RU2732297C2 - Derivatives of non-steroid anti-inflammatory agents - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention relates to a compound of general formula (I) applicable in medicine:

where R is a non-steroidal anti-inflammatory agent residue containing a carboxyl group, attached by an amide bond and representing diclofenac, ibuprofen, naproxen, ketorolac, aceclofenac, indometacin, ketoprofen or sulindac, and X is CH₂, (CH₂)₃CONHCH₂ or CH₂CH₂O(CO)NHCH₂, wherein when X is CH₂, R cannot be a residue of ibuprofen, ketoprofen or sulindac.

EFFECT: invention discloses novel compounds which are effective for use as an agent possessing anti-inflammatory, cytotoxic and/or neuroprotective activity.

2 cl, 4 tbl, 10 ex

Description

The invention relates to the field of medicinal chemistry and can be used in medicine as anti-inflammatory, neuroprotective agents, and also as cytostatic agents for use in oncology by using derivatives of non-steroidal anti-inflammatory drugs containing an additional functional structural element.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a large group of drugs used to reduce pain, inflammation of various etiologies and as an antipyretic agent. NSAIDs differ in the chemical structure of the active ingredient, the enzyme they inhibit, and the duration of action.

There are several subgroups of NSAIDs:

• salicylates acetylsalicylic acid (aspirin) - one of the best-selling OTC drugs, like other derivatives of salicylic acid - salicylamide, diflunisal, etc .;

• derivatives of phenylacetic acid - diclofenac, aceclofenac, sulindac, keterolac;

• derivatives of indoleacetic acid - indomethacin;

• derivatives of propionic acid, have the ending "-profen" - ketoprofen, ibuprofen, flunoxaprofen, fenoprofen, etc .;

• derivatives of anthranilic acid, have the ending "-fenamic acid" - mefenamic acid, flufenamic acid, meclofenamic acid, etc .;

• drugs from the "oxicam" group - droxicam, meloxicam, piroxicam, tenoxicam, etc .;

• pyrazolones - metamizole (analgin), clofeson, kebuzon, phenylbutazone, etc .;

• coxibs - selective inhibitors of cyclooxygenase-2 - lumiracoxib, celecoxib, valdecoxib, etc.

The range of drug use in treatment tactics is quite extensive: rheumatoid arthritis, osteoarthritis, gout, dysmenorrhea, migraine, metastatic bone pain, intestinal obstruction, renal colic, postoperative pain, dental procedures, etc. Recently, NSAIDs have been used in combination chemotherapy regimens for cancer (Umar, A., Steele, V.E., Menter, DG, & Hawk, E.T. (2016). Mechanisms of nonsteroidal anti-inflammatory drugs in cancer prevention. Seminars in Oncology, 43 (1), 65-77.doi: 10.1053 / j.seminoncol.2015.09.010). The principle of action of NSAIDs is based on inhibition of the enzyme responsible for the biosynthesis of prostaglandins - cyclooxygenase (COX-1 or COX-2). Depending on whether the drugs inhibit both enzymes or predominantly only one enzyme (COX-1 or COX-2), all NSAIDs are divided into selective and non-selective. The most famous non-selective NSAIDs, such as aspirin, ibuprofen, diclofenac, etc., are widely available in most countries of the world, are dispensed without a prescription and are well tolerated by patients, but in the case of long-term use (for example, in rheumatoid arthritis), the main side effects must be taken into account. the effects of NSAIDs.

The main side effects include, firstly, the ulcerogenic effect (the ability to damage the mucous membrane of the stomach and duodenum), which occurs due to inhibition of the biosynthesis of gastrocytoprotective prostaglandins. Short-term use of NSAIDs can cause stomach upset, while long-term use, especially in high doses, can lead to stomach ulcers and gastric bleeding. In addition, enteric dosage forms of NSAIDs can cause serious intestinal damage (Sostres C., Carla J., Gargallo C. J., Lanas A. Nonsteroidal anti-inflammatory drugs and upper and lower gastrointestinal mucosal damage. Arthritis Res. Ther., 2013.15 (suppl3) : p. S3).

Secondly, with selective inhibition of COX-2, serious complications of the cardiovascular system, up to and including death, occur. That is why a number of drugs (for example, Rofecoxib) were withdrawn from production and use, and in relation to others (for example, Celecoxib, Valdecoxib, Etoricoxib, etc.), additional clinical studies are being conducted to study the safety of their use.

Thirdly, the toxic effect on the kidneys - vasoconstriction (vasoconstriction) and a reversible form of renal failure due to nonspecific blockage of cyclooxygenase, which, with prolonged use of NSAIDs, leads to acute renal failure and acute tubular necrosis.

One of the most effective ways to reduce the side effects of NSAIDs is to chemically modify their structure. On the one hand, temporary protective groups are used that block the free carboxyl group of a number of NSAIDs, which reduces gastrotoxicity, and on the other hand, bifunctional multitarget drugs based on NSAIDs are synthesized. The peculiarity of the second group of modified NSAIDs is the use of several pharmacological targets to increase the effectiveness and safety of such a drug. The substances claimed in the present invention belong to the second group of modified NSAIDs.

Derivatives of ibuprofen and amino alcohols are known, in particular the amide of ibuprofen and aminoethanol (Zhong, Guangxiang; Chen, Lulu; Jiang, Jiansong, Assignee: Zhejiang University of Technology, Peop. Rep. China, CN 101531611, CN 2009-10096554 Mar 6, 2009) treatment of ibuprofen with thionyl chloride, followed by condensation of the formed acid chloride with aminoethanol. Also described in open sources amides of ibuprofen and indomethacin and aminoethanol, which were further converted into methacrylate derivatives followed by controlled polymerization (Davaran, S., & Entezami, A.A. (1997). Acrylic type polymers containing ibuprofen and indomethacin with difunctional spacer group : synthesis and hydrolysis.Journal of Controlled Release, 47 (1), 41-9.doi: 10.1016 / s0168-3659 (96) 01614-8).

Ibuprofen amide with gamma-aminobutyric acid was used to synthesize bifunctional ibuprofen derivatives with anti-inflammatory properties and the ability to inhibit the pain receptor TRPV1 (Yan L., Pan M, Fu M., Wang J., Huang W., Qian H. Design, synthesis and biological evaluation of novel analgesic agents targeting both cyclooxygenase and TRPV1. BioorgMedChem. 2016 Feb 15; 24 (4): 849-57. doi: 10.1016 / j.bmc. 2016.01.009).

Also known are dopamine derivatives attached through a linker consisting of gamma-aminobutyric acid or aminoethanol to prostaglandin E2 (Bezuglov V.V., Gretskaya N.M., Akimov M.G., Zinchenko G.N., Tukhovskaya E.A. ., Murashev AN Prostaglandin derivatives possessing anti-inflammatory and analgesic activity. RF patent No. 2568603, application No. 2015100913, priority from 15.01.2015, published 20.11.2015, bull. No. 32.)

However, neither the patent nor the open literature contains information about bifunctional derivatives of non-steroidal anti-inflammatory drugs containing a dopamine residue attached through a linker, which is a residue of aminoethanol and gamma-aminobutyric acid, the significant cytotoxic and neuroprotective effect of which in combination with NSAIDs was unexpectedly found in the process of research.

The CAS register lists the structures of nonsteroidal anti-inflammatory drugs and dopamine amides (in particular, for ibuprofen amide and dopamine CAS No. 1007698-71-6), however, there are no references to sources that describe the synthesis and biological activity of compounds of this type. Thus, any biological activity of these compounds, including anti-inflammatory activity, is not known from the prior art.

The objective of this invention is to expand the range of biologically active derivatives of non-steroidal anti-inflammatory drugs with anti-inflammatory effects. The problem is solved by the synthesis of amide derivatives of non-steroidal anti-inflammatory drugs (NSAIDs) containing a carboxyl group, for example, but not limited to, diclofenac, ibuprofen, naproxen, ketoprofen, sulindac, ketorolac, aceclofenac, indomethacin, in which the amide moiety or amine-ethanol is present acid substituted with a dopamine residue.

Thus, the subject of this invention is amide derivatives of non-steroidal anti-inflammatory drugs of the general formula

(I):

where R = non-steroidal anti-inflammatory drug (NSAID) residue containing a carboxyl group, for example, but not limited to, diclofenac, ibuprofen, naproxen, ketoprofen, sulindac, ketorolac, aceclofenac, indomethacin linked by an amide bond, and XHCH = (CH ₂ ) ₃ CON ₂ or CH ₂ CH ₂ O (CO) NHCH ₂ .

Unless otherwise indicated, all technical and scientific terms used in this document have the same meanings as are generally accepted in the field to which the invention belongs, and can be understood by a person skilled in the art.

"Substituted" means that a specific group or residue contains one or more substituents other than a hydrogen atom.

"Residue" means a portion of a molecule with one or more hydrogen atoms removed.

"Mix" means the joint inclusion of two or more substances without the formation of chemical bonds, and the physical properties of each of the components remain unchanged.

"Pharmaceutically acceptable excipient" means that the component used to prepare the dosage form of the claimed substances will not harm the human body.

"Pharmaceutically acceptable solvent" means that this solvent, when forming a solution of the claimed substance, will not harm the human body.

In general, the substances of the present invention can be synthesized by methods known in the field of chemistry. Some processes for the production of specific substances of the present invention are illustrated by the schemes given in the examples.

The inventive substances can be used in the form of a mixture with pharmaceutically acceptable excipients for the formation of tablets, capsules, pills, as well as in the form of gels or solutions in pharmaceutically acceptable solvents. The substances according to the invention in these forms can be administered orally, intranasally or parenterally.

The biological effects of the claimed substances include anti-inflammatory effects, cytotoxic effects on tumor cells, and neuroprotective effects in neurotoxicity models.

Thus, the object of the present invention is solved by the synthesis of amide derivatives of non-steroidal anti-inflammatory drugs containing a carboxyl group, for example, but not limited to, diclofenac, ibuprofen, naproxen, ketoprofen, sulindac, ketorolac, aceclofenac, indomethacin, in which the amide moiety or is represented by aminoethanol aminobutyric acid substituted with a dopamine residue, which have anti-inflammatory, cytotoxic and neuroprotective effects.

The following examples are provided for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way.

Example 1. Ibuprofen-Gamma-Aminobutyric Acid-Dopamine Conjugate (IBU-GABA-DA)

i - E ₃ N, iBuClF, MeCN, 1 hour, room temperature; ii - GABA (silylated form), MeCN, 18 hours, room temperature; iii - E ₃ N, iBuClF, MeCN, 1 hour room temperature; iv - Et ₃ N, MeCN, DA * HCl (silylated form), room temperature.

53 mg of gamma-aminobutyric acid (GABA, 0.51 mmol) was dissolved in acetonitrile (0.5 ml) and 250 μL of BSTFA was added and stirred until GABA was completely dissolved.

Triethylamine (81 μL, 0.58 mmol) was added to ibuprofen 110 mg (0.53 mmol) in 2000 μL of acetonitrile and stirred at room temperature for 5-10 min, then 76 μL (0.58 mmol) of isobutyl chloroformate was added. After 1 hour, the precipitate of triethylamine hydrochloride was filtered off, and silylated gamma-aminobutyric acid in acetonitrile was added to the resulting solution of mixed ibuprofen anhydride and left at room temperature for 18 hours. Acetonitrile was evaporated from the reaction mixture, the residue was diluted with water, acidified with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was evaporated, the residue was dissolved in chloroform and applied to a SilicaGel 60 column. The product was eluted from the column with a chloroform-methanol system. Yield of IBY-GABA 124 mg (93%) as a colorless oil.

104 mg (0.55 mmol) of dopamine hydrochloride was dissolved in 1200 μL of acetonitrile with 435 μL of BSTF and 100 μL of pyridine. Stirred until dopamine was completely dissolved.

To 124 mg (0.5 mmol) IBU-GABA in 1200 μL of acetonitrile were added 76 μL (0.49 mmol) of triethylamine and 70 μL (0.54 mmol) of isobutyl chloroformate. After 1 hour, a silylated dopamine solution was added. The mixture was left at room temperature for 18 hours. The reaction mixture was diluted with methanol, kept for 30 minutes, evaporated, the residue was diluted with water acidified with bisulfate, and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was evaporated, the residue was dissolved in chloroform and applied to a SilicaGel 60 column. The product was eluted from the column with a chloroform-methanol system. Yield 105 mg (50%). Yellow viscous oil. R _f = 0.37 (chloroform-methanol, 10: 1). Mass spectrum: calc. 426.252, C ₂₅ H ₃₄ N ₂ O ₄ , found 427.27 [M + H] ⁺ .

Example 2. Aceclofenac-Gamma-Aminobutyric Acid-Dopamine Conjugate (ACEK-GABA-DA)

i - DiPCD, СН2С12, 1-OH-BZT, 1 hour, room temperature; ii - GABA DA * CF3COOH, Et ₃ N, DMF, 1 h, room temperature

To 150 mg (0.43 mmol) of aceclofenac dissolved in 3000 μL of methylene chloride, 72 μL (0.46 mmol) of diisopropyl carbodiimide (DiPrCD), 65 mg (0.42 mmol) of 1-hydroxybenzotriazole (1-OH-BZT) were successively added. 193 mg (0.55 mmol) of GABA-DA in the form of a salt with trifluoroacetic acid, 100 μl of Et ₃ N (0.55 mmol) in 2 ml of DMF were added to the obtained activated derivative of aceclofenac. The reaction mixture was left for 1 hour with stirring at room temperature. Then the organic solvents were evaporated, the residue was diluted with water, acidified with bisulfate, and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was evaporated, the residue was dissolved in chloroform and applied to a SilicaGel 60 column. The product was eluted from the column with a chloroform-methanol system. Yield 193.8mg (79.8%). Slightly yellow viscous oil. R _f = 0.27 (chloroform-methanol, 10: 1). Mass spectrum: calc. 573.1433, C28H29Cl2N3O6; found 574.153 [M + H] +, 596.129 [M + Na] +.

Diclofenac-gamma-aminobutyric acid dopamine conjugate (DIK-GABA-DA)

Received similarly to that described in example 2. Yield 50%. Brownish viscous oil. R _ƒ = 0.37 (chloroform-methanol, 10: 1). Mass spectrum: calc. 515.1379, C ₂₆ H ₂₇ Cl ₂ N ₃ O ₄ ; found 516.147 [M + H] ⁺ , 540.119 [M + Na] ⁺ .

Ketorolac Gamma Aminobutyric Acid Dopamine Conjugate (KTR-GABA-DA)

Received similarly to that described in example 2. Yield 40%. Colorless oil. R _ƒ = 0.36 (chloroform-methanol, 10: 1). Mass spectrum: calc. 475.2107, C ₂₇ H ₂₉ N ₃ O ₅ ; found 476.221 [M + H] ⁺ , 498.201 [M + Na] ⁺ .

Naproxen-Gamma-Aminobutyric Acid-Dopamine Conjugate (NAP-GABA-DA)

Received similarly to that described in example 2. Yield 82%. Beige oil. R _ƒ = 0.43 (chloroform-methanol, 10: 1). Mass spectrum: calc. 450.2155, C ₂₆ H ₃₀ N ₂ O ₅ ; found 451.233 [M + H] ⁺ , 473.208 [M + Na] ⁺ .

Example 3. Conjugate of ketoprofen, gamma-aminobutyric acid and dopamine (KET-GABA-DA)

i - 1,1'-CDI, MeCN, 1 hour, room temperature; ii - GABA-DA * CF ₃ COOH, Et ₃ N, DMF, 1.5 hours, room temperature.

To 100 mg (0.39 mmol) of ketoprofen dissolved in 5000 μL of acetonitrile was added 77 mg (0.47 mmol) of 1,1'-carbonyldimidazole (1,1'-CDI). The mixture was stirred at room temperature for 1 h. Then 166 mg (0.47 mmol) of GABA-DA in the form of a salt with trifluoroacetic acid and 65 μL (0.47 mmol) Et ₃ N in 1 ml of DMF were added, the mixture was stirred for 1.5 h at room temperature.

The organic solvents were evaporated, the residue was diluted with water, acidified with bisulfate, and was extracted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was evaporated, the residue was dissolved in chloroform and applied to a SilicaGel 60 column. The product was eluted from the column with a chloroform-methanol system. Yield 82 mg (44%). R _ƒ = 0.36. Brown oil. Mass spectrum: calc. 474.2155, C ₂₈ H ₃₀ N ₂ O ₅ ; found 475.226 [M + H] ⁺ , 497.212 [M + Na] ⁺ .

Sulindac-Gamma-Aminobutyric Acid-Dopamine Conjugate (SUL-GABA-DA)

Received similarly to that described in example 3. Yield 41%. R _ƒ = 0.41 (chloroform-methanol, 10: 1). Yellow-brown oil. Mass spectrum: calc. 576.2094, C ₃₂ H ₃₃ FN ₂ O ₅ S; found 577.219 [M + H] ⁺ , 599.209 [M + Na] ⁺ .

Indomethacin-gamma-aminobutyric acid dopamine conjugate (INDO-GABA-DA)

Received similarly to that described in example 3. Yield 31%. R _ƒ = 0.33 (chloroform-methanol, 10: 1). Slightly yellowish crystals, T pl. 184-186 ° C. Mass spectrum: calc. 577.1980 (C ₃₁ H ₃₂ ClN ₃ O ₆ ); found 578.200 [M + H] ⁺ , 600.192 [M + Na] ⁺ .

Example 4. Ibuprofen Ethanolamine Dopamine Conjugate (IBU-EA-DA)

i - E ₃ N, iBuClF, MeCN, 1 hour, room temperature; ii - mono-ethanolamine, MeCN, 18 hours, room temperature; iii - a solution of 2M phosgene in toluene, THF, Et3N, 0 ° C, 20 min., 1 h at room temperature; iv - DA (silylated), MeCN, 18 hours, room temperature.

To ibuprofen 205.7 mg (0.99 mmol) in 4000 μL of acetonitrile was added 152 μL (1.1 mmol) of triethylamine, stirred at room temperature for 5-10 min, and 142 μL (1.1 mmol) of isobutyl chloroformate were added. After 15 minutes. The reaction mixture was filtered, and to the resulting solution was added 90 μL (1.5 mmol) of mono-ethanolamine in 0.5 mL of acetonitrile and left at room temperature for 18 h. The reaction mixture was evaporated, the residue was diluted with water acidified with bisulfate, and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was evaporated, the residue was dissolved in chloroform and applied to a SilicaGel 60 column. The product was eluted from the column with a chloroform-methanol gradient system. Yield IBU-EA 209 mg (83%) as a colorless oil.

51 mg (0.25 mmol) of dopamine hydrochloride was dissolved in 1000 μL of acetonitrile containing 200 μL of BSTF and 100 μL of pyridine. Stirred at room temperature until dopamine was completely dissolved.

A solution of 80 mg (0.23 mmol) IBU-EA in 4000 μL of tetrahydrofuran and 45 μL (0.32 mmol) of triethylamine was gradually added to 1 ml of a solution of 2M phosgene in toluene cooled to 0 ° C. Allow the mixture to warm to room temperature over 20 minutes. and filtered. A silylated dopamine solution was added to the resulting solution. The reaction mixture was kept at room temperature for 18 h. The reaction mixture was diluted with methanol and held for 30 min. The mixture was evaporated at room temperature, the residue was diluted with water, acidified with bisulfate, and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous Na ₂ SO ₄ . The organic layer was evaporated, the residue was dissolved in chloroform and applied to a SilicaGel60 column. The product was eluted from the column with a chloroform-methanol system. Yield 65.6 mg (65%). Brownish oil. R _ƒ = 0.24 (chloroform-methanol, 15: 1). Mass spectrum: Calculated, 428.2311 (C ₂₄ H ₃₂ N ₂ O ₅ ); found 429.1300 [M + H] ⁺ .

Indomethacin Ethanolamine Dopamine Conjugate (INDO-EA-DA)

Was prepared analogously to that described in example 4 from indomethacin, ethanolamine and dopamine hydrochloride. R _ƒ = 0.28 (chloroform-methanol, 15: 1). Yield 60%, yellowish oil. Mass spectrum: Calculated 579.1772 (C ₃₀ H ₃₀ ClN ₃ O ₇ ); found 580.1823 [M + H] ⁺ .

Example 5. Conjugate of gamma-aminobutyric acid with dopamine (GABA-DA)

i - Boc ₂ O, NEt ₃ , MeCN-H ₂ O (1: 1), 2 hours, 0 ° C; ii - E ₃ N, isobutyl chloroformate, MeCN, 1 hour, room temperature; iii - silylated dopamine, NEt ₃ , MeCN, 18 hours, room temperature; iv -CF ₃ CO ₂ , CH ₂ Cl ₂ , 1 hour, room temperature.

To a solution of 1.11 g (10.76 mmol) of γ-aminobutyric acid in 10 ml of a mixture of acetonitrile-water (1: 1), add 2.91 ml (12.68 mmol) (Boc) ₂ O and 1.75 ml (12.68 mmol) of triethylamine and stir for 2 h at 0 ° C. The reaction mixture was washed with hexane (2 × 100 ml), the acetonitrile was evaporated, the aqueous fraction was acidified to pH 3 and extracted with ethyl acetate (2 × 100 ml). The combined organic extract was washed with water (2 x 50 ml), brine, dried over anhydrous sodium sulfate. The drying agent is filtered off, the filtrate is evaporated in a water-jet vacuum pump. 1.56 g of N-Boc-GABA are obtained, yield 80%, colorless oil.

5.8 ml of BSTF and 1.5 ml of pyridine are added to a solution of 1.57 g (8.3 mmol) of dopamine hydrochloride in 10 ml of acetonitrile, and the mixture is stirred at 25 ° C until dopamine is completely dissolved. 1.18 ml (8.5 mmol) of triethylamine and 1.09 ml (8.5 mmol) of isobutyl chloroformate are added to a solution of 1.56 g (7.69 mmol) of N-Boc-GABA in 15 ml of acetonitrile, the mixture is stirred for 1 h at 25 ° С, a solution of silylated dopamine is added, and the mixture is stirred 18 h. The reaction mixture is evaporated in vacuo, the residue is dissolved in 200 ml of ethyl acetate and washed with 2N sodium bisulfate solution (200 ml), water (2 × 100 ml), brine, dried over anhydrous sodium sulfate. The drying agent is filtered off, the filtrate is evaporated in a water-jet vacuum pump. The residue was purified by column chromatography on silica gel (SilicaGel 60) in a chloroform-methanol gradient system. The fractions containing the product (control by TLC) are combined, the solvent is evaporated off in vacuo. 1.61 g of Boc-GABA-DA are obtained, yield 62%.

To a solution of 1.61 g of Boc-GABA-DA in 10 ml of methylene chloride, 1.0 ml of trifluoroacetic acid is added and the mixture is stirred for 1 h at 25 ° C. The reaction mixture is evaporated in a water-jet vacuum pump. 1.64 g of γ-aminobutyric acid-dopamine conjugate trifluoroacetate is obtained, yield 98%, colorless oil. Mass spectrum: Calculated 238.1317 (C ₁₂ H ₁₈ N ₂ O ₃ ); found 239.1327 [M + H] ⁺ , 261.1326 [M + Na] ⁺ .

Example 6. Ibuprofen Dopamine Conjugate (IBU-DA)

i - E ₃ N, isobutyl chloroformate, MeCN, 1 hour, room temperature; ii - silylated dopamine, NEt ₃ , MeCN, 18 hours, room temperature.

To a solution of 110 mg (0.58 mmol) of dopamine hydrochloride in 1 ml of acetonitrile, 400 μL of BSTF and 100 μL of pyridine are added and stirred until the complete dissolution of dopamine at 25 ° C. To a solution of 200 mg (0.97 mmol) ibuprofen in 3 ml of acetonitrile add 147 μL (1.07 mmol) of triethiamine and 137 μL (1.07 mmol) of isobutyl chloroformate, stirring for 1 h at 25 ° С. A solution of silylated dopamine is added to the resulting mixed ibuprofen anhydride and stirred for 18 hours at 25 ° C. The reaction mixture was evaporated, the residue was dissolved in 50 ml of ethyl acetate and washed with 2N sodium bisulfate solution (20 ml), water (2 × 20 ml), brine, dried over anhydrous sodium sulfate. The drying agent is filtered off, the filtrate is evaporated on a rotary evaporator in a water-jet vacuum pump. The residue was purified by column chromatography on silica gel (SilicaGel 60) in a chloroform-methanol gradient system. The fractions containing the product (control by TLC) are combined, the solvent is evaporated off in vacuo. 123 mg of ibuprofen-dopamine conjugate is obtained, yield 61%, colorless viscous oil, Rf = 0.36 chloroform-methanol, 19: 1). Mass spectrum: Calculated 341.1991 (C21H27NO3); found 342.2098 [M + H] +, 364.2 [M + Na] +.

Diclofenac dopamine conjugate (DIC-DA)

Prepared analogously to that described in example 6 from diclofenac and dopamine hydrochloride. Yield 62%, brown oil, Rf = 0.7 (chloroform-methanol, 9: 1). Mass spectrum: Calculated 430.0851 (C22H20Cl2N2O3); found 431.095 [M + H] +, 453.072 [M + Na] +.

Ketorolac Dopamine Conjugate (KTR-DA)

Prepared analogously to that described in example 6 from ketorolac and dopamine hydrochloride. Yield 63%, yellow oil, Rf = 0.7 (chloroform-methanol, 9: 1). Mass spectrum: Calculated 390.1580 (C23H22N2O4); found 391.1681 [M + H] +, 413.1513 [M + Na] +.

Ketoprofen Dopamine Conjugate (KET-DA)

Received analogously to that described in example 6 from ketoprofen and dopamine hydrochloride. Yield 59%, crystallizing colorless oil, Rf = 0.4 (chloroform-methanol, 9: 1). Mass spectrum: calculated 389.1627 (C24H23NO4); found 390.1723 [M + H] +, 412.1581 [M + Na] +.

Sulindac Dopamine Conjugate (SUL-DA)

Received analogously to that described in example 6 from sulindac and dopamine hydrochloride. Yield 54%, yellow crystals, Tm = 138-142 ° C. Rf = 0.72 (chloroform-methanol, 9: 1). Mass spectrum: Calculated 491.1567 (C28H26FNO4S); found 492.1670 [M + H] +, 514.1447 [M + Na] +.

Indomethacin Dopamine Conjugate (INDO-DA)

Received similarly to that described in example 6 from indomethacin and dopamine hydrochloride. Yield 80%, yellowish oil. Rf = 0.65 (chloroform-methanol, 9: 1). Mass spectrum: Calculated 492.1452 (C27H25ClN2O5); found 493.1552 [M + H] +, 515.1322 [M + Na] +.

Naproxen Dopamine Conjugate (NAP-DA)

Was prepared analogously to that described in example 6 from naproxen and dopamine hydrochloride. Yield 80%, beige crystals, Tm = 143-145 ° C. Rf = 0.75 (chloroform-methanol, 9: 1). Mass spectrum: Calculated 365.1627 (C22H23NO4); found 366.1729 [M + H] +, 388.1592 [M + Na] +.

Example 7. Conjugate of aceclofenac and dopamine

i - diisopropylcarbodiimide, 1-hydroxybenzotriazole, 1 hour, room temperature; ii - dopamine hydrochloride, DMF, NEt ₃ , 1 h, room temperature.

To a solution of 200 mg (0.56 mmol) of aceclofenac in 4 ml of methylene chloride, 86.5 mg (0.56 mmol) of 1-hydroxybenzenetriazole and 96 μL (0.62 mmol) of diisopropylcarbodiimide are added and the mixture is stirred for 1.5 h at 25 ° С. The precipitate that formed was filtered off, 117.8 mg (0.62 mmol) of a solution of dopamine hydrochloride in 1.5 ml of dimethylformamide and 30 μL (0.68 mmol) of triethylamine were added, and the mixture was stirred for 1.5 h at 25 ° С. The reaction mixture was evaporated, the residue was dissolved in 50 ml of ethyl acetate and washed with 2M sodium bisulfate solution (20 ml), water (2 × 20 ml), brine, dried over anhydrous sodium sulfate. The drying agent is filtered off, the filtrate is evaporated on a rotary evaporator in a water-jet vacuum pump. The residue was purified by column chromatography on silica gel (SilicaGel 60) in a chloroform-methanol gradient system. The fractions containing the product (control by TLC) are combined, the solvent is evaporated off in vacuo. 135 mg of aceclofenac-dopamine conjugate is obtained, yield 49%. Yellow-brown light crystals, T pl. = 89-92 ° C. Mass spectrum: calculated, 488.0906 (C24H22Cl2N2O5); found 489.099 [M + H] +, 511.081 [M + Na] +.

Example 8. Anti-inflammatory effect of NSAID derivatives on RAW-264.7 cells

The RAW-264.7 cell line is a monocyte / macrophage isolated from the ascites fluid of AMLV-induced mouse leukemia. When the bacterial cell wall is stimulated by lipopolysaccharide, the cells respond in an inflammatory manner, releasing various mediators into the environment - indicators of inflammation, in particular, nitric oxide. The change in the level of this mediator (measured by the amount of nitrite ion in the cell incubation medium) is an indicator of the pro- or anti-inflammatory effect of the compounds.

RAW-264.7 cells were purchased from the cell bank of the Institute of Cytology, St. Petersburg. The cells were incubated at 95% humidity, in an atmosphere of 5% CO ₂ , at a temperature of 37 ° C. Culturing was carried out in DMEM medium containing 7% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B. Cells were subcultured every 48-72 hours. For suspension, the cells were incubated in Versene's solution in within 2-3 minutes. in Trypsin-EDTA solution (0.25%).

Anti-inflammatory activity testing

Cells were plated 100 thousand in the wells of a 96-well plate in 100 μl of medium. The induction of an inflammatory response was performed using 1 μg / ml lipopolysaccharide in the culture medium. An aliquot of the standard solution of the test substances in DMSO was added to the medium for the induction of an inflammatory response with stimulants and the culture medium in the wells was replaced with the resulting solution. It was incubated for 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 a 0.04% aqueous solution of sulfanilamide was added and kept at room temperature for 10 min. protected from light, 12.5 μL of a 2% solution of naphthylethylenediamine in 3M HCl was added, and the mixture was kept for another 10 min. at room temperature with protection from light, after which the optical absorption was determined at a wavelength of 540 nm.

The most active in this test were dopamine derivatives of ketoprofen (KET-DA, KET-GABA-DA) EC5028.86 ± 1; 33.65 ± 0.5 and keterolac (KTR-DA, KTR-GABA-DA) EC50 34.73 ± 1; 37.77 ± 2. It should be noted that these derivatives had no cytotoxic effect on these cells.

Other NSAID derivatives also exhibited moderate anti-inflammatory activity (Table 1).

In this model, derivatives of NSAIDs with dopamine, linked through the aminoethanol residue, showed high anti-inflammatory activity. Thus, for IBU-EA-DA, the EC50 value was 11 ± 3 μM.

Derivatives of NSAIDs with dopamine also had an anti-inflammatory effect on the culture of BV-2 microglia cells. The substances were tested as described above. The results are shown in Table 2.

DIK-DA, DIK-GABA-DA, and KET-GABA-DA (EC50 53.0; 65.2; 52.6, respectively) exhibited the highest activity on this cell line. At the same time, the activity of NSAID derivatives with dopamine exceeded the activity of the initial NSAID, in some cases almost 2 times. Thus, the EC50 value for diclofenac was 126.8 μM, and for its derivatives with dopamine (DIK-DA, DIK-GABA-DA) - 53.0 and 65.2 μM, respectively.

Example 9. Cytotoxic effect of NSAID derivatives with dopamine on cancer cell lines

A549 lung carcinoma cells were purchased from the cell bank of the Institute of Cytology of St. Petersburg. The cells were incubated at 95% humidity, 5% CO2, at 37 ° C. Cultivation was carried out in DMEM medium containing 7% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B. Cells were subcultured every 48-72 hours.

Assessment of the effect of substances on cells

The cells were plated 10 thousand in the wells of a 96-well plate in 100 μl of medium. After 24 h of cultivation, 100 μL of the test substances were added to the cells in the form of a DMSO solution in a cell culture medium with a final DMSO concentration of 0.5%. The cells were incubated with the test substances for 24 hours. Cells were used as a positive control, to which 100 μl of medium with 0.5% DMSO was added.

Evaluation of cell survival in culture using the MTT test

After the end of incubation, the medium was removed from each well and 100 μl of MTT reagent (0.5 mg / ml MTT, 3.5 mg / ml D-glucose in Hanks solution) was added. It was incubated in a CO2 incubator for 1.5 h. The medium with MTT was removed, and 100 μL of DMSO was added to each well, and the mixture was stirred for 2 min. The optical density of the solution was determined at wavelengths of 576 nm and 620 nm. The results are shown in Table 3.

The most active compound in this test was ibuprofen amide with dopamine (IBU-DA), for which the half-lethal concentration was 29.81 ± 0.5 μM. Amides of diclofenac and indomethacin also exhibited cytotoxic activity against this type of cancer cells in the LC50 range, 40-60 μM, while the original NSAIDs were inactive in this test.

Indomethacin derivatives on rat C6 glioma cells were significantly more active than the initial indomethacin. The LC50 values for INDO-DA and INDO-GABA-DA were 72.1 ± 1.5 and 32.3 ± 2 μM, respectively, while for indomethacin this value significantly exceeded 100 μM. It should be noted that the introduction of a fragment of gamma-aminobutyric acid into the structure of the dopamine derivative of indomethacin more than doubled the cytotoxic activity of INDO-GABA-DA towards C6 cells.

Example 10. Neuroprotective effect of NSAID derivatives with dopamine

SH-SY5Y human neuroblastoma cells are used as a model to test the neuroprotective potential of chemical compounds.

The cells were purchased from the ATCC collection of cell cultures. The cells were incubated at 95% humidity, 5% CO2, at 37 ° C. The cultivation was carried out in DMEM medium containing 7% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B.

Assessment of the effect of substances on cells

The cells were plated 20 thousand in the wells of a 96-well plate in 100 μl of medium. After 24 h of cultivation, 100 μL of the test substances were added to the cells in the form of a DMSO solution in a cell culture medium with a final DMSO concentration of 0.5%. The neurotoxin MPP ⁺ and the NSAID derivative were added either simultaneously or in the sequence shown in Table 4. The cells were incubated with the test substances for 24 hours. Cells were used as a positive control, to which 100 μl of medium with 0.5% DMSO was added. The results are shown in Table 4.

The dopamine derivatives of ibuprofen and diclofenac showed the greatest neuroprotective activity in all variants of the experiment.

Dopamine derivatives of ibuprofen were also active in a model of chemical hypoxia on SH-SY5Y cells induced by the addition of CoCl ₂ × 6H ₂ O (425 μM) to the incubation medium. For IBU-DA and IBU-GABA-DA, EC ₅₀ values were 18.5 and 55.8 μM, respectively.

Thus, the above examples show that the claimed substances have anti-inflammatory, cytotoxic activity towards cancer cells, and also have a neuroprotective effect on cells of the nervous system.

It should be understood that while the invention is described in conjunction with its detailed description, the above description is intended to be illustrative and not limit the scope of the invention, which is defined by the scope of the appended claims. Others:

Claims (4)

1. Compound of general formula (I):

wherein R represents the residue of a nonsteroidal anti-inflammatory agent containing a carboxyl group, an adjoint amide bond and constituting diclofenac, ibuprofen, naproxen, ketorolac, aceclofenac, indomethacin, ketoprofen and sulindac, and X represents CH _2, (CH _{2) 3} CONHCH _2, or CH ₂ CH ₂ O (CO) NHCH ₂ , and in the case when X is CH2, R cannot be an ibuprofen, ketoprofen or sulindac residue. 2. The use of a compound according to claim 1 as an agent having anti-inflammatory, cytotoxic and / or neuroprotective activity.