CN104224781B - The pyridine compounds and their and its new application of one group of 2- amide groups -3- alkoxy substitution - Google Patents

Abstract

The invention discloses the pyridine compounds and their preparation method of 2 amide groups, 3 alkoxy substitution and its applications in treatment and/or prevention of arterial Atherosclerotic cardiovascular disease.It is demonstrated experimentally that compound of the present invention or pharmaceutical composition, which have, raises or stablize ABCA1, SR BI/CLA 1, macrophage foam cell formation or enhancing Macrophage cholesterol outflow can be inhibited.Show that such compound has lipid-loweringing, norcholesterol effect, there is the application prospect for becoming the disease medicaments such as treatment reducing blood lipid, atherosclerosis and cardiovascular disease.

Description

The pyridine compounds and their and its new application of one group of 2- amide groups -3- alkoxy substitution

Technical field

The present invention relates to a kind of 2- amide groups -3- alkoxy pyridines class compounds, are applied to Atherosclerotic cardiovascular The treatment and/or prevention of disease, belong to pharmaceutical field；The present invention relates to the compounds to raise or stablize ABCA1, SR-BI/ CLA-1, lipid-loweringing, cholesterol-lowering drug purposes；The present invention further relates to the preparation method of the compound；The invention further relates to institute State the pharmaceutical composition of compound.

Background technology

Angiocardiopathy is the primary killers for endangering human health in developed country and most developing countries, in recent years With the raising of people's living standard, the incidence of cardiovascular and cerebrovascular disease is in apparent ascendant trend, and cardiovascular and cerebrovascular disease is dead The composition for accounting for population in the world death is died up to 1/3.Atherosclerosis (Atherosclerosis) is a variety of serious cardiovascular diseases The pathologic basis of sick (such as coronary heart diseases and angina pectoris, myocardial infarction, cerebral apoplexy).

Peripheral cells inner cholesterol dysbolism is the important pathogenesis of atherosclerosis, and excess cholesterol is outside liver Removing in tissue is to prevent and treat the committed step of atherosclerosis.High-density lipoprotein (HDL) will be in peripheral tissues Cholesterol transport be metabolized to liver or drained in the form of cholic acid again, this process is referred to as reverse cholesterol transport (RCT) 【1,2】.It is to treat the effective means of atherosclerosis that RCT and cholesterol, which exclude approach,.

The atherosclerosis drug therapy clinically applied at present includes vasodilator drug, adjustment hypolipidemic medicine, resists Antiplatelet drug etc..The Statins being widely used at present is the first-line drug for treating relevant disease, and mechanism of action is to inhibit body Interior HMG-CoA reductase activity inhibits the biosynthesis of cholesterol and enhancing peripheral cells to absorb cholesterol Ldl receptor adjusts access to realize antiatherosclerosis, but can only reduce by 20%~40% cardiovascular event, for a long time Take that also result in gastrointestinal dysfunction syndrome, fash, mental depression, rhabdomyolysis and hepar damnification etc. serious bad Reaction.Therefore, there is an urgent need to develop new cardiovascular drugs【3-5】.

Scavenger receptor BI (scavenger receptor class B type I, SR-BI) be functional HDL by The HDL receptor of body, people is referred to as CLA-1.SR-BI/CLA-1 is one of the main attemperator of HDL metabolism, Key effect is played during RCT, it is considered to be finds the potential new target drone of Novel cardiovascular drug【6】.The enhancing of RCT will have Conducive to AS lesions savings cholesterol reduce and lesion reverse, therefore can by increase SR-BI/CLA-1 expressions come Accelerate RCT, promotes the removing of body cholesteryl ester more than needed.

ATP binding cassette transporter bodies A1 (ATP-binding cassette transporter A1, ABCA1) is that ATP is combined One of cassette transporter body superfamily member is a kind of important to transport the cell metabolites such as various ions, lipid by the energy of ATP Protein called membrane transporters.ABCA1 phosphatide and free cholesterol can be transported to poor fat or the apolipoprotein A-1 without fat in mediated cell (apoA-I), to promote high-density lipoprotein (HDL) to generate, start reverse cholesterol transport process, extra fat is removed in body It plays a significant role during matter.ABCA1 gene mutations can cause serious HDL to lack syndrome-Tangier Disease (TD), patient with blood plasma HDL and apoA-I there is a serious shortage of increasing as main feature with tissue macrophages inner cholesterol ester, normal companion There are hypercholesterolemia, angiocardiopathy and AS【7】.To transgenic mice research shows that ABCA1 overexpressions make apoA-I The Cholesterol Efflux of mediation obviously increases, and plasma HDL levels significantly increase, and ABCA1 protein levels increase with Cholesterol Efflux Add and HDL levels increase apparent correlation.Therefore, SR-BI/CLA-1 and/or ABCA1 can be used as treatment hyperlipidemia and artery The novel targets of cardiovascular and cerebrovascular disease based on atherosis.SR-BI/CLA-1 and/or ABCA1 gene expressions or work(can be increased The compound of energy promises to be the effectively treatment angiocardiopathies newtype drug primer such as hyperlipidemia and atherosclerosis.

In short, there is also heavier side effects for the Statins medicine clinically used, still lack treatment artery at present The specific medicament of atherosis.For obtain tissue specificity it is stronger, by adjust RCT key receptors to promote outside cholesterol Row reduces lipid accumulation, to play prevention and/or treatment atherosclerosis newtype drug, utilizes the Chinese Academy of Medical Sciences Prescription sieve on the ABCA1 and SR-BI/CLA-1 of country of Institute of Medicinal Biological Technique new drug (microorganism) screening experiment room structure Modeling type is screened extensively, it is found that the pyridine compounds and their of one group of 2- amide groups -3- alkoxy substitution has apparent up-regulation The activity of ABCA1 and SR-BI/CLA-1, and determine confirm that it has the work of antiatherosclerosis angiocardiopathy in vitro Property.2- amide groups -3- alkoxies substitution pyridine compounds and their had no in terms of study of anti-atherogenic effect it has been reported that It is the discovery for the first time of present patent application.It the pyridine compounds and their of this group of 2- amide groups -3- alkoxy substitution and clinically uses Statins are compared, and structure does not have similitude, and play study of anti-atherogenic effect mechanism difference, are expected to become special Property adjust RCT key receptors, reduce lipid, reduce cholesterol, to as antiatherosclerosis or even Cardiovascular Medicine.Therefore, the pyridine compounds and their of this group of 2- amide groups -3- alkoxy substitution is likely to be with novel mechanism Antiatherosclerosis compound has wide development prospect.

Invention content

The pyridine compounds and their of -3 alkoxy of 2- amide groups substitution of the present invention is being prepared with upper mediator ATP combinations Application in the drug of cassette transporter body A1 (ABCA1) and/or scavenger receptor BI (CLA-1/SR-B1) expression activity, structure As shown in logical formula (I)：

Wherein, n=0-4 represents the linear paraffin base for including the atom number including hetero atom between 0-4；R1 represents H The alkyl of atom or C1-C4；The independent alkyl for representing C1-C3 of R2, R3, R4, the oxyl of C1-C3, halogen atom or H atom； The independent representatives of R5 include H, C1-C4 alkyl, C1-C3 alcoxyls (sulphur) base, halogen atom, the substitution of 1,3- dioxy (sulphur) pentamethylene base Phenyl ring, pyridine ring, pyrimidine ring, piperidine ring, piperazine ring, the cycloalkane of C3-C6,1,2,3- triazoles, 1,2,4- triazoles, pyrrole Cough up ring, furan nucleus, oxazole rings, imidazole ring, thiphene ring, thiazole ring and benzimidazole, benzothiazole, benzoxazole, benzofuran, Benzothiophene or indole ring.

In compound shown in formula (I), R1 is preferably H, and alkyl is preferably methyl or tertiary butyl, and alcoxyl (sulphur) base is preferably Trifluoromethoxy or trifluoromethylthio, the ring system representated by R5 are preferably phenyl ring, furan nucleus, pyrrole ring, imidazole ring or benzo Imidazole ring；N is preferably 0, that is, represents benzoic acids structure fragment.

Prepare the universal method using amides compound of the compound.It has main steps that, is replaced with different ring systems Carboxylic acid (III) and substitution 2- amino -3- alkoxy pyridines class compounds (II) be used as raw material, using EDCI as condensing agent, Under solvent appropriate and temperature condition, final synthesis obtains corresponding compound in logical formula (I), and general formula synthetic route is as follows：

Route and method as described above can be stablized, repeatable synthesize to obtain all chemical combination of the present invention Object.

Upper mediator ATP binding cassette transporter bodies A1 (ABCA1) of the present invention and/or scavenger receptor BI (CLA-1/SR- B1) expression drug is the drug for the treatment of and/or prevention of cardiovascular disease.

Upper mediator ATP binding cassette transporter bodies A1 (ABCA1) of the present invention and/or scavenger receptor BI (CLA-1/SR- B1) drug of expression activity is the drug for reducing Lipid pharmaceutical or reducing cholesterol.

Upper mediator ATP binding cassette transporter bodies A1 (ABCA1) of the present invention and/or scavenger receptor BI (CLA-1/SR- B1) drug of expression activity is the drug of antiatherosclerosis.

The present invention also provides the compositions of the compound to prepare with upper mediator ATP binding cassette transporter bodies A1 (ABCA1) and/or the application in the drug of scavenger receptor BI (CLA-1/SR-B1) expression activity.

Mediator ATP binding cassette transporter bodies A1 (ABCA1) and/or scavenger receptor on the composition of compound of the present invention The drug that BI (CLA-1/SR-B1) expressed or increased stabilizing active is the drug for reducing Lipid pharmaceutical or reducing cholesterol.

Mediator ATP binding cassette transporter bodies A1 (ABCA1) and/or scavenger receptor on the composition of compound of the present invention The drug of BI (CLA-1/SR-B1) expression activity is the drug of antiatherosclerosis.

Mediator ATP binding cassette transporter bodies A1 (ABCA1) and/or scavenger receptor on the composition of compound of the present invention The drug of BI (CLA-1/SR-B1) expression activity is treatment and/or prevention of cardiovascular disease medicament.

The various dosage forms of pharmaceutical composition of the present invention can be prepared, example according to the conventional production process of pharmaceutical field So that active constituent is mixed with one or more carriers, is then made into required dosage form.

The pharmaceutical composition of compound of the present invention, it is characterised in that it is with the compound containing therapeutically effective amount For active constituent, and contain one or more pharmaceutically acceptable carriers.

People ABCA1, CLA-1/SR-B1 and CLAp-3 ' UTR screening models that the present inventor is established using this laboratory are to special The activity of sharp compound is evaluated, and selection 0.1%DMSO is negative control, and 9CRA is positive control, measures all compounds Up-regulation rate under 10 μ g/ml concentration in model (ABCA1, CLA-1, CLAp-3 ' UTR).Multiple determination of activity experimental result It proves, all compounds of the present invention have up-regulation effect, the activity of part of compounds and survey on ABCA1CLAp-3 ' UTR models It is fixed that the results are shown in Table 1.Table 1 is only to help those skilled in the art to more fully understand the present invention, but do not limit in any way The present invention.

Present invention discover that LX-1 in logical formula (I), 3,5,7,10,19,20,22,23 wait with up-regulation ABCA1's and CLA-1 Effect.Wherein, especially LX-1, LX-3, LX-5, LX-7 shows the activity of good up-regulation ABCA1 and CLA-1, and provides Their applications in terms for the treatment of atherosclerosis develop into a kind of novel antiatherosclerosis for the analog derivative Drug is laid a good foundation.LX-1 on tri- models of ABCA1, CLA-1, CLAp-3 ' UTR can dose dependent up-regulation ABCA1, The expression of CLA-1, CLAp-3 ' UTR, EC50 are respectively 0.089,0.027 and 3.5 μ g/ml (Fig. 1).By LX-1 (0.24,1.2, 6.0 and 30.0 μ g/ml) RAW264.7 or HepG2 cells 18-24h, western blot is acted on the results show that the two are changed Closing object can be in the horizontal apparent expression for raising ABCA1 and SR-BI in RAW264.7 cells of albumen (Fig. 2).Thus it proves, the present invention The compound or composition can be used as the sub- ABCA1 or HDL receptor CLA-1/SR- of up-regulation ATP binding cassette transporters The drug of B1 expression activities.

In the present invention, the inhibition macrophage foam cell formation refers to utilizing people or mouse nascent generation or immortal monocyte After induced denaturation forms macrophage, a large amount of neutral fats for being formed after absorbing a large amount of denatured lipoprotein or elecrtonegativity phosphatide The accumulation of matter, a large amount of red fat drips formed after oil red O stain substantially reduce.When LX-1, LX-3, LX-5, LX-7 (compound Concentration is 10 μ g/ml) be respectively acting on mouse monokaryon-macrophage RAW264.7 of rich fat after, oil red O stain result table Bright, the compound can significantly reduce accumulation of the lipid in macrophage, have significant inhibition macrophage foam cell formation (figure 3) activity reduces lipid content.Thus it proves, compound or composition of the present invention can be as reduction lipid accumulation Fat-reducing medicament.

LX-1, LX-3, LX-5, LX-7 (compound concentration is 10, μ g/ml) are respectively acting on the small of Cholesterol Accumulation After mouse monocytes/macrophages RAW264.7, using apoA-I or HDL as cholesterol acceptor, scintiloscope measures cell respectively The content of interior and extracellular cholesterol calculates Cholesterol Efflux rate (ratio of intracellular and the total cholesterol of cell amount, total courage Sterol be intracellular and it is extracellular and).Compared with blank control, LX-1, LX-3, LX-5, LX-7 can promote Cholesterol Efflux to arrive Extracellularly, and cholesterol efflux amount can be increased to 150% or more, illustrates that LX-1, LX-3, LX-5, LX-7 have and promotes cholesterol The effect (Fig. 4) of outer row reduces cholesterol level.Thus it proves, compound or composition of the present invention can be used as and reduce The cholesterol lowering drug object of cholesterol.

ApoE-/- mouse high fat diet two months builds Atherosclerosis Model, and LX-1 is administered 2 months, blood fat result It has been shown that, LX-1 can significantly reduce the protein cholesterol (LDL-C) of Triglycerides in Serum (TG), total cholesterol (TC), low-density Horizontal (table 2).Thus it proves, compound or composition of the present invention can reduce cholesterol in animal body, can conduct Antiatherosclerosis cardiovascular drugs.

Advantageous effect of the invention

1) novelty of action target spot：Current study show that ABCA1 and CLA-1 are the novel targets of antiatherosclerosis, There is great influence in generation, the development of atherosclerosis, but there has been no the drug appearance for acting on this target spot.

2) novelty of compound：The present invention elaborates the pyridines chemical combination of 2- amide groups -3- alkoxies substitution for the first time Definite effect of the object in terms of artereosclerotic cardiovascular, and from the original for illustrating its anti arteriosclerosis effect from molecular mechanism for the first time Cause, this at home and abroad belongs to for the first time, this is for China's Antiatherosclerosis medicine of the exploitation with independent intellectual property right with weight The meaning wanted.Meanwhile the present invention also illustrates that by target spot of ABCA1 and CLA-1 new, effective anti-atherogenic can be screened The lead compound of hardening.

3) prevention and treatment of artereosclerotic cardiovascular disease：Vascular diseases especially atherosclerosis has seriously threatened To the life and health of the mankind, existing drug is early, and oneself cannot meet the needs of clinical, cannot fundamentally treat.Present invention discover that Compound in animal body have good anti-atherosclerotic effect, for exploitation anti arteriosclerosis drug provide very Good lead compound, has broad application prospects.

1 part of compounds of table is in 10 μ g/ml concentration to the rate of change of CLAp-3 ' UTR, ABCA1 and CLA-1/SR-B1

Influences (mg/dL) of the table 2LX-1 to high fat diet apoE-/- lipid of mice of high fat diet

Description of the drawings

Fig. 1 is amount effect relation curves of the LX-1 in ABCA1, CLA-1, CLAp-3 ' UTR screening models.

Fig. 2 is ABCA1 and SR-BI/CLA-1 expression in LX-1 increase HepG2 cells, and wherein LX-1 concentration is respectively 0.24,1.2,6.0 and 30.0 μ g/ml.

Fig. 3 compounds inhibit macrophage foam cell formation, wherein a：Blank control;b：Positive control (Ox-LDL)；C-f is successively For：LX-1, LX-3, LX-5, LX-7 (compound concentration is 10 μ g/ml).

Fig. 4 compounds promote Cholesterol Efflux, wherein LX-1, LX-3, LX-5, LX-7 to induce courage in RAW264.7 cells solid Alcohol flows out to ApoA-I, and compound concentration is 10.0 μ g/ml.

Specific implementation mode

The preparation embodiment of following part of compounds can make professional technician be more completely understood the present invention, but not with Any mode limits the present invention, and the structure of all compounds is determined through 1H NMR and MS (ESI).

Embodiment 1N- (3- hydroxyl -2- pyridyl groups) -4- (1,3- bis- thio -2- pentamethylene base) benzamide (LX-1) It prepares

According to the operating method of embodiment 1, with 0.23g (0.001mol) 4- (1,3- bis- thio -2- pentamethylene base) benzene first Acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feed intake, and obtain LX-1 sterling 0.31g white solids, yield 97%.MS (ESI, m/z)：319 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：10.568 (s, 1H, NH-CO), 9.856 (s, 1H ,-OH), 7.977 (m, 2H, Ph-H), 7.655 (m, 2H, Ph-H), 7.341 (dd, 1H, J=8.0Hz, 1.5Hz, Py-6H), 7.224 (dd, 1H, J=8.0Hz, 5.0Hz, Py-4H), 5.843 (s, 1H, SCHS), 5.337 (t, 1H, J=5.0Hz, Py-5H), 3.57 (m, 2H, CH2), 3.396 (m, 2H, CH2)

The preparation of embodiment 2N- (3- hydroxyl -2- pyridyl groups) -4- trifluoromethoxies benzamide (LX-3)

According to the operating method of embodiment 1, with 0.21g (0.001mol) 4- trifluoro-methoxy-benzoic acids and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feeds intake, and obtains LX-3 sterling 0.25g yellow solids, yield 84%.MS (ESI, m/z)： 299 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：8.151 (m, 1H, Ph-H), 7.960 (d, 1H, J=5.0Hz, Py- 6H), 7.530 (m, 1H, Ph-H), 7.326 (d, 1H, J=7.5Hz, Py-4H), 7.203 (m, 1H, NH-CO), 6.654 (s, 1H ,- OH), 5.337 (t, 1H, J=5.0Hz, Py-5H),

The preparation of embodiment 3N- (3- hydroxyl -2- pyridyl groups) -4- iodobenzamides (LX-5)

According to the operating method of embodiment 1, with 0.25g (0.001mol) 4- iodo-benzoic acids and 0.11g (0.001mol) 2- Amino -3- pyridine alcohol feeds intake, and obtains LX-5 sterling 0.36g white solids, yield 90%.MS (ESI, m/z)：341 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：7.957 (s, 1H, NH), 7.924 (m, 2H, Ph-2H), 7.650 (s, 1H, Py-6), 7.323 (m, 2H, Ph-2H), 7.199 (s, 1H, OH), 5.337 (m, 2H, Py-4,5H),

The preparation of embodiment 4N- (3- hydroxyl -2- pyridyl groups) -4- trifluoromethylthios benzamide (LX-7)

4- trifluoromethylthio benzoic acid 0.25g (0.0011mol) are dissolved in 30mL dichloromethane, 0.23g is continuously added After being stirred at room temperature 30 minutes, 2- amino -3- pyridine alcohol 0.11g (0.001mol) are added, room temperature is anti-in (0.0012mol) EDCI 12h is answered, the reaction was complete for TLC detections, stops reaction.Reaction solution uses saturated sodium bicarbonate solution, saturated common salt water washing, nothing successively Aqueous sodium persulfate is dried.It is evaporated off after solvent and elutes to obtain LX-7 sterling 0.27g white solids, yield 96% through silica gel column chromatography post separation. MS (ESI, m/z)：315 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：8.126 (m, 2H, Ph-H), 7.968 (d, 1H, J=5.0Hz, Py-6H), 7.885 (m, 2H, Ph-H), 7.340 (d, 1H, J=8.0Hz, Py-4H), 7.223 (m, 1H, NH-CO), 6.654 (s, 1H ,-OH), 5.337 (t, 1H, J=5.0Hz, Py-5H)

The preparation of embodiment 5N- (3- hydroxyl -2- pyridyl groups) -5- methylfuran -2- formamides (LX-9)

According to the operating method of embodiment 1, with 0.13g (0.001mol) 5- methylfuran -2- formic acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feeds intake, and obtains LX-9 sterling 0.32g brown solids, yield 89%.MS (ESI, m/z)： 357 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：10.239 (s, 1H, NH), 7.951 (d, 1H, J=4.5Hz), 7.381 (d, 1H, J=3.0Hz, Py-4H), 7.319 (d, 1H, J=8.0Hz, Py-6H), 7.202 (d, 1H, J=4.5Hz, 3-H), 7.186 (d, 1H, J=4.5Hz, 4-H), 6.657 (s, 1H, OH), 6.354 (d, 1H, 3.0Hz, Py-5H), 5.342 (m, 2H), 2.383 (s, 3H, CH3),

The preparation of embodiment 6N- (3- hydroxyl -2- pyridyl groups) -4- tert-butyl benzenes acetamide oxide (LX-11)

According to the operating method of embodiment 1, with 0.21g (0.001mol) 4- tert-butyl benzenes fluoroacetic acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feeds intake, and obtains LX-11 sterling 0.27g light yellow solids, yield 90%.MS (ESI, m/ z)：301 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：7.674 (d, 1H, J=7.0Hz, Ph-H), 7.445 (d, 1H, J =7.0Hz, Ph-H), 7.353 (d, 1H, J=5.0Hz, Py-6H), 7.251 (m, 2H, Ph-2H), 7.201 (m, 1H, NH), 7.036 (d, 1H, J=5.0Hz, Py-4H), 6.953 (d, 1H, J=5.0Hz, Py-5H), 6.558 (s, 1H, OH), 3.524 (s, 2H, CH2O), 2.006 (m, 9H, CH3*3)

The preparation of embodiment 7N- (3- hydroxyl -2- pyridyl groups) furans -3- formamides (LX-13)

According to the operating method of embodiment 1, with 0.11g (0.001mol) furans -3- formic acid and 0.11g (0.001mol) 2- Amino -3- pyridine alcohol feeds intake, and obtains LX-13 sterling 0.18g pink solids, yield 88%.MS (ESI, m/z)：205 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：10.556 (s, 1H, NH), 8.494 (s, 1H, Fu-2H), 7.977 (d, 1H, J= 3.5Hz, Fu-5H), 7.824 (s, 1H, OH), 7.351 (dd, 1H, J=8.0Hz, 1.5Hz, Py-4H), 7.226 (dd, 1H, J= 5.0Hz, 1.5Hz, Py-6H), 7.085 (d, 1H, J=1.0Hz, Fu-4H), 5.337 (t, 1H, J=5.0Hz, Py-5H)

The preparation of the chloro- 5- brombenzamides (LX-15) of embodiment 8N- (3- hydroxyl -2- pyridyl groups) -2-

According to the operating method of embodiment 1, with the chloro- 5- bromobenzoic acids of 0.25g (0.0011mol) 2- and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feeds intake, and obtains LX-15 sterling 0.31g yellow solids, yield 94.5%.MS (ESI, m/ z)：327 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：7.910 (s, 1H, Py-6H), 7.815 (s, 1H, 6-H), 7.705 (d, 1H, J=8.5Hz, 4-H), 7.514 (d, 1H, J=8.5Hz, 3-H), 7.320 (d, 1H, J=7.5Hz, Py-4H), 7.201 (m, 1H, NH-CO), 6.654 (s, 1H ,-OH), 5.337 (t, 1H, J=5.0Hz, Py-5H)

The preparation of embodiment 9N- (3- hydroxyl -2- pyridyl groups) -2,4- dibromo-phenoxies acetamide (LX-18)

According to the operating method of embodiment 1, with 0.31g (0.001mol) 2,4- dibromo-phenoxies acetic acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feeds intake, and obtains LX-18 sterling 0.37g light yellow solids, yield 92%.MS (ESI, m/ z)：401 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：7.809 (d, 1H, J=2.5Hz, 3-H), 7.515 (dd, 1H, J =9.0Hz, 2.5Hz, 5-H), 7.201 (br-s, 1H, Py-6H), 7.106 (br-s, 1H, Py-4H), 6.985 (d, 1H, J= 9.0Hz, 6-H), 6.656 (s, 1H ,-OH), 5.336 (t, 1H, J=5.0Hz, Py-5H), 4.822 (s, 2H, CH2)

Embodiment 10N- (3- hydroxyl -2- pyridyl groups) the chloro- 3- of 4- (N- allyl sulfonamides base) benzamide (LX-20) It prepares

According to the operating method of embodiment 1, with the chloro- 3- of 0.28g (0.001mol) 4- (N- allyl sulfonamides base) benzene first Acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feed intake, and obtain LX-20 sterling 0.35g white solids, yield 95%.MS (ESI,m/z)：368 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：9.847 (s, 1H, NH-SO2), 8.538 (s, 1H, OH), 8.254 (t, 1H, J=5.0Hz, Py-6H), 8.209 (d, 1H, J=9.0Hz, Py-4H), 7.964 (d, 1H, J=4.0Hz, 2- H), 7.838 (d, 1H, J=8.0Hz, 6-H), 7.343 (d, 1H, J=8.0Hz, 5-H), 7.229 (m, 1H, NH-CO), 5.680 (m, 1H ,=CH), 5.337 (t, 1H, J=5.0Hz, Py-5H), 5.145 (d, 1H, J=15.0Hz ,=CH), 5.007 (d, 1H, J= 10.0Hz ,=CH), 3.182 (d, 2H, J=5.0Hz, CH2)

Embodiment 11N- (3- hydroxyl -2- pyridyl groups) -4- (N- acetoxyl groups -2,6- lupetidine) benzamide (LX- 22) preparation

According to the operating method of embodiment 1, with 0.29g (0.001mol) 4- (N-2,6- lupetidine) phenoxy acetamide Yl benzoic acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feed intake, and obtain LX-22 sterling 0.35g yellow solids, yield 91%.MS (ESI, m/z)：384 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：10.539 (s, 1H, NH), 8.030 (m, 2H, J=8.5Hz, Ph-2H), 7.978 (d, 1H, J=4.5Hz, Py-4H), 7.346 (d, 1H, J=7.0Hz, Py-6H), 7.221 (dd, 1H, J=7.0Hz, 4.5Hz, Py-5H), 7.028 (m, 2H, J=8.5Hz, Ph-2H), 6.555 (s, 1H, OH), 5.335 (s, 2H, OCH2), 2.006 (m, 2H, CH2), 1.561 (m, 2H, CH2), 1.468 (t, 3H, J=6.5Hz, CH3), 1.163 (m, 2H, CH2), 0.888 (t, 3H, J=6.5Hz, CH3)

Embodiment 12N- (3- hydroxyl -2- pyridyl groups) -4- (5- trifluoromethyl -2- pyridines oxygroup) benzamide (LX-25) Preparation

According to the operating method of embodiment 1, with 0.28g (0.001mol) 4- (5- trifluoromethyl -2- pyridines oxygroup) benzene first Acid and 0.11g (0.001mol) 2- amino -3- pyridine alcohol feed intake, and obtain LX-25 sterling 0.36g white solids, yield 96%.MS (ESI, m/z)：376 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：8.137 (m, 2H, Ph-H), 7.975d, 1H, J= 5.0Hz, Py-6H), 7.846 (m, 2H, Ph-H), 7.643 (d, 1H, J=8.0Hz, Py-4 ' H), 7.546 (s, 1H, Py-6 ' H), 7.352 (d, 1H, J=8.0Hz, Py-4H), 7.226 (m, 1H, NH-CO), 6.852 (d, 1H, Py-3 ' H), 6.654 (s, 1H ,- OH), 5.336 (t, 1H, J=5.0Hz, Py-5H)

The preparation of embodiment 13N- (3- hydroxyl -4- chloro-2-pyridyls) -4- chlorobenzamides (LX-27)

According to the operating method of embodiment 1, with 0.16g (0.001mol) 4- chlorobenzoic acids and 0.15g (0.001mol) 2- Amino -4- chloro-3-pyridyl alcohol feeds intake, and obtains LX-27 sterling 0.25g light green solids, yield 88%.MS (ESI, m/z)：283[M] +, 1H NMR (500MHz, d6-DMSO) δ (ppm)：10.523 (s, 1H, NH), 7.978 (m, 2H, Ph-2H), 7.724 (d, 1H, J =5.0Hz, Py-5H), 7.656 (m, 2H, Ph-2H), 6.855 (d, 1H, J=5.0Hz, Py-6H), 6.524 (s, 1H, OH)

The preparation of embodiment 14N, N- bis- (3- hydroxyl -2- pyridyl groups)-Isosorbide-5-Nitrae-hexamethylene diformamide (LX-28)

According to the operating method of embodiment 1, with 0.17g (0.001mol) Isosorbide-5-Nitrae-cyclohexyldicarboxylic acids and 0.22g (0.002mol) 2- amino -3- pyridine alcohol feeds intake, and obtains LX-28 sterling 0.32g yellow solids, yield 89%.MS (ESI, m/z)： 357 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：7.912 (s, 1H, NH), 7.294 (d, 1H, J=7.5Hz, Py- 6H), 7.199 (s, 1H, NH), 7.166 (m, 1H, Py-5H), 6.656 (s, 1H, OH), 5.337 (t, 1H, J=6.0Hz, Py-6 ' H), 4.379 (5 ' H of t, 2H, J=6.0Hz, Py-4 '), 3.524 (s, 1H, OH), 3.438 (m, 1H, CH), 3.382 (m, 1H, CH), 2.006 (m, 4H, CH2CH2), 1.661 (m, 2H, CH2) 1.468 (m, 2H, CH2)

The preparation of embodiment 15N- (3- hydroxy-5-methyl oxygroup -2- pyridyl groups) -4- chlorobenzamides (LX-30)

According to the operating method of embodiment 1, with 0.16g (0.001mol) 4- chlorobenzoic acids and 0.14g (0.001mol) 2- Amino -5- methoxyl group -3- pyridine alcohol feeds intake, and obtains LX-30 sterling 0.26g light yellow solids, yield 93%.MS (ESI, m/z)： 279 [M]+, 1H NMR (500MHz, d6-DMSO) δ (ppm)：10.521 (s, 1H, NH), 7.976 (m, 2H, Ph-2H), 7.744 (d, 1H, J=1.5Hz, Py-6H), 7.656 (m, 2H, Ph-2H), 6.823 (d, 1H, J=1.5Hz, Py-4H), 6.524 (s, 1H, OH), 3.843 (s, 3H, OCH3)

Activity of 16 the compounds of this invention of embodiment to adjustment screening model on ABCA1

ABCA1-LUC HepG2 cells are inoculated in 5 × 104/hole in 96 porocyte culture plates, about 6h waits for that cell pastes After wall, the culture medium containing serum is removed, gently rinses that cell is primary with PBS, the MEM- without serum is added in each experimental port 200 μ l of EBSS culture mediums, then it is separately added into 2 μ l compound samples to be measured (final concentration 2.5 or 10 μ g/ml), final concentration 0.1% The hole of DMSO culture mediums is as blank control.37 DEG C are continued at, after cultivating 18-24h under the conditions of 5%CO2, PBS (200 holes μ l/) Board-washing 2 times, abandons PBS.Cell pyrolysis liquid (20 holes μ l/) (Promega) is added, after 15-30min, microscopically observation cell is split After solution is complete, luciferase (60 holes μ l/) is added, measures uciferase activity (microplate reader reading) immediately.

With following equation calculation sample to be tested to the rate of change of uciferase activity：

Rate of change (%)=A/B × 100

Wherein, A is that the cell fluorescence element enzymatic activity (RLU) measured after sample to be tested is added, and B is that blank control sample is added (DMSO) the cell fluorescence element enzymatic activity (RLU) measured afterwards.

Activity of 17 the compounds of this invention of embodiment to adjustment screening model on CLA-1

CLAP-LUC HepG2 cells are inoculated in 5104 cells/wells in 96 porocyte culture plates, about 6h waits for cell After adherent, the culture medium containing serum is removed, gently rinses that cell is primary with PBS, the MEM- without serum is added in each experimental port 200 μ l of EBSS culture mediums, then it is separately added into 2 μ l compound samples to be measured (final concentration 2.5 or 10 μ g/ml), final concentration 0.1% The hole of DMSO culture mediums is as blank control.37 DEG C are continued at, after being cultivated 18 hours under the conditions of 5%CO2, PBS (200 holes μ l/) Board-washing 2 times, abandons PBS.Cell pyrolysis liquid (20 holes μ l/) (Promega) is added, after 15-30min, microscopically observation cell is split After completely, luciferase (60 holes μ l/) is added, measures uciferase activity (microplate reader reading) immediately.

With following equation calculation sample to be tested to the rate of change of uciferase activity：

Rate of change (%)=A/B × 100

Wherein, A is that the cell fluorescence element enzymatic activity (RLU) measured after sample to be tested is added, and B is that blank control sample is added (DMSO) the cell fluorescence element enzymatic activity (RLU) measured afterwards.

Activity of 18 the compounds of this invention of embodiment to CLA-1p-3 ' UTR screening models

CLA-1p-3 ' UTR HepG2 cells are inoculated in 5104 cells/wells in 96 porocyte culture plates, about 6h is waited for After cell is adherent, the culture medium containing serum is removed, gently rinses that cell is primary with PBS, each experimental port is added without serum 200 μ l of MEM-EBSS culture mediums, then it is separately added into 2 μ l compound samples to be measured (final concentration 2.5 or 10 μ g/ml), final concentration The hole of 0.1%DMSO culture mediums is as blank control.37 DEG C are continued at, after being cultivated 18 hours under the conditions of 5%CO2, PBS (200 μ The holes l/) board-washing 2 times, abandon PBS.Cell pyrolysis liquid (20 holes μ l/) (Promega) is added, after 15-30min, microscopically observation is thin After born of the same parents are split completely, luciferase (60 holes μ l/) is added, measures uciferase activity (microplate reader reading) immediately.

With following equation calculation sample to be tested to the rate of change of uciferase activity：

Rate of change (%)=A/B × 100

Embodiment 19 inhibits macrophage foam cell formationization experiment

The monocytes/macrophages RAW264.7 of the mouse sugared culture solution adhere-wall cultures of the DMEM- high containing 10%FBS.Cell It is inoculated in 96 porocyte culture plates with 6 × 104/hole, in 37 DEG C, after being incubated overnight under the conditions of 5%CO2, is changed to serum-free DMEM- high glucose mediums (100 holes μ l/).Cell is divided into control group, foam cells group and sample-adding group (each final compound concentration 10 μ g/ml), the Ox-LDL of final concentration of 80mg/L is added to foam cells group and sample-adding group, sample-adding group will be added centainly simultaneously The sample to be tested of concentration.37 DEG C, after being cultivated for 24 hours under the conditions of 5%CO2, carry out oil red O stain.

96 orifice plates are taken out from CO2 incubators, 4% paraformaldehyde fixes (15 holes μ l/) 10min, abandons solution, distilled water is washed Twice, 60% isopropanol (150 holes μ l/) is added, 5min is placed, discards solution.Oil red O is added using liquid in each hole, 150 1h is dyed in the holes μ l/.Solution is discarded, with 60% isopropanol (150 holes μ l/) hole flushing, then washes two with distilled water (150 holes μ l/) It is secondary, it is last to add 150 μ l distilled waters to be placed in microscopically observation, take pictures per hole.

Embodiment 20 [3H] Cholesterol Efflux is tested

DMEM- high glucose mediums (500 μ l/ hole) of the mouse monokaryon-macrophage RAW264.7 containing 10%FBS, with 2 × It 105/hole, is inoculated in 24 porocyte culture plates, in 37 DEG C, is incubated overnight under the conditions of 5%CO2.

Cell liquid is abandoned, the DMEM- high glucose mediums (500 holes μ l/) containing 0.2% (w/v) BSA are changed to, 1,2- [3H] is added Cholesterol simultaneously makes its final concentration of 1 μ Ci/ml, 37 DEG C, is incubated for 24 hours under the conditions of 5%CO2.

Cell is washed with PBS (holes 1ml/) 2 times, and the measurement culture of compound containing a certain concentration (10 μ g/ml of final concentration) is added Base (0.2%BSA, 0.1%DMSO, 25mM HEPES, pH7.4 is added in DMEM), 37 DEG C of incubation 18-24h.

Cell is washed with PBS (holes 1ml/) 2 times, and culture medium is added, and (0.2%BSA, 0.1%DMSO, 25mM is added in DMEM HEPES, pH7.4), with or without the apoA-I of 10 μ g/ml, it is incubated 4h.

Culture medium is collected, 10000 × g centrifuges 5min, takes supernatant to be measured.

With 0.1M NaOH0.5ml lysis at room temperature cell 30min, it is to be measured to collect lysate.

It measures：Sample to be tested is transferred to respectively on 3MM filter paper, 75 DEG C of drying, the scraps of paper, which are placed on liquid, to be dodged in cup, is added 10ml liquid dodges liquid, and (mass concentration is 0.5%PPO (2,5- diphenyloxazole) and the 0.05%POPOP (bis- -2-15- phenyl evils of Isosorbide-5-Nitrae - Azoles benzene) with volume fraction be 55% dimethylbenzene and 45% glycol dimethyl ether solvent mixed preparing, be placed in brown container memory Put, use overnight), liquid scintillation counter counts.Entire experimental cell is divided into control group and (is not added with cholesterol but adds apoA-I, add Cholesterol) and sample-adding group (while cholesterol, apoA-I and certain density sample to be tested [15,20] is added).

Cholesterol Efflux rate %=culture solution cpm values/total cpm value × 100%

=culture solution cpm values/(culture solution cpm values+cell cpm values) × 100%

Pharmacodynamic evaluations of the embodiment 21LX-1 in apoE-/- Mice Body

ApoE-/- mouse, 7 week old, normal diet are fed one week；

ApoE-/- mouse is weighed, it is random to be grouped, be divided into 5 groups (model group, high dose group (10mg/Kg), middle dose Amount group (30mg/Kg), low dose group (90mg/Kg), negative control group, positive drug group (5mg/Kg)), every group of 6-8 is only；

Since 8 week old, model group and administration group feed high lipid food, and negative control group continues to feed normal diet；It fills Stomach is administered；

By administration or 8 weeks apoE- of carboxymethyl cellulose/- mouse fasting 6h；It plucks eyeball and takes blood, with heparin rinse EP pipes collect blood, turn upside down, be placed on ice, 4000rpm/min, 4 DEG C centrifugation 3min, by the serum of separation according to the world With illustrate to measure serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein The content of white cholesterol (LDL-C).The above detection kit is Chen Bei controls clinical reagent Products in Beijing.

Bibliography

[1]Fielding CJ and Fielding PE：Molecular physiology of reverse cholesterol transport.J Lipid Res1995；36：211-228.

[2]Oram JF and Yokoyama S：Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.J Lipid Res1996；37：2473-2491.

[3] Alan D.Attie, John P.Kastelein and Michael R.Hayden.Pivotal role of ABCA1in reverse cholesterol transport influencing HDL levels and susceptibito atherosclerosis.J Lipid Res2001；42：1717-1726.

[4] S Soumian, C Albrecht, AH Davies and RGJ Gibbs.ABCA1and atherosclerosis.Vascular Medicine2005；10：109-119.

[5]John F.Oram and Jay W.Heinecke.ATP-binding cassette transporter A1：a cell cholesterol exporter that protects against cardiovascular disease.Physiol Rev2005；85：1343-1372.

[6] Silver DL, Tall AR.The cellular biology of scavenger receptor class B type I.Curr Opin Lipidol2001；12(5)：497-504.

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Claims (6)

1. structure compound as shown in logical formula (I) is being prepared with upper mediator ATP binding cassette transporter body A1 and/or scavenger receptor Application in the drug of B1 expression activities,

（I）

In logical formula (I),

R1 is represented：H atom；

The independent representative of R2, R3, R4：H atom；

N=0,1,2；

And when n=0, R5 is：

（1）4- trifluomethoxybenzenes；

（2）4- iodobenzenes；

（3）4- trifluoromethylthio benzene；

（4）5- methyl -2- furans；

（5）3- furyls；

（6）The chloro- 5- bromophenyls of 2-；

（7）The chloro- 3- allyls amido sulfoamido benzene of 4-；

（8）N- benzene oxygen acetyl group -2,6- lupetidines；

（9）4- (5- trifluoromethyl -2- pyridines oxygroup) benzene；

（10）4- chlorobenzenes；

When n=1, R5 is：

（11）4- (1,3- bis- sulphur pentamethylene base) phenoxy group；

When n=2, R5 is：

（12）4- tertiary butyl Phenoxymethyls；

（13）2,4- dibromo-phenoxy methyl；

（14）N-（3- hydroxyl -2- pyridines）Cyclohexyl formamide.

2. application according to claim 1, which is characterized in that the drug is：

（1）The drug for the treatment of and/or prevention of cardiovascular disease；

（2）It reduces lipid or reduces the drug of cholesterol；

Or（3）The drug of antiatherosclerosis.

3. the composition comprising the compound described in claim 1 formula of (I) is being prepared with upper mediator ATP binding cassette transporters Application in the drug of body A1 and/or scavenger receptor B1 expression activities.

4. application according to claim 3, which is characterized in that the drug is：

（1）It reduces lipid or reduces the drug of cholesterol；

（2）The drug of antiatherosclerosis；

Or（3）Treatment and/or prevention of cardiovascular disease medicament.

5. the application described in claim 3 or 4, which is characterized in that the various dosage forms of composition according to pharmaceutical field routine It is prepared by production method.

6. the application described in claim 3 or 4, which is characterized in that the composition contains in the claim 1 of therapeutically effective amount Compound described in logical formula (I) is active constituent and one or more pharmaceutically acceptable carriers.