CN109758459A - The new application of substituted pyridine - Google Patents

Abstract

The present invention relates to the new applications of substituted pyridine, and in particular to formula (I) compound represented or its officinal salt are preparing the purposes in the drug for treating hypertension caused by hypertension especially diabetes, wherein R₁For by C_1‑10- the CH that alkyl optionally replaces₂Or by C_1‑10- the NH- that alkyl optionally replaces；And R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1‑10Alkyl.

Description

The new application of substituted pyridine

Technical field

The present invention relates to a kind of new applications of substituted pyridine.

Background technique

N2-1H- indazole -5- base-N6- methyl-3-nitro -2,6- pyridine diamines is commonly referred to as KRIBB11, and molecular weight is 284.27g/mol is the inhibitor of known Heat shock factor1 (HSF1), and structural formula is as follows:

Studies have shown that the compound inhibits the activity of HSF1 in a manner of concentration dependent.It can lower HSP70 and HSP27 can inhibit the proliferation of cancer cell, will prevent the cell cycle in G2/M phase and apoptosis-induced.

It to the hsp70 promoter of heat-inducible to the no inhibiting effect of the recruitment of HSF1, to the 230th serine of HSF1 Phosphorylation also without inhibiting effect, but inhibit the hsp70 promoter of heat-inducible to the recruitment of pTEFb and depend on p- The phosphorylation of the 2nd serine of polymerase II CTD of TEFb.

PARP and caspase-3 cracking increases in the cell handled with KRIBB11.RKO and KRIBB11 are incubated with, Show that about 10 μM of toxicity threshold, IC50 are 20-30 μM.

To sum up, the effect for KRIBB11 in terms for the treatment of tumour is existing in the prior art largely reports.

However, there is no the reports about the compound in terms for the treatment of other diseases in the prior art.

Diabetic angiopathy is the highest disease of lethality in diabetes B complication, can directly induce heart infarction and The generation of the acute cardiovascular and cerebrovascular disease such as cerebral infarction.It probes into pathogenesis therein and how to alleviate hypertension and Atherosclerosis Change etc., it is important scientific problems urgently to be resolved.

FAM3A is a new mitochondrial protein, the present inventor's early-stage study discovery, by promoting ATP synthesis secretion to swash P2Y receptor living, and then activate PI3K-Akt and ERK1/2 signal transduction pathway stimulated vascular smooth muscle cell (Vascular Smooth Muscle Cells, VSMCs) it is proliferated and migrates.When injury of blood vessel, prostaglandin E2 receptor EP2 expression is obvious Increase, inhibit FAM3A expression, prevent VSMCs hyper-proliferative and neointimal hyperplasia, thus to prevention and treatment atherosclerosis and Postangioplasty restenosis important in inhibiting.

Hypertensin 2 (AngiotensinII, AngII) and homocysteine (Homocyteine, Hcy) are in glycosuria Disease often increases when occurring, and is the Important cause of disease for leading to diabetic angiopathy.Whether FAM3A has intervened AngII and Hcy is lured The vascular smooth muscle cell proliferation migration led and vascular remodeling process, are also not yet disclosed.

Summary of the invention

In one aspect, this disclosure relates to formula (I) compounds represented or its officinal salt to prepare for treating Purposes in the drug of hypertension caused by hypertension especially diabetes,

Wherein:

R₁For by C_1-10- the CH that alkyl optionally replaces₂Or by C_1-10- the NH- that alkyl optionally replaces；And

R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1-10Alkyl.

In another aspect, this disclosure relates to pharmaceutical composition, it includes formula as described above (I) compound or its Officinal salt and pharmaceutically acceptable carrier, the pharmaceutical composition is for treating high blood caused by hypertension, especially diabetes Pressure.

In another aspect, this disclosure relates to treat the method for hypertension caused by hypertension especially diabetes, It includes applying formula (I) compound or pharmaceutically acceptable salt thereof as described above or pharmaceutical composition to individual in need.

Detailed description of the invention

It hereafter will only by way of example and the embodiment that describes the application with reference to the accompanying drawings, in which:

Figure 1A shows Loxp and FAM3A when not being transfused AngII^VSMC-/-The blood pressure of mouse.

Figure 1B show infusion AngII 28 days after Loxp and FAM3A^VSMC-/-The blood pressure of mouse.

Before Fig. 1 C shows intraperitoneal injection KRIBB11 or control DMSO, the blood pressure of two groups of SHR rats.

Fig. 1 D shows intraperitoneal injection KRIBB11 or after control DMSO 7 day, the blood pressure of two groups of SHR rats.

Fig. 1 E, which is shown, to be injected intraperitoneally before KRIBB11 or control DMSO and after 7 days, the heart rate of two groups of SHR rats.

The endothelium-dependent relaxation that Fig. 2A shows the mouse aorta pectoralis of PE induction is shunk.

Fig. 2 B shows preincubate L-NAME (no inhibitor, 100 μM) the mouse aorta pectoralis that PE is induced after 1 hour Endothelium dependent/non-dependent is shunk.

Fig. 2 C shows the endothelium-dependent relaxation of the mouse aorta pectoralis of Ach induction.

Fig. 2 D shows preincubate L-NAME (no inhibitor, 100 μM) the mouse aorta pectoralis that SNP is induced after 1 hour Endothelium dependent/non-dependent diastole.

The endothelium-dependent relaxation that Fig. 2 E shows the SHR rat of PE induction is shunk.

Fig. 2 F shows the endothelium dependent/non-dependent diastole of the SHR rat of SNP induction.

Fig. 2 G shows the endothelium-dependent relaxation of artery on the SHR rat mesentery of Ach induction.

Fig. 3 A shows the immunofluorescence (400X) of aorta pectoralis in SHR and SD rat, which is by DAPI (dye core), α- The fluorogram of SMA (dye myofilament) and HSF1 three merge the picture of display.

Fig. 3 B shows the immunofluorescence (400X) of superior mesenteric artery in SHR and SD rat, which is by DAPI (dye Core), the fluorogram of α-SMA (dye myofilament) and HSF1 three merge the picture shown.

Fig. 4 A is shown in Primary rat VSMCs, under Ang II and Hcy (Bioss) processing of various concentration, FAM3A, The representative figure of EP2 protein expression and Akt activation levels.

Fig. 4 B is shown in Primary rat VSMCs, and the Ang II and Hcy of various concentration handle lower FAM3A, EP2 albumen The statistical chart of expression and Akt activation levels.

When Fig. 4 C shows plasmid overexpression HSF1, when adding or GW9662 (PPAR gamma inhibitors, 10 μM) are not added, primary The representative figure and statistical chart of FAM3A protein expression and Akt activation levels in rat VSMCs.

Fig. 4 D is shown under (0.5 μM) of Ang II processing, when adding or being not added KRIBB11 (HSF1 inhibitor, 3 μM) The representative figure and statistical chart of FAM3A, PPAR γ protein expression level.

Fig. 4 E is shown under free serum culture, and suramin (P2 acceptor inhibitor, 100 μM) handles institute after Primary rat VSMCs The variation of caused cell Proliferation.

Fig. 5 A shows Ad-GFP, Ad-FAM3A or Ad-FAM3A+ suramin processing or primary big without any processing Free Ca in mouse VSMCs²⁺It is horizontal.

Fig. 5 B shows the processing of Ang II or Ang II+ suramin or the middle reaches Primary rat VSMCs without any processing From Ca²⁺It is horizontal.

Fig. 5 C shows Ang II or Ang II+KRIBB11 processing or the middle reaches Primary rat VSMCs without any processing From Ca²⁺It is horizontal.

Fig. 6 shows the mechanism general introduction figure that FAM3A participates in blood pressure control.

Specific embodiment

In the following description, including certain concrete details are to provide comprehensive reason to each disclosed embodiment Solution.However, those skilled in the relevant art are not, it will be recognized that use one or more of these concrete details, and use other Embodiment can be achieved in the case where method, component, material etc..

Unless required in addition of the application, in entire disclosure and claims, word " comprising " should be interpreted that out Formula, meaning including formula are put, i.e., " including but not limited to ".

" embodiment " mentioned in entire this specification or " embodiment " or " in another embodiment " or " in certain embodiments " mean include in an at least embodiment it is relevant to described in the embodiment with specific reference to Element, structure or feature.Therefore, throughout the specification different location occur phrase " in one embodiment " or " in reality Apply in scheme " or " in another embodiment " or " in certain embodiments " same embodiment need not be all referred to.In addition, Key element, structure or feature can combine in one or more embodiments in any suitable manner.

It is also understood that term "or" is usually with it includes the meaning of "and/or" and uses, unless in text in addition clearly Regulation.

Definition

Name herein is indicated by the simplification symbol of the total number of carbon atoms for showing to find in shown chemical group in front Certain chemical groups.For example, C_1-10It is 1 to 10 carbon atom such as undefined alkyl that alkyl description, which has sum,.Simplify The total number of carbon atoms and the carbon that is likely to be present in the substituent group of the group is not included in symbol.

Therefore, unless otherwise opposite explanation, otherwise following term used in specification and claim has following The meaning:

" alkyl ", which refers to, to be only made of carbon and hydrogen atom, without unsaturated bond, and by the rest part of singly-bound and molecule Connected linear chain or branched chain hydrocarbon chain radical, such as methyl, ethyl, n-propyl, 1- Methylethyl (isopropyl), normal-butyl, positive penta Base, 1,1- dimethyl ethyl (tert-butyl) etc..

In one aspect, this disclosure relates to formula (I) compounds represented or its officinal salt to prepare for treating Purposes in the drug of hypertension caused by hypertension especially diabetes,

Wherein:

R₁For by C_1-10- the CH that alkyl optionally replaces₂Or by C_1-10- the NH- that alkyl optionally replaces；And

R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1-10Alkyl.

In some specific embodiments, R₁For by C_1-6- the CH that alkyl optionally replaces₂Or by C_1-6Alkyl optionally takes - the NH- in generation.

In some specific embodiments, R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1-6Alkane Base.

In some more particular embodiments, R₁For by C_1-3- the CH that alkyl optionally replaces₂Or by C_1-3Alkyl is optional - the NH- replaced.

In some more particular embodiments, R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1-3 Alkyl.

In some more particular embodiments, R₂For C_1-3Alkyl.

In some more particular embodiments, R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is H.

In some more particular embodiments, R₁For-NH-, R₂For-CH₃, and R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀ It is H.

In another aspect, this disclosure relates to pharmaceutical composition, it includes formula as described above (I) compound or its Officinal salt and pharmaceutically acceptable carrier, described pharmaceutical composition is for treating height caused by hypertension, especially diabetes Blood pressure.

Term " officinal salt " refers to the salt for retaining the expectation biological activity of above compound, including pharmaceutically acceptable acid addition salts And base addition salts.The suitable pharmaceutically acceptable acid addition salts of formula (I) compound can be prepared by inorganic acid or organic acid.These nothings The example of machine acid is hydrochloric acid, sulfuric acid and phosphoric acid.Suitable organic acid can be selected from aliphatic, cycloaliphatic, aromatic series, heterocyclic carboxylic acid And sulfonic classes of organic acids, the example are formic acid, acetic acid, propionic acid, succinic acid, hydroxyacetic acid, gluconic acid, lactic acid, malic acid, wine Stone acid, citric acid, fumaric acid, maleic acid, alkyl sulfonic acid, aryl sulfonic acid.The suitable Pharmaceutically acceptable base addition salts of formula (I) compound It is prepared including the metal salt prepared by lithium, sodium, potassium, magnesium, calcium, aluminum and zinc, and by organic base such as choline, diethanol amine, morpholine Organic salt.Other examples of organic salt are ammonium salt, quaternary salt such as tetramethyl ammonium；Amino acid addition salt for example with glycine and Salt formed by arginine.The other information of officinal salt is found in Remington ' s Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995.In the case of these substances are solid, ability Field technique personnel it should be understood that the compounds of this invention and salt can be different crystal or polymorphic exist, all these shapes Formula, which is intended to, to be fallen within the scope of the present invention and specific formula.

Pharmaceutical composition can be granule, powder, tablet, coated tablet, capsule, suppository, solution, syrup, Fruit juice, suspension, emulsion, drops, injectable solutions etc..For example, in order to be prepared into tablet or capsule, it can be by active constituent It is combined with nontoxic pharmaceutically acceptable inert carrier such as ethyl alcohol, glycerol, water etc..In addition, it is if desired or necessary, it may include foot Enough adhesive, lubricant, disintegrating agent or colorants.Described adhesive may include starch, gelatin, natural sugar (such as glucose or Beta lactose), corn sweetener, natural or synthetic glue (such as gum arabic, bassora gum) or enuatrol, odium stearate, stearic acid Magnesium, sodium benzoate, sodium acetate, sodium chloride etc., but it is not limited to these.Disintegrating agent may include starch, methylcellulose, agar, Bentonite, xanthan gum etc., but it is not limited to these.

When composition is prepared into liquid solution, the pharmaceutically acceptable carrier of sterilizing is suitble to can be selected from salt water, nothing Bacterium water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerol, ethyl alcohol or its Mixture.If desired, composition may include other representative additives, such as antioxidant, buffer or bacteriostatic agent.In addition, Diluent, dispersing agent, surfactant, adhesive or lubricant can be added additionally composition is prepared into injection (such as water Solution, suspension or emulsion), pill, capsule, granule or tablet.

In addition, can be according to Remington ' s Pharmaceutical Science (Mack Publishing Company, Easton, PA) disclosed in method composition is prepared by suitable form according to specified disease or component.

The pharmaceutical composition of present disclosure oral or parenteral can be applied.It, can intravenous, skin when parenteral administration Under, in intramuscular, abdomen or transdermal administration.It specifically, can oral administration.

The sufficient dosage of the pharmaceutical composition of present disclosure, such as preparation method can be determined according to many factors, applied Method, age, weight and the gender of patient, pathological state, diet, administration time, administration method, excretion rate and response are sensitive Degree.Those skilled in the art can readily determine that for expectation prevention or treat effective dosage.The one of present disclosure In a exemplary implementation scheme, the daily dose of the pharmaceutical composition of present disclosure is 0.001g/kg to 10g/kg.

Pharmaceutically acceptable carrier and/or excipient can be used the disclosure according to method usually used in this field The pharmaceutical composition of content is prepared into single dose form or multiple dose form.Preparation can be the shape of oil or the solution in aqueous medium The form of formula, suspension, emulsion, extract, powder, granule, tablet or capsule, and also may include dispersing agent or stabilization Agent.

Comprising formula (I) compound as active constituent composition can oral, rectum, in intravenous, intra-arterial, abdomen, flesh In interior, breastbone, percutaneous, part, intraocular or intradermal administration.

In another aspect, this disclosure relates to treat the method for hypertension caused by hypertension especially diabetes, It includes applying formula (I) compound or pharmaceutically acceptable salt thereof as described above or pharmaceutical composition to individual in need.

Hereinafter, the application more fully understands each side of the invention by being explained in detail by following examples Face and its advantage.It will be appreciated, however, that embodiment below, which is non-limiting, is simply used for illustrating certain realities of the invention Apply scheme.

Embodiment

Abbreviation:

AngII: Angiotensin II；

FAM3: the type cytokines family that protein sequence similarity is 3；

FAM3A^VSMC-/-Mouse: FAM3A smooth muscle specific knockdown mouse；

DMSO: dimethyl sulfoxide；

SHR rat: spontaneous hypertensive rat；

PE: phenylpropyl alcohol adrenaline；

Ach: acetylcholine；

SNP: sodium nitroprussiate；

L-NAME:L- L-NAME；

PFA: paraformaldehyde；

PBS: phosphate buffered saline solution；

OCT:Optimum Cutting Temperature, optimum Cutting temperature；

DAPI:4,6 '-diamidinos -2-phenylindone；

FBS: fetal calf serum；

S:Suramin, suramin；

Hcy: homocysteine；

VSMC: vascular smooth muscle cells.

Ordinary test scheme

Animal

Use C57BL/6 background, male, the FAM3A in 8 to 12 weeks^VSMC-/-Mouse and Loxp mouse without any processing. FAM3A is constructed in Cyagen Biosciences Inc (China) using Cre-Loxp technology^VSMC-/-Mouse.

Use male, Wistar the and SHR rat that weight is 150 to 200g.

Mouse and rat are housed in standart animal test room, temperature is maintained at 24 DEG C, and carries out artificial 12 hours Cycle light and dark does not limit food and drinking-water.

All animal cares and experimental program meet Ministry of Health of the People's Republic of China's the care of animal rule and Beijing is big Learn experimental animal nursing and guide for use.

The implantation of permeability mini-pump and AngII infusion

Pass through Alzet osmotic mini-pump (Alzet Model#2004；Durect；Cupertino, CA, the U.S.) subcutaneously infuse Enter AngII (1,000ng/kg/min；Cat#H-1706；Bachem；Torrance, CA, the U.S.).It, will be micro- before being subcutaneously implanted Type pump is incubated for 24 hours in 37 DEG C of physiological saline.In Loxp and FAM3A^VSMC-/-AngII (1000ng/kg/ is transfused in mouse min).By mouse amobarbital calmness, and the back for being subcutaneously implanted every mouse will be pumped, subsequent experimental is carried out after 28 days.Make Cutting part is closed with surgical staples.

SHR rats by intraperitoneal injection KRIBB11

Male, the SHR rat (12) that weight is 200g are divided into two groups, two groups are not significantly different in terms of blood pressure. KRIBB11 (HSF1 inhibitor, 5mg/kg/day is injected intraperitoneally in experimental group；Cat#S8402；Selleck), sham-operation group abdominal cavity It injects equivalent DMSO (Sigma), continuous injection 7 days.

Embodiment one, tail sleeve method measuring blood pressure

Laboratory apparatus

CODA, Kent, USA (U.S. Kent company animal non-invasive blood pressure measuring system CODA)

Experimental method

Loxp mouse and FAM3A are measured by tail sleeve method^VSMC-/-Mouse bury pump before and bury pump 28 days after blood pressure and the heart Rate.Pass through the blood pressure and heart rate before tail sleeve method measurement SHR rats by intraperitoneal injection KRIBB11 and after continuous injection 7 days.When mouse and When rat is stablized, blood pressure and heart rate are measured.Take the average value of 10 measurements.

Experimental result is shown in Figure 1A -1E

It can be seen in fig. 1 that in physiological conditions, FAM3A^VSMC-/-The blood pressure of mouse is significantly lower than Loxp mouse.

It can be seen from figure 1b AngII (1000ng/kg/min) buried pump after 28 days, FAM3A^VSMC-/-The systolic pressure of mouse It is substantially less than control group with mean arterial pressure, and does not have significant difference between two groups of diastolic pressure.

Before intraperitoneal injection be can be seen that from Fig. 1 C, the blood pressure of two groups of SHR rats does not have significant difference.

It can be seen that the DMSO solution (5mg/kg/ of continuous intraperitoneal injection KRIBB11 (KRI is abbreviated as in figure) from Fig. 1 D It) after 7 days, control group of the processing group blood pressure significantly lower than injection DMSO.Although compared with the 0th day figure C integral level on It rises, but after be in injection together compared with the 7th day control group, KRIBB11 still significantly reduces blood pressure.

It can be seen that intraperitoneal injection front and back from Fig. 1 E, the heart rate between two groups of SHR rat does not have notable difference.

Embodiment two, antiotasis experiment

By injecting anaesthetized with pentobarbital adult male mice [FAM3A^VSMC-/-(5), Loxp (5)] and SHR rat (5) take out artery on mouse aorta pectoralis and SHR rat mesentery, be placed in it is pre- first pass through binary gas 30 minutes, pre-cooling Krebs solution in: 119mmol/L NaCl, 4.7mmol/L KCl, 2.5mmol/L CaCl₂、1mmol/L MgCl₂、 25mmol/LNaHCO₃、1.2mmol/L KH₂PO₄With 11mmol/L D-Glucose.It removes the adipose tissue of artery adherency and cuts At the ring segment of 2mm long.Pay special attention to avoid damage to endothelium during shelling blood vessel.The aorta pectoralis of mouse, with and without It is incubated for 10 minutes in the case where AngII (100nmol/L), and the superior mesenteric artery of SHR rat, with and without KRIBB11 (HSF1 inhibitor, 12 μm of ol/L；Cat#S8402；Selleck it is incubated for 6 hours in the case where).All arteries are being contained 10% It is incubated in DMEM (Gibco, Grand Island, NY) culture medium of FBS (Gibco), 100IU penicillin and 100 μ g/mL streptomysins It educates, is placed in 95%O₂+ 5%CO₂CO₂It is cultivated in incubator.Vascular circle is suspended on myograph The variation of isometric tension is recorded in (MyoTechnology, Alhuse, Denmark).In short, by 2 steel wires (40 μm of diameter) It is inserted into the inner cavity of container, and each wire is fixed on the jaw of instrument.The addition 5mL Krebs solution in cell, and 37 DEG C of (pH=7.4), 95%O₂+ 5%CO₂It is cultivated under environment.Each vascular circle zeroing post-tensioning is extended into this initial tension of 3mN, Then stablize 30 minutes before each experiment starts.

By the way that stabilization and repeatable contraction caused by potassium chloride (KCl, 60mM) is added, to check vasoactive.So It is washed three times with Kreb liquid afterwards, and carries out 30 minutes balances.Then, by the phenylpropyl alcohol adrenaline of these arteries and accumulation addition (10^-9To 10^-5Mol/L) (every kind of concentration each 3 minutes or so) are incubated with, and record in each container blood vessel in each concentration Under antiotasis.After reaching maximum and stable contraction, acetylcholine (10 is added by accumulation^-9To 10^-5Mol/L) make Angiogenic endothelial dependence diastole.Utilize sodium nitroprussiate (10^-9To 10^-5Mol/L the diastole of endothelium dependent/non-dependent) is studied.

Experimental result is shown in Fig. 2A -2G.

It can be seen that from Fig. 2A to 2D and the endothelium-dependent relaxation or dependent/non-dependent of PE induction shunk, FAM3A^VSMC-/-It is small The convergent force of the aorta pectoralis of mouse is substantially less than Loxp mouse, and the vasodilation power between two groups does not have significant difference.

It can be seen that compared with the control group from Fig. 2 E, KRIBB11 (12 μM) can inhibit artery on SHR rat mesentery Vessel retraction power.

It can be seen that compared with the control group from Fig. 2 F, KRIBB11 (12 μM) can improve artery on SHR rat mesentery Arterial dilation.

It can be seen that from Fig. 2 G when Ach concentration is 10^-8M and 10^-7When M, compared with the control group, KRIBB11 (12 μM) can To improve the arterial dilation of SHR rat.Since SHR rat blood vessel endothelium itself is had damage, Ach is lured instead in higher concentrations Lead contractile response.

Embodiment three, immunofluorescence

Wistar the and SHR rat aorta for being first 200g with PBS perfusion male, weight, then uses 4%PFA perfusion again, Blood vessel is peeled after perfusion is complete, is fixed overnight in 4%PFA in 4 DEG C, is then transferred in 30% sucrose and is incubated at 4 DEG C Until tissue sinks (3 days or so).Then blood vessel is embedded in OCT glue (Tissue-Tek) and frozen section is for dyeing. Nonspecific binding site is closed using 10% lowlenthal serum, is closed 1 hour.Then it is incubated with following antibody: mouse anti alpha- SMA (1: 200, Abcam), rabbit-anti HSF1 (1: 200, Abcam), at 4 DEG C overnight.It second day, will after being washed three times with PBS Blood vessel and secondary antibody: FITC- goat anti-rabbit igg (Invitrogen) and CY3- goat anti-mouse IgG (Invitrogen) exist It is incubated at 37 DEG C 2 hours, then marks nucleus with DAPI (Invitrogen).Fluorescent image is obtained with Laser Scanning Confocal Microscope.

Experimental result is shown in Fig. 3 A and 3B.

As can be seen from Figure 3A, the expression of SHR rat chest aorta HSF1 is significantly higher than SD rat.It can be with from Fig. 3 B Find out, the expression of artery HSF1 is significantly higher than SD rat on SHR rat mesentery.Therefore, suppression of the KRIBB11 as HSF1 Preparation can be used as the potential drug for the treatment of hypertension.

Example IV, cell experiment

Rat primary vascular smooth muscle cells culture

Aortic smooth muscle cell is separated from male SD (Sprague Dawley) rat, and is having 20%FBS, 2mM It is cultivated in the DMEM culture medium of L-Glutamine, 100U/mL penicillin and 10mg/mL streptomysin.Culture medium updates weekly twice. All experimental procedures are in CO₂Incubator (37 DEG C of temperature, 95%O₂+ 5%CO₂In environment) culture.

Western blotting

Using the lysis buffer containing fresh protein enzyme and inhibitors of phosphatases (Beijing Puli's Lay company) from rat serum Protein is extracted in pipe and cell.By cell lysate and homogenate with 12000rpm centrifugation 10 minutes at 4 DEG C.Use BCA Protein Assay Kit quantifies the protein content in supernatant.By SDS-PAGE separating sample proteins and it is transferred to nitre Acid cellulose film.Immunoblotting is carried out using the primary antibody for target gene.With first antibody: rabbit-anti HSF1 (1: 1000, Abcam) or rabbit-anti EP2 (1: 1000, Abcam) or rabbit-anti FAM3A (1: 500, Bioss) or the anti-PPAR γ of mouse (1: 1000, Santa Cruz) or rabbit-anti Akt, pAkt (1: 1000, Cell Signaling Technology, CST) anti-GAPDH of mouse (1: 1000, Zhong Shan Golden Bridge) be incubated overnight after, washing film simultaneously incubates together with the secondary antibody of HRP conjugation, and is tried using chemiluminescence The detection of agent box.

Adenovirus is overexpressed FAM3A in rat primary VSMCs

Before further analysis, with 50MOI Ad-GFP or Ad-FAM3A infection cell 36 hours.

Plasmid is overexpressed HSF1 in rat primary VSMCs

Cell should be 24 hours hungry before transfection.Transfection changes liquid in first 1 hour, with 2 μ g pGFP or pHSF1 (Invitrogen) transfect cell, after 6-8 hour, replacement culture medium and be added AngII (0.5 μM, Cat#H-1706；Bachem； Torrance, CA, USA), add or be not added GW9662 (PPAR gamma inhibitors, 10 μM, Santa Cruz), suramin (P2 receptor suppression Preparation, 100 μM, Santa Cruz), culture received cell after 24 hours.

Experimental result is shown in Fig. 4 A-4E.

It can be seen that from Fig. 4 A and 4B when the concentration of Ang II is 0.5 μM, the concentration of Hcy is 500 μM, FAM3A albumen Expression increase it is the most obvious.

It can be seen that from Fig. 4 C after inhibiting PPAR γ, HSF1 can be significantly inhibited to the FAM3A up-regulation expressed and to Akt Activation.

It can be seen that after inhibiting HSF1 from Fig. 4 D, Ang II promotes the up-regulation of PPAR γ and FAM3A expression significantly to be pressed down System.

From Fig. 4 E can be seen that suramin processing after, HSF1 significantly presses down the up-regulation of Primary rat VSMCs proliferation level System.

Embodiment five, cytoplasm calcium ion measurement

By Primary rat VSMCs (P4-P8) inoculation and (in 6 orifice plates, each hole is pre-placed secondary culture in 6 orifice plates 24 × 24mm coverslip).Culture medium is replaced after cell is adherent, is added 50MOI Ad-GFP or Ad-FAM3A 36 hours, Huo Zhejia Enter AngII processing, cell should hungry 24 hours before AngII is added, while add or be not added suramin or KRIBB11 to handle 24 small When.1 hour before receipts cell, after being washed three times with PBS, 1 μM of Fura-2AM (Invitrogen) dye liquor is added into each hole, And 6 orifice plates are placed in CO₂It is incubated for 30 minutes in incubator.Then it is washed 3 times with tyrode, and 1ml platform is added into each hole Formula liquid (wraps up plate tinfoil paper to be measured to be protected from light).With 71 fluorescence microscope of Olympus ix be imaged, record 340nM per second and Luminous intensity at 380nM and F340/F380 in 200 seconds (the intracellular basic free calcium ions of ratio reflection are horizontal).

Experimental result is shown in Fig. 5 A-5C.

As can be seen from Figure 5A, in Primary rat VSMCs, overexpression FAM3A can increase Intracellular free calcium and contain Amount, and suramin (P2 acceptor inhibitor, 100 μM) can inhibit this process.

As can be seen from Figure 5B, in Primary rat VSMCs, (0.5 μM) processing of Ang II can increase intracellular free Ca2+ Ion concentration, and suramin can inhibit the process.

It can be seen that in Primary rat VSMCs from Fig. 5 C, Ang II processing can increase Intracellular free calcium and contain Amount, and KRIBB11 (HSF1 inhibitor, 3 μM) can inhibit the process.

In order to probe into the generation whether FAM3A participates in hypertension, it is small that the present inventor constructs VSMC specificity FAM3A knockout Mouse (FAM3A^VSMC-/-).Compared to Loxp mouse, FAM3A^VSMC-/-Mouse blood pressure under base state is substantially reduced；With AngII packet After burying processing one month, FAM3A^VSMC-/-The blood pressure of mouse is also significantly lower than the Loxp mouse (Figure 1A-B) of same treatment.Blood vessel Tension experiment discloses FAM3A^VSMC-/-The vessel retraction function of mouse is significantly reduced compared with Loxp mouse blood vessel, and diastolic function is without bright Significant difference is not (Fig. 2A-D).This discloses FAM3A and takes part in blood pressure control process, the hypertension and blood vessel for having mediated AngII to induce Reconstruct.

In order to explore the molecular mechanism of AngII and Hcy regulation FAM3A expression, the present inventor selects the primary blood of normal rat Pipe smooth muscle cell carries out experiment in vitro.After using AngII and Hcy processing respectively, EP2 expression is remarkably decreased in cell, and FAM3A protein expression obviously increases, while Akt is significantly activated (Fig. 4 A-B).

There is the binding site of a PPRE in the gene promoter area FAM3A, is nuclear receptors PPAR's γ (Peroxisome Proliferatro-activated receptor γ, peroxisome proliferators activated receptor γ) direct target gene. Early-stage study shows in VSMCs breeding that EP2 expression increases with PPAR γ expression decline, in addition, numerous studies are It proves, the vascular diseases such as PPAR γ and atherosclerosis have correlation, but whether PPAR γ intervenes EP2 to FAM3A's It adjusts and participates in VSMCs proliferation and migration regulation waits to study.

The present inventor predicts that there are multiple in the gene promoter area PPAR γ of rat and people for discovery by biological information HSF1 binding site.Immunofluorescence shows (Fig. 3 A-B) that SHR rat chest aorta and superior mesenteric artery HSF1 expression are equal It is apparently higher than control SD rat, this illustrates that HSF1 may intervene the generating process of hypertension.In order to further study, the present invention People gives SHR rats by intraperitoneal injection using HSF1 inhibitor KRIBB11, to observe the case where injecting front and back blood pressure.The results show that even After continuous injection seven days, compared with control group (injection equivalent DMSO) blood pressure, SHR rat blood pressure is decreased obviously, and two groups of hearts rate do not have Marked difference (Fig. 1 C-E).Antiotasis experiment discloses, and the blood vessel that HSF1 inhibitor KRIBB11 can obviously reduce SHR rat is received Contracting power, while improving arterial dilation (Fig. 2 E-G).Plasmid is overexpressed after HSF1, and FAM3A protein expression obviously increases, together When Akt be significantly activated, the inhibitor GW9662 of PPAR γ can inhibit this process (Fig. 4 C)；Meanwhile plasmid is overexpressed HSF1 may additionally facilitate VSMCs proliferation, and P2 acceptor inhibitor suramin can inhibit this process (Fig. 4 E).In AngII processing Afterwards, PPAR γ and FAM3A protein expression level rises in VSMCs, and HSF1 inhibitor KRIBB11 can inhibit this process (Fig. 4 D).These results suggest that inhibit or activation HSF1 expression after, PPAR γ-FAM3A-ATP-Akt signal shaft be suppressed or Activation.

After adenovirus is overexpressed FAM3A, cytosolic free calcium ion concentration increases, and P2 acceptor inhibitor suramin can inhibit this One process (Fig. 5 A)；After AngII processing, VSMCs cytosolic free calcium ion concentration is significantly increased, this process can be pressed down by P2 receptor Preparation suramin and HSF1 inhibitor KRIBB11 invert (Fig. 5 B-C).

After HSF1-PPAR γ-FAM3A-ATP-Akt signal shaft is activated, cytosolic free calcium ion concentration can be increased, promoted Vessel retraction promotes the proliferation and migration of VSMCs, so as to cause the generation of vascular remodeling and hypertension.When diabetes occur, Raised AngII and Hcy in blood plasma inhibits EP2 expression by AngII receptor pathway, and then activates HSF1-PPAR γ- FAM3A-Akt signal shaft, stimulates the proliferation and migration of vascular smooth muscle, to mediate the generation of hypertension and vascular remodeling. In diabetic angiopathy treatment, is expressed and blocked downstream with FAM3A in HSF1 inhibitor KRIBB11 inhibition vascular smooth muscle Access, it is possible to the new Intervention Strategy as diabetic hypertension and vascular lesion.The mechanism that Fig. 6 is shown helps to understand above-mentioned Disclosure.

From the foregoing it is appreciated that although in order to which the purpose of exemplary illustration describes the specific embodiment of the application, But under conditions of without departing from spirit and scope, technical staff described in this field can make various modifications or change Into.These deformations or modification should all fall into the application scope of the appended claims.

Claims (9)

1. formula (I) compound represented or its officinal salt are in preparation for treating high blood caused by hypertension especially diabetes Purposes in the drug of pressure,

Wherein:

R₁For by C_1-10- the CH that alkyl optionally replaces₂Or by C_1-10- the NH- that alkyl optionally replaces；And

R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1-10Alkyl.

2. purposes as described in claim 1, wherein R₁For by C_1-6- the CH that alkyl optionally replaces₂Or by C_1-6Alkyl is optional - the NH- replaced.

3. purposes as claimed in claim 1 or 2, wherein R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is each independently H or C_1-6 Alkyl.

4. purposes as claimed any one in claims 1 to 3, wherein R₁For by C_1-3- the CH that alkyl optionally replaces₂Or by C_1-3- the NH- that alkyl optionally replaces.

5. purposes according to any one of claims 1 to 4, wherein R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀Each independently For H or C_1-3Alkyl.

6. the purposes as described in any one of claims 1 to 5, wherein R₂For C_1-3Alkyl.

7. such as purposes described in any one of claims 1 to 6, wherein R₃、R₄、R₅、R₆、R₇、R₈、R₉And R₁₀It is H.

8. the purposes as described in any one of claims 1 to 7, wherein R₁For-NH-, R₂For-CH₃, and R₃、R₄、R₅、R₆、R₇、 R₈、R₉And R₁₀It is H.

9. pharmaceutical composition, it includes compound or pharmaceutically acceptable salt thereofs of any of claims 1-8 and drug to connect The carrier received, described pharmaceutical composition is for treating hypertension caused by hypertension, especially diabetes.