CN107074839A - Composition and method for treating the metabolism illness related to body weight - Google Patents

Composition and method for treating the metabolism illness related to body weight Download PDF

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Publication number
CN107074839A
CN107074839A CN201480058587.XA CN201480058587A CN107074839A CN 107074839 A CN107074839 A CN 107074839A CN 201480058587 A CN201480058587 A CN 201480058587A CN 107074839 A CN107074839 A CN 107074839A
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Prior art keywords
compound
cancer
srebp
alkyl
phenyl
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CN201480058587.XA
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Chinese (zh)
Inventor
上杉元成
萨利赫·J·瓦基尔
卢特福·阿布-艾-海格
渡边湛
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Baylor College of Medicine
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Baylor College of Medicine
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Priority claimed from US14/013,918 external-priority patent/US9085566B2/en
Priority claimed from US14/052,074 external-priority patent/US9187485B2/en
Priority claimed from US14/270,130 external-priority patent/US9233941B2/en
Application filed by Baylor College of Medicine filed Critical Baylor College of Medicine
Publication of CN107074839A publication Critical patent/CN107074839A/en
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Abstract

The present invention relates to compound, pharmaceutical composition and the preparation for being coupled structure A B C with three, the three connections structure A B C have piperidyl, pyrrolinyl or pyridine radicals A rings, thiazole B rings and phenyl C rings, and they further can independently be substituted.Also provide and utilize compound as described herein, pharmaceutical composition and preparation for treating metabolic disorder or the method for cell hyperproliferative diseases.Also provide and lose weight or increase the method for themogenesis during weight loss, methods described utilizes structure A B C compound or the compound of such structural formula, the R in the structural formula as mentioned1It is H, Et, OMe or n-propyl；Y is CH or (formula)；R2It is OH, OMe or NH i Pr；R3It is H, F or Cl；And R4It is H, Me, Cl, Br, F, OH, OBz, OCH2COOMe、OCH2COOH、NH2、NH‑i‑Pr、NHCOMe、NHSO2Me, NHBn (formula), OMe, NHBoc, (formula), NHTs, (formula) or (formula)；Its pharmaceutical salts or stereoisomer or the two.

Description

Composition and method for treating the metabolism illness related to body weight
The cross reference of related application
The copending application U.S. Serial No that this international application requires to submit on May 5th, 2014 according to 35U.S.C. § 120 The interests of 14/270,130 priority, its pending Shen for requiring to submit on October 11st, 2013 according to 35U.S.C. § 120 Please U.S. Serial No 14/052,074 priority interests, it requires to submit within 29th in August in 2013 according to 35U.S.C. § 120 Copending application U.S. Serial No 14/013,918 priority interests, all these applications are entirely incorporated in this by reference Wen Zhong.
Federal funds explanation
Present invention uses from NIH (National Institutes of Health) grubstake GM-63115 and Ministry of National Defence grubstake DAMD 17-03-1-0228 Federal funds.U.S. government possesses some of the present invention Right.
Background of invention
Invention field
In short, the present invention relates to the medicine of metabolic disorder and biology field.In specific aspect, this hair Bright field is related to for treating illness as partially due to fat particular composition caused by the up-regulation of themogenesis.Some In aspect, the composition includes 125B11 (fatostatin A) and the like or derivative.
The description of association area
Metabolic syndrome is related to many cardiovascular risk factors, including hypertension (hypertension), dysiipidemia (dyslipidaemia), fat (obesity), diabetes B (diabetes of type 2), pancreatic β cell dysfunction (pancreatic β-cell dysfunction) and atherosclerosis (atherosclerosis).Fat or carbon hydrate The different diet of thing content is conducive to the energetic supersession of animal (including people).Long chain fatty acids are that main energy is originated and constituted The important component of the lipid of cell membrane.They derive from food, and by acetyl coenzyme A de novo formation.Cholesterine is also derived from Food and synthesized by acetyl coenzyme A.Carbohydrate is converted into acylglycerol by aliphatic acid and cholesteric de novo formation Ester relates separately at least 12 kinds and 23 kinds of enzyme reactions.The expression of the gene of encoding such enzymes is referred to as sterol regulation member Three kinds of transcription factor SREBP-1a, -1c of part associated proteins (SREBP) and SREBP-2 control.These embrane-associated proteins are to turn The member for recording a class of the basic helix-loop-helix leucine zipper family of the factor.With other leucine zippers member's Transcription factor is different, and SREBP is synthesized into ER- film combination precursors, and the precursor needs to combine the albumen of Golgi membrane by two kinds Enzyme (site -1 and the protease of site -2) is discharged by proteolysis, with the transcription of the target gene in active cell core.
SREBP Proteolytic activation is by the tight regulatory control of sterol, and the regulation and control are by activating egg with SREBP cuttings The interaction of (SCAP) (SREBP ER- film combinations escort albumen) in vain.When sterol is accumulated in ER films, SCAP/SREBP Compound can not leave ER and reach golgiosome, and therefore SREBP proteolysis processing is suppressed.SREBP is control The key fat generation transcription factor of fat metabolism homeostasis.
Prior art lacks available for the new compositions and method for treating a variety of metabolic disorders.Present invention accomplishes ability This long-standing needs and wishes in domain.
Summary of the invention
Present invention teach that compound, its preparation and pharmaceutical composition and its application for treating metabolic disorder, the generation Disease decline office, invitation, etc. on account of illness such as, but is not limited to, is related to the related patient's condition of the disease (for example, cancer) or body weight of cell hyperplasia.
The present invention relates to the compound with following general structures：
A-B-C
A rings are pyridines or substituted pyridine, piperidines or substituted piperidines, pyrrolidines or substituted pyrrolidines, thiazole or take Thiazole, phenyl ring or the substituted phenyl ring in generation.B rings are thiazoles or substituted thiazole, piperazine or substituted piperazine, phenyl ring or substituted Phenyl ring.C rings are phenyl ring or substituted phenyl ring, pyridine or substituted pyridine, thiazole or substituted thiazole.
The invention further relates to the compound with following chemical constitutions：
R1Substituent is H, halogen ,-OH ,-O-C1-3Alkoxy ,-OC (O) R3；R3It is C1-C3Alkyl or aryl ,-OCH2-C (O)OR4；R4It is H or C1-C3Alkyl ,-NHR5；R5It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-C3Acid amides ,- NHC(O)O-R6Carbamate；R6It is the tert-butyl group or benzyl ,-NH-SO2-R7Sulfonamide, and R7It is alkyl or aryl.R2Take Can be alkyl or R for base8OC (O)-, and R8It is C3-C5Alkyl or aryl.
The invention further relates to the compound with following chemical constitutions：
R1Substituent is H, halogen ,-OH ,-O-C1-3Alkoxy ,-OC (O) R3；R3It is C1-C3Alkyl or aryl ,-OCH2-C (O)OR4；R4It is H or C1-C3Alkyl ,-NHR5；R5It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-C3Acid amides ,- NHC(O)O-R6Carbamate；R6It is the tert-butyl group or benzyl ,-NH-SO2-R7Sulfonamide, and R7It is alkyl or aryl.R2Take Can be alkyl or R for base8OC (O)-, and R8It is C3-C5Alkyl or aryl.
The invention further relates to the compound with following chemical constitutions：
R9It is H, halogen ,-OH ,-O-C1-C3Alkoxy ,-OC (O) R11；R11It is C1-C3Alkyl or aryl ,-OCH2-C(O) OR12；R12It is H or C1-C3Alkyl ,-NHR13；R13It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-3Acid amides ,- NHC(O)O-R14Carbamate；R14It is the tert-butyl group or benzyl ,-NH-SO2-R15Sulfonamide；R15It is alkyl or aryl or-SO2- NH-R16Sulfonamide, and R16It is alkyl or aryl.R10It is nitrogen or methylene.N is 0 or 1, and when n is 1, Z is-C=O. A can have following structures：
Wherein R17It is H or C1-C3Alkyl.
The present invention relates to compound as described herein, it is formulated into the pharmaceutical composition in pharmaceutical excipient, or It is formulated into the preparation for including food, ani-mal feed substance or medicine.The invention further relates to kit, the kit includes this Compound or its combination or its pharmaceutical composition or its other preparation and the container for accommodating the compound described in text.
The invention further relates to following compounds：N- (4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) phenyl) Methanesulfonamide, 2- (4- (4- bromophenyls) thiazol-2-yl) pyrrolidines -1- t-butyl formates, 2- (4- (4- bromophenyls) thiazole - 2- yls) pyrrolidines -1- benzyl formates, 4- (4- bromophenyls) -2- (pyrrolidin-2-yl) thiazole, 4- (4- bromophenyls) -2- (1- third Base pyrrolidin-2-yl) thiazole, 3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- t-butyl formates, 3- (4- (4- bromophenyls) Thiazol-2-yl) piperidines -1- benzyl formates, 3- (4- (4- bromophenyls) thiazol-2-yl) -1- propylpiperdines, 4- (4- (4- bromobenzenes Base) thiazol-2-yl) piperidines -1- benzyl formates, (R) -2- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) pyrrolidines - 1- benzyl formates, 3- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates, 4- (4- (4- (methyl Sulfoamido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates, 4- (3- (pyridine -2- bases)-[1,2,4] triazol [4,3-b] Pyridazine -6- bases)-N- tosyl aniline, (4- (5- chloro-2-methyls phenyl) piperazine -1- bases) (4- (tosyl amino) Phenyl) ketone, 4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases)-N- tosyl aniline, The chloro- 4- methyl-N- of 3- (6- (4- (3- (trifluoromethyl) benzyl) piperazine -1- bases) pyridin-3-yl) benzsulfamide, the chloro- N- (4- of 4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases) phenyl) benzsulfamide, (Z) -4- (3- cyano group -3- (4- (2,4- 3,5-dimethylphenyl) thiazol-2-yl) pi-allyl)-N- (thiazol-2-yl) benzsulfamide, N- (3- (H- imidazos [1, 2-a] pyridine -2- bases) phenyl) -4- methyl -2- phenyl thiazole -5- formamides, N- (3- (benzo [d] thiazol-2-yl) phenyl) is different Niacinamide, 3- (4- chlorphenyls) -4,5- dihydro -1- phenyl -5- (2- phenyl thiazole -4- bases) -1H- pyrazoles, N- (4- (6- methyl Benzo [d] thiazol-2-yl) phenyl) -2- (tolylmethyl sulfoamido between N-) acetamide, N- (4- (6- methyl benzo [d] thiophenes Azoles -2- bases) phenyl) -2- (N- p-methylphenyl sulfonyloxy methyls amido) acetamide；Its pharmaceutical salts；Stereoisomer；And its any group Close.
The invention further relates to treat the method for the metabolic disorder in animal, methods described includes giving individual administration Therapeutically effective amount it is at least one have chemical constitution A-B-C's or compound or pharmaceutically acceptable salt thereof as specifically described herein or vertical The step of body isomers or combinations thereof.In preferred in terms of, the metabolic disorder is cancer or the related illness of body weight. The present invention relates to correlation technique the step of further comprising providing the second therapy.Second therapy includes dietetic treatment, thing Physiotherapy method, behavior therapy, operation, medicinal treatment and combinations thereof.In certain aspects, the metabolic disorder is diabetes, And other therapy includes dietetic treatment, physical therapy and medicinal treatment.
The invention further relates to treat the method for the cell hyperproliferative diseases in patient in need.It is described Method includes at least one compound or pharmaceutically acceptable salt thereof as described herein or three-dimensional different to patient therapeuticallv's effective dose The step of structure body or combinations thereof.
The invention further relates to the correlation technique for the body weight for mitigating the animal for having this to need.Methods described include to At least one compound or pharmaceutically acceptable salt thereof or stereoisomer as described herein of patient therapeuticallv's effective dose or their group Close.
The invention further relates to the method for the body weight for mitigating the animal for having this to need.Methods described is included to described Animal be applied in the therapeutically effective amount in medicinal medium have structure A-B-C's compound as specifically described herein or its Pharmaceutical salts or stereoisomer or combinations thereof.A rings can be pyridine or substituted pyridine, piperidines or substituted piperidines, pyrrole Cough up alkane or substituted pyrrolidines, thiazole or substituted thiazole, phenyl ring or substituted phenyl ring.B rings can be thiazole or substituted thiophene Azoles, piperazine or substituted piperazine, phenyl ring or substituted phenyl ring.C rings can be phenyl ring or substituted phenyl ring, pyridine or substituted pyrrole Pyridine, thiazole or substituted thiazole.
The invention further relates to for during mitigating in the weight of animals increase themogenesis without reduce lean body mass method, It the described method comprises the following steps：The change with following structures for the therapeutically effective amount being applied in the animal in medicinal medium Compound：
Or its pharmaceutical salts or stereoisomer or combinations thereof.R1Substituent can be with H, Et, OMe or n-propyl；Y is CH OrR2Substituent can be OH, OMe or NH-i-Pr.R3Substituent can be H, F or Cl.R4Substituent be H, Me, Cl、Br、F、OH、OBz、OCH2COOMe、OCH2COOH、NH2、NH-i-Pr、NHCOMe、NHSO2Me、NHBn、 OMe、NHBoc、NHTs、The compound can be its pharmaceutical salts Or stereoisomer or combinations thereof.
The feature and technological merit of the present invention have rather broadly been described above, it is following so as to more fully understand The detailed description of the present invention.Other features and advantages of the present invention will be described below, and this constitutes the claims in the present invention Theme.It will be understood by those skilled in the art that disclosed concept and specific embodiment can be easily used as improving or designing use In the basis for the other structures for implementing identical purpose of the invention.It should also be understood by those skilled in the art that these equivalent constructions are not The spirit and scope of the present invention proposed away from appended claims., will more by following description when being considered in conjunction with the accompanying The new feature of feature of present invention in the institutional framework and operating method of the present invention is understood well, with and other objects and advantages. But it will be clearly understood that every width accompanying drawing is only purpose of illustration and description, the limitation of the present invention should not be defined as.
Brief description
For a more complete understanding of the present invention, following description will be carried out with reference to accompanying drawing now.
Figure 1A -1B show the checking of the microarray results by RT-PCR.By DU145 cells DMSO (swimming lane 1) and 5 μM 125B11 (swimming lane 2) handles 6 hours (Figure 1A).Then extract total serum IgE and carry out RT-PCR.(Figure 1B) RT-PCR and micro- The summary of array data.ACL, ATP citrate-lyase；HMG CoAR, 3- hydroxy-3-methyl-glutaryl-CoA-reductase； LDLR, LDL receptor, MVD, mevalonate pyrophosphate decarboxylase；SCD, stearoyl-coa desaturase； INSIG1, insulin-induced gene 1；GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig. 2A -2C show that 125B11 suppresses the ability that endogenous SREBP activates reporter.Driven with SRE-1- β-gal reporters (Fig. 2 B) cotransfection under luciferase reporter (pSRE-Luc) (Fig. 2A) and actin promoter control HEK293 cells.In comprising the culture medium without lipid serum transfection is handled with the 125B11 of various concentration or only with DMSO Cell.After cultivating 20 hours, uciferase activity is determined, data are standardized by betagalactosidase activity.In fig. 2 c, With pCMV-SREBP-1c (1-436) and pSRE-Luc transfected HEK 293s, used in comprising the culture medium without lipid serum or The cell of transfection is handled without 20mM 125B11s.Each value represents the average value of three independent experiments.
Fig. 3 A-3H show 125B11 to SREBP-1 and -2 effect.By DU145 cells single DMSO or method (1 and 5 μM) of figure statin A is handled 6 hours.SREBP-1 (Fig. 3 A) or SREBP-2 (Fig. 3 B) precursor are checked by Western blotting With the level of mature form.The figure of downside shows the Western blotting of actin, is used as loading control.(Fig. 3 C-3H) passes through Immunostaining examines SREBP-1 positioning.Cell single DMSO (Fig. 3 C-3E) or 5 μM of 125B11 (Fig. 3 F-3H) places Reason, (Fig. 3 C and Fig. 3 F) is then dyed with DAPI or (Fig. 3 D and Fig. 3 G) is dyed with anti-SREBP-1.
Fig. 4 A-4G are shown to be struck low SREBP-1 to suppress insulin-induced fat generation by siRNA.Set up two The clone of the stable transfection of low 3T3-L1 cells is struck in SREBP-1 expression, and induces it to be differentiated to form fat cell.Strike low Cell it is undifferentiated (Fig. 4 D and Fig. 4 F), and with empty vectors (neo) transfect 3T3-L1 cells be largely differentiated to form fat Cell (Fig. 4 B).Fig. 4 A, 4C and 4E are shown without insulin-induced cell.Fig. 4 G are the western blot analysis of clone, Show successfully to have struck low SREBP-1.
Fig. 5 A-5B demonstrate siRNA and strike the not dependent growth of serum that low SREBP-1 blocks DU145 prostate gland cancer cells. In fig. 5, set up the clone that the stable transfection of low DU145 cells is struck in two SREBP-1 expression, and without serum, contain Cultivated 3 days in the MEM culture mediums of 2% hyclone (FBS), 2% fat-free hyclone or 1 μ g/mL IGF1.Pass through WST-1 Determine measurement growth rate.Low cell is struck in the MEM culture mediums without serum, containing 2% fat-free FB or 1 μ g/mL IGF1 Do not grow, but show in the presence of serum the growth with control cell as many.Test to carry out in triplicate.Fig. 5 B are aobvious Show and clone the Western blotting that SREBP-1 in 1 and 2 strikes low degree.
Fig. 6 A-6G demonstrate effect of the 125B11 to mouse after fasting/give again fat-free diet.Whole During experiment (then fasting is fed with fat-free diet up to 48 hours in 48 hours again after starting one day), noted daily in mouse peritoneum Penetrate 30mg/kg 125B11s.Weight loss (Fig. 6 A) and food intake (Fig. 6 B) are measured at the end of being fed with 48 hours.Fig. 6 C The serum constituent of display processing and control mice.Fig. 6 D are representational from 2 compareed with 125B11 treatment group The SREBP-1 of the liver extract of different mouse Western blotting.The applied sample amount of protein is standardized.Fig. 6 E are display livers The representational Western blotting of the FAS expression (above) of dirty extract and the Coomassie-stained gels (figure below) of loading control. Fig. 6 F-6G show FAS and ACC activity in liver extract.Data are average value+SD (n=5)；* P ＜ 0.05.
Fig. 7 A-7E show that 125B11 handles the effect to mouse in two weeks.The mouse at 5-6 monthly ages is injected into 30mg/ daily Kg 125B11s or 10%DMSO, continue two weeks.Fig. 7 A show the body weight with 125B11 before and after the processing, Fig. 7 B display processings The amount of weight loss afterwards.The serum levels of glucose, cholesterine and triglycerides (TG) in Fig. 7 C display processings and control mice. Fig. 7 D show the FAS activity in liver extract.Fig. 7 E are the generations of the liver extract of control group and each three mouse for the treatment of group The western blot analysis of table.The applied sample amount of protein is standardized.Data are expressed as average value+SD (n=5)；* P ＜ 0.05。
Fig. 8 A-8C show influence of the 125B11 to body weight and food consumption.By two groups of ob/ob male mices (n= 5) daily intraperitoneal injection 125B11 or the 10%DMSO (control group) in PBS, continue surrounding.Mouse chow diet Raise, testing first day and subsequent every day, the quantity of food of measurement mice weights and consumption.Fig. 8 A are representational right Take the picture of the mouse handled with 125B11.Fig. 8 B show the weight of every mouse in each group determined daily.Show weight The average value and variance of amount.In Fig. 8 C, measure food intake daily, and during being expressed as 28 days in every mouse accumulation Food intake.
Fig. 9 A-9H demonstrate the serum constituent for the ob/ob mouse that control and 125B11 are handled.From overnight fasting Mouse tail vein collection blood, separation cell after collect serum.Constituent is determined as described below.Data are expressed as average Value ± SD, every group of n=5 mouse.
Figure 10 A-10D show influence of the method figure statin to the liver and adipose tissue of ob/ob mouse.Figure 10 A explicit representations The mouse (left side) of figure statin A processing and the liver for compareing (right side).Figure 10 B show with Oil-Red O dyeing to detect that fat drips are used in combination The histologic analysis of the freezing microtome section of the control of Mayer haematoxylin redyeing and the liver of 125B11 ob/ob mouse.Usage The fat drips that the liver of three different mouse of figure statin A processing shows to dye red substantially reduce (above), small with processing Mouse is compared, and control display is rich in the fat drips (figure below) for dying red.Figure 10 C show the ob/ob mouse from 125B11 processing (left side) and the epididymal adipose tissues pad of control (right side) separation.Figure 10 D show to compare from ob/ob and separated with the mouse of 125B11 processing Liver and epididymal adipose tissues pad average weight.Data are expressed as average value ± SD, every group of n=5 mouse (* P ＜ 0.05).
Triglycerides (Figure 11 A) in the liver for the ob/ob mouse that Figure 11 A-11B displays control and 125B11 are handled With cholesterine (Figure 11 B) level.Lipid is extracted from liver, triglycerides and cholesterine are quantified as described below.Data are expressed as Average value ± SD, every group of n=5 mouse (* P=0.0004；)。
Figure 12 A-12D demonstrate the expression and activity that 125B11 reduces fat generation enzyme.Figure 12 A show as follows The activity of the acetyl-CoA carboxylase (ACC) determined described in text in the liver extract of ob/ob mouse, Figure 12 B display fat The activity of acid synthase.Figure 12 C are separated by 4-12%NuPAGE MES gels, are detected with different antibodies, and detected with ECL The western blot analysis of liver crude extract from three individual ob/ob mouse.Figure 12 D are shown to be standardized to actin Afterwards, ratio of the 125B11 relative to the intensity of the particular bands of the difference fat generation enzyme of control mice.Data are expressed as putting down Mean value ± SD, every group of n=5 mouse ( *P<0.05)。
Figure 13 shows that liver fat generates the transcriptional level of enzyme in control relative to 125B11 ob/ob mouse.Often The mRNA level in-site for planting gene is standardized for actin.Mouse (n=5) separation handled from control and 125B11 RNA, and measured by real-time quantitative RT-PCR.Relative to control, * P ＜ 0.05.
Figure 14 A-14F show the exemplary compound 1-66 of the present invention.
Figure 15 is shown and determined using the exemplary analog 2-18 plain enzyme reporter of standard fluorescence.
Figure 16 is shown and determined using the exemplary analog 19-34 plain enzyme reporter of standard fluorescence.
Figure 17 is provided to be determined using the exemplary analog 35-44 plain enzyme reporter of standard fluorescence.
Figure 18 A-18D show that method figure statin blocks SREBP activation.Pass through in comprising the culture medium without lipid serum Method figure statin prevents the ability that endogenous SREBP activates luciferase reporter gene.CHO-K1 cell transfectings are driven with SRE-1- Dynamic luciferase reporter (pSRE-Luc) (Figure 18 A).The cell of transfection is used in comprising in the culture medium without lipid serum Various concentrations method figure statin processing.In comprising the culture medium without lipid serum method figure statin to cotransfection with pCMV- The influence (Figure 18 B) of SREBP-1c (1-436) and pSRE-Luc CHO-K1 cells.In the CHO-K1 cells of transfection PLAP-BP2 keeps film combination, unless it is cracked and be secreted into culture medium (left figure) by S1P in golgiosome.With EtOH Control is compared, usage figure statin (20 μM) or sterol (10 μ g/mL cholesterine and 1 μ g/mL 25- hydroxycholesterol oxycholesterols) processing influence PLAP-BP2 cracking (Figure 18 C).The western blot analysis of the CHO-K1 cells of usage figure statin processing.P and N are represented respectively The uncracked film precursors of SREBP-2 and the nuclear forms (Figure 18 D) of cracking.
Figure 19 A-19B explicit representation figures statin blocks SREBP to be displaced to golgiosome from ER.Figure 19 A are Western blottings point Analysis, it shows brefeldin A (brefeldin A) to only using EtOH, sterol (10 μ g/ml cholesterine and 1 μ g/ml 25- Hydroxycholesterol oxycholesterol) or the processing of 20 μM of method figure statins CHO-K1 cells influence.Figure 19 B are to be with or without 20 μM of method figure statins Or the anti-SCAP IgG- of utilization of the cell grown in the presence of sterol (10 μ g/mL cholesterine and 1 μ g/mL 25- hydroxycholesterol oxycholesterols) 9D5 western blot analysis.The number on the right represents the sugar chain number of N- connections present on the SCAP fragments of albumen enzyme protection.
Figure 20 A-20D show dansyl method figure statin (dansyl fatostatin), method figure statin-polyproline joint- The structure and the cell using their processing of biotin conjugate and polyproline joint-biotin conjugate.Figure 20 A show why Sample inserts polyproline joint with preferably Protection Code figure statin molecule (Sato et al., 2007).In Figure 20 B, dansyl method is used Figure statin and the CHO-K1 cells of ER-tracker red processing show that dansyl method figure statin is positioned in ER.The μ of engineer's scale=10 m.The interaction of Figure 20 C explicit representation figure statins and SCAP, this is by the way that in CHO-K1 film extracts, biotin is used with being incorporated into It is the anti-SCAP of the protein of neutravidin (Neutravidine)-agarose beads of the method figure statin saturation of change, anti- The western blot analysis of SREBP-1, anti-SREBP-2 and anti-ATF6 antibody are shown.Figure 20 D are shown for competition assay, are incited somebody to action Film extract carries out preincubate with single EtOH, cholesterine or method figure statin.
Figure 21 shows effect of the method figure statin to the liver and adipose tissue of ob/ob mouse.It is small that method figure statin is handled The liver section display dyeing of mouse and control mice is red fat drips.
Figure 22 shows the western blot analysis of the CHO-K1 cells of usage figure statin processing.P and N are represented respectively The uncracked film precursors of SREBP-1 and the nuclear forms of cracking.
Figure 23 provides the exemplary of method figure statin, dansyl method figure statin and Fa Tu statins-polyproline joint-biotin Synthetic schemes.
Figure 24 shows that in containing the culture medium for whetheing there is lipid serum method figure statin analog suppresses endogenous SREBP and swashed The ability of luciferase reporter gene living.CHO-K1 cells are transfected with pSRE-Luc.In containing the culture medium for whetheing there is lipid serum With the cell of method figure statin, dansyl method figure statin or isopropyl amine derivative the processing transfection of various concentrations.
Figure 25 A-25D explicit representation figures statin reduces the expression and activity of fat generation enzyme.Measure ob/ob mouse livers The activity of acetyl-CoA carboxylase (ACC) (Figure 25 A) and fatty acid synthase (FAS) (Figure 25 B) in extract.Carry out liver thick The western blot analysis (Figure 25 C) of extract.Show that method figure statin is small relative to compareing after being standardized for actin The ratio (Figure 25 D) of the intensity of the particular bands of different fat generation enzymes in mouse.Data are expressed as average value ± SD, every group of n=5 Mouse (*P<0.05)。
Figure 26 provides the synthetic schemes of compound 53.
Figure 27 provides the synthetic schemes of compound 19 and compound 17.
Figure 28 displays are determined using exemplary analog 45-55 and 19 standard SREBP activation.
Figure 29 displays are determined using exemplary analog 56-61 and 19 standard SREBP activation.
Figure 30 displays are determined using exemplary analog 62-66 standard SREBP activation.
Inhibition concentration (sub 50) data of Figure 31 display examples compound 53.
Inhibition concentration (sub 50) data of Figure 32 display examples compound 58 and 61.
Figure 33 A-33B show that compound 19 suppresses breast cancer cell line SUM 159 growth.Cell is thin with 10000 The density in born of the same parents/hole is seeded in 96 hole plates includes serum (the charcoal stripped that 2% activated carbon is handled in 100ul Serum in culture medium).After 24 hours, the compound 19 of shown concentration is added into the cell, then continues 48 hours.Make Determined with WST-1 and determine cells viability.Figure 33 A:The effect of the cell growth of compound 19 of various concentrations is shown, by Absorbance change at A450nm is clearly illustrated.Figure 33 B:As analyzed and determined by RT PCR, 10 μM of processing HePG2 cells show Write the expression (black bar) for lowering fat generation gene；Value is expressed as average value ± SD；*P<0.05.
Figure 34 A-34C show that compound 19 suppresses Bel7402 HePG2 growth.Cell is thin with 100000 The density in born of the same parents/hole is seeded in 16 hole plates in the culture medium of the serum handled comprising 2% activated carbon.After 24 hours, to institute The compound 19 of concentration shown in being added in cell is stated, then continues 48 hours.Figure 34 A displays control and with 25,50 and 100 μM of chemical combination The photo of the HePG2 cells of the processing of thing 19.Figure 34 B show representational western blot analysis, and it shows, and untreated right Photograph ratio, the cell (T) of processing has the maturation of reduction level and the SREBP-1 and higher levels of precursor of activity form.Figure 34C shows that, as analyzed and determined by RT PCR, 10 μM of processing HePG2 cells significantly lower the expression water of fat generation gene It is flat, and do not influence INSIG2 (known its is not regulated and controled by SREBP).Value is expressed as average value ± SD；*P<0.05.
Figure 35 A-35F show that compound 19 suppresses people acute lymphoblastic leukemia cell line MOLT-4 and people is multiple Property myeloma cell line RPMI8226 growth.By 10,000 MOLT-4 cells (Figure 35 A-35C) and 20,000 RPMI8226 cells (Figure 35 D-35F) are seeded in 96 hole plates is handling serum (FF-FBS) comprising 5%FBS or without fat carbon In the culture mediums of RPMI 1640,24 hours are continued in 37C.By the compound 19 of MOLT-4 and RPMI8226 cell various concentrations (control (None control) without anything without DMSO, has DMSO excipient, in DMSO to reprocessing within 48 hours In 1,2,5,10 and 20 μM；RPMI8226 cells are also used in 3 μM of processing in DMSO).At the end of 48 hours, cell is carried out MTT is determined, so that it is determined that viability.Value is expressed as average value ± SD；*P<0.05.
Figure 36 A-36F show that compound 17 suppresses people acute lymphoblastic leukemia cell line MOLT-4 and people is multiple Property myeloma cell line RPMI8226 growth.By 20,000 MOLT-4 cells (Figure 36 A-36C) and 20,000 RPMI8226 cells (Figure 36 D-36F) are seeded in 96 hole plates to be handled comprising 5% hyclone (FBS) or 5% without fat carbon In the RPMI1640 culture mediums of serum (FF-FBS), 24 hours are continued in 37C.Then by the compound 17 of cell various concentrations (control (None control) without anything without DMSO, has DMSO excipient, in DMSO to reprocessing within 48 hours In 1,2,3,5,10 and 20 μM).At the end of the processing of 48 hours, cell is subjected to MTT measure, so that it is determined that viability.Value It is expressed as average value ± SD (n=3)；**P<0.001；***P<0.0001.
Figure 37 A-37D:After feeding RD and HFHC diet three weeks, the body weight and composition (fat and thin) of SD rats.Will be initial Body weight is about the male SD rat feeding full diet (RD) or higher fatty acid high-carbon water of the 6-7 week old of 193 ± 7.0 gram/rats Compound diet three weeks.Use ECHO MRI methods15Measure body weight (Figure 37 B), fat weight (Figure 37 C) and lean muscle mass (lean Weight) (Figure 37 D) content.(*P<0.05)
Figure 38 A-38B are shown in handled eight weeks with compound 19 after the increased weight (Figure 38 A) accumulated and food intake (scheme 38B).By SD rat feeding HFHC diet up to three weeks, begin through afterwards oral gavage various dose compound 19 or Cottonseed oil excipient is reprocessed eight weeks.Body weight and food intake are measured, and calculates accumulated value.(value is average value+S.E.M；*P <0.05；Processing compared with control vehicle group).
Figure 39 A-39E show the body composition fat and lean body mass (lean mass) determined by ECHO MRI methods.Figure 39A is shown in using the total weight after compound 19 in grams.Figure 39 B, which are shown in, to be applied after compound 19 in grams Total body fat.Figure 39 C are shown in the fatty percentage using every animal after compound 19.Figure 39 D, which are shown in, applies compound Total lean body mass after 19 in grams.Figure 39 E are shown in using every gram of percentage lean body mass after compound 19.It is different real The rat for testing group carries out a Body composition analysis every two weeks.By relative to body weight fat and quality weight calculate fat and The percentage of lean body mass.Mark in Figure 39 B-39E corresponds to identical group in Figure 39 A.Asterisk represents treatment group and control group Between significant difference (* P<0.05).Data are average value ± S.E.M (n=15).
Figure 40 A-40D show the effect of 19 couples of feeding HFHC of compound SD rat livers.Figure 40 A:(2.5 Hes of processing 10mg/kg) with the representational liver of control rats (every group of n=5 is only).Figure 40 B:The Oil Red O dyes of liver freezing microtome section Color.Oil droplet is shown in red.Bar represents 200 microns.Figure 40 C:TG and courage in the hepatic tissue of animal from control and processing Sterol levels；Figure 40 D:Measured, compared with the control, controlled in the rat of processing by SREBP-1 and 2 by real-time PCR The multiple change of gene expression.ACC:Acetyl-CoA carboxylase；ACL:ATP citrate-lyase；SCD:Stearoyl-coacetylase is gone Saturation enzyme, MVD mevalonic acid decarboxylases；LDLR；Ldl receptor；INSIG-1:Insulin-induced gene 1；Average value ± SE；*P< 0.05。
Figure 41 shows the multiple change of the UCP2 gene expression in the rat handled with compound 19 compared with the control.From Total serum IgE is extracted in the hepatic tissue for the rat that control and the compound 19 of dosage shown in are handled, and is determined using real-time PCR UCP mRNA level.
Detailed description of the invention
The general embodiment of invention
Use the compounds for treating and/or prevention metabolic disorder of the present invention.For example, aliphatic acid and the cholesteric life of imbalance Thing is synthesized and the Excess free enthalpy of dietary fat is related to plurality of medical complication, at least including obesity, diabetes, hypertension and the heart Angiosis, and in certain aspects, these patient's condition are treated and/or prevented using the compound of the present invention.Epidemiology Evidence shows that the metabolic disease including fat also promotes to develop the cancer of intrusion form, includes, but not limited to prostate Cancer.
During fat consumption, sterol controlling element associated proteins (SREBP) are hydrolyzed from memebrane protein discharges and translocates to nucleus Interior, they activate the transcription for the gene for participating in cholesterine and fatty acid biological synthesis in core.The present invention will be known to resistance before The synthesized micromolecule of disconnected Adipogenesis and growth of cancer cells is accredited as the selective depressant of SREBP activation, and additionally provides The analogs and derivatives of the molecule.Drug-like molecule 125B11 weakens SREBP Proteolytic activation, so as to reduce it The transcription of responsive genes (responsive genes) in the cell.In mouse, 125B11 blocks SREBP-1 in liver Activation, loses weight, reduction blood cholesterol and glucose level, and lowers fat generation enzyme.In treated mouse Fatty acid synthase and the reduction of activity of acetyl coenzyme A carboxylase and its expression in liver.In certain aspects, 125B11 is used Work understands the instrument of cellular pathways, and provides the starting point that shared molecule is used as the pharmaceutical intervention of at least metabolic disease.
In certain embodiments, the small molecule of regulation and control metabolism Relevant phenotype is used as the work for dissecting complicated correlation Tool.125B11 causes two kinds of different phenotypes in the mammalian cell of culture：The pancreas of 3T3-L1 l cells The lipogenetic complete inhibition of island element induction；With the insulin-like growth independent of serum of DU145 Human Prostate Cancer Cells The selectivity of the factor 1 (IGF1) dependence growth is prevented.
In certain aspects of the invention, 125B11 selective exclusion SREBP activation, SREBP is that activation participates in cholesteric The key fat generation transcription factor of alcohol and the specific gene of aliphatic acid synthesis.125B11 is accredited as SREBP inhibitor It is consistent with its anti-cellulite generation property, and indicate effects of the SREBP in the IGF1- dependences growth of prostate cancer.This hair It is bright to be related to the 125B11 for blocking at least SREBP-1 activation (such as shown in experiment mice), preferably its analog And derivative compound.Applying 125B11 to fat ob/ob mouse causes substantially reducing for weight loss and interior fat. Also, during weight loss, the expression increase of uncoupling proteins 1, uncoupling protein-3 and Uncoupling protein-4 gene, themogenesis Increase, and lean body mass is not reduced.
The present invention relates to the treatment and/or prevention of at least one symptom of metabolic disorder.Metabolic disorder can be any class Type, as long as being improved with the compound of the present invention or preventing one of its symptom.Specifically, metabolic disease is derived from one or more Inborn errors of metabolism (it can be described as hereditary conditions), such as by defect metabolic enzyme (for example with it is one or more mutation or Unordered enzyme, including the mutation in modulin and transporting mechanism) caused by inhereditary feature.
Generally, metabolic disorder may be defined as the illness for influenceing cellular energy to produce.Although most of metabolic disorders are heredity Property, some illnesss are probably acquired disease caused by one or more factors, and these factors include diet, toxin, infection Deng.Inherited metabolic illness is probably that caused by genetic defect, the genetic defect causes a certain step in cellular process The missing or improper structure of enzyme necessary to rapid.The other metabolic disorder of maximum kind includes following：1) glycogen storage disease (glycogen Storage diseases) (also referred to as glycogenosis (glycogenosis) or dextrinosis (dextrinosis)), including shadow Ring the illness of carbohydrate metabolism；2) fat oxidation illness, it influences fat metabolism and the metabolism of fatty ingredient；With 3) line grain Body illness, it influences mitochondria.The example of glycogen storage disease (GSD) at least includes I type GSD (glucose-6-phosphatase deficiencies； Feng's gierke's disease (von Gierke ' s disease))；II type GSD (acid-maltase deficiencies；Pompe's disease (Pompe's disease))；Type III GSD (glycogen debrancher deficiencies；Cori's disease (Cori's disease) or glycogen storage disease type III (Forbe ' s disease))；(glycogen branching enzyme lacks IV types GSD；Andersen's disease (Andersen disease))；V-type GSD (muscle glycogen phosphorus It is acidified azymia；McArdle disease (McArdle disease))；VI types GSD (hepatic phosphorylase deficiency, hers' disease (Hers's disease))；VII type GSD (muscle phosphofructokinase deficiencies；Tarui's disease (Tarui's disease))；IX type GSD (phosphorylations Kinase enzyme lacks)；(glucose transporter lacks with XI types GSD；Fanconi-than hyperkeratosis follicularis et parafollicularis in cutem penetrans (Fanconi-Bickel disease))。
In certain embodiments, fatty acid metabolism, which lacks, can be described as fat oxidation illness or lipoidosis.For example, They may relate to influence body in such as muscle, liver and other cell type internal oxidition aliphatic acid to produce the ability of energy Azymia caused by one or more inborn errors of metabolisms.The example that fatty acid metabolism lacks at least includes：Coacetylase takes off Hydrogen azymia；Other coacetylase azymias；Carnitine associated conditions；Or lipoidosis.The example of CoA dehydrogenase azymia is extremely Include less：Pole long acyl-CoA dehydrogenase azymia (VLCAD)；Long-chain 3- hydroxyls acyl-CoA dehydrogenase lacks (LCHAD)；Medium chain acyl-CoA dehydrogenase azymia (MCAD)；Short chain acyl-CoA dehydrogenase azymia (SCAD)；And short chain L-3- hydroxyls acyl-CoA dehydrogenase lacks (SCHAD).The enzymoprivic example of other coacetylases at least includes：2,4- diene acyls Coenzyme A reductase azymia；3- hydroxy-3-methyl glutaryls-coacetylase cracking azymia；With malonyl coenzyme A OMP decarboxylase deficiency OMP. The example that carnitine correlation lacks at least includes：Primary meat poisoning base deficit；The indexable azymia of carnitine-acylcarnitines；Meat Malicious alkali palmitoyl transferase I lacks (CPT)；Lack (CPT) with carnitine palmitoyltransferase II.The example bag of lipoidosis Include acid lipase disease；Primary familial xanthomatosis (Wolman disease)；Cholesterol ester storage disorders (cholesteryl ester storage disease)；Gaucher disease (Gaucher disease)；Niemann-Pick disease (Niemann-Pick disease)；Method In cloth sick (Fabry disease)；Farber's disease (Farber ' s disease)；Gangliosidosis (gangliosidoses)；Krabbe disease (Krabb é disease)；And metachromatic leukodystrophy (metachromatic leukodystrophy).Other disorder of fatty acid metabolism at least include the functional protein of mitochondria three Lack；Electron transfer flavoprotein (ETF) dehydrogenase deficiency (GAII and MADD)；Tangier disease (Tangier disease)；With Acute fatty liver of pregnancy.The example of mitochondriopathy at least includes progressive ophthalmoplegia externa (progressive external Ophthalmoplegia, PEO)；Diabetes and deafness (DAD)；The primary hereditary optic neuropathy of thunder (Leber hereditary Optic neuropathy, LHON), mitochondrial encephalomyopathy (Mitochondrial encephalomyopathy), lactate acid Poisoning (lactic acidosis) and apoplexy sample syndrome (stroke-like syndrome, MELAS)；Lafora's disease Disease and ragged red fibers (Myoclonic epilepsy and ragged-red fibers, MERRF)；Leigh's syndrome (Leigh syndrome)；Subacute sclerosing encephalopathic (subacute sclerosing encephalopathy)；Neuropathy, Incoordination, retinitis pigmentosa and sagging (Neuropathy, ataxia, retinitis pigmentosa, and Ptosis, NARP)；Kearns-Sayre syndrome (Kearns-Sayre syndrome, KSS)；Muscular nerve source property stomach and intestine encephalopathic (Myoneurogenic gastrointestinal encephalopathy, MNGIE).In specific aspect of the invention, generation The disease that declines office, invitation, etc. on account of illness be it is following in one or more or with the one or more in following as one of its complication：It is fat, high Pionemia (hyperlipemia), diabetes, fatty liver, hypertension and cardiovascular disease.
The present invention relates to the treatment of the disease related to cell hyperplasia.In specific example, cell hyperplasia It can be caused by cancer or other tumor diseases or illness.Do not limit, the hyperproliferative disease can be breast cancer, Respiratory cancer, the cancer of the brain, genital cancer, prostate cancer, digestive system cancer, carcinoma of urethra, cancer eye, liver cancer, cutaneum carcinoma, head and neck cancer, first Shape gland cancer, accessory thyroid glands cancer, lymthoma, sarcoma, melanoma, leukaemia, the DISTANT METASTASES IN of Huppert's disease or solid tumor.
The general compound of invention
Usually, the present invention provides compound or pharmaceutically acceptable salt thereof and stereoisomer with following formulas：
A-B-C
Wherein A, B and C may be the same or different, and can be each the bicyclic system of 5-, 6- or 7- yuan of rings or fusion, institute It is heterocycle or non-heterocycle, the ring or non-substituted ring of substitution to state ring, A, B and C be directly connected to or the atomic link by insertion or Joint is connected, and the atomic link or joint are the carbochains or undersaturated with or without the saturation of other functional group Carbochain.
Preferably, A rings are that have heteroatomic 6 circle heterocycles.A rings can be substituted.In preferred embodiments, should Ring is pyridine ring；It is highly preferred that the nitrogen-atoms of pyridine ring is relative to 4 of B ring positions or 2.Most preferably, pyridine ring exists Replaced on carbon in the α positions relative to nitrogen heteroatom by n-propyl.In other preferred embodiments, there is 1-5 on A rings The side chain of individual atom, the side chain of more preferably 1-5 carbon.In other exemplary and non-limiting embodiments, A rings can be with E.g. phenyl, pyrroles, thiophene, furans, pyrimidine, isoquinolin, quinoline, benzofuran, indoles,Azoles, naphthyl, piperidines, pyrroles Alkane, imidazoles, imidazoles [1,2-a] pyridine, benzimidazole, thiazole or benzothiazole.
In one aspect of the invention, A rings are piperidine rings.Preferably, the nitrogen-atoms on piperidine ring is relative to B rings position 4 put.In other related aspects, nitrogen-atoms on piperidine ring is relative to 3 of B ring positions.The nitrogen-atoms of piperidines Can further it be substituted, and wherein described substitution is selected from group consisting of the following：Alkyl, sulfoxide, sulfone, alkyl or aryl sulphur Acid esters, sulfonic acid, and their any combination.For example, substitution can be propyl group, t-butyloxycarbonyl (BOC) or benzyl Base Epoxide carbonyl group.
In another aspect of the invention, A rings are pyrrolidine rings；Preferably, the nitrogen-atoms on pyrrolidine ring is relative to B rings 2 of position.The nitrogen-atoms of pyrrolidines can be further substituted, and wherein described substitution is selected from group consisting of the following： Alkyl, sulfoxide, sulfone, alkyl or aryl sulphonic acid ester, sulfonic acid, and their any combination.For example, substitution can be propyl group base Group, t-butyloxycarbonyl (BOC) or benzyloxycarbonyl groups.
Preferably, B rings are that have at least two heteroatomic 5- yuan of rings.B rings can be substituted.In preferred embodiment In, B rings are thiazole rings.In other exemplary and non-limiting embodiments, B rings can be for exampleIt is azoles, imidazoles, differentAzoles, imidazoles, thiophene, furans, pyrimidine, pyrazoles, isothiazole, thiazole and pyridazine, aryl or pyrazoles.B rings can also be with two Individual heteroatomic 6- yuan of rings.For example, B rings are piperazine rings.Preferably, C rings are 6- yuan of rings, most preferably phenyl ring.C rings can be by Substitution.In preferred embodiments, C rings are methyl substituted.In other exemplary and non-limiting embodiments, C Ring may, for example, be phenyl, pyridine, pyrroles, thiophene, furans, pyrimidine, isoquinolin, quinoline, benzofuran, indoles,Azoles or naphthalene Base.
Exemplary compound is provided in Figure 14 A-14F and table 3 and 4.Reference compound 1, the pyridine of n-propyl substitution Ring corresponds to the A rings of formula, and the thiazole ring of 2,4- substitutions corresponds to the B rings of formula, and methyl substituted phenyl ring is corresponding to logical The C rings of formula.It should be understood that substitution allow in A, B and C ring any one optional position, and any substitution can with it is any Other replace identical or difference.In addition to those shown in Figure 14 A-14F, under substituted non-limitative example also includes Group：H (that is, unsubstituted)；Hydroxyl；C1-10Alkyl；C2-10Alkenyl；C2-10Alkynyl；C3-6Cycloalkyl；Aryl；Heteroaryl；Wherein institute Alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl is stated optionally to be taken by the 1-5 groups selected from group consisting of the following Generation：Hydroxyl ,-(C=O) Ra；- (C=O) ORa,-(C=O) H ,-(C=O) OH, O (CH2)nCOORa, wherein n=1-10, and its Middle RaIt is C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, or C3-6Cycloalkyl, aryl, heteroaryl, fluorine, chlorine, bromine, iodine, cyano group, carboxylic Base, amino, mono-substituted amino and dibasic amino, mono-substituted acylamino- and dibasic acylamino- and their times Meaning combination；- (C=O) Ra；- (C=O) ORa,-(C=O) H；- (C=O) OH；-O(CH2)nCOORa, wherein n=1-10, and its Middle RaIt is C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, or C3-6Cycloalkyl, aryl or heteroaryl, fluorine, chlorine, bromine, iodine；Cyano group；Carboxylic Base；Amino；Amide groups, with the substituted monosubstituted and dibasic amino selected from group consisting of the following：C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, C3-6Cycloalkyl, aryl, heteroaryl, sulfoxide, sulfone, sulphonic acid ester, alkyl sulfonic ester, sulfonic acid and they Any combination；Wherein described alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl are optionally selected from by following by 1-5 The group of the group of composition is replaced：Hydroxyl ,-(C=O) Ra,-(C=O) ORa,-(C=O) H ,-(C=O) OH ,-O (CH2)nCOORa, wherein n=1-10, and wherein RaIt is C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, or C3-6Cycloalkyl, aryl and miscellaneous Aryl；Fluorine；Chlorine；Bromine；Iodine；Cyano group；Carboxyl；Amino；Mono-substituted and dibasic ammonia with one or more following radicals Base：C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl group, and their any combination；And, with selected from consisting of the following The substituted monosubstituted and disubstituded amide base of group：C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, C3-6Cycloalkyl, aryl is miscellaneous Aryl, sulfoxide, sulfone, sulphonic acid ester, alkyl sulfonic ester, sulfonic acid, sulphonic acid ester, alkyl sulfonic ester, sulfonic acid and their any combination； The base that wherein described alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl are optionally selected from group consisting of the following by 1-5 Group is replaced：Hydroxyl；- (C=O) Ra；- (C=O) ORa,-(C=O) H；- (C=O) OH ,-O (CH2)nCOORa, wherein n=1- 10, and wherein RaIt is C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, or C3-6Cycloalkyl, aryl or heteroaryl；Fluorine；Chlorine；Bromine； Iodine；Cyano group；Carboxyl；Amino；Monosubstituted and disubstituded amide base with one or more following radicals：C1-10Alkyl, C2-10 Alkenyl, C2-10Alkynyl group, and their any combination.
In the present invention, there are compound or pharmaceutically acceptable salt thereof or stereoisomer with following formulas：A-B-C, wherein A, B It is identical or different with C, and wherein each include the bicyclic system of 5-, 6- or 7- yuan of rings or fusion, the ring is heterocycle or non- Heterocycle, the ring or non-substituted ring of substitution, wherein A, B and C are directly connected to or the atomic link by insertion or joint connection, and Wherein described atomic link or joint are the carbochain or undersaturated carbochain with or without the saturation of other functional group, wherein In described A, B and C any one, any two or all three be all unsubstituted or with one or more substitutions, and its In any substituent can be identical or different with any other substitution, and wherein it is described substitution it is consisting of the following：A) hydroxyl, b) C1-10 alkyl, c) C2-10 alkenyls, d) C2-10 alkynyls, e) C3-6 cycloalkyl, f) aryl, g) heteroaryl, wherein b), c), d), E), f) and/or g) in the substitution optionally further by 1-5 substituent group consisting of the following：1) hydroxyl, 2)-(C =O) Ra, 3) and-(C=O) ORa, 4) and-(C=O) H, 5)-(C=O) OH, 6)-O (CH2)nCOORa, wherein n=1-10,7) and halogen, 8) cyano group, 9) carboxyl, 10) amino, 11) mono-substituted amino, 12) dibasic amino, 13) amide groups, 14) mono-substituted acyl Amido；15) disubstituded amide base, and their any combination, wherein 2), 3) or 6) in, RaIt is C1-10 alkyl, C2- 10 alkenyls, C2-10 alkynyls, C3-6 cycloalkyl, aryl or heteroaryl, h)-(C=O) Ra, i)-(C=O) ORa, j)-(C=O) H, K)-(C=O) OH；l)-O(CH2)nCOORa, wherein n=1-10, wherein h), i) or l) in, RaIt is C1-10Alkyl, C2-10Alkene Base, C2-10Alkynyl, C3-6Cycloalkyl, aryl or heteroaryl, m) halogen, n) cyano group, o) carboxyl, p) amino, q) mono-substituted ammonia Base, r) dibasic amino, s) amide groups, t) mono-substituted amide groups, and u) disubstituded amide base, wherein described monosubstituted One or more of amino, dibasic amino, mono-substituted amide groups and disubstituded amide base have and be selected under State the substitution of the group of composition：C1-10 alkyl, C2-10 alkenyls, C2-10 alkynyls, C3-6 cycloalkyl, aryl, heteroaryl, sulfoxide, Sulfone, sulphonic acid ester, alkyl sulfonic ester, sulfonic acid, and their any combination, wherein in u), the alkyl, alkenyl, alkynyl, ring The substituent group that alkyl, aryl or heteroaryl are optionally further selected from group consisting of the following by 1-5：I) hydroxyl, ii)- (C=O) Ra, iii) and-(C=O) ORa, iv) and-(C=O) H, v)-(C=O) OH, vi)-O (CH2)nCOORa, wherein n=1-10, its In in ii), iii) or vi) in, RaIt is C1-10Alkyl, C2-10Alkenyl, C2-10Alkynyl, C3-6Cycloalkyl, aryl or heteroaryl, vii) Halogen, viii) cyano group, ix) carboxyl, x) amino, xi) mono-substituted amino, xii) dibasic amino, xiii) amide groups； Xiv) mono-substituted amide groups, xv) disubstituded amide base, and their any combination.
Exemplary compound, composition, preparation and application method
In one embodiment of the invention there is provided the compound with chemical constitution A-B-C, wherein A be pyridine or Substituted pyridine, piperidines or substituted piperidines, pyrrolidines or substituted pyrrolidines, thiazole or substituted thiazole, phenyl ring or substitution Phenyl ring；B is thiazole or substituted thiazole, piperazine or substituted piperazine, phenyl ring or substituted phenyl ring；And C is phenyl ring or taken The phenyl ring in generation, pyridine or substituted pyridine, thiazole or substituted thiazole.
In a preferred aspect, the chemical constitution is：
Wherein R1It is H, halogen ,-OH ,-O-C1-3Alkoxy ,-OC (O) R3；R3It is C1-C3Alkyl or aryl ,-OCH2-C(O) OR4；R4It is H or C1-C3Alkyl ,-NHR5；R5It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-C3Acid amides ,-NHC (O)O-R6Carbamate；R6It is the tert-butyl group or fluorenyl methyl or-NH-SO2-R7Sulfonamide；R7It is alkyl or aryl, R2It is alkane Base or R8OC (O)-, and R8It is C3-C5Alkyl or aryl.Especially, the halogen can be bromine.
In in terms of another is preferred, the chemical constitution is：
Wherein R1It is H, halogen ,-OH ,-O-C1-3Alkoxy ,-OC (O) R3；R3It is C1-C3Alkyl or aryl ,-OCH2-C(O) OR4；R4It is H or C1-C3Alkyl ,-NHR5；R5It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-C3Acid amides ,-NHC (O)O-R6Carbamate；R6It is the tert-butyl group or fluorenyl methyl or-NH-SO2-R7Sulfonamide；R7It is alkyl or aryl, R2It is alkane Base or R8OC (O)-, and R8It is C3-C5Alkyl or aryl.Especially, the halogen can be bromine.
In in terms of another is preferred, the chemical constitution is：
Wherein R9It is H, halogen ,-OH ,-O-C1-C3Alkoxy ,-OC (O) R11；R11It is C1-C3Alkyl or aryl ,-OCH2-C (O)OR12；R12It is H or C1-C3Alkyl ,-NHR13；R13It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-3Acyl Amine ,-NHC (O) O-R14Carbamate；R14It is the tert-butyl group or fluorenyl methyl ,-NH-SO2-R15Sulfonamide；R15It is alkyl or virtue Base or-SO2-NH-R16Sulfonamide；R16It is alkyl or aryl, R10It is nitrogen or methylene, n is 0 or 1, and when n is 1, Z is- C=O；And A is
Wherein R17It is H or C1- C3Alkyl.
In in terms of another is preferred, the chemical constitution is：
There is provided the pharmaceutical composition for including aforesaid compound and pharmaceutical excipient in related embodiment.Another There is provided the aforesaid compound for being formulated into food, animal feed material or medicine in individual related embodiment.In another phase There is provided the kit for including aforesaid compound and the container for accommodating the compound in the embodiment of pass.The container can be with It is to be adapted to store medicine known in the art and commercially available any appropriate container.
In another embodiment of the invention, compound preferably be N- (4- (2- (2- propyIpyridine -4- bases) thiazole - 4- yls) phenyl) Methanesulfonamide, 2- (4- (4- bromophenyls) thiazol-2-yl) pyrrolidines -1- t-butyl formates, 2- (4- (4- bromines Phenyl) thiazol-2-yl) pyrrolidines -1- benzyl formates, 4- (4- bromophenyls) -2- (pyrrolidin-2-yl) thiazole, 4- (4- bromobenzenes Base) -2- (1- propyl pyrrole alkane -2- bases) thiazole, 3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- t-butyl formates, 3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- benzyl formates, 3- (4- (4- bromophenyls) thiazol-2-yl) -1- propylpiperdines, 4- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- benzyl formates, (R) -2- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazole - 2- yls) pyrrolidines -1- benzyl formates, 3- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates, 4- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates, 4- (3- (pyridine -2- bases)-[1,2,4] Triazol [4,3-b] pyridazine -6- bases)-N- tosyl aniline, (4- (5- chloro-2-methyls phenyl) piperazine -1- bases) (4- (first BENZENESUFONYLAMINO) phenyl) ketone, 4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases)-N- first Benzenesulphonanilide, the chloro- 4- methyl-N- of 3- (6- (4- (3- (trifluoromethyl) benzyl) piperazine -1- bases) pyridin-3-yl) benzene sulfonyl Amine, the chloro- N- of 4- (4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases) phenyl) benzsulfamide, (Z) -4- (3- cyano group -3- (4- (2,4- 3,5-dimethylphenyl) thiazol-2-yl) pi-allyl)-N- (thiazol-2-yl) benzsulfamide, N- (3- (H- imidazos [1,2-a] pyridine -2- bases) phenyl) -4- methyl -2- phenyl thiazole -5- formamides, N- (3- (benzo [d] thiophenes Azoles -2- bases) phenyl) Pyrazinamide, 3- (4- chlorphenyls) -4,5- dihydro -1- phenyl -5- (2- phenyl thiazole -4- bases) -1H- pyrroles Azoles, N- (4- (6- methyl benzo [d] thiazol-2-yl) phenyl) -2- (tolylmethyl sulfoamido between N-) acetamide, N- (4- (6- methyl benzo [d] thiazol-2-yl) phenyl) -2- (N- p-methylphenyl sulfonyloxy methyls amido) acetamide；Its pharmaceutical salts；It is three-dimensional Isomers；Or their any combination.
In another embodiment of the present invention there is provided the method for treating the metabolic disorder in animal, the side Method include to the animal apply therapeutically effective amount at least one aforesaid compound or its pharmaceutical salts or stereoisomer or it Combination the step of.
To the embodiment further, the step of methods described includes providing the second therapy to the animal.Enter at this In the embodiment of one step, second therapy includes dietetic treatment, physical therapy, behavior therapy, operation, medicinal treatment, change Treat or its combination.Especially, second therapy can be lifestyle change, antihyperglycemic agents, insulin, hyperglycemic factor Sample peptide (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinedione, hypolipidemic compounds or combination two or more in them.
In two embodiments, the metabolic disorder is the related patient's condition of body weight, the disease related to cell hyperplasia Disease, hyperlipidemia, diabetes or its complication, fatty liver, hypertension or cardiovascular disease.Especially, the metabolic disorder can be with It is fat, hypertension, artery sclerosis (arteriosclerosis), asthma (asthma), hyperlipidemia (hyperlipidemia), hyperinsulinemia (hyperinsulinemia), NASH (non-alcoholic Fatty liver) and the diabetes B caused by insulin resistance.In the one side of these embodiments, the metabolism Illness is the related illness of body weight, wherein the compound increase uncoupling proteins 1 of the therapeutically effective amount, uncoupling protein-3 or The expression of Uncoupling protein-4 gene.In addition, in this aspect, during the weight loss of animal, the chemical combination of the therapeutically effective amount Thing increases themogenesis, without reducing lean body mass.In another aspect, the disease related to cell hyperplasia is cancer. Especially, the cancer is breast cancer, respiratory cancer, the cancer of the brain, genital cancer, prostate cancer, digestive system cancer, carcinoma of urethra, eye Cancer, liver cancer, cutaneum carcinoma, head and neck cancer, thyroid cancer, accessory thyroid glands cancer, lymthoma, sarcoma, melanoma, leukaemia or solid tumor DISTANT METASTASES IN.
There is provided the side for treating the cell hyperproliferative diseases in patient in need in another embodiment Method, methods described includes at least one aforesaid compound or its pharmaceutical salts or three-dimensional different to patient therapeuticallv's effective dose The step of structure body or combinations thereof.Especially, the cell hyperproliferative diseases can be previously described cancer.
There is provided the method for treating the cancer in patient in need, methods described in related embodiment The step of including one or more foregoing pharmaceutical compositions to patient therapeuticallv's effective dose.The cancer can be such as this It is literary described.
There is provided the method for the body weight for mitigating the animal for having this to need, methods described bag in another embodiment The step of including the one or more aforesaid compounds for the therapeutically effective amount being applied in the animal in medicinal medium.
There is provided for increase themogenesis during mitigating in the weight of animals in another embodiment of the present invention The method for not reducing lean body mass, methods described includes the compound for the therapeutically effective amount being applied in the animal in medicinal medium Or the step of its pharmaceutical salts or stereoisomer or combinations thereof, the compound has following structures：
Wherein R1Substituent is H, Et, OMe or n-propyl；Y be CH orR2It is OH, OMe or NH-i-Pr；R3Be H, F or Cl；R4It is H, Me, Cl, Br, F, OH, OBz, OCH2COOMe、OCH2COOH、NH2、NH-i-Pr、NHCOMe、NHSO2Me、 NHBn、OMe、NHBoc、NHTs、Use the present invention Method, after the compound is applied, total body fat of animal is reduced.
In all these embodiments and its aspect, those of ordinary skill in the art can be according to the generation to be treated Decline office, invitation, etc. on account of illness disease or needs result be readily determined the present invention compound useful dosage, the metabolic disorder such as, but do not limit In the disease related to cell hyperplasia or the related illness of body weight.Typically, the compound with about 1mg/kg to about 100mg/kg dosage is applied.The compound can be in the composition existed with food, animal feed material or medicament forms It is middle to apply.In preferred in terms of, by increasing unrelated in the case where being reduced with or without lean body mass simultaneously Themogenesis mitigates the body weight of animal.Typically, the compound increases the expression of UCPS.The generation of UCPS Table example includes uncoupling proteins 1, uncoupling protein-3 and Uncoupling protein-4 gene.The method of the present invention can be used for a variety of situations, Include, but not limited to wherein animal and suffer from the related patient's condition of the body weight selected from group consisting of the following：Obesity, hypertension, artery Hardening, asthma, hyperlipidemia, hyperinsulinemia, NASH and the diabetes B caused by insulin resistance. Also, those of ordinary skill in the art will readily appreciate that the purposes that the second therapy is provided to the animal, described second treats Method includes, but not limited to lifestyle change, antihyperglycemic agents, insulin, glucagon-like peptide (GLP), dipeptidyl peptidase The inhibitor of enzyme -4, thiazolidinedione, hypolipidemic compounds and combination two or more in them.
When used as contemplated in this specification, " one " (" a " or " an ") can mean one or more.When used in claims When middle, when being used in combination with word " comprising ", word " one " (" a " or " an ") can mean one or more than one.With When this paper, " another " can mean at least two or more.In specific embodiments, for example, the side of the present invention Face " substantially can be made up of " or " being made up of one or more sequences of the present invention " one or more sequences of the present invention.This Some embodiments of invention can be made up of or substantially one or more elements of the invention, method and step and/or method It is made up of one or more elements of the invention, method and step and/or method.It is expected that any means of the present invention or combination Thing can be realized according to any other methods as described herein or composition.
Term " animal " is used herein to refer to mammal, preferably people, patient, subject or individual, its receive or Through applying one or more compounds as described herein, composition or preparation.
Term " alkyl " is used herein to be referred to remove a hydrogen atom institute from the acyclic saturated hydrocarbons of straight or branched is conceptual Obtained substituted monoradical (that is ,-CH3,-CH2CH3,-CH2CH2CH3,-CH (CH3)2,-CH2CH2CH2CH3,-CH2CH (CH3)2,-C (CH3)3Deng).
Term " alkenyl " is used herein to be referred to from the acyclic unsaturation of the straight or branched containing at least one carbon-to-carbon double bond Conceptual substituted monoradical (that is ,-CH=CH removed obtained by a hydrogen atom of hydrocarbon2,-CH=CHCH3,-C=C (CH3)2,-CH2CH=CH2Deng).
Term " alkynyl " is used herein to be referred to from the acyclic unsaturation of the straight or branched containing at least one carbon-to-carbon triple bond Conceptual substituted monoradical (that is ,-C ≡ the CH ,-C ≡ CCH removed obtained by a hydrogen atom of hydrocarbon3,-C ≡ CCH (CH3)2,-CH2C ≡ CH etc.).
Term " aryloxy group " is used herein to refer to the aryl with bridge joint oxygen atom, such as phenoxy group (- OC6H5) or benzene first Acyloxy (- OCH2C6H5)." arylamino " means the aryl with bridge joint amine functional group, such as-NHCH2C6H5." aryl amide Base " means the aryl with bridge joint amide group, for example-(C=O) NHCH2C6H5。
Term " alkylidene " is used herein to be referred to go from the same carbon atom of the acyclic saturated hydrocarbons of straight or branched is conceptual Except substituted divalent group (i.e.=CH obtained by two hydrogen atoms2,=CHCH3,=C (CH3)2Deng).
Term " cycloalkyl " is used herein to be referred to from taking obtained by saturation monocyclic hydrocarbon one hydrogen atom of conceptual removal The monoradical (that is, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl or suberyl) in generation.
Term " aryl " is used herein to be referred to obtained by monocyclic or bicyclic aromatic hydrocarbon one hydrogen atom of conceptual removal Substituted monoradical.The example of aromatic yl group has phenyl, indenyl and naphthyl.
Term " heteroaryl " is used herein refer to from containing 1,2,3 or 4 heteroatomic monocyclic selected from N, O or S or The conceptual substituted monoradical removed obtained by a hydrogen atom of bicyclic aromatic ring system system.The example of heteroaryl groups includes But it is not limited to：Pyrrole radicals, furyl, thienyl, imidazole radicals, pyrazolyl,It is oxazolyl, differentIt is oxazolyl, thiazolyl, pyridine radicals, phonetic Piperidinyl, pyrazinyl, benzimidazolyl, indyl and purine radicals.Heteroaryl substituent may connect to carbon atom or by hetero atom Connection.The example of single ring heteroaryl group includes：Pyrrole radicals, furyl, thienyl, pyrazolyl,It is oxazolyl, differentOxazolyl, thiophene Oxazolyl and pyridine radicals.The example of bicyclic heteroaryl group includes：Pyrimidine radicals, pyrazinyl, benzimidazolyl, indyl and purine Base.Single ring can have 5 or 6 atoms.Therefore, this includes 4 unit monocycle heteroaryl groups and 5- unit monocycle heteroaryl groups. Including the bicyclic heteroaryl group with a 5- yuan of rings and 6- yuan of rings and the bicyclic heteroaryl base with 2 6- yuan of rings Group.
Term " halogen " includes iodine, bromine, chlorine and fluorine.
Term " substituted " is understood to include the multiple substitution value of substituent.Original on chemical group or structure division Sub- valency replaces when being met by the atom outside dehydrogenation or functional group.If multiple substitution, substituted compound can be by one Substituent structure part that is individual or multiple disclosed or claiming independently is replaced with singular or plural form.Independent substitution is represented (two or more) substituent can be with identical or different.
Term " pharmaceutical salts " in this article refers to the salt of the compound of the pharmacological activity with required parent compound. This salt includes：(1) acid-addition salts, with the inorganic acid such as formation such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid；Or and organic acid Such as acetic acid, propionic acid, caproic acid, pentamethylene propionic acid, glycolic, pyruvic acid, lactic acid, malonic acid, butanedioic acid, malic acid, maleic acid, prolong Fumarate, tartaric acid, citric acid, benzoic acid, 3- (4- (2-hydroxybenzoyl)s) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, second Sulfonic acid, 1,2- ethane-disulfonic acid, 2- ethylenehydrinsulfonic acids, benzene sulfonic acid, 4- chlorobenzenesulfonic acids, 2- naphthalene sulfonic acids, 4- toluenesulfonic acids, camphor Sulfonic acid, 4- methyl bicycles [2.2.2]-oct-2-ene -1- formic acid, glucoheptonic acid, 3- phenylpropionic acids, trimethylace tonitric, tert-butyl group second The formation such as acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid；Or (2) parent chemical combination The salt that acid proton present in thing is formed by metal ion when for example alkali metal ion, alkaline-earth metal ions or aluminium ion replace； Or the salt with the organic base such as coordination such as monoethanolamine, diethanol amine, triethanolamine, tromethamine, N-METHYL-ALPHA-L-GLUCOSAMINE formation.
Term " stereoisomer " means that atom connectivity is identical with other one or more molecules but steric arrangement Different isomery molecules.It is different that this definition includes enantiomter, diastereoisomer, cis-isomer, transisomer, conformation Structure body.
Term " unsubstituted " means all valences on chemical group or structure division by hydrogen saturation.
Term " carbochain of saturation " acyclic saturated hydrocarbons (that is ,-CH used herein for referring to straight or branched3,-CH2- ,- CH2CH3,-CH2CH2--CH2CH2CH3,-CH2CH2CH2,-CH2CH2CH2CH3,-CH2CH2CH2CH3- ,-CH2CH(CH3)2,-C (CH3)3Deng).
Term " undersaturated carbochain " is used herein to be referred to include at least one carbon-to-carbon double bond (that is ,-CH=CH2,-CH =CH- ,-CH=CHCH3,-C=C (CH3)2,-CH2CH=CH2,-CH2CH=CH- etc.) or at least one carbon-to-carbon triple bond (that is ,- C ≡ CH ,-C ≡ C- ,-C ≡ CCH3,-C ≡ CCH- ,-C ≡ CCH (CH3)2,-CH2C ≡ CH etc.) the acyclic insatiable hunger of straight or branched And hydrocarbon.
Present invention additionally comprises the shielded derivative of compounds as disclosed herein.For example, when the compound bag of the present invention During group containing such as hydroxyl or carbonyl, these groups can use appropriate protection group to be protected.The list of suitable protection group Referring to T.W.Greene, Protective Groups in Organic Synthesis (protection group in organic synthesis), John Wiley＆Sons, Inc.1981, the disclosure of which is hereby incorporated by reference completely.The compounds of this invention is protected Derivative can be prepared by method known in the art.
The compound of the present invention can have asymmetric center, chiral axis and a chiral planes, and as racemate, outside disappear Revolve mixture and single diastereoisomer and its all possible isomers and mixture is present, including optical isomer, It is included within the scope of the present invention.In addition, compounds as disclosed herein can exist with tautomeric forms, and two kinds mutual Tautomeric forms thereof is all covered within the scope of the invention, even if only describing a kind of structure of dynamic isomer.
In one embodiment of the invention, composition targeting sterol controlling element associated proteins of the invention (SREBP) one or more members of approach.The approach is related to film combination transcription factor SREBP proteolysis release, in tool In terms of body, this promotes the transhipment from endochylema to nucleus.In nucleus, the base of SREBP and the related enzyme of coding lipid generation The element for being referred to as sterol controlling element (SRE) present in the control region of cause is combined.After SREBP is combined with DNA, target base is adjusted The transcription of cause, for example, raise.
Pharmaceutical preparation
The pharmaceutical composition of the present invention includes this one or more hair for the effective dose being dissolved or dispersed in pharmaceutical carrier Bright composition (other reagent is also included when appropriate).Phrase " the available carrier of medicinal or pharmacology " refers to suitably be administered to Animal (such as people) will not produce the molecular entity and composition of side effect, allergy or other adverse reactions.According to the present invention's Disclosure, the preparation of the pharmaceutical composition containing at least one 125B11 analog or derivative or other active components It is well known by persons skilled in the art, such as Remington's Pharmaceutical Sciences (Remington pharmacopeia), the 18th Version .Mack Printing Company, exemplified by 1990.Moreover, being administered for animal (such as people), it should be understood that preparation should Meet aseptic, pyrogenicity, overall security and purity rubric, as required by FDA biological standards office.
As used herein, " pharmaceutical carrier " include any and all solvent, decentralized medium, coating, surfactant, resist Oxidant, preservative (such as antibacterial agent, antifungal agent), isotonic agent, absorption delaying agent, salt, preservative, medicine, drug substance stable Agent, gel, adhesive, excipient, disintegrant, lubricant, sweetener, flavor enhancement, dyestuff, similar substance and their group Close, as known to persons of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (Lei Ming Pharmacopeia), the 18th edition .Mack Printing Company, 1990, the 1289-1329 pages).At present except it is any with it is active into Outside point incompatible conventional carrier, it is contemplated that its application in pharmaceutical composition.
Depending on its be with solid, liquid or aerosol form apply, and its whether need to such as inject to Medicine approach is sterile, and 125B11 analog or derivative can include different types of carrier.The present invention can be with vein Interior, intradermal, percutaneous, intrathecal, intra-arterial, intraperitoneal, intranasal, intravaginal, rectum are interior, local, intramuscular, subcutaneous, mucous membrane interior, mouth Clothes, part, regionality (locally), suction (for example, aerosol suction), injection, infusion, continuous infusion, direct invasion target are thin The positioning of born of the same parents is irrigated, by conduit, by lavation, with emulsion, lipid composition (such as liposome) or common by this area Other method known to technical staff or foregoing any combination are administered (for example, with reference to Remington's Pharmaceutical Sciences (Remington pharmacopeia), the 18th edition .Mack Printing Company, 1990).
125B11 analog or derivative can be configured to free alkali, the composition of neutral or salt form.Pharmaceutical salts bag Include acid-addition salts, for example the salt with the free amine group formation of proteinaceous compositions, or and inorganic acid, such as hydrochloric acid or phosphorus The salt that acid, or organic acid, such as acetic acid, oxalic acid, tartaric acid or mandelic acid are formed.Salt with free carboxyl groups formation can source From inorganic base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or iron hydroxide；Or organic base, it is such as different Propylamine, trimethylamine, histidine or procaine.After preparation, solution is carried out with the mode compatible with formulation and therapeutically effective amount Administration.Preparation easily can be administered with various formulations, for example, be formulated for parenteral, such as Injectable solution, or The aerosol of lung is delivered to, or is formulated for digestion administration, such as drug release capsules.
Further, according to the present invention, it is adaptable to which the composition of the invention of administration is provided with or without inert diluents In the pharmaceutical carrier of agent.Carrier be able to should assimilate, and including liquid, semisolid (i.e. paste) or solid carrier.At present except right Outside the harmful any conventional media of the therapeutic effect of recipient or the composition included in it, reagent, diluent or carrier, Its application for being used for the administration composition for implementing the inventive method is suitable.The example of carrier or diluent includes：Fat, Oil, water, salting liquid, lipid, liposome, resin, adhesive, filler etc., or its combination.Composition can also include various antioxygens Agent is to delay the oxidation of one or more components.Furthermore, it is possible to the effect of microorganism is prevented by preservative, it is such as, a variety of Antibacterial agent and antifungal agent, it includes but is not limited to, parabens (such as methyl p-hydroxybenzoate, to hydroxyl Propyl benzoate), methaform, phenol, sorbic acid, thimerosal or its combination.According to the present invention, with any routine and practical ways By composition and carrier combinations, the mode such as by solution, suspension, emulsification, mixing, encapsulating, absorption.These methods are these Known to art personnel.
In one embodiment of the invention, composition is fully combined or mixed with semi-solid or solid carrier Close.Mixing can be carried out in any usual manner, for example, grind.Also stabilizer can be added in mixed process to protect the composition from Therapeutic activity is lost, i.e., be denatured under one's belt.The example of workable stabilizer includes in composition：Buffer solution, amino acid are for example sweet Propylhomoserin and lysine, sugar such as dextrose, mannose, galactolipin, fructose, lactose, sucrose, maltose, D-sorbite, mannitol Deng.
In other embodiments, the present invention includes 125B11 analog or derivative, Yi Zhonghuo it is contemplated that using The medicine lipid vehicle composition of a variety of lipids and aqueous solvent.As used herein, term " lipid " is defined as being included in water Middle characteristic is insoluble and any of the extensive material that can be extracted with organic solvent.The extensive types of compound is ability Known to field technique personnel, when term used herein " lipid ", it is not limited to any specific structure.Example includes Compound and its derivative containing long-chain fat hydrocarbon.Lipid can be naturally occurring or synthesized (i.e. artificial design or system Standby).However, lipid is typically a kind of biological substance.Biological lipid is known in the art, it may for example comprise neutral fat, Phosphatide, phosphoglyceride, steroids, terpene, lysolipin, glycosyl sphingolipid, glycolipid, thioester (sulphatides), contain ether and ester and connect The lipid and polymerization lipid and combinations thereof of the aliphatic acid connect.
Those of ordinary skill in the art are familiar with the technical scope that can be used for being distributed to composition in lipid excipients.Example Such as, 125B11 analog or derivative can be distributed to containing fat by any mode known to persons of ordinary skill in the art In the solution of matter, with Lipid dissolution, emulsified, mixed with lipid with lipid, combined with lipid, be covalently bonded in lipid, as outstanding Supernatant liquid be included in lipid in, included with micella or liposome or with micella or lipid bluk recombination, or otherwise with lipid Or lipid conformation is combined.Dispersion may cause or not cause the formation of liposome.
Giving the actual dose of the composition of the invention of animal sufferer can be determined by physics and physiologic factor, for example Body weight, patient's condition seriousness, type, past or the parallel Results means for the treatment of disease, sufferer idiopathic and administration way Footpath.According to dosage and approach, dosage and/or the administration number of times of effective dose preferably can become according to the response of subject Change.
In certain embodiments, pharmaceutical composition can include for example, at least about 0.1% reactive compound.Other In embodiment, reactive compound can such as component unit weight about 2% to about 75%, or about 25% to about 60%, And any range that can wherein derive.Certainly, the amount of reactive compound can be with such in each treatment compositions useful Prepared by mode, make it that in any given unit dose of compound suitable dosage can be obtained.Such as solubility, biological utilisation The factors such as degree, biological half-life, method of administration, product shelf life and other pharmacology, which are considered, prepares the pharmaceutical preparation What the technical staff in field was expected, as such, it is desirable to a variety of dosage and therapeutic scheme.
In other nonrestrictive examples, the dosage being administered every time can also be comprising about 1 microgram/kg/ body weight, about 5 micro- Gram/kg/ body weight, about 10 micrograms/kg/ body weight, about 50 micrograms/kg/ body weight, about 100 micrograms/kg/ body weight, about 200 micrograms/kg/ Body weight, about 350 micrograms/kg/ body weight, about 500 micrograms/kg/ body weight, about 1 milligram/kg/ body weight, about 5 milligrams/kg/ body weight, about 10 Milligram/kg/ body weight, about 50 milligrams/kg/ body weight, about 100 milligrams/kg/ body weight, about 200 milligrams/kg/ body weight, about 350 milligrams/ Kg/ body weight, about 500 milligrams/kg/ body weight, to about 1000mg/kg/ body weight or higher, and any scope that can be derived. In the non-limitative example for the scope that the numeral listed from there is derived, based on above-mentioned numeral, about 5mg/kg/ body weight can be given To about 100mg/kg/ body weight, the scope of about 5 micrograms/kg/ body weight to about 500 milligrams/kg/ body weight etc..
Digest composition and preparation
125B11 analog or derivative are configured to be administered through digestion approach.Digestion approach includes composition with disappearing Change all possible method of administration that road is directly contacted.Specifically, pharmaceutical composition disclosed herein can it is oral, buccal, Per rectum or sublingual administration.So, these compositions can be prepared with inert diluent or with assimilable edible carrier, or it Can be encapsulated in duricrust or soft shell gelatin capsules, either they can it is tabletted or they can be directly incorporated into dietetic food In.
Reactive compound can be combined with excipient, and can absorb tablet, it is buccal piece, lozenge, capsule, elixir, mixed The forms such as suspension, syrup, paper wafer use (Mathiowitz et al., 1997；Hwang et al., 1998；U.S. Patent number 5, 641,515；5,580,579；With 5,792,451).Tablet, lozenge, pill, capsule etc. can also include following：Adhesive, such as Such as gum tragacanth, Arabic gum, cornstarch, gelatin or its combination；Excipient, such as Dicalcium Phosphate, mannitol, breast Sugar, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate or its combination；Disintegrant, such as cornstarch, potato Starch, alginic acid or its combination；Lubricant, such as magnesium stearate；Sweetener, such as sucrose, lactose, saccharin or its group Close；Flavor enhancement, such as peppermint, wintergreen, cherry flavoring, orange flavor etc..When unit dosage forms are capsules, except upper State outside the material of type, can also include liquid-carrier.Various other materials can have or change in addition dosage list with coating form The physical form of position.For example, can be coated with shellac, sugar or both to tablet, pill or capsule.When dosage form is capsule When, in addition to materials of the above type, it can also include carrier, such as liquid-carrier.Intestines can be carried out to gelatine capsule, tablet or pill It is coated.Enteric coating prevents composition in denaturation of the pH for acid stomach or upper intestines.See, for example, U.S. Patent number 5,629, 001.When reaching small intestine, alkaline pH therein dissolving is coated, and allows composition to discharge and absorbed by specific cells, for example on Skin enterocyte and peyer's patches M cells (Peyer's patch M cell).Elixir syrup can include and be used as sweet tea The active compound sucrose of taste agent, as the methyl p-hydroxybenzoate and propyl ester of preservative, dyestuff and flavor enhancement, such as cherry Or orange flavor.Certainly, any material for preparing any unit dosage forms should be that pharmacy is pure and in amount used Under it is substantially nontoxic.In addition, reactive compound can be coupled in extended release preparation and formula.
For being administered orally, composition of the invention can alternatively combine one or more excipient, with formed collutory, Toothpaste, buccal piece, oral spray or sublingual oral Preparation.For example, the desired amount of active component can be mixed properly Solvent such as dobell's solution (many Bei Ershi liquid (Dobell's Solution)) in prepare collutory.Alternatively, active component In the solution that oral administration solution such as boronic acid containing sodium, glycerine and saleratus can be mixed, or it is distributed in toothpaste, or it is effective to treat Amount is added in the composition that can include water, adhesive, abrasive material, flavor enhancement, foaming agent and Humectant.Alternatively, it will can combine Thing is configured to that tablet that is sublingual or otherwise dissolving in the oral cavity or solution form can be placed in.
Include suppository suitable for other preparations of other digestion mode of administration.Suppository is the solid formulation of various weight and shape Type, for example, being typically inserted into rectally.After insertion, suppository softens in chamber liquid, melts or dissolved.Generally, for suppository, Traditional carrier may include, for example, PAG, triglycerides or its combination.In certain embodiments, suppository can be with Formed by the mixture comprising e.g., from about 0.5% to about 10% and preferably from about 1% to about 2% active component.
Parenteral composi and preparation
125B11 analog or derivative can pass through Parenteral.As used herein, " parenteral " bag of term Include and bypass gastral approach.Specifically, the method for administration of pharmaceutical composition disclosed herein can be, such as, but not limited to： Intravenous, intradermal, intramuscular, intra-arterial, intrathecal, subcutaneous or intraperitoneal U.S. Patent number 6,537,514,6,613,308,5, 466,468,5,543,158；5,641,515；With 5,399,363.
It can be prepared in the water of appropriate mixed surfactant such as hydroxypropyl cellulose as free alkali or pharmacologically The solution of the reactive compound of acceptable salt form.Also it can be prepared in glycerine, liquid macrogol and its mixture and oil Dispersion.Under ordinary conditions of storage and use, these preparations include preservative to prevent microorganism from growing.Suitable for injection The medicament forms used include aseptic aqueous solution or dispersion and faced with preparing the sterile of sterile injectable solutions or dispersion Powder (United States Patent (USP) 5,466,468).In all cases, form must be sterile, and it is stream to a certain extent that must be Body is easily to inject.Under conditions of manufacture and storage, it must be stable, it has to be possible to combating microorganisms (for example bacterium and Fungi) contamination and preserve.Carrier can be solvent or decentralized medium, it may for example comprise water, ethanol, polyalcohol are (that is, sweet Oil, propane diols and liquid macrogol etc.), their suitable compounds, and/or vegetable oil.For example, using coating such as lecithin Fat, for dispersion, by the granularity needed for maintenance, and uses surfactant, can maintain appropriate mobility.Prevent micro- Biological effect can be realized by various antiseptics and antifungal agent, for example, parabens, anesin, benzene Phenol, sorbic acid, thimerosal etc..In many cases it is preferred to including isotonic agent, such as sugar or sodium chloride.Injectable composition Extension absorption can be realized by the composition using delay absorption reagent such as aluminum monostearate and gelatin.
For the parenteral carried out with the aqueous solution, for example, solution is because of the appropriate buffering if necessary, and with enough Salt solution or glucose first make liquid diluent isotonic.These specific aqueous solution are particularly suited for intravenous, intramuscular, subcutaneous and peritonaeum Interior administration.On this, workable sterile aqueous media is those skilled in the art according to known to the disclosure. For example, a dosage can be dissolved in 1 milliliter of isotonic NaCl solution, and it is added in 1000ml hypodermoclysis fluids, or Person is injected (see, for example, " Remington's Pharmaceutical Sciences in proposed infusion site《Thunder Bright pharmacopeia》" the 15th edition, the 1035-1038 pages and the 1570-1580 pages)., will according to the state of the subject of required treatment Need to carry out dosage some changes.In any event, the people for being responsible for administration will determine to be used for the appropriate agent of individual subjects Amount.
Sterile injectable solutions are prepared by following：The desired amount of reactive compound is attached in appropriate solvent, needed Will when be combined with above-mentioned various other compositions, then filtration sterilization.Generally, dispersion is prepared by following：Will be various degerming Active component is attached in the sterile carrier containing basic decentralized medium and from the other compositions needed for those described above. In the situation of aseptic powdery agent for preparing sterile injectable solutions, preparation method preferably is vacuum drying and lyophilized skill Art, produces the powder of any other required composition of activating agent and the solution from its aseptic filtration previous.Powder composition Combined with the liquid-carrier with or without stabilizer such as water or saline solution.
Reactive compound 125B11 analog or derivative can be configured to be administered through various approach, for example Local (i.e. percutaneous) administration, mucosa delivery (intranasal, vagina etc.) and/or inhalation.The pharmaceutical composition of local administration can be wrapped Reactive compound is included, it is formulated for medication coating, such as ointment, paste, emulsifiable paste or powder.Ointment includes all parts The grease of coating, absorbent, emulsion and based on water miscible composition, and emulsifiable paste and lotion are those for only including emulsion bases Composition.The medicine of local administration can promote the endermic absorption of active component comprising penetration enhancers.Suitable infiltration increases Strong agent includes glycerine, alcohol, alkyl methyl sulfoxides, pyrrolidones and luarocapram.Possibility for the composition of local coating Matrix include：Polyethylene glycol, lanolin, cold cream and vaseline and any other suitable absorbent, emulsion or water-soluble soft Cream base matter.When needing, topical formulations can also include emulsifying agent, gelling agent and anti-microbial preservative, to preserve active component simultaneously Homogeneous mixture is provided.The percutaneous dosing of the present invention can also include using " paster ".For example, paster can be when certain Between during in provide one or more active materials with continuous mode at a predetermined rate.
Pharmaceutical composition can be passed by eye drops, intranasal spray, inhalant and/or other aerosol delivery carriers Send.Composition is set directly to be delivered to the method for lung in such as U.S. Patent number 5,756,353 and 5,804 by nose aerosol, Described in 212.Similarly, with the medicine delivery of intranasal microparticle resins and lysophosphatidyl-glycerol compound (U.S. Patent number 5, 725,871) it is also that pharmaceutical field is well-known.Transmucosal delivery medicine is in the U.S. in the form of Haloport F matrix Described in the patent No. 5,780,045.
Term aerosol refers to the colloid system for the finely-divided solid for being dispersed in liquefaction or the liquid particles in pressurized gas propellant System.The present invention is used for the typical aerosol that sucks by active component in liquid propellant or in liquid propellant and appropriate molten Supensoid agent composition in the mixture of agent.Suitable propellant includes hydrocarbon and hydrocarbyl ether.
Combination treatment
In order to improve the validity of composition of the invention, other therapies can be delivered to the individual with metabolic disorder. For example, in addition to another therapy for being used for obesity, the composition of the present invention can also be applied to obese individuals.For example, another Outer obesity therapeutic includes dietetic treatment, physical therapy (motion), medicinal treatment, operation and behavior therapy.Exemplary medicine Therapy is included for example： (Xenical), Phentermine (Phentermine) and west Cloth Qu Ming (Sibutramine)Exemplary operation includes such as liposuction and gastric bypass.
For the individual with diabetes, for example, exemplary other treatment compound includes following one kind or many Kind：Ai Ketuo (Actos) (Pioglitazone (pioglitizone))；ACTOSPlus Met；Ya Moli (Amaryl) (Ge Liemei Urea (glimepiride))；Avandaryl (Avandia (Avandia)+Glimepiride)；Avandia (Avandia) (Rosiglitazone (rosiglitazone))；Wonder quick (Avandamet) (Rosiglitazone Maleate (rosiglitazone maleate) and salt Sour melbine (metformin hydrochloride))；Byettap；Duetact (PIOGITAZONE HYDROCHLORIDEs (pioglitazone HCl) and Glimepiride)；Cover your not (Galvus) (vildagliptin (Vildagliptin))；Glipizide (Glipizide) (sulfonylureas (Sulfonlyurea))；Glucophage (Glucophage) (melbine (metformin))；Lattice The U.S. urea (Glimepiride) of row；Glucovance (glibenclamide (glyburide)/melbine)；Glipizide XL (Glucotrol XL) (agent of Glipizide sustained release)；Glibenclamide (Glyburide)；Glyset (Miglitol (miglitol)) glycosidase Inhibitor；Januvia (phosphoric acid Xi Gelieting (sitagliptin phosphate))；Glipizide-melbine preparation (Metaglip) (Glipizide+melbine)；Melbine-biguanide (Metformin-biguanide)；Prandin is (auspicious Ge Lienai (repaglinide))；Precose (acarbose (acarbose))；Rezulin (troglitazones (troglitazone))；Starlix (Nateglinide (nateglinide)).Other diabetotherapies include improving diet and fortune It is dynamic.
The measurement of exemplary metabolic disorder treatment
In certain aspects of the present disclosure, the composition of individual one or more present invention is given, and assesses the individual The improvement of at least one symptom of metabolic disorder.For example, in the specific embodiment that metabolic disorder is fat, it may be determined that use Fat improvement during the composition treatment of one or more present invention and/or after treatment.Fat improvement can pass through any mark Quasi- mode is measured, but in particular aspects, for example, passing through measured body weight, body mass index (BMI) measurement, and/or body part Dimensional measurement (such as waistline measurement) determines fat improvement.Calculating BMI illustrative methods includes：The body weight of one people is (with thousand Gram be unit) divided by his height (in units of rice) square (body weight [kg] height [m]2).The BMI for thinking more than 30 is fertilizer Fat, the BMI between 25 to 29.9 is overweight.In other aspects of the present invention, the treatment of the present invention is being applied to diabetic individual The individual improvement is detected after method.In a specific embodiment, diabetes are monitored by blood testing.For example, blood Liquid detection can determine chemical substance A1C.Blood glucose is higher, and A1C levels will be higher.In some cases, measurement cholesterine (including HDL and/or LDL cholesterine) and/or triglycerides, such as pass through the standard mode in this area.In specific situation, carry out Empty stomach lipoprotein distribution profile (fasting lipoprotein profile), is such as carried out by the standard mode in this area.
The kit of the present invention
Arbitrary composition as described herein may be embodied in kit.In non-limitative example, kit includes suitable Composition for treating and/or preventing one or more metabolic disorders.In other embodiments of the present invention, kit bag Include one or more apparatuses for being used to sample from individual.For example, the apparatus can be swab (such as cotton wool swabs), toothpick, solution One or more in cut open cutter, spatula, syringe etc..In another embodiment, other compounds, example are provided with kit Such as it is used for other compounds for treating and/or preventing metabolic disorder.For example, any combinations thing provided in kit can be in water Packed in property medium or with lyophilized form.The container of kit generally includes at least one bottle, test tube, flask, bottle, syringe Or other vessel forms, component can be placed in one, and preferably appropriate decile.When there is more than one component in kit When, kit generally also includes second, third or other other container, and these extra components can be respectively placed in wherein.
Including the following examples, to illustrate the preferred embodiments of the invention.It will be understood by those skilled in the art that below Embodiment described in technology represent the inventors discovered that operational excellence in an embodiment of the present invention technology, thus can To be considered to constitute the preference pattern of its implementation.However, according to the disclosure, it will be understood by those skilled in the art that Many changes can be carried out to disclosed specific embodiment, and still obtain similar or like result without departing from this hair Bright spirit and scope.
Embodiment 1
125B11 reduces the expression of SREBP- response genes
The allelic expression of drug-treated and untreated cell relatively can reveal that the spy influenceed by 125B11 Determine molecular pathways.DU145 cells are handled with 125B11 or only DMSO, and the mRNA samples of extraction are passed through into Affimetrix DNA microarray is analyzed, and 33,000 gene (table 1) (mapping) in positioning.
Shown in the microarray results of table 1. adjusted by 125B11 known to or the gene that may be controlled by SREBP
As a result show, 55% is lowered in the gene of (0.7 times of ＜) by 125B11 is known or is probably to be regulated and controled by sterol Element conjugated protein (SREBP) control, including ldl receptor, HMG- CoA-reductases and fatty acid synthase (Horton et al., 2003).Being tested by RT-PCR confirms the downward (Figure 1A -1B) of representational SREBP- responses gene.These results indicate that 125B11 is the selective depressant of SREBP approach.
In order to illustrate that 125B11 weakens SREBP function, presence or absence of in the case of 125B11 The ability (Fig. 2A -2B) of the transcription of endogenous SREBP activation SREBP- response reporters is determined in HEK293 cells.Method figure Statin A with concentration dependant manner reduce reporter activation, wherein the expression of luciferase by sterol controlling element three Individual Repetitive controller.On the contrary, 125B11 can not weaken mature form (amino acid/11-500) activation of SREBP-1 heterogenous expression The ability (Fig. 2 C) of reporter gene activity.These results indicate that in 125B11 energy selective exclusion cell SREBP activation Process.
Embodiment 2
125B11 blocks SREBP Proteolytic activation
In order to examine whether 125B11 influences SREBP Proteolytic activation, with resisting for the NH2 ends for SREBP-1 Body, the full cell lysate (Fig. 3 A) of the DU145 cells handled by western blot analysis with 125B11.125B11 The amount for the 68 KDa mature forms that SREBP-1 is reduced with dosage-dependent manner is handled, and the amount of 125 KDa precursor forms increases Plus.The antibody for its COOH end is used to obtain similar results (Fig. 3 B) for SREBP-2.These result showing method figure statins A Directly or indirectly weaken the Proteolytic activation of two kinds of SREBP isotypes.
The Proteolytic activation for suppressing SREBP will destroy SREBP nuclear transfer.With resisting for the NH2 ends for SREBP-1 Body, influence of the 125B11 to SREBP-1 Subcellular Localizations is analyzed by immunofluorescence microscopy.Only cell is handled with DMSO When, in the culture medium of serum-free (fat-free), SREBP-1 is almost exclusively positioned in core (Fig. 3 C-3E).On the contrary, with During 125B11 Incubate cells, SREBP-1 immunofluorescence reduces and accordingly increases (Fig. 3 F-3H) outside core in core, and this shows 125B11 suppresses SREBP-1 nuclear location.
Embodiment 3
By striking low SREBP-1 methods of ascertainment figure statin phenotype
125B11 causes two kinds of phenotypes in culture cell：(i) the insulin-induced fat generation of 3T3-L1 cells Suppression and (ii) DU145 prostate gland cancer cells serum dependent/non-dependent grow prevent.The first phenotype and following conclusions are complete It is complete consistent：Because known actions of the SREBP-1 in fat generation, 125B11 is SREBP-1 blocking agent (Tontonoz Et al., 1993).In order to confirm under cell culture condition, by transfecting the specific siRNAs of SREBP-1 (siRNA) The expression (Fig. 4 G) for the SREBP-1 that expression vector comes in silence 3T3-L1 cells, and detect silence to insulin-induced fat The effect of fat generation.As expection, strike low SREBP-1 expression blocked completely the oil droplet of 3T3-L1 cells formed (Fig. 4 D-4F, Clone 1 and 2), and with empty vectors (neo；Fig. 4 A) transfection control cell show and parent 3T3-L1 cells (Fig. 4 B) one Accumulation of fat more than sample.These results indicate that the phenotype that 125B11 is induced in 3T3-L1 cells is by suppressing SREBP-1 Mediation.
In order to test whether suppression of the 125B11 to SREBP-1 mediates what the serum dependent/non-dependent of DU145 cells grew Prevent, the expression vector similarly by transfection SREBP-1 specific siRNAs makes the expression silencing of SREBP-1 in DU145 cells (Fig. 5 B).The control cell transfected with empty vectors (neo) grows in the presence of serum or IGF1, as parent DU145 is thin Born of the same parents are such.On the contrary, the low cell (clone 1 and 2) that strikes that SREBP-1 expression is silenced shows reduced serum dependent/non-dependent The growth of IGF1 drivings, and it is few (Fig. 5 A) to the growth effect of its serum dependency.
It is probably because of outside fat source in serum free medium to SREBP-1 demand in the growth of serum dependent/non-dependent Lack.When ectogenous fat acid is not present in serum, cell needs synthetic fatty acid and cholesterine (membrane structure unit) to tie up Hold cell growth.In order to test importance of the aliphatic acid in cell growth, monitor SREBP-1 in fat-free blood serum medium and strike The growth (Fig. 5 A) of low cell.The cell growth that SREBP-1 silences are damaged in fat-free culture medium, degree contains with it in serum-free As in IGF1 culture medium.These results indicate that 125B11 blocks the serum of cancer cell non-by suppressing SREBP-1 Dependence grows.
Embodiment 4
125B11 mitigates mouse weight, reduction cholesterine and glucose level and lowers fat generation enzyme
The medicine sample chemical constitution of 125B11 promotes inventor to study the SREBP- in the liver of its suppression intact animal 1 ability.Detect and be followed by the fat-free high carbohydrate meals of feeding and the fat of 48 hours in long-time fasting (48 hours) Under fat formation condition, effect of the 125B11 to liver SREBP-1.Since the previous day of 48 hours fasting phases, with 30mg/kg/ It continues 5 days to injecting 125B11 in mouse peritoneum.After fasting 48 hours, relative to control group, treatment group weight loss It is more that (6.12 ± 0.6 relative to 4.9 ± 0.3 grams/mouse；P=0.01) (Fig. 6 A).Do not observe that food intake subtracts during processing Less or overt toxicity (Fig. 6 B).It is interesting that fat-free high carbohydrate meals is fed with again after 48 hours, 125B11 Occurring reduction glucose level in the serum of the mouse of processing, (110 ± 23 relative to 137 ± 14mg/dl；) and cholesteric P=0.06 (93 ± 20 relative to 120 ± 19mg/dl for alcohol；P=0.12 trend (Fig. 6 C)).HDL and LDL is reduced in processing mouse group. However, the reduction of LDL levels seems significantly (16 ± 5 relative to 30 ± 6mg/d1) (Fig. 6 C).
The expression of SREBP-1 in liver extract is detected by Western blotting.With the result one of cell culture Cause, the liver extract of the mouse handled with 125B11 shows the SREBP-1 of decrement 68KDa mature forms and increasing The 125KDa precursor forms (Fig. 6 D) of dosage.The liver expression of fatty acid synthase (FAS), fatty acid synthase are also determined after processing (FAS) it is a kind of representational SREBP-1 responses fat generation enzyme (Boizard et al., 1998).The albumen of liver extract Matter engram analysis show that 125B11 processing makes FAS expression reduce up to 30% (Fig. 6 E).Phase one is reduced with expression Cause, its enzymatic activity in extract is also similarly reduced.(Fig. 6 G) such as observed to FAS, in liver extract also by The activity reduction (Fig. 6 F) of the acetyl-CoA carboxylase (ACC) of SREBP-1 regulation and control.These results indicate that with the cell in culture As middle discovery, 125B11 has blocked the activation of SREBP-1 in mouse liver.
The mouse of processing (2 weeks) another group of feeding normal diet of long period causes weight loss 10%, and control group Body weight it is unchanged (Fig. 7 A-7B).Between two groups food intake it is similar (processing and control mice be respectively 3.8 and 3.5g/ small Mouse/day).The result of mouse with being fed with the feeding fat-free diet of fasting/again is consistent, and the mouse for being fed with normal diet exists The trend (Fig. 7 C) of the gentle relatively low triglycerides (TG) of significantly lower G/W and cholesterol levels is shown in blood.FAS Activity and its protein level also reduce about 30% (Fig. 7 D and 7E).
Embodiment 5
The significance of the present invention
It is the valuable instrument for exploring complex cell process (including metabolic pathway) to be proved bioactive small molecule.Fat A kind of key regulatory of matter stable state and insulin action is SREBP transcription factor families (Brown and Goldstein, 1997). The small molecule of regulation SREBP functions can be used for treating metabolic disease, and may be used as further from molecular level understanding The instrument of disease.As shown by data based on cell and animal, 125B11 is by lowering the amounts of maturation SREBP-1 forms in core Reduce the expression of fat generation gene.
Report activation SREBP-1 and -2 small molecule.These reductions LDL molecule is by stimulating SREBP albumen water Deactivate and raise the expression of ldl receptor.Although the molecular mechanism of effect is not yet fully apparent from, Notes of Key Data SCAP is this point The major target class of son.Different from these molecules, 125B11 suppresses SREBP activation and lowers SREBP- responses gene (bag Include ldl receptor gene) expression (table 1).
The animal data of 125B11 is consistent with cell culture result.Again feeding fat-free diet under usage figure he In the liver extract of the mouse of spit of fland A processing, with notable lower level ripe SREBP-1 forms and higher levels of precursor Form.On the other hand and as expection, in the liver extract of control group, the level of mature form is higher than precursor forms (Horton et al., 1998).It is interesting that the total amount of combining form seems unchanged, unique difference is core (maturation) and born of the same parents Distribution between matter form (precursor).These as shown by data, 125B11 may not change SREBP-1 expression, but carry The cutting process of high precursor, causes the reduction of its amount and the increase of ripe and activity form.It is small by using SCAP deficiencies The experiment of mouse is proved SREBP-1 and cuts the importance in Fatty synthesis：Under the conditions of being fed with again, mouse liver can not be lured Lead ACC and FAS expression (Liang et al., 2002).
In order to evaluate the physiology significance of core SREBP-1 levels reduction, ACC and FAS level and activity are determined.Ring Should be handled in 125B11, they in liver extract activity lowered.These results are used as aliphatic acid with SREBP-1 The effect of route of synthesis regulator is consistent (Shimano, 2000).Shimano et al. is disclosed, and is raised with high carbohydrate meals Support SREBP-1-/-FAS and ACC level can not be induced during mouse, this confirms SREBP-1 in the expression of regulation fat generation enzyme Effect.
Compared with the control, a kind of interesting observation in the mouse of 125B11 processing is the reduction of body weight and blood glucose.Body It is probably the fatty generating rate because fat generation enzyme such as ACC and FAS relatively low caused by lowering to reduce again.In addition, being used as ACC Product and carnitine palmitoyltransferase potent inhibitor, the reduction of malonyl coenzyme A can cause increased aliphatic acid oxygen Change and fat combustion.In a specific embodiment, 125B11 suppresses SREBP-1 cuttings and lowers fat generation enzyme, increases Plus fatty acid oxidation, lose weight and increase insulin sensitivity, so as to cause glucose to reduce.
Embodiment 6
Material
Degreasing serum (Goldstein et al., 1983) is prepared as described.Not fatty FBS is purchased from Fisher.Rabbit-anti- SREBP-1 (sc-8984) and the anti-actin of goat (sc-1616) polyclonal antibody are purchased from Santa Cruz Biotechnology.The anti-SREBP-2 polyclonal antibodies of mouse and the anti-FAS antibody of mouse are derived from BD Biosciences. Anti- goat IgG HRP and anti-rabbit igg HRP are derived from Promega.The anti-reagent acquisitions of fading of ProLong Gold containing DAPI From Molecular Probes Invitrogen Detection Technologies.Anti- rabbit igg FITC is derived from Chemicon International.Dexamethasone (Dexamethasone, DEX) and 1- methyl -3- isobutyl group lutein (MIX) it is derived from Sigma.
The preparation of 125B11
While stirring, by the bromo- 4 '-methyl acetophenones of 2- (1.22g, 5.70mmol) and protionamide (1.03g, 5.70mmol) mixture in ethanol (20ml) is heated 0.5 hour at 70 DEG C, is subsequently cooled to 0 DEG C.The yellow of formation is sunk Form sediment and filter, washed with cold ethanol, the 125B11 hydrobromate (1.78g, 83%) of yellow needles is obtained after drying：1H NMR(DMSO-d6, 600MHz) and dH8.88 (d, J=6.2Hz, 1H), 8.54 (s, 1H), 8.46 (d, J=1.4Hz, 1H), 8.36 (dd, J=1.4,6.2Hz,1H), 7.99 (d, J=7.6Hz, 2H), 7.31 (d, J=7.6Hz, 2H), 3.03 (t, J=7.6Hz, 2H), 2.35 (s, 3H), 1.80 (m, 2H), 0.96 (t, J=7.6,3H)；On C18H18N2The HRMS (FAB) that S+H is calculated is accurate Quality needs m/z 295.1269, observation m/z 295.1269.
Cell culture
In 37 DEG C, 5%CO2It is lower that DU145 people's androgen dependent/non-dependent prostate gland cancer cells (ATCC) are maintained at containing 2mM L- glutamines, 1.0mM Sodium Pyruvates, 0.1mM nonessential amino acid and 1.5g/L sodium acid carbonates and 10% hyclone, In Eagle ' the s minimum essential mediums of 100 units/mL penicillin and 100 μ g/mL streptomycin sulphates.At 37 DEG C by 3T3-L1 Fibroblast (ATCC) is maintained at containing 5.5mM glucose, 10% hyclone, 50 μ g/mL gentamicins, 0.5mM paddy amine In Eagle ' the s culture mediums of Dulbecco ' the s improvement of acid amides and 0.5 μ g/mL amphotericin Bs (fungizone).37 DEG C, 5%CO2It is lower that human embryo kidney 293 cells (ATCC) are maintained at containing 10% hyclone, 100 units/mL penicillin and 100 μ g/ In Eagle ' the s culture mediums of Dulbecco ' the s improvement of mL streptomycin sulphates.
Oligonucleotide microarray is analyzed
In the presence of 1 μ g/mL IGF1, in serum free medium, with 5mM 125B11s or only with DMSO processing DU145 prostate gland cancer cells 6 hours, by Total RNAs extraction to TRI reagents (Molecular Research Center), and lead to Cross RNeasy Mini Kit (Qiagen) further separation.In Baylor College Medicine microarray core mechanism (Baylor College Of Medicine Microarray Core Facility) pass through Affymetrix Human Genome U133 Plus The mRNA of 2.0 Array analysis purifying, the Array of Affymetrix Human Genome U133 Plus 2.0 are by almost 45,000 probe groups are constituted, and the probe groups, which are represented, derives from about 33,000 certified human gene more than 39,000 Plant transcript (Affymetrix, Inc.).
Luciferase reporter is determined
At the 0th day, by HEK293 cells with 5x 103The triplicate bed board of density in/hole is to containing 10% on 96 orifice plates Eagle ' the s culture mediums of Dulbecco ' the s improvement of hyclone, 100 units/mL penicillin and 100 μ g/mL streptomycin sulphates In.2nd day, following matter is used by using Lipofectamine reagents (Lipofectamine reagent) (Invitrogen) Grain transient cotransfection cell：A kind of 0.4 μ g/ hole pSRE-Luc (luciferase reporter construct of SRE-1 drivings) and 0.1 μ G/ holes b-gal reporters, wherein β-gal expression is controlled by actin promoter, and final volume is 150mL.At 37 DEG C Culture 5 hours after, with phosphate-buffered salt water washing cell, then under conditions of being not present or there is 125B11, containing 10% 100 μ l Dulbecco ' the s improvement without lipid serum, 100 units/μ L penicillin and 100 μ g/mL streptomycin sulphates Cultivated in Eagle ' s culture mediums.After culture 20 hours, with 20 μ L 1x Reporter Lysis Buffer (Promega) The cell in each hole is cracked, aliquot is used to determine luciferase (10 μ L) and beta galactosidase (10 μ L) activity.It is right In luciferase assay, detection photon is produced in the ARVOsx multiple labelings counters (PerkinElmer) of Wallac 1420, with Per second count represents.Determine, after 37 DEG C are cultivated 0.5 hour, existed by microplate reader (Tecan) for beta galactosidase The hydrolysis of O- nitrobenzophenones-beta-D-galactosidase is determined at 405nm wavelength.It is right by the activity (OD units) of beta galactosidase Uciferase activity (counting per second) is standardized., will for the overexpression of SREBP-1c N-terminal mature form PCMV-SREBP-1c (1-436) and pSRE-Luc cotransfections.PSRE-Luc and pCMV-SREBP-1c (1-436) by J.L.Goldstein (University of Texas Southwestern Medical Center) is provided.
RT-PCR is tested
It will be used in combination from DU145 cells in Total RNAs extraction to TRI reagents (Molecular Research Center) RNeasy Mini Kit are separated.RT-PCR is carried out to RNA sample using Access RT-PCR System.RT-PCR is anti- Liquid is answered to include total serum IgE, 1 μM of each primer, 0.2mM dNTP, 1mM MgSO4, AMV reverse transcriptase (2 unit) and Tf1 DNA are poly- Synthase (2 unit), the μ L of final volume 25.Primer pair used is as follows：It is 5'-TCA for LDL receptor (LDLR) GAC CGG GAC TGC TTG GAC GGC TCA GTC-3'(SEQ ID NO:1) with 5'-CCA CTT AGG CAG TGG AAC TCG AAG GCC G-3'(SEQ ID NO:2)；It is 5'-GCC TGC for stearyl-coenzyme A desaturase (SCD) TTG ATA ATA TAT AAA C-3'(SEQ ID NO:3) with 5'-CAC TTG AAT TGA GCT TTA G-3'(SEQ ID NO:4)；It is 5'-AAG AAA AAG TGT CAG ACA GCT GG-3'(SEQ ID for ATP citrate-lyase (ACL) NO:5) with 5'-TGG ACT GAA GGG GTG TTA GC-3'(SEQ ID NO:6)；For 3- hydroxy-3-methyl glutaryls- CoA-reductase (HMG CoA R) is 5'-GCC CGA CAG TTC TGA ACT GGA ACA-3'(SEQ ID NO:7) and 5'-GAA CCT GAG ACC TCT CTG AAA GAG-3'(SEQ ID NO:8)；It is 5'- for mevalonate kinase (MVD) CTG CCT GAC TGC CTC AGC-3'(SEQ ID NO:9) with 5'-ACC TCT CCT GAC ACC TGG G-3'(SEQ ID NO:10)；It is 5'-AAG ACT TCA GGG TAA GTC ATC A-3' for insulin-induced gene 1 (INSIG1) (SEQ ID NO:11) with 5'-CGT GTA TAA TGG TGT CTA TCA G-3'(SEQ ID NO:12).Amplification condition is such as Under：Continue 4 minutes at 94 DEG C, then 1 circulation is denatured 40 seconds at 94 DEG C, is annealed 40 seconds at 50 DEG C, extended 2 minutes at 68 DEG C, 22 circulations are (for SCD and HMG CoA R), and in 58 DEG C of annealing, 24 circulations are (for LDLR and INSIG1), or at 60 DEG C Annealing, 24 circulations are (for ATP citrate-lyase (ACL)), and in 55 DEG C of annealing, 30 circulations are (for MVD).Pass through agar The DNA of sugared gel analysis amplification is simultaneously quantified with Scion-image (edition 4 .02) software.
Western blotting
By DU145 prostate gland cancer cells with 2x 105The density of individual cells/well is inoculated into 6 orifice plates, in serum-free MEM In 37 DEG C of overnight incubations.Then in the presence of IGF1 (1 μ g/mL), cell is handled with DMSO or 125B11 (1 or 5mM).Training Educate after 6 hours, cell is collected into PBS and cracked in SDS buffer solutions.Sample is separated on 10%SDS-PAGE gels, And produce trace with rabbit-anti-SREBP-1 and anti-SREBP-2 antibody.With enhanced chemiluminescence (ECL) detection reagent (Amersham) specific band is shown.
Immunofluorescence experiment
DU145 prostate gland cancer cells are inoculated on cover glass, then used in containing IGF1 in serum-free MEM overnight 5mM 125B11s or only DMSO processing in the serum-free MEM of (1 μ g/mL).After culture 6 hours, by cell at -20 DEG C in first 20 minutes are fixed in alcohol, and is closed 1 hour in the PBS containing 5% milk and 0.1% polysorbas20.By sample and many grams of rabbit Grand anti-SREBP-1 (Santa Cruz：Sc-8984) it is incubated together, the anti-rabbit igg being then conjugated with fluorescein isothiocyanate Antibody (Chemicon Inc) is incubated together.Using the filter plate suitable for fluoroscopic examination, existed with × 400 multiplication factor Cover glass is visualized under Nikon TE200 fluorescence microscopes.
SREBP siRNA strikes low
The complementation of sequence (512-531) of the insertion from SREBP-1 genes is few in pSUPER carriers (OligoEngine) Nucleotides：5'-GAT CCC CGC CAC ATT GAG CTC CTC TCT TCA AGA GAG AGA GGA GCT CAA TGT GGC TTT TTG GAAA-3'(SEQ ID NO:, and 5'-AGC TTT TCC AAA AAG CCA CAT TGA GCT 13) CCT CTC TCT CTT GAA GGA GGA GCT CAA TGT GGC GGG-3'(SEQ ID NO:14).With Fugene 6 (Roche) by resulting plasmid transfection into 3T3-L1 or DU145 cells.To set up the clone of stable transfection, concentration For 500 μ g/mL neomycin derivative G418 (Gibco), and set up stable transformant.Pass through Western blotting evaluation SREBP-1 expression.For fat generation experiment, 3T3-L1 cells are inoculated into 96 orifice plates, are containing 10% tire ox In the DMEM of serum, and 2 days are further cultured for converging completely.0th day, culture medium is converted into inducing culture：Containing 10% tire Cow's serum, 5 μ g/mL insulin, 0.5mM 1- methyl -3- isobutyl groups lutein (MIX) and the DMEM of 1 μM of dexamethasone (DEX). 2nd day, inducing culture is removed, and be replaced with the DMEM culture mediums containing 10% hyclone and 5 μ g/mL insulin.10th My god, fatty oil droplet is dyed with Oil-Red O.For cell growth assay, by DU145 cells with 2,000 cells/wells Density be inoculated on 96 orifice plates serum-free or containing 1 μ g/mL IGF1,2% without fat hyclone or 2% hyclone In MEM.Pass through WST-1 evaluation of measuring cell growths after 3 days.The experiment is carried out in triplicate.
Utilize the zooscopy of 125B11
Make male mice (129Sv backgrounds) in Baylor College Medicine animal care center (Animal Care Center at Baylor College of Medicine) controlled condition under (12 hours light dark cycles；25 DEG C) to live, it can arbitrarily connect Nearly standard lab chow (Purina Mills) and water.Using male mice of two kinds of different schemes to the 5-6 monthly ages (129Sv backgrounds) intraperitoneal applies 125B11 (30mg/kg；150μL).The first scheme includes：Mouse fasting 48 is small When, fat-free diet is then fed with again 48 hours again.In addition to SREBP, the processing can also induced lipolysis generation enzyme such as ACC and FAS Activity and level.Start within 24 hours before fasting to apply 125B11 or the 10%DMSO to control group (n=in PBS 5), and continue daily apply until experiment terminate.
In second scheme, 30mg/kg 125B11s or 10%DMSO two groups of males of processing daily in PBS Mouse (n=5), continues 2 weeks.Food intake and body weight are determined daily.At the end of experiment, of short duration fasting 4-5 hours of mouse is right They carry out taking blood to detect serum composition.Then mouse is put to death, their liver is quickly removed and ground to form in liquid nitrogen Powder.The tissue suspension of powdered is being contained into 0.1mM PMSF, 5mM benzenecarboximidamide and 5mg/mL protease inhibitor cocktails (Roche) in 10ml PBS, with Polytron (3x30 seconds, at a high speed) homogenization, and carry out of short duration ultrasonically treated to drop Solve DNA.By clarifying extract within 20 minutes in 16,000xg centrifugations.Then with the commercially available antibody for FAS and SREBP-1 Western blot analysis are carried out to sample.It is active (Mao et al., 2006) that FAS and ACC is determined as before.
Embodiment 7
In ob/ob mouse, 125B11 prevents fatty liver, mitigates hyperglycemia and cause weight loss
Although making great efforts to explore the network of regulation food intake and energy balance, do not understand obesity is how to cause this completely yet A little diseases.Study effect of the 125B11 to male ob mouse, especially its by reducing white adipose size to prevent body The effect of increase, diabetic condition and fatty liver again.As previously mentioned, 125B11 is the inhibitor for transcribing main control, its It is the effect by suppressing SREBP-1.The normal mouse weight loss handled with 125B11, and with relatively low grape Sugar and cholesterol levels.Compared with the control, 125B11 reduces the active mature form in the liver of the mouse of processing SREBP-1。
SREBP-1 to -2 aliphatic acid to play related but different effect in cholesteric biosynthesis.SREBP-1 is excellent Gene needed for first activation aliphatic acid is synthesized, and SREBP-2 is conducive to cholesterine to generate.Because 125B11 blocks SREBP-1 With possible SREBP-2 activation, aliphatic acid and cholesteric life are instantaneously adjusted using 125B11 to fat ob/ob mouse Thing synthesizes and discloses phenotype interesting in obesity mice.
125B11 is to body weight and the influence of food intake
Research is using the male ob/ob mouse of 4-5 week old, and its average weight is about 23g/ mouse.Daily intraperitoneal delivery 125B11 (30mg/kg/ days), and measure body weight and food intake.As shown in figures 8 a-8b, the increasing of the mouse weight of processing Plus it is substantially less than control.At the end of first week is handled, injection DMSO average 4.82g/ mouse of ob control mices increase (from 23.58 ± 0.62 to 28.40 ± 1.45), and method figure statin treatment group increase be about 3.37g/ mouse (from 23.08 ± 1.53 to 26.45 ± 1.2g/ mouse), (p=0.03).After processing 28 days, 125B11 treatment group weight ratio control light about 12% (32.1 ± 1.4 for method figure statin 36.2 ± 2.2g/ mouse) (P=0.02).Accumulation food intake in two groups is similar (Fig. 8 C). On average, in treatment group, food intake is with compareing no significant difference, respectively, 5.4 ± 1.5 compared to 5.9 ± 1.4g/ mouse days.
Influence of the 125B11 to glucose in blood and lipid characteristic
One of phenotype most unique is the hyperglycemia caused by insulin resistance symptoms in ob/ob mouse.In order to determine Influence of the 125B11 to blood glucose and lipid, standard diet raise ob/ob mouse in analysis glucose, triglycerides and Cholesteric serum levels.
As shown in Fig. 9 A-9H, after overnight fasting, the glucose level in the animal blood serum through processing is lower about than compareing 70%；Respectively 153.2 ± 30.5 and 429.4 ± 87mg/dl (P=0.003).G/W in animal blood serum through processing It is flat to become suitable with the wild-type mice with feature ob genes, and DMSO control mice is given as expected blood glucose mistake It is high.It is interesting that compared with the control, ketoboidies (ketond bodies) (beta-hydroxy-butanoic acid ester) increase in the animal through processing About 7 times；Respectively 3.62 ± 1.41 and 0.5 ± 0.37mg/dl (P=0.004).The high level of ketoboidies in 125B11 animal Illustrate that fatty acid oxidation is dramatically increased in liver, the primary product in the oxidation is the ketoboidies being secreted into blood.Also, warp Increased blood constituent is the free fatty (NEFA) of the no esterification determined in serum in the mouse of processing, and it is higher about than compareing 70%；Respectively 1.93 ± 0.26 and 0.7 ± 0.2mEq/l (P=0.028).This increase of NEFA levels is probably because right Lipolysis increase from adipose tissue caused by the increase of fatty acid oxidation demand.Known FFA and pancreas in animal and people Insulin resistance is relevant.But, although FFA levels are improved in the serum of the ob/ob mouse of 125B11 processing, but glucose Level is substantially less than control, and this shows the improvement of insulin sensitivity, and this is probably because improved insulin signal transduction institute Cause.In addition, it has recently been found that due to mutant acetyl-CoA carboxylase mouse (Acc2-/-Mutant mice) mouse tissue In (liver, fat and muscle) fatty acid oxidation increase, cause in the blood caused by increased lipolysis in fat cell compared with High ketoboidies and increased NEFA.Compared with the control, triglycerides (TG) level in the mice serum through processing is improved about 30%, respectively 115 ± 11 and 79 ± 12 (P=0.006), this shows that 125B11 improves secretion and migration of the TG from liver. Total cholesteric serum levels are shown as compared with low tendency in method figure statin processing animal, and 183 ± 16 compared to 219 ± 18mg/ Dl (P=0.06).However, LDL significantly reduces about 35%, (31 ± 3 compared to 48 ± 8；P=0.02), and HDL reduction it is less, about For 22% (144 ± 11 and 183 ± 12；P=0.02).Due in the serum for the mouse that 125B11 is handled, LDL levels ratio HDL declines are more, and this shows the result needed for being handled with 125B11.Based on TG level calculations transhipment triglycerides, phosphatide With cholesteric VLDL level, increase by about 50% (23.1 ± 2.3 compared to 15.8 ± 2.4mg/dl).
125B11 reduces epididymal adipose tissues size and improves fatty liver
Due to uncontrolled food intake, ob/ob mouse become morbid obesity, and in adipose tissue and Different Organs As have accumulated too high fat level in liver, cause non-alcoholic fatty liver disease condition and insulin resistance.In about 8-9 week old, Compared with those mouse handled with 125B11, control untreated mice display becomes big liver size and the fat accumulated, (Figure 10 A) as shown in pale color.The average weight of the mouse liver of 125B11 processing is fewer by about 32% than compareing (1.59 ± 0.2 relative to 2.34 ± 0.15；P=0.06) (Figure 10 D).The control mice liver section bag of fat drips is dyed with oil red Containing abundant fat drips, and those of the mouse of 125B11 processing are cut into slices no fat drips, and fat drips are mainly triglycerides (figure 10B).It has been shown that fatty liver occurs for the transgenic mice for being overexpressed SREBP 1.However, lacking SREBP-1 ob/ob mouse (lepob/ob X Srebp 1-/-) in, the fatty liver patient's condition significantly improves, and this shows that SREBP 1 is fat in ob/ob mouse The major contributor of liver development.
At the end of 125B11 is handled 4 weeks, the mouse weight of processing is lighter than compareing.It is used as primary white fat by determining The epididymal adipose tissues pad of fat tissue finds that the mouse of 125B11 processing has significantly smaller fat pad (Figure 10 C).Fat pad Average weight is fewer by about 20% than control, and (2.7 ± 0.1 relative to 3.6 ± 0.2；P=0.02) (Figure 10 D).Less fat pad can Can be because lipid reserves are reduced and/or fat generates fatty acid oxidation increase in reduction and fat.Acc2 is used before-/-It is prominent The research of variant mouse shows, lacking ACC2 also causes fatty, less epididymal adipose tissues pad less in liver and including liver Increased fatty acid oxidation in different tissues inside.It is noted herein, due to lacking the malonyl coenzyme A of ACC2 generations to meat The suppression of malicious alkali palmitoyl transferase, fatty acid oxidation increase, mouse becomes height insulin sensitivity and avoids diet induced Fat and diabetes.At specific aspect, 125B11 lowers ACC enzymes increase fatty acid oxidation and to suppress different tissues (all Such as, such as liver, fat and muscle) in aliphatic acid synthesis.Determine the TG in the ob/ob mouse livers of 125B11 processing And cholesterol levels, and compare and be compared with ob/b.As shown in Figure 11 A, the TG levels in the mouse liver of processing decline about 65% (is respectively 14.8 ± 3.7 and 38.7 ± 6.0mg/ grams of liver；P=0.0004).125B11 can also make the courage in liver Sterol levels decrease beyond 20% (2.8 ± 0.5 and 3.6 ± 0.1；P=0.03) (Figure 11 B).These results further demonstrate Oil Red O are dyed, and are shown, partly the fatty liver in the ob/ob mouse caused by liver fat generation increase can pass through 125B11 treatment is entirely prevented.The decline of these lipids is because synthesis TG and cholesterine in the ob/ob mouse livers of processing Or the fat needed for its precursor generates significantly inhibiting for enzyme.Further, since different mouse tissues (including liver) are to aliphatic acid oxygen The raising of change demand, lipase liver activity is improved, and also increases these lipids from hepatic metastasis to circulation, with different Fatty acid oxidation tissue (such as heart and muscle) utilize.At specific aspect, this ob/ob mouse handled with 125B11 The TG of higher level is relevant in blood.
125B11 lowers the fat generation enzyme in ob/b mouse livers
Enzyme in fatty constructive ways is adjusted by transcription factor such as PPAR and SREBP.Examine small in the ob/ob through processing Influence of the 125B11 to fat generation enzyme level and activity in mouse.Determine the second for performing the rate-limiting step in aliphatic acid synthesis The activity of acyl coenzyme A carboxylases (ACC).ACC is catalyzed the carboxylation of acetyl coenzyme A and produces malonyl coenzyme A (in aliphatic acid synthesis Construction unit), and the synthesis of aliphatic acid is performed by another multifunctional enzyme fatty acid synthase (FAS).Malonyl coenzyme A except Outside being worked in aliphatic acid synthesis, also by suppressing carnitine palmitoyltransferase 1 (CPT 1) in fatty acid oxidation Play a significant role.Fat generation enzyme is significantly induced in ob/ob mouse, partly explains the morbid obesity of these mouse Phenotype.125B11 processing mouse liver extract in ACC activity decrease about 40% (3.44 ± 0.44 relative to 5.55 ± 0.57n mol/min.mg) (Figure 12 A).In the liver extract of the ob/ob mouse of 125B11 processing, fat Acid synthase activity is also significantly down-regulated.In the mouse through processing FAS activity decreases more than 70% (8.64 ± 1.91 relative to 22.6 ± 1.37n mol/min.mg) (Figure 12 B).Under the decline of ACC and FAS activity is all due to two kinds of expression of enzymes levels Drop, as shown in the western blot analysis of two kinds of enzymes (Figure 12 C).Its product fatty acid, C14:0 and C16:0, at method figure statin Than in the liver of untreated control mice notable lower (about 50%) (table 2) in the liver of the ob/ob mouse of reason.
ACC is caused the suppression and activation of enzyme respectively by the acute regulation of phosphorylation/dephosphorylation mechanism.Such as Figure 12 C institutes Show, phosphorus-ACC level is higher in control group, but because ACC expression is equally higher and reach identical degree, this Illustrate that 125B11 does not change specific phosphorylation level (P-ACC/ACC albumen).These results indicate that under ACC activity Adjust only because enzyme level decline, rather than phosphorylation state decline (Figure 12 C-12D).Show exist in liver before Two kinds of ACC isotypes；ACC1 (the advantage isotype in liver) and ACC2 (the advantage isotype in muscle), they are being adjusted respectively Not same-action is played in section lipid synthesis and oxidation.The decline of ACC and FAS activity shows that fat generation is reduced, and in Fa Tuta Fat combustion is significantly improved in the liver of the mouse of spit of fland A processing, this ob/ob handled with the 125B11 as shown in Fig. 9 A-9H 7 times of increases of ketoboidies are consistent in the blood of mouse.Determine also auxiliary in SREBP-1, ATP citrate-lyase (ACL) and stearic acid The level of two kinds of key enzymes in fatty acid metabolism under the transcriptional control of enzyme A desaturases 1 (SCD1).It is small in 125B11 In the liver extract of mouse, the protein level that kytoplasm citrate is converted into the ACL of acetyl coenzyme A is set to decline about 70%, acetyl Coacetylase is substrates of the ACC to produce the malonyl coenzyme A synthesized for aliphatic acid.This downward of ACL levels is further expanded Big influence of the 125B11 to the reduction of fatty generating process in fat generation tissue such as liver.SCD1 passes through in fatty acyl Base coacetylase such as palmityl-CoA and stearoyl-CoA Δ 9 introduce cis-double bonds catalysis monounsaturated fatty acids biosynthesis In rate-limiting step.Product palmitoleoyl coacetylase (16:1) with oleyl coenzyme A (18:1) be triglycerides and cholesterol ester weight Composition is wanted, also, causes metabolic rate to increase in the missing including the SCD1 in the mouse including ob/ob mouse, obesity mitigates, The diabetes for preventing fatty liver and avoiding diet from inducing.As indicated in fig. 12d, compared with the control, the mouse that 125B11 is handled SCD1 protein level declines about 50% in liver extract.This can be confirmed by following result：Monounsaturated fatty acids C16: 1、C18:1 and C20:1 declines about 70%, and it extends product (C18:2)N-6、(C18:3)N-6、(C20:2) N-6 and (C20-3) N-6 desaturation reduces by about 50% (table 2).In specific embodiments of the present invention, this reduction to SCD1 Influence is the key factor of weight loss, and reduces the TG levels in liver and avoid fatty liver.It is interesting that method figure statin A processing does not change the protein level of FADS1 or the desaturase of Δ 5.
Table 2：The mouse of OB/OB processing and the gas chromatography-mass spectrum of the aliphatic acid in the liver of their untreated controls (GC-MS) analyze
The ob/ob mouse and untreated control mice that liver specimens (100mg) are handled from 125B11 obtain, and In -80 DEG C of storages until carrying out Analysis of Fatty Acid Content.Aliphatic acid is extracted according to Folch scheme and gas chromatography-mass spectrum is used (GC-SM) quantitative analysis is carried out.As shown above, aliphatic acid de novo formation product C14:0 and C16:0 declines about 50%, and list is not Saturated fatty acid C16:1、C18:1 and C20:1 and its extension desaturation product (C18:2)N-6、(C18:3)N-6、(C20:2)N- 6th, (C20-3) N-6 and (C20:5) N-3 declines about 70%.FAS product myristinate (C14:0) with palmitate (C16: 0) about 50% (P ＜ 0.05) is declined.Prolong from not only the aliphatic acid de novo formation by FAS but also from food and chain The C18 levels of long system do not change.It is interesting that there is C20:0 declines about 15% (P+0.059) and C24:0 increase by 30% (P=0.05) strong trend.It is parallel with substantially reducing for chain saturated fatty acids, as a result show monounsaturated fatty acids C16: 1、C18:1 and C20:1 level substantially reduces about 70% (P ＜ 0.004).Also, polyunsaturated long-chain fatty acids (18:2)N- 6、(18:3)N-6、(20:2)N-6、(20:3) N-6 and (20:5) N-3 level declines 30-60%.These reductions are due to fat Downward of the key enzyme (FAS, ACC and SCD, ACL) in transcription and translation level in fat constructive ways.These results contribute to 125B11 is explained in the triglyceride levels prepared in liver by reducing to improve the effect in the fatty liver patient's condition.
The downward of the mRNA level in-site of fat generation enzyme
Protein level, which declines, may be attributed to transcription or translational control.It is real in addition to fat generation transcription factor PPAR γ When PCR can also be used to determining representational fat generation Gene A CC1, FAS and SCD1 mRNA level in-site level.ACC1、FAS Decline about 80% (Figure 13) with SCD1 mRNA level in-site.These results are consistent with relatively low zymoprotein level and activity, and Strong indication 125B11 is generated by suppressing SREBP-1 ripe reduction fat.In a particular embodiment, fat generation The downward of enzyme is related to one of its central transcription factor, i.e. PPAR γ.In the extract of the mouse of 125B11 processing, this turn The mRNA level in-site for recording the factor declines about 40% (Figure 13).Because the ob/ob mouse handled with 125B11 alleviate hyperglycemia And preventing fatty liver, this prompting, the PPAR γ in specific aspect, liver are possible influence these pathological conditions several One kind in the factor of kind.In a word, in the ob/ob mouse of processing, 125B11 is subtracted by it to acting through for SREBP-1 Few liver TG reserves improve fatty liver, mitigate obesity and reduce hyperglycemia.These research promptings, 125B11 and the like It is the medicament of useful confrontation such as fat, fatty liver and diabetes.
Make 4-5 week old fat (ob/ob) mouse of homozygous males (C57BL/6J, The Jackson Laboratory, Bar Harbor, ME) (12 hours light dark cycles under controlled conditions of living；25℃).Per 5 mouse of cage, and arrived at it After reaching, mouse is freely near the mark laboratory chow (Purina Mills, Richmond IN) and water, continues one week.In experiment First day and subsequent every day, determine the quantity of food of mice weights and consumption.Mice weights are determined between daily 3-5p.m. With remaining food, afterwards ip inject 125B11 (30mg/kg；150μL).Continue to apply 125B11 or in PBS daily In 10%DMSO to control group (n=5), continue 4 weeks until research terminate.
Injection 125B11 after 28 days daily, by mouse overnight fasting and takes blood, and uses Glucometer Precision Xtra (Abbott) measure full blood glucose and beta-hydroxy-butanoic acid ester.In order to determine serum composition, pass through Comparative Pathology Laboratory (Baylor College of Medicine) carry out glucose, glycerine three Ester and cholesterine measurement.Determine serum non-esterified by using NEFA C kits (Wako Chemicals, Richmond, VA) Aliphatic acid (NEFA).
Put to death mouse and determine the weight of liver and epididymal adipose tissues pad.The liver from each animal is dyed with Oil Red O The freezing microtome section of dirty section cut into slices with visible liver in fat drop (TG), as discussed previously (Abu-Elheiga et al., 2001).Remaining liver organization is chilled in liquid nitrogen, and is preserved at -80 DEG C in case further analysis.As in the literature described (Chandler et al., 2003), uses the Cholesterol E Kit suitable for carrying out colorimetric analysis with 96 well plate formats (Wako) and Infinity Triglyceride Kit (Thermo Electron, Melbourne, Australia) carry out liver Dirty triglycerides and cholesterol content.
Enzymatic activity and western blot analysis
By the hepatic portion grind into powder of freezing in liquid nitrogen.By powdered tissue suspension containing 0.1mM PMSF, In the 10ml PBS of 5mM benzenecarboximidamides and 5mg/ml protease inhibitor cocktails (Roche), and with Polytron (3x30 seconds, it is high Speed) homogenization, and carry out of short duration ultrasonically treated with degradation of dna.By making extract clear within 20 minutes with 16,000x g centrifugations Clearly.The protein concentration in supernatant is determined, and western blot analysis are carried out with the commercially available antibody for following enzyme：FAS(BD Biosciences), citrate lyase SCD1, FADS1, ACC and phosphorus-ACC antibody.With Amersham ECL PlusTMAlbumen Matter blotting detection reagent visualizes protein.Scan the intensity of the particular bands of destination protein and for beta-actin standard Change is quantified.It is active (Mao et al., 2006) that FAS and ACC from liver extract are determined as described above.
Quantitative real-time PCR
With TRIzol reagents (Invitrogen) total serum IgE is prepared from mouse tissue.Equivalent RNA from 5 mouse is converged Close, and handled with DNA enzymatic I (Turbo DNA-free, Ambion, Inc.).Inverted with Superscript II RNAase H- Record enzyme (Invitrogen) and synthesize the first chain cDNA from the 2 μ g DNA enzymatic I- total serum IgEs handled using random hexamers.It is real When PCR come from 20 μ l final volume comprising total serum IgE, 0.5 μM of forward and reverse primer and the 10 μ l of 10ng reverse transcriptions The 2x main mixtures of DyNAmo HS SYBR Green qPCR kits (Finnzymes).Using DNA Engine Opticon System (MJ Research, Inc), performing PCR is entered in 96 orifice plates.All reactions are triplicate to be carried out, and with comparing C (t) Method calculates mRNA relative quantity.Cycle threshold C (t) is calculated with Opticon monitoring of software 2.02 (MJ Research).Mouse β- Actin mRNA is used as internal contrast.Data are expressed as average value ± SD.Examined with unpaired couple of tail Student t (unpaired two-tailed Student t-test) assesses two group differences.
Embodiment 8
The identification of the target molecules of 125B11 and the like or derivative
In certain aspects of the invention, one or more targets of 125B11 or its analog or derivative are identified. Although this identification can be carried out using any suitable method, in a particular embodiment, to 125B11 or its is similar Thing or derivative are marked.Exemplary mark includes such as biotin.
Embodiment 9
Exemplary compound and its modification
Figure 14 A-14F show the exemplary compounds of the present invention, and their given title is provided in table 3 and table 4.Figure 15-17 shows the plain enzyme of exemplary fluorescence by these exemplary compounds for 20mM of the same procedure shown in Fig. 2A Reporter is determined.Fat generation is carried out as described determines (Choi et al., 2003).Oil droplet formation in cell will be completely inhibited Analog scoring is fat generation inhibitory analogues.
Table 3：The exemplary compounds of the present invention
Table 4
Moreover, technical staff understands, it is adapted to the one or more aspects of modification exemplary compounds to identify other Suitable compound.For example, when determine specific compound for one or more metabolic disorders treatment and/or prevention it is appropriate During property, the compound can be modified to identify other related compounds for identical or different metabolic disorder.Specific In embodiment, these changes can be carried out according to exemplary chemical group described herein.
Embodiment 10
Fatty synthesis is blocked by suppressing SREBP activation
In cell after fat consumption, sterol controlling element associated proteins (SREBP) are hydrolyzed by memebrane protein to be discharged and is transferred to In core, they activate the transcription for the gene for participating in cholesterine and fatty acid biological synthesis in core.In the present invention, it was demonstrated that resistance The lipogenetic small synthetic molecules that break are the selective depressants of SREBP activation.The diaryl thiazole for being referred to as method figure statin spreads out Biology weakens SREBP Proteolytic activation, so as to reduce the transcription of fat generation gene in cell.The molecular target of method figure statin Seemingly SREBP cuts activator protein (SCAP) to mark.Method figure statin blocks increased weight, blood glucose and liver in obesity ob/ob mouse Fat accumulation increase, or even under the conditions of uncontrolled food intake it is also such.
As described herein, method figure statin suppresses insulin-induced fat generation and the DU145 cells of 3T3-L1 cells Serum dependent/non-dependent growth (Choi et al., 2003).Compare the allelic expression through drug-treated and untreated cell with Obtain on the information of the specific molecular approach influenceed by method figure statin.Usage figure statin or only DMSO processing DU145 cells, and And the mRNA samples extracted are analyzed by the Affymetrix DNA microarray that located 33,000 genes.In these genes (all can be from the GenBank numbers of the National Center for Biotechnology Information on WWW Obtained according to storehouse), in response to the processing of method figure statin, the transcriptional level of 63 kinds of genes declines at least 35% (table 5).Impacted gene In 34 kinds synthesize directly related with fat or sterol, such as the gene of encoding biosynthetic enzymes, and reported impacted base Because in 18 kinds by SREBP controls (Horton et al., 2003).The downward of impacted SREBP- response genes passes through RT- PCR experiment is confirmed.SREBP responses gene and fat in the list of down-regulated gene/cholesterine biosynthesis gene High rate suggestion method figure statin acts on SREBP approach.The result of the display microarray of table 5 analysis.18 kinds of underscores are reported The gene of display is by SREBP controls, and the gene that runic is shown is related to fat or sterol synthesis.
Table 5
In order to which method of verification figure statin weakens SREBP function, in the presence of method figure statin is with or without, determine Endogenous SREBP activates the ability (Figure 18 A) of SREBP- response reporters in CHO-K1 cells.Method figure statin reduces report The expression of the activation of gene, wherein luciferase is controlled by sterol controlling element.The expression of method figure statin exogenous The influence of the ability of SREBP-1 mature form (amino acid/11-436) activation reporter is limited (Figure 18 B), shows Fa Tuta Spit of fland weakens SREBP activation.
In order to determine whether method figure statin influences the transfer of ER- golgiosomes and SREBP proteolysis to process, use The reporter of Sakai et al. (1998) exploitations is determined.PLAP-BP2 in the CHO-K1 cells of transfection keeps film combination, unless its Cut and be secreted into culture medium by the S1P in golgiosome.In the measure, with lacking NH2- ends DNA- binding structural domains The alkaline phosphatases (PLAP-BP2513-1141) of secretion of SREBP-2 segment compositions allow the phosphatase by producing fluorescence The change in fluorescence monitoring transfer and processing (Figure 18 C) of substrate.When the plasmid corotation with coding PLAP-BP2513-1141 and SCAP When contaminating cell, PLAP phosphatases are secreted, fluorescence signal is produced.Secretion (figure can be similarly reduced in addition method figure statin or sterol 18C).The suppression of the SREBP activation of method figure statin mediation is confirmed by SREBP western blot analysis.Usage figure he The amount of spit of fland processing CHO-K1 cell reductions SREBP-2 68KDa mature forms simultaneously increases the amount (figure of 125KDa precursor forms 18D).On SREBP-1, similar result (Figure 22) is obtained.These result collectively show thats, method figure statin blocks two kinds of SREBP The activation of isotype.
Inventors believe that, the SREBP proteolysis that method figure statin weakens in golgiosome are cut or SCAP/SREBP is multiple The ER- golgiosomes transfer of compound.A kind of known brefeldin A (Brefeldin A), blocking protein turns from ER direct motions The natural products of golgiosome is moved to, SREBP can be caused unresponsive to sterol, and by making S1P again fixed from golgiosome Position causes SREBP composition processing (DeBose-Boyd et al., 1999) in ER to ER.In the presence of brefeldin A Under, method figure statin is processed without influence (Figure 19 A) on SREBP, and this shows that method figure statin is unable to blocks protein hydrolysis in itself.
In order to which whether determination method figure statin blocks the ER- of SCAP/SREBP compounds to-golgiosome transfer, the present inventor Analyze the glycosylated degree of N connections of SCAP in golgiosome.SCAP includes glycosylated chamber ring (luminal loop), its quilt The proteolysis of trypsase are protected against, and recognize anti-SCAP IgG-9D5.When SCAP is located in ER, two in ring Individual oligosaccharides is sensitive to endoglycosidase H.When SCAP is transported to golgiosome, its sugar becomes to be resistant to disappearing for endoglycosidase H Change.Compared with the SCAP that ER is combined, the SCAP of transfer has higher level of glycosylation, and to endoglycosidase H tolerance Property is stronger.Sterol by suppress the transfer of ER- golgiosomes prevent SCAP become tolerance endoglycosidase H (Nohturfft etc., 1998).Cell is grown under conditions of being not present or there is method figure statin or sterol, trypsase and inscribe glucosides are used in succession Enzyme H handles film fraction.In the cell grown in the case of without method figure statin and sterol, SCAP tryptic fragment is more Endoglycosidase H is resistant to, and with one or two sugar chains (Figure 19 B, swimming lane 1).Cell is deposited method figure statin or sterol In lower growth, SCAP fragments are smaller to endoglycosidase H tolerance, and no or only one sugar chain (Figure 19 B, swimming Road 2 and 3).Therefore, method figure statin shows as suppressing transfers of the SCAP from ER to golgiosome.
The research of the structure-activity relation of method figure statin shows, when modifying its first with various alkyl or aryl sulfoamidos During benzene structure division, the biological activity of molecule is retained or even improved.A kind of fluorescent derivative, dansyl method figure statin (Figure 20 A) retains the ability (Figure 24) that it blocks SREBP activation, and as microprobe.Confocal microscopy is disclosed With the positioning (Figure 20 B) of the dansyl method figure statin of ER-tracker red (ER specific marker thing) located coincident.On the contrary, Any organelle can not be navigated to by lacking the control dansyl molecule of method figure statin.Selective ER positioning suggestion method figure statin combinations ER In protein；Most possible candidate is SCAP, and it is the cholesteric target for controlling SREBP (Radhakrishnan et al., 2004).To examine this it is assumed that being incorporated into method figure statin-poly- dried meat ammonia from cell lysate purifying The protein (Figure 20 A) (Sato et al., 2007) of sour joint-biotin conjugate, and with for SCAP, SREBP-1, SREBP- A kind of 2 and ATF6 (incoherent ER combinations transcription factor) antibody is analyzed (Ye et al., 2000) by Western blotting. As a result show, method figure statin is incorporated into SCAP, but do not combine other protein (Figure 20 C).When adding excessive method figure statin, institute State with reference to losing, and excessive cholesterine is without so (Figure 20 D), this proposes that such possibility, i.e. method figure statin may Interacted in the site different from cholesterine and SCAP.
Key effects of the SREBP in fat generation is had determined that, then, is examined under conditions of uncontrolled food intake Test pharmacotoxicological effect of the method figure statin to ob/ob mouse (a kind of Mice model of obesity).Daily intraperitoneal delivery method figure statin, prison Survey food intake and body weight.The average daily food intake of the mouse of processing (is respectively 5.4+1.5 with compareing that there was no significant difference Relative to 5.9+1.4g/ mouse/day, p>0.05) overt toxicity is not observed, and during processing.The processing of usage figure statin After 28 days, the mouse lighter than untreated control about 12% of processing (is respectively 32.1 ± 1.4 and 36.22 grams/mouse, p= 0.02).One of phenotype most unique is hyperglycemia caused by insulin resistance in ob/ob mouse.Blood constituent inspection is disclosed, The average glucose levels of the mouse of processing are lower than untreated mouse by about 70%, and (respectively 153.2 ± 30.5 relative to 429.4 ± 87mg/dl, p=0.003), it is in the range of normal glucose levels.The SREBP-1c of these results and report is in liver pancreas Effect in insulin resistance pathogenesis is consistent (Ide et al., 2004).
Another phenotype of ob/ob mouse is fatty excess accumulation in organ, including NASH.Untreated Ob/ob mouse in, substantially show liver expand and fatty by their pale colors, and usage figure statin processing Mouse liver is acted normally.Compared with untreated mouse, the liver of the mouse of processing is light~and 32%, and fat pad is smaller. The Oil red dyeing of liver section shows that the liver of untreated ob/ob mouse contains abundant fat drop, and processing is small Mouse liver includes the accumulation of lipid (Figure 21) of reduced levels.Triglycerides and cholesterol levels in the mouse liver of processing also under Drop.Method figure statin prevent ob/ob mouse fatty livers with report SREBP-1 fatty liver generation in effect it is consistent：It is overexpressed Fatty liver occurs for SREBP-1 transgenic mice, and lacks SREBP-1 ob/ob mouse (lepob/ob X Srebp 1-/-) tool The liver (Yahagi et al., 2002) of unsoundness.
It is because the liver expression of SREBP- responses fat generation enzyme to think that the liver fat level in the mouse of processing declines Reduction.Therefore, inspection technique figure statin is to representative SREBP- responses fat generation enzyme, including fatty acid synthase (FAS), acetyl The orgotein level of CoA carboxylase enzyme (ACC), stearyl-coenzyme A desaturase 1 (SCD1) and ATP citrate-lyase (ACL) With the effect of enzymatic activity.Biochemical analysis shows, in the liver extract of the mouse of method figure statin processing, the egg of fat generation enzyme White level and activity decrease (Figure 25 A-25C).Therefore, method figure statin blocks the SREBP-1 processing in liver, lowers fat generation Enzyme and reduce liver triglycerides storage.Method figure statin represents the first the non-sterols synthetic molecules for suppressing SREBP activation.
Luciferase reporter is determined.0th day, by CHO-K1 plating cells to 96 orifice plates in culture medium A (Ham ' s F- 1: 1 mixture of 12 culture mediums and Eagle ' the s culture mediums of Dulbecco ' s improvement, with 5% hyclone, 100 units/mL Penicillin and 100 μ g/mL streptomycin sulphates) in.2nd day, using Lipofectamine reagents (Invitrogen), use pSRE- (β-gal are reported by Luc (a kind of luciferase reporter construct of SRE-1 drivings) (Hua et al., 1995) and pAc- β-gal Son, wherein β-gal expression is by actin promoter control) transient cotransfection cell.It is slow with phosphoric acid after cultivating 5 hours Salt solution (PBS) washing cell is rushed, then under conditions of being not present or there is method figure statin, (Ham ' s F-12 are trained in culture medium B 1: 1 mixture of base and Eagle ' the s culture mediums of Dulbecco ' s improvement is supported, with 5% degreasing serum, 100 units/mL moulds Element, 100 μ g/mL streptomycin sulphates, 50mM compactins (compactin) and 50mM mevalonic acids sodium) in cultivated.Cultivate 20 After hour, the cell in each hole is cracked, and by aliquot for determining luciferase and betagalactosidase activity.By β- The activity normalized uciferase activity of galactosidase.For the overexpression of SREBP-1c N-terminal mature form, pSRE- is used Luc and pAc- β-gal cotransfections pCMV-SREBP-1c (1-436).
The western blot analysis of SREBP processing.0th day, the 100mm of CHO-K1 plating cells to culture medium A is cultivated On ware.2nd day, cell is washed with PBS, then under conditions of being not present or there is method figure statin, is trained in culture medium B Educate.3rd day, cell washed once with cold PBS, then use Tris-HCl containing 10mM, pH 7.6,100mM NaCl, 1% (w/v) SDS and protease inhibitor cocktail (1 μ g/ml Pepstatin As, 10 μ g/ml leupeptins, 200 μM of phenyl methyl sulphurs Acyl fluorides) buffer solution processing.Determine protein concentration (the BCA kits of each total cell extract；Pierce), then by cell The 22-33 μ g aliquots of extract and buffer solution (the 250mM Tris-HCl, pH 6.8,10%SDS, 25% of 0.25 volume Glycerine, 0.2% (w/v) bromophenol blue and 5% (v/v) 2 mercapto ethanol) mixing, heated 7 minutes at 95 DEG C.Sample is in 10%SDS- Separated on PAGE gels, with mouse monoclonal antibody (IgG-7D4) trace (Yang et al., 1995) for SREBP-2. Specific band is visualized with enhanced chemiluminescence (ECL) detection reagent (Amersham).
The modification of SCAP oligosaccharides.Cellular membrane fractions are prepared as described elsewhere herein.By film granule be resuspended in containing 10mM HepesKOH (pH 7.4), 10mM KCl, 1.5mM MgCl2, 1mM EDETATE SODIUMs and 100mM NaCl 0.1mL delays In fliud flushing.Then, by the aliquot of protein 30 DEG C, be not present or in the presence of the 1 μ g pancreas eggs in 58 μ L cumulative volume Cultivated 30 minutes under conditions of white enzyme.By adding 2 μ L (400 unit) soybean pancreatin inhibitor terminating reaction.For with inscribe Glycosidase H subsequent treatment, each sample receives 10 μ l and contains 3.5% (weight/volume) SDS and 7% (volume/volume) 2- mercaptos The solution of base ethanol.After 100 DEG C are heated 10 minutes, each sample receives to add 9 μ l 0.5M sodium citrates (pH in succession 5.5), 5 μ L contain solution (the concentration 1x, corresponding to 10 μ g/mL leupeptins, 5 μ g/mL pepstatins of 17 ' protease inhibitors A and 2 μ g/mL AKOLINEs), followed by 1 μ L (5 unit) endoglycosidase H.Reaction is stayed overnight at 37 DEG C, and by adding 20 μ L contain 0.25M TrisHCl (pH 6.8), 2%SDS, 10% (volume/volume) glycerine, 0.05% (weight/volume) The buffer solution terminating reaction of bromophenol blue and 4%2- mercaptoethanols.Then mixture is heated 5 minutes at 100 DEG C, and carries out SDS/ PAGE (12% gel).
Confocal microscopy.By the CHO-K1 cells and 0.2 on~70% orifice plate of glass bottom 96 (Grainer) converged μM ER-tracker Red (Invitrogen) and 5mM dansyl method figures statin are cultivated 1 hour.It is burnt with the copolymerization of CSU10 rotating disks is equipped with Scanner (Yokogawa Electric Corporation) and ORCA-CCD cameras (Hamamatsu Photonics) The Laser Scanning Confocal Microscopes of Carl Zeiss LSM 510 are obtained and analysis of fluorescence image.With IPLab softwares (Solution Systems image) is analyzed.
Combination mensuration.Cellular membrane fractions are prepared as described in compensation process.With containing 0.1%FOS-Choline 10 The PBS of (Hampton Research) extracts film fraction.By extract and the neutral chain with biotinylated method figure statin saturation Avidin (Neutravidine)-agarose beads (10 μ L) is mixed and cultivated 1 hour.With reference to albumen with containing 0.1% FOS-Choline 10 PBS is washed four times, is boiled in 25 μ L SDS sample buffers, and carry out Western blotting.For competing Measure is striven, the cholesterine of saturation capacity or method figure statin are added in film extract, then cultivated together with the globule.
Zooscopy step.Make fat (ob/ob) mouse (C57BL/6J, the The Jackson of homozygous males of 4-5 week old Laboratory, Bar Harbor, ME) (12 hours light dark cycles under controlled conditions of living；25℃).Every cage supports 5 Animal, and they reach after, the animal can freely be near the mark laboratory chow (Purina Mills, Richmond, ) and water was up to one week IN.First day and subsequent every day are being tested, 3:00 to 5:Every mouse is measured between 00p.m. Weight and food intake.After weight measurement, the mouse of processing receives ip injection figure statins (30mg/kg；150 μ L), it is right Receive the 10%DMSO in PBS according to mouse.Injection continues 4 weeks daily, until research terminates.
Blood constituent.Daily injection figure statin, by mouse fasting 5-6 hours, uses Glucometer after 28 days Precision Xtra (Abbott) determine full blood glucose and beta-hydroxy-butanoic acid ester.Pass through Comparative Pathology Laboratory (Baylor College of Medicine) carries out serum composition, glucose, triglycerides and cholesteric Determine.Serum non-esterified fatty acid (NEFA) is determined using NEFA C kits (Wako Chemicals, Richmond, VA).
Liver is analyzed.Mouse is put to death, and determines liver and weight of epididymal fat pad.Each is come from Oil Red O dyeing The freezing microtome section of the liver section of animal, the fat drop (triglycerides) in being cut into slices with visible liver, as described (Abu-Elheiga et al., 2001).Remaining liver organization is freezed in liquid nitrogen, and is preserved at -80 DEG C in case further dividing Analysis.
Tissue triglycerides and cholesterol content.Using Cholesterol E Kit (Wako) and Infinity Triglyceride Kit (Thermo Electron, Melbourne, Australia), such as Chandler et al. (2003) institute Liver tg and cholesteric content are determined as stating.
Synthetic method figure statin 1, dansyl method figure statin and Fa Tu statins-polyproline joint-biotin conjugate
Figure 23 shows method figure statin 1, dansyl method figure statin, method figure statin-polyproline joint-biotin conjugate Synthesis and the intermediate of synthesis.
The synthesis of method figure statin 1
Under agitation, by protionamide (1.03g, 5.70mmol) and the bromo- 4 '-methyl acetophenones of 2- (1.22g, 5.70mmol) mixture in ethanol (20ml) is heated 0.5 hour at 70 DEG C, is subsequently cooled to 0 DEG C.Yellow to formation is sunk Shallow lake is filtered, and is washed with cold ethanol, and the 2- propyl group -4- (4- p-methylphenyls thiazol-2-yl) of yellow needles is obtained after drying Pyridine (method figure statin) 1HBr salt (1.78g, 83%).mp:190-193℃；1H NMR (600MHz, DMSO-d6):δ 8.88 (d, J =6.2Hz, 1H), 8.54 (s, 1H), 8.46 (d, J=1.4Hz, 1H), 8.36 (dd, J=1.4,6.2Hz, 1H), 7.99 (d, J =7.6Hz, 2H), 7.31 (d, J=7.6Hz, 2H), 3.03 (t, J=7.6Hz, 2H), 2.35 (s, 3H), 1.80 (m, 2H), 0.96 (t, J=7.6Hz, 3H)；13C NMR (150MHz, DMSO-d6):D161.3,158.5,156.9,146.2,143.2, 138.4,130.4,129.5,126.3,122.3,120.3,119.5,35.0,22.4,20.9,13.4；HRMS(m/z):[M+H ]+For C18H19N2S calculated values 295.1269；Actual measurement 295.1269.
The synthesis of 4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) aniline 16
Load 2 (1.08g, 3.0mmol), benzophenone imine (benzophenone imine) in pressure pipe (0.57g, 3.3mmol), Pd2(dba)3(86mg, 0.15mmol), BINAP (280mg, 0.45mmol), sodium tert-butoxide (1.44g, Dry toluene (30mL) and purged 9.0mmol) and with argon gas.Heated 20 hours in being bathed by the pressure seal of tube and at 100 DEG C.Cooling To room temperature, reactant mixture handles (SiO through chromatography2, 4: 1 hexanes: EtOAc), obtain 1.35g yellow oils 8 (98%).Then, 2N HCl/waters solution (15mL) is added in 8 (1.35g, 2.9mmol) THF (20mL) solution.Room temperature is stirred Mix after 2 hours, reactant mixture is concentrated under reduced pressure, then diluted with EtOAc (100mL), and use saturation Na2CO3(50mL) Solution is washed.Aqueous cleaning solution is extracted with EtOAc (3x 40mL), and the EtOAc layers of merging are used into Na2SO4It is dried and concentrated.Slightly Product handles (SiO through chromatography2, 4: 1 hexanes: EtOAc), obtain the 4- (2- (2- propyIpyridines -4- that 0.73g is white crystal Base) thiazole-4-yl) aniline 16 (82%).1H NMR (600MHz, CDCl3):δ 8.61 (d, J=4.8Hz, 1H), 7.80 (d, J= 8.9Hz, 2H), 7.75 (d, J=1.4Hz, 1H), 7.67 (dd, J=1.4,4.8Hz, 1H), 7.36 (s, 1H), 6.75 (d, J= 8.9Hz, 2H), 3.82 (brs, 1H), 2.85 (t, J=7.6Hz, 2H), 1.83 (m, 2H), 1.01 (t, J=7.6Hz, 3H)；13C NMR (150MHz, CDCl3):δ 164.9,163.4,157.3,150.0,146.8,140.8,127.7,124.8,119.2, 117.8,115.1,111.3,40.4,23.1,13.9；HRMS(m/z):[M+H]+For C17H18N3S calculated values 296.1221；It is real Survey 296.1228.
The synthesis of dansyl method figure statin
To 16 (50mg, 0.17mmol) and pyridine (27mg, 0.34mmol) in CH2Cl2Magnetic agitation in (5mL) it is molten Liquid adds dansyl chloride (50mg, 0.18mmol).After stirring 17 hours, reactant mixture is concentrated under reduced pressure, and residue is existed It is allocated between EtOAc (50mL) and saturation NH4Cl solution (20mL).Aqueous phase is extracted with EtOAc (2x 20mL).The extraction of merging Take thing saturation NaHCO3Solution is washed, and uses Na2SO4It is dried and concentrated.Crude product handles (SiO through chromatography2, 2: 1 hexanes: EtOAc), the dansyl method figure statin (65mg, 73%) for yellow crystals is obtained.1H NMR (600MHz, CDCl3):δ 8.60 (d, J =4.8Hz, 1H), 8.50 (d, J=8.2Hz, 1H), 8.36 (d, J=8.3Hz, 1H), 8.21 (d, J=8.3Hz, 1H), 7.76 (d, J=8.6Hz, 2H), 7.70 (d, J=1.5Hz, 1H), 7.62 (dd, J=1.5,4.8Hz, 1H), 7.61 (t, J=8.3Hz, 1H) 7.44 (s, 1H), 7.43 (dd, J=7.5,8.2Hz, 1H), 7.19 (d, J=7.5Hz, 1H), 7.04 (d, J=8.6Hz, 2H), 6.97 (brs, 1H), 2.87 (s, 6H), 2.84 (t, J=7.6Hz, 2H), 1.81 (m, 2H), 1.00 (t, J=7.6Hz, 3H)；13C NMR (150MHz, CDCl3):δ 165.5,163.5,156.1,152.2,150.0,140.5,136.7,134.0, 131.1,131.0,130.5,129.8,129.7,128.7,127.3,123.1,121.6,119.2,118.3,117.8, 115.3,113.8,45.4,40.4,23.1,13.9；HRMS(m/z):[M+H]+For C29H29N4O2S2Calculated value 529.1732； Actual measurement 529.1733.
The synthesis of 4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) phenylcarbamate 40
To 16 (0.57g, 1.92mmol) and 4- (dimethylamino) pyridine (5mg, 0.4mmol) in THF (20mL) The solution of magnetic agitation adds di-tert-butyl dicarbonate (0.49g, 2.21mmol).After stirring 17 hours, reactant mixture is subtracted Pressure concentration, and by residue in EtOAc (100mL) and saturation NH4It is allocated between Cl solution (30mL).Aqueous phase EtOAc (2x 50mL) extract.The extract of merging saturation NaHCO3Solution is washed, and uses Na2SO4It is dried and concentrated.Crude product through chromatography at Manage (SiO2, 2: 1 hexanes: EtOAc), obtain 4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) benzene for yellow foam Carbamate 40 (0.33g, 43%).1H NMR (300MHz, CDCl3):δ 8.68 (d, J=5.1Hz, 1H), 7.93 (d, J=8.4Hz, 2H), 7.79 (s, 1H), 7.72 (d, J=5.1Hz, 1H), 7.52 (s, 1H), 7.47 (d, J=8.4Hz, 2H), 6.58 (s, 1H), 2.90 (t, J=7.5Hz, 2H), 1.87 (m, 2H), 1.55 (s, 9H), 1.03 (t, J=7.5Hz, 3H)；13C NMR (75MHz, CDCl3):δ 163.1,156.5,152.3,149.6,140.6,138.5,128.7,128.1, 127.0,118.3,117.7,114.3,112.9,80.6,41.0,28.4,24.1,13.7；HRMS(m/z):[M+H]+For C22H26N3O2S calculated values 396.1746；Actual measurement 396.1738.
R=N (Boc) CH2CH2CH2The synthesis of COOH intermediates
In N2In atmosphere, by 40 (200mg, 0.51mmol) add NaH (60% dispersion liquid in mineral oil, 24mg, 0.6mmol) the suspension in DMF (5mL), and the mixture is stirred at room temperature 2 hours.Then, add at DMF (2mL) In NaI (91mg, 0.6mmol) and 4- bromobutyrates (0.12g, 0.6mmol).After stirring 18 hours, the reaction is mixed Thing is poured into water (20mL), and is extracted with EtOAc (2x 50mL).The extract Na of merging2SO4It is dried and concentrated.Crude product (SiO is handled through chromatography2, 2: 1 hexanes: AcOEt), obtain 11 (35mg, 13%) for yellow oil.Then to 11 The solution of (30mg, 2.9mmol) in THF (1mL) and MeOH (0.5mL) adds the 2N NaOH aqueous solution (0.2mL).Room temperature is stirred Mix after 18 hours, the reactant mixture is concentrated under reduced pressure, then diluted with EtOAc (10mL), use saturation NH4Cl solution (5mL) Washing.Aqueous cleaning solution is extracted with EtOAc (2 × 10mL), and the EtOAc layers of merging are used into Na2SO4It is dried and concentrated.Crude product (SiO is handled through chromatography2, EtOAc), obtain R=N (Boc) CH that 14mg is white foam2CH2CH2COOH intermediates (50%).1H NMR (300MHz, CDCl3):δ 8.69 (d, J=5.1Hz, 1H), 7.95 (d, J=8.4Hz, 2H), 7.80 (s, 1H), 7.73 (d, J=5.1Hz, 1H), 7.53 (s, 1H), 7.51 (d, J=8.4Hz, 2H), 6.58 (s, 1H), 2.92 (m, 4H), 2.33 (m, 2H), 1.90 (m, 4H), 1.50 (s, 9H), 1.02 (t, J=7.5Hz, 3H)；13C NMR (75MHz, CDCl3):δ 174.2,162.9,156.3,152.9,149.6,140.6,138.5,129.3,128.9,127.2,119.0,117.9, 114.8,113.3,80.8,41.0,40.3,32.1,28.4,24.1,21.1,13.7；HRMS(m/z):[M+H]+For C26H32N3O4S calculated values 482.2114；Actual measurement 482.2120.
The synthesis of 4- (4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) phenyl amino)-N- butanamides
To R=N (Boc) CH2CH2CH2COOH intermediates (17mg, 0.035mmol), triethylamine (17 μ l, 0.14mmol) and HATU (16mg, 0.042mmol) is added in solution of the isopropylamine (4 μ L, 0.046mmol) in DMF (0.5mL).Stir 18 small Shi Hou, the reactant mixture is concentrated under reduced pressure, and by residue in EtOAc (20mL) and saturation NH4Enter between Cl solution (10mL) Row distribution.Aqueous phase is extracted with EtOAc (2x 10mL).The extract of merging saturation NaHCO3Solution is washed, and uses Na2SO4Dry And concentrate.Crude product handles (SiO through chromatography2, 2: 1 hexanes: EtOAc), obtain the 4- of the N-Boc protections for yellow oil (4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) phenyl amino)-N- butanamides (14mg, 77%).Then, to Solution of 13 (12mg, the 2.9mmol) in THF (1mL) adds TFA (0.2mL).It is stirred at room temperature after 18 hours, the reaction is mixed Compound is concentrated under reduced pressure, and is then diluted with EtOAc (20mL), and use saturation NH4Cl solution (10mL) is washed.Aqueous cleaning solution is used EtOAc (2 × 5mL) is extracted, and the EtOAc layers of merging are used into Na2SO4It is dried and concentrated.Crude product handles (SiO through chromatography2, 2: 1 hexanes: EtOAc), obtain 4- (4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) benzene that 6.2mg is yellow foam Base amino)-N- butanamides (63%).1H NMR (300MHz, CDCl3):δ 8.69 (d, J=5.4Hz, 1H), 8.04 (1H, s), 7.96 (d, J=8.7Hz, 2H), 7.96 (s, 1H), 7.72 (d, J=5.4Hz, 1H), 7.51 (s, 1H), 6.65 (d, J =8.7Hz, 2H), 3.95 (m, 1H), 3.03 (m, 4H), 2.35 (m, 2H), 1.92 (m, 4H), 1.25 (d, J=6.3Hz, 6H), 1.03 (t, J=7.5Hz, 3H)；13C NMR (75MHz, CDCl3):δ 171.6,162.9,156.3,152.9,140.6,138.5, 129.3,128.9,127.2,119.0,117.9,114.8,113.3,42.2,41.0,40.3,32.1,24.1,23.2,21.1, 13.7；HRMS(m/z):[M+H]+For C24H31N4OS calculated values 423.2219；Actual measurement 423.2216.
The synthesis of method figure statin-polyproline joint-biotin conjugate
Method figure statin-KPGQFLYELKKPPPPPPPPPKK (SEQ ID NO:15)-aminocaproic acid-biotin.
On Rink-Amide mbha resins by be coupled N- α-Fmoc protection amino acid, N- ε-Fmoc-e- amino oneself Acid, R=N (Boc) CH2CH2CH2COOH intermediates, biotin and synthesize conjugate, purified as described by reversed-phase HPLC (Sato et al., 2007).Conjugate 3:For C155H238N35O29S2 +Calculated value needs to be 3119.9.Survey (MALDI-TOF- MS) it is 3119.7 [M+H]+。
Plasmid.PSRE-Luc, pCMV-SREBP-1c (1-436), pCMV-PLAP-BP2 (513-1141) and pCMV-SCAP Presented by J.L.Goldstein and M.S.Brown (University of Texas Southwestern Medical Center) Give (Sakai et al., 1998；Hua et al., 1995).
Antibody.The anti-SREBP-1IgG of monoclonal (2A4), anti-SCAP IgG (9D5) and anti-ATF6 IgG (H-280) purchases From Santa Cruz Biotechnology.The anti-SREBP-2IgG-7D4 of monoclonal is by J.L.Goldstein and M.S.Brown (University of Texas Southwestern Medical Center) is presented.It is Anti-TNF-α-FA, anti-ACC, anti- SCD1 and anti-ACL IgG is purchased from BD Biosciences.
Cell culture.Chinese hamster ovary cell K1 (CHO-K1) cell is maintained at 5% hyclone, 100 lists The Eagle's culture mediums of the Dulbecco's improvement of position/mL penicillin and 100 μ g/mL streptomycin sulphates/Ham ' s F12 cultures At 37 DEG C in 5%CO in base [1: 1]2Under.People's androgen dependent/non-dependent prostate gland cancer cell (DU145) is maintained at containing 2mM L- glutamines, 1.0mM Sodium Pyruvates, 0.1mM nonessential amino acid and 1.5g/L sodium acid carbonates and 10% hyclone, In Eagle ' the s minimum essential mediums of 100 units/mL penicillin and 100 μ g/mL streptomycin sulphates, at 37 DEG C in 5%CO2 Under.
The preparation of cellular membrane fractions.Collect cell, be then resuspended in buffer solution (10mM HepesKOH (pH 7.4), 10mM KCl, 1.5mM MgCl2With 1mM EDETATE SODIUMs) in, by No. 22 syringe needles, and 1,000 × g is centrifuged 5 minutes.Then will Supernatant is centrifuged 30 minutes in 15,000xg after core, removes supernatant.
Oligonucleotide microarray is analyzed.In the presence of 1 μ g/mL IGF1,5mM method figure statins are used in serum free medium Or only DMSO handles DU145 prostate gland cancer cells 6 hours, extracted in TRI reagents (Molecular Research Center) Total serum IgE is simultaneously further separated by RNeasy Mini Kit (Qiagen).In Baylor College of Medicine Microarray Core Facility analyze pure by the Array of Affymetrix Human Genome U133 Plus 2.0 The mRNA of change, the Array of Affymetrix Human Genome U133 Plus 2.0 are by almost 45,000 kinds of probe groups Constitute, the probe groups, which are represented, derives from about 33, more than 39,000 kinds transcripts of 000 kind of certified human gene (Affymetrix, Inc.).
RT-PCR is tested.From DU145 cells by Total RNAs extraction to TRI reagents (Molecular Research Center) In and separated with RNeasy Mini Kit (Qiagen).Using Access RT-PCR System (Promega) to RNA Sample carries out RT-PCR.RT-PCR reactions include total serum IgE, 1 μM of various primers, 0.2mM dNTP, 1mM MgSO4, AMV reversions Record enzyme (2 unit) and Tf1 archaeal dna polymerases (2 unit), the μ L of final volume 25.Primer pair used is as follows：For low-density lipoprotein Polymeric immunoglobulin receptor (LDLR) is 5'-TCA GAC CGG GAC TGC TTG GAC GGC TCA GTC-3'(SEQ ID NO:16) and 5'-CCA CTT AGG CAG TGG AAC TCG AAG GCC G-3'(SEQ ID NO:17)；Go to satisfy for stearoyl-CoA It is 5'-GCC TGC TTG ATA ATA TAT AAA C-3'(SEQ ID NO with enzyme (SCD):18) with 5'-CAC TTG AAT TGA GCT TTA G-3'(SEQ ID NO:19)；It is 5'-AAG AAA AAG TGT for ATP citrate-lyase (ACL) CAG ACA GCT GG-3'(SEQ ID NO:20) with 5'-TGG ACT GAA GGG GTG TTA GC-3'(SEQ ID NO: 21)；It is 5'-GCC CGA CAG TTC TGA ACT for 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) GGA ACA-3'(SEQ ID NO:22) with 5'-GAA CCT GAG ACC TCT CTG AAA GAG-3'(SEQ ID NO: 23)；It is 5'-CTG CCT GAC TGC CTC AGC-3'(SEQ ID NO for mevalonate pyrophosphate decarboxylase (MVD): 24) with 5'-ACC TCT CCT GAC ACC TGG G-3'(SEQ ID NO:25)；For insulin-induced gene 1 (INSIG1) it is 5'-AAG ACT TCA GGG TAA GTC ATC A-3'(SEQ ID NO:26) with 5'-CGT GTA TAA TGG TGT CTA TCA G-3'(SEQ ID NO:27).Amplification condition is as follows：In 94 DEG C of 4 minutes 1 time circulations, then 94 DEG C denaturation 40 seconds, 50 DEG C anneal 40 seconds, 68 DEG C extend 2 minutes, 22 times circulation (for SCD and HMG CoA R), at 58 DEG C Anneal and carry out 24 circulations (for LDLR and INSIG1), or annealed at 60 DEG C and carry out 24 circulations (for ACL), 55 DEG C of 30 circulations of annealing and carry out are (for MVD).The DNA that is expanded by agarose gel analysis is simultaneously soft with Scion-image Part is quantified.
PLAP-BP2 is cut.0th day, by CHO-K1 plating cells to 96 orifice plates in culture medium A.2nd day, use Lipofectamine reagents (Invitrogen), with pCMV-PLAP-BP2 (513-1141), pCMV-SCAP and pAc- β-gal Transient cotransfection cell.After cultivating 5 hours, then cells rinsed with PBS is being not present or there is method figure statin (20 μM) or steroid Under conditions of alcohol (10 μ g/mL cholesterine and 1 μ g/mL 25- hydroxycholesterol oxycholesterols), cultivated in culture medium B.After culture 20 hours, Determine the alkaline phosphatase activities of the secretion of culture medium aliquot.By the cell cracking in every hole, for determining beta galactose Glycosides enzymatic activity.Pass through the activity normalized alkaline phosphatase activities of beta galactosidase.
Enzymatic activity and western blot analysis.By the hepatic portion grind into powder of freezing in liquid nitrogen.By powdered group Knit and be suspended in the 10ml PBS containing 0.1mM PMSF, 5mM benzenecarboximidamide and 5mg/ml protease inhibitor cocktails (Roche) In, and with Polytron (3x30 seconds, at a high speed) homogenization, it is ultrasonic with degradation of dna in short-term.By extract by with 16,000xg from The heart is clarified for 20 minutes.The protein concentration in supernatant is determined, and albumen is carried out with the antibody for FAS, ACC, SCD1 and ACL Matter engram analysis.The intensity of the particular bands of scanning protein interested is simultaneously standardized with quantitative for b- actins.Such as The foregoing description (Mao et al., 2006) determines FAS and ACC activity in liver extract.
Embodiment 11
(R) synthesis of -2- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) pyrrolidines -1- benzyl formates 53
It is as shown in Figure 26, in room temperature, to solution of the L- prolineamides (1.14g, 10.0mmol) in acetonitrile (100mL) Add benzyl chloroformate (1.55mL, 11.0mmol) and triethylamine (2.79mL, 20mmol).Mixture is stirred at room temperature 12 small When, and diluted with AcOEt.Pour the mixture into 0.1M HCl solutions, and separated between organic layer and water layer.Will be organic The NaHCO of layer saturation3The aqueous solution and salt water washing, dry (Na2SO4), filtering, and concentrate.Residue passes through silica gel column chromatography Method is purified, and is produced as 67 (2.26g, 91%) of colorless oil.
67 (586mg, 2.36mmol) and Lawesson reagents (1.05g, 2.60mmol) are mixed in toluene (30mL) Compound is stirred 1 hour at 90 DEG C.It is cooled to after room temperature, by evaporation solvent.Residue is purified by silica gel column chromatography, is produced as 68 (495mg, 79%) of white, amorphous solid.
By the mixing of 68 (300mg, 1.13mmol) and 4- bromobenzenes acylbromide (318mg, 1.13mmol) in EtOH (10mL) Thing is stirred 2 hours under reflux.After evaporation, residue is purified by silica gel column chromatography, the 48 of white solid are produced as (265mg, 53%).
By 48 (230mg, 0.519mmol), Pd2(dba)3(9.50mg, 2mol%), BINAP (97%, 20.0mg, 6mol%), sodium tert-butoxide (69.8mg, 0.727mmol) and benzophenone imine (0.105mL, 0.623mmol) are in toluene Mixture in (5.0mL) 8 hours under reflux.It is cooled to after room temperature, mixture is filtered by diatomite (Celite), and And evaporate filtrate.Residue is purified by silica gel column chromatography, is produced as 69 (228mg, 81%) of yellow amorphous solid.
HCl/water solution (4M, 0.3mL) is added to solution of 69 (175mg, the 0.321mmol) in THF (3mL), and will Mixture is stirred at room temperature 1 hour.By the NaHCO of mixture saturation3The aqueous solution is neutralized, and is extracted with AcOEt.By organic layer Salt water washing is used, (Na is dried2SO4), filtering, and concentrate.Residue is purified by silica gel column chromatography, is produced as colorless oil 70 (113mg, 93%) of shape thing.
By 70 (92mg, 0.24mmol), mesyl chloride (0.021mL, 0.27mmol) and pyridine (0.043mL, 0.53mmol) in CH2Cl2Mixture in (2mL) is stirred at room temperature 8 hours.Add after MeOH, mixture is dilute with AcOEt Release, and with the NaHCO of saturation3The aqueous solution and salt water washing.Organic layer is dried into (Na2SO4), filtering, and concentrate.Will residual Thing is purified by silica gel column chromatography, is produced as 53 (91mg, 83%) of white solid.
1H NMR(CDCl3, 600MHz, two rotational isomers, ratio=1:0.95) δ 7.80-7.86 (m, 2H), 7.14- 7.41 (m, 7H), 6.64 (s, 1H, main rotational isomers), 6.83 (s, 1H, secondary rotational isomers), 5.07-5.36 (m, 3H), 3.73 (m, 1H), 3.59 (m, 1H), 3.03 (s, 3H, main rotational isomers), 3.00 (s, 3H, secondary rotation Isomers), 2.28-2.39 (m, 2H), 2.00-2.10 (m, 2H)；13C NMR(CDCl3, 150MHz, two rotational isomers, show Show main peak) 174.7,155.4,154.4,136.4,131.9,128.5,128.3,127.9,127.8,127.7,127.7, 120.8,112.3,67.1,59.4,47.2,39.4,32.9,23.1；HRMS(FAB):For C22H24N3O4S2[M+H]+Accurate matter Measure calculated value 458.1203；Actual measurement 458.1200.
Embodiment 12
The synthesis of N- (4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) phenyl) Methanesulfomide (19)
As shown in figure 27, in reaction (a), by protionamide (9.01g, 50.0mmol) and 4- bromobenzene acylbromides 6a The mixture of (13.9g, 50.0mmol) in EtOH (400mL) is stirred 1 hour at 70 DEG C.It is cooled to after room temperature, by sediment Filtered out with EtOAc, and with EtOAc and the NaHCO of saturation3The aqueous solution is washed.Organic layer salt water washing, uses Na2SO4Dry, And concentrate, be produced as 4- (4- bromophenyls) -2- (2- propyIpyridine -4- bases) thiazole 2 (14.3g, 79%) of light yellow solid.
Reaction (b) in, in pressure pipe load 2 (1.08g, 3.0mmol), benzophenone imine (0.57g, 3.3mmol), Pd2dba3(86mg, 0.15mmol), BINAP (280mg, 0.45mmol), sodium tert-butoxide (1.44g, 9.0mmol) and dry toluene (30mL), and purged with argon gas.Heated 20 hours in being bathed by the pressure seal of tube and at 100 DEG C.It is cooled to after room temperature, reaction mixing Thing handles (SiO through chromatography2, 4:1 hexane:EtOAc), 1.35g yellow oils 71 (98%) are obtained.
Then, in reaction (c), 2M HCl/waters are added to solution of 71 (1.35g, the 2.9mmol) in THF (20mL) molten Liquid (15mL).It is stirred at room temperature after 2 hours, reactant mixture is concentrated under reduced pressure, is then diluted with EtOAc (100mL), and Use saturation Na2CO3(50mL) solution is washed.Aqueous cleaning solution is extracted with EtOAc (3x 40mL), and the EtOAc layers of merging are used Na2SO4It is dried and concentrated.Crude product handles (SiO through chromatography2, 4: 1 hexanes: EtOAc), it is white crystal to obtain 0.73g 4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) aniline 16 (82%).
1H NMR (600MHz, CDCl3):δ 8.61 (d, J=4.8Hz, 1H), 7.80 (d, J=8.9Hz, 2H), 7.75 (d, J =1.4Hz, 1H), 7.67 (dd, J=1.4,4.8Hz, 1H), 7.36 (s, 1H), 6.75 (d, J=8.9Hz, 2H), 3.82 (brs, 1H), 2.85 (t, J=7.6Hz, 2H), 1.83 (m, 2H), 1.01 (t, J=7.6Hz, 3H)；13C NMR (150MHz, CDCl3):δ 164.9,163.4,157.3,150.0,146.8,140.8,127.7,124.8,119.2,117.8,115.1,111.3,40.4, 23.1,13.9；HRMS(m/z):[M+H]+for C17H18N3S calculated values 296.1221；Actual measurement 296.1228.
In reaction (d), in 0 DEG C to 16 (800mg, 2.71mmol) and pyridine (0.66mL, 8.1mmol) in CH2Cl2 The solution of stirring in (20mL) adds mesyl chloride (0.23mL, 2.97mmol).After stirring 0.5 hour, pour the mixture into In 2M citric acid solutions, and extracted with EtOAc.By the NaHCO of the extract saturation of merging3Solution and salt water washing, are used Na2SO4Dry, and concentrate, be produced as N- (4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) phenyl) first of yellow colored foam thing Sulfonamide 19 (880mg, 87%).
1H NMR (300MHz, CD3OD):δ 8.55 (d, J=5.2Hz, 1H), 8.02 (d, J=8.8Hz, 2H), 7.95 (s, 1H), 7.90 (d, J=1.9Hz, 1H), 7.84 (dd, J=1.9,5.2Hz, 1H), 7.34 (d, J=8.8Hz, 2H), 3.00 (s, 3H), (t, J=7.3Hz, the 3H) .m/z=374 [M+H] of 2.86 (t, J=7.7Hz, 2H), 1.80 (m, 2H), 1.01+。
Embodiment 13
The synthesis of N- isopropyls -4- (2- (2- propyIpyridine -4- bases) thiazole-4-yl) aniline (17)
As shown in figure 27, synthesis compound 17 method utilize with for identical described in the compound 19 in embodiment 12 Step a-c.For compound 17, in step e, to 16 (1.02g, 3.45mmol) in CH2Cl2The solution of stirring in (20mL) Add acetone (2.5mL, 34.5mmol) and acetic acid (2.0mL, 34.5mmol).Stir the mixture for after 1 hour, add Na (OAc)3BH (1.5g, 6.9mmol).The mixture is stirred 20 hours.The mixture is poured into the NaHCO of saturation3In solution, And extracted with EtOAc.By the extract Na of merging2SO4It is dried and concentrated.Chromatography processing (the SiO of crude product2, 4:1 oneself Alkane/EtOAc) it is produced as 17 (845mg, 73%) of white foam thing.1H NMR (300MHz, CDCl3):δ 8.61 (d, J= 5.2Hz, 1H), 7.81 (d, J=8.5Hz, 2H), 7.76 (d, J=1.4Hz, 1H), 7.68 (dd, J=1.4,5.2Hz, 1H), 7.34 (s, 1H), 6.65 (d, J=8.5Hz, 2H), 3.70 (m, 1H), 2.86 (t, J=7.6Hz, 2H), 1.83 (m, 2H), 1.25 (d, J=6.0Hz, 6H), 1.02 (t, J=7.4Hz, 3H) .m/z=338 [M+H]+。
Embodiment 14
Compound 45-66 SREBP activation is determined
According to the method for 125B11 is used in embodiment 1, the exemplary compounds to the identification in table 4 carry out standard SREBP activation is determined.In CHOK1 cells, in the compound 45-55 and 19 (Figure 28), compound presence or absence of 5 μM Under conditions of 56-61 and 19 (Figure 29) and compound 62-66 (Figure 30), measurement endogenous SREBP activation SREBP- response reports The ability of the transcription of dao gene.Wherein, compound 61 shows about 25% SREBP activation suppression, and compound 53 is shown about 30% suppression, compound 58 shows about 42% suppression, and compound 19 shows about 45% suppression.Determine compound 53 The inhibition concentration of (Figure 31) and compound 58 and 61 (Figure 32).
Embodiment 15
Compound 19 suppresses human breast cancer cell line SUM159 growth and lowers fatty constructive ways
Cause within 48 hours cell growth inhibition and toxic action (figure with the processing SUM159 cells of compound 19 of various concentrations 33A).Cell is inoculated on 96 orifice plates with the density of 5000 cells/wells and includes the serum that 2% activated carbon is handled in 100 μ l Culture medium in.After 24 hours, the compound 19 of various concentrations is added, then up to 48 hours.Use WST-1 Cell Viability and Proliferation Assay (ScienCell Research Laboratory, Carlsbad CA) Measure cells viability.The change of fat generation mrna expression is determined in the cell with 10 μM of processing.In the presence of place In the notable downward of the gene under transcription factor SREBP controls.It is known not by these transcription factors adjust Insig2 genes not (Figure 33 B) affected by this treatment.
Also confirm effect of the compound 19 to human hepatocellular carcinoma cell line (HePG2).As shown in Figure 34 A-34B, compound 19 produce high toxicity level, as shown in the significant changes and growth inhibition of the form as the cell through processing.When with 25 μM When compound 19 handles HePG2 cells, the level of ripe and activity form is substantially reduced, and the increase of SREBP-1 precursors, and phase Ying Di, (Figure 34 A-34B) is increased by the expression of the SREBP genes controlled.These results meet and support compound 19 to lead to Cross suppression SREBP activation and as the conceptual effect of lipogenetic inhibitor.
Compound 19 suppresses people's acute lymphoblastic leukemia cell line MOLT-4 growth
MOLT-4 cells are grown under two kinds of different conditions：It is under 5% hyclone (FBS) and fat-free in 5%FBS Under serum (serum of activated carbon processing).10,000 cells were inoculated with 96 orifice plates up to 24 hours, then start to use various concentrations Compound 19 handle.The compound 19 being dissolved in DMSO is added into cell, continues 48 hours, then determines true with MTT Determine cell growth.As shown in Figure 35 A, cell is similar with the growth under fat-free FBS in 5%FBS.Under two kinds of growth conditions, Compound 19 suppresses about 75-80% cell growth at 10 and 20 μM.These results indicate that at 10 and 20 μM, or even in external source In the presence of property lipid, compound 19 is equally effective, and this shows that the suppression of from the beginning lipid synthesis causes the life in the cancer cell system It is long to suppress.It is interesting that under fat-free FBS, in the case of 2 and 5 μM of compounds 19, there is 30-50% growth inhibition (Figure 35 C), and under the conditions of 5%FBS, with the suppression trend (Figure 35 B) of 5 μM of presence strongly.
Compound 19 suppresses people's multiple myeloma cell line RPMI8226 growth
RPMI8226 cells are cultivated under the same conditions with MOLT-4 cells.20,000 cells are inoculated into 96 holes Up to 24 hours in plate, then handled with the compound 19 of increase concentration, as described on MOLT-4 cells.As shown in Figure 35 D, Cell is similar with the growth under fat-free FBS in 5%FBS, and difference is, compared with the corresponding amount in 5%FBS, 5% The growth that 5 μM of compounds 19 in FF FBS suppress is about more 1.5 times.10 μM and 20 μM in 5%FBS and 5%FF FBS The validity of compound 19 is similar.Start at 3 μM (Figure 35 E) strong suppression trend occur under the conditions of 5%FBS, and Start the strong suppression trend of appearance at 2 μM (Figure 35 F) under the fat-free serum of FBS.
Embodiment 16
Compound 17 suppresses people's acute lymphoblastic leukemia cell line MOLT-4 growth
MOLT-4 cells grow under two kinds of different conditions：Under 5% hyclone (FBS) and in the fat-free blood of 5%FBS Clearly under (serum of activated carbon processing).20,000 cells were seeded in 96 orifice plates up to 24 hours, then started with different dense The compound 17 of degree is handled.The compound 17 being dissolved in DMSO is added into cell, continues 48 hours, is then determined with MTT Determine cell growth.As shown in Figure 36 A, 5, the life of 10 and 20 μM of strong inhibition cells under the fat-free FBS of 5%FBS and 5% It is long.Under 5%FF FBS, 3 μM of suppression shown are about 2.5 times under the conditions of 5%FBS.Under two kinds of growth conditions, change The cell growth that compound 17 is 5,10 and 20 μM suppress about 85-100%.Under the fat-free serum of 5%FBS and 5%FBS, from 1 μM Start all the presence of strong suppression trend (Figure 36 B-36C).
Compound 17 suppresses people's multiple myeloma cell line RPMI growth
RPMI8226 grows under the same conditions with MOLT-4 cells.20,000 cells are inoculated on 96 orifice plates and reached 24 hours, then handled with the compound 19 of increase concentration, as described on MOLT-4 cells.As shown in Figure 36 D, cell exists Growth under 5%FBS and fat-free FBS is initially similar, and difference is, compared with the corresponding amount in 5%FBS, 5% In FF FBS, 5, that 10 and 20 μM of compound 17 suppresses growth is many about 1.5-6 times.In 5%FBS conditions (Figure 35 E) and 5%FBS Under fat-free serum (Figure 35 F), since 3 μM, there is strong suppression.
Embodiment 17
Compound 19 raises uncoupling protein-3 (UCP2) and reduces body fat
SD male rats feeding Western diet (high fat Hi CHO (HFHC)) is after 3 weeks, and the diet is rich in carbon Hydrate (35%kcal) and fat (45%kcal).The result of MTD researchs shows that maximum effective dose may be or be less than 100mg/kg.Therefore, 1/40 and the 1/10 of selection 100mg/kg is used for effect disquisition.The rat for being fed with HFHC diet is divided into four Group, every group of 15 animals：(1) control group of any processing is not received；(2) control group of 1ml cottonseed oils is given；(3) use The experimental group of the processing of 2.5mg/kg compounds 19；(4) with the experimental group of the processing of 10mg/kg compounds 19.Finally, one group (n=5) It is fed with routine rat feed (RD).
Compound 19 effectively loses weight and total body fat
Compared with being fed with the rat of conventional feed, feeding HFHC diet makes the total weight increase about 12% of rat for three weeks, point Wei not 332 ± 6.5 relative to 375 ± 4.0 grams (Figure 37 A).As expected, the total body fat and % fat of the rat of feeding conventional feed Fat be substantially less than feeding HFHC diet those (be respectively 44.0 ± 2.5 relative to 62.4 ± 2.4g/ rats and 13.95 ± 0.5 Relative to 18.48 ± 0.48%).Compared with the rat that HFHC is fed with, the also higher (figure of lean meat percentage in the rat of RD feedings 37B, 37C and 37D).
Effect of the compound 19 to body weight and composition
Body weight, and food intake of measurement per 2-3 days are determined daily.Until Friday since Sunday, to rat with Compound 19 in cottonseed oil was up to six days.Body weight based on that day for applying medicine and control vehicle calculates dosage.As schemed Shown in 38, after feeding HFHC diet three weeks and before beginning the process, about 200 grams (Figure 38 A) of animal weightening.It is fed with HFHC drinks Eat but be not given to cottonseed oil control group and the second control group (control vehicle) weightening it is similar, this shows excipient to body weight Change is without effect (Figure 38 B).After start to process two weeks, in the group that 2.5mg/kg and 10mg/kg is handled, under body weight is notable Drop.At the end of eight weeks are handled, compared with control vehicle, it (is respectively 357.7 ± 10.4g/ that 2.5mg/kg, which reduces and weighs about 15%, Rat is relative to 420.9 ± 2.4g/ rats) (Figure 38 A).In the rat of 10mg/kg processing, increased weight declines more, (being respectively 321.4 ± 10.3g/ rats) lower than control vehicle group by about 24%.Accumulative food intake is in control and 2.5mg/ (respectively 1370 ± 21 relative to 1320 ± 18g/ rats) similar between kg groups, and slightly reduced in 10mg/kg groups (1245 ± 21, see Fig. 2 B).When calculating total weight increase ratio relative to food intake, compared with the control, in 2.5 Hes In 10mg/kg groups increased body weight less about 13% and 16% (be respectively 0.326 ± 0.011,0.313 ± 0.014 relative to 0.373±0.011).Under conditions of the food intake in the absence of reduction, the body weight mitigated in 2.5mg/kg groups can be solved It is interpreted as meaning that the medicine loses weight in the case where not reducing food intake, as observed in ob/ob mouse.
The increased reduction of weight is mainly due to relatively low body fat
In order to determine that 19 pairs of compound reduces the effect of total body fat composition, a body is determined every two weeks using ECHOMRI methods Body is constituted.After processing two weeks, with the total weight of the 2.5 and 10mg/kg rats handled less than control and control vehicle group (figure 39A).This reduction is mainly due to relatively low body fat, and lean body mass does not have significant changes (Figure 39 B).Handle after eight weeks, 2.5 Weight ratio control group low 8% and 15% with the rat of 10mg/kg processing (is respectively 545.6 ± 9.7,502.8 ± 7.8 Hes 592.9 ± 5.4g/ rats, see Figure 39 A).The body weight of 10mg/kg groups reaches the value similar to the rat for being fed with regular diet.More Importantly, the difference of body weight can be mainly due to the difference (Figure 39 B) of fat content between treatment group and control group.Processing After eight weeks, compared with control-excipient, in 2.5 and 10mg/kg, total body fat weight low about 25 and 43% (be respectively 114.7 ± 5.2,86.5 ± 3.2 and 150.9 ± 4.2gr/ rats).Total % fat is also significantly lower, and wherein 2.5mg/kg groups have and feeding The % that the rat group of regular diet is similar is fatty, and 10mg/kg groups have the percentage lower than regular diet；26.6± 0.53 (control group)；21.9 ± 0.82 (2.5mg/kg groups)；18.0 ± 0.6 (10mg/kg) and 21.2 ± 0.82 (regular diets) (Figure 39 C).Although compared with the lean body mass percentage of control group, the lean body mass percentage in 2.5 and 10mg/kg is higher, Between all groups, lean body mass does not have significant difference (Figure 39 D and 39E).
Compound 19 improves the fatty liver patient's condition and downward from the beginning lipogenetic gene expression caused by HFHC diet And it is potentially by raising UCPS and themogenesis
Sprague-Dawley rats not only inducing obesity is fed with high fat diet, also causes the fatty liver patient's condition, at this The triglycerides of accumulated high levels in a little livers.After processing eight weeks, the SD rats of 19 pairs of feeding HFHC diet of compound are examined Liver effect.As shown in fig. 40, compared with the rat of processing, control rats have developed bigger more fertile liver.This enters one Walk by using Oil Red O decoration methods dye the freezing from every group of five animals liver section confirm, wherein, with to photograph Than the lipopexia in all treatment groups (n=5) is all lower (Figure 40 B).These results are by determining to compare and handling in rat TG and cholesterol levels confirm.As shown in figure 40 c, in the animal groups of two processing, TG and cholesterol levels are low by 40 respectively With 25% (Figure 40 C).Use real-time PCR methodology, it has been found that the gene (ACC, SCD and ACL) that is controlled by SREBP-1 and by The gene (MVD, LDLR and INSIG-1) of SREBP-2 controls is significantly down-regulated 40-80% (Figure 40 D).These results indicate that changing Compound 19 not only reduces total body fat, and improves the fatty liver patient's condition, and is the medicine of promising treatment people's Fatty Liver Disease.
The up-regulation of uncoupling protein-3 (UCP2) in the rat liver handled with compound 19
Fully demonstrate the up-regulation that UCP participates in themogenesis.They are made metabolic energy by reducing mitochondrial membrane potential Dissipated for heat.Liver is very active metabolic organ, especially in lipid synthesis.The present invention is it has been shown that in control Food consumption between the rat handled with compound 19 is not significantly different.Although the fatty generative nature diet of feeding, processing Animal still accumulate significantly lower body fat, including liver.Liver is examined, as the example of the possible uncoupling of energy, And determine the UCP2 in liver (main UCP) level.As shown in figure 41, using real-time PCR methodology, in 2.5 and 10mg/kg In the group of processing, UCP2 levels are significantly higher, increase about 1.5 and 3 times respectively.These results show that lipid contains in liver strongly The reduction of amount is due to that reduced synthesis and uncoupling cause, and wherein energy part passes through the themogenesis of uncoupling and dissipated.
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Claims (38)

1. compound, it has following chemical constitutions：
A-B-C
Wherein A is pyridine or substituted pyridine, piperidines or substituted piperidines, pyrrolidines or substituted pyrrolidines, thiazole or substitution Thiazole, phenyl ring or substituted phenyl ring；
B is thiazole or substituted thiazole, piperazine or substituted piperazine, phenyl ring or substituted phenyl ring；And
C is phenyl ring or substituted phenyl ring, pyridine or substituted pyridine, thiazole or substituted thiazole.
2. the compound of claim 1, wherein the chemical constitution is：
Wherein R1It is H, halogen ,-OH ,-O-C1-3Alkoxy ,-OC (O) R3；
R3It is C1-C3Alkyl or aryl ,-OCH2-C(O)OR4；
R4It is H or C1-C3Alkyl ,-NHR5；
R5It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-C3Acid amides ,-NHC (O) O-R6Carbamate；
R6It is the tert-butyl group or fluorenyl methyl or-NH-SO2-R7Sulfonamide；
R7It is alkyl or aryl,
R2It is alkyl or R8OC (O)-, and
R8It is C3-C5Alkyl or aryl.
3. the compound of claim 2, wherein the halogen is bromine.
4. the compound of claim 1, wherein the chemical constitution is：
Wherein R1It is H, halogen ,-OH ,-O-C1-3Alkoxy ,-OC (O) R3；
R3It is C1-C3Alkyl or aryl ,-OCH2-C(O)OR4；
R4It is H or C1-C3Alkyl ,-NHR5；
R5It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-C3Acid amides ,-NHC (O) O-R6Carbamate；
R6It is the tert-butyl group or fluorenyl methyl or-NH-SO2-R7Sulfonamide；
R7It is alkyl or aryl,
R2It is alkyl or R8OC (O)-, and
R8It is C3-C5Alkyl or aryl.
5. the compound of claim 4, wherein the halogen is bromine.
6. the compound of claim 1, wherein the chemical constitution is：
Wherein R9It is H, halogen ,-OH ,-O-C1-C3Alkoxy ,-OC (O) R11；
R11It is C1-C3Alkyl or aryl ,-OCH2-C(O)OR12；
R12It is H or C1-C3Alkyl ,-NHR13；
R13It is H, C1-C4Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C1-3Acid amides ,-NHC (O) O-R14Carbamate；
R14It is the tert-butyl group or fluorenyl methyl ,-NH-SO2-R15Sulfonamide；
R15It is alkyl or aryl or-SO2-NH-R16Sulfonamide；
R16It is alkyl or aryl,
R10It is nitrogen or methylene,
N is 0 or 1, and when n is 1, Z is-C=O；And
A is
Wherein R17It is H or C1-C3Alkyl.
7. the compound of claim 1, wherein the chemical constitution is：
8. the compound of claim 1, wherein the chemical constitution is：
9. the compound of claim 1, wherein the chemical constitution is：
10. the compound of claim 1, wherein the chemical constitution is：
11. the compound of claim 1, wherein the chemical constitution is：
12. the compound of claim 1, wherein the chemical constitution is：
13. the compound of claim 1, wherein the chemical constitution is：
14. treating the method for the metabolic disorder in animal, methods described comprises the steps：
The compound or pharmaceutically acceptable salt thereof or stereoisomer of at least one claim 1 of therapeutically effective amount are applied to the animal Or combinations thereof.
15. the method for claim 14, it also comprises the steps：
The second therapy is provided to the animal.
16. the method for claim 15, wherein second therapy include dietetic treatment, physical therapy, behavior therapy, operation, Medicinal treatment or its combination.
17. the method for claim 16, wherein second therapy is lifestyle change, antihyperglycemic agents, insulin, pancreas height Blood glucose element sample peptide (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinedione, hypolipidemic compounds or group two or more in them Close.
18. the method for claim 14, wherein the metabolic disorder is the disease related to cell hyperplasia, body weight correlation The patient's condition, hyperlipidemia, diabetes or its complication, fatty liver, hypertension or cardiovascular disease.
19. the method for claim 18, wherein the metabolic disorder be obesity, hypertension, artery sclerosis, asthma, hyperlipidemia, Hyperinsulinemia, NASH and the diabetes B caused by insulin resistance.
20. the method for claim 18, wherein the disease related to cell hyperplasia is cancer.
21. the method for claim 20, wherein the cancer is breast cancer, respiratory cancer, the cancer of the brain, genital cancer, prostate It is cancer, digestive system cancer, carcinoma of urethra, cancer eye, liver cancer, cutaneum carcinoma, head and neck cancer, thyroid cancer, accessory thyroid glands cancer, lymthoma, sarcoma, black The DISTANT METASTASES IN of plain knurl, leukaemia or solid tumor.
22. the method for claim 18, wherein the metabolic disorder is the related illness of body weight, the therapeutically effective amount it is described The expression of the albumen 3 of compound increase uncoupling proteins 1, uncoupling protein-3 or uncoupling.
23. the method for claim 18, wherein the metabolic disorder is the related illness of body weight, process is mitigated in the weight of animals In, the compound increase themogenesis of the therapeutically effective amount, without reducing lean body mass.
24. the method for treating the cell hyperproliferative diseases in patient in need, methods described comprises the steps：
To the compound or pharmaceutically acceptable salt thereof or stereoisomer of at least one claim 1 of patient therapeuticallv's effective dose Or combinations thereof.
25. the method for claim 24, wherein the cell hyperproliferative diseases are breast cancer, respiratory cancer, the cancer of the brain, genitals Official's cancer, prostate cancer, digestive system cancer, carcinoma of urethra, cancer eye, liver cancer, cutaneum carcinoma, head and neck cancer, thyroid cancer, accessory thyroid glands cancer, pouring Bar knurl, sarcoma, melanoma, the DISTANT METASTASES IN of leukaemia or solid tumor.
26. the method for the body weight for mitigating the animal for having this to need, methods described comprises the steps：Applied to the animal The compound of one or more claims 1 of therapeutically effective amount in medicinal medium.
27. the method for claim 26, wherein the animal suffers from obesity, hypertension, artery sclerosis, asthma, hyperlipidemia, height Insulinemia, NASH or diabetes B or its combination caused by insulin resistance.
28. preparation, compound of the preparation comprising claim 1 and food, ani-mal feed substance or medicine.
29. for method of the increase themogenesis without reducing lean body mass during the weight of animals mitigation, methods described includes Following step：The preparation of the claim 28 of therapeutically effective amount is applied to the animal.
30. pharmaceutical composition, compound of the described pharmaceutical composition comprising claim 1 and pharmaceutical excipient.
31. the method for treating the cancer in patient in need, methods described comprises the steps：Applied to the patient With the pharmaceutical composition of one or more claims 30 of therapeutically effective amount.
32. kit, it is included：
The compound of claim 1, and
Accommodate the container of the compound.
33. compound, it is：
2- (4- (4- bromophenyls) thiazol-2-yl) pyrrolidines -1- t-butyl formates,
2- (4- (4- bromophenyls) thiazol-2-yl) pyrrolidines -1- benzyl formates,
4- (4- bromophenyls) -2- (pyrrolidin-2-yl) thiazole,
4- (4- bromophenyls) -2- (1- propyl pyrrole alkane -2- bases) thiazole,
3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- t-butyl formates,
3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- benzyl formates,
3- (4- (4- bromophenyls) thiazol-2-yl) -1- propylpiperdines,
4- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- benzyl formates,
(R) -2- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) pyrrolidines -1- benzyl formates
3- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates
4- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates
4- (3- (pyridine -2- bases)-[1,2,4] triazol [4,3-b] pyridazine -6- bases)-N- tosyl aniline,
(4- (5- chloro-2-methyls phenyl) piperazine -1- bases) (4- tosyls amino) phenyl) ketone,
4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases)-N- tosyl aniline,
The chloro- 4- methyl-N- of 3- (6- (4- (3- (trifluoromethyl) benzyl) piperazine -1- bases) pyridin-3-yl) benzsulfamide
The chloro- N- of 4- (4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases) phenyl) benzsulfamide
(Z) -4- (3- cyano group -3- (4- (2,4- 3,5-dimethylphenyls) thiazol-2-yl) pi-allyl)-N- (thiazol-2-yl) benzene sulfonyl Amine
N- (3- (H- imidazos [1,2-a] pyridine -2- bases) phenyl) -4- methyl -2- phenyl thiazole -5- formamides,
N- (3- (benzo [d] thiazol-2-yl) phenyl) Pyrazinamide,
3- (4- chlorphenyls) -4,5- dihydro -1- phenyl -5- (2- phenyl thiazole -4- bases) -1H- pyrazoles,
N- (4- (6- methyl benzo [d] thiazol-2-yl) phenyl) -2- (tolylmethyl sulfoamido between N-) acetamide；Or
N- (4- (6- methyl benzo [d] thiazol-2-yl) phenyl) -2- (N- p-methylphenyl sulfonyloxy methyls amido) acetamide.
34. for method of the increase themogenesis without reducing lean body mass during the weight of animals mitigation, methods described includes The step of compound for the therapeutically effective amount being applied in the animal in medicinal medium, the compound has following structures：
Wherein R1It is H, Et, OMe or n-propyl；
Y be CH or
R2It is OH, OMe or NH-i-Pr；
R3It is H, F or Cl；And
R4It is H, Me, Cl, Br, F, OH, OBz, OCH2COOMe、OCH2COOH、NH2、NH-i-Pr、NHCOMe、NHSO2Me、NHBn、OMe、NHBoc、NHTs、Or its pharmaceutical salts or solid Isomers or combinations thereof.
35. the method for claim 34, wherein the animal is with the related patient's condition of following body weight：Obesity, hypertension, artery are hard Change, asthma, hyperlipidemia, hyperinsulinemia, NASH and the diabetes B caused by insulin resistance.
36. the method for claim 36, it also includes providing the second therapy to the animal, and second therapy includes life side Formula change, antihyperglycemic agents, insulin, glucagon-like peptide (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinedione, drop Compound or combination two or more in them.
37. the method for claim 1 wherein the compound is configured to food, animal feed material or medicine.
38. the method for claim 38, wherein compound increase uncoupling proteins 1, uncoupling protein-3 and UCPS 3 expression.
CN201480058587.XA 2013-08-29 2014-08-29 Composition and method for treating the metabolism illness related to body weight Pending CN107074839A (en)

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US14/052,074 US9187485B2 (en) 2007-02-02 2013-10-11 Methods and compositions for the treatment of cancer and related hyperproliferative disorders
US14/270,130 US9233941B2 (en) 2007-02-02 2014-05-05 Methods and compositions for the treatment of body weight related disorders
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