RU2474426C1 - Prostamides and their analogues possessing neuroprotective action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry and concerns an agent possessing neuroprotective action representing prostamide of formula

(1) which can find application in medicine in therapy of ischemic cerebral damages and neurodegenerative diseases.

EFFECT: composition shows high efficacy.

16 ex

Description

The invention relates to the field of biochemistry and may find application in medicine in the treatment of ischemic brain lesions and neurodegenerative diseases by introducing substances with a neuroprotective effect.

Neuroprotectors (synonym cerebroprotectors) are drugs that stop and limit damage to brain tissue that develops as a result of acute ischemia (hypoxia). Compounds of various classes of both natural origin and chemically synthesized are used as neuroprotective substances. Among these drugs, the following drugs used in the clinic can be noted (B. Vilensky (2010) Principles of evidence-based medicine in relation to the appointment of neuroprotective therapy for ischemic stroke. Clinic. 2010. No. 2. P.28-32).

Cerebrolysin - a brain hydrolyzate of young pigs containing 85% amino acids and 15% peptides; the molecular weight of the peptides that make up this drug does not exceed 10 kDa, which ensures the rapid penetration of cerebrolysin through the blood-brain barrier.

Cortexin is a preparation containing a complex of levorotatory amino acids and polypeptides with a molecular weight of 1 to 10 kDa and a balanced vitamin and mineral composition, is extracted from the cerebral cortex of calves (pigs), has an organ-specific effect, freely penetrates the BBB; it regulates the ratio of inhibitory and excitatory amino acids, the level of serotonin and dopamine, has antioxidant activity, reduces the content of antibodies to the total myelin protein and helps to normalize the bioelectric activity of the brain.

Citicoline - a metabolic precursor of phospholipids, stabilizes neurometabolic function; the leading mechanism of the neuroprotective activity of the drug is the interruption of the leading links of the "ischemic cascade" and the safety of the penumbra, that is, the blockade of the main mechanism of cell death and the formation of neurological deficit.

Semax - a synthetic neuropeptide with a pronounced nootropic effect; increases the adaptive capabilities of the brain, increases its resistance to stress damage, hypoxia and ischemia, has a pronounced antioxidant, antihypoxic, angioprotective and neurotrophic effects.

These and other drugs can improve the condition of patients with ischemic stroke, but they are ineffective in the treatment of other neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases, etc.

This invention solves the problem of expanding the range of substances with neuroprotective effect. The problem is solved by using prostamides and their analogues with neuroprotective action, the general formula:

where X = CH ₂ -CH ₂ or cis-CH == CH

and R is an amide-linked residue of 2-hydroxyethylamine, or 2-nitroxyethylamine, or glycine, or gamma-aminobutyric acid, or 4-hydroxyphenylethylamine (tyramine), or 3,4-dihydroxyphenylethylamine (dopamine).

Close in structure to the claimed compounds is natural prostaglandin E ₂ , which is formed in the body from arachidonic acid under the action of cyclooxygenase and prostaglandin E synthase enzymes. Prostaglandin E ₂ has a multiple physiological effect, activating specific receptors EP1, EP2, EP3 and EP4. When interacting with the EP2 receptor, prostaglandin E ₂ exhibits neuroprotective properties (McCullough L, et al. (2004) Neuroprotective function of the PGE ₂ EP ₂ receptor in cerebral ischemia. J Neurosci 24: 257-268). However, its interaction with another receptor subtype - EP1 has a neurotoxic effect (Kawano T, et al. (2006) Prostaglandin E ₂ EP1 receptors: downstream effectors of COX-2 neurotoxicity. Nature Med. 12: 225-229), which excludes the possibility of using this compound as a neuroprotective agent.

Prostamide E ₂ , which is ethanolamide of prostaglandin E ₂ , is a product of the metabolism of ethanolamide arachidonic acid - an endogenous ligand of cannabinoid receptors. Unlike prostaglandin E ₂ , prostamide E ₂ does not bind to EP receptors and its action is mediated by unknown mechanisms. The well-known and well-documented biological activity of prostamide E ₂ is its ability to cause contraction of the iris in experimental animals (Matias J., et al. (2004) Prostaglandin Ethanolamides (Prostamides): In Vitro Pharmacology and Metabolism // J. Pharmacol. Exp. Ther. 309: 745-757). The property of prostamide E ₂ to have a neuroprotective effect was not known. Neuroprotective properties of prostaglandin E ₂ amide and gamma-aminobutyric acid and glycinamide prostaglandin E _{2 have} also not been described. Amides of prostaglandin E ₂ and nitroethanolamine, tyramine and dopamine are new compounds with neuroprotective properties.

All of these compounds are prepared by standard chemical synthesis methods, including activation of the carboxyl group with appropriate reagents and condensation with an amine or alcohol. The biological activity of the claimed compounds is confirmed in experiments on cultures of nerve cells exposed to various toxic factors that mimic the processes occurring in the body during the development of neurodegenerative processes. In all experiments, these compounds showed a significant neuroprotective effect in the concentration range of 1–20 μM. In addition, these compounds at the indicated concentrations did not have toxic effects on nerve cells in primary and linear culture models.

Thus, the claimed compounds solve the problem of expanding the range of substances with neuroprotective action, which can be used in medicine as neuroprotective agents.

The invention is illustrated by examples.

Example 1. Amide of prostaglandin E ₂ and monoethanolamine (PGE ₂ -EA) (X = cis-CH = CH, R = 2-hydroxyethylamine). 43 μl (0.3 mmol) of triethylamine was added to 100 mg (0.28 mmol) of prostaglandin E ₂ in 2.5 ml of acetonitrile and cooled to + 4 ° C over 15 minutes, then 38 μl (0.3 mmol) of isobutyl chloroformate was added and stirred at + 4 ° C within 30 minutes To the resulting mixed anhydride, 26 μl (0.42 mmol) of monoethanolamine was added 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. The fractions containing the product (control by TLC) were combined, the solvent was evaporated in vacuo. 71 mg of PGE ₂ -EA were obtained, yield 64%. Mass spectrum: m / z 396.113 [M + H], 413.132 [M + H ₂ O], 791.171 [2M + H].

Example 2. Amide of prostaglandin E ₂ and dopamine (PGE ₂ -DA) (X = cis-CH = CH, R = 3,4-dihydroxyphenylethylamine). To 121 mg (0.34 mmol) of prostaglandin E ₂ in 5 ml of acetonitrile was added 52.2 μl (0.377 mmol) of triethylamine and cooled with salt with hummock for 5 min, then 48.8 μl (0.377 mmol) of isobutyl chloroformate was added. It was stirred for 20 minutes, then 78 mg (0.41 mmol) of dopamine hydrochloride in 600 μl of DMF and 52 μl (0.377 mmol) of triethylamine were added and stirred for another 18 hours. The precipitate was filtered off, the solvent was evaporated. The residue was dissolved in 10 ml of ethyl acetate, washed with 2 N aqueous NaHSO ₄ solution, water, saturated aqueous NaCl solution, 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. The fractions containing the product (control by TLC) were combined, the solvent was evaporated in vacuo. Received 103 mg of PGE ₂ -DA, yield 61%. Mass spectrum: m / z 488.077 [M + H], 505.096 [M + H ₂ O].

Example 3. Amide of prostaglandin E ₂ and gamma-aminobutyric acid (GABA) (PGE ₂ -GABA) (X = cis-CH = CH, R = gamma-aminobutyric acid). The synthesis of PGE ₂ -GABA was carried out as described in example 2. Yield 79%. Mass spectrum: m / z 438.094 [M + H], 460.021 [M + Na].

Example 4. Amide of prostaglandin E ₁ and monoethanolamine (PGE ₁ -EA) (X = CH ₂ -CH ₂ , R = 2-hydroxyethylamine). The synthesis of PGE ₁ -EA was carried out as described in example 1. Yield 76%. Mass spectrum: m / z 398.103 [M + H], 415.112 [M + H ₂ O], 793.169 [2M + H].

Example 5. Amide of prostaglandin E ₂ and nitroethanolamine (PGE ₂ -NEA) (X = cis-CH = CH, R = 2-nitroxyethylamine). The synthesis of PGE ₂ -NEA was carried out as described in example 1. Yield 75%. Mass spectrum: m / z 396.103 [M-NO ₂ ], 441.065 [M + H], 458.084 [M + H ₂ O].

Example 6. Amide of prostaglandin E ₁ and nitroethanolamine (PGE ₁ -NEA) (X = CH ₂ -CH ₂ , R = 2-nitroxyethylamine). The synthesis of PGE ₁ -NEA was carried out as described in example 2. Yield 74%. Mass spectrum: m / z 398.103 [M-NO ₂ ], 443.065 [M + H], 460.084 [M + H ₂ O].

Example 7. Amide of prostaglandin E ₁ and dopamine (PGE ₁ -DA) (X = CH ₂ -CH ₂ , R = 3,4-dihydroxyphenylethylamine). The synthesis of PGE ₁ -DA was carried out as described in example 2. Yield 69%. Mass spectrum: m / z 490.077 [M + H], 507.096 [M + H ₂ O].

Example 8. Amide of prostaglandin E ₂ and tyramine (PGE ₂ -TA) (X = cis-CH = CH, R = 4-hydroxyphenylethylamine). The synthesis of PGE ₂ -TA was carried out as described in example 2. Yield 66.5%. Mass spectrum: m / z 472.017 [M + H], 494.953 [M + Na].

Example 9. Amide of prostaglandin E ₁ and tyramine (PGE ₁ -TA) (X = CH ₂ -CH ₂ , R = 3,4-dihydroxyphenylethylamine). The synthesis of PGE ₁ -TA was carried out as described in example 2. Yield 58.3%. Mass spectrum: m / z 474.017 [M + H], 496.953 [M + Na].

Example 10. Amide of prostaglandin E ₂ and glycine (PGE ₂ -Gly) (X = cis-CH = CH, R = glycine). The synthesis of PGE ₂ -Gly was carried out as described in example 2. Yield 83%. Mass spectrum: m / z 410.106 [M + H], 432.073 [M + Na], 448.029 [M + K].

Example 11. Amide of prostaglandin E ₁ and glycine (PGE ₁ -Gly) (X = CH ₂ -CH ₂ , R = glycine). The synthesis of PGE ₁ -Gly was carried out as described in example 2. Yield 65%. Mass spectrum: m / z 412.106 [M + H], 434.074 [M + Na], 450.03 [M + K].

Example 12. The amide of prostaglandin E ₁ and gamma-aminobutyric acid (PGE ₁ -GABA) (X = CH ₂ -CH ₂ , R = gamma-aminobutyric acid). The synthesis of PGE ₁ -GABA was carried out as described in example 2. Yield 77%. Mass spectrum: m / z 440.096 [M + H], 462.023 [M + Na].

Example 13. Isolation and cultivation of granular rat cerebellar neurons.

Cells were isolated from the cerebellum of 7-day old rat pups. The isolated cerebellum was freed from the external membranes, crushed and placed in a Trypsin-EDTA 1: 250 solution without Ca ²⁺ and Mg ²⁺ . Incubated for 10 min at a temperature of 37 ° C. MEM medium (PanEco) containing 10% calf serum was added to the resulting suspension and carefully resuspended. From the suspension, cells were pelleted by centrifugation for 10 minutes with an acceleration of 1000g. Cells were resuspended in MEM medium with KCl (25 mM) and 10% calf serum; after that, the cells were placed in tablets pretreated with polyethyleneimine at a density of 350 thousand cells / cm ² .

Example 14. Evaluation of the neuroprotective effect in the model of glutamate toxicity. After culturing for 7 days, the cells of the primary culture of rat cerebellar granular neurons obtained as described in Example 10 were incubated for 15 minutes in buffer (NaCl 154 mm, KCl 25 mm, CaCl ₂ 3 mm, MgCl ₂ 2 mm, Na ₂ HPO ₄ 0.4 mM, HEPES 5 mM, D-glucose 10 mM, NaHCO ₃ 3.6 mM) containing 100 μM sodium glutamate. After that, the solutions of the studied substances in the conditioned medium were added to the culture. After 3 hours, the number of living cells was assessed using an MTT test.

Among the studied compounds, the prostamide PGE ₂ -EA had a pronounced protective effect, which at a concentration of 2 μM reached 50% of the surviving cells compared to the control, and at 5 μM, 100%. The nitro derivative PGE ₂ -EA (PGE ₂ -NEA) also protected cortical neurons from the toxic effects of glutamate. Moreover, at a concentration of PGE ₂ -NEA 2 μM, survival of 90% of the cells was observed, which is almost 2 times higher than at the same concentration of PGE ₂ -EA. The PGE ₂ derivative with glycine (PGE ₂ -Gly) showed an activity profile similar to PGE ₂ -EA: at concentrations of 5 μM and 10 μM, 60% and 100% of surviving cells were observed, respectively. The activity of PGE ₂ -GABA was 50% of surviving cells at 10 μM. Amide PGE ₂ with tyramine protected neurons starting at a concentration of 2 μM (40% of surviving cells). At a concentration of 5 μM, a survival of 70% of the cells was observed; at 10 μM, 100% survival of neurons was observed. Derivatives of prostaglandin E ₁ showed similar neuroprotective activity with amides of prostaglandin E ₂ . In control experiments without the addition of glutamate, all of the claimed compounds showed no toxic effects up to a concentration of 50 μM.

Example 15. Evaluation of the neuroprotective effect of substances in the K ⁺ - deprivation model.

The K ⁺ - deprivation model is applicable for granular neurons of the brain, since they need an increased concentration of K ⁺ ions (25 mM). A decrease in the concentration of K ⁺ ions causes apoptosis in this cell culture.

A primary culture of rat cerebellar granular neurons was obtained as described in Example 10 and cultured for 7 days. The conditioned medium was collected, diluted 5 times with serum-free medium (MEM). The final concentration of K ⁺ ions was 9 mm, serum - 2%. Under these conditions, after about 24 hours, approximately 50% of the cells of the neuronal culture died. Initial solutions of substances were prepared in a medium with an ion content of K ⁺ 9 mM. The number of living cells was evaluated using the MTT test.

Prostamide E ₂ (PGE ₂ -EA) showed a pronounced protective effect in the concentration range of 0.1-1 μM (the maximum effect of 77% of surviving cells was observed at a concentration of 1 μM). Nitroprostamide PGE ₂ -NEA at a concentration of 1 μM showed a 100% protective effect.

Example 16. Assessment of toxicity in a culture of transformed glial cells of rat C6.

The rat C6 glioma cell line was obtained by cloning from induced rat glioma. Cells were plated in the wells of a 96-well plate, 2 × 10 ⁴ cells in 100 μl of medium. The bottom of the wells was preliminarily coated with a solution of polyethyleneimine in water (0.5 mg / ml) for at least 30 minutes, after which the wells were washed with sterilized water. After 24 hours of cultivation, test substances were added to the cells, one concentration per three wells. Stock solutions of the compounds were prepared in a medium used for cell cultivation. Serum content in the medium (10%) promotes better dissolution of lipid compounds. The incubation with substances was carried out for 48 hours. At the end of the incubation, cell survival was assessed using the MTT test.

Almost all of the claimed compounds, for example, amides of prostaglandin E ₂ and ethanolamine (prostamide E ₂ ), nitroethanolamine, gamma-aminobutyric acid, etc., did not show a cytotoxic effect up to a concentration of 50 μM. The only compound that had a cytotoxic effect on C6 glioma cells was prostaglandin E ₂ amide and dopamine (reduction of cell viability by 93% at a concentration of 50 μM), however, this substance was non-toxic in primary cultures of cerebellar neurons.

Claims (1)

A tool with a neuroprotective effect, which is a prostamide of the general formula:

,
where X = CH ₂ -CH ₂ or cis-CH = CH
and R is an amide-linked residue of 2-hydroxyethylamine, or 2-nitroxyethylamine, or glycine, or gamma-aminobutyric acid, or 4-hydroxyphenylethylamine (tyramine), or 3,4-dihydroxyphenylethylamine (dopamine).