RU2792376C1 - Method for producing a biologically active substance having antibacterial activity, and a substance obtained by this method - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a biologically active substance with antibacterial activity and a method for its production. A new derivative of 6-aminopenicillanic acid is obtained — a new, previously undescribed (Z)-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (I), which has a high antibacterial effect, and a method for its production is revealed. The compound (I) specified exhibits low toxicity and can be used in medicine.

EFFECT: new derivative of 6-aminopenicillanic acid is obtained that exhibits low toxicity and can be used in medicine.

2 cl, 1 ex, 1 tbl

Description

The invention relates to biologically active substances and can be used in medicine as an antibacterial drug.

Known methods of obtaining both derivatives of penicillin [Patent 547176 France. Method for obtaining penicillins / Jean Bouchodon, Pierre Le Roy, Mayer Naum Messer (France). - No. 2000645/04; dec. 02/15/1974; publ. 02/15/1977, Bull. No. 6], and ampicillin from the group of semi-synthetic antibiotics, widely used in medical practice [Patent 568369 France. The method of obtaining ampicillin / Jean Bouchodon, Pierre Le Roy, Mayer Naum Messer (France). - No. 2000645/2112821/04; dec. 03/18/1975; publ. 08/05/1977, Bull. No. 29; A.s. 490804 USSR. The method of obtaining anhydrous ampicillin / S.S. Smirnova, T.A. Voloshin, S.S. Shaikhiev, G.M. Mitkokh (USSR). - No. 1930391/23-4; dec. 06/07/1973; publ. 11/05/1975, Bull. No. 41; Patent 525431 Denmark. Method for obtaining crystalline pivaloyloxymethyl ether of ampicillin / Ernst Thorndal Binderup, Hans Jürgen Petersen, Sven Lisberg (Denmark). - No. 1963763/04; dec. 10/05/1973; publ. 08/15/1976, Bull. No. 30].

The literature describes a method for obtaining ampicillin from 6-aminopenicillanic acid by chemical or enzymatic acylation. Chemical methods are based on the acylation of 6-aminopenicillanic acid with a phenylglycine derivative whose amino group is blocked by a protective group, or with a phenylacetic acid derivative containing a group capable of converting into an amino radical. At the end of the acylation, the protecting group must be removed from the side chain of the intermediate penicillin under mild enough conditions so as not to affect the rest of the molecule [Patent 547176 France. Method for obtaining penicillins / Jean Bouchodon, Pierre Le Roy, Mayer Naum Messer (France). - No. 2000645/04; dec. 02/15/1974; publ. 02/15/1977, Bull. No. 6].

However, the methods known in the literature for the synthesis of 6-aminopenicillanic acid derivatives are complex and are accompanied by losses of the target product. In the case of obtaining ampicillin by acylation of 6-aminopenicillanic acid with a derivative of phenylglycine, the amino group of which is blocked by a protective group and activated at the carboxyl group, it is accompanied by heating of the reaction mass, which reduces the efficiency of protection of the amino group of the phenylglycyl residue, leads to the formation of mixtures and the need to separate the reaction products, which reduces the yield of the target product.

The technical objective of the invention is to simplify the method for obtaining penicillin derivatives, as a result of which a biologically active substance with high antibacterial activity was synthesized.

For this, a method is proposed for obtaining a biological substance with antibacterial activity by synthesizing ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3-dimethyl-7-oxo-4-thia- 1-azabicyclo[3.2.0]heptane-2-carboxylic acid ( I ), carried out by the interaction of 5-phenyl-2,3-dihydro-2,3-furandione ( II ) with 6-aminopenicillanic acid in absolute acetonitrile for 40 minutes according to the scheme:

Synthesis of the original 5-phenyl-2,3-dihydro-2,3-furandione ( II ) was carried out according to the previously described methods [5-Aryl-2,3-furandiones / Yu.S. Andreichikov, Yu.A. Nalimova, G.D. Plakhina, R.F. Saraeva, S.P. Tendryakova // Chemistry of heterocyclic compounds. - 1975. - No. 11. - S. 1468-1470; Reactions of 5-aryl-furan-2,3-diones with acylmethylenetriphenylphosphoranes: Synthesis and biological activity of 3(2 H )-furanone derivatives / VO Kozminykh, NM Igidov, EN Kozminykh, ZG Aliev // Pharmazie. - 1993. - Bd. 48, H. 2. - S. 99-106].

The closest analogue in structure and action of the claimed compound is 6-aminopenicillanic acid ( Ia ) [Rolinson, GN 6-Aminopenicillanic Acid / GN Rolinson, Betchworth // Chemotherapia. - 1961. - Vol. 2, #1. - P. 52-59] formulas:

Structural analogue ( Ia ) has low antibacterial activity (table 1).

In medical practice, dioxidine is widely used as an antibacterial drug, similar in pharmacological action to the claimed compound ( I ), which we took as a comparison standard [Mashkovsky, M.D. Medicines: a guide for doctors / M.D. Mashkovsky. - 16th ed., revised, corrected. and additional - M.: New wave, 2012. - S. 848].

The technical objective of the invention is a derivative of 6-aminopenicillanic acid - a new, previously undescribed ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3-dimethyl-7-oxo-4-thia -1-azabicyclo[3.2.0]heptane-2-carboxylic acid ( I ), which has a high antibacterial effect.

To solve this problem, we propose a biologically active substance with high antibacterial activity, a derivative of 6-aminopenicillanic acid, namely - ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3 -dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid of formula ( I ):

The essence of the invention is illustrated by example 1.

Example 1. Obtaining the claimed compound (I). A mixture of 0.35 g (0.002 mol) of compound II and 0.43 g (0.002 mol) of 6-aminopenicillanic acid in 20 ml of anhydrous acetonitrile was stirred on a magnetic stirrer at 70°C for 40 minutes. The resulting solution was cooled, the precipitate formed during cooling was filtered off and recrystallized from anhydrous acetonitrile. Yield 0.55 g (70%). T. pl. 165-166 ^about C. Found, %: C 55.50; H 4.61; N 7.13; S 8.28. C ₁₈ H ₁₈ N ₂ O ₆ S. Calculated, %: C 55.38; H 4.65; N 7.18; S 8.21. IR spectrum (FSM-1201, in vaseline oil, ν, cm ^-1 ): 3360 (NH), 1751 (CO), 1692 (CO), 1674, 1597 (C=C, C=O). ^1H NMR spectrum (Bruker Avance III HD, operating frequency 400 MHz, DMSO- d ₆ , TMS, d, ppm): 1.51 s (3H, CH ₃ ), 1.62 s (3H, CH ₃ ), 4.38 s (1H, CH), 5.54-5.64 m (2H, 2CH), 7.11 s (1H, CH), 7.57-8.78 (m, 5H _arom ), 8.78 d (1H, NH, J 7.7), 13.21 br. with (1H, OH). ^13C NMR spectrum (Bruker Avance III HD, operating frequency 100 MHz, DMSO- d6 , TMS, d, ppm): 26.51, 31.16, 58.58 _, 63.94, 67.02, 70.23, 94.72, 127.60, 133.90, 160.93, 168.71, 171.83, 176.94, 186.07.

The claimed compound ( I ) is a colorless crystalline substance, soluble in DMF, acetic acid, when heated - in ethyl alcohol and acetonitrile.

The proposed method makes it possible to obtain the target product - a derivative of 6-aminopenicillanic acid ( I ) in high yield, and is also more convenient and easy to use, since protection of the carboxyl group and amino group is not required, as in the case of the synthesis of ampicillin, and, therefore, upon completion acylation does not require the removal of protective groups, and is also not accompanied by the formation of by-products.

Study of antibacterial activity and acute toxicity of the claimed compound (I).

Acute toxicity (LD ₅₀ , mg/kg) of compound ( I ) was studied in white mice (females) weighing 16-18 g with a single intraperitoneal injection [Guidelines for preclinical studies of drugs / ed. A.N. Mironova, N.D. Bunyatyan, A.N. Vasilyeva, O.L. Verstakova, M.V. Zhuravleva, V.K. Lepakhina, N.V. Korobova, V.A. Merkulova, S.N. Orekhova, I.V. Sakaeva, D.B. Utesheva, A.N. Yavorsky. - M.: Grif and K, 2012. - Part 1. - 944 p.]. The animals were observed for 10 days, recording the behavior, intensity and nature of motor activity, the presence of convulsions, coordination of movements, skeletal muscle tone, reactions to tactile, sound and light stimuli, frequency and depth of respiratory movements, heart rate, condition of the hair and skin, color of visible mucous membranes, water and food intake, changes in body weight.

Expected acute toxicity, following the recommendations of the State Pharmacological Committee for the study of general toxic effects of biologically active substances [Guidelines for the study of general toxic effects of pharmacological agents. Approved 25. 12. 97 / Vestn. pharmacopoe. committee. - 1998. - No. 1. - S. 27-32].

It was found that for compound ( I ) LD ₅₀ is >1200 mg/kg. Therefore, the claimed compound ( I ) is less toxic than the reference standard dioxidine, the LD ₅₀ of which is 750 mg/kg [US National Library of Medicine [Electronic resource]. - Access mode: https://chem.nlm.nih.gov/chemidplus/name/dioxydine] 1.6 times (Table 1).

According to the drug toxicity classification, compound ( I ) belongs to the class of practically non-toxic substances [Sidorov, K.K. On the classification of toxicity of poisons in parenteral methods of administration / K.K. Sidorov // Toxicology of new industrial chemicals. - 1973. - Issue. 13. - S. 47-51; Berezovskaya, I.V. Classification of chemicals according to the parameters of acute toxicity in parenteral administration / I.V. Berezovskaya // Chemical and Pharmaceutical Journal. - 2003. - T. 37, No. 3. - S. 32-34].

Antibacterial activity the claimed compound(I)was determined by the test-tube method of double serial dilutions in a liquid nutrient medium [Guidelines for conducting preclinical studies of drugs / ed. A.N. Mironova, N.D. Bunyatyan, A.N. Vasilyeva, O.L. Verstakova, M.V. Zhuravleva, V.K. Lepakhina, N.V. Korobova, V.A. Merkulova, S.N. Orekhova, I.V. Sakaeva, D.B. Utesheva, A.N. Yavorsky. - M.: Grif and K, 2012. - Part 1. - 944 p.]. Screening for antibacterial activity was carried out against type strainsStaphylococcus aureusATCC 6538-PEscherichia coliATCC 25922. The test compounds weighing 0.05 g were dissolved in 5 ml of DMSO, obtaining a stock solution of the substance at a concentration of 10⁴mcg/ml. This solution served as the basis for the working solution, having a concentration of 2 × 10³ μg/ml, which was serially diluted twice in liquid nutrient medium in a row of 10 test tubes. The concentration of the studied compounds in the first tube of a series of dilutions in the nutrient medium was 1000.0 μg/ml. Nutrient broth (Federal Scientific Research Center of PMB, Obolensk) and typical daily cultures grown on nutrient agar were used to determine the antibacterial activity. The concentration of microbial cells in the experiment was 2-5×10⁵ cfu/ml As a positive control, a nutrient medium with the studied culture was used. An intact nutrient medium was used as a negative control. The cultures were incubated in a thermostat at a temperature of 35±2°C. Bacterial growth was assessed visually after 18-24 h of incubation. As the value of the MIC (minimum inhibitory concentration) was taken the lowest concentration of the compound, at which there is no visible growth of the test organism.

The results of the claimed compound (I) on antibacterial activity were compared with the experimental data of dioxidine and its analogue in structure ( Ia ) (table 1). The results of tests of antibacterial activity and acute toxicity are presented in table 1.

Table 1
Antibacterial activity and acute toxicity of compound ( I ) connection Acute toxicity of compounds
LD ₅₀ , mg/kg,
(v/b) Antibacterial activity
MIC, mcg/ml S. aureus
ATCC 6538-P E. coli
ATCC 25922 Claimed compound ( I ) >1200 7.8 31.2 Structural analogue ( Iа ) - 1000.0 1000.0 Dioxidine
(1% solution) 750* 62.5 31.2 *-literature data:
*US National Library of Medicine [Electronic resource]. - Mode of access: https://chem.nlm.nih.gov/chemidplus/name/dioxydine.

As can be seen from table 1, the structural analog ( Ia ) has a low antibacterial activity. Acylation of 6-aminopenicillanic acid ( Ia ) with 5-phenyl-2,3-dihydro-2,3-furandione to form ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)- 3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid ( I ) leads to a sharp increase in the biological action against the studied strains of microorganisms.

In addition, the claimed compound (I) in terms of antibacterial activity exceeds the effect of the dioxidine reference standard against Staphylococcus aureus ATCC 6538-P ~ 8 times (IPC 62.5 / 7.8) and is comparable to the effect of dioxidine against Escherichia coli ATCC 25922, and is also less toxic than dioxidine.

Thus, ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane -2-carboxylic acid ( I ) was synthesized in a simple and convenient way, exhibits higher antibacterial activity compared to the structural analog ( Ia ) and reference standard (dioxidine solution 1%), and is also practically non-toxic, which makes it possible to use the compound ( I ) to create a new drug with antibacterial activity.

Claims (3)

1. A method for obtaining a biological substance with antibacterial activity by synthesizing ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3-dimethyl-7-oxo-4-thia-1 -azabicyclo[3.2.0]heptane-2-carboxylic acid (I), carried out by the interaction of 5-phenyl-2,3-dihydro-2,3-furandione with 6-aminopenicillanic acid in absolute acetonitrile for 40 minutes. 2. A biologically active substance with high antibacterial activity, a derivative of 6-aminopenicillanic acid, namely - ( Z )-6-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3,3-dimethyl- 7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid of formula (I):

.