RU2784215C1 - (1-BENZYL-1H-1,2,3-TRIAZOL-4-YL)METHYL (Z)-2-((3S,4S,8S,10S,11R,14S,16S)-16-ACETOXY-3,11 -DIHYDROXY-4,8,10,14-TETRAMETHYLHEXADECAHYDRO-17H-CYCLOPENTA[a]PHENANTHRENE-17-YLIDENE)-6-METHYLHEPT-5-ENOATE EXHIBITING ANTIBACTERIAL AND FUNGICIDAL ACTIVITY - Google Patents

Abstract

FIELD: medicinal chemistry.

SUBSTANCE: invention relates to the field of medicinal chemistry, namely to (1-Benzyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3S,4S,8S,10S,11R,14S, 16S)-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene-17-ylidene)-6-methylhept-5-enoate (1), which has antimicrobial activity.

EFFECT: invention also relates to the use of said compound as an agent with antimicrobial activity for combating human and animal diseases caused by Gram-positive Staphylococcus aureus (MRSA) bacteria and pathogenic fungal cultures of Cryptococcus neoformans.

2 cl, 2 tbl, 2 ex

Description

The invention relates to the field of medicinal chemistry, namely to a new 1,2,3-triazole-containing fusidic acid derivative of formula (1), which is (1-Benzyl-1H-1,2,3-triazol-4-yl)methyl (Z )-2-((3S,4S,8S,10S,11R,14S,16S)-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene -17-ylidene)-6-methylhept-5-enoate, showing antibacterial activity against Staphylococcus aureus (MRSA) and fungicidal activity against Cryptococcus neoformans.

N,N'-(1,4-phenylene)bis(2-(4-R-1H-1, 2,3-triazol-1-yl)acetamide) is known as bis-1,2,3- triazole (2) and N,N'-(1,4-phenylene)bis(2-(4-(2-(4-R-1H-1,2,3-triazol-1-yl)ethyl)-1H -1,2,3-triazol-1-yl)acetamide) in the form of tetrakis-1,2,3-triazole (3), containing cyclohexylmethyl or cyclopentyl substituents in the molecule, exhibiting antibacterial and antifungal properties (Scheme 1) [Y. Nural, S. Ozdemir, M. S. Yalcin, B. Demir, H. Atabey, Z. Seferoglu, A. Ece. New bis- and tetrakis-1,2,3-triazole derivatives: Synthesis, DNA cleavage, molecular docking, antimicrobial, antioxidant activity and acid dissociation constants. Bioorg. Med. Chem. Lett. 2022. 55. 128453].

Scheme 1

In vitro screening of the antimicrobial activity of these compounds against gram-positive (E. hirae, B. cereus, S. aureus) and gram-negative (P. aeruginosa, L. pneumophila subsp. pneumophia, E. coli) microorganisms, as well as two fungal cultures of C. albicans and C. tropicalis showed that the bis-1,2,3-triazole derivative (3) showed the highest antibacterial activity against E. coli and E. hirae strains with MIC values of 4 and 8 µg/ml, respectively. Compound (4) also proved to be active against E. hirae bacteria, which also showed a fungicidal effect against the fungal culture C. tropicalis, showing an MIC value of 4 μg/ml for both pathogens. The tetrakis-1,2,3-triazole derivative (5) was fungicidal against C. albicans and C. tropicalis strains (MIC 8 and 4 µg/ml, respectively).

Known are 1,2,3-triazole derivatives of betulinic (6-9) and oleanolic (10-13) acids containing aryl-substituted fragments in the triazole ring (Scheme 2).

Scheme 2

An in vitro study of the antitumor activity of compounds (6-13) showed that betulinic acid conjugates (6-8) inhibited the growth of cancer cells by more than 50%. Betulinic acid analog (6) containing a fluorophenyl fragment was noted as the leader, demonstrating moderate cytotoxic activity against four human cancer cell lines (HL-60, Mia PaCa-2, PC-43, A549) with IC ₅₀ ranging from 5 to 7 μM [I. Khan, SK Guru, SK Rath, PK Chinthakindi, B. Singh, S. Koul, S. Bhushan, PL Sangwan. A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in human leukemia HL-60 Cells. Eur. J. Med. Chem. 2016. 108. 104-116]. Among oleanolic acid derivatives (10-13), compound (11) with a p-nirophenyl substituent at the triazole fragment showed the best inhibitory activity against A372-S2 melanoma and HT1080 fibrosarcoma cells with IC _{50 of} 4.97 μM and 3.51 μM, respectively [G. Wei, W. Luan, S. Wang, S. Cui, F. Li, Y. Liu, Y. Liu, M. Cheng. A Library of 1,2,3-triazole-substituted oleanolic acid derivatives as anticancer agents: design, synthesis, and biological evaluation. Org. Biomol. Chem. 2015. 13. 1507-1514]. The study of antibacterial activity was not conducted.

A 1,2,3-triazole derivative of dihydroquinopimaric acid methyl ester (14) containing a benzyl substituent in the triazole ring is known (Scheme 3).

Scheme 3

An in vitro study of the antibacterial action against gram-positive (S. aureus) and gram-negative (E. coli, K. Pneumoniae, A. baumannii, P. aeruginosa) microorganisms, as well as two fungal cultures (C. albicans, C. neoformans) showed that that the resulting diterpene derivative (14) does not exhibit antimicrobial activity against these pathogens [E.V. Tret'yakova, E.V. Salimova, L.V. Parfenova. Synthesis, modification, and biological activity of propargylated methyl dihydroquinopimarates. Nat. Prod. Res. 2022.36(1). 79-86].

Thus, the 1,2,3-triazole-containing fusidic acid derivative (1) and its antimicrobial activity have not been described in the literature.

The objective of the invention is the synthesis and study of antimicrobial activity in vitro (1-benzyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3S,4S,8S,10S,11R,14S ,16S)-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene-17-ylidene)-6-methylhept-5-enoate (1) in against gram-positive and gram-negative microorganisms (S. aureus, E. coli, K. Pneumoniae, A. baumannii, P. aeruginosa), as well as two fungal cultures (C. albicans, C. neoformans) and evaluation of results in accordance with the antimicrobial screening system substances adopted in CO-ADD (http://www.co-add.org).

-2.7 (c 1.18, CHCl₃). Структура соединения (1) установлена с помощью 1D и 2D спектроскопии ЯМР ¹Н и ¹³С и масс-спектрометрии MALDI TOF/TOF.The claimed compound (1) was synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) according to the method described in [EV Tret'yakova, EV Salimova, LV Parfenova. Synthesis, modification, and biological activity of propargylated methyl dihydroquinopimarates. Nat. Prod. Res. 2022.36(1). 79-86]. At the first stage, equimolar amounts of benzyl bromide, triethylamine, and sodium azide were mixed at room temperature in methylene chloride, as a result of which benzyl azide was obtained in situ, which then reacted with fusidic acid propargyl ester (15) in the presence of CuI as a catalyst (Scheme 4). As a result of the reaction, 1,2,3-triazole derivative of fusidic acid (1) was obtained with a yield of 70%, which was a white powder (mp. 108-110°C),

-2.7 (c 1.18, CHCl ₃ ). The structure of compound ( ¹ ) was determined using 1D and 2D 1H and 13C NMR spectroscopy and MALDI ^TOF /TOF mass spectrometry.

Scheme 4

The essence of the invention is illustrated by the following examples.

-2.7 (c 1.18, CHCl₃).Example 1 A mixture of 1 mmol (0.7 ml) of triethylamine, 1 mmol (0.07 g) of sodium azide and 1 mmol (0.25 ml) of benzyl bromide in 5 ml of CH ₂ Cl ₂ was vigorously stirred for 30 min at room temperature . 0.5 mmol (0.44 g) of compound (15) in ₅ ml _of CH2Cl2 and 0.11 mmol (0.02 g) of copper(I) iodide were added to the mixture, and the mixture was stirred for 12 h at room temperature (TLC monitoring). The reaction mixture was poured into 50 ml of water, the products were extracted with chloroform (2×50 ml), the extract was washed with water until neutral pH, dried with CaCl ₂ . The solvent was evaporated, the residue was chromatographed on a column of silica gel, eluting with a mixture of chloroform : methanol 40:1. Yield of compound (1) 0.38 g (70%), white powder. So pl. 108-110°С,

-2.7 (c 1.18, CHCl ₃ ).

Spectral characteristics of (1-benzyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3S,4S,8S,10S,11R,14S,16S)-16-acetoxy-3 ,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene-17-ylidene)-6-methylhept-5-enoate (1) ¹ .

1H NMR spectrum (CDCl ₃ ), δ, ^ppm : 0.89 s (3H, H ¹⁸ ), 0.91 d (3H, H ²⁸ , ^3J 7.5 Hz), 0.96 s (3H, H ¹⁹ ), 1.02- 1.13 m (1Н, Н ⁶ ), 1.07-1.15 m (1Н, Н ⁷ ), 1.25 d (1Н, Н ¹⁵ , J 14.0 Hz), 1.35 s (1Н, Н ³⁰ ), 1.45-1.51 m (1Н, Н ¹ ), 1.49 s (3H, H ²⁷ ), 1.50–1.58 m (1H, H ⁵ ), 1.50–1.66 m (1H, H ⁶ ), 1.51–1.57 m (1H, H ⁹ ), 1.61 s (3H, H ²⁶ ), 1.67–1.76 m (1H, H ⁷ ), 1.69–1.76 m (1H, H ² ), 1.76–1.84 m (1H, H ¹² ), 1.78–1.87 m (1H, H ² ), 1.91 s (3Н, OSOS Н ₃ ), 1.91-1.97 m (1Н, Н ²³ ), 2.03-2.12 m (1Н, Н ²³ ), 2.06-2.15 m (1Н, Н ⁴ ), 2.09-2.18 m (1Н, Н ¹⁵ ), 2.11-2.19 m (1Н, Н ¹ ), 2.28 d (1Н, Н ¹² , J 13.0 Hz), 2.34-2.53 m (2Н, Н ²² ), 3.02 d (1Н, Н ¹³ , J 11.5 Hz), 3.70-3.75 m (1Н, Н ³ ), 4.29-4.35 m (1Н, Н ¹¹ ), 5.02 t (1Н, Н ²⁴ , J 7.0 Hz), 5.05 d (1Н, Н ^1' , J 12.8 Hz), 5.22 d (1Н, Н ^1' , J 12.8 Hz), 5.52 s (2Н, Н ^4' ), 5.81 d (1Н, Н ¹⁶ , J 8.5 Hz), 7.25-7.31 m (1Н, Н ^8' ), 7.27 d (2Н, Н ^6',10' , J 6.0 Hz), 7.32-7.37 m (2Н, Н ^7',9' ), 7.60 s (1Н, Н ^3' ). ^13С NMR spectrum (CDCl3), δ, ppm: 15.99 ( _С28 ), 17.68 ( ^С27 ), 17.77 ( ^С18 ), 20.90 (С6), 20.96 ( ^{ОСО СН3} ), _23.09 ( C ¹⁹ ), 23.85 (C ³⁰ ), 25.69 (C ²⁶ ), 28.29 (C ²³ ), 28.93 (C ²² ), 29.92 (C ² ), 30.09 (C ¹ ), 32.03 (C ⁷ ), 35.14 (C ⁴ ), 35.54 (C ¹² ), 36.45 (C ⁵ ), 36.87 (C ¹⁰ ), 38.99 (C ¹⁵ ), 39.44 (C ⁸ ), 44.11 (C ¹³ ), 48.66 (C ¹⁴ ), 49.28 (C ⁹ ), 54.17 (С ^4' ), 57.86 (С ^1' ), 68.12 (С ¹¹ ), 71.36 (С ³ ), 74.35 (С ¹⁶ ), 122.97 (С ²⁴ ), 123.93 (С ^3' ), 128.07 (С ^6' , С ^10' ), 128.76 (С ^8' ), 129.11 (С ^7' , С ^9' ), 129.92 (С ²⁰ ), 132.52 (С ²⁵ ), 134.50 (С ^5' ), 143.04 (С ^2' ), 149.08 (C ¹⁷ ), 169.80 (C ²¹ ), 170.48 (O C OCH ₃ ). Mass spectrum (MALDI TOF/TOF), m/z (I _rel. , %): 710 (30.5) [M+Na] ⁺ , 726 (100) [M+K] ⁺ .

¹ The reaction was monitored by TLC on Sorbfil plates (Sorbpolimer, Krasnodar, Russia), developed with 10% sulfuric acid solution. The melting point was determined on a RNMK 80/2617 instrument. 1D ( ¹ H, ¹³ C) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra were taken on a Bruker Avance 500 spectrometer (125.78 MHz for ¹³ C and 500.17 MHz for ¹ H) using standard Bruker pulse sequences, internal standard Me ₄ Si, solvent - CDCl ₃ . Optical angles were measured on a Perkin-Elmer 341 polarimeter. MALDI TOF/TOF mass spectra were obtained on a Bruker Autoflex TM III Smartbeam spectrometer using a 3-(4-hydroxy-3,5-dimethoxyphenyl) prop-2-enoic acid matrix (sinapinic acid ).

Example 2 Antimicrobial screening of compound (1) was carried out in CO-ADD (The Community for Antimicrobial Drug Discovery) funded by the Wellcome Trust (UK) and the University of Queensland (Australia) on five bacterial strains: Escherichia coli (E. coli) ATCC 25922 , Klebsiella pneumoniae (K. pneumoniae) ATCC 700603, Acinetobacter baumannii (A. baumannii) ATCC 19606, Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 and Staphylococcus aureus (S. aureus (MRSA)) ATCC 43300. Antifungal activity was determined on two fungal strains: Candida albicans (C. albicans) ATCC 90028 and Cryptococcus neoformans (C. neoformans) ATCC 208821.

Primary screening for antimicrobial activity was performed by cell proliferation inhibition tests using samples at a single (32 µg/mL) concentration. An aliquot of each sample in DMSO was placed in a 384-well plate and treated with the appropriate bacterial culture. Bacterial growth inhibition was determined by measuring absorbance at 600 nm (OD600) using a Tecan M1000 Pro monochrome microplate reader. Percent growth inhibition was calculated for each well using a negative control (medium only) and a positive control (bacteria without inhibitors) on the same plate. All tests are duplicated. If growth inhibition ≥80% was observed one or both times, the compound was considered active.

The primary screening revealed the presence of antimicrobial activity in (1-benzyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3S,4S,8S,10S,11R,14S,16S )-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene-17-ylidene)-6-methylhept-5-enoate (1) in relation to bacterial culture S aureus (MRSA) and C. neoformans fungal culture (Table 1).

Table 1. % Inhibition of microorganism growth by compound (1) and fusidic acid at a concentration of 32 μg/ml. Gram positive Gram negative Mushrooms Sa (MRSA) ¹ Ec ² Kp ³ Pa ⁴ Ab ^{5 Ca6} Cn ⁷ (one) 100.3 1.3 23.1 1.2 16.2 13.3 54.9 Fusidic acid 97.9 21.6 14.1 45.2 22.7 1.1 2.1 Vancomy
qing 100 - - - - - - Colistin - 100 100 100 100 - - Flucon
angry - - - - - 100 100

¹ S. aureus (MRSA); ² E. coli; ³ K. pneumonia; ⁴ P. aeruginosa; ⁵ A. baumannii;

⁶ C. albicans; ⁷ C. neoformans.

For compound (1), the minimum inhibitory concentration was determined in relation to the above cultures, and cytotoxic activity was also studied (table 2).

Table 2. Antibacterial, fungicidal and cytotoxic activities of (1-benzyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3S,4S,8S,10S,11R,14S,16S )-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene-17-ylidene)-6-methylhept-5-enoate (1) and fusidic acid. Compound Antibacterial activity Fungicidal activity Cytotoxic activity Staphylococcus aureus (MRSA) Cryptococcus neoformans HEK293 (ATCC CRL-1573) MIC*, μg/ml MIC, µg/ml CC ₅₀ **, µg/ml (one) 0.25
0.25 32
32 32
32 Fusidic acid 0.25
0.25 32
32 32
32 Vancomycin one - Fluconazole eight Tamoxifen - 9

*MIC (Minimum Inhibitory Concentration) is the lowest concentration at which complete inhibition of bacteria was found.

**CC ₅₀ is the concentration of the test substance at which 50% of the human embryonic kidney cell line HEK293 is killed.

The minimum inhibitory concentration (MIC; µg/mL) was determined according to the Clinical and Laboratory Standards Institute (CLSI, https://clsi.org/) guidelines, determining the lowest concentration at which complete inhibition of bacteria or fungi was found. The tests were carried out in duplicate. The maximum percentage of growth inhibition was designated as D _max . Compounds were classified as active at MIC≤16 μg/ml or MIC≤10 μM in any replica (n=2 on different plates).

The cytotoxic effect (Hk; CC ₅₀ (μg/ml)) was determined on the human embryonic kidney cell line HEK293 by determining the concentration causing 50% cell death. Growth inhibition of HEK293 cells was determined by measuring fluorescence after addition of 5 μl of 25 μg/ml resazurin (final concentration 2.3 μg/ml) and after incubation for another 3 h at 37°C in 5% CO ₂ . Fluorescence intensity was measured using a Tecan M1000 Pro monochrome microplate reader using automatic gain calculation. The maximum percentage of cytotoxicity was designated as D _max . The compound was considered toxic at CC ₅₀ ≤32 µg/mL or CC ₅₀ ≤10 µM. In addition, samples were marked as partial cytotoxic if D _max ≥50%, even at CC ₅₀ above the maximum test concentration.

"Colistin" and "Vancomycin" were used as positive standards for assessing bacterial inhibition for gram-negative and gram-positive bacteria, respectively. Fluconazole has been used as the standard fungicide for C. albicans and C. neoformans. "Tamoxifen" was used as a positive standard for assessing cytotoxicity. Methods for testing the in vitro antimicrobial, fungicidal, and cytotoxic activity of compounds are available at http://www.co-add.org.

Analysis of the results of antimicrobial screening showed that the 1,2,3-triazole-containing fusidic acid derivative (1) at a concentration of 0.25 μg/ml exhibited high antibacterial activity, inhibiting the growth and reproduction of > 100% of Gram-positive Staphylococcus aureus (MRSA) bacteria. In addition, compound (1) also had a moderate fungicidal effect against C. neoformans strain, inhibiting the growth of this fungal culture by 54.9% at a concentration of 32 µg/ml, and showed low toxicity against the HEK293 cell line at the maximum tested concentration of 32 µg /ml

Thus, compound (1) exhibits antimicrobial activity against pathogenic microorganisms S. aureus (MRSA) and C. neoformans and has low toxicity at the maximum concentration tested.

Claims (3)

1. (1-Benzyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3S,4S,8S,10S,11R,14S,16S)-16-acetoxy-3 ,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthrene-17-ylidene)-6-methylhept-5-enoate (1)

. 2. Use of the 1,2,3-triazole-containing fusidic acid derivative of claim 1 as an antimicrobial agent for combating human and animal diseases caused by gram-positive Staphylococcus aureus (MRSA) bacteria and pathogenic fungal cultures of Cryptococcus neoformans.