RU2793336C1 - Pyrimidine derivative with antimicrobial and immunotropic activity - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: invention relates to the fields of organic chemistry and medicine, namely to a new pyrimidine derivative of the specified formula, which has antimicrobial and immunotropic activity and is characterized by safety.

EFFECT: obtaining pyrimidine derivative of the specified formula.

1 cl, 3 ex

Description

The tool belongs to the field of medicine and can be used as a medicine in patients suffering from infectious and inflammatory diseases of staphylococcal etiology, which, along with antimicrobial (antistaphylococcal) and immunotropic action.

Staphylococci are a permanent part of the skin microbiota and can cause various infections of the skin and internal organs with the possible development of sepsis. The problem of increasing resistance of staphylococci and, in particular, S. aureus, to the most actively used antibiotics raises concerns about the effectiveness of chemotherapy. In addition, it should be noted that the use of antibiotics, as a rule, leads to a decrease in the body's immune defense, which, along with progressive antibiotic resistance, emphasizes the relevance of developing new antibacterial agents that also exhibit immunotropic properties.

Currently existing antimicrobials that have a bacteriostatic or bactericidal effect on staphylococci are potent drugs, the use of which is accompanied by undesirable side effects. The clinical efficacy of current antistaphylococcal antibiotics does not always correlate with in vitro susceptibility data; often accompanied by the formation of secondary foci of infection (soft tissue, bone, heart valves, lungs); often there is inadequate dosing and duration of therapy, which is accompanied by complications and recurrence of infection. An important negative effect of synthetic antimicrobials is also hepato- and nephrotoxicity, as well as immunosuppressive effects and the rapid development of antibiotic resistance in staphylococcus to this class of drugs (https://www.rigla.ru/about/news/2020/antibiotiki-penitsillmovogo-ryada; https://www.rlsnet.ru/fg_index_id_261.htm; https://www.vidal.ru/vracham/antibiotikikoterapiya/obzor-antibiotik/antibiotiki-penitsilliny; http://antibiotics.ru/ab/013-22. shtml).

A compound is known according to RU 2148580 C1 dated May 10, 2002, which can be used as an antimicrobial drug. The substance according to RU 2198166 C2 dated February 10, 2003 has antiviral, immunostimulatory, antichlamydial, antituberculous, analgesic and hepatoprotective effects. Known invention RU 2130023 C1 dated May 10, 1999, which describes an agent with antiflaviral activity, which suggests the possibility of its use in medicine as a drug for the treatment of tick-borne encephalitis.

The closest analogue to the claimed agent and method of its use is the compound according to RU 2451683 dated May 27, 2012, which has antiviral, antibacterial and fungicidal activity. The main disadvantage of this compound is the lack of proven safety and complex antimicrobial and immunotropic action.

The technical objective of the claimed invention is to expand the chemical groups of compounds considered as the basis for creating highly effective, non-toxic antimicrobial agents with immunotropic activity.

The problem is solved by the proposed pyrimidine derivative with the formula

sodium 3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-carboxylate, the synthesis of which was carried out in three stages aimed at obtaining the quinazolinone system, a thiazine fragment condensed with an acetamide component, and introducing it into the C3 position of the quinazolinone. The rest of quinazolinone in the salt form according to the scheme proposed by Alper was obtained using imidoyl chloride, carbon monoxide and ortho-iodaniline substituted with a carboxyl group in an alkaline medium as starting materials. The increase in the speed of the process was achieved by using a mixture of palladium acetate and triphenylphosphine as a catalyst and by carrying out the process at reduced temperature and pressure. The interaction of β-aminoethanethiol and 1,2 dichloroethane gave a 1,4 thiazine fragment, which was subsequently subjected to condensation with the N-derivative as a result of the reaction of 2-chloropropionic acid perchloric anhydride with hydroxylamine.

Pyrimidine bases are an integral part of nucleic acids, and therefore their derivatives combine several types of pharmacological activity. In a number of experiments, it was found that the compounds of this group have anabolic activity, have an anti-inflammatory effect, accelerate the processes of reparative regeneration, stimulate cellular and humoral immunity factors, activate leukogenesis and erythropoiesis, and are also effective as antiviral, antitumor and other agents. Pyrimidine derivatives have found their application in the treatment of infectious, surgical, neurological, oncological and many other diseases and represent a group of a wide variety of chemicals with a wide range of pharmacological activity.

New in the present invention is that as an active ingredient with antimicrobial (antistaphylococcal) and immunotropic action, pyrimidine derivative sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1 -oxopropan-2-yl)-4-oxo-3,4-dihydroquinazoline-6-carboxylate.

The proposed solution meets the invention criterion "Novelty", which is due to the uniqueness of the structure due to the presence of a pyrimidine component and a heterocyclic thiazine residue in it, each of which causes the manifestation of a wide range of pharmacological activity of medicinal substances, including antimicrobial and immunotropic. It has been established that the mechanism of the antimicrobial action of pyrimidine derivatives is based on their ability to suppress DNA synthesis by inhibiting the work of bacterial DNA gyrase and type IV topoisomerase, which leads to the death of the pathogen cell. The participation of thiazines in the formation of the active center of antibiotics, such as cephalosporins, allows them to be considered as a pharmacologically significant fragment of the drug molecule. The simultaneous presence of nitrogen atoms in the thiazine molecule makes it possible for them to participate in biochemical processes as multidentate ligands and allows them to be used in coordination chemistry to obtain new scaffolds with potential bioactivity. The presence in the structure of oxygen and nitrogen atoms, which exhibit an electron-donor function when bound to target molecules with the formation of transition complex compounds via hydrogen bonds, is important in the activation of nonspecific immunity factors. The salt form of the substance determines its polarity and increases the degree of absorption and biodistribution, and also provides variability in the development of dosage forms, including those using nanotechnology.

The compliance of the invention with the conditions of the inventive step has been established, since the claimed agent differs from its analogue by introducing a pyrimidine derivative as the main active ingredient, which has safety, antimicrobial (antistaphylococcal) and immunotropic effects.

In an experiment on laboratory animals, the effectiveness of the application of the proposed agent, which has a safety, antimicrobial (antistaphylococcal) and immunotropic effect, has been proven.

The essence of the claimed invention and the achievement of the technical result is illustrated by the following examples.

Example 1

A safe agent with antimicrobial (antistaphylococcal) and immunotropic activity based on a pyrimidine derivative has been obtained.

The acute toxicity of the studied pyrimidine compound was assessed on 50 non-linear male rats aged 5-6 months weighing from 220 to 250 g.

All animals were divided into groups:

- the first group (control) - animals treated intragastrically with an equivolume of distilled water;

- experimental groups, including animals treated with a pyrimidine compound at doses of 500; 1000; 2000; 4000 mg/kg intragastric suspension.

Possible manifestations of acute toxicity were assessed daily for 14 days after the administration of the compound based on an assessment of the general condition, motor activity, body weight, condition of the hair, skin and mucous membranes.

During pathomorphological examination of the internal organs, a macroscopic analysis and their weighing were performed.

In assessing the acute toxicity of the pyrimidine derivative sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6 -carboxylate, it was found that the studied substance in doses of 500 and 1000 mg/kg did not cause changes in the general condition, hair and skin and mucous membranes. The new compound at doses of 2000 and 4000 mg/kg contributed to a decrease in the motor activity of animals during the first day, but on the next day these manifestations leveled off.

It should be noted that in the study of acute toxicity, no death of laboratory animals was noted, and therefore the level of the semi-lethal dose cannot be determined. Taking into account the obtained results, the maximum dose of 4000 mg/kg was adopted as the LD ₅₀ . Thus, the new pyrimidine derivative sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4 dihydroquinazolin-6- carboxylate is a low-toxic substance (safe) and belongs to the 4th class of toxicity.

Example 2

Study of the antimicrobial activity of the pyrimidine derivative sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6- carboxylate was carried out in vitro on a test culture of the Staphylococcusaureus strain, which was isolated from the sputum of patients undergoing treatment at the State Budgetary Institution of Healthcare JSC "City Clinical Hospital No. CM. Kirov, Astrakhan. The evaluation of the antimicrobial activity of the pyrimidine derivative was carried out by the method of serial dilutions in meat-peptone broth (MPB) with the formation of rows with different concentrations of the compound: 128 μg/ml; 64 µg/ml; 32 µg/ml; 16 µg/ml; 8 µg/ml; 4 µg/ml; 2 µg/ml; 1 µg/ml; 0.5 µg/ml; 0.25 µg/ml. Cultures of microorganisms were inoculated into test tubes, after which they were incubated for 24 hours at 37°C, centrifuged, and the sediment was inoculated onto nutrient agar for cultivating microorganisms (HMF-agar). Then, a visual assessment was made of the presence or absence of a visible characteristic growth (CR), followed by the establishment of the minimum inhibitory concentration of the substance. Test tubes containing BCH with introduced microorganisms (positive control) were used as controls.

Study of the antimicrobial activity of the pyrimidine compound sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6- carboxylate in vivo resulted, modeling a generalized infection by intraperitoneal administration of Staphylococcus aureus at a dose of ×10 ⁸ microbial bodies to mice of 5 weeks of age. Animals were divided into several groups: control I - received an equivalent volume of water for injection; control II - animals infected with Staphylococcusaureus and not treated; experimental animals treated with the test compound at a dose of 39 mg/kg, starting from the day of infection for 7 days. During the treatment of animals, their survival was assessed. After removing the surviving mice from the experiment, the index of contamination of blood and internal organs (spleen and liver) was calculated.

When studying the in vitro antimicrobial activity of a pyrimidine derivative, it was found that at a concentration of 128 μg/ml, the substance has a high activity against Staphylococcus aureus, while at concentrations of 64 μg/ml, 32 μg/ml and 16 μg/ml, a decrease in the intensity of the characteristic growth of the microorganism from 25% to 50% in comparison with the positive control. In dilutions from 8 µg/ml to 0.25 µg/ml, growth of Staphylococcus aureus was noted over 50% of the area of inoculation of the nutrient medium, which indicates the absence of anti-staphylococcal activity.

When determining growth-inhibiting concentrations of the pyrimidine compound sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin- 6-carboxylate, it was found that the studied substance had bactericidal activity against Staphylococcus aureus at a concentration of 128 μg/ml.

Pyrimidine derivative sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-carboxylate, administered at a dose of 39 mg/kg significantly reduced the lethality of laboratory animals, resulting in their 100% survival rate.

When studying the contamination of internal organs and blood, it was found that in animals in control group I, no growth of Staphylococcus aureus was observed. With the introduction of the pyrimidine compound sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-carboxylate at a dose of 39 mg/kg, no growth of microorganisms was observed either in the internal organs or in the blood.

The pyrimidine compound sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-carboxylate promotes the formation of an antimicrobial immunity and has a pronounced therapeutic effect of antimicrobial orientation in the conditions of experimental staphylococcal generalized infection.

Example 3

Experiments on the study of immunotropic activity were carried out on SBA mice of both sexes 3-4 months of age.

The experimental study was carried out in two series. In series 1, in order to study dose-dependent immunotropic properties, the animals were divided into the following groups (n=9-10): control I received an equivalent volume of distilled water; control II (animals with experimental immunosuppression) received cyclophosphamide (Cyclophosphamide (CFA), OOO Mediapharm, Russia) at a dose of 100 mg/kg, intraperitoneally, once; experimental animals with experimental immunosuppression received intraperitoneally pyrimidine compound sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4- dihydroquinazoline-6-carboxylate at doses of 19 mg/kg (experiment No. 1), 38 mg/kg (experiment No. 2), 76 mg/kg (experiment No. 3) and 152 mg/kg (experiment No. 4) for 3 days. The dose range was formed on the basis of information about the molecular weight of the substance.

Series 2 was devoted to the study of the severity of the immunotropic properties of the compound depending on the timing of administration in relation to the induction of experimental immunosuppression. The following groups of animals were formed: control I received an equivalent volume of distilled water; control II received cyclophosphamide at a dose of 100 mg/kg, intraperitoneally, once. In the first experimental group (experiment No. 1), daily prophylactic administration of the substance sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo- 3,4-dihydroquinazoline-6-carboxylate was started 3 days before CFA, the course was 3 injections. In animals of the second experimental group (experiment No. 2), daily therapeutic administration of the substance sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo -3,4-dihydroquinazoline-6-carboxylate was started the next day after the injection of cyclophosphamide, the course also amounted to 3 injections. The immunotropic effect of the pyrimidine derivative was evaluated on the basis of an organometric analysis of the mass and cellularity of immunocompetent organs (thymus and spleen), as well as delayed-type hypersensitivity reactions (DHRT) and direct hemagglutination (DHA).

For organometric analysis, cell suspensions were prepared in medium 199 at the rate of 50 mg/ml for the spleen and 10 mg/ml for the thymus, filtered, washed twice with medium 199 to remove particles of adipose tissue (for 10 min at 1500 rpm), after which resuspended in medium 199 to the initial concentration. Suspensions of lymphoid organs for counting were preliminarily mixed 1:1 with 3% acetic acid stained with methylene blue, and the number of nucleated cells (splenocytes and thymocytes) was counted in the Goryaev chamber. When setting RGZT animals were immunized subcutaneously with sheep erythrocytes (EB) at a dose of 1 × 10 ⁷ in 100 µl of saline. A resolving dose of EB 1 × 10 ⁷ was injected in a volume of 20 μl on day 5 after sensitization under the aponeurotic plate of one of the hind limbs (“experimental” paw), in the contralateral (“control” paw) - saline. Accounting for the intensity of the local reaction was carried out after 24 hours, calculating the index of RGZT according to the formula (Mo - Mk) / Mk × 100%, where Mo is the mass of the “experimental” paw, Mk is the mass of the “control” paw.

For RPHA, mice were immunized with EB at a dose of 5 × 10 ⁶ in 100 µl of saline. Serum was obtained 7 days after immunization. To inactivate complement, the serum was heated at t=56°C for 30 min. The hemagglutination reaction was carried out in 96-well plates. To suppress nonspecific binding of antibodies, the reaction was placed in 50 μl of a diluting liquid (0.5% solution of bovine serum albumin prepared in saline), in which the test sera were serially diluted twice. After dilution of the sera, 25 µl of 1% EB suspension was added to the wells. Preliminary accounting of the results of RPGA was made after 1 hour of incubation at t=37°C, then the plates were rearranged in a refrigerator at t=+4°C, and after 18 hours the reaction was taken into account finally. The antibody titer (the highest serum dilution at which EB agglutination is observed) was expressed as geometric mean values.

An experimental study of the immunotropic properties of a new pyrimidine compound sodium-3-(1-((4H-1,4-thiazin-4-yl)amino)-1-oxopropan-2-yl)-4-oxo-3,4-dihydroquinazoline -6-carboxylate indicates the presence of immunomodulatory properties, manifested at a dose of 39 mg/kg, when administered at different times relative to the induction of experimental immunosuppression.

Thus, the achievement of the present invention is to obtain an agent based on a pyrimidine derivative characterized by safety, antimicrobial (antistaphylococcal) and immunotropic activity.

Claims (3)

A pyrimidine derivative with the formula:

, characterized by safety, antimicrobial and immunotropic activity.