Thiazole inner salt compounds, and preparation methods and uses thereof

Abstract

The present invention pertains to field of pharmaceutical chemicals, and relates to thiazole inner salt compounds, preparation methods and uses thereof. Specifically, the present invention relates to a compound of Formula I, hydrates or pharmaceutically acceptable salts thereof. The compound of Formula I of the present invention is a potent cross-linking protein cleavage agent, has a stable structure, good physical and chemical properties, and good pharmacological activities, and is suitable for large scale production to obtain samples with stable, controllable and reliable quality, thereby being suitable for pharmaceutical development. ##STR00001##

Claims

1. A method for preparing the compound of Formula I, comprising the following steps: Compound A is reacted with 1,2-epoxypropane to obtain Compound B, ##STR00009## wherein, in the structure of Compound A, X is chlorine, bromine or iodine; ##STR00010## wherein: n is 0, 1, 2 or 3; wherein when n is 0 the compound of Formula I is Compound B; and wherein when n is 1, 2, or 3 the compound of Formula I is prepared from Compound B through the following steps: Compound B is dissolved in methanol followed by dropwise addition of ethyl acetate and the mixture is left to stand to obtain monocrystals of the compound of Formula I.

2. The method according to claim 1, wherein Compound A is prepared via the following steps: 4-methylthiazole is reacted with chloroacetic acid, bromoacetic acid or iodoacetic acid to obtain Compound A, ##STR00011##

3. The method according to claim 2, wherein Compound A is separated and purified via recrystallization.

4. A method for preparing monocrystals of the compound of Formula I, comprising the following steps: 3-methylcarbonyloxy-4-methyl-thiazole inner salt is dissolved in methanol followed by dropwise addition of ethyl acetate and the mixture is left to stand to obtain monocrystals; ##STR00012## wherein: n is 0, 1, 2 or 3.

5. The method according to claim 3, wherein the solvent used for recrystallization is any one independently selected from the group consisting of acetone, methanol, ethanol, ethyl ether, petroleum ether, and n-hexane, or any mixture thereof.

6. The method according to claim 4, wherein for 1 mg of 3-methylcarbonyloxy-4-methyl-thiazole inner salt, 0.05 mL methanol and 0.3 mL ethyl acetate are used.

7. The method according to claim 1, wherein Compound B is separated and purified via recrystallization.

8. The method according to claim 7, wherein the solvent used for recrystallization is any one independently selected from the group consisting of acetone, methanol, ethanol, ethyl ether, petroleum ether, and n-hexane, or any mixture thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: comparison of appearances of the compound of Formula A (M=Na, K═Br) before and after three months of storage, in which FIG. 1A is of the newly prepared compound of Formula A; FIG. 1B is of the compound of Formula A after three months of storage (temperature: 40° C., humidity: 75%, atmospheric pressure).

(2) FIG. 2: the X-single crystal diffraction structure diagram of 3-methylcarbonyloxy-4-methyl-thiazole inner salt monohydrate (n=1).

(3) FIG. 3: the molecular crystal packing diagram of 3-methylcarbonyloxy-4-methyl-thiazole inner salt monohydrate (n=1).

(4) FIG. 4: the X-ray powder diffraction diagram of 3-methylcarbonyloxy-4-methyl-thiazole inner salt (n=0).

(5) FIG. 5: the X-ray powder diffraction diagram of 3-methylcarbonyloxy-4-methyl-thiazole inner salt monohydrate (n=1).

(6) FIG. 6: systolic pressure dynamic curves of different groups during time period 10:00-20:00 (mean±standard deviation, n=12).

(7) FIG. 7: systolic pressure variable coefficients of different groups during time period 10:00-20:00 (mean±standard deviation, n=12.*P<0.05, .sup.#P<0.01 vs. normal group; **P<0.01 vs. model).

(8) FIG. 8: diastolic pressure dynamic curves of different groups during time period 10:00-20:00.

(9) FIG. 9: pulse pressure dynamic curves of different groups during time period 10:00-20:00.

(10) FIG. 10: heart rate dynamic curves of different groups during time period 10:00-20:00.

(11) FIG. 11: systolic pressure dynamic curves of different groups during time period 10:00-20:00 (M mean±standard deviation, n=12. .sup.##P<0.01 vs. model group; **P<0.01 vs. Nifedipine group).

(12) FIG. 12: systolic pressure drop scope dynamic curves of different groups during time period 10:00-20:00 (mean±standard deviation, n=12. .sup.##P<0.01 vs. model group; *P<0.05, **P<0.01 vs. Nifedipine group).

(13) FIG. 13: systolic pressure variable coefficient dynamic curves of different groups during time period 11:00-15:00 (mean±standard deviation, n=12. **P<0.01 vs. model group).

(14) FIG. 14: diastolic pressure dynamic curves of different groups during time period 10:00-20:00 (mean±standard deviation, n=12. .sup.##P<0.01 vs. model group; *P<0.05, **P<0.01 vs. Nifedipine group).

(15) FIG. 15: pulse pressure dynamic curves of different groups during time period 10:00-20:00 (mean±standard deviation, n=12. **P<0.01 vs. Nifedipine group).

(16) FIG. 16: heart rate dynamic curves of different groups during time period 10:00-20:00.

(17) FIG. 17: ejection time dynamic curves of different groups during time period 10:00-20:00 (mean±standard deviation, n=12. *P<0.05 vs. Nifedipine group).

(18) FIG. 18: muscular oxygen uptake index dynamic curves of different groups during time period 10:00-20:00 (mean±standard deviation, n=12. *P<0.05 vs. Nifedipine group).

(19) FIG. 19: plasma 6-ketone-prostaglandin content of different groups (mean±standard deviation, n=9-11. *P<0.05 vs. normal group; **P<0.01 vs. model group; ##P<0.05 vs. Nifedipine group).

(20) FIG. 20: plasma TXB.sub.2 content of different groups (mean±standard deviation, n=9-11. *P<0.01 vs. normal group; **P<0.01 vs. model group).

(21) FIG. 21: TXB.sub.2/6-Keto-PGI1a values of different therapeutic groups (mean±standard deviation, n=9-11. *P<0.01 vs. normal group; **P<0.01 vs. model group; .sup.$$P<0.05 vs. Nifedipine group).

(22) FIG. 22: plasma MCP-1 contents of different therapeutic groups (mean±standard deviation, n=9-11. *P<0.05 vs. normal group; .sup.##p<0.01 vs. model group).

(23) FIG. 23: plasma BNP content of different therapeutic groups (mean±standard deviation, n=9-11. *P<0.05 vs. normal group; .sup.ΔΔp<0.05, **p<0.01 vs. model group; .sup.##p<0.01 vs. Nifedipine group).

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

(24) The embodiments of the present invention are described in details in conjunction with the following examples, but those skilled in the art would understand the examples are used for illustrating the present invention only, rather than for limiting the scope of the present invention. Any specific conditions that were not given in the examples are conventional conditions or conditions suggested by the manufacturers. The reagents or instruments which manufacturers were not given were all conventional products commercially available in markets.

(25) Melting points of compounds were measured with SRY-1 type melting point apparatus, which was not subjected to temperature calibration. 1H-NMR and 13C-NMR spectra were measured with BrukerARX400 type nuclear magnetic resonance spectrometer; mass spectra were measured with API-150EX LC/MS high resolution mass spectrometer; X-ray single crystal diffraction was measured with Rigaku Saturn944 CCD diffractometer; X-ray powder diffraction was measured with Bruker D8 Advance diffractometer.

Example 1: Preparation of 3-carboxymethyl-4-methyl-thiazolium bromide (Compound A)

(26) 15.6 g 4-methylthiazole was dissolved in 50 mL anhydrous acetone, added with 21 g bromoacetic acid, stirred for 3 h, filtered to obtain a solid, recrystallized with ethanol to obtain a white solid, dried to obtain 26 g of product, yield 72%, mP=240.6-241.6° C.

(27) MS[M].sup.+=158.2 m/e; .sup.1H-NMR (400 MHz, DMSO-d.sub.6), 2.48 (d, 3H); 5.55 (s, 2H); 8.09 (d, 1H); 10.25 (d, 1H); 14.05 (brs, H).

Example 2: Preparation of 3-methylcarbonyloxy-4-methyl-thiazole Inner Salt (n=0)

(28) 10 g 3-carboxymethyl-4-methyl-thiazolium bromide white solid was dissolved in 50 mL distilled water, added then with 7.31 g 1,2-epoxypropane, stirred at room temperature for 12 h, after the end of reaction, the reaction solution was extracted with 30 mL of dichloromethane, for 3 times, dichloromethane layer was discarded; the water layer was evaporated at a reduced pressure to obtain a light yellow oily substance. A defined amount of acetone was added into the oily substance, and light yellow particles were obtained via precipitation; recrystallization was performed with ethanol-ethyl ether system (wherein the most preferable recrystallization ratio was: 1 g of yellow particles was heated and dissolved in 4.5 mL ethanol, then added with 2 mL ethyl ether), to obtain white crystal 5.15 g, yield 78%, mP=169° C.

(29) MS: 158 [M+H].sup.+, 315 [2M+H].sup.+, 472 [3M+H].sup.+; .sup.1H-NMR (400 MHz, DMSO-d.sub.6), 2.41 (d, 3H), 4.76 (s, 2H), 7.90 (d, 1H), 9.97 (d, 1H); .sup.13C-NMR (Methanol-d.sub.4), δ 12.98, 56.71, 121.47, 148.36, 169.71;

(30) Elemental analysis: Anal. Calcd for C.sub.6H.sub.7NO.sub.2S (157.2): C, 45.85; H, 4.49; N, 8.91%

(31) Found: C, 45.74; H, 4.51; N, 8.87%.

(32) The crystal structure was measured with X-single crystal diffraction.

(33) It was a crystal form compound 3-methylcarbonyloxy-4-methyl-thiazole inner salt (n=0), and its X-ray powder diffraction spectra showed characteristic diffraction peaks at 12.6, 13.3, 14.9, 18.5, 19.1, 27.0, 27.7, 28.8, 29.8, 32.1, 40.8, 42.8, 45.2, 47.9, 52.6, 54.8, 55.6, 59.0 (2θ/° C.) (see details in FIG. 4).

Example 3: Preparation of 3-methylcarbonyloxy-4-methyl-thiazole Inner Salt Monohydrate (n=1)

(34) 2 g of the 3-methylcarbonyloxy-4-methyl-thiazole inner salt (n=0) as prepared in Example 2 was dissolved at 20° C. in a mixture solvent of 100 mL methanol and 1 mL water, after complete dissolution, 300 mL ethyl acetate solution was added slowly; after mixing homogeneously, standing was performed at 5° C. for 12 h, the precipitated crystal was 3-methylcarbonyloxy-4-methyl-thiazole inner salt monohydrate (n=1).

(35) Test of crystal structure determination with X-ray single crystal diffraction:

(36) 2 mg of 3-methylcarbonyloxy-4-methyl-thiazole inner salt white crystal was added with 0.1 mL of anhydrous methanol, after the particles were dissolved, 0.6 mL ethyl acetate was added drop-wisely, standing was carried out until crystal particles grew slowly to form monocrystals (3-methylcarbonyloxy-4-methyl-thiazole inner salt monohydrate, n=1). Crystal structure was determined with X-single crystal diffraction.

(37) It was a crystal form of the compound 3-methylcarbonyloxy-4-methyl-thiazole inner salt monohydrate (n=1), and its X-ray powder diffraction spectra showed characteristic diffraction peaks at 11.8, 15.2, 16.7, 18.9, 19.3, 19.8, 21.0, 23.8, 24.5, 25.2, 26.4, 26.9, 28.6, 29.3, 31.3, 31.9, 32.1, 34.1, 34.7, 35.0, 35.6, 38.9, 40.1, 40.6, 43.1, 45.9, 46.7, 48.1, 49.0 (2θ/° C.) (see details in FIG. 5).

(38) Crystallographic data: C.sub.6H.sub.7NO.sub.2S.H.sub.2O, Mr=175.20, orthorhombic system, space group P-1, crystallographic parameters: a=5.6082(11) Å, alpha=90 deg., b=8.4615(17) Å, beta=90 deg., c=16.064(3) Å, gamma=90 deg.

(39) The single crystal structure diagram was shown in FIG. 2. Molecular crystal packing diagram was shown in FIG. 3.

Example 4: Stability Test

(40) Three batches of sample (prepared according to Example 2) were taken according to the Chinese Pharmacopoeia, 2010 Edition, packaged like packaging for sale (high-density polyethylene bags were used for drug packaging), placed under conditions of RT40° C., RH75% (NaCl saturated solution) to carry out accelerated test, after 1, 2, 3, 6 months, sampled for observation of the compound of Formula I and the compound of Formula A, compared to the data of the 0.sup.th day, and the results were shown in Table 1 and Table 2.

(41) One batches of sample as prepared according to Example 3 were taken, packaged like packaging for sale (high-density polyethylene bags were used for drug packaging), placed under conditions of RT40° C., RH75% (NaCl saturated solution), after 1, 2, 3 months, sampled and compared to the data of the 0.sup.th day, and the results were shown in Table 3.

(42) Three batches of sample of the compound of Formula A were taken according to the Chinese Pharmacopoeia, 2010 Edition, packaged like packaging for sale (high-density polyethylene bags were used for drug packaging), placed under conditions of RT40° C., RH75% (NaCl saturated solution), after 1, 2, 3 months, sampled and compared to the data of the 0.sup.th day, and the results were shown in Table 1 and Table 4.

(43) TABLE-US-00001 TABLE 1 Comparison of quality stabilization between the compound of Formula I (n = 0) as prepared in Example 2 to the compound of Formula A (M = Na, K = Br) Name of compound Elemental analysis Accelerated test, 3 months C: 45.72, H: 4.51, N: 8.87%. (standard values: C: 45.85, H: 4.49, N: 8.91%) C: 45.75, H: 4.52, N: 8.88% C: 45.83, H: 4.52, N: 8.92%. no obvious appearance change     no obvious appearance change no obvious appearance change C: 30.79, H: 2.78, N: 5.46, Br: 27.63%. (standard values: C: 27.71, H: 2.71, N: 5.39, Br: 30.72%) C: 29.57, H: 2.80, N: 5.50, Br: 28.30%.   C: 29.09, H: 2.68, N: 5.45, Br: 28.68%. caking, appearance changing into dark brown   caking, appearance changing into dark brown caking, appearance changing into dark brown

(44) TABLE-US-00002 TABLE 2 Purity observation results of the compound of Formula I as prepared in Example 2 in the accelerated tests Melting Weight Appearance Purity* point gain Batch Time and color (%) (° C.) (%) 1 0.sup.th faint yellow to 99.8 168.3-169.8 0 month yellow powder 1.sup.st faint yellow to 99.7 168.1-169.7 0.11 month yellow powder 2.sup.nd faint yellow to 99.6 168.2-169.9 0.12 month yellow powder 3.sup.rd faint yellow to 99.8 168.2-169.8 0.15 month yellow powder 6.sup.th faint yellow to 99.9 168.1-169.9 0.18 month yellow powder 2 0.sup.th faint yellow to 100.0 168.4-170.2 0 month yellow powder 1.sup.st faint yellow to 99.8 168.3-169.8 0.12 month yellow powder 2.sup.nd faint yellow to 99.2 168.4-169.9 0.12 month yellow powder 3.sup.rd faint yellow to 99.8 168.2-170.0 0.14 month yellow powder 6.sup.th faint yellow to 99.6 168.3-169.9 0.15 month yellow powder 3 0.sup.th faint yellow to 99.6 168.3-169.9 0 month yellow powder 1.sup.st faint yellow to 99.8 168.4-169.9 0.11 month yellow powder 2.sup.nd faint yellow to 99.6 168.3-170.0 0.13 month yellow powder 3.sup.rd faint yellow to 99.3 168.3-170.2 0.13 month yellow powder 6.sup.th faint yellow to 99.8 168.2-170.1 0.15 month yellow powder

(45) TABLE-US-00003 TABLE 3 Purity observation results of the compound of Example 3 in accelerated test Melting Weight Appearance Purity* point gain Batch Time and color (%) (° C.) (%) 1 0.sup.th faint yellow to 99.9 172.3-171.8 0 month yellow powder 1.sup.st faint yellow to 99.8 172.1-171.7 0.04 month yellow powder 2.sup.nd faint yellow to 99.7 172.2-171.9 0.06 month yellow powder 3.sup.rd faint yellow to 99.7 172.2-171.8 0.07 month yellow powder

(46) TABLE-US-00004 TABLE 4 Purity observation results of the compound of Formula A (M = Na, K = Br) in the accelerated test Melting Weight Appearance Purity* point gain Batch Time and color (%) (° C.) (%) 1 0.sup.th faint yellow to 98.7 >220.0 0 month yellow powder 1.sup.st brown, tacky 85.1 unmeasured 18.0 month 2.sup.nd dark brown, 60.6 unmeasured 32.6 month caking 3.sup.rd dark brown, 51.2 unmeasured 41.5 month caking 2 0.sup.th faint yellow to 98.8 >220.0 0 month yellow powder 1.sup.st brown, tacky 82.7 unmeasured 18.5 month 2.sup.nd dark brown, 59.8 unmeasured 33.6 month caking 3.sup.rd dark brown, 48.5 unmeasured 42.1 month caking 3 0.sup.th faint yellow to 99.1 >220.0 0 month yellow powder 1.sup.st brown, tacky 81.6 unmeasured 19.0 month 2.sup.nd dark brown, 60.3 unmeasured 33.8 month caking 3.sup.rd dark brown, 54.6 unmeasured 44.0 month caking

(47) It can be seen in the above data that the compounds of Examples 2 and Example 3 showed stabilization significantly superior to that of the known compound of Formula A, and thus had better potential to be medicament.

Example 5: Test of In Vitro Breaking Erythrocytic Surface Cross-Linked IgG (Immune Globulin G)

(48) Because erythrocytic surface cross-linked IgG is a typical AGEs cross-linked structure, the determination of degree that a compound breaks erythrocytic surface cross-linked IgG is well known as a better method for evaluating the ability of the compound in breaking AGEs cross-linked structure (Bruceh. R. Wolffenbuttel, Breakers of advanced glycation end products restore large artery properties in experimental diabetes, Natl. Acad. Sci. U.S.A 1998, 95, 4630).

(49) Method for treating blood cells: 16 weeks diabetic rats were narcotized, blood samples were got from their carotid arteries, added with heparin for anticoagulation, centrifuged at 4° C. and 1000 g for 3 min, the lower layer RBC (red blood cells) was taken; washed with 0.1 mol/L PBS (pH7.4) for 3 times, centrifuged each time at 4° C. and 1000 g for 3 min; and the lower layer RBC was used for test.

(50) In vitro administration method: 0.1 mol/L isotonic PBS (phosphate buffer) (pH7.4) was used as negative control, each of the compounds to be tested formed solutions with different concentrations using the buffer. Into per 900 μL of solution or solvent, 100 μL RBC was added, slightly shook at 37° C. for 16-18 h; centrifuged at 1000 g, 4° C. for 3 min, the supernatant was discarded, the plate was washed with 0.1 mol/L PBS (pH7.4) for 4 times to remove residual compound; centrifuged at 1000 g, 4° C. for 3 min, the lower layer RBC was diluted in ratio of 1:100 for ELISA determination.

(51) Procedure for determination of RBC surface cross-linked IgG content via immunoadsorption method: Multiscreen-HA 0.45 μm 96-well plate (Millipore), sealed with Superblock (300 μL/well), 37° C., 1 h; then drained under 5 mmHg of negative pressure, the whole plate was washed with PBST for 3 times, washed with 0.1M PBS (pH7.4) for twice, shaking plate each time for 1 min; added with RBC (50 μL/well) to be tested, another PBS background control well (OD.sub.0) was set; drained under negative pressure; washed with 150 μL of 0.1 mol/L PBS (pH7.4) for 4 times, shaking plate each time for 1 min. After being drained under negative pressure, the 1:500 diluted goat-anti-mouse IgG-HRP (50 μL/well) was added, stood at room temperature for 2 h, drained; washed with 0.1 mol/L PBS (pH7.4), 150 μL/well, for 3 times, shaking plate each time for 1 min; drained; added with o-phenylenediamine (OPD) substrate developing solution (100 μL/well), stood at room temperature in dark for 30 min, the reaction was terminated with 2 mol/L H.sub.2SO.sub.4 (100 μL/well); the reaction solution (150 μL/well) was sucked out quickly and transferred to a normal 96-well ELISA plate, and OD values were determined under 490 nm.

(52) Calculation of Lytic Rates of the Compounds to be Tested:

(53) Corrected OD=Average OD of RBC sample to be tested−Average OD of RBC-free PBS background well, the lytic rate is expressed in percentage of decrease of OD.sub.490 nm value: (OD.sub.490 nm of PBS well−OD.sub.490nm of compound to be tested)/OD.sub.490nm of PBS well×100%. The test results were shown in Table 5:

(54) TABLE-US-00005 TABLE 5 Lytic rates of RBC surface cross-linked IgG caused by the compound of Example 2 Lytic rate (decrease of OD value, %) Compound 1 μM 10 μM 30 μM 100 μM Compound of 27.1 ± 2.2 27.5 ± 2.9 23.2 ± 5.1 27.9 ± 3.0 Example 2

(55) The results of Table 5 shows that the compound of Example 2 has high lytic rate to RBC surface cross-linked IgG.

Example 6: Effects of the Compound of Example 2 on 24 h Urine Volume/Water Intake in Rats with Diabetes Accompanied by Hypertension

(56) 1. Test Method:

(57) (1) Grouping and Administration Method

(58) Rats were grouped according to bodyweight and blood pressure: group of rats with diabetes accompanied by hypertension that were not administered with a drug (model group), Nifedipine group, Example 2 compound+Nifedipine group. In the meantime, pure diabetes group and normal control group of the same week-age were also set. The compound of Example 2 (36 mg/kg) was dissolved in distilled water just before use, administered intragastrically, once per day, for 5 weeks. After 3 weeks of administration, an implant was embedded in abdominal aorta, restored for week, blood pressure was monitored for 3 days, until the blood pressure become stable, then Nifedipine (0.75 mg/kg) was intragastrically administered at 10:00 am, once per day, for consecutive 7 days.

(59) (2) Preparation of Nifedipine Solution

(60) Nifedipine powder was placed in 5 mL EP tube, added with a defined amount of CMC-Na, added with 4 steel balls, eddied for 5-10 min, after Nifedipine was completely suspended, volume was metered, and suspension was performed again.

(61) (3) Determination of Water-Intake and Urine Volume

(62) In the third week of administration, the rats of each group were solely placed and fed in metabolism cage, and the water intake and urine volume within 24 hours on the 19.sup.th, 20.sup.th and 21.sup.st day were recorded.

(63) (4) Blood Pressure Monitoring of the Compound of Example 2 in Combination with Nifedipine

(64) The operation procedures were the same of (1), (2), (3), after 1 week of postoperative recovery, the rat cages were placed on DSI receiver, the parameters to be monitored and channels were set, the implants were turned on by using magnetic switches; after debugging, recording biological signals was started; after consecutive 3 days, Nifedipine and the compound of Example 2 were administered at 10:00 am per day, the cardiovascular parameters of rats during time period of 10:00-20:00 were dynamically recorded, for consecutive 7 days (in this period, the saline concentration was strictly controlled at 1%).

(65) (5) Method for Content Determination of Vasoactive Substances in Rats

(66) Method for determination of TXB.sub.2, 6-Keto-PGF1a: whole blood samples were taken, added with 40 μL of Indometacin EDTA-Na for anticoagulation, centrifuged at 4° C., 3500 rmp/min, 15 min, to obtain plasma, stored at −70° C. The determination was carried out via radioimmunoassy by Beijing Huaying Biotechnology Co., Ltd.

(67) Method for determination of BNP, MCP-1: whole blood samples were taken, added with 40 μL EDTA-Na for anticoagulation, centrifuged at 1000 g/min, 10 min, to separate plasma, stored at −70° C. The determination was carried out via radioimmunoassy by Beijing Huaying Biotechnology Co., Ltd.

(68) (6) Statistical Method

(69) The test data were expressed in form of mean±SD (mean±standard deviation), SPSS2.0 software was used for processing data, statistical treatment was performed by using one-way analysis of variance, and significant difference was determined when P<0.01.

(70) In the 3.sup.rd week of administering the compound of Example 2, V.sub.urine/V.sub.water-intake values of rats in 24 hours were calculated.

(71) 2. Test Results

(72) The results were shown in Table 6:

(73) TABLE-US-00006 TABLE 6 Measurement results of water-intakes and urine volumes in different groups (Mean ± SD, n = 12) Model Compound of Example 2 V.sub.water-intake (mL) 369.3 ± 122.7 375.0 ± 123.6 V.sub.urine (mL) 302.9 ± 101.8 318.3 ± 106.0 V.sub.urine/bodyweight (mL/g) 0.82 ± 0.31 0.88 ± 0.31 V.sub.urine/bodyweight (mL/g) 1.00 ± 0.78 1.03 ± 0.34 V.sub.urine/V.sub.water-intake 0.79 ± 0.06  0.86 ± 0.05* *P < 0.01 vs. model.

(74) The test results of Example 6 shows that the compound of Example 2 is capable of significantly increasing water-intake and urine volume in rats.

Example 7: Effects of the Compound of Example 2 on Rats with Diabetes Accompanied by Hypertension

(75) After 4 weeks of administering the compound of Example 2, the rats of model group showed an average systolic pressures during time period 10:00-20:00 significantly higher than that of the normal control group (NC group). The compound of Example 2 showed an average systolic pressures during time period 10:00-20:00 significantly lower than that of the model group (169.8:15.8 mmHg vs. 181±14.9 mmHg, P<0.05) (see details in FIG. 6).

(76) After 4 weeks of administering the compound of Example 2, the rats of diabetes group showed a systolic pressure variable coefficient (CV) during time period 10:00-20:00 significantly higher than that of the NC group (P<0.05), while the rats of the model group showed a further significantly increase in systolic pressure CV (P<0.01). In comparison with the model group, the rats of the group of the compound of Example 2 showed a significantly decrease in systolic pressure CV (P<0.01) (see details in FIG. 7).

(77) After 4 weeks of administering the compound of Example 2, the rats of model group showed a significant increase in average systolic pressure during time period 10:00-20:00 in comparison with the NC group. In comparison with the model group, the group of the compound of Example 2 showed a significantly decrease in average systolic pressure during time period 10:00-20:00 (123.1±13.4 mmHg vs. 132.3±12.7 mmHg) (see details in FIG. 8).

(78) After 4 weeks of administering the compound of Example 2, the rats of model group showed a significant increase in average pulse-pressure difference during time period 10:00-20:00 in comparison with the NC group (P<0.01). In comparison with the model group, the group of the compound of Example 2 showed no significant change (46.5±5.7 mmHg vs. 49.7±3.5 mmHg) (see details in FIG. 9).

(79) After 4 weeks of administering the compound of Example 2, the rats of model group showed a significant decrease in average heart rate during time period 10:00-20:00 in comparison with the NC group (P<0.01). In comparison with the model group, the group of the compound of Example 2 showed no significant change (255±17 vs. 257±13 beats/min) (see details in FIG. 10).

(80) The results of Example 7 show that the compound of Example 2 has effects of stabilizing blood pressure, conforming to the therapeutic principle of diabetes accompanied by hypertension, and may act as an adjuvant drug for enhancing blood pressure stability in anti-hypertension drug combination.

Example 8: Effects of the Compound of Example 2 in Combination with Nifedipine on Rats with Diabetes Accompanied by Hypertension

(81) After administration of Nifedipine at 10:00, the systolic pressures of the administration groups dropped quickly, and reached the maximum of pressure drop after 1.5 h; the Nifedipine group showed the systolic pressure stated to rise after 2 h of administration, and the systolic pressure was substantially close to that of the model group after 10 h; the Example 2 compound+Nifedipine group showed the systolic pressure could keep stable for 5 h after reaching the lowest value, and then slowly recovered. In comparison with the Nifedipine group, the Example 2 compound+Nifedipine group showed a significant decrease in average systolic pressure after administration for 1 h (135.8±12.5 mmHg vs. 155.2±14.9, P<0.01); 5 h (135.0±11.4 mmHg vs. 166.0±15.0 mmHg, P<0.01), 10 h (152.2±10.4 mmHg vs. 179.0±14.1 mmHg, P<0.01) (see details in FIG. 11).

(82) After administration for 1.5 h, the Example 2 compound+Nifedipine group showed a pressure drop ΔSBP significantly higher than that of the Nifedipine group (37.1±13.5 mmHg vs. 25.3±9.3 mmHg, P<0.05). After administration for 5 h, the Example 2 compound+Nifedipine group showed a ΔSBP significantly higher than that of the Nifedipine group (30.9-12.5 mmHg vs. 15.9±9.3 mmHg, P<0.01), and after administration for 10 h, the Example 2 compound+Nifedipine group showed a ΔSBP significantly higher than that of the Nifedipine group (19.4±16.4 mmHg vs. 1.19±3.5 mmHg, P<0.01) (see details in FIG. 12).

(83) After administration for 1-5 h, in comparison with the model group, the Nifedipine group showed no significant systolic pressure variable coefficient (CV) (0.047±0.017 vs. 0.051±0.012), the Example 2 compound+Nifedipine group showed a significant decrease in CV (0.019±0.006 vs. 0.051±0.012, P<0.01) (see details in FIG. 13).

(84) After administration at 10:00, the systolic pressures of the administration groups showed a quick decrease, and almost reached the maximum pressure drop range after 1.5 h, then slowly recovered. After administration for 1.5 h, the Example 2 compound+Nifedipine group showed a significant decrease in average systolic pressure in comparison with the Nifedipine group (95.8±14.5 mmHg vs. 111.2±15.3, P<0.05). After administration for 5 h, the Example 2 compound+Nifedipine group showed a significant decrease in average systolic pressure in comparison with the Nifedipine group (96.6±12.3 mmHg vs. 115.9±15.7 mmHg, P<0.01). After administration for 10 h, the Nifedipine group showed a diastolic pressure close to that of the model group, the Example 2 compound+Nifedipine group showed a significant decrease in average systolic pressure in comparison with the Nifedipine group (106.1±16.4 mmHg vs. 130.1±14.8 mmHg, P<0.01) (see details in FIG. 14).

(85) After administration at 10:00, the Example 2 compound+Nifedipine group showed a rapid decrease in pulse-pressure difference (PH), which reached the maximum of pressure drop at 11:00; the Nifedipine group showed a slight decrease of PH, which started to rise after 1 h, and almost reached that of the model group after 10 h; the Example 2 compound+Nifedipine e group reached the lowest value of PH after 1 h, which started to rise slowly since then. After administration for 1 h, the Example 2 compound+Nifedipine group showed a significant decrease in average pulse-pressure difference in comparison with the Nifedipine group (40.2±4.5 vs. 46.9-2.9 mmHg, P<0.01). After administration for 5 h, the Example 2 compound+Nifedipine group showed a significant decrease in average pulse-pressure difference in comparison with the Nifedipine group (40.6±4.9 vs. 48.1±5.1 mmHg, P<0.01). After administration for 10 h, the Example 2 compound+Nifedipine group showed no change in average pulse-pressure difference in comparison with the Nifedipine group (45.6±5.8 vs. 48.7±5.2 mmHg) (see details in FIG. 15).

(86) After administration at 10:00, the administration groups showed a rapid increase in heart rate (HR), which reached the maximum after 20 min, then started to drop slowly, after administration for 8 h, the heart rate of the administration groups substantively recovered the level before administration. After administration, the Example 2 compound+Nifedipine group showed no change in heart rate in comparison with the Nifedipine group (see details in FIG. 16).

(87) After administration at 10:00, the administration groups showed a rapid decrease in ejection time (ET), which reached the lowest value after 20 min, then started to rise slowly; after administration for 5 h, the ET of the Nifedipine group substantively recovered the level before administration; after administration for 10 h, the Nifedipine group showed a ET value substantively equivalent to that of the MC group. After administration for 20 min, the Example 2 compound+Nifedipine group showed a significant decrease in ET in comparison with the Nifedipine group (67.3±5.3 ms vs. 71.8±4.2 ms), then the ET started to rise slowly, and showed a rapid increase stage after 8-10 h, and reached to a ET level substantively equivalent to that of the MC group after 10 h; after administration for 5 h, the Example 2 compound+Nifedipine group showed a significant decrease in ET in comparison with the Nifedipine group (74.2±5.2 ms vs. 81.1±5.0 ms, P<0.05), and this effect kept for 4 h (see details in FIG. 17).

(88) Myocardial oxygen consumption index (MOCI) reflects total myocardial oxygen consumption. After administration for 1 h, in comparison with the model group, the Nifedipine group showed a decrease in MOCI, without significant difference (3772.7±444.7 vs. 4255.0±416.1, P=0.36), while the Example 2 compound+Nifedipine group showed a significant decrease in MOCI (3128.4±238.7 vs. 4255.0±416.1, P<0.05). After administration for 5 h, in comparison with the model group, the Nifedipine group showed a decrease in MOCI, without significant difference, the Example 2 compound+Nifedipine group showed a significant decrease in MOCI (P<0.05). After administration for 10 h, the Nifedipine group showed a MOCI recovered to the level before administration, the Example 2 compound+Nifedipine group showed a MOCI lower than that of the model group and the Nifedipine group, without significant difference (see details in FIG. 18).

(89) The results of Example 8 showed that the compound of Example 2 can significantly enhance the effects of Nifedipine on heart; and the compound of Example 2 in combination with Nifedipine could significantly reduce the blood pressure of rats with diabetes accompanied by hypertension.

Example 9: Effects of the Compound of Example 2 in Combination with Nifedipine on Vascular Active Factors Content in Rats with Diabetes Accompanied by Hypertension

(90) (1) Effects of the Compound of Example 2 in Combination with Nifedipine on Plasma 6-Ketone Prostaglandin Content in Rats with Diabetes Accompanied by Hypertension

(91) Plasma 6-ketone-prostaglandin is a metabolite of prostacyclin (PG1.sub.2), reflecting the PG1.sub.2 content in plasma. In comparison with the diabetes group, the rats of the model group showed a significant decrease in plasma 6-ketone-prostaglandin content in comparison with the normal group (78.15±5.6, 77.62±8.67 vs. 85.41±4.36 pg/mL, P<0.05). The rats of the Example 2 compound+Nifedipine group showed a significant increase in comparison with the model group (87.21±6.90 vs. 77.62±8.67 pg/mL, P<0.01), and a significant increase in comparison with the Nifedipine group as well (P<0.05) (see details in FIG. 19).

(92) (2) Effects of AGEs Breaker in Combination with Nifedipine on TXB.sub.2 Content in Plasma of Rats with Diabetes Accompanied by Hypertension

(93) TXB.sub.2 is a metabolite of TXA.sub.2, and reflects TXA.sub.2 contents in plasma. In comparison with the normal group, the rats of the diabetes group and the model group showed a significant increase in TXB.sub.2 content (93.14±10.99, 104.19±11.68 vs. 64.88±7.24, P<0.01). The Example 2 compound+Nifedipine group and the Nifedipine group showed a significant decrease in TXB.sub.2 content in comparison with the model group (73.64±12.27, 80.88±15.31 vs. 104.19±11.68 pg/mL, P<0.01); The Example 2 compound+Nifedipine group showed no significant difference in TXB.sub.2 content in comparison with the Nifedipine group (see details in FIG. 20).

(94) (3) Effects of the Compound of Example 2 in Combination with Nifedipine on Plasma TXB.sub.2/6-Keto-PGI1a Ratio of Rats with Diabetes Accompanied by Hypertension

(95) TXB.sub.2/6-Keto-PGI1a ratio reflects the level of plasma TXA.sub.2/PGI.sub.2. In comparison with the normal group, the rats of the diabetes group and the model group showed significant increase in TXB.sub.2/6-Keto-PGI1a (1.15±0.15, 1.18±0.16 vs. 0.80±0.10, P<0.01); the Example 2 compound+Nifedipine group showed a significant decrease in TXB2/6-Keto-PGI1a in comparison with the model group (0.93±0.13 vs. 1.18-0.16, P<0.01); the Nifedipine group showed a slightly decrease in comparison with the model group (1.06±0.14 vs. 1.18±0.16), without significant difference; the Example 2 compound+Nifedipine group showed a significant decrease in TXB.sub.2/6-Keto-PGI1a in comparison with the Nifedipine group (0.93±0.13 vs. 1.06±0.14, P<0.05) (see details in FIG. 21).

(96) (4) Effects of the Compound of Example 2 in Combination with Nifedipine on Plasma MCP-1 Content in Rats with Diabetes Accompanied by Hypertension

(97) In comparison with the normal group, the rats of the diabetes groups and the model group showed a significant increase in plasma MCP-1 content (75.9±9.7, 77.4±9.5 vs. 66.9±7.3 pg/mL, P<0.05); the Example 2 compound+Nifedipine group showed a significant decrease in plasma MCP-1 content in comparison with the model group (64.0±14.2 vs. 77.4-9.5 pg/mL, P<0.01); the Nifedipine group showed a slight decrease in plasma MCP-1 content in comparison with the model group (70.7-8.8 vs. 77.4-9.5 pg/mL), without significant difference; the Example 2 compound+Nifedipine group showed a significant decrease in plasma MCP-1 content in comparison with the Nifedipine group (P<0.05) (see details in FIG. 22).

(98) (5) Effects of the Compound of Example 2 in Combination with Nifedipine on Plasma BNP Content in Rats with Diabetes Accompanied by Hypertension

(99) In comparison with the normal group, the rats of the model group showed a significant increase in plasma BNP content (16.7±2.0 vs. 14.3±2.1 pg/mL, P<0.05); the Example 2 compound+Nifedipine group showed a significant decrease in plasma BNP content in comparison with the model group (12.2±3.5 vs. 16.7±2.0 pg/ml, P<0.01); the Nifedipine group showed a significant decrease in plasma BNP content in comparison with the model group (14.6±2.4 vs. 16.7-2.0 pg/mL, P<0.05); the Example 2 compound+Nifedipine group showed a significant decrease in plasma MCP-1 content in comparison with the Nifedipine group (P<0.05, P<0.01)(see details in FIG. 23).

(100) The results of Example 9 showed that the compound of Example 2 resulted in decrease in TXA.sub.2/PGI.sub.2 ratio, vascular dilatation, reduction of thrombosis, delaying atherosclerosis procedure, and vascular protection. The AGEs breaker can also reduce plasma BNP content in rats with diabetes accompanied by hypertension, and significantly reduce plasma MCP-1 content in rats with diabetes accompanied by hypertension.

(101) Although the specific models of the present invention have been described in details in the specific models of the present invention, the skilled in the art would understand those details could be modified and replaced according the disclosures, and all of these changes fall within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents thereof.