RU2135474C1 - Salts of 1-deoxy-1-n-methylaminohexaalcohols with n-acridone acetic acid showing immunomodulating activity and drugs on their base - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention relates to synthetic biologically active compounds of class belonging to derivatives of 1-deoxy-1-N-methylaminohexaalcohols and N-acridone acetic acid. Invention describes new compounds - salts of 1-deoxy-1-N-methylaminohexaalcohols with N-acridone acetic acid of the general formula (I)

Description

 The invention relates to medicine, namely to synthetic biologically active compounds belonging to the class of derivatives of 1-deoxy-1-N-methylaminohexaspiols and N-acridonoacetic acid, as well as dosage forms based on them.

 The proposed compounds have pronounced immunomodulating properties: sharply increase the factors of specific protection of the animal organism and cause the induction of interferon. The compounds have low toxicity, high stability in aqueous solutions and organic compositions.

 The main component (cation) of the claimed compounds are 1-deoxy-1-methylaminohexols (1-deoxy-1-methylamino-D-sorbitol, 1-deoxy-1-methylamino-D-mannitol, 1-deoxy-1-methylamino-D -galactitol).

 The second biologically active component of the compounds (anion) is N-acridonoacetic acid (10-methylene-carboxy-9-acridone).

 It is known that amino alcohols are called organic molecules that have amino and hydroxyl functional groups in their composition. The simplest representative of aminoalcohols is aminoethanol.

В настоящее время особое внимание исследователей привлекли 1-дезокси-1-метиламино-D-гексолы общей формулы

различающиеся положением гидроксильных групп у четырех асимметрических атомов углерода C₂-C₅.A special group of amino alcohols, which has important biological significance, are 1-deoxy-1-aminopolyols. The most important representative of this group is vitamin B2 (riboflavin), which controls almost all oxidative enzymatic reactions in cells (Metzler D., Biochemistry, Moscow, Mir, 1980, v. 2, pp. 529-535):

Currently, special attention of researchers has attracted 1-deoxy-1-methylamino-D-hexols of the general formula

differing in the position of hydroxyl groups in four asymmetric C ₂ -C ₅ carbon atoms.

Впервые в 1972 году Fryer и Grunberg обнаружили, что некоторые производные акридонов обладают выраженными противовирусными свойствами [4] Fryer and Grunberg, US patent N 3681360, МКИ C 07 D 219/06, 01.08.72.These compounds are important for the normal functioning of biological membranes (Ca ion transport) [2] -Adam O.SidKi and Kenneth G. Baimbridge, J. of Physiology (1997) 504.2, pp 579-590, and are also often used as harmless additives in various medical compositions [3] -Kappes at all, US patent N 5508394. The second biologically active component (anion) is acridonoacetic acid of the formula

For the first time in 1972, Fryer and Grunberg discovered that some acridone derivatives have pronounced antiviral properties [4] Fryer and Grunberg, US patent N 3681360, MKI C 07 D 219/06, 01.08.72.

 Subsequently, Jnglot A.D. et al. showed that the sodium salt of N-acridonoacetic acid has interferon-inducing activity in experiments in mice [5] Jnglot A D. et al, Arh. Jmmunol. et Ther., Exp., 63, 1985, p. 275-285.

 Recent studies conducted in recent years have allowed the creation of acridonoacetic acid highly active drugs: comedone and cycloferon [6-7] RF Patents N 2020941 (07/06/92), N 2029764 (16.11.92), N 2033413 (28.01.93) patent US B 5658886 (12/07/94).

 The work carried out in 1991-1994 revealed an extremely interesting feature: N-acridonoacetic acid and / or its salts, when combined with various monosaccharides, both with free functional groups and provided with protection, sharply change their properties, in some cases acquiring new types of pharmacological activity that are not characteristic of the components taken separately [8] RF Patent N 2080108 (05.27.97).

В 1996 году были заявлены соли 2-дезокси-2-амино(или 2-метиламино)-D-глюкозы с N-акридонуксусной кислотой общей формулы

Из приведенной формулы видно, что прототип идентичен заявленному нами своим акридоновым фрагментом, но резко отличается строением катиона. Действительно, в прототипе использованы производные аминосахаров (2-дезокси-2-амино (или -2-метиламино)-D-глюкозы, в то время как в заявленном нами изобретении в качестве катиона используются значительно более дешевые производные аминоспиртов.An interesting attempt by the authors of RF patent N 2036198 to obtain a compound of acridonoacetic acid with 1-deoxy-1-methylaminohexose. The authors patent N-methyl-N- / a, Δ-glucopyranosyl / (ammonium-2-) acridon-9-one-10 yl / acetate, the cycloferon named by them, which, according to their data, has interferonogenic, antiviral, including anti HIV, antiparasitic, antipromotor, radioprotective activity of the formula

In 1996, salts of 2-deoxy-2-amino (or 2-methylamino) -D-glucose with N-acridonoacetic acid of the general formula were declared

From the above formula it can be seen that the prototype is identical to what we claimed for its acridone fragment, but it differs sharply in the structure of the cation. Indeed, the prototype used derivatives of amino sugars (2-deoxy-2-amino (or -2-methylamino) -D-glucose, while in the claimed invention we use significantly cheaper derivatives of amino alcohols as the cation.

 The compounds obtained in this way not only possess all the properties of the prototype, but also have a number of significant advantages, namely, patented substances have a lasting effect on the factors of specific protection of the body.

At present, one of the fragments of the molecule: 1-deoxy-1-methylamino-D-sorbitol and 1-deoxy-1-methylamino-D-galactitol and others, is obtained commercially by treating the corresponding D-hexoses with gaseous methylamine, followed by restoration of the Schiff bases obtained [10] Kochetkov N. K. et al. Chemistry of carbohydrates, M., Chemistry, 1967. The physicochemical and biological properties of these aminopolyols are well studied [11] Handbook of Biochemist, ed. R. Dawson et al. M., Mir, 1991. For example, 1-deoxy-1-methylamino-D-sorbitol (meglumine), has a melting point of 129-131.5 ^o C, [α] ²⁵ = 16.5 ^o (C = 10; H ₂ O); solubility 1000 g / l (25 ^o C); molecular weight 195.22; gross formula C ₇ H ₁₇ NO ₅ .

The second fragment of the claimed compounds (anion):
N-acridonoacetic acid is also produced on an industrial scale and is approved for medical use (FS-42-2481-95, Neovir-12.5% solution for injection).

 The synthesis of the claimed compounds is carried out by salt formation due to various acid-base properties of the starting compounds. The starting compounds, due to weakly expressed acid-base properties (weak acids and bases), are usually unstable in aqueous solutions, if one of the components is a water-insoluble substance (in our case, N-acridonoacetic acid is practically insoluble in water), therefore, the manufacture of drugs their basis was considered almost impossible. In the course of the studies carried out by the applicants, it was unexpectedly found that the aminopolyols used by us are weak bases, but have extremely high hydrophilicity (due to the linear structure and the presence of five hydroxyl groups), which allows them to form stable water-soluble salts with antimicrobial activity (Pat. RF N 2093510).

 The authors of this solution proposed the structural formula of the synthesized compounds, presented the methods of synthesis and presented the results of biological tests on models of bacteria, DNA and RNA viruses.

 These substances, the formula of which is given above, are the closest structural analogues of the claimed compounds, and we have adopted as a prototype. The prototype is described in detail in RF patent N 2093510, MKI 6 C 07 D 219/06, from 26/09/96, publ. BI N - 98 (prototype).

 The objective of the invention is to obtain new chemical compounds - derivatives of salts of 1-deoxy-1-methylamino-D-hexols with N-acridonoacetic acid, which have a pronounced long-term effect on the factors of specific protection of the body from pathogens and harmful environmental influences, as well as causing pronounced induction of interferon.

 In this case, all the useful properties of the prototype must be fully preserved.

 The synthesized compounds should have high stability in dosage forms, easier in manufacturing technology than the prototype.

Кроме того, поставленная задача решена путем создания лекарственных средств, обладающих иммуномодулирующей активностью, включающих указанное выше синтезированное соединение со слабыми нерастворимыми в воде кислотами (N-акридонуксусной кислотой).The problem is solved by the synthesis of new chemical compounds, namely salts of 1-deoxy-1-methylamino-D-hexols with N-acridonoacetic acid of the general formula

In addition, the task was solved by creating drugs with immunomodulating activity, including the above synthesized compound with weak water-insoluble acids (N-acridonoacetic acid).

 Moreover, the synthesis methods developed by the applicants are more economical than that of the prototype.

 The invention is illustrated by two examples of the synthesis of the claimed salts and three examples of the manufacture of finished dosage forms based on them, as well as experiments to determine their biological (immunomodulating) activity with three measurement tables.

H ¹ NMR spectra were measured on a Bruker 200 spectrometer and confirm the structure of the obtained compounds.

 Synthesis Example 1 1000 g of N-Acridonoacetic acid are added to 1500 ml of water, in small portions with vigorous stirring, 770 g of 1-deoxy-1-methylamino-D-sorbitol (meglumine) is added. The mixture is refluxed for 20 minutes (while complete dissolution of the starting components is observed).

The resulting solution was cooled to room temperature and mixed with 3000 ml of acetone with vigorous stirring. The precipitated caramel-like precipitate was kept at 0-5 ^° C. for 5-7 hours, then filtered. The filter cake was washed with 300 ml of cold acetone, dried in vacuum at 85-90 ^o C. Receive 1600 g (yield 90.5%) of a light yellow coarse-grained substance - salt 1 - deoxy-1-methylamino-D-sorbitol with N- acridonoacetic acid.

 The salt of 1-deoxy-1-methylamino-D-sorbitol with N-acridonoacetic acid does not have a clear melting point.

To characterize the purity of the synthesized compound, the most convenient method is UV spectroscopy. Our studies showed that with a purity of 99%, the specific extinction coefficient E _1cm ^1% is 198 ± 1.

Molecular weight 448.47;
gross formula: C ₂₂ H ₂₈ N ₂ O ₈ ;
elemental analysis:
calculated: C, 58.86; H 6.24; N, 6.24;
Found: C, 58.65; H 6.28; N, 6.27.

NMR data (characteristic signals) H1 DMSO d6:
7.3-8.4 ppm signal region of the aromatic heterocyclic system of acridone (8H);
4.85 ppm (s, 2H) is the proton signal of the N-methylenecarboxy substituent;
2.95 ppm (d, 2H) —proton signal at the first C-atom of methylaminosorbitol;
2.45 ppm (s; 3H) is the signal of the protons of the methyl group of methylaminosorbitol;
LD ₅₀ (intravenous, white mice) is 650 mg / kg
Synthesis Example 2 100 g of N-Acridonoacetic acid are dissolved by boiling in 500 ml of 95% ethanol. With stirring, 77 g of 1-deoxy-1-methylamino-D-galactiol are added. The resulting mixture was refluxed for 15 minutes, then cooled to room temperature. In this case, a large-crystalline precipitate forms, which is filtered, washed with absolute ethanol, then dried in vacuum at 70-90 ^° C. 171 g (96.5% yield) of 1-deoxy-1-methylamino-D-galactiol salt product with N are obtained -acridonoacetic acid.

The specific extinction coefficient E _1cm ^1% = 198 ± 1;
gross formula: C ₂₂ H ₂₈ N ₂ O ₈
molecular weight 448.47;
elemental analysis (%):
calculated: C, 58.86; H 6.24; N, 6.24;
Found: C, 58.91; H 6.18; N, 6.22.

NMR data (characteristic signals) H1 DMSO d6:
7.3-8.4 ppm signal region of the aromatic heterocyclic system of acridone (8H);
4.9 ppm (s, 2H) is the proton signal of the N-methylenecarboxy substituent;
2.98 ppm (d, 2H) —proton signal at the first C-atom of methylaminosorbitol;
2.46 ppm (s; 3H) is the signal of the protons of the methyl group of methylaminosorbitol;
LD ₅₀ (intravenous, white mice) is 600 mg / kg.

 The salt of N-acridonoacetic acid with 1-deoxy-1-methylamino-D-mannitol is obtained in a similar manner with a yield of more than 90%.

 Example 1 of the manufacture of a medicinal product.

 100 g of Salts of 1-deoxy-1-methylamino-D-sorbitol with N-acridonoacetic acid are dissolved in 800 ml of pyrogen-free water, the solution volume is adjusted to 1000 ml with pyrogen-free water, filtered through bacterial filters. After filling into ampoules, sealing and sterilization, a 10% solution of the dosage form suitable for parenteral use (intravenously, intramuscularly) is obtained. The resulting dosage form is stable during storage, has physiologically optimal pH values of 6.8-7.8.

 Example 2 of the manufacture of a medicinal product.

 100 g of finely ground N-acridonoacetic acid (purity not less than 98%) is added to 300 ml of pyrogen-free water, 77 g of 1-deoxy-1-methylamino-D-sorbitol (meglumine) (purity not less than 99%) are added in small portions with vigorous stirring. . The mixture was stirred for 1.5 hours at room temperature. After complete dissolution of the components, the volume of the solution was adjusted with pyrogen-free water to 1400 ml. The solution is filtered through bacterial filters and poured into ampoules or vials, sterilized, receiving a dosage form in the form of a 12.5% sterile solution suitable for parenteral administration.

 Example 3 of the manufacture of a medicinal product.

50 g of Salts of 1-deoxy-1-methylamino-D-galactitol with N-acridonoacetic acid are added to 100 g of glycerol. The resulting suspension at 70 ^o C, mixed with 50 g of castor oil and 800 g of lanolin.

 Get the finished dosage form in the form of 5% ointment, suitable for external use. The ointment does not cause side effects, does not exfoliate during storage in a wide temperature range, is well absorbed through the skin.

Industrial applicability
In experiments to determine the biological activity used 10% sterile aqueous solutions of drugs:
salts of 1-deoxy-1-methylamino-D-sorbitol with N-acridonoacetic acid (as an example), and 2-deoxy-2-methylamino-D-glucose as reference preparations.

The study of interferon-inducing activity of the claimed compounds for example:
(1) salts of 1-deoxy-1-methylamino-D-sorbitol with N-acridonoacetic acid; and
(2) salts of 2-deoxy-2-methylamino-D-glucose with N-acridonoacetic acid (prototype)
studied on mice. Compounds (1) and (2) at a dose of 100 mg / kg were administered once intramuscularly to CBA mice weighing 15-16 g. The results of the experiment are presented in table 1.

 From the presented data it follows that compounds (1) and (2) have approximately the same ability to induce the formation of high titers of interferon in the first hours after administration.

 To study the effect of the administration of drugs (1) and (2) on the immunological reactivity of the body, experiments were conducted to evaluate the state of T and B immunity systems.

 The studies were carried out on 30 white rats (males), weighing (180 ± 5) g. Experimental animals were injected with preparations (1) and (2), at a dose of 20 mg / kg, 1 time per day, 3 days in a row. The control group was injected with saline in the same volume. The study of the immunological status of the organism was carried out 10 days after the last injection. The results of the action of drugs on the number and function of T and B lymphocytes in the experimental groups, compared with the control are presented in tables 2 and 3.

 As can be seen from the data in tables 2 and 3, the relative number of T and B lymphocytes does not change significantly under the influence of drugs.

 On the contrary, characterizing the functioning activity in RBTL of mature forms of T-lymphocytes (spontaneous inclusion test with PHA) and young forms of T-lymphocytes (test with KoH-A) increased in the case of the claimed compounds in 2.3 - 2.5 times compared with control.

 Under the influence of compound (1), B-lymphocyte function was activated more than 1.5 times in the LPS test compared to the control. That is, the claimed drugs significantly increase the functional activity of the cellular and humoral immunity (specific protective forces of the body).

 However, as follows from the data in Table 3, the function of T-lymphocyte-suspension cells in the SS and IS tests was significantly reduced under the influence of the drug (1).

 In the case of compound (2) (prototype), all the observed changes are practically not expressed.

 From the presented data it follows that the claimed compounds are almost identical with the prototype in their ability to induce early interferon, but differ sharply in their ability to increase the functional activity of cellular and humoral immunity.

 The claimed compounds in their antiviral and antimicrobial properties are similar to the activity of the prototype.

Thus, from the above examples on the synthesis of the claimed compounds: salts of 1-deoxy-1-methylaminohexols with N-acridonoacetic acid and the manufacture of dosage forms based on them, as well as the results of biological tests, it follows that the advantages of the claimed drugs are:
- obtaining the claimed compounds and medicines based on them is easily feasible in an industrial environment at existing pharmaceutical industries.

- a sharp increase in the factors of specific protection of the body for a long time (at least 10 days),
- medicines based on the compounds declared by us are stable during long-term storage, highly effective and convenient to use.

 Thus, the claimed compounds are new, non-obvious and industrially applicable, i.e. meet all the requirements for the invention.

Claims (2)

1. Salts of 1-deoxy-1-N-methylaminohexaspertols with N-acridonoacetic acid of the general formula

possessing immunomodulatory activity.  2. A drug having immunomodulatory activity, characterized in that it includes a compound according to claim 1.

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