KCNQ potassium channel agonists, method of preparation and method of use thereof

Abstract

The present invention provides a compound represented by general formula I or a pharmaceutical acceptable salt thereof, the preparation method therefor and the use thereof in preparing a medicine for treating a neurological disease, such as epilepsy, convulsion, neuropathic pain, acute ischemic stroke, and a neurodegenerative disease. The compound according to present invention has a better absorption in brain tissue when compared with RTG. In addition, the compound provided by present invention has not only a greatly enhanced efficacy, but also a neurotoxicity greatly lower than that of RTG, and thus possesses a wider safety window. ##STR00001##

Claims

1. A compound having the structure of formula I or a pharmaceutical acceptable salt thereof ##STR00047## wherein, X is selected from a group consisting of oxygen and sulfur; n is 1, 2 or 3; R.sub.1 is H or halogen; R.sub.2 and R.sub.3 are each independently selected from a group consisting of H, D and C.sub.1-C.sub.3 alkyl; or R.sub.2 and R.sub.3 together with the carbon atom to which they attached form C.sub.3-C.sub.6 saturated ring; R.sub.4 and R.sub.5 are each independently selected from a group consisting of H; C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.4 alkoxyl; C.sub.1-C.sub.6 alkyl substituted by halogen; C.sub.1-C.sub.4 alkoxy substituted by halogen; provided that R.sub.4 and R.sub.5 are not simultaneously hydrogen; R.sub.6 is selected from a group consisting of C.sub.1-C.sub.6 alkoxy; C.sub.1-C.sub.6 alkylamino; C.sub.1-C.sub.6 alkyl; C.sub.3-C.sub.6 cycloalkyl; C.sub.2-C.sub.6 alkenyl; C.sub.2-C.sub.6 alkynyl; C.sub.6-C.sub.10 aryl; C.sub.1-C.sub.6 alkyl optionally substituted by halogen, cyano, hydroxy, C.sub.1-C.sub.6 alkoxyl, di(C.sub.1-C.sub.4 alkyl) amino, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkylcarbonylamino, or C.sub.1-C.sub.6 alkoxycarbonyl; C.sub.3-C.sub.6 cycloalkyl optionally substituted by halogen; C.sub.2-C.sub.6 alkenyl optionally substituted by halogen; C.sub.2-C.sub.6 alkynyl optionally substituted by halogen; tetrahydrofuranyl; and ##STR00048## wherein, R.sub.7 and R.sub.8 are each independently selected from a group consisting of C.sub.1-C.sub.4 alkyl.

2. The compound or pharmaceutical acceptable salt thereof according to claim 1, wherein the compound has the structure represented by formula II: ##STR00049## wherein, X is selected from a group consisting of oxygen and sulfur; R.sub.1 is H or halogen; R.sub.2 and R.sub.3 are each independently selected from a group consisting of H, D and C.sub.1-C.sub.3 alkyl; or R.sub.2 and R.sub.3 together with the carbon atom to which they attached form C.sub.3-C.sub.6 saturated ring; R.sub.4 and R.sub.5 are each independently selected from a group consisting of H; C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.4 alkoxyl; C.sub.1-C.sub.6 alkyl substituted by halogen; C.sub.1-C.sub.4 alkoxy substituted by halogen; provided that R.sub.4 and R.sub.5 are not simultaneously hydrogen; R.sub.6 is selected from a group consisting of C.sub.1-C.sub.6 alkoxy; C.sub.1-C.sub.6 alkylamino; C.sub.1-C.sub.6 alkyl; C.sub.3-C.sub.6 cycloalkyl; C.sub.2-C.sub.6 alkenyl; C.sub.2-C.sub.6 alkynyl; C.sub.6-C.sub.10 aryl; C.sub.1-C.sub.6 alkyl optionally substituted by halogen, cyano, hydroxy, C.sub.1-C.sub.6 alkoxyl, di(C.sub.1-C.sub.4 alkyl) amino, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkylcarbonylamino, or C.sub.1-C.sub.6 alkoxycarbonyl; C.sub.3-C.sub.6 cycloalkyl optionally substituted by halogen; C.sub.2-C.sub.6 alkenyl optionally substituted by halogen; C.sub.2-C.sub.6 alkynyl optionally substituted by halogen; tetrahydrofuranyl; and ##STR00050## wherein, R.sub.7 and R.sub.8 are each independently selected from a group consisting of C.sub.1-C.sub.4 alkyl.

3. The compound or pharmaceutical acceptable salt thereof according to claim 1, wherein the compound has a structure selected from the following formula III to V: ##STR00051## wherein, R.sub.2 and R.sub.3 are each independently selected from a group consisting of H, D and C.sub.1-C.sub.3 alkyl; or R.sub.2 and R.sub.3 together with the carbon atom to which they attached form C.sub.3-C.sub.6 saturated ring; R.sub.4 and R.sub.5 are each independently selected from a group consisting of H; C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.4 alkoxyl; C.sub.1-C.sub.6 alkyl substituted by halogen; C.sub.1-C.sub.4 alkoxy optionally substituted by halogen; provided that R.sub.4 and R.sub.5 are not simultaneously hydrogen; R.sub.9 is selected from a group consisting of C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.6 cycloalkyl; R.sub.10 is selected from a group consisting of C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.6 alkyl optionally substituted by halogen, cyano, hydroxy, C.sub.1-C.sub.6 alkoxyl, di(C.sub.1-C.sub.4 alkyl)amino, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkylamido, or C.sub.1-C.sub.6 alkoxycarbonyl; C.sub.3-C.sub.6 cycloalkyl optionally substituted by halogen; tetrahydrofuranyl; and ##STR00052## wherein, R.sub.7 and R.sub.8 are each independently selected from a group consisting of C.sub.1-C.sub.4 alkyl.

4. The compound or pharmaceutical acceptable salt thereof according to claim 1, wherein one of R.sub.4 and R.sub.5 is methyl, and the other is H or methyl.

5. A compound or pharmaceutical acceptable salt thereof, wherein the compound is selected from the following compounds: ##STR00053## ##STR00054## ##STR00055## ##STR00056##

6. The compound or pharmaceutical acceptable salt thereof according to claim 1, wherein the pharmaceutical acceptable salts are salts formed by the compound with an acid, and for example, the acid is selected from a group consisting of maleic acid, succinic acid, citric acid, tartaric acid, fumaric acid, formic acid, acetic acid, propanoic acid, propandioic acid, oxalic acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, camphoric acid, camphor sulfonic acid, salicylic acid, acetyl salicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, amygdalic acid, malic acid, sorbic acid, trifluoroacetic acid, taurine, homotaurine, isethionic acid, cinnamic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and perchloric acid.

7. A pharmaceutical composition comprising the compound or pharmaceutical acceptable salt according to claim 1 as an active ingredient and a pharmaceutical acceptable adjuvant.

8. The compound or pharmaceutical acceptable salt thereof according to claim 2, wherein one of R.sub.4 and R.sub.5 is methyl, and the other is H or methyl.

9. The compound or pharmaceutical acceptable salt thereof according to claim 2, wherein the pharmaceutical acceptable salts are salts formed by the compound with an acid.

10. A pharmaceutical composition comprising the compound or pharmaceutical acceptable salt according to claim 2 as an active ingredient and a pharmaceutical acceptable adjuvant.

11. The compound or pharmaceutical acceptable salt thereof according to claim 1, wherein, R.sub.1 is H or fluorine; R.sub.2 and R.sub.3 are each independently selected from a group consisting of H and D, or R.sub.2 and R.sub.3 together with the carbon atom to which they attached form cyclopropyl; one of R.sub.4 and R.sub.5 is C.sub.1-C.sub.4 alkyl, and the other is H or C.sub.1-C.sub.4 alkyl.

12. The compound or pharmaceutical acceptable salt thereof according to claim 2, wherein, R.sub.1 is H or fluorine; R.sub.2 and R.sub.3 are each independently selected from a group consisting of H and D, or R.sub.2 and R.sub.3 together with the carbon atom to which they attached form cyclopropyl; one of R.sub.4 and R.sub.5 is C.sub.1-C.sub.4 alkyl, and the other is H or C.sub.1-C.sub.4 alkyl.

13. The compound or pharmaceutical acceptable salt thereof according to claim 3, wherein, R.sub.2 and R.sub.3 are each independently selected from a group consisting of H and D, or R.sub.2 and R.sub.3 together with the carbon atom to which they attached form cyclopropyl; one of R.sub.4 and R.sub.5 is C.sub.1-C.sub.4 alkyl, and the other is H or C.sub.1-C.sub.4 alkyl; R.sub.9 is selected from a group consisting of C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.6 cycloalkyl; R.sub.10 is selected from a group consisting of C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.6 alkyl optionally substituted by halogen, cyano, hydroxy, C.sub.1-C.sub.6 alkoxyl, di(C.sub.1-C.sub.4 alkyl)amino, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkylamido, or C.sub.1-C.sub.6 alkoxycarbonyl; C.sub.3-C.sub.6 cycloalkyl optionally substituted by halogen; tetrahydrofuranyl; and ##STR00057## wherein, R.sub.7 and R.sub.8 are each independently selected from a group consisting of C.sub.1-C.sub.4 alkyl.

14. The compound or pharmaceutical acceptable salt thereof according to claim 3, wherein, R.sub.9 is selected from a group consisting of methyl, ethyl and propyl; R.sub.10 is selected from a group consisting of C.sub.1-C.sub.3 alkyl optionally substituted by halogen, cyano, hydroxy, C.sub.1-C.sub.3 alkoxyl, di(C.sub.1-C.sub.3 alkyl)amino, C.sub.1-C.sub.3 alkylcarbonyl, C.sub.1-C.sub.3 alkylamido, C.sub.1-C.sub.3 alkoxycarbonyl; C.sub.3-C.sub.6 cycloakyl optionally substituted by halogen; tetrahydrofuranyl; and ##STR00058## wherein, R.sub.7 and R.sub.8 are each independently selected from a group consisting of C.sub.1-C.sub.3 alkyl.

15. The compound or pharmaceutical acceptable salt thereof according to claim 9, wherein the acid is selected from a group consisting of maleic acid, succinic acid, citric acid, tartaric acid, fumaric acid, formic acid, acetic acid, propanoic acid, propandioic acid, oxalic acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, camphoric acid, camphor sulfonic acid, salicylic acid, acetyl salicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, ascorbic acid, gallic acid, amygdalic acid, malic acid, sorbic acid, trifluoroacetic acid, taurine, homotaurine, isethionic acid, cinnamic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and perchloric acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the dose-response curves of K43 according to present invention and RTG on KCNQ2 homotetramer channel;

(2) FIG. 2 is a graph showing the dose-response curves of K43 according to present invention and k21 on KCNQ2/3 heterotetramer channel;

(3) FIG. 3 is a graph showing the dose-response curves of K43 and K41 according to present invention and RTG against IVIES in vivo in mice;

(4) FIG. 4 is a graph showing the dose-response curves of K43 and K41 according to present invention and RTG on the athletic capability of mice.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(5) The present invention will be further illustrated based on the following examples, but the present invention will not be limited thereto.

(6) I. Preparation Examples for Compounds

(7) In following preparation examples, NMR was conducted on a Mercury-Vx 300M instrument manufactured by Varian with calibration of H 7.26 ppm (CDCl.sub.3), 2.50 ppm (DMSO-d6) and 3.15 ppm (CD3OD). The reagents were mainly provided by Shanghai Chemical Reagent Co. Ltd., and the silica gel plate (model HSGF 254) for thin layer chromatography (TLC) was manufactured by Huiyou Silica gel Development Co. Ltd., Yantai, Shandong. The compounds were purified by normal phase column chromatography with a silica gel (model zcx-11) of 200-300 mesh, manufactured by Branch of Qingdao Haiyang Chemical Co. Ltd.

Preparation Example 1

Preparation Example 1.1 Synthesis of methyl 4-(N-parafluorobenzyl-N-propargyl-amino)-2,6-dimethylphenylaminoformate (K43)

(8) ##STR00023## ##STR00024##

(9) 2,6-dimethylaniline K43-a (1.2 g, 10 mmol) was dissolved in dichloromethane (4 mL) and pyridine (25 mL), p-toluenesulfonyl chloride (p-TsCl) (2.29 g, 12 mmol) was added thereto and the obtained mixture was refluxed for 6 h. After cooled to room temperature, the reaction system was poured into a 3M HCl solution (20 mL), and then dichloromethane (20 mL) was added thereto. The organic phase obtained by phase separation was washed with water (20 mL) twice, and then concentrated. The residue was recrystallized in ethanol to give K43-b as a white solid (2.1 g, yield: 76%). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.58 (d, J=8.4 Hz, 2H), 7.25 (d, J=8.4 Hz, 2H), 7.00-7.11 (m, 3H), 5.96 (s, 1H), 2.42 (s, 3H), 2.04 (s, 6H).

(10) The obtained K43-b (2.10 g, 7.6 mmol) was dissolved in glacial acetic acid (AcOH) (40 mL), and then water (40 mL) and sodium nitrate (1.3 g, 15.2 mmol) were added thereto. The mixture was cooled to 0 C. by ice bath, and concentrated nitric acid was added thereto, and then the reaction mixture was heated to reflux for 4 h. After the completion of reaction was monitored by TLC, water (20 mL) was added and the mixture was cooled to 0 C. A large amount of yellowish solid was precipitated, and suction-filtered to give K43-c (19 g, yield: 78%), which was directly used in the next step.

(11) K43-c (1.9 g, 5.9 mmol) and water (0.75 mL) were added into a round-bottomed flask, concentrated sulfuric acid (10 mL) was added thereto, and the mixture was kept under 40 C. overnight. The mixture was cooled to room temperature, and crushed ice and 2M aqueous sodium hydroxide solution (15 mL) were poured thereto. The mixture was extracted with ethyl acetate (50 mL), and the organic phase obtained was washed with water (20 mL) twice, and then with saturated saline (20 mL) once, dried with anhydrous sodium sulfate and concentrated to give K43-d as a yellow sold (980 mg, yield: 100%), which was directly used in the next step.

(12) K43-d (1.52 g, 5.9 mmol) and dichloromethane (DCM) (40 mL) were added into a flask and dissolved under stirring. After the mixture was cooled to 0 C. by ice-water bath, di-t-butyl dicarbonate (Boc.sub.2O) was added thereto (2.58 g, 11.8 mmol). Triethylamine (TEA) (1.77 mL, 12.9 mmol) and 4-Dimethylaminopyridine (DMAP) (722 mg, 5.9 mmol) were added under slowly stirring. After half an hour, the temperature was rise to room temperature and the reaction continued overnight. The reaction mixture was washed with 1M HCl (30 mL) once, and with water (50 mL) twice, and dried with anhydrous sodium sulfate. The crude product obtained by concentration was purified with silica gel column chromatography (petroleum ether/ethyl acetate=6:1) to give K43-e as a yellowish solid (1.84 g, yield: 85%), which was directly used in the next step.

(13) K43-e (1.84 g, 5.0 mmol) obtained above was dissolved in ethyl acetate (EtOAc, 20 mL), 10% Pd/C (55 mg, 0.5 mmol) was added under nitrogen atmosphere. Hydrogen was purged three times and the reaction was performed under stirring at room temperature for 4 h. Hydrogen was removed, and nitrogen was purged three times. The reaction mixture was filtered, and the filtrate was concentrated to give K43-f (quantitative yield). .sup.1H NMR (300 MHz, CDCl.sub.3): 6.38 (s, 1H), 3.52 (s, 2H), 2.05 (s, 6H), 1.39 (s, 18H).

(14) K43-f (366 mg, 1.0 mmol) obtained above and p-fluorobenzaldehyde (108 L, 1.0 mmol) were added into a three-necked flask, toluene (10 mL) was added thereto, and after a water segregator was equipped onto the three-necked flask, the mixture was heated to reflux for 3 hour. The mixture was cooled to room temperature, and toluene was removed by vacuum concentration. The obtained crude K43-g was re-dissolved in methanol (20 mL), and sodium borohydride (NaBH.sub.4) (76 mg, 2.0 mmol) was added thereto in batch under vigorous stirring. After the addition, the mixture continued to react for 2 h at room temperature. Crushed ice was added to quench the reaction, and then most of methanol was removed by vacuum concentration. The residue was dissolved in ethyl acetate (20 mL), washed with water (15 mL) twice and with saturated saline (10 mL) once, dried with anhydrous sodium sulfate. After concentration, the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give a benzyl substituted product K43-h (320 mg, yield: 72%; yellow solid).

(15) K43-h (320 mg, 0.72 mmol) obtained above was dissolved in N,N-dimethylformamide (DMF, 5 ml), N,N-diisopropylethylamine (DIPEA) (257 L, 1.44 mmol) and propargyl bromide (84 L, 1.08 mmol) were added thereto. The reaction system was reacted at 65 C. for 4 h. Then, ethyl acetate (50 mL) was dropwisely added thereto. The resultant was washed with water (25 mL) twice, and then with saturated saline (20 mL) once, and then dried with anhydrous sodium sulfate. The residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=15:1) to give a propargyl-substituted intermediate K43-i (312 mg, yield 90%).

(16) The intermediate K43-i (48 mg, 0.1 mmol) was dissolved in dichloromethane, and trifluoroacetic acid was added thereto under ice bath, and the temperature was kept for 2 h. After vacuum concentration, the resultant crude K43-i was redissolved in dichloroethane (1 mL), and DIPEA (35.0 L, 0.2 mmol) and methyl chloroformate (11.7 L, 0.15 mmol) were added thereto under ice-water bath. After the ice-water bath was removed, the reaction was kept at room temperature for 1 h. After concentration, the residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to give K43 (31 mg, yield 92%). .sup.1H NMR (300 MHz, CDCl.sub.3): M 7.27 (dd, J=8.4, 5.4 Hz, 2H), 7.02 (t, J=9.0 Hz, 2H), 6.59 (s, 2H), 5.87 (brs, 1H), 4.48 (s, 2H), 3.96 (d, J=2.1 Hz, 2H), 3.73 (brs, 3H), 2.13 (s, 6H), 2.26 (t, J=2.1 Hz, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3): R 163.6 (J=248.4 Hz), 155.2, 138.3, 137.5, 135.2, 133.7 (J=9.2 Hz), 125.2, 122.0, 116.1 (J=22.4 Hz), 80.4, 73.0, 58.7, 53.0, 47.0, 21.0. HR-ESIMS (m/z): calculated for C.sub.20H.sub.22FN.sub.2O.sub.2 [M+H].sup.+: 341.1665. found: 341.1656.

Preparation Example 1.2 the Following Compounds were Prepared in a Similar Manner as that in Preparation Example 1

(17) TABLE-US-00001 Compound Formula .sup.1H NMR (CDCl.sub.3, 300 MHz) data, K40 7.35 (dd, J.sub.1 = 8.4 Hz, J.sub.2 = 5.7 Hz, 2H), 7.17-7.26 (m, 3H), 6.97-7.03 (m, 2H), 6.75 (s, 1H), 4.15 (s, 2H), 3.76 (s, 3H), 3.56 (d, J = 2.1 Hz, 2H), 2.33 (s, 3H), 2.25 (t, J = 2.1 Hz, 1H). K41 7.41 (brs, 1H), 7.26-7.31 (m, 2H), 6.99-7.05 (m, 2H), 6.73-6.76 (m, 2H), 6.23 (s, 1H), 4.46 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 3.75 (s, 3H), 2.22-2.24 (m, 4H). K42 7.88 (d, J = 8.7 Hz, 1H), 7.27-7.34 (m, 2H), 6.98-7.17 (m, 4H), 6.93 (s, 1H), 4.46 (s, 2H), 3.97 (d, J = 2.1 Hz, 2H), 3.78 (s, 3H), 2.27 (t, J = 2.1 Hz, 1H). K44 8.10 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.35 (dd, J.sub.1 = 1.2 Hz, J.sub.2 = 8.4 Hz, 1H), 7.28 (ddd, J.sub.1 = 3.0 Hz, J.sub.2 = 4.8 Hz, J = 8.1 Hz, 2H), 7.18 (dd, J.sub.1 = 1.2 Hz, J.sub.2 = 7.8 Hz, 1H), 7.00 (ddd, J.sub.1 = 2.7 Hz, J.sub.2 = 3.9 Hz, J.sub.3 = 11.4 Hz, 2H), 4.11 (s, 2H), 3.79 (s, 3H), 3.56 (d, J = 2.7 Hz, 2H), 2.27 (t, J = 2.4 Hz, 1H). K45 7.37 (dd, J.sub.1 = 6.0 Hz, J.sub.2 = 8.7 Hz, 2H), 7.24 (brs, 1H), 7.04 (d, J = 8.7 Hz, 1H), 6.98 (t, J = 8.7 Hz, 2H), 6.69 (dd, J.sub.1 = 2.4 Hz, J.sub.2 = 8.4 Hz, 1H), 6.63 (brs, 1H), 4.22 (s, 2H), 3.88 (s, 2H), 3.76 (s, 3H), 2.24 (t, J = 2.1 Hz, 1H). K46 0 7.89 (brs, 1H), 7.31 (dd, J.sub.1 = 6.0 Hz, J.sub.2 = 8.1 Hz, 2H), 6.94-7.05 (m, 3H), 6.53 (dd, J.sub.1 = 2.4 Hz, J.sub.2 = 9.0 Hz, 1H), 6.47 (s, 1H), 4.45 (s, 2H), 3.95 (d, J = 2.1 Hz, 2H), 3.79 (s, 3H), 3.72 (s, 3H), 2.58 (t, J = 2.1 Hz, 1H). K48 7.92 (brs, 1H), 7.25-7.30 (m, 2H), 7.03 (d, J = 8.7 Hz, 2H), 6.81 (dd, J = 8.7, 2.7 Hz, 1H), 6.77 (s, 1H), 6.63 (s, 1H), 4.46 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 3.78 (s, 3H), 2.26 (t, J = 2.1 Hz, 1H). K49 8.40 (brs, 1H), 7.24 (dd, J = 8.4, 5.4 Hz, 2H), 7.11 (d, J = 9.3 Hz, 2H), 7.02 (d, J = 9.3 Hz, 2H), 6.82-6.85 (m, 2H), 4.50 (s, 2H), 4.10 (s, 3H), 3.93 (d, J = 2.1 Hz, 2H), 2.24 (t, J = 2.1 Hz, 1H).

Preparation Example 2 Synthesis of methyl 4-(N-parafluorobenzyl-N-propargyl-amino)-2,6-dimethylphenylaminoformate (K43)

(18) ##STR00033##

(19) 2,6-dimethylaniline (40 g, 0.33 mol) was dissolved in dichloromethane (250 mL), and then DIPEA (115 mL, 0.66 mol) was added thereto. Methyl chloroformate (38.35 ml, 0.5 mol) was dropwisely added thereto under ice-water bath. After the addition, the reaction system was naturally warmed to room temperature, and stirred overnight. TLC showed that the reactants were completely reacted. 1% HCl (60 mL) was slowly added into the reaction system, and the reaction system was stirred and layered. The water layer was extracted with dichloromethane. The organic phase was combined, washed with saturated saline, dried with anhydrous sodium sulfate, filtrated and concentrated. The crude product was dissolved in a small amount of dichloromethane, and petroleum ether was dropwisely added thereto to precipitate a solid as the intermediate K43-k (59 g, yield: 88%). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.08 (s, 3H), 6.03 (s, 1H), 3.76 (s, 3H), 2.27 (s, 6H).

(20) The intermediate K43-k (20 g) was dissolved in acetic acid (90 mL), and water (80 mL) and sodium nitrite (19.0, 0.223 mol) were added thereto. Concentrated nitric acid (65%, 55 mL) was dropwisely added under ice-water bath, and the internal temperature of the reaction system was controlled to be not higher than 5 C. After the dropwise addition, the reaction system was slowly warmed to room temperature and stirred for 30 mins, and then heated to 140 C. to reflux for 4 h. After cooled to room temperature, the reaction system was poured into ice-water to quench the reaction. The solid precipitated was filtrated and dried to give an intermediate K43-l (17.8 g, yield 71%). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.964 (s, 1H), 7.082 (s, 1H), 3.786 (s, 3H), 2.364 (s, 3H), 2.265 (s, 3H).

(21) The intermediate K43-l (15 g, 0.067 mol) was dissolved in ethyl acetate (200 mL), and 10% Pd/C (1.5 g) was added thereto under nitrogen atmosphere. After hydrogen was purged three times, the reaction was performed overnight at room temperature. The reaction system was filtrated, and the filtrate was concentrated to give an intermediate K43-m at quantitative yield, which was directly used in the next step.

(22) The intermediate K43-m (13 g, 0.067 mol) was dissolved in toluene (100 mL), p-toluenesulfonic acid (0.38 g, 0.002 mol) and p-fluorobenzaldehyde (12.45 g, 10.8 ml) were added thereto. After the water segregator was equipped, the reaction system was heated to reflux and separate water for 4-5 h. TLC showed that the reaction was completed. The reaction system was concentrated under vacuum to give a crude of the intermediate K43-n, which was directly used in next step. The obtained crude K43-n was dissolved in methanol (150 mL), and sodium borohydride (5.07 g, 0.13 mol) was added thereto in batch under ice-water bath. Then the ice-water bath was removed, and the reaction was performed at room temperature for 1-2 h. TLC showed that the reaction was completed. The reaction system was poured into ice-water bath, and stirred. The solid precipitated was filtrated and dried to give a crude of the intermediate K43-o. The obtained crude of the intermediate K43-o was dissolved in a small amount of dichloromethane, and petroleum ether was dropwisely added thereto to precipitate a nearly white solid, which was filtrated and dried to give the intermediate K43-o (11.1 g, yield in two steps: 55%). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.335-7.306 (dd, J=8.7 Hz, 1H), 7.306-7.287 (dd, J=5.7 Hz, 1H), 7.049-7.027 (dd, J=6.6 Hz, 1H), 7.027-6.991 (dd, J=10.8 Hz, 1H), 6.331 (s, 2H), 5.892 (s, 1H), 4.257 (s, 2H), 3.933 (s, 1H), 3.744 (s, 3H), 2.164 (s, 6H).

(23) The intermediate K43-o (10 g, 0.033 mol) was dissolved in DMF (80 mL), and DIPEA (8.54 g, 0.066 mol) and propargyl bromide 2.74 mL, 0.036 mol) were added thereto. The reaction system was reacted at 60 C. for 5 h, and TLC showed that the reaction was completed. The reaction system was poured into water and stirred to precipitate a solid. The solid was filtrated, dried to give a crude of K43, which was dissolved in a small amount of dichloromethane, and petroleum ether was added thereto to precipitate a solid. The solid was filtrated to give K43 (9.8 g, yield: 87%). The .sup.1H NMR thereof was consistent with that of K43 prepared in Preparation Example 1.

Preparation Example 3

Preparation Example 3.1 Synthesis of methyl 4-(N-parafluorobenzyl-propargyl-amino)phenylaminothioformate (K43)

(24) ##STR00034##

(25) p-nitroaniline (2.76 g, 20.0 mmol) and p-fluorobenzaldehyde (2.1 mL, 20.0 mmol) were added into a 150 mL three necked flask, toluene (60 mL) was added thereto and the reaction was refluxed and water was separated with water segregator for 3 h. After cooled to room temperature, the reaction system was concentrated under vacuum to remove toluene. The obtained intermediate K49-a was redissolved in methanol (40 mL), NaBH.sub.4 (1.52 g, 40.0 mmol) was added thereto in batch under vigorously stirring, and the reaction was preformed at room temperature for 3 h. Crushed ice was added to quench the reaction, and water (30 mL) was added under vigorously stirring to precipitate a large amount of solid, which was suction-filtrated. The filter cake was washed with anhydrous ethylether (10 mL) twice to give a product K49-b (3.4 g, yield: 70%, yellow solid). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.08 (d, J=9.3 Hz, 2H), 7.31 (dd, J.sub.1=5.4 Hz, J.sub.2=8.4 Hz, 2H), 7.06 (t, J=8.7 Hz, 2H), 6.57 (d, J=9.3 Hz, 2H), 4.86 (s, 1H), 4.41 (d, J=2.7 Hz, 2H).

(26) K49-b (3.4 g, 14.0 mmol) was dissolved in DMF (40 mL), NaH (616 mg, 15.4 mmol) was quickly added under ice bath, and the reaction was preformed at room temperature for 0.5 h. Propargyl bromide (1.22 mL, 15.4 mmol) was added, and the reaction was preformed at 65 C. for 4 h. Ethyl acetate (80 mL) was dropwisely added into the reaction system. The mixture was transferred to a separating funnel and washed with water (40 mL) twice. The organic phase was combined and washed with saturated saline (30 mL) once, dried with anhydrous sodium sulfate, and concentrated. and the residue was purified with column chromatography (PE/EA=8:1) to give K49-c (3.4 g, yield: 86%). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.14 (d, J=9.0 Hz, 2H), 7.26 (dd, J=5.4, 8.4 Hz, 2H), 7.04 (t, J=8.7 Hz, 2H), 6.79 (d, J=9.3 Hz, 2H), 4.68 (s, 2H), 4.17 (d, J=2.1 Hz, 2H), 2.32 (t, J=2.1 Hz, 1H).

(27) The obtained K49-c (3.4 g, 12.0 mmol) was dissolved in anhydrous ethanol (50 mL), glacial acetic acid (3.0 mL) and iron powder (1.3 g) were added thereto and the mixture was refluxed for 4 h. The mixture was filtrated to remove the unreacted iron powder. The filtrate was concentrated to nearly dryness and the residue was redissolved in ethyl acetate (70 mL) and transferred to a separating funnel. The mixture was washed with saturated aqueous sodium bicarbonate solution (20 mL) once, and with water (40 mL) twice. The organic phase was combined and washed with saturated saline (30 mL) once, dried with anhydrous sulfate, and concentrated to remove solvent. The residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=4:1, 3:1) to give K49-d (1.95 g, yield: 64%, brown solid). .sup.13C NMR (75 MHz, CDCl.sub.3): 162.3 (J=243.3 Hz), 142.4, 140.2, 134.6, 129.8 (J=8.0 Hz), 118.9, 116.5, 115.5 (J=21.1 Hz), 80.0, 73.0, 55.6, 41.4.

(28) K49-d (508 mg, 2.0 mmol) was dissolved in dichloromethane (10 mL), triethylamine (750 L, 5.2 mmol) was added, thiophosgene (300 L, 2.6 mmol) was dropwisely added under ice bath and the mixture was reacted at room temperature for 3 h. The mixture was concentrated to remove solvent, and the residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give K49-e (576 mg, yield: 96.0%, yellow oil). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.24 (dd, J=8.4, 5.4 Hz, 2H), 7.12 (d, J=9.3 Hz, 2H), 7.03 (t, J=8.7 Hz, 2H), 6.78 (d, J=9.3 Hz, 2H), 4.53 (s, 2H), 4.03 (d, J=2.1 Hz, 2H), 2.25 (t, J=2.1 Hz, 1H).

(29) K49-e (150 mg, 0.5 mmol) was dissolved in methanol (5 mL) and the mixture was refluxed overnight. The mixture was concentrated to remove solvent, and the obtained crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to give K49 (142 mg, yield: 87.0%, yellow oil). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.40 (brs, 1H) 7.24 (dd, J=8.4, 5.4 Hz, 2H), 7.11 (d, J=9.3 Hz, 2H), 7.02 (d, J=9.3 Hz, 2H), 6.82-6.85 (m, 2H), 4.50 (s, 2H), 4.10 (s, 3H), 3.93 (d, J=2.1 Hz, 2H), 2.24 (t, J=2.1 Hz, 1H).

Preparation Example 3.2 Synthesis of methyl 4-(N-parafluorobenzyl-N-propargyl-amino)-3-fluorophenylaminothioformate (K50)

(30) ##STR00035##

(31) K50 was prepared in the similar manner as that in Preparation Example 3.2 .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.25 (brs, 1H), 7.38 (dd, J=5.4, 8.4 Hz, 2H), 7.09-7.15 (m, 2H) 7.02 (t, J=9.3 Hz, 2H), 4.28 (s, 2H), 4.12 (brs, 3H), 3.93 (d, J=2.1 Hz, 2H), 2.27 (t, J=2.1 Hz, 1H).

(32) ##STR00036##

(33) K49-d (508 mg, 2.0 mmol) was dissolved in anhydrous toluene (10 mL), triethylamine (1.07 mL, 6.0 mmol) and triphosgene (356 mg, 1.2 mmol) were added thereto, and the mixture was reacted under refluxing for 3 h. The mixture was concentrated to remove solvent, and the obtained residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give K51-a (536 mg, yield: 96%, yellow solid).

(34) K51-a (84 mg, 0.3 mmol) and N,O-dimethylhydroxylamine hydrochloride (35 mg, 0.36 mmol) was dissolved in anhydrous toluene (5 mL), triethylamine (82 L, 0.6 mmol) was further added thereto and the mixture was reacted at room temperature overnight. The mixture was concentrated to remove solvent, and the obtained residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to give K51 (90 mg, yield: 88%, yellow oil). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.54 (brs, 1H), 7.32 (dd, J=8.4, 5.4 Hz, 2H), 7.24 (d, J=9.3 Hz, 2H), 7.01 (t, J=8.4 Hz, 2H), 6.89 (d, J=9.3 Hz, 2H), 4.45 (s, 2H), 3.94 (d, J=2.1 Hz, 2H), 3.75 (s, 3H), 3.17 (s, 3H), 2.21 (t, J=2.1 Hz, 1H).

Preparation Example 4

Preparation Example 4.1 Synthesis of N-[4-(N-p-fluorobenzyl-N-propargyl-amino)-phenyl]-2-methoxyacetamide (K52)

(35) ##STR00037##

(36) K49-d (100 mg, 0.4 mmol) and methoxyacetic acid (34 L, 0.44 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 92 mg, 0.48 mmol) were dissolved in anhydrous dichloromethane (5 mL), DIPEA (107 L, 0.6 mmol) was further added thereto and the mixture was reacted at room temperature for 4 h. After 20 mL of ethyl acetate was added, the reaction system was transferred to a separating funnel and washed with water (10 mL) twice. The organic phase was combined and washed with saturated saline (10 mL) once, dried with anhydrous sulfate, and concentrated to remove solvent. The obtained residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to give K52 (104 mg, yield: 80%). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.10 (s, 1H), 7.43 (d, J=9.0 Hz, 2H), 7.27 (dd, J=5.4, 8.4 Hz, 2H), 7.02 (t, J=8.7 Hz, 2H), 6.86 (d, J=9.0 Hz, 2H), 4.47 (s, 2H), 4.00 (s, 3H), 3.96 (d, J=2.1 Hz, 2H), 3.49 (s, 3H), 2.22 (t, J=2.1 Hz, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3): 167.4, 162.3 (J=243.6 Hz), 146.1, 134.1, 134.0, 129.2 (J=8.0 Hz), 121.7, 115.8 (J=21.6 Hz), 115.6, 79.6, 72.7, 59.5, 54.9, 40.5. HR-ESIMS (m/z): calculated for C.sub.19R.sub.20FN.sub.2O.sub.2 [M+H].sup.+327.1509. found: 327.1501.

Preparation Example 4.2, the Following Compounds were Prepared in the Similar Manner as that in Preparation Example 1 by Reacting with an Acid Corresponding to the Product, Starting from K49-d

(37) TABLE-US-00002 K53 8.15 (s, 1H), 7.43 (d, J = 9.3 Hz, 2H), 7.28 (dd, J = 8.4, 5.4 Hz, 2H), 7.02 (t, J = 8.7 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 4.47 (s, 2H), 4.04 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 3.64 (q, J = 6.9 Hz, 2H), 2.22 (t, J = 2.1 Hz, 1H), 1.30 (t, J = 6.9 Hz, 3H). K54 8.32 (s, 1H), 7.45 (d, J = 9.0 Hz, 2H), 7.29 (dd, J = 8.4, 5.4 Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.86 (d, J = 9.0 Hz, 2H), 4.46 (s, 2H), 3.96-4.03 (m, 5H), 2.32-2.38 (m, 1H), 2.14-2.23 (m, 2H), 1.90-1.95 (m, 2H). K55 0 8.92 (s, 1H), 7.46 (d, J = 9.0 Hz, 2H), 7.28 (dd, J = 8.7, 5.4 Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 4.46 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 3.05 (s, 2H), 2.36 (s, 6H), 2.22 (t, J = 2.1 Hz, 1H). K56 8.51 (s, 1H), 7.38 (d, J = 9.0 Hz, 2H), 7.26 (dd, J = 8.7, 5.4 Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 6.71 (s, 1H), 4.45 (s, 2H), 4.08 (d, J = 5.1 Hz, 2H), 3.95 (d, J = 2.1 Hz, 2H), 2.21 (t, J = 2.1 Hz, 1H), 2.01 (s, 3H). K57 8.95 (s, 1H), 7.42 (d, J = 9.0 Hz, 2H), 7.29 (dd, J = 8.4, 5.4 Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 4.47 (s, 2H), 3.97 (d, J = 2.1 Hz, 2H), 3.79 (s, 3H), 3.46 (s, 2H), 2.22 (t, J = 2.1 Hz, 1H). K58 7.37 (d, J = 9.3 Hz, 2H), 7.27 (dd, J = 8.4, 5.4 Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 4.46 (s, 2H), 3.96 (d, J = 2.1 Hz, 2H), 2.18-2.36 (m, 4H), 1.68-2.06 (m, 6H).

Preparation Example 5 Synthesis of methyl 4-(N-parafluorobenzyl-N-3,3-dideuteriumpropargyl-amino)phenylaminothioformate (K47)

(38) ##STR00044##

(39) K47-a (0.51 mL, 5.0 mmo) was dissolved in anhydrous tetrahydrofuran (THF) (10 mL), aluminium lithium deuteride solution (157.5 mg, 3.75 mmol) was slowly added thereto under dry ice-acetone bath, and the mixture was warmed to 40 C. and kept at such temperature for 5 h. The reaction was quenched by using 0.5 mL of methanol, and warmed to room temperature, and then quenched with aqueous ammonium chloride solution. The reaction system was extracted with ethylether (20 mL), and the obtained organic phase was washed with water (15 mL) twice, and with saturated saline (10 mL) once, dried with anhydrous sodium sulfate and carefully concentrated to about 1 mL. The obtained intermediate K47-b was directly used in the next step.

(40) The intermediate K47-b obtained above was dissolved in dichloromethane (10 mL), p-toluenesulfonyl chloride (1.15 g, 6 mmol) and triethylamine (0.82 mL, 6 mmol) were added under ice bath, and the mixture was kept at the temperature for 2 h. The reaction system was poured into crushed ice, and extracted with ethylether. The obtained organic phase was washed with water (20 mL) twice and concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to give an intermediate, 4-toluenesulfonate K47-c (80 mg, yield: 13%, yellow solid). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.82 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 2H), 2.45 (s, 3H), 2.04 (s, 1H).

(41) K1 was prepared according to the method of Preparation method 1 of WO2013060097. 4-toluenesulfonate K47-c (40 mg, 0.19 mmol) and K1 (52 mg, 0.19 mmol) were dissolved in DMF (3 mL), and then DIPEA (0.175 mL, 1.0 mmol) was added thereto. After the reaction was preformed at 65 C. for 4 h, ethyl acetate (20 mL) was dropwisely added into the reaction system. The mixture was transferred to a separating funnel and washed with water (15 mL) twice. The obtained organic phase was further washed with Saturated saline (10 mL) once, dried with anhydrous sodium sulfate and concentrated. The obtained K47 crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to give K47 (55 mg, yield: 92%, yellow oil). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.25-7.31 (m, 4H), 7.02 (t, J=8.7 Hz, 2H), 6.85 (d, J=8.7 Hz, 2H), 6.68 (s, 1H), 4.44 (s, 2H), 3.75 (s, 3H), 2.23 (s, 1H).

Preparation Example 6, Synthesis of methyl 4-(N-parafluorobenzyl-N-3-cyclopropyl propargyl-amino) phenylaminothioformate (K59)

(42) ##STR00045##

(43) Methyl aminocyclopropylcarboxylate (690 mg, 6 mmol), cesium carbonate (3.90 g, 12 mmol), 2,2-bis(diphenylphosphino)-1,1-binaphthalene (BINAP, 124 mg, 0.2 mmol), and p-bromonitrobenzene (1.2 g, 6 mmol) were dissolved in anhydrous toluene (50 mL). After the inside atmosphere of the reactor was completely replaced with argon, Bis(dibenzylideneacetone)palladium (Pd(dba).sub.2, 182 mg, 0.2 mmol) as a catalyst was quickly added, and the mixture was heated to reflux for 6 h. After the reaction mixture was cooled to room temperature, ethyl acetate (60 mL) was dropwisely added thereto. The obtained organic phase was washed with water (30 mL) twice, and with saturated saline (10 mL) once, dried with anhydrous sodium sulfate, and concentrated to remove solvent. The obtained crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give an intermediate K59-a (1.06 g, yield: 75%, brown solid). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.10 (d, J=8.7 Hz, 2H), 6.67 (d, J=8.7 Hz, 2H), 5.11 (brs, 1H), 3.74 (s, 3H), 1.68-1.72 (m, 2H), 1.20-1.24 (m, 2H).

(44) The intermediate K59-a (1.06 g, 4.5 mmol) was dissolved in DMF (20 mL), NaH (216 mg, 5.4 mmol) was quickly added thereto under ice bath and then the ice bath was removed and the mixture was reacted at room temperature for 1 h. Then p-fluorobenzyl bromide (0.622 mL, 5.0 mmol) was added, and the obtained mixture was reacted at 65 C. for 3 h. Ethyl acetate (40 mL) was added into the reaction system, and the obtained organic phase was washed with water (30 mL) twice, and with saturated saline (30 mL) once, dried with anhydrous sodium sulfate and concentrated to remove solvent. The obtained crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=12:1) to give K59-b (1.20 g, yield: 83%).

(45) The intermediate K59-b (340 mg, 1.0 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and diisobutylaluminum hydride (DIBAL-H, 1M solution in THF, 1.6 mL, 1.6 mmol) was dropwisely added thereto under dry ice-acetone bath, and the obtained mixture was kept at the temperature for 5 h. After that, 0.5 mL of methanol was added into the mixture to quench the reaction. After the mixture was warmed to room temperature, 20 mL of ethyl acetate was dropwisely added thereto. The obtained mixture was washed with 1M aqueous HCl solution (10 mL) once, with water (15 mL) twice and with saturated saline (10 mL) once, respectively. Then the organic phase was dried with anhydrous sulfate, and concentrated to remove solvent. The crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=6:1) to give an intermediate K59-c (177 mg, yield: 56%, oil). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.99 (d, J=9.3 Hz, 2H), 6.96-7.02 (m, 4H), 6.74 (d, J=9.7 Hz, 2H), 4.73-4.94 (m, 2H), 4.21 (brs, 1H), 3.48 (brs, 1H), 1.23-1.27 (m, 4H).

(46) The intermediate K59-c (177 mg, 0.56 mmol) was dissolved in dichloromethane (5 mL), and Dess-Martin periodinane (DMP, 367 mg, 0.84 mmol) was added thereto. After kept at room temperature for 4 h, the mixture was concentrated, and the residue was purified with silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give an intermediate K59-d (150 mg, yield: 85%, yellow solid). .sup.1H NMR (300 MHz, CDCl.sub.3): 9.11 (s, 1H), 8.05 (d, J=10.5 Hz, 2H), 6.99-7.11 (m, 4H), 6.68 (d, J=10.5 Hz, 2H), 4.87 (d, J=17.4 Hz, 1H), 4.65 (d, J=17.4 Hz, 1H), 1.95-2.00 (m, 1H), 1.51-1.63 (m, 3H).

(47) The intermediate K59-d (150 mg, 0.48 mmol) and anhydrous potassium carbonate (K.sub.2CO.sub.3) (132 mg, 0.96 mmol) were dissolved in methanol (5 mL), and Bestmann reagent (dimethyl diazomethylphosphonate, 110 mg, 0.58 mmol) was added thereto. After kept at room temperature overnight, the mixture was concentrated and the obtained residue was redissolved in ethyl acetate (20 mL). The obtained solution was washed with water (15 mL) twice, and with saturated saline (10 mL) once respectively, and dried with anhydrous sodium sulfate, and concentrated to remove solvent. The obtained crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=8:1) to obtain an intermediate K59-e (106 mg, yield: 71%, brown solid). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.10 (d, J=9.3 Hz, 2H), 7.09-7.12 (m, 2H), 6.98-7.04 (m, 2H), 6.92 (d, J=9.3 Hz, 2H), 4.75 (s, 2H), 2.19 (s, 1H), 1.21-1.25 (m, 4H).

(48) The intermediate K59-e (106 mg, 0.34 mmol) was dissolved in anhydrous ethanol (5 ml), glacial acetic acid (0.2 mL) and iron powder (60 mg) were added thereto, and the reaction system was refluxed for 3 h. The mixture was filtrated to remove unreacted iron powder, and the filtrate was sufficiently concentrated and the residue was redissolved in 20 mL of ethyl acetate. The obtained solution was washed with saturated aqueous sodium bicarbonate solution (10 mL) once, with water (15 mL) twice, and with saturated saline (10 mL) once respectively, and dried with anhydrous sodium sulfate. and concentrated to remove solvent. The obtained crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=6:1, 4:1) to obtain an intermediate K59-f (82 mg, yield: 86%, yellow solid). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.20 (dd, J=8.4, 5.4 Hz, 2H), 6.98 (t, J=8.4 Hz, 2H), 6.84 (d, J=9.3 Hz, 2H), 6.60 (d, J=9.3 Hz, 2H), 4.50 (s, 2H), 3.36 (brs, 2H), 2.14 (s, 1H), 1.18-1.20 (m, 2H), 1.04-1.07 (m, 2H).

(49) The intermediate K59-f (82 mg, 0.28 mmol) was dissolved in dichloromethane (5 mL), DIPEA (0.10 mL, 0.56 mmol) was added thereto, and methyl chloroformate (34 L, 0.44 mmol) was dropwisely added under ice bath. After addition, the mixture was kept at room temperature for half an hour. Into the above reaction system, 10 mL of ethyl acetate was added dropwisely, and the obtained organic phase was washed with water (10 mL) twice, and with saturated saline (10 mL) once, respectively, dried with anhydrous sodium sulfate, and then concentrated to remove solvent. The obtained crude was purified with silica gel column chromatography (petroleum ether/ethyl acetate=6:1) to give K59 (82 mg, yield: 91%, yellow oil). .sup.1H NMR (300 MHz, CDCl.sub.3): 7.15-7.21 (m, 4H), 6.98 (t, J=8.4 Hz, 2H), 6.88 (d, J=9.0 Hz, 2H), 6.38 (s, 1H), 4.59 (s, 2H), 3.74 (s, 3H), 2.13 (s, 1H), 1.26-1.29 (m, 2H), 1.10-1.14 (m, 2H). .sup.13C NMR (75 MHz, CD.sub.3OD): 162.1 (J=147.8 Hz), 155.2, 144.6, 135.9, 131.3, 128.3 (J=6.0 Hz), 120.2, 115.8, 114.9 (J=21.0 Hz), 84.8, 68.4, 55.9, 51.3, 42.6, 18.8. HR-ESIMS (m/z): calculated for C.sub.20H.sub.20FN.sub.2O.sub.2 [M+H].sup.+339.1509. found: 339.1505.

Preparation Example 7 Synthesis of methyl 4-(N-parafluorobenzyl-N-propargyl-amino)-2,6-dimethylphenylaminoformate hydrochloride (K43.HCl)

(50) ##STR00046##

(51) Methyl 4-(N-parafluorobenzyl-N-propargyl-amino)-2,6-dimethylphenylaminoformate (K43, 510 mg, 1.5 mmol) was dissolved in dichloromethane (5 mL), and a hydrogen chloride solution in ethyl acetate (5N, 1 mL) was added thereto. The mixture was stirred for 10 mins, and then concentrated to remove solvent to give methyl 4-(N-parafluorobenzyl-N-propargyl-amino)-2,6-dimethylphenylaminoformate hydrochloride (K43.HCl) (565 mg).

(52) The hydrochlorides of other compounds can be obtained by using a method similar to that of Preparation example 7.

II. Electrophysiological Experimental Examples

Electrophysiological Experimental Example 1: the Cell Lines Used in the Electrophysiological Experiment was Chinese Hamster Ovary Cell Line; KCNQ cDNA was Transformed into E. coli and Expressed in E. coli, and then Confirmed by Plasmid Extraction and Sequencing

(53) 1. Cell Culture and Transfection

(54) The culture medium for Chinese hamster oocytes (CHO-K1) (Culture Collection of Chinese Academy of Sciences): 50/50 DMEM/F-12 (Cellgo, Manassas, Va.), added with 10% fetal bovine serum (FBS) (Gibco Australia) and 2 mM L-glutamic acid (Invitrogen). Expression of KCNQ channels: 24 h before transfection, CHO-K1 spreaded on a dish with a diameter of 60 mm. Lipofectamine2000 agent (Invitrogen) was used for Transfection according to the protocol thereof. GFP (green fluorescent protein) was cotransfected to be used as a indication of successful converting into KCNQ plasmid.

(55) 2. Electrophysiological Recordings in CHO Cells

(56) Whole-cell voltage-clamp recording was performed on an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, Calif.) at room temperature. The electrode was made by drawing borosilicate glass capillary (World Precision Instruments, Sarasota, Fla.). The electric resistance of the electrode filled with an intracellular fluid is 3 to 5 M. The composition of the intracellular fluid (1 L) was as follows: 145 mM KCl (Sigma), 1 mM MgCl.sub.2(Sigma), 5 mM EGTA (Sigma), 10 mM HEPES(Sigma) and 5 mM MgATP (Sigma) (pH was adjusted to 7.3 with KOH). During the recording, an extracellular fluid was continuously perfused by using a BPS perfusion system (ALA Scientific Instruments, Westburg, N.Y.). The composition of the extracellular fluid (1 L) was as follows: 140 mM NaCl (Sigma), 5 mM KCl (Sigma), 2 mM CaCl.sub.2(Sigma), 1.5 mM MgCl.sub.2(Sigma), 10 mM HEPES(Sigma) and 10 mM glucose (Sigma) (pH was adjusted to 7.4 with NaOH). Electric signal was filtered at 1 kHz and further converted into digital signal by using pClamp 9.2 software (Molecular Devices, Sunnyvale, Calif.) in DigiData 1322A. Series resistors compensate 60 to 80%. Up to date, multivoltage scheme was generally adopted, wherein the clamp voltage was set at 80 mV, stimulating voltage was a gradient voltage from 90 mV to 60 mV with interval of 10 mV, and the stimulating time for each voltage was 2000 ms.

(57) 3. Experimental Results

(58) V.sub.1/2 is the voltage at which 50% of cells were activated, V.sub.1/2 is the shift amount of V.sub.1/2, negative sign() represents a left-ward shift of the current activation curve. I/I.sub.0 represents the current enhancement factor, wherein, I.sub.0 is the maximum induced current produced under stimulation of 10 mV testing voltage after administrating cells with blank extracellular fluid, I is the maximum induced current produced under stimulation of 10 mV testing voltage after administrating the drug (the compound concentration was 10 M), I/I.sub.0>1 represents activating activity, and I/I.sub.0<1 represents inhibiting activity. N is the number of tested cells. NT represents non-tested.

(59) TABLE-US-00003 Compound V.sub.1/2(mV) I/I.sub.0 N K40HCl 3.42 1.54 1.40 0.04 3 K41HCl 23.28 1.37 4.8 0.73 3 K42HCl 5.2 0.7 0.52 0.01 3 K43HCl 38.52 1.67 4.75 1.29 3 K44HCl 13.47 2.13 0.72 0.05 3 K45HCl 5.1 0.9 0.57 0.01 3 K46HCl 6.5 2.1 1.52 0.55 3 K47HCl 18.10 0.03 2.10 0.08 3 K48HCl 13.2 1.5 1.24 0.16 3 K49HCl 22 2.5 4.4 1.6 3 K50HCl 9.06 0.15 1.39 0.12 3 K51HCl 8.89 1.12 1.61 0.08 5 K52HCl 10.93 1.86 5.80 0.52 5 K53HCl 14.81 0.92 7.31 1.09 5 K54HCl 30.9 4.1 7.55 1.92 5 K55HCl 10.76 4.51 1.72 0.14 3 K56HCl 2.0 1.3 1.10 0.10 3 K57HCl 31.55 0.86 8.13 2.57 3 K58HCl 33.68 1.05 1.78 0.19 3 K59HCl 0.14 2.33 1.01 0.13 3

(60) Results and discussion: from the above electrophysiological Experimental results, it can be seen that the compounds disclosed in present invention not only well retains the agonistic activity on KCNQ potassium channel, but also some compounds according to present invention have a significantly improved current enhancement factor (I/I.sub.0) than that of K21 disclosed in WO2013060097 (I/I.sub.0=1.530.15).

Electrophysiological Experimental Example 2: Comparison of Agonistic Activity of K43 and RTG on KCNQ2 Homotetramer Channel

(61) The experimental procedure was the same as that in Electrophysiological Example 1. The assay for KCNQ2 channel dose-response curve (DRC) was performed on CHO-K1 cells transfected with KCNQ2 plasmid; and the assay for KCNQ2/3 heterotetramer channel DRC was performed on CHO-K1 cells cotransfected with KCNQ2 and KCNQ3 plasmids. Dose-response curve was fitted by using Boltzmann equation (Boltzmann sigmoidal), and the results were shown in FIG. 1.

(62) Results and discussion: in FIG. 1, EC.sub.50=1.53 nM (K43), EC.sub.50=1.32 M (RTG). From the comparison results of DRCs in FIG. 1, it can be seen that the agonistic activity of K43 on KCNQ2 homotetramer channel was more than 800 times of that of RTG, and the agonistic activity of K43 was much higher than that of RTG.

Electrophysiological Experiment Example 3: Comparison of Agonistic Activity of K43, CF341 (i.e., K21 Disclosed in WO2013060097) and RTG on KCNQ2/3 Heterotetramer Channel

(63) The experimental procedure was the same as that in Electrophysiological Example 1. KCNQ2/3 heterotetramer channel is based on 4 ng plasmid per 10 L Lipo2000. KCNQ2 and KCNQ3 plasmid were cotransfected at a mass ratio of 1:1. 24 h after transfection, CKO-K1 cells were lysed with trypsin(Sigma, China) and re-spreaded on a dish with a diameter of 60 mm which was laid with poly-L-lysine (Sigma)-immersed slides thereon. The assay for KCNQ2/3 heterotetramer channel DRC was performed on CHO-K1 cells cotransfected with KCNQ2 and KCNQ3 plasmids. DRC was fitted by using Boltzmann equation (Boltzmann sigmoidal), and results were shown in FIG. 2.

(64) Results and discussion: in FIG. 2, EC.sub.50=49 nM (K43), EC.sub.50=1.9 M (K21). From the comparison of DRCs in FIG. 2, it can be seen that the agonistic activity of K43 on KCNQ2/3 heterotetramer channel (the main mediate channel for in vivo M current) was 20 times or more and 30 times or more of that K21 (EC.sub.50=990 nM) and RTG (it is reported that EC.sub.50=1.6 M, Sanker R, et al., Epilepsia, 2012, 53, 412-424), respectively, and the agonistic activity of K43 on KCNQ2/3 heterotetramer channel was also much higher than those of K21 and RTG.

III. Examples for Evaluation on Pharmacodynamical Effects of Compounds In Vivo

In Vivo Pharmacodynamical Effects Example 1: Preventive and Therapeutic Effects of Compounds K41.HCl and K43.HCl Administered by Oral Perfusion on Animal Model Induced by MES (Maximum Electroshock)

(65) YLS-9A model physiological pharmaceutical electronic stimulator was used in the experiment to induce convulsion of mice, and specific parameters were set as follows: configuration 8, stimulating voltage: 160V, period of stimulation: 5.4 sec. Healthy KM mice (SPF level, male, body weight: 18 to 22 g) were selected for the experiment. After the ear tip of the mice was sufficiently wetted with physiological saline, the mice was electrically stimulated once with ear clip electrode, and tonic hind-limb seizure was deemed as indication of convulsion. The mice were screened one day before the experiment to weed out the dead mice and the ones without generalized tonic seizure. The qualified mice were caged randomly with access to water at liberty. Before the experiment started, the mice were fasted for 8 h.

(66) The compounds to be tested were freshly formulated on the day of experiment. RTG hydrochloride was dissolved with ultrapure water to obtain a solution with desired concentration. K41.HCl and K43.HCl were formulated with 5% DMSO+95% of 1% Tween80, that is, a proper amount of the compound to be tested was weighted and, first, sufficiently dissolved in 5% DMSO, and then added with a desire volume of 1% Tween80 to be sufficiently suspended to form a suspension with a certain drug concentration. The mice screened one day before were randomly grouped with each group having 10 mice, marked, weighted and then administered with the compound to be tested or solvent (5% DMSO+95%(1% Tween 80)) by oral perfusion with administered volume of 0.2 ml/10 g. The dose range for each compound was 1 to 56 mg/kg for RTG, 1 to 40 mg/kg for K41.HCl, and 0.5 to 4 mg/kg for K43.HCl. 30 mins after administration, MES experiment was performed and the experimental parameters were the same as the above. The number of mice with generalized tonic-clonic convulsion in each group was recorded, the protection rate of each of the compounds to be tested on convulsive mice induce by MES was calculated and dose-response curve for each compound was plotted. The dose-response curve for each compound was obtained according to the analysis of Graphpad Prism 5 software, as shown in FIG. 3.

(67) Results and discussion: the MES experimental results showed that each of the orally administered RTG hydrochloride, K41.HCl and K43.HCl shows a dose dependent protective effect on convulsive mice induce by MES. ED.sub.50 (50% effective dose) of each of the compound was 21.80 mg/kg for RTG hydrochloride with a 95% CI (95% confidence interval) of: 19.03 to 24.97 mg/kg; 4.80 mg/kg for K41.HCl with a 95% CI of 3.42 to 6.74 mg/kg; and 1.60 mg/kg for K43.HCl with a 95% CI of 1.35 to 1.88 mg/kg. The efficacies of K43 and K31 against MES were higher than that of RTG.

IV. Examples for Evaluating TD50 of Compound

Rotarod Experimental Example 1: Influence of K41.HCl and K43.HCl Administered by Oral Perfusion on Motor Coordination Ability of Mice

(68) YLS-4C Rotarod system was used in the experiment, wherein the diameter of the rod was 3 cm and the rotate speed was set at 6 rpm. Healthy KM mice (SPF level) were selected, wherein male and female were equal, the body weight was 18 to 22 g. One day before experiment, the mice were place on the rotarod for training and screening. The tail tip of mice was held to make them creep on the rotarod during the training. After creeping for a while, the tail tip of mice was gradually relaxed, and completely released when they did not rely on the tail to balance their body. The mice that jumped on the rotarod or griped the rotarod were weed out. In addition, three time periods were set with 1 min for each. The mice that did not fall off in all of the three time periods were qualified for testing. The qualified mice were randomly caged according to the gender, and allowed to eat and drink freely.

(69) On the day of the experiment, each compound to be tested was freshly formulated by the same method as described in MES experiment. If the compound was not sufficiently suspended, the suspension was further mixed by ultrasonication for 20 min so as to make the compound sufficiently suspended. The qualified mice screened one day before were randomly grouped with each group having 10 mice, marked, weighted and then administered with the compound to be tested or solvent (5% DMSO+95%(1% Tween 80)) by oral perfusion with a administered volume of 0.2 ml/10 g. The dose range for each compound was 30 to 150 mg/kg for RTG, 30 to 180 mg/kg for K41.HCl, and 90 to 210 mg/kg for K43.HCl. 30 mins after administration, Rotarod experiment was performed and the experiment parameters were the same as the above. The number of mice that fell off from the rorarod was recorded, and the influence of each compound on motor coordination ability of mice was analyzed. The curve for dose of each compound verse motor deficit (percentage) of mice for each compound was obtained according to the analysis of Graphpad Prism 5 software and was shown in FIG. 4.

(70) Results and discussion: TD.sub.50 (50% acute neurotoxicity dose) of each compound obtained from the Rotarod experiment was 74.21 mg/kg for RTG with a 95% CI of 69.65 to 79.07 mg/kg; 103.40 mg/kg for K41.HCl with a 95% CI of 72.62 to 147.20 mg/kg; and 152.40 mg/kg for K43.HCl with a 95% CI of 104.30 to 222.60 mg/kg. P.I values of RTG, K41.HCl and K43.HCl in MES experiment, which were calculated according to the following equation: P.I (Protective index)=TD.sub.50/ED.sub.50, were 3.40, 21.54 and 95.25, respectively, indicating that K43 and K41 has a weaker neurotoxicity than RTG, thus possess a wider safety window.

V. Pharmacokinetical Examples

Pharmacokinetical Example 1, Research on Distribution of the Compounds K41.HCl and K43.HCl in Mice's Brain Tissue

(71) Formulation of the compound: K41.HCl was formulated to a 0.5 mg/ml solution for intragastric perfusion by using 16% DMSO/20% Tween 80/64% physiological saline; to a 0.2 mg/ml solution for intravenous injection by diluting the above solution for intragastric perfusion with physical saline containing 1% Tween 80. K43.HCl was formulated to a 0.5 mg/ml solution for intragastric perfusion by using 5% DMSO/5% Tween80/80% physiological saline; to a 0.2 mg/ml solution for intravenous injection by diluting the above solution for intragastric perfusion with physical saline containing 1% Tween 80.

(72) Experimental Design

(73) 84 healthy ICR mice (male, body weight, 18-20 g) were fasted for 8 h with access to water at liberty before the experiment. The mice were fed at the same time 2 h after administration of the compound. Specific arrangement was shown in the following table.

(74) TABLE-US-00004 Number Administration Administration of Administration dose volume Group animals Compound manner (mg/kg) (ml/kg) Sampling time(h) 1 27 K41HCl Intragastric 5 10 0.25, 0.5, 1, 2, 3, 4, perfusion 6, 8 and 24 h 2 15 K41HCl intravenous 2 10 5 min, 0.25, 0.5, 1, injection 2, 4, 6, 8 and 24 h 3 27 K43HCl Intragastric 5 10 0.25, 0.5, 1, 2, 3, 4, perfusion 6, 8 and 24 h 4 15 K43HCl Intravenous 2 10 5 min, 0.25, 0.5, 1, injection 2, 4, 6, 8 and 24 h

(75) Sample Collection

(76) After administration by intragastric perfusion, mice were sacrificed by cutting abdominal aorta at the time points set as the above with 3 mice for each time point. 0.5 mL of whole blood was collected for each animal, placed into a heparinized test tube and subjected to centrifugation at 11000 rpm for 5 min to separate plasma, which was then cryopreserved in a 20 C. refrigerator. After the animal was sacrificed, its whole brain was collected, washed with ice-cooled physiological saline to remove residual blood, absorbed to dryness, labeled and cryopreserved in a 20 C. refrigerator. After administration by intravenous injection, 0.5 mL of venous blood was collected from venous plexus behind eyeball, placed into a heparinized test tube and subjected to centrifugation at 11000 rpm for 5 min to separate plasma, which was then cryopreserved in a 20 C. refrigerator. The concentration of each compound in plasma and brain tissue was determined by LC-MS/MS.

(77) Experimental Results

(78) Pharmacokinetic parameters after administration were calculated according to the obtained data of plasma concentration, using the non-compartmental model of Phoenix 1.3 software (Pharsight Co., USA).

(79) Pharmacokinetic parameters after administrating K41.HCl by intragastric perfusion at 5 mg/kg and intravenous infection at 2 mg/kg

(80) TABLE-US-00005 Intragastric Intravenous perfusion injection 5 mg/kg 2 mg/kg Parameters Unit Plasma Brain Plasma T.sub.1/2 (h) 0.64 0.63 0.49 T.sub.max (h) 0.25 0.25 / C.sub.max (ng/ml or ng/g) 606 1467 / AUC.sub.0-t (ng .Math. h/ml or ng .Math. h/g) 646 1680 281 AUC.sub.0- (ng .Math. h/ml or ng .Math. h/g) 654 1684 294 MRT.sub.0- (h) 1.04 1.10 0.45 CL (L/h/kg) / / 6.81 Vss (L/kg) / / 3.04 F (%) 91.9 / / AUC.sub.0-t 2.6 ratio(brain/plasma)

(81) Pharmacokinetic parameters after administrating K43.HCl by intragastric perfusion at 5 mg/kg and intravenous infection at 2 mg/kg

(82) TABLE-US-00006 Intragastric intravenous perfusion 5 injection mg/kg 2 mg/kg Parameters Unit Plasma Brain Plasma T.sub.1/2 (h) 0.62 1.06 0.40 T.sub.max (h) 0.25 0.25 / C.sub.max (ng/ml or ng/g) 350 1429 / AUC.sub.0-t (ng .Math. h/ml or ng .Math. h/g) 184 1329 326 AUC.sub.0- (ng .Math. h/ml or ng .Math. h/g) 189 1346 334 MRT.sub.0- (h) 0.94 1.29 0.43 CL (L/h/kg) / / 6.00 Vss (L/kg) / / 2.61 F (%) 22.5 / / AUC.sub.0-t 7.2 ratio(brain/plasma)

(83) Experimental Conclusion

(84) After mice were administered with K41.HCl by intragastric perfusion at 5 mg/kg, the time for achieving the maximum concentrations (T.sub.max) of K41.HCl in plasma and brain tissue was 0.25 h; the concentration of K41.HCl in brain tissue was 2.6 time of that in plasma. Oral bioavailability of K41.HCl in ICR mice was 91.9%.

(85) After mice were administered with K43.HCl by intragastric perfusion at 5 mg/kg, the time for achieving the maximum concentrations (T.sub.max) of K43.HCl in plasma and brain tissue was 0.25 h; the concentration of K43.HCl in brain tissue was 7.2 time of that in plasma. Oral bioavailability of K43.HCl in ICR mice was 22.5%.

(86) It is reported in WO2013060097 that, under the same experimental conditions, the exposure amount of RTG in mice's brain was only 16% of that in plasma when RTG was administered by intragastric perfusion at 20 mg/kg, and the exposure amount of RTG was only 14% of that in plasma when RTG was administered by intravenous injection at 20 mg/kg. Thus, from the results of the in vivo pharmacokinetic experiment in mice, it can be seen that K41.HCl and K43.HCl have a better concentration distribution in brain tissue than RTG, and possess an equal or even higher distribution coefficient than K21 disclosed in WO2013060097 in brain tissue of mice.

Pharmacokinetical Example 2: Research on Distribution of K43 in Rats' Brain Tissue after Administration Via Intravenous Injection

(87) Experimental Purpose:

(88) After K43 was administered to Sprague Dawley rats via intravenous injection ororal administration, blood sample and brain tissue were collected at different time points. the concentration of K43 in plasma and brain of rats after administration of the compounds was analyzed by LC-MS/MS and used to calculate the pharmacokinetic parameters, evaluating the oral bioavailability and distribution in brain tissue of K43 in rats.

(89) Experimental Design

(90) 24 SD rats, which were provided by SHANGHAI SLAC LABORATORY ANIMAL CO. LTD., were used to perform the experiments according to the following table.

(91) TABLE-US-00007 Administration information Number of Administration animals Administration concentration* Administration Sampling Administration Group Male Compound dose (mg/kg) (mg/mL) volume (mL/kg) manner manner 1 3 K43 0.500 0.500 1.00 Plasma IV 2** 3 K43 5.00 0.500 10.0 Plasma PO 3** 3 K43 5.00 0.500 10.0 Brain PO 4** 3 K43 5.00 0.500 10.0 Brain PO 5** 3 K43 5.00 0.500 10.0 Brain PO 6** 3 K43 5.00 0.500 10.0 Brain PO 7** 3 K43 5.00 0.500 10.0 Brain PO 8** 3 K43 5.00 0.500 10.0 Brain PO *The concentration of the compound was calculated according to the free radicals. **The second group was for collecting brain tissue at 24 h; the third group to the eighth group were for collecting brain tissue at the time point of 0.25, 0.5, 1, 2, 4, 6 hr, respectively.

(92) Sample Collection:

(93) About 0.15 mL of blood was collected through orbit per animal every time, and EDTAK2 was used for anticoagulation. For IV group, the time points for collection were before administration (0 hr), and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after administration; and for PO group, the time points for collection were before administration (0 hr), 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h after administration. The time points for collecting brain tissue were 0.25, 0.5, 1, 2, 4, 6 and 24 h. The collected blood samples were placed on icen, and plasma was centrifuged within 1 h (centrifugal condition: 12000 rpm, 2 min, 4 C.). The collected plasma was cryopreserved in a 20 C. refrigerator before analysis.

(94) Experimental Results:

(95) Pharmacokinetic parameters after administration were calculated according to the obtained data of plasma concentration using the non-compartmental model of WinNonlin V6.3.

(96) Main pharmacokinetic parameters after intravenously administrating K43 (intravenous injection (IV), 0.500 mg/kg) to SD rats

(97) TABLE-US-00008 PK parameters IV-1 IV-2 IV-3 Mean SD RSD (%) Dose mg .Math. kg.sup.1 0.500 K.sub.cl h.sup.1 0.552 0.427 0.516 0.498 0.0641 13 t.sub.1/2 h 1.26 1.62 1.34 1.41 0.191 14 AUC.sub.0-t h .Math. ng .Math. mL.sup.1 219 232 239 230 10.4 4.5 AUC.sub.0-inf h .Math. ng .Math. mL.sup.1 221 236 242 233 10.9 4.7 AUMC.sub.0-t h .Math. h .Math. ng .Math. mL.sup.1 287 258 270 271 14.6 5.4 AUMC.sub.0-inf h .Math. h .Math. ng .Math. mL.sup.1 304 295 293 297 6.17 2.1 CL mL .Math. kg.sup.1 .Math. min.sup.1 37.8 35.3 34.5 35.8 1.70 4.8 MRT.sub.IV h 1.38 1.25 1.21 1.28 0.0879 6.9 Vd.sub.SS L .Math. kg.sup.1 3.13 2.65 2.51 2.76 0.324 12

(98) Main pharmacokinetic parameters after administrating K43 (oral administration, PO, 5.00 mg/kg) to SD rats

(99) TABLE-US-00009 PK parameters PO-4 PO-5 PO-6 Mean SD RSD (%) Dose mg .Math. kg.sup.1 5.00 K.sub.cl h.sup.1 0.186 0.0857 0.371 0.214 0.145 68 t.sub.1/2 h 3.74 8.09 1.87 4.56 3.19 70 t.sub.max h 2.00 4.00 1.00 2.33 1.53 65 C.sub.max ng .Math. mL.sup.1 62.7 138 107 103 37.8 37 AUC.sub.0-t h .Math. ng .Math. mL.sup.1 467 1379 379 742 554 75 AUC.sub.0-inf h .Math. ng .Math. mL.sup.1 471 1605 407 828 674 81 AUMC.sub.0-t h .Math. h .Math. ng .Math. mL.sup.1 2360 10030 957 4449 4884 110 AUMC.sub.0-inf h .Math. h .Math. ng .Math. mL.sup.1 2497 18103 1256 7285 9389 129 MRT.sub.PO h 5.30 11.3 3.09 6.55 4.236 65 F % 20.3 59.9 16.5 32.2 24.1 75

(100) Main pharmacokinetic parameters in brain tissue after administrating K43 (oral administration, PO, 5.00 mg/kg) to SD rats.

(101) TABLE-US-00010 PK parameters Brain Dose mg .Math. kg.sup.1 5.00 K.sub.cl h.sup.1 0.0542 t.sub.1/2 h 12.8 t.sub.max h 2.00 C.sub.max ng .Math. g.sup.1 366 AUC.sub.0-t h .Math. ng .Math. g.sup.1 2895 AUC.sub.0-inf h .Math. ng .Math. g.sup.1 4314 AUMC.sub.0-t h .Math. h .Math. ng .Math. g.sup.1 25306 AUMC.sub.0-inf h .Math. h .Math. ng .Math. g.sup.1 85535 MRT.sub.PO h 19.8

(102) Experimental Results:

(103) The results of research on pharmacokinetics and distribution in brain tissue of K43 in rats showed that the half life in rats after intravenous injection (IV) (dose: 0.5 mg/kg) was 1.410.191 hr, clearance (CL) was 35.81.70 mL/kg/min, and Vss was 2.760.324 L/kg.

(104) The average time for achieving the maximum plasma concentration in rats after oral administration (PO) K43 (dose: 5.00 mg/kg) was 2.331.53 hr, the maximum plasma concentration was 10337.8 ng/mL, AUC.sub.0.fwdarw.24 hr was 742554 hr*ng/mL, and the oral bioavailability of K43 in SD rats was 32.224.1%.

(105) The average time for achieving the maximum concentration in brain tissue of rats after oral administration (PO) K43 (dose: 5.00 mg/kg) was 2.00 hr, the average maximum concentration was 366 ng/g, AUC.sub.0.fwdarw.24 hr was 2895 hr*ng/g, AUC.sub.0.fwdarw.24 hr of K43 in brain tissue of SD rats is 3.9 times of that in plasma.