): 1 H NMR(400MHz,DMSO-d6)δ8.97(s,1H),8.35(s,1H),7.79(s,1H),7.75(s,1H),7.48(d,J=8.5Hz,1H),7 .24(t,J=8.1Hz,1H),7.04(t,J=8.8Hz,2H),6.97(dd,J=7.9,2.1Hz,1H),6.77(dd,J=9.0,4.5Hz,2H); 13 CNMR(100MHz,DMSO-d6)δ157.79,156.22(d,J=151.9Hz),146.24,141.89,133.32,130.52,121. 80,118.14(d,J=97.4Hz),115.82,115.59,114.27(d,J=7.7Hz).HRMS(ESI)m / z[M+H]+Calcdfor C 13 H 11 ClFN3O:280.0647.found 280.0645.

[0072] 1-m-Chlorophenyl-4-p-Chlorophenylaminourea (C9): 1H NMR (400MHz, DMSO-d6) δ8.75(s,1H),8.25(s,1H),8.01(s,1H),7.25–7.09(m,4H),6.79-6.70(m,3H),6.55-6.50(m,1H); 13 C NMR(100MHz,DMSO-d6)δ159.98,156.68,151.67,141.39,133.94,130.92,129.7 2,118.73,112.21,111.55,111.47,107.84,104.89.HRMS(ESI)m / z[M+H]+Calcd for C 13 H 11 Cl2N3O:296.0352.found 296.0353.

[0073] 1,4-Dichlorophenylaminourea (C10): 1 H NMR (400MHz, DMSO-d6) δ8.91(s,1H),8.31(s,1H),7.91(s,1H),7.58(d,J=8.5Hz,2H),7.24(dd,J=21.0,8.8Hz,4H),6.77(d,J=8.8Hz,2H); 13 C NMR(100MHz,DMSO-d6)δ156.86,148.85,139.26,130.44,129.52,129.01,128.7 5,125.80,125.42,122.71,121.50,120.83,114.49.HRMS(ESI)m / z[M+H]+Calcd forC 13 H 11 Cl2N3O:296.0352.found 296.0356.

[0074] 1-m-Fluorophenyl-4-p-chlorophenylaminourea (C11): 1 H NMR (400MHz, DMSO-d6) δ8.92(s,1H),8.33(s,1H),8.03(s,1H),7.59(d,J=8.9Hz, 2H),7.31-7.25(m,2H),7.23-7.15(m,1H),6.63-6.59(m,1H),6.56-6.47(m,2H); 13C NMR(100MHz,DMSO-d6)δ163.62(d,J=240.7Hz),156.77,152.20(d,J=10.2Hz),139.24,130.86(d,J=9.9Hz ),128.77,125.86,120.85,108.94,105.45(d,J=21.2Hz),99.47(d,J=25.6Hz).HRMS(ESI)m / z[M+H]+Calcd forC 13 H 11 ClFN3O:280.0647.found280.0643.

[0075] 1-p-Fluorophenyl-4-p-chlorophenylaminourea (C12): 1 H NMR (400MHz, DMSO-d6) δ8.91(s,1H),8.30(s,1H),7.75(s,1H),7.61(d,J=8.5Hz,2H),7.31-7.24(m,2H),7.03(t,J=8.8Hz,2H),6.81-6.74(m,2H); 13 CNMR(100MHz,DMSO-d6)δ157.77,156.23(d,J=158.1Hz),146.30,139.29,128.73,12 5.79,120.84,115.70(d,J=22.4Hz),114.24(d,J=7.4Hz).HRMS(ESI)m / z[M+H]+Calcd for C 13 H 11 ClFN3O:280.0647.found 280.0647.

[0076] 1-p-Chlorophenyl-4-p-fluorophenylaminourea (C17): 1 H NMR (400MHz, DMSO-d6) δ8.81(s,1H),8.24(s,1H),7.90(s,1H),7.54(dd,J=8. 9,5.0Hz,2H),7.22(d,J=8.8Hz,2H),7.06(t,J=8.9Hz,2H),6.82-6.74(m,2H); 13 C NMR (100MHz, DMSO-d6) δ158.01 (d, J = 198.7Hz), 156.63, 148.89, 136.51 (d, J = 2. 4Hz),129.00,122.69,115.37(d,J=21.9Hz),114.47.HRMS(ESI)m / z[M+H]+Calcd for C13 H 11 ClFN3O:280.0647.found 280.0644.

[0077] II. Antibacterial Activity of 1,4-Diphenylaminourea Compounds

[0078] 1. Test strain

[0079] Standard Candida albicans ATCC10231;

[0080] Clinical isolate C. albicans SC5314;

[0081] Clinically resistant strain C. albicans 4935;

[0082] Clinically resistant strain C. albicans 5122;

[0083] Clinically resistant strain C. albicans 5172;

[0084] Clinically resistant strain C. albicans 5272.

[0085] 2. Experimental Methods

[0086] RPMI 1640 medium (containing 800 mg / L sodium bicarbonate), 3-morpholinopropanesulfonic acid (MOPS) buffer (concentration 0.36 g / mL), and 96-well plates, sterilized with medical alcohol, were placed in a clean bench and dried under ultraviolet light. Four 50 mL Erlenmeyer flasks, along with several 10 μL, 200 μL, 300 μL, and 5 mL pipette tips, were placed in an autoclave for sterilization. 0.85 mL of MOPS buffer was added to 50 mL of RPMI 1640 medium to adjust the pH to acidity. The test compound was prepared as a 3.2 mg / mL solution. During the test, an appropriate amount of Candida albicans was picked from the solid medium and mixed into the liquid medium until its turbidity was the same as that of McFarland turbidity. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the specified values, the concentrations of the test solution were adjusted using the microdilution method to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL. After drug addition, the samples were incubated in a sterile incubator at 28°C for 24 hours. When the absorbance of the blank group at 620 nm wavelength was 0.6, the data were recorded and processed. Three control groups were set up for all the above experiments.

[0087] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A培养基对照 Hole) × 100%

[0088] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0089] 3. Examples of preliminary screening for the antibacterial activity of target compounds

[0090] In vitro inhibitory activity of compound A1 against standard Candida albicans ATCC10231

[0091] RPMI 1640 medium (containing 800 mg / L sodium bicarbonate), 3-morpholinopropanesulfonic acid (MOPS) buffer (concentration 0.36 g / mL), and a 96-well plate, sterilized with medical alcohol, were placed in a clean bench and dried under ultraviolet light. Four 50 mL Erlenmeyer flasks, along with several 10 μL, 200 μL, 300 μL, and 5 mL pipette tips, were placed in an autoclave for sterilization. 0.85 mL of MOPS buffer was added to 50 mL of RPMI 1640 medium to adjust the pH to acidity. The test compound A1 was prepared as a 3.2 mg / mL solution. During the test, an appropriate amount of Candida albicans was picked from the solid medium and mixed into the liquid medium until the turbidity was the same as the McFarland turbidity. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solution was incubated in a sterile incubator at 28°C for approximately 24 hours. When the absorbance of the blank group at 620 nm wavelength reached 0.6, the data were recorded and processed. Three control groups were included in all experiments.

[0092] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0093] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0094] Compound A1 inhibits the MIC of standard Candida albicans ATCC10231 80 Value > 64 μg / ml.

[0095] In vitro inhibitory activity of compound A3 against standard Candida albicans ATCC10231

[0096] As described for compound A1, compound A3 was prepared as a 3.2 mg / mL test solution. During the test, an appropriate amount of Candida albicans was picked from the solid culture medium and mixed into the liquid culture medium until the turbidity was the same as that of the McFarland turbidity solution. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and then the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solutions were incubated in a sterile incubator at 28°C for 24 hours. When the absorbance of the blank group at 620 nm was 0.6, the data were recorded and processed. Three control groups were set up for all experiments.

[0097] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0098] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0099] Compound A3 inhibits the MIC of clinical isolate C. albicans SC5314 80 The value is 2 μg / mL.

[0100] Compound B3 exhibits in vitro inhibitory activity against clinical isolate C. albicans SC5314.

[0101] As described for compound A1, compound B3 was prepared as a 3.2 mg / mL test solution. During the test, an appropriate amount of Candida albicans was picked from the solid culture medium and mixed into the liquid culture medium until the turbidity was the same as that of the McFarland turbidity solution. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and then the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solution was incubated in a sterile incubator at 28°C for approximately 24 hours. When the absorbance of the blank group at 620 nm wavelength reached 0.6, the data were recorded and processed. Three control groups were included in all experiments.

[0102] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0103] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound.80 value.

[0104] Compound B3 inhibits the MIC of clinical isolate C. albicans SC5314 80 The value was 2 μg / ml.

[0105] Compound C4 exhibits in vitro inhibitory activity against clinical isolate C. albicans SC5314.

[0106] As described for compound A1, compound C4 was prepared as a 3.2 mg / mL test solution. During the test, an appropriate amount of Candida albicans was picked from the solid culture medium and mixed into the liquid culture medium until the turbidity was the same as that of the McFarland turbidity solution. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and then the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solution was incubated in a sterile incubator at 28°C for approximately 24 hours. When the absorbance of the blank group at 620 nm wavelength reached 0.6, the data were recorded and processed. Three control groups were included in all experiments.

[0107] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0108] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0109] Compound C4 inhibits the MIC of clinical isolate C. albicans SC5314 80 The value is 4 μg / mL.

[0110] In vitro inhibitory activity of compound C12 against clinically resistant strain C. albicans SC5314

[0111] As described for compound A1, compound C12 was prepared as a 3.2 mg / mL test solution. During the test, an appropriate amount of Candida albicans was picked from the solid culture medium and mixed into the liquid culture medium until the turbidity was the same as that of the McFarland turbidity. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and then the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solutions were incubated in a sterile incubator at 28°C for 24 hours. When the absorbance of the blank group at 620 nm was 0.6, the data were recorded and processed. Three control groups were set up for all experiments.

[0112] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0113] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0114] Compound C12 inhibits the MIC of clinically resistant strain C. albicans SC5314 80 The value was 0.5 μg / mL.

[0115] In vitro inhibitory activity of compound C12 against clinically resistant strain C. albicans 4935

[0116] As described for compound A1, compound C12 was prepared as a 3.2 mg / mL test solution. During the test, an appropriate amount of Candida albicans was picked from the solid culture medium and mixed into the liquid culture medium until the turbidity was the same as that of the McFarland turbidity. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and then the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solution was incubated in a sterile incubator at 28°C for approximately 24 hours. When the absorbance of the blank group at 620 nm was 0.6, the data were recorded and processed. Three control groups were set up for all experiments.

[0117] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0118] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0119] Compound C12 inhibits the MIC of clinically resistant strain C. albicans 4935. 80 The value is 0.5

[0120] μg / mL.

[0121] Compound C17 exhibits in vitro inhibitory activity against clinical isolate C. albicans SC5314.

[0122] As described for compound A1, compound C12 was prepared as a 3.2 mg / mL test solution. During the test, an appropriate amount of Candida albicans was picked from the solid culture medium and mixed into the liquid culture medium until the turbidity was the same as that of the McFarland turbidity. For the initial screening of antibacterial activity, 8 μL of the test solution was added to each well, and then the volume was replenished with 192 μL of bacterial culture. The MIC of the highly active compound was determined. 80 At the time of measurement, the concentrations of the test solution were adjusted to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using the microdilution method. After drug addition, the solution was incubated in a sterile incubator at 28°C for approximately 24 hours. When the absorbance of the blank group at 620 nm wavelength reached 0.6, the data were recorded and processed. Three control groups were included in all experiments.

[0123] Inhibition rate calculation formula: Inhibition rate % = (A 菌液对照孔 -A 化合物孔 ) / (A 菌液对照孔 -A 培养基对照 Hole) × 100%

[0124] The minimum concentration at which the inhibition rate is >80% is the MIC of the compound. 80 value.

[0125] Compound C17 inhibits the MIC of clinical isolate C. albicans SC5314 80 The value was 0.5 μg / mL.

[0126] Table 2A series compounds antifungal activity test results (MIC) 80 (μg / mL)

[0127]

[0128] Table 3. Results of antifungal activity tests for Series B compounds (MIC) 80 (μg / mL)

[0129]

[0130] Table 4. Antifungal activity test results of C-series compounds (MIC) 80 (μg / mL)

[0131]

[0132]

[0133] This invention employed a microdilution method to determine the in vitro inhibitory activity of 48 synthesized target compounds against six tested strains: standard Candida albicans ATCC10231, clinical isolate C. albicans SC5314, and clinically resistant strains C. albicans 4935, C. albicans 5122, C. albicans 5172, and C. albicans 5272. The results showed that all compounds exhibited varying degrees of inhibitory activity against the tested strains. Overall, compounds A5 and B3 showed significantly higher inhibitory activity against the standard Candida albicans ATCC10231, clinical isolate C. albicans SC5314, and resistant strain C. albicans 5272 than the positive control drug, fluconazole. Compounds C4, C6, C9, C10, C11, C12, and C17 showed superior inhibitory activity against all tested strains compared to fluconazole, with C12 and C17 exhibiting the best activity.

[0134] These results fully demonstrate that the tested samples—1,4-diphenylaminourea compounds—have antibacterial effects.

[0135] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1,1,4-Diphenylaminourea compounds, characterized in that, The molecular structural formula is: Wherein, R1 and R2 are hydrogen atoms, halogen atoms, cyano groups, methyl groups, ethyl groups, propyl groups, methoxy groups, trifluoromethyl groups, and trifluoromethoxy groups.

2. The 1,4-diphenylaminourea compound according to claim 1, characterized in that, The synthetic routes for each series of compounds are as follows: (1) Synthetic routes of A-series compounds (2) Synthetic routes of B-series compounds (3) Synthetic routes of C-series compounds 3. The use of the 1,4-diphenylaminourea compound according to claim 1 in the preparation of antibacterial drugs for Candida albicans.

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