RU2730604C1 - (2z)-2-[(3β, 4α, 8α, 11α, 14β, 16β)-16-(acetyloxy)-3-({3-[(4-aminobutyl)amino]propyl}amino)-11-hydroxy-4,8,10,14-tetramethyl gonane-17-ylidene]-6-methylhept-5-enoic acid with antimicrobial and fungicidal activity and method for production thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to synthesis of biologically active analogues of natural compounds, specifically to production of a new representative of nitrogen-containing derivatives of fusidic acid - (2Z)-2-[(3β,4α,8α,11α,14β,16β)-16-(acetyloxy)-3-({3-[(4-aminobutyl)amino]propyl}amino)-11-hydroxy-4,8,10,14-tetramethyl-gonan-17-ylidene]-6-methylhept-5-enoic acid (compound (1)), having antibacterial activity on Staphylococcus aureus (MRSA) and inhibiting growth of a fungal culture of Cryptococcus neoformans. Compound (1) is obtained based on the corresponding dioxo-derivative of fusidic acid by reacting with 6 equivalents of spermidine in the presence of titanium (IV) isopropoxide in dry methanol medium while stirring for 2 hours. Output of compound (1) is 90 %.

EFFECT: compound (1) shows fungicidal activity with respect to the pathogenic fungal culture of Cryptococcus neoformans with low toxicity and has minimal haemolytic action at the maximum tested concentration.

2 cl, 2 tbl, 1 ex

Description

The invention relates to the field of synthesis of biologically active analogs of natural compounds, namely to the production of (2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (acetyloxy) -3 - ({3 - [( 4-aminobutyl) amino] propyl} amino) -11-hydroxy-4,8,10,14-tetramethylgonan-17-ylidene] -6-methylhept-5-enoic acid (1) with antibacterial activity against Staphylococcus aureus (MRSA) and the growth-inhibiting fungal culture of Cryptococcus neoformans:

Known examples of obtaining derivatives of fusidic acid containing a branched polyamine fragment in the molecule by means of a reductive amination reaction using NaBH (OAc) ₃ as a reducing agent. The method consists in the interaction of 1 eq. 3-ketofusidic acid (2) with 3 eq. polyamine linear or branched structure and 3 EQ. NaBH (OAc) ₃ in methanol solution. The reaction proceeds at room temperature in the presence of acetic acid for 6-16 hours and leads to the formation of a mixture of α- and β-isomeric amines (3-8) with a yield of 70 to 85%:

The biological activity of the resulting derivatives was studied against gram-positive and gram-negative bacteria (Staphylococci, Streptococci, Corynebacteriae, Mycobacteriae, Proteus, Propionibacterium, Pseudomonas, Neisseriae, E. coli), as well as fungal strains (Candida, Aspergillus) and found that they do not have antimicrobial and fungicidal action against strains of pathogens of bacterial and fungal infections of humans and animals ([1] T. Duvold. Blanched polyamine steroid derivatives. Patent WO 03/087121 A1, 2003; [2] T. Duvold. Novel fusidic acid derivatives. Patent WO 02/077007 A2, 2002).

Known examples of the synthesis of derivatives of fusidic acid containing N- (3-aminopropyl) butane-1,4-diamine substituent in the molecule. The method consists in the interaction of derivatives of N-succinimide ester of tetrahydrofusidic acid (9) with 3 equiv. N- (3-aminopropyl) butane-1,4-diamine (spermidine) in dry chloroform. The reaction proceeds at room temperature for 16 hours and leads to the formation of amides (10-15) with yields from 60 to 90%:

The study of the biological activity of the obtained derivatives showed that compounds (10) and (14) exhibit antibacterial action against strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and also inhibit the growth of fungal cultures of Candida albicans and Aspergillus niger [T. Duvold. Novel fusidic acid derivatives. Patent WO 02/077007 A2, 2002].

Thus, the synthesis and biological properties of 3β-N- (3-aminopropyl) butane-1,4-diamine derivative of fusidic acid (1) are not described in the literature.

The objective of the present invention is to develop a method for producing a new compound - ((2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (acetyloxy) -3 - ({3 - [(4-aminobutyl) amino] propyl} amino) -11-hydroxy-4,8,10,14-tetramethylgonan-17-ylidene] -6-methylhept-5-enoic acid, which has antimicrobial and antifungal activity.

The synthesis of the claimed compound (1) was carried out as follows: the 3,11-dioxo derivative of fusidic acid (16) was involved in the interaction with 3 equiv. N- (3-aminopropyl) butane-1,4-diamine (spermidine) and 0.3 eq. titanium (IV) isopropoxide in dry methanol, followed by treatment of the reaction mass with 2 eq. sodium borohydride. After isolation and purification by chromatography gave derivative (1), the structure of which is set via 1D and 2D NMR spectroscopy, ¹ H and ¹³ C and mass spectrometry MALDI TOF / TOF:

The advantages of the proposed method:

In the known method, amino derivatives (3-8) are obtained in the form of a mixture of α- and β-stereoisomers in a ratio of 25:75, respectively.

In contrast to the known method, the proposed method allows one to obtain 3β-N- (3-aminopropyl) butane-1,4-diamine derivative (1) in the form of an individual stereoisomer with a quantitative yield.

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

(с 1.13, МеОН). Структура соединения (1) подтверждена с помощью 1D и 2D спектроскопии ЯМР ¹Н и ¹³С и масс-спектрометрии MALDI TOF/TOF. На основании спектральных данных установлено, что реакция проходит с высокой хемо- и стереоселективностью по 3 положению молекулы, в то время как кето-группа в 11-положении в реакцию аминирования не вступает и восстанавливается до гидроксильной. Аминогруппа в полученных соединениях имеет β-конфигурацию, что подтверждается спектрами NOESY.Example 1. A mixture of diketone (16) (0.26 g, 0.5 mmol), titanium (IV) isopropoxide (55 mg, 0.15 mmol) and 3β-N- (3-aminopropyl) butane-1,4-diamine (0.22 g, 1.5 mmol) in 5 ml of absolute methanol was stirred in an argon atmosphere at room temperature for 2 hours. The reaction mixture was cooled to -70 ° C and sodium borohydride (0.04 g, 1.0 mmol) was added. The resulting mixture was stirred for 0.5 hour at -70 ° C, after which the temperature was gradually increased to room temperature and 5 ml of water was added to the reaction mixture. The formed precipitate was filtered off, washed with methanol. The mother liquor was concentrated in vacuo to give the crude amine, which was purified by column chromatography on silica gel eluting with methanol. After chromatographic purification, the triamino derivative (1) was obtained in a yield of 90%, which was a light yellow crystalline substance, so pl. 248-250 ° C,

(from 1.13, MeOH). The structure of compound (1) was confirmed using 1D and 2D NMR spectroscopy, ¹ H and ¹³ C and mass spectrometry MALDI TOF / TOF. Based on the spectral data, it was established that the reaction proceeds with high chemo- and stereoselectivity at the 3-position of the molecule, while the keto-group at the 11-position does not enter into the amination reaction and is reduced to hydroxyl. The amino group in the obtained compounds has the β-configuration, which is confirmed by the NOESY spectra.

Spectral characteristics of ((2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (acetyloxy) -3 - ({3 - [(4-aminobutyl) amino] propyl} amino) -11 -hydroxy-4,8,10,14-tetramethylgonan-17-ylidene] -6-methylhept-5-enoic acid (1) ¹ .

¹ H NMR spectrum (CDCl ₃ ), δ, ppm: 0.94-0.98 m (1H, H ⁶ ), 0.96 s (3H, H ¹⁸ ), 1.07 s (3H, H ¹⁹ ), 1.08 d (3H, H ²⁸ , ³ J 6.4 Hz), 1.14-1.27 m (1H, H ⁷ ), 1.19-1.28 m (1H, H ⁶ ), 1.21-1.30 m (1H, H ¹⁵ ), 1.36 s (3H, H ³⁰ ) , 1.60-1.67 m (1H, H ⁹ ), 1.63 s (3H, H ²⁷ ), 1.69 s (3H, H ²⁶ ), 1.72-1.84 m (1H, H ⁴ ), 1.74-1.84 m (1H, H ⁵ ), 1.74-1.86 m (2H, H ³⁴ ), 1.75-1.84 m (1H, H ² ), 1.76-1.87 m (1H, H ⁷ ), 1.83-1.93 m (1H, H ¹² ), 1.92-1.99 m (1H, H ¹ ), 2.00 s (3H, H ³² ), 2.04-2.11 m (1H, H ² ), 2.06-2.19 m (2H, H ²³ ), 2.10-2.20 m (2H, H ³⁸ ), 2.10 -2.20 m (1H, H ¹⁵ ), 2.13-2.22 m (1H, H ¹ ), 2.21-2.34 m (2H, H ³⁷ ), 2.29-2.36 m (1H, H ¹² ), 2.33-2.43 m (1H, H ²² ), 2.51-2.61 m (1H, H ²² ), 2.77-2.82 m (1H, H ³⁶ ), 2.96-3.06 m (2H, H ³³ ), 3.01-3.08 m (1H, H ¹³ ), 3.01- 3.15 m (2H, H ³⁵ ), 3.06-3.14 m (2H, H ³⁹ ), 3.11-3.23 m (1H, H ³⁶ ), 5.16 t (1H, H ²⁴ , ³ J 7.5 Hz), 5.84 d (1H, H ¹⁶ , ³ J 8.2 Hz).

¹³ C NMR spectrum (CDCl ₃ ), δ, ppm: 14.36 (C ²⁸ ), 16.55 (C ¹⁸ ), 16.55 (C ²⁷ ), 19.52 (C ³² ), 21.08 (C ⁶ ), 22.53 (C ³⁰ ), 22.82 (C ¹⁹ ), 22.92 (C ³⁷ ), 22.99 (C ³⁸ ), 24.06 (C ² , C ³⁴ ), 24.51 (C ²⁶ ), 27.87 (C ²³ ), 28.85 (C ²² ), 31.69 (C ⁷ ), 33.35 (C ¹ ), 34.95 (C ⁴ ), 35.80 (C ¹² ), 36.05 (C ¹⁰ ), 36.62 (C ³⁹ ), 38.65 (C ¹⁵ ), 38.71 (C ³³ ), 39.12 (C ⁸ ) , 42.98 (C ⁵ ), 43.15 (C ¹³ ), 44.58 (C ³⁶ ), 46.90 (C ³⁵ ), 48.46 (C ¹⁴ ), 48.84 (C ⁹ ), 63.03 (C ³ ), 66.82 (C ¹¹ ), 74.33 (C ¹⁶ ), 123.31 (C ²⁴ ), 131.85 (C ²⁰ ), 131.56 (C ²⁵ ), 133.56 (C ¹⁷ ), 171.56 (C ³¹ ), 175.66 (C ²¹ ). Mass spectrum (MALDI TOF / TOF), m / z (I _rel ,%): 644 (100) [M + H] ⁺ , 683 (55.3) [M + K] ⁺ .

¹ The reaction was monitored by TLC on Sorbfil plates (Sorbpolymer, Krasnodar, Russia), developed with 10% sulfuric acid solution. For column chromatography, silica gel L (50-160 μm) KSKG was used. The melting point was determined on a RNMK 80/2617 apparatus. 1D ( ¹ H, ¹³ C) and 2D (COZY, NOESY, HSQC, HMBC) NMR spectra were recorded on a Bruker Avance 500 spectrometer (125.78 MHz for ¹³ C and 500.17 MHz for ¹ H) using standard pulse sequences from Bruker, internal standard Me ₄ Si, solvent - CD ₃ OD. 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 matrix of 3- (4-hydroxy-3,5-dimethoxyphenyl) prop-2-enoic acid (sinapic acid ).

Antimicrobial screening of compound (1) was performed in CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (Great Britain) 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) ATCC 43300. Antifungal activity was determined on two fungal strains (Candida albicans albicans) ATCC 90028 and Cryptococcus neoformans (C. neoformans) ATCC 208821.

Initial 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. The inhibition of bacterial growth was determined spectrophotometrically at 600 nm on 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. In the event that growth inhibition> 80% was observed one or both times, the compound was considered active (Table 1).

At the initial screening, the presence of antimicrobial and fungicidal activity was revealed in (2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (acetyloxy) -3 - ({3 - [(4-aminobutyl) amino] propyl} amino) -11-hydroxy-4,8,10,14-tetramethyl-gonan-17-ylidene] -6-methylhept-5-enoic acid (1) against the culture of the bacteria Staphylococcus aureus and the fungal culture of Cryptococcus neoformans ... For compound (1), the minimum inhibitory concentration was determined in relation to the above cultures, and also studied the cytotoxic and hemolytic activities.

The minimum inhibitory concentration (MIC; μg / ml) was set in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI), USA), determining the lowest concentration at which complete inhibition of bacteria or fungi was found. Screening was performed using the serial dilution method. Samples were prepared in DMSO at a test concentration of 32 μg / ml. All bacteria were cultured on Mueller Hinton agar at 37 ° C overnight. A sample of each culture was then diluted 40-fold and incubated at 37 ° C for 1.5-3 hours.The resulting cultures were added to each well of a 384-well plate containing the test sample (cell density 5 × 10 ⁵ CFU / ml and total volume 50 μl). All plates were covered and incubated at 37 ° C for 18 h without shaking. Bacterial growth inhibition was determined by measuring absorbance at 600 nm using a Tecan M1000 Pro monochrome microplate reader. The percentage of growth inhibition was calculated for each well using a negative control (medium only) and a positive control (bacteria without inhibitors) on the same plate. Inhibition values were determined using modified Z-scores calculated using the median and median absolute deviation (MAD) of samples (no control) on the same plate. Samples with an inhibition value greater than 80% and a Z-score greater than 2.5 for both replicas were classified as active substances. Samples with inhibition rates of 50 to 80% and a Z-score above 2.5 for both replicas were classified as partially active.

The tests were carried out in duplicate. The maximum percentage of growth inhibition was designated DMax. Hits were graded at MIC ≤ 16 μg / ml or MIC ≤ 10 μM in any replica (n = 2 on different plates).

The cytotoxic effect was determined on the human embryonic kidney cell line HEK293 by determining the concentration causing 50% cell death (Hk CC ₅₀ ). Growth inhibition of HEK293 cells was determined by measuring fluorescence after adding 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 DMax. The compound was considered toxic at CC ₅₀ ≤ 32 μg / ml or CC ₅₀ ≤ 10 μM. In addition, samples were noted as partial cytotoxic if DMax ≥ 50%, even with a CC ₅₀ above the maximum tested concentration.

Hemolytic activity (Hm НС ₁₀ and НС ₅₀ - concentration at 10% and 50% hemolysis, respectively) was determined by measuring the absorption at 405 mm of the supernatant - the supernatant liquid formed after incubation for 1 h at 37 ° С of plates containing samples of compounds with added to them washed human blood cells, and subsequent centrifugation at 1000 rpm for 10 minutes. Absorbance was measured using a Tecan M1000 Pro monochrome microplate reader.

The maximum percentage of hemolysis is presented as DMax. A low DMax at HC ₁₀ > 32 μg / ml (maximum tested concentration) indicates samples without hemolytic activity. Samples with hemolytic activity were characterized at HC ₁₀ ≤ 32 μg / ml. In addition, samples were labeled as partially hemolytic if DMax ≥ 50%, even at HC ₁₀ > maximum test concentration.

Colistin and Vancomycin were used as positive bacterial inhibition standards for gram-negative and gram-positive bacteria, respectively. Fluconazole has been used as a standard fungal inhibitor for C. albicans and C. neoformans. Tamoxifen was used as a positive cytotoxicity standard. Melittin was used as a positive hemolytic standard.

The methodology for testing the antimicrobial, fungicidal, cytotoxic and hemolytic activity in vitro of compounds is also given at http://www.co-add.org.

Sample (2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (acetyloxy) -3 - ({3 - [(4-aminobutyl) amino] propyl} amino) -11-hydroxy -4,8,10,14-tetramethyl-gonan-17-ylidene] -6-methylhept-5-enoic acid (1) at a concentration of 8.0 μg / ml showed fungicidal activity that is absent in fusidic acid, inhibiting growth and reproduction> 100 % of the fungal culture Cryptococcus neoformans. At a concentration of 32.0 μg / ml, compound (1) inhibits the growth and reproduction of gram-positive bacteria Staphylococcus aureus, which, according to the CO-ADD criteria, is a weakly expressed antibacterial activity. The hemolytic activity of compound (1) does not exceed 5.5% even at the maximum tested concentration> 32 μg / ml, which is 1.8 times lower than that of fusidic acid, and the cytotoxicity of compound (1) is 1.5 times lower than that of the native antibiotic (Table 2) ...

Thus, compound (1) exhibits fungicidal activity against the pathogenic fungal culture Cryptococcus neoformans at low toxicity and has a minimal hemolytic effect at the maximum tested concentration.

Claims (4)

1. The method of obtaining (2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (acetyloxy) -3 - ({3 - [(4-aminobutyl) amino] propyl} amino) - 11-hydroxy-4,8,10,14-tetramethyl-gonan-17-ylidene] -6-methylhept-5-enoic acid (1),

consisting in the interaction (2Z) -2 - [(4α, 5α, 8α, 9β, 13α, 14β, 16β) -16- (acetyloxy) -4,8,10,14-tetramethyl-3,11-dioxogonane-17- ylidene] -6-methylhept-5-enoic acid with 6 equivalents of N- (3-aminopropyl) butane-1,4-diamine and 0.3 equivalents of titanium (IV) isopropoxide for 2 hours. 2. (2Z) -2 - [(3β, 4α, 8α, 11α, 14β, 16β) -16- (Acetyloxy) -3 - ({3 - [(4-aminobutyl) amino] propyl} amino) -11- hydroxy-4,8,10,14-tetramethylgonan-17-ylidene] -6-methylhept-5-enoic acid having antibacterial activity against Staphylococcus aureus (MRSA) and fungicidal activity against the fungal culture of Cryptococcus neoformans.