The present invention relates to new alkanolamide derivatives of cinnamic acid and use of alkanoloamide derivatives of cinnamic acid for preparation of drugs. The subject compounds possess pharmacological activity, especially in treatment and/or prophylaxis of neurological symptoms or diseases.

Epilepsy is one of the most frequent neurological disease. The estimated prevalence of epilepsy, depending of world's region, is 0.2-4.1 % [1]. As a result, the number of epilepsy patients reaches 50 million all over the world, and 400 000 in Poland [2, 3]. The most common epilepsy treatment option is pharmacotherapy used in at least 95% of patients. The main aim of the pharmacotherapy of epilepsy is controlling the seizures, which means eradication of them or at least limitation of their number. As a consequence patients gain increase of life quality. Moreover, there is less risk of organic damage of brain [4]. Despite of remarkable improvement in the field of therapy of epilepsy and the increase of number of antiepileptic drugs (AEDs) available in the market there are still about 30% of epilepsy patients who are not sufficiently treated. About 40% of patients with partial seizures (both simple and complex) are resistant to known AEDs [5]. The literature data clearly indicate that one of the reason of resistance is insufficiency of currently available antiepileptic drugs. Those facts are important reasons for research conducted within the proposed invention.

At the Department of Bioorganic Chemistry Jagiellonian University Medical College in Krakow the searching of new anticonvulsant agents has been performed for many years now. One of the interesting group are amide derivatives of cinnamic acid but previous results were not promising. The synthesized compounds showed only moderate anticonvulsant activity [6].

Other published results of pharmacological evaluation in standard tests of compounds from that group were also rather not promising [7, 8, 9]. Although some papers contain information about anticonvulsant activity, they do not bring any data from resistant seizures models as well as about analgesic activity [10, 1 1].

Patent search of new anticonvulsant agents revealed Patent US4107320-A which described /V-(3-chlorcinnamoyl)-pyrolidinewith the following formula.

Patent US4175924-A describedA/-allyl-3-flurcinnamamide, showed in the formula below, which was found to possess anticonvulsant activity, preferably in configuration trans.

Patent US4190674-A described /V-cyclopropyl-3-fluorcinnamamide, showed in formula below, as new anticonvulsant agent, preferably in configuration trans.

In Patent US4309444-A one can find N-isobutyl-3-chlorcinnamamide, showed in the formula below, reported as a new anticonvulsant agent.

Alkanolamide derivatives of cinnamic acid (cinnamamide derivatives), which are subject of present patent claim were proved to act as anticonvulsant and/oranalgesic agents while tested in animal models. The results were unexpected, especially while considering previous data. The compounds constitute promising group as potential drugs in treatment and/or prophylaxis of neurological symptoms or diseases.

According to the present invention alkanolamide derivatives of cinnamic acid are defined by the general formula

R Formula 1.

The compounds possess preferably configuration trans. In the general structure R1 is (f?)-2-aminopropan-1-ol, (S)-2-aminopropan-1-ol, (SJ-l-aminopropan^-ol, ( ^SJ-l-aminobutan^-ol, (R,S)-2-amino-3-methylbutan-1-ol, and R2 is hydrogen atom or methyl group in appropriate position in phenyl ring, preferably 2.

Moreover, the invention reports also use of alkanolamide derivatives of cinnamic acidof the general formula 1 in which R1 is (/?)-2-aminopropan-1-ol, (S)-2- aminopropan-1-ol, (S)-1 -aminopropan-2-ol, (f?,S)-1-aminobutan-2-ol, (R,S)-2- amino-3-metylobutan-1-ol, (R,S)-2-aminopropan-1 -ol, (R,S)-1-aminopropan-2-ol, (R,S)-2-aminobutan-1-ol, (R)-2-aminobutan-1-ol, (frans)-4-aminocyclohexanol, 3- hydroxypiperidine, R2 is hydrogen atom, chloride or methyl group in appropriate position in phenyl ring, preferably 4 for chloride, 2 for methyl group, for preparation of drugs fortreatment and/or prophylaxis of neurological symptoms or diseases, especially in epilepsy and/or neuropathic pain.

Compounds of the general formula 1 or drugs containing those compounds as active ingredients could be administered orally or intraperitoneally, preferable route of administration would depend on clinical manifestation of the disease. Compounds of the general formula 1 could be used alone or in combination with other auxiliary substances.

The specialist will be able choose appropriate auxiliary substances for specific form of administration based on scientific knowledge. Among possible auxiliary substances one can mention solvents, gel forming substances, carriers, antioxidants, emulsifying agents, antifoaming agents, flavouring substances, preservatives or colorants. The dosage of compounds with general formula 1 would depend on potency and time of action as well as treated syndrome, patient's gender, age, body weight, and individual reaction to the drug. The details of the invention aresummarized in examples below, in which following abbreviations were used.

Table.

Compound R1 R2

KM-513 (R)- 2-aminopropan-1 -ol H

KM-512 (S)-2-aminopropan-1 -ol H

KM-568 (S)-1 -aminopropan-2-ol H

KM-626 (R, S)-2-amino-3-methylbutan-1 -ol H

KM-584 (R,S)-1 -aminobutan-2-ol 2-CH ₃

KM-604 (S)- 2-aminopropan-1 -ol 2-CH3

KM-488 (R, S)-2-aminopropan-1 -ol H

KM-530 (f?,S)-1 -aminopropan-2-ol H

KM-517 (R,S)-2-aminobutan-1 -ol H

KM-578 (/?,S)-1 -aminobutan-2-ol H

KM-616 (frans)-4-aminocyclohexanol 4-CI

KM-570 (f?)-2-aminobutan-1 -ol H

KM-608 3-hydroxypiperidine H KM-610 (ft,S)-1-aminopropan-2-ol 4-CI

KM-612 (f?,S)-2-aminobutan-1-ol 4-CI

Compounds KM-488, KM-530, KM-517, KM-578, KM-616, KM-570, KM-608, KM- 610, and KM-612 are already known but their usefulness for treatment and/or prophylaxis of neurological symptoms or diseases, especially epilepsy and/or neuropathic pain is not obvious.

The invention is illustrated by following examples.

Example 1. Compound KM-513

In a250 ml Erlenmeyer flask a K2CO3 solution was prepared by dissolving 2.78 g (0.02 M) K2CO3 in 50 ml distilled water. Then 1.13 g (0.015 M) of (R)-2- aminopropan-1-ol was added to the solution. Then 10 ml of toluene was added. The flask was placed on magnetic stirrer in room temperature. The solution of 2.5 g (0.015 M) frans-cinnamoyl chloride in 30 ml of toluene was being added dropwise for about 20 minutes. The reaction mixture was mixed constantly. After the reaction was completed, the reaction mixture was cooled down, the precipitate was filtered and washed with 20 ml of 5%K ₂ C03and 50 ml of distilled water. Obtained compound was dried and crystallized with mixture/i-hexane:toluene (3:1 v/v). Compound KM-513 of theformula 1 was obtained(R1 : (R)-2-aminopropan-1- ol, R2:H). ¹ H NMR (δ, ppm,CDCI ₃ with internal standard TMS) (CDCI ₃ ): 1.25 (dd, J=2.6; J=6.9; 3H, -CH ₃ ); 2.93 (bs, 1 H, OH); 3.58-3.64 (m, 1 H, -CHH-OH); 3.73- 3.77 (m, 1 H, -CHH-OH); 4.17-4.24 (m, 1 H, CH-(CH ₂ OH)); 5.85 (bs, 1 H, NH); 6.40 (dd,J=2.6; J=15.6; 1 H, Ar-CH=CH-); 7.25-7.50 (m, 5H, Ar-H); 7.63 (dd, J=2.6; J=15.6; 1 H, Ar-CH=CH), specific rotation [a] _D ²⁰ =-6.62; ESI-MS [M+H] z calc./found 206.11/206.18. Melting point: 128-130°C, elemental analysis: Ccaic./Cfound=70.22/70.09; H _C aic./Hf _OU nd=7.37/7.40; N _ca | _C /N _foU nd=6.82/6.79. Example 2. Compound KM-512

In a250 ml Erlenmeyer flask a K ₂ CO ₃ solution was prepared by dissolving 2.78 g (0.02 M) K ₂ CO ₃ in 50 ml distilled water. Then 1.13 g (0.015 M) of (S)-2- aminopropan-1-ol was added to the solution. Then 10 ml of toluene was added. The flask was placed on magnetic stirrer in room temperature. The solution of 2.5 g (0.015 M) frans-cinnamoyl chloride in 30 ml of toluene was being added dropwise for about 20 minutes. The reaction mixture was mixed constantly. After the reaction was completed, the reaction mixture was cooled down, the precipitate was filtered and washed with 20 ml of 5%K ₂ C03and 50 ml of distilled water. Obtained compound was dried and crystallized with mixturen-hexane:toluene (3:1 v/v). Compound KM-512 of the formula 1 was obtained(R1 : (S)-2-aminopropan-1- ol, R2:H). H NMR (δ, ppm,CDCI ₃ with internal standard TMS) (CDCI ₃ ): 1.25 (d, J=6.7; 3H, -CH ₃ ); 2.55 (bs, 1 H, OH); 3.61 (dd, J=6.2; J=11.0; 1 H, -CHH-OH); 3.75 (dd, =3.6; J=10.9; 1 H, -CHH-OH); 4.18-4.26 (m, 1 H, CH-(CH ₂ OH)); 5.89 (bs, 1 H, NH); 6.41 (d,J=15.6; 1 H, Ar-CH=CH-); 7.26-7.37 (m, 3H, Ar-H); 7.46-7.50 (m, 2H, Ar-H); 7.63 (d,J=15.6; 1 H, Ar-CH=CH);specific rotation [a] _D ²⁰ =6.85; ESI-MS [M+H] z calc./found 206.11/206.12. Melting point: 128-130°C, elemental analysis: Ccaic./Cf _O und=70,22/70,12;

Example 3. Compound KM-568

In a250 ml Erlenmeyer flask a K2CO3 solution was prepared by dissolving 2.78 g (0.02 M) K2CO3 in 50 ml distilled water. Then 1.13 g (0.015 M) of (S)-1- aminopropan-2-ol was added to the solution. Then 10 ml of toluene was added. The flask was placed on magnetic stirrer in room temperature. The solution of 2.5 g (0.015 M) rans-cinnamoyl chloride in 30 ml of toluene was being added dropwise for about 20 minutes. The reaction mixture was mixed constantly. After the reaction was completed, the reaction mixture was cooled down, the precipitate was filtered and washed with 20 ml of 5%K2CO ₃ and 50 ml of distilled water. Obtained compound was dried and crystallized with mixturen-hexane:toluene (3:1 v/v). Compound KM-568 of the formula 1 was obtained (R1 : (S)-1-aminopropan-2- ol, R2:H). ¹ H NMR (δ, ppm,CDCI ₃ with internal standard TMS) (CDCI ₃ ): 1.24 (d, J=3.4; 3H, -CH ₃ ); 2.64 (d, J=4.4; 1 H, OH); 3.21-3.30 (m, 1 H, NH-CHH-); 3.56-3.63 (m, 1 H, NH-CHH-); 3.97-4.04 (m, 1 H, CH(OH)-CH ₃ ); 6.12 (bs, 1 H, NH); 6.43 (d, J=15.6; 1 H, Ar-CH=CH-); 7.34-7.41 (m, 3H, Ar-H); 7.47-7.52 (m, 2H, Ar-H); 7.64 (d, J=15.6; 1 H, Ar-CH=CH-);specific rotation [a] _D ²⁰ =22.27; ESI-MS [M+H] ⁺ m/z calc./found206.11/206.18. Melting point: 121-123°C, elemental analysis: Ccaic./Cfound=70.22/70.06; H _C aic./Hf ₀ und=7.37/7.47; N _ca i _c /Nf _O und=6.82/6.80.

Example 4. Compound KM-626 In a250 ml Erlenmeyer flask a K2CO3 solution was prepared by dissolving 2.78 g (0.02 M) K2CO3 in 50 ml distilled water. Then 1.55 g (0.015 M) of (/?,S)-2-amino- 3-methylbutan-1-ol was added to the solution. Then 10 ml of toluene was added. The flask was placed on magnetic stirrer in room temperature. The solution of 2.5 g (0.015 M) frans-cinnamoyl chloride in 30 ml of toluene was being added dropwise for about 20 minutes. The reaction mixture was mixed constantly. After the reaction was completed, the reaction mixture was cooled down, the precipitate was filtered and washed with 20 ml of 5%K ₂ C0 ₃ and 50 ml of distilled water. Obtained compound was dried and crystallized with mixture i-hexane:toluene (3:1 v/v). Compound KM-626of the formula 1 was obtained (R1 : (f?,S)-2-amino-3- methylbutan-1-ol, R2:H). ¹ H NMR (δ, ppm,CDCI ₃ with internal standard TMS) (CDCI3): 0.99 (d, J=5.4; 3H, -CH ₃ ); 1.01 (d, J=5.4; 3H, -CH ₃ ); 1.97 (dqv, J=13.7; J=6.9; -CH(CH ₃ ) ₂ ); 2.53 (bs, 1 H, OH); 3.70-3.83 (m, 2H, -CH2 _. -OH); 3.83-3.94 (m, 1 H, -NH-CH); 5.84 (d, J=8.0; NH); 6.45 (d, J=15.6; 1 H, Ar-CH=CH-); 7.31-7.42 (m, 3H, Ar-H); 7.45-7.56 (m, 2H, Ar-H); 7.65 (d, J=15.6; 1 H, Ar-CH=CH-);ESI-MS [M+H] z calc./found234.14/234.24. Melting point: 98-100°C, elemental analysis:

Example 5. Compound KM-584

Step 1. Synthesis of irans-2-methylcinnamoyl chloride

In a 250 ml round bottom flask there were placed: 5 g frans-2-methylcinnamic acid, 30 ml toluene and 5 ml thionyl chloride (d=1.63 g/ml). The reaction mixture was refluxed for 4 hours, then solvents were evaporated under reduced pressure, 10 ml of toluene was added twice and evaporated.

Step 2. A/-acylation

In a250 ml Erlenmeyer flask a 5% NaOH solution was prepared at volume of 50 ml. Then 2.01 g (0.015 M) of (f?,S)-1-aminobutan-2-ol oxalate was added to the solution. The flask was placed on magnetic stirrer in room temperature and was being mixed for 10 min. Then 10 ml of toluene was added.

The solution of 2.7 g (0.015 M) frans-2-methylcinnamoyl chloride in 30 ml of toluene was being added dropwise for about 20 minutes. The reaction mixture was mixed constantly. After the reaction was completed, the reaction mixture was cooled down, the precipitate was filtered and washed with 20 ml of 5%K ₂ CO ₃ and 50 ml of distilled water. Obtained compound was dried and crystallized with mixturen-hexane:toluene (3:1 v/v). Compound KM-584of theformula 1 was obtained (R1 : (R,S)-1-aminobutan-2-ol, R2:CH ₃ in position 2). ¹ H NMR (δ, ppm.CDC with internal standard TMS) (CDCI ₃ ): 0.98 (t, J=7.4; 3H, -CH ₂ -CH ₃ ); 1.49-1.59 (m, 2H, -CH ₂ -CH ₃ ); 2.42 (s, 3H, Ar-CH ₃ ); 2.64 (bs, 1H, OH); 3.23-3.59 (m, 1 H, -CH(OH)-CH ₂ -); 3.60-3.76 (m, 2H, NH-CH2-); 6.26 (bs, 1 H, NH); 6.34 (d, J=15.4; 1 H, Ar-CH=CH-); 7.14-7.27 (m, 3H, Ar-H); 7.48 (d, J=7.7; 1 H, Ar-H); 7.92 (d, J=15.4; 1H, Ar-CH=CH-); ESI-MS [M+H] ⁺ m/z calc./found234.14/234.24. Melting point: 89-91 °C, elemental analysis: Ccaic./Cf _OU nd=72.07/71.67; N _C aic./Nf _O und=6.00/5.94.

Example 6. Compound KM-604

Step 1. Synthesis of frans-2-methylcinnamoyl chloride

In a 250 ml round bottom flask there were placed: 5 g frans-2-methylcinnamic acid, 30 ml toluene and 5 ml thionyl chloride (d=1.63 g/ml). The reaction mixture was refluxed for 4 hours, then solvents were evaporated under reduced pressure, 10 ml of toluene was added twice and evaporated.

Step 2. A/-acylation

In a250 ml Erlenmeyer flask a K2CO3 solution was prepared by dissolving 2.78 g (0.02 M) K2CO3 in 50 ml distilled water. Then 1.55 g (0.015 M) of (S)-2- aminopropan-1-ol was added to the solution. Then 10 ml of toluene was added. The flask was placed on magnetic stirrer in room temperature. The solution of 2.7 g (0.015 M) frans-2-methylcinnamoyl chloride in 30 ml of toluene was being added dropwise for about 20 minutes. The reaction mixture was mixed constantly. After the reaction was completed, the reaction mixture was cooled down, the precipitate was filtered and washed with 20 ml of 5%K ₂ CO ₃ and 50 ml of distilled water. Obtained compound was dried and crystallized with mixturen-hexane:toluene (3:1 v/v). Compound KM-604 of the formula 1 was obtained (R1 : (S)-2-aminopropan-1- ol, R2: CH ₃ in position 2). ¹ H NMR (δ, ppm,CDCI ₃ with internal standard TMS) (CDCI3): 1.26 (d, J=6.9; 3H, -CH ₃ ); 2.43 (s, 3H, Ar-CH ₃ ); 2.50 (bs, 1 H, OH); 3.58- 3.66 (m, 1 H, -CHH-OH); 3.72-3.80 (m, 1 H, -CHH-OH); 4.18-4.30 (m, 1 H, NH-CH); 6.00 (bs, 1H, NH); 6.32 (d, J=15.5; 1 H, Ar-CH=CH-); 7.14-7.28 (m, 3H, Ar-H); 7.50 (d, J=7.7; 1 H, Ar-H); 7.93 (d, J=15.4; 1 H, Ar-CH=CH-); specific rotation [α]ο ²⁰ =3.14; ESI-MS [M+H] ⁺ m/z calc./found220.13/220.14. Melting point: 152- 154°C, elemental analysis: Ccaic./Cfound=71.21/71.30;

Ncalc./Nfound=6.39/6.27.

Pharmacological activity evaluation. Pharmacological activity evaluation of the compounds was performedat National Institute of Neurological Disorders and Stroke, National Institutes of Health, Rockville, USA within Anticonvulsant Drug Development Program (ADD, Anticonvulsant Screening Program, ASP). The tests were carried out according to published procedures, which were previously used to prove activity of currently used anticonvulsant and/or analgesic drugs. In general, rodents (mice, rats) were used in the tests. In animals there were provoked pathological states similar to those which occur in humans in central nervous system disability, especially inepilepsy or neuropathic pain [12].

For example, activity in MES test, described below, performed in rats or mice is characteristic for compounds active in humans in tonic-clonic seizures. Compounds active in scMet test in rodents are usually efficient in absence seizures. 6-Hz test corresponds with seizures resistant to currently used antiepileptic drugs as well as psychogenic seizures. Proven activity in 6-Hz test as well as kindling models may result in influencing on epileptogenesis (which is development of epilepsy). Activity in pilocarpine induced status model is usually connected with status epilepticus prevention. Activity in formalin test is characteristic for compounds which prevent pain caused by neurological dysfunction (first stage of test) or pain caused by inflammation (second stage of test) while activity in sciatic nerve ligation model suggest efficiency in chronic neuropathic pain [12, 13].

Screening

The first stage of the pharmacological evaluation was screening of the tested compounds in maximal electroshock test (MES) and subcutaneous pentetrazole test (scMet). Additionally, neurotoxicity evaluation was performed to check safety of the tested compounds in central nervous system. For screening tests rodents were used, Carworth Farms No 1 mice (intraperitoneal administration, i.p.) and Sprague-Dawley rats (oral administration, p.o.). The tested compounds were dissolved or suspended in 0.5% methylcellulose. The samples were prepared to obtain certain dose and at the same time total volume of the samples were 0.01 ml/g for mice and 0.04 ml/10 g for rats. Standard doses used in screening were 30, 100, and 300 mg/kg body weight for mice and 30 or 50 mg/kg body weight for rats. The tests were performed 0.5 and 4 hours after administration of the compounds (sometimes additionally 0.25 and 1 hour after administration of the compound) [12].

Neurotoxicity evaluation For neurotoxicity evaluation in mice rotorod test was used. In the test, a mouse was placed on knurled plastic rod rotating at 6 rpm. Standard mouse is able to stay on the rod for a long time. If the tested animal was not able to stay on the rod in each of three trials after administration of the compound the neurotoxicity was reported.

In rats neurotoxicity can be reported according to observation of the animals after administration of the compounds. The signs of the neurotoxicity are among others circular or zigzag gait, ataxia, abnormal spread of the legs, abnormal body posture, tremor, hyperactivity, somnolence, stupor, lack of exploratory behavior, catalepsy. Another performed examination is gently lowering over the edge of the table of the hind leg of the animal. Normal animal quickly lifts its leg back to a proper position. If it does not neutotoxicity is noted. Neurotoxicity is also noted when loss of skeletal muscle tone and hypotonia are observed [12].

Maximal electroshock model (MES)

In MES test seizures were provoked by means of alternate electric current with frequency60 Hz and 50 mA (mice) or 150 mA (rats). The current was being delivered for 0.2 s via corneal electrodes. Protection in the MES test was defined as the abolition of the hindlimb tonic extension component of the seizure. Activity in MES is commonly accepted predictor for efficacy in generalized tonic-clonic seizures in humans [12, 13]. Subcutaneous pentetrazole model (scMet) The scMet was conducted by subcutaneous administration of pentetrazole dissolved in 0.9% NaCI solution at the dose of 85 mg/kg (for mice) or 70 mg/kg body weight (for rats). A failure of observing even a threshold seizure (a single episode of clonic spasm which remain at least 5 s) was classified as protection.lt is generally accepted that thescMet test is predictive of anticonvulsant activity againstnonconvulsiveseizures (e.g. myoclonic, absence) [12, 13].

The results of the performed tests are presented in the tables below.

Table 1. Results in MES, scMet and neurotoxicity evaluation of the tested compounds in mice after intraperitonealadministration

Compound Dose MES ^a) scMef TOX ^D '

[mg/kg] 0.25 0.5 1.0 4,0 0.25 0.5 1.0 4.0 0.25 1 0.5 1.0 4.0 h h h h h h h h h h h h

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-488 100 - 3/3 - 0/3 2/5 0/1 0/5 0/1 0/5 0/8 0/5 0/4

300 - 1/1 - 1/1 - 1/1 - 0/1 - 4/4 - 0/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-512 100 - 3/3 - 0/3 - 0/1 - 0/1 - 2/8 - 0/4

300 - 1/1 - 1/1 - 0/1 - 0/1 - 4/4 - 0/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-513 100 - 3/3 - 0/3 - 0/1 - 0/1 - 1/8 - 0/4

300 - 1/1 - 0/1 - 1/1 - 0/1 - 4/4 - 0/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-517 100 - 1/3 - 0/3 - 0/1 - 0/1 - 3/8 - 0/4

300 - 1/1 - 0/1 - 1/1 - 0/1 - 4/4 - 0/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-530 100 - 2/3 - 0/3 - 0/1 - 0/1 - 1/8 - 0/4

300 - 1/1 - 0/1 - 1/1 - 0/1 - 4/4 - 1/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-568 100 - 3/3 - 0/3 - 0/1 - 0/1 - 0/8 - 0/4

300 - 1/1 - 1/1 - 1/1 - 0/1 - 4/4 - 0/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-570 100 - 3/3 - 0/3 - 0/1 - 0/1 - 0/8 - 0/4

300 - 1/1 - 0/1 - 0/1 - 0/1 - 4/4 - 0/2

30 - 0/1 - 0/1 - 0/1 - 0/1 - 0/4 - 0/2

KM-578 100 - 2/3 - 0/3 - 0/1 - 0/1 - 0/8 - 0/4

300 - 1/1 - 0/1 - 1/1 - 0/1 - 4/4 - 0/2

KM-584 3 - 0/4 - - - - - - 0/4 - -

- t e compoun was not teste n t e particu ar case.

Table 2. Results in MES, scMet and neurotoxicity evaluation of the tested compounds in rats after oral administration

Dose Activity ³ '

Compound Test

[mg/kg] 0.25 h 0.5 h 1.0 h 2.0 h 4.0 h

MES 30 1/4 1/4 2/4 0/4 0/4

KM-488

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 0/4 2/4 0/4 0/4

KM-512

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 1/4 0/4 0/4 1/4 0/4

KM-513 scMet 50 2/4 0/4 0/4 0/4 1/4

TOX 50 0/4 0/4 0/4 0/4 0/4

KM-530 MES 30 0/4 2/4 2/4 0/4 0/4 TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 1/4 3/4 3/4 0/4 0/4

KM-568

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 0/4 2/4 0/4 0/4

KM-570

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 4/4 3/4 1/4 1/4 1/4

KM-578

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 100 3/4 3/4 1/4 0/4 0/4

KM-584 TOX 100 0/4 0/4 0/4 0/4 0/4

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 3/4 3/4 4/4 2/4 1/4

KM-604

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 2/4 3/4 3/4 1/4 0/4

KM-608

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 1/4 4/4 1/4 1/4 0/4 scMet 50 2/4 1/4 1/4 0/4 0/4

K -610

30 0/4 0/4 0/4 0/4 0/4

TOX

50 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 2/4 2/4 1/4 0/4

KM-612

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 0/4 0/4 4/4 2/4

KM-616

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 0/4 0/4 0/4 1/4

KM-626

TOX 30 0/4 0/4 0/4 0/4 0/4 ^a )Number of animals protected/number of animals tested in the MES or scMettest or number of animals displaying neurological deficit/number of animals tested

Further pharmacological evaluation Quantitative evaluation

For most active and promising compounds quantitative evaluation was performed. ED ₅ oS (dose effective in 50% of tested animals) and TD50S (dose neurotoxic in 50% of tested animals) were calculated according to pharmacological tests results in which different doses of compounds were used. The results of quantitative evaluation are presented in Tables 4 - 5. Table 3. Quantitative data of the tested compounds determined

afterintraperitoneal administration

)time = 0.25 h; )time = 0.5 h; )time = 1 .0 h; )time = 2.0 h Table 4. Quantitative data of the tested compounds determined in rats after oral administration

. .

Table 5. Results in MES, scMet and neurotoxicity evaluation of the tested compounds in rats after intraperitoneal administration

Dose Activity ³ '

Compound Test

[mg/kg] 0.25 h 0.5 h 1.0 h 2.0 h 4.0 h

MES 30 3/4 2/4 0/4 0/4 0/4

KM-568

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 4/4 2/4 0/4 0/4 0/4

KM-584

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 2/4 2/4 1/4 0/4 0/4

KM-604

TOX 30 0/4 0/4 0/4 0/4 0/4 MES 30 2/4 0/4 0/4 0/4 0/4

KM-608

TOX 30 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 1/4 0/4 0/4 0/4

KM-610 scMet 50 2/4 3/4 1/4 1/4 0/4

TOX 50 0/4 0/4 0/4 0/4 0/4

MES 30 4/4 4/4 2/4 0/4 0/4

KM-612 scMet 50 2/4 3/4 3/4 1/4 0/4

TOX 50 0/4 0/4 0/4 0/4 0/4

MES 30 0/4 0/4 0/4 0/4 2/4

KM-616

TOX 30 0/4 0/4 0/4 0/4 0/4 ^a )Number of animals protected/number of animals tested in the MES or scMet test or number of animals displaying neurological deficit/number of animals tested

Table 6. Quantitative data of the tested compounds determined in rats afterintraperitoneal administration

6-Hz test

6-Hz test was conducted in mice. The seizures were provoked by means of pulses of current at frequency of 6 Hz via corneal electrodes. The protection in the test is defined as continuation of normal activity of the animal for 10 s since electrical stimulation. Activity in 6-Hz may be concerned as efficacy in psychomotor seizures occurring in human limbic epilepsy. Moreover, 6-Hz test was proposed as a model of therapy-resistant seizures [13]. Table 7. Results of 6-Hz evaluation of the tested compounds in mice after i.p. administration

Dose Activity

Compound Test

[mg/kg] 0.25 h 0.5 h 1.0 h 2.0 h 4.0 h

6-Hz ^a ' 100 3/4 2/4 0/4 0/4 0/4

KM-488

TOX°' 100 0/4 0/4 0/4 1/4 0/4 6-Hz ^a ' 100 4/4 4/4 4/4 0/4 0/4

KM-530

TOX ^D> 100 2/4 0/4 0/4 0/4 0/4

6-Hz ^a ' 100 4/4 4/4 2/4 1/4 0/4

KM-568

TOX ^D> 100 1/4 0/4 0/4 0/4 0/4

6-Hz ^a ' 100 4/4 2/4 1/4 1/4 0/4

KM-578

TOX"' 100 0/4 0/4 0/4 0/4 0/4

6-Hz ^a ' 100 4/4 4/4 2/4 1/4 0/4

KM-584

JOX ^D> 100 4/4 3/4 0/4 0/4 0/4

6-Hz ^a) 100 4/4 4/4 4/4 0/4 0/4

KM-604

TOX"' 100 4/4 4/4 0/4 0/4 0/4

6-Hz ^a ' 100 4/4 4/4 2/4 0/4 0/4

KM-608

TOX ⁰ ' 100 0/4 0/4 0/4 0/4 0/4

6-Hz ^a ' 100 4/4 4/4 2/4 0/4 0/4

KM-612

TOX°' 100 4/4 1/4 0/4 0/4 0/4

6-Hz ^a ' 100 3/4 0/4 3/4 3/4 4/4

KM-616

TOX ⁰ ' 100 1/4 0/4 2/4 4/4 2/4

6-Hz ^a) 100 4/4 4/4 0/4 0/4 0/4

KM-626

TOX ⁰ ' , 100 0/4 0/4 0/4 0/4 0/4 ^a ) Number of animals protected/number of animals tested in the 6-Hz test;

) Number of animals displaying neurological deficit/number of animals tested

For compounds KM-488and KM-626 quantitative evaluation in 6-Hz test was performed and ED ₅₀ s were calculated. Results were as followed (with 95% confidence interval):

KM-488 ED ₅₀ = 60.9 (44.8-75.1 ) mg/kg (i.p., time=0.25 h);

KM-626ED ₅₀ =105.88 (90.59-119.38) mg/kg (i.p., time=0.25 h).

Some currently used antiepileptic drugs (carbamazepine, gabapentin, lamotrigin, phenytoin, topiramate) are inactive in 6-Hz test. Data for active drugs are as follow ethosuximideED ₅₀ =167 (114-223); felbamate ED ₅₀ =73.8 (36.3-119); levetiracetam ED ₅₀ =19.4; phenobarbital ED ₅₀ =14.8 (8.9-23.9); tiagabine ED ₅₀ =0.66; valproic acid ED ₅₀ =126 (94.5-152); zonisamide ED ₅₀ =97.4 (74.6-136) (values in mg/kg body weight, tests in mice, i.p.) [14].

Pilocarpine induced status prevention (PISP) Tests were conducted in Sprague Dawley rats. Tested compounds were administered intraperitoneally, then at certain time pilocarpine was administered at the dose of 50 mg/kg. Immediately after pilocarpine injection (time=0.0 h), the animal was observed and the compound was classified as "active" or "non-active" depending of the behavior of animal. The animal was observed constantly for 30 min (time=0.5 h) and compound was again classified according to its activity in prevention of pilocarpine induced seizures. Compounds which were active in screening tests were subjected to quantitative evaluation in which at least 10 doses in 8 rats were used.

PISP test is considered as animal model of epileptogenesis, activity in that model is potential prognosis for efficacy in prevention of development of epilepsy [13]. Table 8. Results in pilocarpine induced status prevention of the tested compoundsafter i.p. administration in rats

Number Changes in body weight [g]

ComDose

Time [h] Activity ³ ' of Protected rats Non protectedrats pound [mg/kg]

deaths

450 0.0 6/8 4 + 7.3+/-1.5 - 22.0+/-0.0

KM-488

900 0.5 2/8 0 - 17.5+/-1.3 - 20.0+/-2.2

200 0.0 5/7 1 - 7.0+/-7.5 - 20+/-0.0

200 0.5 0/8 0 - 28.8+/-1.3

KM-512 250 0.5 5/8 0 - 6.0+/-7.8 - 25.0+/-1.8

300 0.5 6/7 0 - 10.0+/-5.4 - 35.0+/-0.0

400 0.5 7/8 1 - 22.5+/-2.7 - 25.0+/-0.0

200 0.0 7/8 0 + 0.0+/-1.4 - 25+/-0.0

200 0.5 0/8 1 - 27.1 +/-1.4

KM-513 275 0.5 3/7 1 - 30.0+/-0.0 - 31.7+/-1.0

350 0.5 6/8 0 - 30+/-1.1 - 22.5+/-3.8

400 0.5 8/8 0 - 27.5

200 0.0 7/8 0 - 5.0+/-4.8 + 5.0+/-0.0

200 0.5 1/8 0 - 10.0+/-0.0 - 27.1 +/-2.9

KM-517

300 0.5 5/7 0 + 4.0+/-1.9 - 10.0+/-5.0

400 0.5 8/8 0 - 26.9+/-4.4

200 0.0 7/7 0 + 1 -4+/-1.3

KM-530

400 0.5 0/8 0 - 26.3+/-0.8

200 0.0 8/8 0 + 2.5+/-1.3

KM-568 200 0.5 1/8 0 - 35.0+/-0.0 - 30.7+/-0.7

300 0.5 5/8 2 - 33.8+/-0.9 - 30.0+/-0.0 400 0.5 7/8 0 - 20.7+/-5.5 - 30.0+/-0.0

450 0.0 8/8 0 + 0.0+/-1.3

KM-570

600 0.5 8/8 0 - 3.1 +/-3.3

KM-610 300 0.5 0/8 1 - 24.3+/-1.9 ^a ) Number of animals protected/number of animals tested in the pilocarpine induced status prevention

Compounds KM-512, KM-513, KM-517 i KM-568 were subjected to quantitative evaluation in which doses ED ₅ oi EDgrieffective doses in 50% and 97% of the tested animals, respectively) were calculated. Results are presented in Table 9.

Table 9. Results of quantitative evaluation in PISP model of selected compounds

'time=0.5 h Kindling model

One of possible kindling model is electric kindling conducted in rats. Electrodes were implanted to rats' brains (hippocampus), then subthreshold stimuli were delivered several times until one could observe changes in behavior of the animals as well as in electric activity of the brain. As a result seizures were observed. Kindling model was design to mimic epileptogenesis, thus activity in that test could be predictor of such efficacy in humans [13].

Compounds KM-488 and KM-530 were tested in kindling model in rats and ED ₅ oS were found as follow (with 95% confidence interval):

KM-488 ED ₅₀ =51.6 (30.8-89.4) mg/kg,

KM-530 ED ₅₀ =50.28 (39.79-66.02) mg/kg body weight.

Presented results obtained in several animal models suggest that the tested compounds possess broad anticonvulsant activity. The activity proved in various models may result in beneficial activity in humans in multiple seizures like tonic- clonic, absence, or psychomotor. Especially promising is the fact of observed activity in those models which are considered as predictive for epileptogenesis.

Analgesic activity

Analgesic activity of the compounds was evaluated by means of formalin test and sciatic nerve ligation test. Because of specific cellular mechanism of action i.e. stabilization of membrane potential, some antiepileptic drugs are useful in several types of pain, especially neuropathic pain, which is pain connected with abnormal function of neurons. As a result, compounds tested for anticonvulsant activity are often subjected to analgesic activity evaluation, which is also part of Anticonvulsant Screening Program conducted at NIH, USA.

Formalin test is one of the animal models of pain. The test was done in mice by injection of 0.5% formalin into hind paw. In response of formalin injection animals lick affected paw, and the number or time of licking is measured as a proxy for perceived pain. The first (acute) phase is thought to correspond to the direct stimulation of peripheral fibres, while second (inflammatory) phase is caused by the release of inflammatory mediators from damaged tissue and nerve endings. Simultaneously control tests were conducted which involved administration into animals pure solvent (e.g.methylcellulose). Then comparison of time of licking in control and test group of animals was done. Statistically significant reduction of time of licking was considered as analgesic activity [15].

Compounds KM-488, KM-604, KM-608, KM-616 i KM-626 were tested in formalin test.

Results obtained in formalin test for compound KM-488 are presented in Table 10 and in Figure 1.

Table 10.

Dose AUC (Area Under Curve)

Phase

[mg/kg] Control KM-488 % Control SEM pvalue

Acute 200.8 69.52 34.63 5.872 < 0.01

85

Inflamatory 491.4 192.0 39.09 15.51 < 0.05 Results obtained in formalin test for compound KM-604 are presented in Table 11 and in Figure 2.

Table 11.

Results obtained in formalin test for compound KM-608 are presented in Table 12 and in Figure 3.

Table 12.

Results obtained in formalin test for compound KM-616 are presented in Table 13 and in Figure 4.

Table 13.

Results obtained in formalin test for compound KM-626 are presented in Table 14 and in Figure 5.

Table 14.

Dose AUC (Area Under Curve)

Phase

(mg/kg) Control KM-626 % Control SEM pvalue

Acute 200.01 103.01 51.5 8.72 < 0.05

106

Inflamatory 737.56 580.24 78.67 8.93 > 0.05 As a conclusion, the tested group of compounds proved to possess broad anticonvulsant as well as some analgesic activity. Especially promising results were found for compound KM-616 which showed strong analgesic activity in both phases of formalin test. Sciatic Nerve Ligation model was performed in rats. Sciatic nerve was isolated and tied off by passing a needle and nylon suture through the nerve.As a result inflammation was observed in affected paw with subsequent damage of the nerve and occurrence of neuropathic pain.

In sciatic nerve ligation model compound KM-604 was tested at the dose of 50 mg/kg body weight. The compound significantly increased pain threshold in the tested animals (maximal effect was 254% when compared to control after 2 hours of administration of the compound). In the test mechanical stimuli (by means of von Frey filaments) were delivered. Detailed results of the test is shown in Table 15 and Figure 6. Table 15. Results in sciatic nerve ligation model of compound KM-604

*data significantly different form control

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