Substituted acethydrazide derivative, preparation method and use thereof

Abstract

The present invention pertains to the field of pharmaceutical chemicals, and relates to a substituted acethydrazide derivative, preparation method and use thereof; preferably, relates to a compound of Formula I or a pharmaceutically acceptable salt thereof. The compound of the present invention or a pharmaceutically acceptable salt thereof can effectively inhibit proline hydroxylase, stabilize HIF-, particularly HIF-1, and has potency in the manufacture of a medicament for treatment and/or prophylaxis and/or adjuvant therapy of anemia, acute ischemic reperfusion injury. ##STR00001##

Claims

1. A method for inhibiting proline hydroxylase or stabilizing HIF-1 in vivo or in vitro, comprising a step of administering to a subject or a cell in need thereof an effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof; ##STR00094## wherein: n is 1, 2, 3, 4 or 5; A is O or S; R.sub.3 is selected from hydrogen, and C.sub.1-C.sub.4 alkyl; Ar.sup.1 is an aryl substituted with R.sub.1, wherein R.sub.1 is selected from: hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, trifluoromethyl and cyano; Ar.sup.2 is an aryl substituted with R.sub.2, R.sub.4, and R.sub.5, wherein R.sub.2, R.sub.4, and R.sub.5 are independently selected from: hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, allyl, halogen and C.sub.1-C.sub.4 dialkylamino; wherein R.sub.2, R.sub.4 and R.sub.5 are not hydrogen simultaneously.

2. The method according to claim 1, wherein the proline hydroxylase is prolyl-4-hydroxylase or EC 1.14.11.2.

3. The method according to claim 1, wherein the HIF-1 is HIF-.

4. The method according to claim 3, wherein the HIF- is HIF-1, HIF-2 or HIF-3.

5. The method according to claim 1, wherein the aryl is independently selected from phenyl, naphthyl, anthracenyl, phenanthrenyl, indenyl, fluorenyl and acenaphthenyl.

6. The method according to claim 1, wherein the C.sub.1-C.sub.6 alkyl is independently C.sub.1-C.sub.4 alkyl.

7. The method according to claim 1, wherein the C.sub.1-C.sub.6 alkoxy is independently C.sub.1-C.sub.4 alkoxy.

8. The method according to claim 1, wherein the C.sub.1-C.sub.4 dialkylamino is C.sub.1-C.sub.3 dialkylamino.

9. The method according to claim 1, wherein the halogen is selected from fluorine, chlorine, bromine and iodine.

10. The method according to claim 1, wherein: n is 1 or 2; A is O; R.sub.3 is hydrogen; Ar.sup.1 is phenyl or naphthyl substituted with R.sub.1, wherein R.sub.1 is selected from: hydrogen, C.sub.1-C.sub.4 alkyl, methoxy, ethoxy, trifluoromethyl, and cyano; Ar.sup.2 is phenyl or naphthyl substituted with R.sub.2, R.sub.4, and R.sub.5, wherein R.sub.2, R.sub.4, and R.sub.5 are independently selected from: hydrogen, hydroxy, C.sub.1-C.sub.4 alkyl, methoxy, ethoxy, allyl, halogen, dimethylamino, diethylamino and methylethylamino; wherein R.sub.2, R.sub.4, and R.sub.5 cannot be hydrogen simultaneously.

11. The method according to claim 1, wherein the compound of Formula I is selected from: (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-bromo-4-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(4-bromobenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(2,3-dihydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-[(3-ethoxy-2-hydroxybenzal)methylene]-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(2,3-dihydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-bromo-6-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3, 5-di-tert-butyl-2-hydroxybenzal)-3-(N-(2-(N-(4-tert-butylbenzyl)-N-methylamino)propyl)N-methylamino)acethydrazide, (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(2,3-dihydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-bromo-6-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-tert-butylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-tert-butylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide, and (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{3-[N-(4-tert-butylbenzyl)-N-methylamino]propyl}N-methylamino}acethydrazide.

Description

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

(1) The embodiments of the present invention are illustrated in details in conjunction with examples. However, those skilled in the art would understand the following examples are merely to illustrate the present invention, rather than to limit the scope of the present invention. When specific conditions are not given in the examples, conventional conditions or conditions recommended by manufacturers are applied. The reagents or instruments which manufactures are not given are all conventional products commercially available in markets.

(2) Melting points of compounds are measured by YRT-3 type melting point instrument, in which temperatures are not calibrated. .sup.1H-NMR spectra are measured by Bruker ARX 400 type NMR spectrometer. FAB mass spectra are measured by Zabspect high resolution magnetic mass spectrometer.

Preparation of Intermediates

Preparation Example 1: Intermediate 1

(3) ##STR00040##

(4) 9.6 mL (0.36 mol) of N,N-dimethylethanediamine and 100 mL of tetrahydrofuran were added to a three-necked bottle equipped with constant pressure funnel, reflux condensing tube and thermometer, mixed evenly and then heated. When the reaction solution was slightly boiled (about 56 C.), 10 g (0.06 mol) of 1-chloromethylnaphthalene was slowly added in dropwise to the reaction solution, white precipitate was gradually generated, and thin-layer chromatography (TLC) was used to monitor the end of reaction. When the spot of 1-chloromethylnaphthalene in the reaction solution disappeared on thin-layer chromatography, heating was stopped, cooling was carried out, solvent was dried out by rotary evaporation, 3 mol/L of sodium hydroxide solution was used for washing, dichloromethane was used for extracting the washing liquid in which pH12 was maintained during extraction, all organic phases were combined, separated by silica column (eluent: dichloromethane/methanol=20/1) to Obtain {[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino, light yellow liquid, yield 86%. 1H-NMR (400 MHz, CDCl3) : 8.18 (7H, m), 4.10 (2H, s), 3.26 (3H, s), 2.50 (4H, m), 2.26 (3H, s); ELMS (m/z): 229.2[M+H].sup.+.

Preparation Example 2: Intermediate 2

(5) ##STR00041##

(6) 3.56 g (0.02 mol) of {[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamine and 2.1 g (0.025 mol) of sodium hydrogen carbonate were added to 100 mL three-necked bottle equipped with constant pressure funnel, reflux condensing tube and thermometer, then 40 mL acetone was added, mixed evenly and then heated to reflux, 2.36 g (0.022 mol) of methyl acetate was slowed added in dropwise to the reaction solution, thin-layer chromatography (TLC) was used to monitor the end of reaction. When the spot of {[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamine in the reaction solution disappeared on thin-layer chromatography, heating was stopped, cooling was carried out, solvent was dried out by rotary evaporation, silica column separation was carried out (eluent: ethyl acetate/petroleum=1/3) to obtain methyl 2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acetate, light yellow liquid, yield 85%. 1H-NMR (400 MHz, CDCl3) : 8.18 (7H, m), 4.10 (2H, s), 3.68 (3H, s), 3.32 (2H, s), 2.37 (4H, m), 2.26 (6H, s); EI-MS (m/z): 301.2[M+H].sup.+.

Preparation Example 3: Intermediate 3

(7) ##STR00042##

(8) 2.1 g (0.06 mol) of 85% hydrazine hydrate and 50 ml of anhydrous ethanol were added to 250 mL three-necked bottle equipped with constant pressure funnel, reflux condensing tube and thermometer, mixed evenly and then heated to reflux, 5.0 g (0.02 mol) of methyl 2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acetate was slowly added to the reaction solution, and reacted for 2 h, then heating was stopped, cooling was carried out, solvent was dried out by rotary evaporation, silica column separation was carried out (eluent: dichloromethane/methanol=10/1) to obtain 2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, white solid, yield 86%. 1H-NMR (400 MHz, CDCl3) : 8.18 (7H, m), 4.10 (2H, s), 3.29 (2H, s), 2.37 (4H, m), 2.26 (6H, s); EI-MS (m/z): 301.2[M+H].sup.+.

Preparation Example 4: Intermediate 4

(9) ##STR00043##

(10) Pare- and meta-trifluoromethylbenzyl chloride was used as raw material, and operations were the same for Intermediate 1. Light yellow liquid product was obtained. MS [M].sup.+=246.1 m/e.

Preparation Example 5: Intermediate 5

(11) ##STR00044##

(12) Intermediate 4 was used as raw material, and operations were the same for Intermediate 2. Light yellow liquid product was obtained. MS [M].sup.+=318.2 m/e.

Preparation Example 6: Intermediate 6

(13) ##STR00045##

(14) Intermediate 5 was used as raw materials, and operations were the same for Intermediate 3. Light yellow liquid product was obtained. MS [M].sup.+=318.2 m/e.

Preparation Example 7: Intermediate 7

(15) ##STR00046##

(16) 4-Isopropylbenzyl chloride was used as raw material, and operations were the same for Intermediate 1. Light yellow liquid product was obtained. MS [M].sup.+=220.2 m/e.

Preparation Example 8: Intermediate 8

(17) ##STR00047##

(18) Intermediate 7 was used as raw material, and operations were the same for Intermediate 2. Light yellow liquid product was obtained. MS [M].sup.+=292.2 m/e.

Preparation Example 9: Intermediate 9

(19) ##STR00048##

(20) Intermediate 8 was used as raw material, and operations were the same for Intermediate 3. Light yellow liquid product was obtained. MS [M].sup.+=292.2 m/e.

Preparation Example 10: Intermediate 10

(21) ##STR00049##

(22) 3-methoxybenzyl chloride was used as raw material, and operations were the same for Intermediate 1. Light yellow liquid product was obtained. MS [M].sup.+=208.2 m/e.

Preparation Example 11: Intermediate 11

(23) ##STR00050##

(24) Intermediate 10 was used as raw material, and operations were the same for Intermediate 2. Light yellow liquid product was obtained. MS [M].sup.+=280.2 m/e.

Preparation Example 12: Intermediate 12

(25) ##STR00051##

(26) Intermediate 11 was used as raw material, and operations were the same for Intermediate 3. Yellow liquid product was obtained. MS [M].sup.+=280.2 m/e.

Preparation Example 13: Intermediate 13

(27) ##STR00052##

(28) 4-(Bromo-methyl)benzonitrile was used as raw material, and operations were the same for Intermediate 1. Light yellow liquid product was obtained. MS [M].sup.+=203.1 m/e.

Preparation Example 14: Intermediate 14

(29) ##STR00053##

(30) Intermediate 13 was used as raw material, and operations were the same for Intermediate 2. Light yellow liquid product was obtained. MS [M].sup.+=275.2 m/e.

Preparation Example 15: Intermediate 15

(31) ##STR00054##

(32) Intermediate 14 was used as raw material, and operations were the same for Intermediate 3. Yellow liquid product was obtained. MS [M].sup.+=275.2 m/e.

Preparation Example 16: Intermediate 16

(33) ##STR00055##

(34) 4-tert-butylbenzyl chloride was used as raw material, and operations were the same for Intermediate 1. Light yellow liquid product was obtained. MS [M].sup.+=234.2 m/e.

Preparation Example 17: Intermediate 17

(35) ##STR00056##

(36) Intermediate 16 was used as raw material, and operations were the same for Intermediate 2. Light yellow liquid product was obtained. MS [M].sup.+=306.2 m/e.

Preparation Example 18: Intermediate 18

(37) ##STR00057##

(38) Intermediate 17 was used as raw material, and operations were the same for Intermediate 3. Yellow liquid product was obtained. MS [M].sup.+=306.2 m/e.

Preparation Example 19: Intermediate 19

(39) ##STR00058##

(40) Para-tert-butylbenzyl chloride and N,N-dimethylpropanediamine were used as raw materials, and operations were the same for Intermediate 1. Light yellow liquid product was obtained. MS [M].sup.+=248.2 m/e.

Preparation Example 20: Intermediate 20

(41) ##STR00059##

(42) Intermediate 16 was used as raw material, and operations were the same for Intermediate 2. Light yellow liquid product was obtained. MS [M].sup.+=320.2 m/e.

Preparation Example 21: Intermediate 21

(43) ##STR00060##

(44) Intermediate 17 was used as raw material, and operations were the same for Intermediate 3. Light yellow liquid product was obtained. MS [M].sup.+=320.2 m/e.

Example 1: (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 1)

(45) ##STR00061##

(46) To 100 mL two-necked bottle equipped with thermometer and reflux condensing tube, 0.5 g (1.7 mmol) of Intermediate 3 and 20 mL of anhydrous ethanol were added, heated to reflux, 0.36 g (0.24 mmol) of ortho-vanilline was added in dropwise to the reaction solution, reacted for 2 h, then heating was stopped, cooling was carried out, solvent was dried out by rotary evaporation, silica column separation was carried out (eluent: dichloromethane/methanol=10/1) to obtain (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide, white solid, yield 85%. .sup.1H-NMR (400 MHz, DMSO-d6) : 11.30 (2H, m), 8.47 (1H, m), 7.84 (2H, m), 7.52 (4H, m), 6.80 (1H, m), 6.66 (1H, m), 6.36 (1H, s), 5.69 (1H, s), 4.01 (2H, s) 3.83 (3H, s), 3.30 (2H, s), 2.79 (4H, s), 2.61 (3H, s), 2.07 (3H, s); ELMS (m/z): 435.5[M+H].sup.+.

Example 2: (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 2)

(47) ##STR00062##

(48) Intermediate 3 and 3,5-di-tert-butylsalicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.48 (1H, s), 11.26 (1H, s), 8.58 (1H, d, J=8.68 Hz), 7.90 (2H, m), 7.47 (4H, m), 7.21 (1H, s), 6.18 (1H, s), 5.75 (1H, s), 4.00 (2H, s), 3.34 (2H, s), 2.78 (4H, s), 2.68 (3H, s), 2.01 (3H, s), 1.32 (9H, s), 1.24 (9H, s); EI-MS (m/z): 517.6[M+H].sup.+.

Example 3: (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 3)

(49) ##STR00063##

(50) Intermediate 3 and 3-ethoxysalicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.25 (2H, m), 8.47 (1H, d, J=8.12 Hz), 7.90 (2H, m), 6.80 (1H, m), 6.62 (2H, m), 6.34 (1H, s), 5.70 (1H, d, J=7.56 Hz), 4.05 (4H, m), 3.30 (2H, m), 2.73 (4H, s), 2.57 (3H, s), 2.06 (3H, s), 1.43 (4H, m); ELMS (m/z): 449.5[M+H].sup.+.

Example 4: (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 4)

(51) ##STR00064##

(52) Intermediate 3 and 2-hydroxy-1-naphthaldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 12.62 (1H, s), 11.81 (1H, s), 8.71 (1H, d, J=8.12 Hz), 7.75 (8H, m), 7.26 (1H, m), 7.09 (3H, m), 6.02 (1H, d, J=8.68 Hz), 4.03 (2H, s), 3.42 (2H, s), 2.90 (2H, s), 2.70 (5H, m), 2.14 (3H, s); ELMS (m/z): 455.4[M+H].sup.+.

Example 5: (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 5)

(53) ##STR00065##

(54) Intermediate 3 and 4-(diethylamino)salicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.08 (1H, s), 10.87 (1H, s), 8.49 (1H, d, J=8.32 Hz), 7.89 (2H, m), 7.48 (4H, m), 6.27 (1H, s), 6.08 (2H, m), 5.84 (1H, s), 3.98 (2H, s), 3.30 (6H, m), 2.72 (4H, s), 2.57 (3H, s), 2.04 (3H, s), 1.18 (6H, m); ELMS (m/z): 476.5[M+H].sup.+.

Example 6: (E)-N-(3-bromo-4-hydroxybenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 6)

(55) ##STR00066##

(56) Intermediate 3 and 3-bromo-4-hydroxybenzaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.02 (1H, s), 8.49 (1H, d, J=8.32 Hz), 7.94 (2H, m), 7.54 (4H, m), 6.92 (3H, m), 6.06 (1H, s), 4.00 (2H, s), 3.32 (2H, s), 2.72 (4H, s), 2.94 (3H, s), 2.03 (3H, s); EI-MS (m/z): 483.3[M+H].sup.+.

Example 7: (E)-N-(4-bromobenzal)-2-{N-{2-[N-(naphthalen-1-yl-methylene)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 7)

(57) ##STR00067##

(58) Intermediate 3 and p-bromobenzaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.08 (1H, s), 8.49 (1H, d, J=8.32 Hz), 7.86 (2H, m), 7.52 (4H, m), 7.31 (2H, m), 6.81 (2H, m), 6.18 (1H, s), 3.98 (2H, s), 3.31 (2H, s), 2.70 (4H, s), 2.61 (3H, s), 2.03 (3H, s); EI-MS (m/z): 467.3[M+H].sup.+.

Example 8: (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 8)

(59) ##STR00068##

(60) Intermediate 6 and 3,5-di-tert-butylsalicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.50 (1H, s), 11.25 (1H, s), 7.94 (1H, s), 7.67 (4H, m), 7.34 (1H, s), 6.69 (1H, s), 3.71 (2H, s), 3.28 (2H, s), 2.64 (2H, s), 2.53 (2H, s), 2.36 (3H, s), 2.25 (3H, s), 1.42 (9H, s), 1.24 (9H, s); ELMS (m/z): 535.5[M+H].sup.+.

Example 9: (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 9)

(61) ##STR00069##

(62) Intermediate 6 and 3-ethoxysalicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.43 (1H, s), 11.05 (1H, s), 8.10 (1H, s), 7.64 (4H, m), 6.90 (2H, m), 6.61 (1H, m), 4.13 (2H, m), 3.66 (2H, s), 3.25 (2H, s), 2.58 (4H, m), 2.29 (6H, m), 1.47 (3H, m); EI-MS (m/z): 467.3[M+H].sup.+.

Example 10: (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 10)

(63) ##STR00070##

(64) Intermediate 6 and 4-(diethylamino)salicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.20 (1H, s), 11.01 (1H, s), 7.87 (1H, s), 7.66 (4H, m), 6.71 (1H, m), 6.22 (2H, m), 6.19 (1H, s), 3.66 (2H, s), 3.38 (4H, m), 3.25 (2H, s), 2.61 (4H, m), 2.33 (6H, m), 1.20 (6H, m); EI-MS (m/z): 494.7[MH].sup.+.

Example 11: (E)-N-(2,3-dihydroxybenzal)-2-{N-{2-[N-(3-trifluoromethylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 11)

(65) ##STR00071##

(66) Intermediate 6 and 2,3-dihydroxybenzaldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.45 (2H, s), 7.80 (1H, s), 7.65 (4H, m), 6.95 (1H, m), 6.74 (1H, m), 6.42 (1H, m), 3.65 (1H, s), 3.28 (2H, s), 2.61 (4H, m), 2.34 (6H, m); EI-MS (m/z): 439.3[M+H].sup.+.

Example 12: (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 12)

(67) ##STR00072##

(68) Intermediate 9 and ortho-vanilline were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.87 (1H, s), 11.39 (1H, s), 7.27 (5H, m), 6.86 (1H, m), 6.74 (1H, m), 6.40 (1H, m), 3.89 (3H, s), 3.57 (2H, m), 3.27 (2H, s), 2.89 (1H, m), 2.61 (4H, m), 2.38 (3H, s), 2.20 (3H, s), 1.25 (6H, s); ELMS (m/z): 427.3[M+H].sup.+.

Example 13: (E)-N-[(3-ethoxy-2-hydroxybenzal)methylene]-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 13)

(69) ##STR00073##

(70) Intermediate 9 and 3-ethoxysalicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.84 (1H, s), 11.30 (1H, s), 7.70 (1H, s), 7.32 (4H, m), 6.89 (1H, m), 6.71 (1H, m), 6.39 (1H, m), 4.12 (2H, m), 3.56 (2H, s), 3.27 (2H, s), 2.90 (1H, m), 2.62 (4H, m), 2.38 (3H, s), 2.20 (3H, s), 1.48 (3H, m), 1.24 (6H, m); EI-MS (m/z): 441.6[M+H].sup.+.

Example 14: (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 14)

(71) ##STR00074##

(72) Intermediate 9 and 2-hydroxy-1-naphthaldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 12.70 (1H, s), 12.18 (1H, s), 8.76 (1H, s), 7.70 (2H, m), 7.38 (8H, m), 3.61 (2H, s), 3.32 (2H, s), 2.75 (1H, m), 2.66 (2H, m), 2.52 (2H, m), 2.41 (3H, s), 2.29 (3H, s), 1.08 (6H, m); EI-MS (m/z): 447.4[M+H].sup.+.

Example 15: (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 15)

(73) ##STR00075##

(74) Intermediate 9 and 3,5-di-tert-butylsalicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.67 (2H, m), 7.78 (1H, s), 7.32 (3H, m), 7.23 (2H, m), 6.69 (1H, s), 3.57 (2H, s), 3.27 (2H, s), 2.87 (1H, m), 2.61 (2H, m), 2.43 (2H, m), 2.35 (3H, s), 2.25 (3H, s), 1.44 (9H, s), 1.27 (9H, s), 1.19 (6H, s); EI-MS (m/z): 509.6[M+H].sup.+.

Example 16: (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 16)

(75) ##STR00076##

(76) Intermediate 9 and 4-(diethylamino)salicylic aldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.40 (1H, s), 11.23 (1H, s), 7.61 (1H, s), 7.32 (5H, m), 6.55 (1H, m), 6.19 (1H, s), 6.10 (1H, m), 3.55 (2H, s), 3.37 (4H, m), 3.23 (2H, s), 2.90 (1H, m), 2.59 (2H, s), 2.50 (2H, s), 2.36 (3H, s), 2.18 (3H, s), 1.25 (12H, m); ELMS (m/z): 468.5[M+H].sup.+.

Example 17: (E)-N-(2,3-dihydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 17)

(77) ##STR00077##

(78) Intermediate 9 and 2,3-dihydroxybenzaldehyde were used as raw materials, and operations were the same for Example 1. White solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.91 (2H, m), 7.49 (1H, s), 7.32 (5H, m), 6.91 (1H, m), 6.68 (1H, m), 6.23 (1H, m), 3.55 (2H, s), 3.28 (2H, s), 2.91 (1H, m), 2.63 (4H, m), 2.42 (3H, s), 2.17 (3H, s), 1.26 (6H, m); EI-MS (m/z): 413.4[M+H].sup.+.

Example 18: (E)-N-(3-bromo-6-hydroxybenzal)-2-{N-{2-[N-(4-isopropylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 18)

(79) ##STR00078##

(80) Intermediate 9 and 3-bromo-6-hydroxybenzaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow liquid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 12.04 (1H, s), 11.31 (1H, s), 7.31 (6H, m), 6.84 (2H, m), 3.54 (2H, s), 3.29 (2H, s), 2.95 (1H, m), 2.64 (2H, m), 2.57 (2H, m), 2.45 (3H, s), 2.18 (3H, s), 1.24 (6H, m); ELMS (m/z): 477.3[M+H].sup.+.

Example 19: (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 19)

(81) ##STR00079##

(82) Intermediate 12 and ortho-vanilline were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.74 (1H, s), 11.38 (1H, s), 7.84 (1H, s), 7.28 (1H, m), 6.96 (5H, m), 6.52 (1H, m), 3.91 (3H, s), 3.80 (3H, s), 3.58 (2H, s), 3.28 (2H, s), 2.62 (4H, m), 2.38 (3H, m), 2.25 (3H, s); EI-MS (m/z): 415.4[M+H].sup.+.

Example 20: (E)-N-(3, 5-di-tert-butyl-2-hydroxybenzal)-3-(N-(2-(N-(4-tert-butylbenzyl)-N-methylamino)propyl)N-methylamino)acethydrazide (Compound 20)

(83) ##STR00080##

(84) Intermediate 12 and 3-ethoxysalicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.70 (1H, s), 11.26 (1H, s), 7.81 (1H, s), 7.27 (1H, m), 6.97 (4H, m), 6.75 (1H, m), 6.51 (1H, m, 4.13 (2H, m), 3.76 (3H, s), 3.56 (2H, s), 3.26 (2H, s), 2.60 (4H, m), 2.36 (3H, s), 2.23 (3H, s), 1.48 (3H, m); ELMS (m/z): 429.4[M+H].sup.+.

Example 21: (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 21)

(85) ##STR00081##

(86) Intermediate 12 and 2-hydroxy-1-naphthaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 12.70 (1H, s), 12.11 (1H, s), 8.76 (1H, s), 7.75 (2H, m), 7.31 (3H, m), 7.21 (4H, m), 6.82 (1H, m), 3.72 (3H, s), 3.61 (2H, s), 3.34 (2H, s), 2.72 (2H, m), 2.58 (2H, s), 2.46 (3H, s), 2.29 (3H, s); EI-MS (m/z): 435.5[M H].sup.+.

Example 22: (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 22)

(87) ##STR00082##

(88) Intermediate 12 and 3,5-di-tert-butylsalicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.64 (2H, m), 7.65 (1H, s), 7.32 (2H, m), 6.99 (2H, m), 6.82 (1H, m), 6.56 (1H, s), 3.74 (3H, s), 3.55 (2H, s), 3.28 (2H, s), 2.64 (4H, m), 2.43 (3H, s), 2.21 (3H, s), 1.44 (9H, s), 1.30 (9H, s); ELMS (m/z): 497.6[MH].sup.+.

Example 23: (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 23)

(89) ##STR00083##

(90) Intermediate 12 and 4-(diethylamino)salicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.29 (2H, m), 7.69 (1H, s), 7.30 (1H, m), 6.97 (3H, m), 6.65 (1H, m), 6.20 (2H, m), 3.78 (3H, s), 3.56 (2H, s), 3.36 (4H, m), 3.23 (2H, s), 2.59 (4H, m), 2.35 (3H, s), 2.22 (3H, s), 1.18 (6H, m); EI-MS (m/z): 456.2 [M H].sup.+.

Example 24: (E)-N-(2,3-dihydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 24)

(91) ##STR00084##

(92) Intermediate 12 and 2,3-dihydroxybenzaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.77 (2H, m), 7.55 (1H, s), 7.29 (1H, m), 6.99 (3H, m), 6.85 (1H, m), 6.75 (1H, m), 6.39 (1H, m), 3.76 (3H, s), 3.57 (2H, s), 3.29 (2H, s), 2.63 (4H, m), 2.41 (3H, s), 2.22 (3H, s); EI-MS (m/z): 401.3[M+H].sup.+.

Example 25: (E)-N-(3-bromo-6-hydroxybenzal)-2-{N-{2-[N-(3-methoxybenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 25)

(93) ##STR00085##

(94) Intermediate 12 and 3-bromo-6-hydroxybenzaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.81 (1H, s), 11.25 (1H, s), 7.34 (3H, m), 6.99 (7H, m), 3.80 (3H, s), 3.54 (2H, s), 3.28 (2H, s), 2.63 (4H, m), 2.44 (3H, s), 2.18 (3H, s); ELMS (m/z): 463.3[M+H].sup.+.

Example 26: (E)-N-[(2-hydroxynaphthalen-1-yl)methylene]-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 26)

(95) ##STR00086##

(96) Intermediate 15 and 2-hydroxy-1-naphthaldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 12.49 (1H, s), 11.46 (1H, s), 9.06 (1H, s), 7.76 (2H, m), 7.65 (2H, m), 7.55 (3H, m), 7.40 (2H, m), 7.22 (1H, m), 3.68 (2H, s), 3.33 (2H, s), 2.68 (2H, m), 2.55 (2H, m), 2.41 (3H, s), 2.29 (3H, s); ELMS (m/z): 430.4[MH].sup.+.

Example 27: (E)-N-(3,5-di-tert-butyl-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 27)

(97) ##STR00087##

(98) Intermediate 15 and 3,5-di-tert-butylsalicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.44 (1H, s), 11.11 (1H, s), 8.11 (1H, s), 7.65 (2H, m), 7.51 (2H, m), 7.36 (1H, s), 6.75 (1H, s), 3.63 (2H, s), 3.27 (2H, s), 2.64 (4H, m), 2.36 (3H, s), 2.24 (3H, s), 1.43 (9H, s), 1.30 (9H, s); ELMS (m/z): 492.5 [M H].sup.+.

Example 28: (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 28)

(99) ##STR00088##

(100) Intermediate 15 and o-vanilline were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.31 (1H, s), 11.10 (1H, s), 8.36 (1H, s), 7.65 (4H, m), 6.92 (2H, m), 6.70 (1H, m), 3.94 (3H, s), 3.66 (2H, s), 3.27 (2H, s), 2.61 (4H, m), 2.31 (6H, m); EI-MS (m/z): 410.5[M+H].sup.+.

Example 29: (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 29)

(101) ##STR00089##

(102) Intermediate 15 and 3-ethoxysalicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.26 (1H, s), 10.90 (1H, s), 8.29 (1H, s), 7.63 (4H, m), 6.90 (3H, m), 4.14 (2H, m), 3.65 (2H, s), 3.25 (2H, s), 2.59 (4H, m), 2.29 (6H, m), 1.48 (3H, m); EI-MS (m/z): 424.4[M+H].sup.+.

Example 30: (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{2-[N-(4-cyanobenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 30)

(103) ##STR00090##

(104) Intermediate 15 and 4-(diethylamino)salicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.13 (1H, s), 10.85 (1H, s), 7.96 (1H, s), 7.64 (4H, m), 6.75 (1H, m), 6.20 (2H, m), 3.63 (2H, s), 3.37 (4H, pi), 3.23 (2H, s), 2.60 (4H, m), 2.33 (3H, s), 2.23 (3H, s), 1.19 (6H, in); ELMS (m/z): 451.5[M+H].sup.+.

Example 31: (E)-N-(3-methoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-tert-butylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 31)

(105) ##STR00091##

(106) Intermediate 18 and o-vanilline were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.92 (1H, s), 10.41 (1H, s), 7.72 (1H, s), 7.42 (4H, m), 6.90 (2H, m), 6.40 (1H, m), 3.90 (3H, s), 3.57 (2H, s), 3.29 (2H, s), 2.63 (4H, m), 2.40 (3H, s), 2.21 (3H, s), 1.32 (9H, m); EI-MS (m/z): 441.5[M+H].sup.+.

Example 32: (E)-N-(3-ethoxy-2-hydroxybenzal)-2-{N-{2-[N-(4-tert-butylbenzyl)-N-methylamino]ethyl}N-methylamino}acethydrazide (Compound 32)

(107) ##STR00092##

(108) Intermediate 18 and 3-ethoxysalicylic aldehyde were used as raw material, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.86 (1H, s), 11.29 (1H, s), 7.69 (1H, m), 7.39 (4H, m), 6.87 (1H, m), 6.72 (1H, m), 6.38 (1H, m), 4.12 (2H, s), 3.56 (2H, s), 3.27 (2H, s), 2.61 (4H, m), 2.38 (3H, s), 2.18 (3H, s), 1.46 (3H, m) 1.32 (9H, m); ELMS (m/z): 455.5[M+H].sup.+.

Example 33: (E)-N-(4-N,N-diethylamino-2-hydroxybenzal)-2-{N-{3-[N-(4-tert-butylbenzyl)-N-methylamino]propyl}N-methylamino}acethydrazide (Compound 33)

(109) ##STR00093##

(110) Intermediate 21 and 4-(diethylamino)salicylic aldehyde were used as raw materials, and operations were the same for Example 1. Light yellow solid product was obtained. .sup.1H-NMR (400 MHz, CDCl3) : 11.13 (1H, s), 10.19 (1H, s), 8.10 (1H, s), 7.29 (2H, m), 7.23 (2H, m), 6.87 (1H, m), 6.18 (2H, m), 3.48 (2H, s), 3.33 (4H, m), 3.16 (2H, s), 2.52 (2H, m), 2.45 (2H, m), 2.33 (3H, s), 2.22 (3H, s), 1.74 (2H, m), 1.27 (9H, s), 1.17 (6H, m); ELMS (m/z): 496.2[M+H].sup.+.

(111) The compounds of the present invention were screened with EGFP-HIF-1-CHOhIR cell model to show pharmacological activity of inhibiting proline hydroxylase and thereby stabilizing HIF-1, which was illustrated as follows:

Experimental Example 1: Evaluation of Activity of the Compounds of the Present Invention in Stabilization of HIF-1

(112) The compounds were formulated with DMSO to form 30 mM stock solutions, and then formulated with analytical nutrient solution to form 2 working solutions, and seven final concentrations, 0.03, 0.1, 0.3, 1, 3, 10, 30 M, were used for screening and obtaining dose-effect relationship. Positive compound bipyridine was formulated with DMSO to form 100 mM mother solution, and then formulated with analytical nutrient solution to form 2 working solution. The positive compound bipyridine in final concentration of 100 M was used as control, and analytic nutrient solution with DMSO in a final concentration of 3 was used as solvent control.

(113) CHOhIR cells (purchased from Thermo Fisher Scientific) which stably expressed EGFP-HIF-1 fusion protein were cultured under 37 C. and 5% CO.sub.2 in F12 culture solution containing 0.5 mg/ml G418 and 10% FBS, transplanted in an amount of 0.810.sup.4 cells/100 l/well on a 96-well culture plate that could be subjected to fluorescence detection, and cultured under 37 C. and 5% CO.sub.2 for 18-24 h. The cells were washed with analytical nutritional solution in amount of 100 l/well, added with analytical nutritional solution in amount of 100 l/well, added with 2 drug in amount of 100 l/well, and each concentration was repeated in 3 wells in parallel way. After the cells were incubated under 37 C. and 5% CO.sub.2 for 3 h, 12% formaldehyde in amount of 100 l/well was added, fixation was carried out at room temperature for 30 min. Culture media was discarded, and the plate was washed with PBS twice, PBS containing 1 M Hoechst was used, staining was performed at room temperature for 1 h. Detection was performed by IN Cell Analyzer 1000 live cell imaging system. Detection conditions were: 20 objective lens, excitation wavelength Ex=460 nm, emission wavelength Em=535 nm, exposure 300 ms to detect blue-fluorescence of cell nucleus passage; excitation wavelength Ex=475 nm, emission wavelength Em=535 nm, exposure 500 ms to detect green-fluorescence EGFP of cytoplasm passage, and pictures were taken continuously in 5 fields of view for each well. IN Cell Analyzer 1000 Multitarget Analysis Module of GE Company was used for analyzing aggregation of green-fluorescence of HIF-1 in cell nucleus, and BP100 M treatment group was used as 100% activation of HIF-1.
Activation rate (%)=(luminance of cell nucleus of drug treatment groupluminance of cell nucleus of solvent control treatment group)/(luminance of cell nucleus of agonist BP treatment groupluminance of cell nucleus of solvent control treatment group)100%

(114) Among 3 wells for parallel detection of each concentration point, average value of 15 fields of view was used for calculation of activation rate.

(115) The results were shown in Table 2.

(116) TABLE-US-00002 TABLE 2 Effects of some example compounds in stabilization of HIF-1 activity (EC.sub.50) Example No. EC.sub.50 (M) 1 2.29 0.27 2 17.8 4.81 3 2.11 0.85 4 0.83 0.18 5 0.78 0.14 8 5.23 1.39 10 0.60 0.18 11 0.17 0.04 13 0.80 0.19 14 1.02 0.34 15 11.20 2.87 17 0.56 0.15 18 2.28 0.67 22 2.77 0.31 23 0.76 0.15 24 0.82 0.09 25 7.19 1.27 26 0.98 0.10 27 7.75 1.33 28 8.42 3.15 29 2.77 0.39 30 0.53 0.02 31 2.76 0.78 32 1.86 0.57 33 0.13 0.02 Bipyridine 41.35 1.60

(117) The results showed that the compounds of the present invention could effectively prevent HIF-1 from being degraded, had effects of stabilizing HIF-1 activity, and their efficiencies were far greater than that of the positive control bipyridine.

(118) The experimental method in this example was a well-known method for detecting degradation of HIF-1 with proline hydroxylase, and when a compound was confirmed to be able to stabilize HIF-1, it must inhibit proline hydroxylase by chelating Fe.sup.2+. Hence, the compounds of the present invention were also confirmed to have effects of inhibiting proline hydroxylase.

(119) In addition, when all of HIF-1, HIF-2 and HIF-3a depend on Fe.sup.2+ catalysis in proline hydroxylase degradation (Hirsila M, Koivunen P, Gunzler V, Kivirikko K I, Myllyharju J. Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor. J. Biol. Chem. 278(33), 30772-30780 (2003).), it could be also determined that the compounds of the present invention had effects of stabilizing HIF-2, HIF-3, or preventing them from being degraded.

(120) Although specific models for carrying out the invention were described in details, those skilled in the art would understand that these details can be modified and changed according to the teachings of disclosures, and all these changes fall into the protection scope of the present invention. The whole protection scope of the present invention is given by the appended claims and any equivalents thereof.