RU2568603C1 - Prostaglandin derivatives having anti-inflammatory and analgesic activity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention may find application in medicine for the treatment of inflammatory conditions accompanied by pain syndrome, by administration of prostaglandin derivatives having anti-inflammatory and analgesic activity, of the general formula (I)

where X=NH, NH-CH₂-CH₂-CH₂-C(O)-NH or NH-CH₂-CH₂-O-C(O)-NH.

EFFECT: agent is highly effective.

3 tbl, 6 ex

Description

The invention relates to the field of biochemistry and can find application in medicine in the treatment of inflammatory conditions accompanied by pain by administering prostaglandin derivatives with anti-inflammatory and analgesic activity.

Inflammation is a universal protective reaction of the body in response to the action of various damaging factors of exogenous and endogenous origin. Such factors include foreign microorganisms and viruses, ionizing radiation, exposure to extreme temperatures, toxic chemical agents, reactive forms of oxygen and nitrogen, as well as decay products of body cells.

The inflammatory process is characterized by four most characteristic signs: hyperemia (redness of the skin due to the expansion of blood vessels and a rush of blood); edema due to increased permeability of the walls of the blood vessels; fever (fever in the whole body); pain arising from irritation of nerve endings under the influence of inflammatory mediators of various nature (Krylov Yu.F., Bobyrev V.M. Pharmacology. Textbook. Moscow, All-Russian Training and Scientific-Methodological Center for Continuing Medical and Pharmaceutical Education (VUNMTS) , 1999. - 115 p.)

With the normal physiological development of the inflammatory reaction, for example, in the case of local damage to the tactic, after the acute phase of inflammation, the resolution, resolving stage occurs, during which proinflammatory mediators and the remains of destroyed cells are removed from the inflammation zone, and all physiological parameters (pH, temperature) return to normal.

Prostaglandins - cyclic carboxylic hydroxylated fatty acids - are formed from eicosapolyenic acids containing three or more methyl-separated double bonds. They are key mediators of inflammation. The amount of cyclooxygenase enzyme, with the help of which the common precursor of all prostaglandins prostaglandin H is formed, increases significantly with inflammation. With the help of other enzymes, the main pro-inflammatory prostaglandins of types E and D are synthesized, as well as thromboxane A ₂ .

The profile of prostaglandin production is determined by the differential expression of their synthesis enzymes inside the cells present in the area of inflammation. So, mast cells mainly generate prostaglandin D ₂ , while macrophages synthesize prostaglandin E ₂ and thromboxane A ₂ . In addition, the profile of prostaglandins depends on the degree of cell activation. For example, resting macrophages synthesize more thromboxane A ₂ than prostaglandin E ₂ , but after activation by bacterial polysaccharide this ratio changes in favor of prostaglandin E ₂ . Despite the fact that the pro-inflammatory mediator effect of prostaglandin E _{2 is} reliably established and cyclooxygenase inhibitors significantly reduce the severity of the inflammatory response, reducing fever and swelling and reducing pain, prostaglandin E ₂ in the stage of resolving inflammation can play a positive role in the substance that contributes to directing the inflammatory process in the opposite direction side (Ricciotti Ε., GA FitzGerald. Prostaglandins and Inflammation. // Arterioscler Thromb Vase Biol. 2011; 31: 986-1000).

Under certain conditions, the inflammatory reaction may become excessive, which forces the use of drugs to reduce the intensity of inflammation and transfer it to the resolution stage. To combat an excessive inflammatory reaction, mainly two groups of drugs are used: steroid and non-steroidal chemical nature.

Among steroidal anti-inflammatory drugs, glucocorticoids (prednisone and related drugs) are most often used, which affect all phases of inflammation. However, glucocorticoids alter the pathogenesis of the inflammatory process during bacterial infection without eliminating its causes, and relapse of the disease can occur after their cancellation. The anti-inflammatory effect of glucocorticoids is due to a direct effect on the focus of inflammation and is fully preserved with local application of their dosage forms (ointments, creams, pastes, etc.) on the skin and mucous membranes. However, with prolonged local use of drugs (especially fluorine-containing), the epidermis, dermis and subcutaneous tissues can atrophy, point hemorrhages, striae appear. Immunobiological reactivity of such tissues is reduced, which can cause pyoderma and candidiasis.

The second group of drugs consists of nonsteroidal anti-inflammatory drugs (NSAIDs) that are diverse in chemical structure, for example, acetylsalicylic acid, sodium salicylate, analgin, paracetamol, butadion, indomethacin, diclofenac sodium, ibuprofen, piroxicam. Individual drugs differ from each other in the strength of anti-inflammatory, antipyretic and analgesic effects. The main mechanism of action of this class of drugs is the inhibition of cyclooxygenase and, as a consequence, a significant decrease in prostaglandin E ₂ biosynthesis. NSAIDs are characterized by side effects: allergies, damage to the gastrointestinal mucosa (ulcerogenicity), edema and bronchospasm. Most of these manifestations are due to the antiprostaglandin effect of the drugs (Krylov Yu.F., Bobyrev V.M. Pharmacology. Textbook. Moscow, All-Russian Educational and Scientific-Methodological Center for Continuing Medical and Pharmaceutical Education (VUNMTS), 1999. - 115 p.) .

These data indicate certain shortcomings of existing anti-inflammatory drugs, which requires the development of new safer drugs that combine anti-inflammatory and analgesic effects. This invention solves the problem of expanding the range of substances with anti-inflammatory and analgesic activity. The problem is solved by derivatives of prostaglandin E ₂ with anti-inflammatory and analgesic activity, of the General formula:

where X = NH, NH — CH ₂ —CH ₂ —CH ₂ —C (O) —NH or NH — CH ₂ —CH ₂ —OC (O) —NH

These compounds are prostaglandin E2 amide and dopamine or prostaglandin E2 amides and ethanolamide (prostamide E2) or gamma-aminobutyric acid, which are functionalized with a dopamine residue. Amides of prostaglandin F1 are known to have anti-inflammatory effects (WO 2011/154754 A1. Use of prostaglandin F1a and its derivatives for reduction of inflammation. L. Puskas et al.). However, prostaglandin F1 is used in this patent, which differs from prostaglandin E2 in the absence of a keto group in the cyclopentane ring and a double bond at position 5, 6 of the side chain.

Also known is the amide of prostaglandin E2 and dopamine with neuroprotective properties (RU 2474426. Prostamides and their analogues with neuroprotective effect. VV Bezuglov and others). However, this patent does not disclose the anti-inflammatory and analgesic properties of this substance, which are new and essential to solve the problem of expanding the range of non-steroidal anti-inflammatory drugs. Two other claimed derivatives of prostaglandin E ₂ and dopamine were not previously known.

All of these compounds are prepared by standard chemical synthesis methods, including activation of the carboxyl group of prostaglandin E _{2 with} appropriate reagents and condensation with an amine or amino alcohol. Condensation with dopamine in the case of prostamide E2 is carried out through the intermediate formation of an activated carbonyl derivative. The biological activity of the claimed compounds was confirmed in experiments on the culture of macrophages in which an inflammatory reaction was caused by a mixture of lipopolysaccharide and phorbol ester. Anti-inflammatory and analgesic activity was also determined in experiments on laboratory mice. In these experiments, these compounds showed a significant anti-inflammatory and analgesic effect. Moreover, in experiments on RAW 264.7 cells, the IC50 values of the anti-inflammatory effect for all of the claimed compounds were in the range of 11-27 μM, while standard NSAIDs (ibuprofen, indomethacin and diclofenac sodium) were not active up to a concentration of 100 μM.

Thus, the claimed compounds solve the problem of expanding the range of substances with anti-inflammatory and analgesic effects, which can be used in medicine as non-steroidal anti-inflammatory drugs.

The invention is illustrated by examples.

Example 1. The synthesis of prostaglandin amide E ₂ and gamma-aminobutyric acid.

A portion of 487 mg (1.38 mmol) of prostaglandin E2 was dissolved in 5000 μl of acetonitrile and 210 μl (1.52 mmol) of triethylamine was added and left for 20 min at + 4 ° C, after which 197 μl (1.52 mmol) of isobutyl chloroformate was added and left at + 4 ° C for 20 minutes After 35 minutes, a solution of a silyl derivative of gamma-aminobutyric acid in acetonitrile prepared from 200 mg of gamma-aminobutyric acid and 1026 μl (3.9 mmol) of bis (trimethylsilyl) trifluoroacetamide in 500 μl of acetonitrile was added to the resulting solution. The reaction was held at room temperature for 60 minutes.

5 ml of ethanol was added to the reaction mixture and left for 30 minutes, after which the reaction mixture was evaporated on a rotary evaporator and dried on an oil pump. The residue was diluted with 30 ml of ethyl acetate, transferred to a separatory funnel and washed with 2 × 30 ml of distilled water, 1 × 20 ml of saturated NaCl solution. The organic layer was dried over anhydrous Na ₂ SO ₄ , the precipitate was filtered off, and the organic layer was evaporated. The yellow oil residue was purified by silica gel column chromatography. Eluted with chloroform-acetone. The fractions containing the product were combined. Received 425 mg (70.3%) of the product as a slightly yellow oil.

R _f = 0.37 (chloroform-methanol, 10: 1), mass spectrum: 438.094 [M + H] +, 460.021 [M + Na] +, ¹ H NMR (CDCl ₃ ; δ ppm; 200 MHz); 0.899 (t; 3H; H20), 1.302-1.311 (m; 6H, H19, H18, H17), 1.448-1.52 (m, 2H, H16), 1.683 (m, 4H, H3, H3 ′), 2.060-2.334 (m, 11H, H2, H4, H7, H8, H10, H3 ′), 2.692-2.784 (m, 1H, H12) ,, 4.031-4.093 (m, 2H, H11, H15), 5.374 (m, 2H, H5, H6), 5.576-5.589 (s, 2H, H13, H14).

Example 2. Synthesis of a dopamine derivative of prostaglandin amide E ₂ and gamma-aminobutyric acid (PGE2-GABA-DA)

A portion of 82 mg (0.19 mmol) of prostaglandin amide E ₂ obtained in Example 1 was dissolved in 200 μl of acetonitrile and 29 μl (0.2 mmol) of triethylamine was added and kept for 20 min at + 4 ° C, after which 27 μl (0.2 mmol) was added isobutylchloroformate and incubated for 20 min at + 4 ° C. 53.5 mg (0.28 mmol) of dopamine hydrochloride in 200 μl of DMF and 39 μl (0.28 mmol) of triethylamine were added to the resulting solution under cooling (an ice bath) and stirring and kept for 18 h at + 4 ° C.

The reaction mixture was evaporated. The residue was suspended in 30 ml of ethyl acetate and washed with 2 × 30 ml of distilled water, 1 × 20 ml of a saturated NaCl solution. The organic layer was dried over anhydrous Na ₂ SO ₄ , the precipitate was filtered off, and the organic layer was evaporated. The residue was purified by silica gel column chromatography. The solvent mixture was eluted with chloroform - acetone. The fractions containing the product were combined. Received 55.5 mg (51.7%) of the product as a white crystalline substance.

R _f = 0.2 (chloroform-methanol, 10: 1), mass spectrum: 571.3262 ([M-H] ^- ), 553.3162 ([M-H-H ₂ O] ^- ) ¹ H-NMR, 200 MHz, CDCl ₃ ): 0.88 (3H, t), 1.30 (6H, m), 1.61 (4H, m), 1.88 (2H, m), 2.91 (7H, m), 2.35 (2H, m), 3.23 (2H, q), 3.43 (2H, m), 4.11 (2H, m), 5.36 (2H, m), 5.66 (2H, m), 6.54 (1H, m), 6.78 (2H, m), 6.97 ( 1H, t), 8.20 (1H, m).

Example 3. Synthesis of dopaminamide prostaglandin E ₂ (PGE2-DA).

To a solution of 67 mg (0.19 mmol) of prostaglandin E2 and 29 μl (0.209 mmol) of triethylamine in 3 ml of acetonitrile, cooled to -15 ° C, 27 μl of isobutyl chloroformate (0.209 mmol) was added with stirring. It was stirred for 20 minutes, a solution of 43 mg of dopamine hydrochloride (0.23 mmol) and 29 μl (0.209 mmol) of triethylamine in 600 μl of dimethylformamide were added. Stirred for 18 hours at 8 ° C. The reaction was processed and the target substance was isolated as described in Example 2. Obtained: 62 mg (67%) of dopaminamide prostaglandin E2 in the form of a white powder. ¹ H-NMR (200 MHz, CDCl ₃ ): 0.89 (3H, t), 1.24-1.77 (8H, m), 2.33 (2H, m), 2.64 (2H, m), 4.09-4.12 (2H, m) , 5.38 (2H, m), 5.65 (2H, m), 6.52 (1H, m), 6.73 (2H, m), 7.21 (1H, t), 7.93 (1H, m). Mass spectrum: m / z 510.280 [M + Na] ⁺ , m / z 470.287 [M + H-H ₂ O] ⁺ .

Example 4. Synthesis of conjugate of ethanolamide prostaglandin E ₂ (prostamide E ₂ ) and dopamine (PGE2-EA-DA).

To a solution of 100 mg (0.28 mmol) of prostaglandin E ₂ cooled to 4 ° C and 43 μl (0.3 mmol) of triethylamine in 5 ml of acetonitrile, 38 μl (0.3 mmol) of isobutyl chloroformate was added and stirred at 4 ° C for 30 min. To the resulting mixed 26 μl (0.42 mmol) of monoethanolamine was added to the anhydride and stirred for 30 minutes. The reaction mixture was diluted with 10 ml of ethyl acetate, washed with water, saturated aqueous NaCl, dried over anhydrous Na ₂ SO ₄ . The drying agent was filtered off, the filtrate was evaporated in vacuo, the residue was purified by silica gel column chromatography in a chloroform-methanol gradient system. Received 88.6 mg (79%) of the product as a yellow oil. A portion of 82 mg (0.24 mmol) of the obtained prostamide PGE2-EA was dissolved in 2 ml of acetonitrile and 73 mg (0.28 mmol) of disuccinimidyl carbonate and 66 μl (0.47 mmol) of Et ₃ 3N were added with stirring and cooling (ice bath) and left for 1.5 h. A portion of 54 mg (0.28 mmol) of dopamine hydrochloride was dissolved in 500 μl of dimethylformamide and added to the mixed carbonate solution obtained above with stirring and cooling (ice bath). The reaction mixture was left for 18 hours. Treated as described in example 2. Received 93 mg (67%) of PGE2-EA-DA as a colorless oil. R _f = 0.27 (chloroform-methanol, 10: 1), mass spectrum: 575.071 [M + H], 636.958 [M + Cu]. ¹ H-NMR: ′ (200 MHz, CDCl3): 0.89 (3H, t), 1.29 (6H, m), 1.39-1.45 (2H, m), 1.63-1.70 (2H, m), 2.02-2.37 (9H , m), 2.59-2.69 (6H, m), 3.06-3.17 (9H, q), 3.31-3.49 (2H, qs), 3.62-3.71 (1H, m), 3.89-4.05 (3H, m ), 4.61 (2H, t), 5.26-5.69 (4H, m), 6.49-6. 86 (3H, m).

Example 5

The ability of the synthesized compounds to influence the inflammatory response was evaluated for the production of nitric oxide (measured as a stable oxidation product — nitrite by the Gris method) in mouse macrophages RAW 264.7. On the eve of the experiment, cell cultures were dispersed into 96-well plates with a density of 100 thousand cells per well. An inflammatory response was induced using 1 μg / ml lipopolysaccharide in combination with 0.5 μg / ml phorbol 12-myristate 13-acetate and in the presence of 2 mM L-arginine in the culture medium (DMEM, 4 mM L-glutamine, 4 g / L D -glucose, 10% bovine fetal serum, 100 units / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / ml amphotericin B) with 2.5% serum. Stock solutions of substances in DMSO were dissolved in a medium for the induction of an inflammatory response with stimulants (anti-inflammatory activity) or without them (pro-inflammatory activity) in a concentration range of 0.01-100 μM and the culture medium in wells was replaced with the resulting solution. Incubation with the substance lasted 20 hours. After that, an aliquot of the medium was taken from each well and the concentration of NO was determined by the modified Gris method. To 75 μl of the analyzed mixture in the wells of a 96-well ELISA plate, 12.5 μl of an aqueous solution of 0.04% sulfanilamide was added, kept at room temperature for 10 min with protection from light, then 12.5 μl of a 2% solution of naphthylethylenediamine in 3M HCl was added, kept for another 10 min at room temperature with protection from light, after which the optical absorption at a wavelength of 540 nm was determined.

The results are presented in table 1.

All tested substances had a pronounced anti-inflammatory effect, but did not stimulate the inflammatory response. IC50 values ranged from 11 to 27 μM. It should be noted that in these experiments, standard NSAIDs (ibuprofen, indomethacin and diclofenac sodium) did not show an anti-inflammatory effect up to a concentration of 100 μM.

Example 6

Experiments to determine anti-inflammatory and analgesic sensitivity were performed on mice of the CD-1 line (outbred albinos). Inflammation was induced by the introduction of Freund's complete adjuvant into the hind paw of the animal. In this case, local inflammation develops, swelling is formed and the paw increases in size. In the case of anti-inflammatory activity of the substance, this size should be statistically different from the size of the control paw into which the solvent was introduced instead of the test compound.

The standard test, which measures the threshold of acute pain sensitivity, is the hot plate test. Pain sensitivity is determined by the delay in the physiological reaction of the animal: withdrawal of the sore paw, sec; licking the front paws, sec; licking the hind legs, sec; bouncing, sec.

Prior to the experiment, all animals were measured with the thickness of the left hind paw using a caliper. This value was the original. Then, a solution of Freund's complete adjuvant (PAF) in water (20%) of 20-30 μl was injected into all the animals in the left hind paw pad. After 20-24 hours, the thickness of the paw was again measured, into which PAF was pricked, after which one of the tested drugs or a solution of 14% alcohol in saline was injected. Then, 20 minutes after administration, the animals were tested in the “hot plate” test for pain sensitivity. Measurement of the thickness of the paw into which PAF was pricked was performed 2, 4, 6, 24, and 48 hours after injection. The results are presented in tables 2 and 3.

Analyzing the data of table 2, you can notice that the effect of the drugs becomes noticeable after 4 hours after administration. However, these changes are not statistically significant. Significant results for all prostaglandin E2 derivatives are observed 48 hours after their administration, and for compounds PGE2-EA-DA and PGE2-GABA-DA, this effect occurs already a day after administration. Thus, all the studied drugs, namely: PGE2-DA, PGE2-C2-DA and PGE2-C4-DA at a dose of 10 mg / kg, show intraperitoneal anti-inflammatory activity

Table 3 presents the results of the pain sensitivity test. In this test, only dopaminamide prostaglandin E ₂ (PGE2-DA) showed a statistically significant ability to reduce pain sensitivity at a dose of 10 mg / kg. Moreover, it can be noted that the effect was noticeable near the injection area. The time of withdrawal of the sore paw increased 2.5 times and the delay in licking the hind legs increased from 28 to 46 seconds. Systemic analgesia was significantly less noticeable. Two other substances also increased the threshold of pain sensitivity in the injection zone. However, in this experiment, this increase was less significant compared to the effect of PGE2-DA.

Tables for the application "Derivatives of prostaglandin with anti-inflammatory and analgesic activity"

Claims (1)

An agent with anti-inflammatory and analgesic effect, which is a derivative of prostaglandin of the general formula:

where X = NH, NH — CH ₂ —CH ₂ —CH ₂ —C (O) —NH or NH — CH ₂ —CH ₂ —OC (O) —NH