CN101035527A - Methods of treating a disorder - Google Patents
Methods of treating a disorder Download PDFInfo
- Publication number
- CN101035527A CN101035527A CN 200580033718 CN200580033718A CN101035527A CN 101035527 A CN101035527 A CN 101035527A CN 200580033718 CN200580033718 CN 200580033718 CN 200580033718 A CN200580033718 A CN 200580033718A CN 101035527 A CN101035527 A CN 101035527A
- Authority
- CN
- China
- Prior art keywords
- chemical compound
- leu
- disease
- ala
- alkyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Compound of formula (I) and methods of treating disorders by administering a compound of formula (I) are described herein. Examples of disorders include neoplastic disorders, fat-cell related disorders, neurodegenerative disorders, and metabolic disorders.
Description
Prioity claim
The application requires the priority of U.S. Patent Application Serial 10/940,269 that JIUYUE in 2004 submitted on the 13rd and the U.S. Patent Application Serial of submitting on March 11st, 2,005 11/077,664, and their whole contents separately are incorporated by reference here.
Background
Sir2 albumen is deacetylase (Imai et al., 2000 of a kind of NAD of use as cofactor; Moazed, 2001; Smith et al., 2000; Tanner et al., 2000; Tanny and Moazed, 2001).Different with other deacetylase (many participation gene silencings wherein), Sir2 is to histone deacetylase inhibitors such as Trichostatin A (TSA) insensitive (Imai et al., 2000; Landry et al., 2000a; Smith et al., 2000).
Summary
The present invention relates to substituted heterocyclic compound, comprise described compound compositions and use the method for described chemical compound and compound composition.Chemical compound can be used for the treatment of disease or disease symptoms with the compositions that comprises them, comprises (those of the deacetylation mediation that for example, SIRT1) mediates by sirtuin.
In one aspect, the present invention relates to treat or the method for the disease (for example, disease as herein described) of object of prevention.This method comprises, uses the chemical compound with formula (I) of effective dose to object:
Wherein,
R
1And R
2With they bonded carbon, form C
5-C
10Cycloalkyl, C
5-C
10Heterocyclic radical, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, C
6-C
10Aryl, or C
5-C
10Heteroaryl, each in them can be randomly by 1-5 R
5Replace; Or R
1Be H, S-alkyl, or S-aryl, and R
2Be aminoalkyl, wherein nitrogen is replaced by alkyl, aryl or aryl alkyl, and each in them is randomly further replaced by alkyl, halogen, hydroxyl or alkoxyl;
R
3And R
4With they bonded carbon, form C
5-C
10Cycloalkyl, C
5-C
10Heterocyclic radical, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, C
6-C
10Aryl, or C
5-C
10Heteroaryl, each in them can be randomly by 1-5 R
6Replace;
Each R
5And R
6Be halogen independently, hydroxyl, C
1-C
10Alkyl, C
1-C
6Haloalkyl, C
1-C
10Alkoxyl, C
1-C
6Halogenated alkoxy, C
6-C
10Aryl, C
5-C
10Heteroaryl, C
7-C
12Aralkyl, C
7-C
12Heteroarylalkyl, C
3-C
8Heterocyclic radical, C
2-C
12Alkenyl, C
2-C
12Alkynyl, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, carboxyl, carboxylate (ester), cyano group, nitro, amino, C
1-C
6Alkyl amino, C
1-C
6Dialkyl amido, sulfydryl, SO
3H, sulfate (ester), S (O) NH
2, S (O)
2NH
2, phosphate (ester), C
1-C
4Alkylene dioxo base, oxo, acyl group, amino carbonyl, C
1-C
6Alkyl amino-carbonyl, C
1-C
6Dialkyl amino carbonyl, C
1-C
10Alkoxy carbonyl, C
1-C
10The thio alkoxy carbonyl, diazanyl carbonyl, C
1-C
6Alkyl hydrazine carbonyl, C
1-C
6Dialkyl group diazanyl carbonyl, the hydroxyl amino carbonyl; The alkoxy amino carbonyl; Or R
5Or R
6One of and R
7Formation contains the cyclic group of 4-6 carbon, a 1-3 nitrogen, a 0-2 oxygen and 0-2 sulfur, and it can be randomly by oxygen or C
1-C
6Alkyl replaces;
X is NR
7, O, or S; Y is NR
7 ', O or S;
The optional two keys of----representative;
R
7And R
7 'Be hydrogen independently of one another, C
1-C
6Alkyl, C
7-C
12Aryl alkyl, C
7-C
12Heteroaryl alkyl; Or R
7With R
5Or R
6One of form the cyclic group contain 4-6 carbon, a 1-3 nitrogen, a 0-2 oxygen and 0-2 sulfur, it can be randomly by oxygen or C
1-C
6Alkyl replaces; And n is 0 or 1.
Embodiment can comprise following one or more.
In certain embodiments, n can be 1.
X can be NR
7And Y can be NR
7 'R7 and R
7 'Can each naturally, for example, hydrogen or CH
3R
7And R
7 'One of can be hydrogen, and another can be CH
3
R
1And R
2Can form C
5-C
10Cycloalkenyl group.
R
1And R
2Can form C
6-C
10Aryl.
R
1And R
2Can form C
5-C
10Cycloalkenyl group, it can be by R
5Replace, and R
3And R
4Can form C
6-C
10Aryl, it can be by R
6Replace.
In certain embodiments, the two keys of cycloalkenyl group can be in conjunction with R
1Carbon and in conjunction with R
2Carbon between.C
5-C
10Cycloalkenyl group, for example, C
6Or C
7Cycloalkenyl group can be by R
5Replace, and C
6-C
10Aryl can be by R
6Replace.
R
6Can be halogen (for example, fluorine or bromine), C
1-C
6Alkyl (for example, CH
3), C
1-C
6Haloalkyl (for example, CF
3) or C
1-C
6Halogenated alkoxy (for example, OCF
3).R
5For example can be, be substituted the C that base replaces
1-C
6Alkyl, for example amino substituent group of described substituent group; Or amino carbonyl (for example substituted amino carbonyl, it is substituted base, and for example aryl, heteroaryl, cycloalkyl, Heterocyclylalkyl, amino carbonyl, alkyl amino-carbonyl, alkoxy carbonyl or other substituent group replace.In each case, substituent group can further be replaced by other substituent group).
N can be 0.
R
1And R
2Can form C
5-C
10Cycloalkenyl group.
R
1And R
2Can form C
6-C
10Aryl.
X can be NR
7, and R
7Can be, for example, hydrogen or CH
3
R
1And R
2Can form C
5-C
10Cycloalkenyl group, it can be by R
5Replace, and R
3And R
4Can form C
6-C
10Aryl, it can be by R
6Replace.
In certain embodiments, the two keys of cycloalkenyl group can be in conjunction with R
1Carbon and in conjunction with R
2Carbon between.C
5-C
10Cycloalkenyl group, for example, C
6Or C
7Cycloalkenyl group can be by R
5Replace, and C
6-C
10Aryl can be by R
6Replace.
R
6Can be halogen (for example, chlorine), C
1-C
6Alkyl (for example, CH
3), C
1-C
6Haloalkyl (for example, CF
3) or C
1-C
6Halogenated alkoxy (for example, OCF
3).R
5It can be amino carbonyl.
N can be 0.
R
1And R
2Can form C
5-C
10Cycloalkenyl group.
R
1And R
2Can form C
6-C
10Aryl.
X can be NR
7, and R
7Can be, for example, hydrogen or CH
3
R
1And R
2Can form C
5-C
10Cycloalkenyl group, it can be by R
5Replace, and R
3And R
4Can form C
6-C
10Aryl, it can be by R
6Replace.
In certain embodiments, the two keys of cycloalkenyl group can be in conjunction with R
1Carbon and in conjunction with R
2Carbon between.C
5-C
10Cycloalkenyl group, for example, C
6Or C
7Cycloalkenyl group can be by R
5Replace, and C
6-C
10Aryl can be by R
6Replace.These chemical compounds can have formula (II) or formula (III):
R
6Can be halogen (for example, fluorine or bromine), C
1-C
6Alkyl (for example, CH
3), C
1-C
6Haloalkyl (for example, CF
3) or C
1-C
6Halogenated alkoxy (for example, OCF
3).R
5It can be amino carbonyl.Chemical compound can be to be selected from Fig. 1 or chemical compound (IV), (V), and (VI), or chemical compound (VII).
In an example, chemical compound can be the chemical compound of formula (VI), and it has the excessive individual isomer of high antimer, and wherein the optical rotation of advantage isomer is minus, for example ,-14.1 (c=0.33, DCM) or, for example, [α]
D 25-41.2 ° of (c0.96, CH
3OH).In second example, chemical compound can be the chemical compound of formula (IV), and it has the excessive individual isomer of high antimer, and wherein the optical rotation of advantage isomer is minus.In some example, use formula (IV), (V), or chemical compound (VII), it has the excessive individual isomer of high antimer, wherein the advantage isomer have with top shown in the identical absolute configuration of negative isomer of the corresponding formula of asterisk carbon (VI) chemical compound.
With respect to non--SIRT1 sirtuin, this chemical compound can preferentially suppress SIRT1, for example, at least 1.5,2,5, or 10 times preferential.This chemical compound can have the Ki to SIRT1, and it is less than 500,100,50, or 40nM.
In some example, chemical compound can reduce the activity of FOXO transcription factor (for example FoxO1 or FoxO3).
Amount can be to improve at least a symptom of disease effectively.Disease or disease can be, for example, and the disease that the age is relevant, infirmities of age, disease with relevant susceptibility factor of age, neoplastic disease, non-neoplastic disease, neurological disorder, cardiovascular disease, metabolism disorder, the disease of dermatological, or the organization factors of dermatological.In one embodiment, disease or disease can be neurodegenerative disease or disease, wherein neurodegenerative disease can be assembled mediation by polyglutamyl amine at least in part, for example, hungtington's chorea, ridge bulbar muscular atrophy (SBMA or Ken Nidishi disease), dentatorubropallidoluysian atrophy (DRPLA), spinocebellar ataxia 1 (SCA1), spinocebellar ataxia 2 (SCA2), Machado-Joseph disease (MJD; SCA3), spinocebellar ataxia 6 (SCA6), spinocebellar ataxia 7 (SCA7) and spinocebellar ataxia 12 (SCA12).Neurodegenerative disease can be parkinson or Alzheimer.
Disease or disease can be relevant with sirtuin or at least in part by its mediation; for example; disease or disease can be relevant with the deacetylation of sirtuin-mediation or at least in part by its mediation; for example; the p53 of the deacetylation of over-drastic sirtuin activity or excessive level; FoxO1, or FoxO3.Sirtuin can be SIRT1, for example, and people SIRT1.
Disease or disease can be cancers.Amount can be, for example, reduces cancer or tumor cell piece effectively, shift risk, or the speed of growth of tumour cell.Amount can be to regulate (for example, increasing) apoptosis effectively.
Disease or disease can be metabolic diseases, for example metabolism syndrome or diabetes (for example, type i diabetes or type ii diabetes).Amount can be for example, to improve insulin sensitivity effectively, increase insulin secretion, perhaps alternate manner or reduction glucose level.In some example, disease or disease are relevant with metabolic disease, for example the relevant diabetes of heart disease.
Disease or disease can be for example obesity or dyslipidemia or hyperlipemias of the relevant disease of fat.Amount can be for example, to reduce the body weight of object or the weight increase of object of prevention effectively.
Disease or disease can be for example Alzheimer or parkinson of neurological disorder.Amount can be for example, to alleviate one or more symptoms of neurological disorder effectively.
Method can comprise, administered compound more than once, for example, the repetitive administration chemical compound.Can one or many inject fast or continuous administration in, administered compound.Can be from outside (from without) (for example,, take in, suck etc.) or internally for example by implanting device by injection, administered compound.
Method can comprise, partly administered compound.
Amount can be the acetylation that increases sirtuin substrate in some cell at least of object (for example, nucleoprotein, for example, histone or transcription factor, for example, p53, FoxO1, or FoxO3) effectively.
Object can be a mammal, for example, and the people.
Object can be differentiated to needing such treatment or prevention.
Method can also comprise, differentiate the object that needs such treatment, for example, by the sirtuin activity in the evaluation object cell, nucleotide homogeneity in the object nucleic acid of evaluation coding sirtuin, the neoplastic cell of evaluation object or superfluous give birth to grow (for example, tumor), genetic constitution or gene expression in the evaluation object cell (for example, tumor biopsy).
Method can also comprise, monitoring target, for example, and with the object imaging, the tumor size in the evaluation object, the sirtuin activity in the evaluation object cell, the side effect in the evaluation object, for example, renal function.
In yet another aspect, the present invention relates to suppress the method for deacetylation of the substrate (for example FoxO transcription factor) of sirtuin-mediation.This method comprises, makes sirtuin contact (I) chemical compound.Inhibition can occur in external, the acellular culture medium, in the cell culture or in the organism, for example, and mammal, preferred people.
In yet another aspect, the present invention relates to estimate the method for multiple chemical compound, this method comprises: the library of compounds that comprises chemical compound lot a) is provided, and every kind of chemical compound has formula (I); And b) for from the chemical compound lot in this library each, i) make chemical compound contact sirtuin test proteins, it comprises the functional deacetylase domain of sirtuin; Ii) estimating is having in the presence of the chemical compound interaction between chemical compound and the sirtuin test proteins.
Embodiment can comprise following one or more:
In one embodiment, the interaction between assessing compound and the sirtuin test proteins comprises, estimates the enzymatic activity of sirtuin test proteins.
In one embodiment, the interaction between assessing compound and the sirtuin test proteins comprises, the binding interactions between assessing compound and the sirtuin test proteins.
Method can also comprise, based on the result who estimates, selects can regulate the active chemical compound of the deacetylase of substrate.Substrate can be acetylizad lysine aminoacid, acetylizad transcription factor (for example, p53, FoxO1, or FoxO3) or their acetylizad peptide, acetylizad histone or their acetylizad peptide.
Method can also comprise, based on the result who estimates, selects to regulate the active chemical compound of sirtuin deacetylase to substrate.
Method can also comprise that based on the result who estimates, selection can be regulated the chemical compound of sirtuin.
In one aspect, the present invention relates to a kind of conjugate, it comprises: targeting agent and chemical compound, wherein targeting agent and chemical compound are covalently bound, and chemical compound has formula (I).
Embodiment can comprise following one or more.
Targeting agent can be an antibody, for example, and the specific antibody of pair cell surface protein (for example, cancer-specific antigen).
Targeting agent can be synthetic peptide.
Targeting agent can be naturally occurring proteic domain.
In yet another aspect, the present invention relates to test kit, it comprises: chemical compound described herein and about treating the operation instructions of disease described herein.Test kit can also comprise printing material, the title of its inclusion compound, the description of structure.
In yet another aspect, the present invention relates to analyze or the method for project organization, this method comprises: provide computer-generation chemical compound described herein (for example, formula I chemical compound) image or structure (preferably 3-D view or structure), second kind of chemical compound that computer-generation is provided (for example, another kind of chemical compound as herein described (for example, formula I chemical compound, NAD) or the target thing (for example, sirtuin (for example, people sirtuin, for example, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7)), or non-target molecule (off-target molecule) (for example, the sirtuin beyond the SIRT1, for example, SIRT2 or SIRT3, or non--sirtuin histone deacetylase)) image or structure (preferably 3-D view or structure); With the structure of first kind and second kind chemical compound of contrast, for example, comparison structure, for example, the parameter relevant, intermolecular or intramolecularly distance, the position of atom or group with bond angle; For example, the chemical compound of the first generation or second filial generation chemical compound-prediction and target thing or non-target molecule interact or suppress their ability.
In a preferred embodiment, further in external, body or computer ground (insilico) with target thing or non-target molecule evaluation structure.
In yet another aspect, the present invention relates to the data base, it comprises: about or identify the information of structure, for example external about the active information of structure, in the body or computer ground, for example, at least 5,10,50, or 100 records.
In one aspect, the present invention relates to the data base, it comprises many records, and each record has: a) about or identify the information of chemical compound with structure as herein described (for example, the structure of formula I); And b) about the information of patient's parameter, this parameter is relevant with neoplastic disease or neurodegenerative disease, for example patient parameter.
In one aspect, the present invention relates to the method for assessing compound, this method comprises: first kind of chemical compound with formula I structure is provided, or has the data record about the information of structure; The second kind of chemical compound that has formula I structure or do not have formula I structure is provided, or has data record about the information of structure; Estimate first kind of chemical compound and second kind of chemical compound, for example external in the body, or computer ground; With second kind of chemical compound of contrast and first kind of chemical compound and sirtuin (for example, SIRT1) interaction () ability for example, inhibition sirtuin, thus estimate second kind of chemical compound and the interactional ability of SIRT1.
In others, the present invention relates to compositions, it comprises the chemical compound and the pharmaceutically acceptable carrier of the arbitrary formula of this paper.Compositions can contain other therapeutic agent, for example, and antitumor agent or neurodegenerative disease medicament.Such compositions is used for the application of the medicine of aforementioned applications in production, also within the scope of the invention.
In yet another aspect, the present invention is the method for the disease (for example, cancer for example, depends on the cancer of p53 or is independent of the cancer of p53) that is characterised in that undesirable cell proliferation in treatment or the object of prevention.This method comprises, uses the SIRT1 antagonist.For example, the SIRT1 antagonist can be following one or more: the antisense of SIRT1, and RNAi, antibody, intrabody and other chemical compound that identifies by method as herein described for example, are induced the apoptotic chemical compound of SIRT1 express cell.
In a preferred embodiment, this method comprises, with the co-administered SIRT1 antagonist of one or more therapeutic agents (therapeutic agent or the medicament that for example, are used for the treatment of undesirable cell proliferation).Therapeutic agent comprises, for example following one or more: chemotherapeutics, radiosiotope, and cytotoxin.The example of chemotherapeutics comprises taxol, cytochalasin B, Gramicidin D, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, busulfan, cisplatin, doxorubicin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, chlorambucil, gemcitabine, D actinomycin D, procaine, tetracaine, lignocaine, Propranolol, puromycin, maytansinoid and its analog or homologue.Other therapeutic agent includes but not limited to, antimetabolite (for example, methotrexate, Ismipur, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, decarbazine), alkylating agent is (for example, chlormethine, plug is for group, chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, mitobronitol, streptozocin, ametycin, and (DDP) cisplatin of cis-dichloro diamidogen platinum (II)), anthracycline antibiotics (for example, daunorubicin (being called daunomycin in the past) and doxorubicin), antibiotic is (for example, more living rhzomorph (being called D actinomycin D in the past), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agent (for example, vincristine, vinblastine, taxol and maytansinoid).Radiosiotope can comprise α, β and/or gamma ray radiator.Radioisotopic example comprises
212Bi,
213Bi,
131I,
211At,
186Re,
90Y and
117Lu.
Can be simultaneously or sequentially use SIRT1 antagonist and therapeutic agent.
Packaging product also within the scope of the invention.Packaging product comprises container, one of aforesaid compound in container, (for example treat disease as herein described with the indication administered compound that accompanies with container, cancer or neurodegenerative disease), the sign (for example, label or inset) of disease or disease symptoms (comprise as herein described those).
Object can be a mammal, preferred people.Object also can right and wrong-people's object, for example, and animal model.In certain embodiments, method can also comprise the discriminating object.Differentiating the object that needs such treatment, can be object or health worker's judgement, and can be subjective (for example, suggestion) or objective (for example, measurable by test or diagnostic method).
Term " mammal " comprises organism, and it comprises mice, rat, cattle, sheep, pig, rabbit, goat, and horse, monkey, Canis familiaris L., cat, and preferred people.
Term " treatment " or " treatment " point at objects are used chemical compound as herein described, its objective is healing, fully recover, alleviate, alleviate, change, treat, improve, improve or influence disease, for example, infect the symptom of disease or to the tendency of disease.
The effective dose scope of above-claimed cpd can be extremely about 500mg/Kg of about 0.1mg/Kg, and perhaps about 1 to about 50mg/Kg.Effective dose also can change with route of administration and with the common probability of using of other reagent.
Term " halo " or " halogen " refer to any group in fluorine, chlorine, the bromine or iodine.
Term " alkyl " refers to contain the hydrocarbon chain of the straight or branched of indicated number purpose carbon atom.For example, C
1-C
12Alkyl represents can have in this group the individual carbon atom of 1-12 (containing endpoints thereof).Term " haloalkyl " refers to wherein one or more hydrogen atoms by the alternate alkyl of halogen, and comprises that wherein all hydrogen are all by the alternate alkyl of halogen (for example, perfluoroalkyl).Term " aryl alkyl " or " aralkyl " refer to that wherein the alkyl hydrogen atom is by the alternate alkyl of aryl.Aralkyl comprises wherein and to surpass a hydrogen atom by the alternate group of aryl.The example of " aryl alkyl " or " aralkyl " comprises benzyl, 2-phenethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl.
Term " alkylidene " refers to divalent alkyl, for example, and-CH
2-,-CH
2CH
2-and-CH
2CH
2CH
2-.
The hydrocarbon chain that term " alkenyl " refers to contain 2-12 carbon atom and has the straight or branched of one or more pairs of keys.Non-limiting examples of alkenyls includes but not limited to, pi-allyl, acrylic, crotyl, 3-hexenyl and 3-octenyl.One of double key carbon can randomly be the attachment point of alkenyl substitutents.Term " alkynyl " refers to contain 2-12 carbon atom and is characterised in that the hydrocarbon chain with one or more triple-linked straight or brancheds.The example of alkynyl includes but not limited to, acetenyl, propinyl and 3-hexin base.One of triple bond carbon can randomly be the attachment point of alkynyl substituted base.
Term " alkyl amino " and " dialkyl amido " refer to respectively-NH (alkyl) and-NH (alkyl) 2.Term " aryl alkyl amino " refers to-NH (aralkyl).Term " alkyl amino alkyl " refer to (alkyl) NH-alkyl-; Term " dialkyl aminoalkyl " refers to (alkyl)
2The N-alkyl-.Term " alkoxyl " refers to-the O-alkyl.Term " sulfydryl " refers to SH.Term " thio alkoxy " refers to-the S-alkyl.Term " thioaryl oxygen " refer to-the S-aryl.
Term " aryl " refers to aromatic monocyclic, bicyclo-or tricyctic hydrocarbon loop systems, and any annular atoms that wherein can replace can be substituted (for example, being replaced by one or more substituent groups).The example of aryl includes but not limited to, phenyl, naphthyl, and anthryl.
Term " cycloalkyl " comprises saturated cyclic, bicyclo-, three ring or the multi-ring alkyls with 3 to 12 carbon as used herein.Annular atoms can be substituted (for example, being replaced by one or more substituent groups) arbitrarily.Cycloalkyl can contain fused rings.Fused rings is the ring with a common carbon atom.The example of cycloalkyl includes but not limited to, cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl and norborneol alkyl.
Term " heterocyclic radical " refers to the first monocycle of non-aromatics 3-10,8-12 unit's dicyclo or 11-14 unit three ring loop systems, if monocycle then has 1-3 hetero atom, if dicyclo then has 1-6 hetero atom, or if three rings then have 1-9 hetero atom, described hetero atom (for example is selected from O, N or S, if be monocycle, dicyclo or three rings respectively, then be carbon atom and 1-3, a 1-6 or 1-9 N, O or S hetero atom).Hetero atom can randomly be the substituent attachment point of heterocyclic radical.Annular atoms can be substituted (for example, being replaced by one or more substituent groups) arbitrarily.Heterocyclic radical can contain fused rings.Fused rings is the ring with a common carbon atom.The example of heterocyclic radical includes but not limited to, tetrahydrofuran base, THP trtrahydropyranyl, piperidyl, morpholino, pyrrolinyl, pyrimidine radicals, quinolyl, and pyrrolidinyl.
Term " cycloalkenyl group " refers to have 5 to 12 carbon, preferably ring-type, the dicyclo, three of the non-aromatics of the fractional saturation of 5 to 8 carbon encircle or multi-ring alkyls.Undersaturated carbon can randomly be the substituent attachment point of cycloalkenyl group.Annular atoms can be substituted (for example, being replaced by one or more substituent groups) arbitrarily.Cycloalkenyl group can contain fused rings.Fused rings is the ring with a common carbon atom.The example of cycloalkenyl group includes but not limited to, cyclohexenyl group, cyclohexadienyl, or norbornene.
The first monocycle of saturated non-aromatics 5-10,8-12 unit's dicyclo or 11-14 unit three ring loop systems are divided in term " heterocycloalkenyl " finger, if monocycle then has 1-3 hetero atom, if dicyclo then has 1-6 hetero atom, or if three rings then have 1-9 hetero atom, described hetero atom (for example is selected from O, N or S, if be monocycle, dicyclo or three rings respectively, then be carbon atom and 1-3, a 1-6 or 1-9 N, O or S hetero atom).Undersaturated carbon or hetero atom can randomly be the substituent attachment points of heterocycloalkenyl.Annular atoms can be substituted (for example, being replaced by one or more substituent groups) arbitrarily.Heterocycloalkenyl can contain fused rings.Fused rings is the ring with a common carbon atom.The example of heterocycloalkenyl includes but not limited to, tetrahydro pyridyl and dihydro pyranyl.
Term " heteroaryl " refers to aromatics 5-8 unit monocycle, 8-12 unit's dicyclo or 11-14 unit three ring loop systems, if monocycle then has 1-3 hetero atom, if dicyclo then has 1-6 hetero atom, or if three rings then have 1-9 hetero atom, described hetero atom (for example is selected from O, N or S, if be monocycle, dicyclo or three rings respectively, then be carbon atom and 1-3, a 1-6 or 1-9 N, O or S hetero atom).Annular atoms can be substituted (for example, being replaced by one or more substituent groups) arbitrarily.
Term " oxo " refers to oxygen atom, and when in conjunction with carbon, it forms carbonyl, and when in conjunction with nitrogen, it forms the N-oxide, and when fault sulphur, it forms sulfoxide or sulfone.
Term " acyl group " refers to alkyl-carbonyl, naphthene base carbonyl, and aryl carbonyl, heterocyclic radical carbonyl, or heteroaryl carbonyl substituted base, wherein any can further be replaced (for example, being replaced by one or more substituent groups).
Term " amino carbonyl, " alkoxy carbonyl, " diazanyl carbonyl and hydroxyl amino carbonyl refer to group-C (O) NH respectively
2,-C (O) O (alkyl) ,-C (O) NHNH
2And-C (O) NHOH.
Term " acylamino-" refers to-NHC (O)-and, wherein N is an attachment point.
Term " substituent group " refers to the group of on the arbitrary atom of alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic radical, heterocycloalkenyl, cycloalkenyl group, aryl or heteroaryl " replacement ".Atom can be substituted arbitrarily.Suitable substituents includes but not limited to, alkyl (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, the alkyl of C12 straight or branched), cycloalkyl, haloalkyl (for example, perfluoroalkyl CF for example
3), aryl, heteroaryl, aralkyl, heteroarylalkyl, heterocyclic radical, alkenyl, alkynyl, cycloalkenyl group, heterocycloalkenyl, alkoxyl, halogenated alkoxy (for example, perfluoro alkoxy OCF for example
3), halogen, hydroxyl, carboxyl, carboxylate (ester), cyano group, nitro, amino, alkyl amino, SO
3H, sulfate (ester), phosphate (ester), methylene-dioxy (O-CH
2-O-, wherein oxygen is attached on the adjacent atom), ethylenedioxy, oxo, sulfo-(for example, C=S), imido grpup (alkyl, aryl, aralkyl), S (O)
nAlkyl (wherein n is 0-2), S (O)
nAryl (wherein n is 0-2), S (O)
nHeteroaryl (wherein n is 0-2), S (O)
nHeterocyclic radical (wherein n is 0-2), amine (single-, two-, alkyl, cycloalkyl, aralkyl, heteroarylalkyl, aryl, heteroaryl and their combination), ester (alkyl, aralkyl, heteroarylalkyl, aryl, heteroaryl), amide (single-, two-, alkyl, aralkyl, heteroarylalkyl, aryl, heteroaryl and their combination), sulfonamides (single-, two-, alkyl, aralkyl, heteroarylalkyl and their combination).In one aspect, the substituent group on the group is any independent aforementioned substituent group independently, or aforementioned substituent random subset.In yet another aspect, substituent group can ownly be replaced by any aforementioned substituent group.
In accompanying drawing below and the detailed description, the details of one or more embodiments of the present invention has been described.From detailed description and accompanying drawing and claim, can understand other features, objects and advantages of the present invention.
All documents that this paper quotes, no matter be printing, electronics, computer-readable recording medium or other form, all clearly whole incorporated by reference, include but not limited to summary, article, periodical, publication, textbook, paper, Internet, data base, patent, patent application and patent disclosure.
Accompanying drawing is described
Fig. 1 is the table of representative compounds and data.
Fig. 2 is the model of computer-generation, and it has shown a kind of possible orientation of the chemical compound 8 of the active site that is combined in SIRT.
Fig. 3 a is the figure that describes the inhibition of 8 couples of mammal SirT1 of chemical compound.
Fig. 3 b is the Western blot of the NCI-H460 cell only handled with etoposide or with etoposide and chemical compound 8.
Fig. 4 is the bar diagram that the enantiomer 8 (-) of description chemical compound 8 causes the acetylizad increase of p53.
Fig. 5 describes the chemical compound that suppresses the SirT catalytic activity also to cause the acetylizad Western blot of p53.
Fig. 6 describes the figure that the enantiomer 8 (-) of comparing chemical compound 8 with enantiomer 8 (+) preferentially suppresses yeast sir2.
Fig. 7 describes the gel determination that chemical compound 8 suppresses the effectiveness of the SirT1 in U2OS cell and the MCF-7 cell.
Fig. 8 is the figure of the influence of the cell survival after 8 pairs of DNA damage of description chemical compound.
Fig. 9 is the figure of influence that describes the cell survival of 8 pairs of NCI-H460 cells of chemical compound.
Figure 10 describes the bar diagram of rise that chemical compound 8 causes abolishing the cell cycle inhibitor p27 of serum starvation-mediation.
Describe in detail
The structure of exemplary compounds
Can be used to put into practice exemplary compounds of the present invention and have general formula (I), and contain substituted 5 or 6 yuan of nucleolus hearts, the latter is contained 1 or 2 oxygen, nitrogen or the sulphur atoms composed atom as ring respectively, for example, and X and Y in the following formula (I).
Ring carbon atom can be substituted arbitrarily.For example, R
1, R
2, R
3, and R
4Can include but not limited to substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic radical, heterocycloalkenyl, cycloalkenyl group, aryl, heteroaryl etc.5 or 6 yuan of nucleolus hearts can be saturated, promptly do not contain two keys, or partially or completely saturated, promptly contain one or two pair key respectively.When n=0, " X " can be oxygen, sulfur or nitrogen, for example, and NR
7Substituent R
7Can be, be not limited to, hydrogen, alkyl, for example, C1, C2, C3, C4 alkyl, SO
2(aryl), acyl group, or ring nitrogen can form the part of carbamate or urea groups.When n=1, X can be NR
7, O, or S; And Y can be NR
7 ', O or S.X and Y can be heteroatomic combination in any, N for example, and N, N, O, N, S, etc.
A preferred subset of formula (I) chemical compound comprises the chemical compound of have one (or preferably 2) and 5 or 6 yuan of condensed rings of the nucleolus heart, for example, and R
1And R
2With they bonded carbon, and/or R
3And R
4With they bonded carbon, for example can form C
5-C
10Cycloalkyl (for example, C5, C6, or C7), C
5-C
10Heterocyclic radical (for example, C5, C6, or C7), C
5-C
10Cycloalkenyl group (for example, C5, C6, or C7), C
5-C
10Heterocycloalkenyl (for example, C5, C6, or C7), C
6-C
10Aryl (for example, C6, C8 or C10), or C
6-C
10Heteroaryl (for example, C5 or C6).Fused rings combination can include but not limited to following one or more:
Preferred combination comprises for example having C by B
6Aryl and C
6Cycloalkenyl group (B1), and C for example have C
6Aryl and C
7Cycloalkenyl group (C1):
In these fused rings systems each can randomly be substituted base and replace, and described substituent group can include but not limited to halogen, hydroxyl, C
1-C
10Alkyl (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), C
1-C
6Haloalkyl (C1, C2, C3, C4, C5, C6), C
1-C
10Alkoxyl (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), C
1-C
6Halogenated alkoxy (C1, C2, C3, C4, C5, C6), C
6-C
10Aryl (C6, C7, C8, C9, C10), C
5-C
10Heteroaryl (C5, C6, C7, C8, C9, C10), C
7-C
12Aralkyl (C7, C8, C9, C10, C11, C12), C
7-C
12Heteroarylalkyl (C7, C8, C9, C10, C11, C12), C
3-C
8Heterocyclic radical (C3, C4, C5, C6, C7, C8), C
2-C
12Alkenyl (C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12), C
2-C
12Alkynyl (C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12), C
5-C
10Cycloalkenyl group (C5, C6, C7, C8, C9, C10), C
5-C
10Heterocycloalkenyl (C5, C6, C7, C8, C9, C10), carboxyl, carboxylate (ester), cyano group, nitro, amino, C
1-C
6Alkyl amino (C1, C2, C3, C4, C5, C6), C
1-C
6Dialkyl amido (C1, C2, C3, C4, C5, C6), sulfydryl, SO
3H, sulfate (ester), S (O) NH
2, S (O)
2NH
2, phosphate (ester), C
1-C
4Alkylene dioxo base (C1, C2, C3, C4), oxo, acyl group, amino carbonyl, C
1-C
6Alkyl amino-carbonyl (C1, C2, C3, C4, C5, C6), C
1-C
6Dialkyl amino carbonyl (C1, C2, C3, C4, C5, C6), C
1-C
10Alkoxy carbonyl (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), C
1-C
10The thio alkoxy carbonyl (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), diazanyl carbonyl, C
1-C
6Alkyl hydrazine carbonyl (C1, C2, C3, C4, C5, C6), C
1-C
6Dialkyl group diazanyl carbonyl (C1, C2, C3, C4, C5, C6), hydroxyl amino carbonyl etc.Preferred substituted comprises halogen (for example, fluorine, chlorine, bromine), C
1-C
10Alkyl (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10), C
1-C
6Haloalkyl (for example, C1, C2, C3, C4, C5, C6, for example, CF
3), C
1-C
6Halogenated alkoxy (for example, C1, C2, C3, C4, C5, C6, for example, OCF
3), or amino carbonyl.Can select 2 substitution patterns on the fused rings as required, for example, a ring can be substituted, and another ring does not replace, and perhaps two rings can be replaced by 1-5 substituent group (1,2,3,4,5 substituent groups).Substituent number on each ring can be identical or different.Shown preferred substitution pattern below:
In certain embodiments, when n be 0 and X be NR
7The time, the nitrogen substituent R
7Can form circulus with one of fused rings that contains for example 4-6 carbon, a 1-3 nitrogen, a 0-2 oxygen and 0-2 sulfur.This circulus can be randomly by oxygen or C
1-C
6Alkyl replaces.
The substituent group that the present invention predicts and the combination of variable only are to cause forming those of stable chemical compound.As used herein, term " stable " refers to such chemical compound, it has is enough to allow the stability of producing, and it can keep the integrity of chemical compound the time period that is enough to be used in purpose described herein (for example, therapeutic or prophylactically be administered to object).
Exemplary compounds comprises those * described in the following table 1:
Table 1: exemplary compounds
| Compound number | Chemical name | Ave.SirT1 p53-382 IC50 |
| 1 | 7-chloro-1,2,3,4-tetrahydrochysene-cyclopenta [b] indole-3-carboxylic acid amide | A |
| 2 | 2,3,4,9-tetrahydrochysene-1H-b-carboline-3-benzoic acid amides | C |
| 3 | 6-bromo-2,3,4,9-tetrahydrochysene-1H-carbazole-2-benzoic acid amides | B |
| 4 | 6-methyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | A |
| 5 | 2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | B |
| 6 | 2-chloro-5,6,7,8,9,10-six hydrogen-cyclohepta [b] indole-6-benzoic acid amides | A |
| 7 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid hydroxy amide | C |
| 8 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | A |
| 9 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-2-benzoic acid amides | C |
| 10 | 1,2,3,4-tetrahydrochysene-cyclopenta [b] indole-3-carboxylic acid amide | B |
| 11 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid (5-chloro-pyridine-2-yl)-amide | B |
| 12 | 1,6-dimethyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | C |
| 13 | 6-trifluoromethoxy-2,3,4,9-tetrahydrochysene-1H-carbazole-2-benzoic acid amides | C |
| 14 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid diacetayl amide | D |
| 15 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid carbamyl methyl-amide | D |
| 16 | 8-carbamyl-6,7,8,9-tetrahydrochysene-5H-carbazole-1-formic acid | D |
| 17 | 6-methyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid | D |
| 18 | 8-carbamyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 19 | [(6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-carbonyl)-amino]-ethyl acetate | D |
| 20 | 9-benzyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | D |
| 21 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-methyl formate | D |
| 22 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid | D |
| 23 | C-(6-methyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-yl)-methylamine | D |
| 24 | 6,9-dimethyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | D |
| 25 | The 7-methyl isophthalic acid, 2,3,4-tetrahydrochysene-cyclopenta [b] indole-3-carboxylic acid amide | D |
| 26 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid buserelin | D |
| 27 | 2-(1-benzyl-3-methyl mercapto-1H-indole-2-yl)-N-p-methylphenyl-acetamide | D |
| 28 | N-benzyl-2-(1-methyl-3-thiophenyl-1H-indole-2-yl)-acetamide | D |
| 29 | N-(4-chloro-phenyl)-2-(1-methyl-3-thiophenyl-1H-indole-2-yl)-acetamide | D |
| 30 | N-(3-hydroxyl-propyl group)-2-(1-methyl-3-thiophenyl-1H-indole-2-yl)-acetamide | D |
| 31 | 2-(1-benzyl-3-thiophenyl-1H-indole-2-yl)-N-(3-hydroxyl-propyl group)-acetamide | D |
| 32 | 2-(1-benzyl-3-methyl mercapto-1H-indole-2-yl)-N-(4-methoxyl group-phenyl)-acetamide | D |
| 33 | 2-(1-benzyl-1H-indole-2-yl)-N-(4-methoxyl group-phenyl)-acetamide | D |
| 34 | 2-(1-methyl-3-methyl mercapto-1H-indole-2-yl)-N-p-methylphenyl-acetamide | D |
| 35 | 2-(1-benzyl-3-methyl mercapto-1H-indole-2-yl)-N-(2-chloro-phenyl)-acetamide | D |
| 36 | 2-(1,5-dimethyl-3-methyl mercapto-1H-indole-2-yl)-N-(2-hydroxyl-ethyl)-acetamide | D |
| 37 | (6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-yl)-[4-(furan-2-carbonyl)-piperazine-1-yl]-ketone | D |
| 38 | 2-(1-benzyl-1H-indole-2-yl)-N-(2-chloro-phenyl)-acetamide | D |
| 39 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 40 | 6-chloro-9-methyl-2,3,4,9-tetrahydrochysene-1H-carbazole-4-Ethyl formate | D |
| 41 | 5,7-two chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 42 | 7-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 43 | 5,7-two chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid | D |
| 44 | 6-chloro-9-methyl-2,3,4,9-tetrahydrochysene-1H-carbazole-4-formic acid | D |
| 45 | 6-chloro-9-methyl-2,3,4,9-tetrahydrochysene-1H-carbazole-4-benzoic acid amides | D |
| 46 | 6-morpholine-4-base-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 47 | 6-morpholine-4-base-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | D |
| 48 | 6-bromo-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 49 | 6-fluoro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-Ethyl formate | D |
| 50 | 3-carbamyl-1,3,4,9-tetrahydrochysene-b-carboline-2-t-butyl formate | D |
| 51 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid (1-phenyl-ethyl)-amide | D |
| 52 | 7,8-two fluoro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides | D |
| 53 | 6-bromo-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid | D |
| 54 | 6-hydroxyl-2,3,4,9-tetrahydrochysene-1H-carbazole-1-formic acid | C |
| 55 | 6-bromo-2,3,4,9-tetrahydrochysene-1H-carbazole-2-Methanamide | B |
| 56 | 6-chloro-2,3,4,9-tetrahydrochysene-1H-pyrido [3,4-b] indole-1-Methanamide | C |
| 57 | 6-bromo-2,3,4,9-tetrahydrochysene-1H-pyrido [3,4-b] indole-1-Methanamide | D |
| 58 | 2-acetyl group-6-chloro-2,3,4,9-tetrahydrochysene-1H-pyrido [3,4-b] indole-1-Methanamide | C |
* have the active chemical compound of being appointed as A and have IC less than 1.0 μ M
50Have the active chemical compound of being appointed as B and have the IC of 1.0 μ M to 10.0 μ M
50Have the active chemical compound of being appointed as C and have IC greater than 10.0 μ M
50Be appointed as chemical compound not test in this mensuration of D.
By external (based on cell and based on acellular) and body in method, can identify and can be used to realize chemical compound of the present invention.The explanation of these methods has been described in an embodiment.
Synthesizing of exemplary compounds
Chemical compound as herein described can be from commercial source (for example, Asinex, Moscow, Russia; Bionet, Camelford, England; ChemDiv, SanDiego, CA; Comgenex, Budapest, Hungary; Enamine, Kiev, Ukraine; IF Lab, Ukraine; Interbioscreen, Moscow, Russia; Maybridge, Tintagel, UK; Specs, The Netherlands; Timtec, Newark, DE; Vitas-M Lab, Moscow Russia) obtains, or uses the raw material and the reagent that can commercially obtain, and is as follows synthetic by conventional method.For example, can be shown in following scheme 1, synthetic exemplary compounds 4.
The 'beta '-ketoester 1 of bromination can with the condensation of 4-chloroaniline, cyclisation subsequently can produce indole 2.The ester saponification can produce acid 3.Last and PyAOP amination can produce amide 4.Other method is known in the art, referring to, for example, United States Patent (USP) 3,859,304, United States Patent (USP) 3,769,298, J.Am.Chem.Soc.1974,74,5495.Top synthesizing can extend to other aniline, for example, 3,5-dichloroaniline, 3-chloroaniline and 4-bromaniline.The aniline that uses N-to replace, for example, 4-chloro-methylphenylamine can obtain the product of regional isomerism, for example, 5.
By such as methods such as column chromatography, high pressure liquid chromatography or recrystallization, can separate chemical compound as herein described and be further purified from reactant mixture.Can understand that as those skilled in the art those of ordinary skill in the art can understand other method of synthetic this paper general formula compound.In addition, can carry out each synthesis step with alternative order or order, to produce target compound.The synthetic chemistry that can be used for synthetic chemical compound as herein described transforms and protecting group method (protect and go and protect), be known in the art, and comprise, for example, be documented in for example following document those: R.Larock, Comprehensive Organic Transformations, VCHPublishers (1989); T.W.Greene and P.G.M.Wuts, Protective Groupsin Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L.Fieser and M.Fieser, Fieser and Fieser ' s Reagents for OrganicSynthesis, John Wiley and Sons (1994); And L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley andSons (1995) and their later release.
Chemical compound of the present invention can contain one or more asymmetric centers, thereby exists as racemic modification and racemic mixture, single enantiomer, independent diastereomer and non-enantiomer mixture.The isomeric forms that all of these chemical compounds are so all clearly comprises in the present invention.But chemical compound of the present invention also can contain the connection (for example, carbon-carbon bond) or the substituent group of limit key rotation, for example because the restriction that the existence of ring or two keys causes.Therefore, all cis/trans and E/Z isomer all clearly comprise in the present invention.Chemical compound of the present invention can also multiple tautomeric form be represented, under these circumstances, the present invention clearly comprises all tautomeric forms of chemical compound described herein, even may (for example only represent a kind of tautomeric form, the alkylation of loop systems may cause the alkylation in a plurality of sites, and the present invention clearly comprises the product that all are such).All such isomeric form of such chemical compound all clearly comprise praises among the present invention.The all crystal forms of chemical compound described herein all clearly comprises in the present invention.
(for example, stereoisomer) technology belongs to the general knowledge of this area, and is documented in: Eliel, E.L. to can be used for the separating isomerism body; Wilen, S.H.; Mander, L.N.Stereochemistry of Organic Compounds, Wiley Interscience, NY, 1994.For example, by forming diastereoisomeric salt, for example use chiral base, for example, (+) or (-) α-Jia Jibianji amine, or by using the high performance liquid chromatography of chiral column, it is excessive (for example chemical compound 3 or 4 can be split into high antimer, 60%, 70%, 80%, 85%, 90%, 95%, 99% or bigger).In some embodiment, direct purification crude product 4 on chiral column provides the chemical compound of enantiomer enrichment.
For the purpose of explaining, shown the enantiomer of chemical compound 4 below.
In some example, use chemical compound disclosed herein, wherein a kind of isomer (for example, R isomer or S isomer) exists so that high antimer is excessive.Usually, with have+32.8 (c=0.38, DCM) or [α]
D 25+ 22.72 ° of (c0.910, CH
3The enantiomer of dextrorotation luminosity OH) is compared, have negative rotation luminosity (for example ,-14.1 (c=0.33, DCM) or [α]
D 25-41.18 ° of (c0.960, CH
3The isomer of chemical compound 4 OH)) has the bigger activity at the SirT1 enzyme.Therefore, in some example, advantageously use chemical compound 4, treat disease with the excessive isomer of high antimer with negative rotation luminosity to object.
Although the enantiomer of chemical compound 4 provides an example of stereoisomer, also predict other stereoisomer, for example as described in following chemical compound 6 and 7.
As the chemical compound of cotype 4, in some cases, advantageously to object use have than its enantiomer bigger to the chemical compound 6 of the affinity of SirT1 or 7 isomer.For example, in some example, advantageously use the chemical compound 7 of enrichment (-) optical isomer, wherein amide (or other substituent group) has the configuration identical with the negative isomer of chemical compound 4.
In some example, advantageously use chemical compound with one of following structure, wherein the stereochemical structure of amide (or other substituent group) conforms to amide in the chemical compound 4 with negative rotation luminosity.
(n is 0 to 4 integer)
Chemical compound of the present invention comprises chemical compound itself, and their salt and their prodrug (if being suitable for).Salt for example, can form between the positively charged substituent group on anion and the chemical compound described herein (for example, amino).Suitable anion comprises chloride ion, bromide ion, iodide ion, sulfate radical, nitrate anion, phosphate radical, citrate, methanesulfonate, trifluoroacetic acid root, and acetate.Similarly, salt also can form between the electronegative substituent group on cation and the chemical compound described herein (for example, carboxylate radical).Suitable cation comprises sodium ion, potassium ion, magnesium ion, calcium ion, and ammonium cation, for example tetramethyl ammonium.The example of prodrug comprises ester and other pharmaceutically acceptable derivates, and it can provide reactive compound after being administered to object.
Can modify chemical compound of the present invention by attached suitable functional group, thereby improve selected biological property, for example targeting particular organization.Such modification is known in the art, and comprise the biology infiltration that can increase in given compartment biology (for example, blood, lymphsystem, central nervous system), increase oral availability, increase dissolubility to allow drug administration by injection, change metabolism and to change those of discharge rate.
In an alternative embodiment, chemical compound as herein described can be used as platform or support, and it can be used for combinatorial chemistry technique, is used to prepare the derivant and/or the chemical library of chemical compound.This derivant of chemical compound and library biologically active, and can be used for differentiating and designing chemical compound with given activity.It is known in the art being fit to use combination of compounds technology described herein, it is exemplified as Obrecht, D. and Villalgrodo, J.M., Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compounds Libraries, Pergamon-ElsevierScience Limited (1998), and comprise following those, for example " split and pool " or " parallel " synthetic technology, solid phase and solution is technology mutually, and coding techniques (referring to, for example, Czarnik, A.W., Curr.Opin.Chem.Bio., (1997) 1,60.Thereby an embodiment relates to uses chemical compound described herein to produce the method in derivant or chemical library, comprising: 1) provide to comprise and permitted porous object; 2) in each hole, provide one or more chemical compounds that identify by methods described herein; 3) in each hole, provide another or multiple chemical compound; 4) from one or more products of each hole resulting separation.An alternative embodiment relates to uses chemical compound as herein described to produce the method in derivant or chemical library, comprising: 1) one or more chemical compounds as herein described that are attached on the solid support are provided; 2) handle by what methods described herein identified with one or more other chemical reagent and be attached to one or more chemical compounds on the solid support; 3) from one or more products of solid support resulting separation.In said method, " label " or identifier or mark part can be attached to and/or break away from chemical compound as herein described or their derivant, to promote tracking, identification or the separation to target product or their intermediate.Such part is known in the art.The chemical reagent that uses in preceding method can comprise, for example, and solvent, reagent, catalyst, protecting group and remove protecting group reagent etc.The example of such chemical reagent is those that occur in various synthetic and the protecting group chemical textbook and the paper of this paper reference.
Sirtuin
Sirtuin is the member of silent message regulator (SIR) family gene.Sirtuin is an albumen, and it comprises the SIR2 domain, and the latter is defined as and marks in Pfam family " SIR2 "-PF02146 to hitting the aminoacid sequence of (hits).This family is called INTERPRO and describes (entry IPR003000) in the INTERPRO data base.In order to differentiate the existence of " SIR2 " domain in protein sequence, and definite target polypeptides or albumen have particular characteristic, can be (for example at the Pfam data base of HMM, the Pfam data base, version 9), use default parameters, search for proteic aminoacid sequence (http://www.sanger.ac.uk/Software/Pfam/HMM_search).The SIR2 domain is indexed as PF02146 in Pfam, and is indexed as INTERPRO description (entry IPR003000) in INTERPRO.For example, the hmmsf program, its part that can be used as HMMER bag of search utility obtains, and is the default program of the family specificity of MILPAT0063, and 15 mark is to determine the default threshold mark that hits.Perhaps, can reduce and determine the threshold score (for example, to 8 bits) hit.Pfam data base's description can be referring to " The Pfam ProteinFamilies Database " Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL (2002) Nucleic Acids Research 30 (1): 276-280 and Sonhammer et al. (1997) Proteins 28 (3): 405-420, and the detailed description of HMM can referring to, for example, Gribskov et al. (1990) Meth.Enzymol.183:146-159; Gribskov et al. (1987) Proc.Natl.Acad.Sci.USA84:4355-4358; Krogh et al. (1994) J.Mol.Biol.235:1501-1531; With Stultz et al. (1993) Protein Sci.2:305-314.
It is conservative to show high sequence in 250 aminoacid core texture territories by member's encoded protein of SIR2 gene family.A gene that better characterizes in this family is Saccharomyces Cerevisiae in S IR2, and it participates in reticent HM locus, and the latter is contained information (Guarente, 1999 of specifying yeast maqting type, telomere position effect and cell senescence; Kaeberlein et al., 1999; Shore, 2000).Yeast Sir2 albumen belong to histone deacetylase family (summary is seen Guarente, 2000; Shore, 2000).Sir2 albumen is a kind of deacetylase, and it can use NAD as cofactor (Imai et al., 2000; Moazed, 2001; Smith etal., 2000; Tanner et al., 2000; Tanny and Moazed, 2001).Different with other deacetylase (many participation gene silencings wherein), Sir2 is to histone deacetylase inhibitors such as Trichostatin A (TSA) relative insensitivity (Imai et al., 2000; Landry et al., 2000a; Smith et al., 2000).Mammal Sir2 congener (for example SIRT1) has the dependent deacetylation enzymatic activity of NAD-(Imai et al., 2000; Smith et al., 2000).
Exemplary mammal sirtuin comprises SIRT1, SIRT2, and SIRT3, for example, people SIRT1, SIRT2, and SIRT3.Chemical compound as herein described can suppress mammal sirtuin, and () one or more activity for example, SIRT1, SIRT2, or SIRT3, for example, its Ki is less than 500,200,100,50, or 40nM.For example, chemical compound can suppress the deacetylation enzymatic activity, for example, and for natural or artificial substrate, for example, substrate as herein described, for example, as follows.
The natural substrate of SIRT1 comprises histone, and p53 and FoxO transcription factor be FoxO1 and FoxO3 for example.SIRT1 albumen can be in conjunction with many other albumen, and they are called " SIRT1 binding partners ".For example, SIRT1 is in conjunction with p53, and works in the p53 approach, for example, the K370 of p53, K371, K372, K381, and/or K382, or comprise one or more peptide in these lysines.For example, the length of peptide can be 5 to 15 aminoacid.SIRT1 albumen also can make histone deacetylation.For example, SIRT1 can make histone H 3 or comprise the lysine 9 or 14 of the one or more little peptide in these lysines deacetylated.Histone deacetylase effect meeting changes partial chromatin Structure, thus near the gene transcription can regulating.Many SIRT1 binding partners are transcription factor, for example, discern the albumen in specific DNA site.For example, SirT1 can make the deacetylated and downward modulation of jaw albumen (being FoxO albumen).Interaction between SIRT1 and the SIRT1 binding partners can be sent SIRT1 to genomic specific region, and can cause the part of substrate (for example, being positioned the histone and the transcription factor of this specific region) to manifest.
The natural substrate of SIRT2 comprises tubulin, for example, and alpha-tubulin.Referring to, for example, North et al.Mol Cell.2003 Feb; 11 (2): 437-44.Exemplary substrate comprises the peptide of the lysine 40 that comprises alpha-tubulin.
Other exemplary sirtuin substrate comprises cytochrome C and its acetylizad peptide.
Term " SIRT1 albumen " and " SIRT1 polypeptide " use in this article interchangeably, and refer to the polypeptide identical with the conservative SIRT1 catalyst structure domain (amino acid residue 258 to 451 of SEQ ID NO:1) of 250 aminoacid at least 25%.SEQ ID NO:1 has described the aminoacid sequence of people SIRT1.In preferred embodiments, the SIRT1 polypeptide can and the amino acid residue 258 to 451 of SEQ ID NO:1 or SEQ ID NO:1 between aminoacid sequence have at least 30,40,50,60,70,80,85,90,95,99% homologys.In other embodiments, the SIRT1 polypeptide can be a fragment, for example, can realize following one or multinomial SIRT1 fragment: in the presence of NAD and/or NAD analog, and deacetylated substrate, and can be in conjunction with target protein, for example, transcription factor.Can estimate such function, for example, by method as herein described.In other embodiments, the SIRT1 polypeptide can be " total length " SIRT1 polypeptide.As used herein term " total length " refers to the to have naturally occurring SIRT1 polypeptide at least polypeptide of length of (or other albumen as herein described)." total length " SIRT1 polypeptide or its fragment also can comprise other sequence, for example, and purification labelling, or other chemical compound that adheres to, for example, the fluorogen that adheres to, or cofactor.With respect to naturally occurring Sir2 family member, term " SIRT1 polypeptide " also can comprise sequence or the variant that comprises one or more displacements (for example, 1 to 10 displacement)." SIRT1 activity " refers to one or more activity of SIRT1, and for example, substrate (for example, aminoacid, peptide, or albumen) deacetylation, for example transcription factor is (for example for substrate, p53) or histone albumen (for example, at cofactor for example in the presence of NAD and/or the NAD analog) and to target thing (for example, target protein, for example, combination transcription factor).
As used herein, proteic " biologically-active moiety " or " functional domain " comprise the interactional fragment of participation of target protein, and described interaction is intramolecularly or intermolecular interaction for example, for example, in conjunction with or catalysis interact.Intermolecular interaction can be specific binding interactions or enzymatic interaction (for example, interaction can be instantaneous and form or destroy covalent bond).Intermolecular interaction can be between albumen and another kind of albumen, between albumen and the another kind of chemical compound, or between proteic first kind of molecule and the second kind of molecule (for example, dimerization interacts).Proteic biologically-active moiety/functional domain comprises such peptide, it comprises with the enough homologies of proteic aminoacid sequence or is derived from its aminoacid sequence, described albumen comprises the aminoacid that lacks than the total length native protein, and shows at least a activity of native protein.By multiple technologies, comprise truncate analysis, direct mutagenesis and Proteolytic enzyme, can identify biologically-active moiety/functional domain.By measuring suitable biochemistry or biology (for example, the hereditism), can measure the activity of mutant or proteolytic fragments.In some embodiment, functional domain is folding independently.Typically, biologically-active moiety comprises (for example, at least a active structures territory or motif SIRT1) that have albumen.The exemplary configurations territory is a SIRT1 core catalyst structure domain.Can to be length be for example 10,25,50,100,200 or more a plurality of amino acid whose polypeptide to proteic biologically-active moiety/functional domain.Proteic biologically-active moiety/functional domain can be regulated the target thing of the reagent of SIRT1 as exploitation.
Be exemplary SIR sequence below:
The dependent deacetylase sirtuin 1 of＞sp|Q96EB6|SIR1_ people NAD-(EC3.5.1.-) is (hSIR2) (SIR2-sample albumen 1)-homo sapiens (Homo sapiens) (people) (hSIRT1).
MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLERSPGEPGGAAPEREV
PAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQGPSREPPLADNL
YDEDDDDEGEEEEEAAAAAIGYRDNLLFGDEIITNGFHSCESDEEDRASHASSSDWTPRP
RIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLWQIVINILSEPPKRKKRKDI
NTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIYARLAVDFPDLPDPQAMFDIE
YFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDTLEQVAGIQRII
QCHGSFATASCLICKYKVDCEAVRGDIFNQVVPRCPRCPADEPLAIMKPEIVFFGENLPE
QFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHEVPQILINREPLPHLHFDVELLG
DCDVIINELCHRLGGEYAKLCCNPVKLSEITEKPPRTQKELAYLSELPPTPLHVSEDSSS
PERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDL
KNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSD
SEDDVLSSSSCGSNSDSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGTD
GDDQEAINEAISVKQEVTDMNYPSNKS (SEQ ID NO：1)
The dependent deacetylase sirtuin 2 of＞sp|Q8IXJ6|SIR2_ people NAD-(EC3.5.1.-) (SIR2-sample) (SIR2-sample albumen 2)-homo sapiens (Homo sapiens) (people).
MAEPDPSHPLETQAGKVQEAQDSDSDSEGGAAGGEADMDFLRNLFSQTLSLGSQKERLLD
ELTLEGVARYMQSERCRRVICLVGAGISTSAGIPDFRSPSTGLYDNLEKYHLPYPEAIFE
ISYFKKHPEPFFALAKELYPGQFKPTICHYFMRLLKDKGLLLRCYTQNIDTLERIAGLEQ
EDLVEAHGTFYTSHCVSASCRHEYPLSWMKEKIFSEVTPKCEDCQSLVKPDIVFFGESLP
ARFFSCMQSDFLKVDLLLVMGTSLQVQPFASLISKAPLSTPRLLINKEKAGQSDPFLGMI
MGLGGGMDFDSKKAYRDVAWLGECDQGCLALAELLGWKKELEDLVRREHASIDAQSGAGV
PNPSTSASPKKSPPPAKDEARTTEREKPQ (SEQ ID NO：2)
The dependent deacetylase sirtuin 3 of＞sp|Q9NTG7|SIR3_ people NAD-, mitochondrion precursor (EC 3.5.1.-) (SIR2-sample albumen 3) (hSIRT3)-homo sapiens (Homosapiens) (people).
MAFWGWRAAAALRLWGRVVERVEAGGGVGPFQACGCRLVLGGRDDVSAGLRGSHGARGEP
LDPARPLQRPPRPEVPRAFRRQPRAAAPSFFFSSIKGGRRSISFSVGASSVVGSGGSSDK
GKLSLQDVAELIRARACQRVVVMVGAGISTPSGIPDFRSPGSGLYSNLQQYDLPYPEAIF
ELPFFFHNPKPFFTLAKELYPGNYKPNVTHYFLRLLHDKGLLLRLYTQNIDGLERVSGIP
ASKLVEAHGTFASATCTVCQRPFPGEDIRADVMADRVPRCPVCTGVVKPDIVFFGEPLPQ
RFLLHVVDFPMADLLLILGTSLEVEPFASLTEAVRSSVPRLLINRDLVGPLAWHPRSRDV
AQLGDVVHGVESLVELLGWTEEMRDLVQRETGKLDGPDK (SEQ ID NO：3)
The dependent deacetylase sirtuin 4 of＞sp|Q9Y6E7|SIR4_ people NAD-(EC3.5.1.-) (SIR2-sample albumen 4)-homo sapiens (Homo sapiens) (people).
MKMSFALTFRSAKGRWIANPSQPCSKASIGLFVPASPPLDPEKVKELQRFITLSKRLLVM
TGAGISTESGIPDYRSEKVGLYARTDRRPIQHGDFVRSAPIRQRYWARNFVGWPQFSSHQ
PNPAHWALSTWEKLGKLYWLVTQNVDALHTKAGSRRLTELHGCMDRVLCLDCGEQTPRGV
LQERFQVLNPTWSAEAHGLAPDGDVFLSEEQVRSFQVPTCVQCGGHLKPDVVFFGDTVNP
DKVDFVHKRVKEADSLLVVGSSLQVYSGYRFILTAWEKKLPIAILNIGPTRSDDLACLKL
NSRCGELLPLIDPC (SEQ ID NO：4)
The dependent deacetylase sirtuin 5 of＞sp|Q9NXA8|SIR5_ people NAD-(EC3.5.1.-) (SIR2-sample albumen 5)-homo sapiens (Homo sapiens) (people).
MRPLQIVPSRLISQLYCGLKPPASTRNQICLKMARPSSSMADFRKFFAKAKHIVIISGAG
VSAESGVPTFRGAGGYWRKWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRA
IAECETRLGKQGRRVVVITQNIDELHRKAGTKNLLEIHGSLFKTRCTSCGVVAENYKSPI
CPALSGKGAPEPGTQDASIPVEKLPRCEEAGCGGLLRPHVVWFGENLDPAILEEVDRELA
HCDLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTETTPATNRFRFHFQGPCGTTLPEA
LACHENETVS (SEQ ID NO：5)
The dependent deacetylase sirtuin 6 of＞sp|Q8N6T7|SIR6_ people NAD-(EC3.5.1.-) (SIR2-sample albumen 6)-homo sapiens (Homo sapiens) (people).
MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASG
TPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHV
RSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGE
LRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVN
LQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPK
EESPTRINGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS (SEQ IDNO：6)
The dependent deacetylase sirtuin 7 of＞sp|Q9NRC8|SIR7_ people NAD-(EC3.5.1.-) (SIR2-sample albumen 7)-homo sapiens (Homo sapiens) (people).
MAAGGLSRSERKAAERVRRLREEQQRERLRQVSRILRKAAAERSAEEGRLLAESADLVTE
LQGRSRRREGLKRRQEEVCDDPEELRGKVRELASAVRNAKYLVVYTGAGISTAASIPDYR
GPNGVWTLLQKGRSVSAADLSEAEPTLTHMSITRLHEQKLVQHVVSQNCDGLHLRSGLPR
TAISELHGNMYIEVCTSCVPNREYVRVFDVTERTALHRHQTGRTCHKCGTQLRDTIVHFG
ERGTLGQPLNWEAATEAASRADTILCLGSSLKVLKKYPRLWCMTKPPSRRPKLYIVNLQW
TPKDDWAALKLHGKCDDVMRLLMAELGLEIPAYSRWQDPIFSLATPLRAGEEGSHSRKSL
CRSREEAPPGDRGAPLSSAPILGGWFGRGCTKRTKRKKVT (SEQ ID NO：7)
For natural or artificial substrate as herein described, exemplary chemical compound as herein described can suppress at least 10,20,25,30,50,80 with the activity of SIRT1 or its functional domain, or 90%.For example, chemical compound can have less than 500,200,100, or the Ki of 50nM.
Chemical compound as herein described also can be regulated the complex that forms between sirtuin and the transcription factor, for example, increases or reduces complex and form, destroy, and/or stability.Exemplary sirtuin-TF complex comprises Sir2-PCAF, SIR2-MyoD, Sir2-PCAF-MyoD, Sir2-p53, Sir2-FoxO1, and Sir2-FoxO3.Chemical compound as herein described also can be regulated the expression of gene that regulated by Sir2, for example, and at the gene described in the table 1 of Fulco et al. (2003) Mol.Cell12:51-62.
External test
In some embodiment, can be in the interaction of external test and SIRT1, for example, in conjunction with.Reactant mixture can comprise SIRT1 cofactor for example NAD and/or NAD analog.
In other embodiments, reactant mixture can comprise the SIRT1 binding partners, for example, and transcription factor, for example, transcription factor beyond p53 or the p53, for example FoxO1 or FoxO3, and can SCREENED COMPOUND, for example, in external test, with the adjusting SIRT1 of evaluation experimental chemical compound and the interactional ability between the SIRT1 binding partners (for example, transcription factor).For example,, make and to determine of the combination of the component of labelling by the labelled compound that detects in the complex, can finish this class and measure to another component by making one of component coupling radiosiotope or enzyme labelling.Component can indicate directly or indirectly
125I,
35S,
14C, or
3H, and by direct count radioactive emission or scintillation counting technique, detection of radioactive isotope.Perhaps, component can be with for example, horseradish peroxidase, and alkali phosphatase, or luciferase carries out enzyme labelling, and, detect enzyme labelling by determining of the conversion of suitable substrate to product.Also can use competition assay to come the physics between evaluation experimental chemical compound and the target thing to interact.
Acellular mensuration comprises, and is being enough to allow under 2 kinds of component interactions and the bonded condition and in the time, and the preparation target protein (for example, SIRT1) and the reactant mixture of experimental compound, thereby forms the complex that can remove and/or detect.
Also can detect 2 kinds of interactions between the molecule, for example, use fluoremetry, wherein at least a molecule is by fluorescent labeling.An example of such mensuration comprise fluorescence energy transfer (FET, or FRET (fluorescence resonance energy transfer) is FRET) (referring to, for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103).Select the fluorogen labelling on first kind of ' donor ' molecule, make its emitted fluorescence energy to be absorbed by the fluorescent labeling on second kind of ' receptor ' molecule, the latter can fluoresce again owing to the energy that absorbs.Perhaps, ' donor ' protein molecular can utilize the natural fluoresence energy of trp residue simply.Selection can be launched the labelling of different wavelengths of light, makes that ' receptor ' molecular marker can separate with the mark zone of ' donor '.Because efficient that the energy between the labelling shifts and the distance dependent between the molecule can be assessed the spatial relationship between the molecule.Under in conjunction with situation about occurring between the molecule, the fluorescent emission of ' receptor ' molecular marker in the mensuration should be maximum.By the fluorescence detection method (for example, using exometer) of standard well known in the art, can measure the FET binding events easily.
Fluorimetric another example is fluorescence polarization (FP).For FP, only need a kind of component of labelling.The variation of the molecular size of the component by labelling detects binding interactions.Size variation can change the upset speed of this component in solution, and detection is the variation of FP.Referring to, for example, Nasir et al. (1999) Comb Chem HTS 2:177-190; Jameson etal. (1995) Methods Enzymol 246:283; Seethala et al.. (1998) AnalBiochem.255:257.Can in porous flat plate, monitor fluorescence polarization, for example, use Tecan Polarion
TMReader.Referring to, for example, Parker et al. (2000) Journal of Biomolecular Screening 5:77-88; And Shoeman, etal.. (1999) 38,16802-16809.
In another embodiment, use real-time biomolecular interaction analysis (BIA) (referring to, for example, Sjolander, S. and Urbaniczky, C. (1991) Anal.Chem.63:2338-2345 and Szabo et al. (1995) Curr.Opin.Struct.Biol.5:699-705), can measure the ability of SIRT1 protein binding target molecule." surface plasma resonance " or " BIA " specific interaction of detection of biological in real time need not any interactant of labelling (for example, BIAcore).The mass change at mating surface place (indicating binding events) can cause the variation (optical phenomena of surface plasma resonance (SPR)) of the optical index of near surface, produces detectable signal, and it can be as the indication of the real time reaction between the biomolecule.
In one embodiment, SIRT1 is anchored on the solid phase.Can when finishing, reaction (for example, association reaction) detect the SIRT1/ experimental compound complex that is anchored on the solid phase.For example, SIRT1 can be anchored on the surface of solids, and can be with detectable labelling discussed in this article, labelling experiment chemical compound (it is not anchored) directly or indirectly.
May need immobilization SIRT1 or anti--SIRT1 antibody, complex form separate promoting, and adapt to the automatization that measures with one of albumen complex form not or both.Can have and not have in the presence of the candidate compound being fit to hold in any container of reactant, finishing experimental compound to the proteic combination of SIRT1, or the interaction of SIRT1 albumen and second kind of component.The example of such container comprises microtitration plate, test tube, and microcentrifugal tube.In one embodiment, can provide fusion rotein, it adds one of permission albumen or both are to the bonded domain of substrate.For example, glutathione-S-transferase/SIRT1 fusion rotein or glutathione-S-transferase/target fusion rotein can be adsorbed onto glutathione agarose pearl (SigmaChemical, St.Louis, MO) or on the deutero-microtitration plate of glutathion, it merges with the target protein or the SIRT1 albumen of experimental compound or experimental compound and not absorption then, and under the condition that helps complex to form (for example, under the physiological condition of salt and pH) incubation mixture.Behind the incubation, washing pearl or micro titer plate well, to remove any unconjugated component, complex is directly or indirectly measured in substrate immobilization under the situation of pearl, for example, as mentioned above.Perhaps, complex can dissociate from substrate, and uses standard techniques, measures SIRT1 combination or active level.
SIRT1 albumen or target molecule immobilization are comprised to other technology on the substrate, use puting together of biotin and streptavidin.(for example use technology known in the art, the biotinylation test kit, Pierce Chemicals, Rockford, IL), can be from biotinylated SIRT1 albumen of biotin-NHS (N-hydroxyl-butanimide) preparation or target molecule, and be immobilized in the hole of 96 hole flat boards (Pierce Chemical) of streptavidin-Bao quilt.
In order to measure, not immobilized component is added to the surface of the bag quilt of the component that contains grappling.After reaction is finished, under the condition on the surface of solids, remove unreacted component (for example, by washing) in being maintained fixed of alloy that can make formation.Can detect the complex that is anchored on the surface of solids in many ways.Under the situation of the previous not immobilized component of labelling in advance, detect and be immobilized in lip-deep labelling, indicating the formation of complex.Under the situation that does not have the previous not immobilized component of labelling in advance, can use indirect labelling to detect and be anchored on lip-deep complex, for example, use is to the antibody (this antibody can for example directly or indirectly be used again, and anti--Ig antibody of labelling comes labelling) of the specific labelling of immobilized component.
In one embodiment, the use meeting is reacted, still can not hindered SIRT1 albumen to the bonded antibody of its target molecule with SIRT1 albumen or target molecule, carries out this mensuration.Such antibody can be derived in the hole of flat board, and put together, unconjugated target thing or SIRT1 albumen are captured in the hole by antibody.Except top about described those methods of the immobilized complex of GST-, the method that detects such complex also comprises, use can be carried out the immune detection of complex with the antibody of SIRT1 albumen or target molecule reaction, and it is fixed to depend on the enzyme translocation that detects the enzymatic activity relevant with SIRT1 albumen or target molecule.
Perhaps, can in liquid phase, carry out acellular mensuration.In such mensuration, by in many standard techniques any, product to be separated with unreacted component, described technology includes but not limited to: differential centrifugation (referring to, for example, Rivas, G., and Minton, A.P., (1993) Trends Biochem Sci 18:284-7); Chromatograph (gel filtration chromatography, ion exchange chromatography); Electrophoresis (referring to, for example, Ausubel, F.et al., eds.CurrentProtocols in Molecular Biology 1999, J.Wiley:New York.); And immunoprecipitation (referring to, for example, Ausubel, F.et al., eds. (1999) CurrentProtocols in Molecular Biology, J.Wiley:New York).Such resin and chromatographic technique be known to those skilled in the art (referring to, for example, Heegaard, N.H., (1998) J Mol Recognit 11:141-8; Hage, D.S., and Tweed, S.A. (1997) J Chromatogr B Biomed Sci Appl.699:499-525).In addition, as described herein, also can use fluorescence energy transfer to detect combination easily, need not to be further purified complex from solution.
In a preferred embodiment, mensuration comprises, make SIRT1 albumen or its biologically-active moiety contact meeting known compound in conjunction with SIRT1, form and measure mixture, make and measure mixture contact experimental compound, and the ability of definite experimental compound and SIRT1 protein-interacting, determine wherein that the experimental compound and the ability of SIRT1 protein-interacting comprise and determine that experimental compound compares with known compound preferentially in conjunction with SIRT1 or its biologically-active moiety or the active ability of regulating target molecule.
Exemplary assay method comprises the SirT1 enzymatic determination of 1536 hole forms, and it is based on " Fluor-de-Lys " measuring principle of the commerce of Biomol, and it is (www.biomol.com/store/Product_data_PDFs/ak500.pdf) that produces fluorescence.In this was measured, with the deacetylation of the e-amido functional group of lysyl-residue and " colour developing " step coupling mutually of generation fluorescence, the latter depended on untight e-amido functional group, and produces fluorescigenic amino methyl coumarin.Can on the macroscopy reader of commerce, read fluorescence.
Other mensuration
Use one of model system of following disease or disease, or other known models of disease described herein or disease, also can estimate the library of chemical compound as herein described or chemical compound.
Be used for the evaluation experimental chemical compound model of amyotrophic effect comprised, for example, 1) the rat medial head of gastrocnemius muscle mass loss that causes of denervation, for example, by cutting off right sciatic nerve at big midleg; 2) the rat medial head of gastrocnemius muscle mass loss that causes of immobilization, for example, by with the crooked fixing right ankle joint of 90 degree; 3) hind leg hangs the rat medial head of gastrocnemius muscle mass loss that causes; (referring to, for example, U.S.2003-0129686); 4) handle the skeletal muscle atrophy cause (T.C.Vary, Shock 7,1-16 (1997) for R.N.Cooney, S.R.Kimball) with cachectic cytokine interleukin element-1 (IL-1); With 5) handle the skeletal muscle atrophy (A.L.Goldberg, J Biol Chem 244,3223-9 (1969)) cause with the glucocorticoid dexamethasone.Model 1,2 comprises by external load to the different degree that changes neural activity and/or muscle experience and induces the generation amyotrophy with 3.Model 4 and 5 is induced atrophy and is not directly influenced these parameters MS (experimental autoimmune encephalomyelitis (EAE)), for example, as Goverman et al., Cell.72:551-60 (1993) is described, with as Brok etal., Immunol.Rev., the primate model summarized of 183:173-85 (2001).
The exemplary animal model of AMD (degeneration of macula that the age is relevant) comprises: simulate the mouse model Bora et al. of the induced with laser of exudative (wetting) degeneration of macula, Proc.Natl.Acad.Sci.USA., 100:2679-84 (2003); Transgenic mice, the cathepsin D that it expresses mutant form causes the feature relevant with the AMD of " provincialism atrophy " form (Rakoczyet al., Am.J.Pathol., 161:1515-24 (2002)); And transgenic mice, it is overexpression VEGF in retinal pigment epithelium, causes CNV.Schwesinger et al.，Am.J.Pathol.158：1161-72(2001)。
Parkinsonian exemplary animal model comprises, by with dopaminergic neurotoxin 1-methyl-4 phenyl 1,2,3, the parkinsonian primate of 6-tetrahydropyridine (MPTP) processing generation (referring to, for example, U. S. application 20030055231 and Wichmann et al., Ann.N.Y.Acad.Sci., 991:199-213 (2003); The rat of 6-hydroxy dopamine-damage (for example, Lab.Anim.Sci., 49:363-71 (1999)); With genetically modified invertebrates model (for example, Lakso et al., J.Neurochem., 86:165-72 (2003) and Link, Mech.Ageing Dev., 122:1639-49 (2001)).
The example molecule model of type ii diabetes comprises: the transgenic mice with deficiency Nkx-2.2 or Nkx-6.1; (US 6,127,598); Fat fa/fa (ZDF) rat (US 6569832) of Zucker diabetes; And macaque, it spontaneously forms obesity, and often makes progress into subsequently tangible type ii diabetes (Hotta et al., Diabetes, 50:1126-33 (2001); And transgenic mice, the IGF-I receptor (KR-IGF-IR) that it has the dominance negative has type ii diabetes-sample insulin resistant.
Neuropathic exemplary animal and cell model comprise: sensation-motor neuron of inductive mice of vincristine (U. S. application 5420112) or rabbit (Ogawa et al., Neurotoxicology, 21:501-11 (2000)); Be used to study streptozocin (STZ)-diabetes rat (Schmidt et al., Am.J.Pathol., 163:21-8 (2003)) of autonomic neuropathy; With carrying out property motor neuron (pmn) mice (Martin et al., Genomics, 75:9-16 (2001)).
Structure-activity relation and based on structure Design.Also may utilization structure-activity relationship (SAR) and generate the interact chemical compound of (for example, antagonism or exciting sirtuin) with sirtuin based on the structure Design principle.SAR can provide about the active information of related compound at least one related assays.Set up the architectural feature of target compound and the association between the activity.For example, may be by estimating the SAR of the chemical compound family relevant, to differentiate the required one or more architectural features of agonist activity with chemical compound described herein.Then, can chemically produce the library of compounds that changes these features.In another embodiment, production forecast can interactional unification compound, and external or body is estimated interiorly.
Can comprise based on structure Design, determine the functional domain of sirtuin and the physical interaction structural model of chemical compound.Structural model can indicate how to transform chemical compound, for example, and to improve interaction or to reduce disadvantageous interaction.Can differentiate the interaction of chemical compound and sirtuin, for example, by the parsing of crystal structure, NMR, or computer based modeling, for example, docking calculation.Referring to, for example, Ewing et al.J Comput AidedMol Des.2001 May; 15 (5): 411-28.
SAR and based on the structure Design scheme, and other method may be used to differentiate pharmacophore.The three-dimensional (3D) that pharmacophore is defined as the uniqueness of chemical group is arranged.The selection of such group has the biological activity of being beneficial to.Because the molecule of pharmaceutical active must interact so that effectively with intravital one or more molecular structures of object, and the objective function character of molecule is derived from these interactions, every kind of reactive compound must contain the unique arrangement of chemical group, and it can make this interaction take place.The chemical group at so-called description center can be expressed as: (a) atom or atom group; (b) puppet-atom, the barycenter at for example Huan center, or molecule; (c) vector, atom pair for example, lone electron pair direction, or planar vertical line.In case set forth, pharmacophore can be used to search for the data base of chemical compound, for example, has those of the structure compatible with pharmacophore.Referring to, for example, U.S.6,343,257; Y.C.Martin, 3D DatabaseSearching in Drug Design, J.Med.Chem.35,2145 (1992); With A.C.Good and J.S.Mason, Three Dimensional Database Searches, Reviews in Comp.Chem.7,67 (1996).Database search queries is not only based on chemical property information, also based on geological information accurately.
The computer based scheme can use database search to find matching template; Y.C.Martin, Database searching in drug design, J.MedicinalChemistry, vol.35, pp 2145-54 (1992), it is incorporated by reference here.The 2-D of existing search chemical compound and 3-D data base's method is suitable for.(Pearl River N.Y.) is molecular shape-search to the Lederle of AmericanCyanamid, the pioneer of trend-vectorial aspect of 3D search and data base.Commercial vendor and other seminar also provide search capability (MACSS-3D, Molecular Design Ltd. (San Leandro, Calif.); CAVEAT, Lauri, G.et al., University of California (Berkeley, Calif.); CHEM-X, Chemical Design, Inc. (Mahwah, N.J.)).The software that is used for these search, can be used to analyze by they significant chemistry and the data base of the potential drug chemical compound of geometry index (for example, Standard Drugs File (DerwentPublications Ltd., London, England), Bielstein data base (Bielstein Information, Frankfurt, Germany or Chicago) and Chemical Registry data base (CAS, Columbus, Ohio)).
In case identify the chemical compound that is complementary with pharmacophore, can be external, in the body or the computer ground activity of testing it, for example, to the combination of sirtuin or its domain.
In one embodiment, can modify chemical compound as agonist or candidate's agonist, for example, at Nature.2003 Sep 11; 425 (6954): the chemical compound described in the 191-196, to differentiate antagonist, for example, use method as herein described.For example, can prepare the library of related compound, and can be in mensuration as herein described, the screening library.
The pharmaceutically acceptable salt of The compounds of this invention comprises those that are derived from pharmaceutically acceptable inorganic and organic bronsted lowry acids and bases bronsted lowry.The example of suitable hydrochlorate comprises acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulphate, butyrate, citrate, camphorate, camsilate, digluconate, lauryl sulfate, esilate, formates, fumarate, the glucose enanthate, oxyacetate, Hemisulphate, enanthate, caproate, hydrochlorate, hydrobromate, hydriodate, the 2-isethionate, lactate, maleate, malonate, mesylate, the 2-naphthalene sulfonate, nicotinate, nitrate, palmitate, pectinate, persulfate, 3-phenylpropionic acid salt, phosphate, picrate, pivalate, propionate, Salicylate, succinate, sulfate, tartrate, rhodanate, toluene fulfonate and hendecane hydrochlorate.Other acid, for example oxalic acid although they itself are not pharmaceutically acceptable, also can be used to prepare the salt as the intermediate that obtains The compounds of this invention and their pharmaceutically-acceptable acid addition.The salt that is derived from appropriate base comprises, alkali metal (for example, sodium) salt, alkaline-earth metal (for example, magnesium) salt, ammonium salt and N-(alkyl)
4 +Salt.The present invention has also expected any alkaline nitrogen-containing group quaternized of chemical compound disclosed herein.By so quaternized, can obtain water or oil-soluble or dispersible products.The salt form of the chemical compound of the arbitrary formula of this paper can be the amino acid salts (for example L-arginine ,-lysine ,-histidine salt) of carboxyl.
The chemical compound of formula described herein can be for example by under injection, intravenous, intra-arterial, the corium, intraperitoneal, intramuscular or subcutaneous administration; Or oral, suck, per nasal, saturating mucosa, part, with the form of ophthalmic preparation or by inhalation, dosage is about 0.5 to about 100mg/kg body weight, perhaps dosage is 1mg to 1000mg/ agent, per 4 to 120 hours, or according to the requirement of specific compound.Methods described herein have been expected chemical compound or the compound compositions of using effective dose, to obtain required or indicated effect.Typically, pharmaceutical composition of the present invention every day about 1 is to about 6 administrations.Perhaps, as continuous infusion.Such administration can be used as chronic or acute treatment.Can with the amount of the active component of the single dosage form of carrier mass combinations produce, change according to host who is treated and specific mode of administration.Typical formulation contains has an appointment 5% to about 95% reactive compound (w/w).Perhaps, such preparation contains and has an appointment 20% to about 80% reactive compound.
May need than top those the lower or higher dosage of enumerating.The concrete dosage and the therapeutic scheme of any particular patient depend on multiple factor, comprise that the order of severity of activity, age, body weight, general health state, sex, diet, administration time, discharge rate, drug regimen, disease, situation or symptom of used particular compound and the course of disease, patient are to the disposal of disease, situation or symptom and treatment doctor's judgement.
Behind patient's the condition improved, if desired, can use chemical compound of the present invention, compositions or the combination of maintenance dose.Subsequently, different with symptom when symptom has been relieved to the level of hope, can reduce the dosage of administration or frequency or both, reach the level that keeps situation about improving.Yet, any repeatedly time the at disease symptoms, patient may be on long-term basis intermittent therapy.
Compositions described herein comprises the chemical compound of formula described herein and other therapeutic agent (if existence), and its amount can realize the adjusting to disease or disease symptoms effectively, comprise as herein described those.
Term " pharmaceutically acceptable carrier or adjuvant " refers to be administered to The compounds of this invention patient's carrier or adjuvant, and when with the dosed administration of the chemical compound that is enough to the delivery treatments amount, can not destroy its pharmacological activity and nontoxic.
Can be used for the pharmaceutically acceptable carrier in the pharmaceutical composition of the present invention, adjuvant and excipient include, but not limited to ion-exchanger, aluminium oxide, aluminium stearate, lecithin, the drug delivery system of self emulsifying (SEDDS) is used for surfactant such as tween or other similar polymer delivery matrices of pharmaceutical dosage form, serum albumin such as human serum albumin as d-alpha-tocopherol cetomacrogol 1000 succinate, buffer substance such as phosphate, glycine, sorbic acid, potassium sorbate, the partial glycerol ester admixture of saturated vegetable fatty acid, water, salt or electrolyte are as protamine sulfate, sodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone is based on cellulosic material, Polyethylene Glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, Polyethylene Glycol and lanoline.Cyclodextrin such as α-, β-and gamma-cyclodextrin, or the derivant of chemical modification such as hydroxy alkyl cyclodextrin comprise 2-and 3-hydroxypropyl-beta-schardinger dextrin-, or other soluble derivant, also can be advantageously used in sending of the chemical compound that improves formula described herein.
Pharmaceutical composition of the present invention can be oral, parenteral, by suck spraying, part, rectum, per nasal, suck, transvaginal or pass through the storage administration of implantation, preferably by oral administration or pass through drug administration by injection.Pharmaceutical composition of the present invention can contain nontoxic pharmaceutically acceptable carrier, adjuvant and the excipient of any routine.In some cases, available pharmaceutically acceptable acid, alkali or buffer are regulated the pH of preparation, thus improve the stability of the chemical compound of preparing or its delivery form.That term used herein " parenteral " comprises is subcutaneous, in the Intradermal, intravenous, intramuscular, intraarticular, intra-arterial, synovial membrane, in the breastbone, in the sheath, in sick the damage and intracranial injection or infusion techniques.
Pharmaceutical composition can be the form of sterile injectable preparation, for example, and sterile injectable aqueous or oleagenous suspension.This suspension can be according to technology known in the art, uses suitable dispersant or wetting agent (as, for example, Tween 80) and suspending agent preparation.Sterile injectable preparation can also be at nontoxic parenteral acceptable diluent or sterile injectable solution or the suspension in the solvent, for example, and the solution in 1,3 butylene glycol.Spendable acceptable excipient and solvent have mannitol, water, Ringer's mixture and isotonic sodium chlorrde solution.In addition, aseptic expressed oi can be used as solvent or suspension medium routinely.For this purpose, the expressed oi of any gentleness be can use, synthetic monoglyceride or diglyceride comprised.Fatty acid as oleic acid and glyceride ester derivatives thereof, is applicable to the preparation ejection preparation, as natural pharmaceutically acceptable oil, as olive oil or Oleum Ricini, particularly with the ethylating form of their polyoxy.These oil solutions or suspension also can comprise long-chain alcohol diluent or dispersant or carboxymethyl cellulose or be usually used in similar dispersant in the preparation of pharmaceutically acceptable dosage form (as emulsion and/or suspension).Be generally used for producing other surfactant such as tween or span and/or other similar emulsifying agent or bioavailability reinforcing agent commonly used of pharmaceutically acceptable solid, liquid or other dosage form, also can be used for the purpose of preparing.
Pharmaceutical composition of the present invention can any oral acceptable forms oral administration, and described dosage form includes, but not limited to capsule, tablet, emulsion and aqueous suspension, dispersion and solution.With regard to the tablet that orally uses, normally used carrier comprises lactose and corn starch.Usually also add lubricant, as magnesium stearate.For with the Capsule form oral administration, useful diluent comprises lactose and dried corn starch., can or be dissolved in the oil phase the active component suspendible when using aqueous suspension and/or emulsion when oral, it can mix with emulsifying agent and/or suspensoid.If desired, can add some sweeting agent and/or flavoring agent and/or coloring agent.
Pharmaceutical composition of the present invention can also be used with the suppository form of rectally.By with The compounds of this invention and suitable non-stimulated mixed with excipients, can prepare these compositionss, described excipient is a solid in room temperature, but is liquid at rectal temperature, therefore can melt in rectum, thereby discharge active component.This class material includes, but not limited to cocoa butter, Cera Flava and Polyethylene Glycol.
When desired therapeutic comprised zone that topical application can be easily approaching or organ, the topical of pharmaceutical composition of the present invention was useful.With regard to being applied topically to skin, pharmaceutical composition should be mixed with the suitable ointment that contains suspendible or be dissolved in the active component in the carrier.The carrier that is used for local application chemical compound of the present invention includes but not limited to, mineral oil, liquid petroleum, white oil, propylene glycol, polyoxyethylene polyoxypropylene chemical compound, emulsifing wax and water.Perhaps, pharmaceutical composition can be mixed with suitable lotion or emulsifiable paste, it contains useful suitable emulsifying agent suspendible or is dissolved in reactive compound in the carrier.Suitable carriers includes but not limited to, mineral oil, Arlacel-60, polysorbate60, cetyl esters wax, spermol (cetearyl alcohol), 2-octyldodecanol, benzyl alcohol and water.By rectal suppository or suitable enema, also pharmaceutical composition of the present invention can be applied to lower intestinal tract partly.The topical transdermal patch is also contained among the present invention.
Pharmaceutical composition of the present invention can pass through nose aerosol or inhalation.Such compositions can prepare according to the technology that pharmaceutical field is known, and can in saline, be prepared into solution, wherein use absorption enhancer, fluorocarbon and/or other solubilizing agent known in the art or the dispersant of benzylalcohol or other suitable antiseptic, raising bioavailability.
Use implantable device, can use the compositions of chemical compound with this paper formula and other reagent (for example, therapeutic agent).Implantable device is known in the art with relevant technology, when needs are sent chemical compound described herein or compositions continuously or regularly with discharging, can be used as delivery system.In addition, the implantable device delivery system can be used for specific site (for example, the site of limitation, organ) the Negrin et al. that target compound or compositions are sent, Biomaterials, 22 (6): 563 (2001).Also can use the timing release tech that comprises substituting delivering method in the present invention.For example, (for example, polymer, liposome) time release formulation also can be used to send chemical compound as herein described and compositions based on polymer technology, lasting release tech and wrapper technology.
Be used to send the patch of the active chemotherapy combination of this paper, also within the scope of the present invention.Patch comprises the chemical compound of material layer (for example, polymer, cloth, gauze, binder) and this paper formula as herein described.One side of material layer can have the protective layer that adheres to above it, to stop passing through of chemical compound or compositions.Patch can comprise binding agent in addition, so that patch is remained on the object.Binding agent is a kind of compositions, comprises those of natural or synthetic source, and when the skin of contact object, it can temporarily adhere on the skin.It can be a waterproof.Binding agent can be positioned on the patch, so that it keeps in touch the long time period of subject's skin.Binding agent can be made of viscosity or adhesion strength, so can remaining on device, it stands accidental position contacting, but, (for example depend on positive effect, draw back, peel off, or other deliberate moving), binding agent is submitted to and is put on originally on one's body external pressure of device or binding agent, and allows to destroy and adhere to contact.Binding agent can be pressure-sensitive, that is to say, allows by pressure (for example, pushing away friction) is put on binding agent or the device binding agent (with the device that will adhere on the skin) to be positioned on the skin.
When the present composition comprises the combination of the chemical compound of formula described herein and one or more additional treatment agent or preventive, chemical compound and additive reagent all should be with common about 1-100% of application dosage in the monotherapy scheme, and the more preferably from about dosage level existence of 5-95%.Additive reagent can be used as the part of multiple dosing scheme, with The compounds of this invention administration dividually.Perhaps, those reagent can be the parts of single dosage form, with the The compounds of this invention mixed together in single compositions.
Neoplastic disease
Chemical compound of the present invention can be used for the treatment of cancer.As used herein, term " cancer ", " hyper-proliferative ", " virulent " and " tumor " are used interchangeably, refer to be characterised in that fast breeding or excrescent those cells, abnormality or situation.This term comprises cell, tissue or the organ of all types of cancerous growths or carcinogenic process, metastatic tissue or vicious transformation, no matter histopathology type or invasion and attack stage." the pathologic hyper-proliferative " cell occurs in the morbid state that is characterised in that malignant growth.
The general medicine implication of term " neoplasia " refers to " new cell growth ", and it produces the reaction of normal Growth Control as forfeiture, for example refers to the neoplastic cell growth." hypertrophy " refers to experience the cell of high-speed unusually growth.But as used herein, term " neoplasia " and " hypertrophy " can be used interchangeably, as they context disclosed, briefly refer to experience the cell of the unusual cell speed of growth.Neoplasia and hypertrophy comprise " tumor ", and it can be benign, premalignant or virulent.
The example of Cancerous disease includes, but not limited to solid tumor, soft tissue neoplasms and shifts sexually transmitted disease (STD) and decrease.The example of solid tumor comprises malignant tumor, the for example sarcoma of various tracts, adenocarcinoma and cancer, as (for example influence lung, mammary gland, lymph, gastrointestinal, colon) and urogenital tract (for example, kidney, urothelium cell), those and adenocarcinoma of pharynx, prostate, ovary, it comprises malignant tumor, as the non-small cell carcinoma of most of colon cancer, rectal cancer, renal cell carcinoma, hepatocarcinoma, lung, carcinoma of small intestine etc.Also can use compounds for treating as herein described or prevent aforementioned cancer metastasis sexually transmitted disease (STD) to become.
Subject methods can be used for the treatment of the malignant tumor of various tracts, as influence those of lung, mammary gland, lymph, gastrointestinal (for example colon) and urogenital tract, prostate, ovary, pharynx, and adenocarcinoma, it comprises non-small cell carcinoma, carcinoma of small intestine and the esophageal carcinoma of malignant tumor such as most of colon cancer, renal cell carcinoma, carcinoma of prostate and/or tumor of testis, lung.Medicable exemplary physical tumor comprises: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell cancer, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, non--small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.
Term " cancer " is that those skilled in the art generally acknowledge, and refers to the malignant tumor of epithelium or endocrine tissue, comprises respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, carcinoma of testis, breast carcinoma, carcinoma of prostate, hormonal system cancer and melanoma.Exemplary cancer comprises those that the tissue by cervix uteri, lung, prostate, mammary gland, head and neck, colon and ovary forms.This term also comprises carcinosarcoma, and for example, it comprises the malignant tumor that is made of carcinous and sarcoma sex organization." adenocarcinoma " refer to derive from the cancer of glandular tissue or wherein tumor cell form the cancer of discernible glandular structure.
Term " sarcoma " is that those skilled in the art generally acknowledge, and refers to the deutero-malignant tumor of mesenchyme.
Subject methods also can be used to suppress the hypertrophy/neoplastic cell in hemopoietic source (for example, coming from medullary system, lymphatic system or erythron) or the propagation of its precursor.For example, the present invention predicts various myeloid treatment of diseases, include but not limited to, acute promyelocyte leukemia (APML), acute myelocytic leukemia (AML) and chronic myelocytic leukemia (CML) (summary is seen Vaickus, L. (1991) Crit Rev.in Oncol./Hemotol.11:267-97).Can include but not limited to acute lymphoblastic leukemia (ALL) by the lymph sample malignant tumor of subject methods treatment, it comprises that B-is that ALL and T-are ALL; Chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Walden Si Telunshi macroglobulinemia (WM).Other form of malignant lymphoma comprises, but be not limited to non_hodgkin lymphoma and modification thereof, peripheral t-cell lymphoma, Adult T-cell leukemia/lymphoma (ATL), skin T-cell lymphoma (CTCL), large granular lymphocyte leukemia (LGF) and Hokdkin disease.
Alzheimer
Alzheimer (AD) is a kind of neurodegenerative disease of complexity, and it can cause neuronic irreversible forfeiture, and is an example that has at least in part the neurodegenerative disease of the symptom that is caused by protein aggregation.Chemical compound as herein described can be used to improve at least a symptom of the object with AD.
The clinical marker of Alzheimer comprises memory, judgement, damaged to the carrying out property of the orientation of physical environment and language.Neuro pathology's sign of AD comprises zone-specific neuron forfeiture, amyloid plaque and neurofibrillary tangles.Amyloid plaque is the extracellular speckle that contains amyloid beta peptide (be also referred to as A β, or A β 42), and described peptide is the cleaved products of amyloid-beta precursor protein (being also referred to as APP).Aggregation in the insoluble cell that neurofibrillary tangles is made up of the fibril of the microtubule-associated protein tau of hyperphosphorylation singularly.Amyloid plaque and neurofibrillary tangles have secondary incident (Clark and Karlawish, Ann.Intern.Med.138 (5): the 400-410 (2003) that helps cause by apoptosis the neuron forfeiture.For example, the dependent apoptosis of neuronic caspase-2-(the Troy et al.J.Neurosci.20 (4): 1386-1392) of amyloid-beta meeting inducing culture.The deposition of speckle may trigger contiguous neuronic apoptosis in a similar fashion in the body.
The sudden change of the gene of coding APP, presenilin-1 and presenilin-2 has participated in early sending out AD (Lendon et al.JAMA 227:825 (1997)).Verified these proteic sudden changes can promote the proteolysis processing of APP by approach in the cell that produces A β.The unusual adjusting of A β processing may be important with subsequently the neuronal damage relevant with speckle to forming amyloid plaque.
Can use multiple standards to come AD in the evaluation object, comprise standard heredity, biochemical, physiological and cognitive.The symptom of AD and diagnosis are that the medical science staff is known.The symptom and the labelling of the certain example of AD are described below.Can use information about these indications and known other indication relevant as " parameter that AD-is relevant " with AD.The parameter that AD-is relevant can comprise qualitatively or quantitative information.The numerical value that example is one or more aspects of quantitative information, for example, proteic concentration or digital subtraction angiography figure.Qualitative information can comprise evaluation, for example, and doctor's evaluation or binary ("Yes"/"No") etc.The parameter that AD-is relevant comprises that denoted object is not made a definite diagnosis AD or do not had the information of the specific indication of AD, for example, is not the typical recognition tests result of AD, or with the irrelevant hereditary APOE polymorphism of AD.
Gradual cognitive impairment is the sign of AD.This is damaged can to show as memory, judge, make decision, to the orientation of physical environment and the decline of language (Nussbaum and Ellis, New Eng.J.Med.348 (14): 1356-1364 (2003)).The eliminating of the dementia of other form can auxiliary diagnosis AD.
Neuronal death can cause AD patient's carrying out property brain atrophy.Imaging technique (for example, nuclear magnetic resonance, or computer x line body section radiography) can be used for detecting brain AD-relevant damage and/or brain atrophy.
It is unusual that AD patient may show the pathological biochemistry that is derived from disease.For example, the rising of the Protein tau level in AD patient's cerebrospinal fluid (Andreasen, N.et al.Arch Neurol.58:349-350 (2001)).The level of the amyloid beta 42 among AD patient's the CSF (A β 42) peptide can reduce (Galasko, D., et al.Arch.Neurol.55:937-945 (1998)).A β 42 levels in AD patient's the blood plasma can raise (Ertekein-Taner, N., et al.Science 290:2303-2304 (2000)).Detection comprises from the biochemical unusual technology in the sample of object, cell method, immunological method and other biological method known in the art.General guide referring to, for example, the technology of putting down in writing in the following document: Sambrook ﹠amp; Russell, Molecular Cloning:A LaboratoryManual, 3rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology (GreenePublishing Associates and Wiley Interscience, N.Y. (1989), (Harlow, E. and Lane, D. (1988) Antibodies:ALaboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY) and their renewal version.
For example, can use antibody, other immunoglobulin and other specific binding partner to come the detection of biological molecule, for example, with AD proteins associated or other antigen.For example, can use one or more specific antibody to survey sample.Multiple form is feasible, for example, ELISA, based on the mensuration of fluorescence, Western blot, and protein arrays.Produce the method for polypeptide array, put down in writing in the literature, for example, De Wildt et al. (2000) .Nature Biotech.18,989-994; Lueking et al. (1999) .Anal.Biochem.270,103-111; Ge, H. (2000) .Nucleic Acids Res.28, e3, I-VII; MacBeath, G., and Schreiber, S.L. (2000) .Science 289,1760-1763; And WO99/51773A1.Use mass spectrum, chromatograph, electrophoresis, enzyme interacting, or use the probe that detects post translational modification (for example, phosphorylation, ubiquitination, glycosylation methylates, or acetylation), also can analyzing proteins.
Can detect from the expression of nucleic acid in the cell of object, for example, by operation, extract, after death or other sampling (for example, blood CSF) obtains.Can estimate one or more expression of gene, for example, by the technology based on hybridization, for example, RNA analyzes, RT-PCR, SAGE, and nucleic acid array.Nucleic acid array can be used for describing a plurality of mRNA kinds in the sample.Nucleic acid array can produce by several different methods, for example, by photoetching method (referring to, for example, U.S. Patent number 5,143,854; 5,510,270; With 5,527,681), mechanical means (for example, as U.S. Patent number 5,384,261 described oriented flow methods) is based on the method (for example, as U.S. Patent number 5,288,514 is described) of pin with based on the technology of pearl (for example, as PCTUS/93/04145 as described in).
In several ways, comprise that the enzyme translocation is fixed, the precursor of usage flag, and nuclear magnetic resonance, NMR (NMR) can detect the metabolite relevant with AD.For example, NMR can be used for determining the relative concentration of sample based on phosphatic chemical compound, for example, and the creatine level.Also can detect other metabolizing parameters, redox state for example, ion concentration (for example, Ca
2+) (for example, using the dyestuff of ion-sensitive), and membrane potential (for example, using the patch clamp technology).
Information about the relevant labelling of AD-can write down and/or be stored in the computer-readable format.Typically, with information with interrelate and also be associated (directly or indirectly) with information about the homogeneity of the one or more nucleotide in the gene of coding sirtuin in object about the reference of object.
In one embodiment, use non--people's animal model (for example, mouse model) of AD, for example, the therapeutic scheme of evaluation Example chemical compound as described herein or chemical compound.For example, US 6,509, and 515 have described a kind of such animal pattern, and it can be used for the learning and memory test natively.This animal with certain horizontal expression amyloid precursor protein (APP) sequence, makes animal carrying out property neurological disorder in short time period after birth in cerebral tissue, usually in back 1 year of birth, preferably after birth in 2 to 6 months.The APP protein sequence is imported animal, or be in brephic animal predecessor, preferably in the oocyte stage unicellular or fertilization, usually earlier than about 8-cell stage.Zygote or embryo grow to mature in pseudo-conceived raising is female then.The amyloid precursor protein gene is imported animal embryo, so that chromosomal integration becomes a kind of state, it causes the super endogenous expression of amyloid precursor protein and carrying out property neurological disorder to take place at brain cortex marginal area, and this brain zone is subjected to appreciable impact in carrying out property neurological disorder state (for example AD).Neurogliosis in the affected transgenic animal and clinical manifestation analog neuron are learned disease.The progress aspect of neurological disorder is characterised in that, the hypertrophy neurogliosis of the 2-deoxyglucose absorption/utilization of the detection of minimizing and/or motor behavior and minimizing and brain cortex marginal area.In addition, observed variation is similar to observed variation in some aged animal.Other animal model also is documented in US5,387,742; 5,877,399; 6,358,752; With 6,187,992.
Parkinson
Parkinson comprises the neural degeneration of the dopaminergic neuron in the black substance, and it causes the degeneration of the nigrostriatum dopamine system of adjustment movement function.This pathology cause again dyskinesia (referring to, for example, Lotharius et al., Nat.Rev.Neurosci., 3:932-42 (2002)).Exemplary motor symptoms comprises: motion can not, stoop posture is walked with difficulty, posture instability, stiff live, muscle rigidity and trembling.Exemplary non-motor symptoms comprises: depression, lack power, and passive, dementia and gastrointestinal dysfunction (referring to, for example, Fahn, Ann.N.Y.Acad.Sci., 991:1-14 (2003) and Pfeiffer, LancetNeurol., 2:107-16 (2003)).In 65 to 69 years old the people of 0.5-1% and 1-3%80 year and older people, observe parkinson (referring to, for example, Nussbaum etal., N.Engl.J.Med., 348:1356-64 (2003)).
Chemical compound as herein described can be used to improve at least a symptom with parkinsonian object.
Parkinsonian molecular marker comprises the minimizing (referring to, for example, U. S. application 20020172664) of aromatics L-amino acid decarboxylases (AADC); The minimizing of DOPAMINE CONTENT IN RABBIT in the nigrostriatum neuron (referring to, for example, Fahn, Ann.N.Y.Acad.Sci., 991:1-14 (2003) and Lotharius et al., Nat.Rev.Neurosci., 3:932-42 (2002)).In some family's case, PD is relevant (for example with the sudden change in coding for alpha-synuclein and the proteic individual gene of parkin (a kind of E3 ubiquitin ligase), Riess et al., J.Neurol.250 Suppl 1:I3-10 (2003) and Nussbaum et al., N.Engl.J.Med., 348:1356-64 (2003)).Missense mutation also relevant (for example, Nussbaum et al., N.Engl.J.Med., 348:1356-64 (2003)) in the terminal ubiquitin hydrolase gene of neuron-specific C-with parkinson.
Can parkinsonian non--estimate the library of chemical compound described herein or chemical compound in people's animal model.Parkinsonian exemplary animal model comprises with dopaminergic neurotoxin 1-methyl-4 phenyl 1,2,3, the parkinsonian primate of 6-tetrahydropyridine (MPTP) processing generation (referring to, for example, U. S. application 20030055231 and Wichmann et al., Ann.N.Y.Acad.Sci., 991:199-213 (2003); The rat of 6-hydroxy dopamine-damage (for example, Lab.Anim.Sci., 49:363-71 (1999)); With genetically modified invertebrates model (for example, Lakso et al., J.Neurochem., 86:165-72 (2003) and Link, Mech.Ageing Dev., 122:1639-49 (2001)).
Estimating polyglutamyl amine assembles
The acellular mensuration of many kinds, measure based on the mensuration of cell and organism and can be used to estimate polyglutamyl amine and assemble, for example, Huntingdon polyglutamyl amine is assembled.Some case history exists, for example, and U.S.2003-0109476.
Measure (for example, acellular, based on cell, or organism) can comprise report albumen, it comprises the polyglutamyl amine duplicate block with at least 35 polyglutamyl amine.Report albumen can be to detect easily, for example, passes through fluorescence.For example, albumen is conjugated on the fluorogen, for example, Fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin phyllochlorin (PerCP), Texas Red, Cy3, Cy5, Cy7, or fluorescence resonance energy series connection fluorogen PerCP-Cy5.5 for example, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7.In another example, albumen is " intrinsic fluorescence ", and promptly it has complete amino acid sequences encoded chromophore by it, and can fluoresce under the situation that does not need cofactor or substrate.For example, this albumen can comprise green fluorescent protein (GFP)-sample chromophore.As used herein, " GFP-sample chromophore " refers to the protein part of intrinsic fluorescence, and it comprises the 11-chain β-bucket with a center alpha-helix, and this center alpha-helix has π-resonator system of puting together, and it comprises 2 aromatic ring systems and the bridge between them.
Can be from the GFP-sample chromophore of naturally occurring albumen, finding, select GFP-sample chromophore, described albumen is A.victoria GFP (GenBank registration number AAA27721) for example, Renilla reniformis GFP, FP583 (GenBank registration number AF168419) (DsRed), FP593 (AF272711), FP483 (AF168420), FP484 (AF168424), FP595 (AF246709), FP486 (AF168421), FP538 (AF168423), and FP506 (AF168422), and only need comprise in the native protein keep chromophoric in required so much of fluorescence.Determining the method in the minimal structure territory that fluorescence is required, is known in the art.Li etal.，J.Biol.Chem.272：28545-28549(1997)。
Perhaps, can from GFP-sample chromophore, select GFP-sample chromophore by those modifications of natural discovery.Typically, carry out such modification, to improve the recombinant production (being with or without the variation of protein sequence) in heterologous expression system, change exciting and/or emission spectra of native protein, promote purification, promote clone or conduct clone's result, or the accidental result of research.Transforming the method for the GFP-sample chromophore of such modification and their fluorescence activity of test (individually and as the part of albumen fusant), is well known in the art.The chromophore that many kinds are modified like this can commercially obtain now, and can easily be used for fusion rotein of the present invention.For example, EGFP (" enhanced GFP "), Cormack et al., Gene173:33-38 (1996); U.S. Patent number 6,090,919 and 5,804,387, be the variant of people's colon-optimization of the red shift of GFP, it has been transformed into brighter fluorescence, higher expression and for use the excitation spectrum of optimizing in flow cytometer in mammalian cell.EGFP can usefully contribute GFP-sample chromophore to fusion rotein, and the latter also comprises polyglutamyl amine zone.Many kinds of EGFP carriers (plasmid and virus) can commercially obtain (ClontechLabs, Palo Alto, Calif., USA).The GFP albumen of other transformation is known.Referring to, for example,, Heim et al., Curr.Biol.6:178-182 (1996); Cormacket al., Gene 173:33-38 (1996), BFP2, EYFP (" enhanced yellow fluorescence protein "), EBFP, Ormo et al., Science 273:1392-1395 (1996), Heikal etal., Proc.Natl.Acad.Sci.USA 97:11996-12001 (2000) .ECFP (" enhanced cyan fluorescent protein ") (Clontech Labs, Palo Alto, Calif., USA).GFP-sample chromophore also can obtain from the GFP of other modification, is included in U.S. Patent number 6,124,128; 6,096,865; 6,090,919; 6,066,476; 6,054,321; 6,027,881; 5,968,750; 5,874,304; 5,804,387; 5,777,079; 5,741,668; With 5,625, those described in 048.
In one embodiment, the report albumen that comprises the polyglutamyl amine duplicate block with at least 35 polyglutamyl amine is used for the mensuration based on cell.
In an example, can use PC12 neuronal cell system, it has the construct of the expression of being transformed into by HD gene extron 1 encoded protein, and described exons 1 contains the alternative repetition codon that is fused on enhanced GFP (green fluorescent protein) gene.Referring to, for example, Boado et al.J.Pharmacol.And Experimental Therapeutics 295 (1): 239-243 (2000) and Kazantsev et al.Proc.Natl.Acad.Sci.USA 96:11404-09 (1999).This expression of gene can cause the appearance of co to the green fluorescence at protein aggregate position.HD gene extron 1-GFP fusion gene is under the control of the inducible promoter that muristerone regulates.A concrete construct has about 46 glutamine and repeats (by CAA or CAG coding).Other construct has, and for example, 103 glutamine repeat.The PC12 cell is grown in DMEM, 5% horse serum (heat-inactivated), and 2.5%FBS and 1% Pen-Strep, and maintain on Zeocin and the G418 in a small amount.With 5 * 10
5The density of cell/ml culture medium in being coated with the 24-hole flat board of poly-L-Lysine coverslip, does not have any selection with the cell plating.After the incubated overnight, add muristerone, to induce the expression of HD gene extron 1-GFP.Can make the cells contacting experimental compound, for example, before or after the plating and before or after inducing.Can be inverted on the 100MAxioskop at the Zeiss that has been equipped with Zeiss 510 LSM confocal microscopes and relevant krypton-argon laser and helium neon laser, obtain data.Sample can be loaded into the coverslip system that is used for improving imaging of Lab-Tek II locellus.In independently experiment (for example, at least 3 or 7 independently experiments), the Huntington protein-GFP accumulative number of counting in the object lens visual field.
Other exemplary approach that is used for assess sample comprises the high throughput device, for example Amersham Biosciences IN cell analysis system and Cellomics
TMThe ArrayScanHCS system, it allows statically and dynamically detects and the Subcellular Localization and the concentration of the part of quantitative fluorescence labelling.Also referring to, U.S. Patent number 5,989,835.
Other exemplary mammal cell line comprises: Chinese hamster ovary celI system and 293 cell lines.For example, (it has about 103Q and repeats to have the HD gene extron 1 of integrating copy, the latter is merged mutually with GFP, become the fusion constructs of coding HD gene extron 1 Q103-GFP) Chinese hamster ovary celI, can produce visible GFP at the nuclear membrane place assembles, it can measure by microscope inspection, can not produce visible GFP gathering at the nuclear membrane place and have the Chinese hamster ovary celI of integrating the fusion constructs (its HD gene extron 1 Q24-GFP that encodes) that copies in Chinese hamster ovary celI.In another example, use 293 cells with HD gene extron 1 (it contains 84 CAG and repeats) of integrating copy.
Can obtain many animal model systems of hungtington's chorea.Referring to, for example, Brouillet, Functional Neurology 15 (4): 239-251 (2000); Ona etal.Nature 399:263-267 (1999), Bates et al.Hum Mol Genet.6 (10): 1633-7 (1997); Hansson et al.J.of Neurochemistry 78:694-703; And Rubinsztein, D.C., Trends in Genetics, Vol.18, No.4, pp.202-209 (to the multiple animal of HD and the summary of non--human model).
In one embodiment, animal is a transgenic mice, it can be expressed (at least one cell) human Huntington protein, its part or comprise human Huntington protein or fusion rotein that it is a part of, they (for example for example have at least 36 glutamine, repeat CAG repeated encoding (perhaps, the CAG of arbitrary number repeat to be CAA) in the section by the CAG of the exons 1 of coding polyglutamyl amine section).
An example of such transgenic mice strain is a R6/2 system (Mangiarini et al.Cell 87:493-506 (1996)).The R6/2 mice is genetically modified hungtington's chorea mice, and it crosses the exons 1 (under the control of endogenesis promoter) of expressing human HD gene.The exons 1 of R6/2 people HD gene has the CAG/ polyglutamyl amine repeat length (average 150 CAG repeat) of elongation.These mices can form carrying out property, final fatal neurological disorder, and it has the choreic many features of Ren Hengtingdunshi.In the R6/2 mice, in Cytoplasm and nucleus, all observed unusual aggregation, it partly constitutes (Davies et al.Cell 90:537-548 (1997)) by the N-end portion (by HD exons 1 coding) of Huntington protein.For example, the human Huntington protein in these transgenic animal is that at least 55 CAG repeat, about 150 multiple gene codes of CAG more preferably by comprising.
These transgenic animal can form the phenotype of hungtington's chorea-sample.These transgenic mices are characterised in that the weight increase of minimizing, and the life-span of shortening and athletic injury, described athletic injury are characterised in that abnormal gait, static tremor, hind leg are rolled up and hyperkinesia (the R6/2 strain for example in 8 to 10 weeks of back of being born; See Mangiarini et al.Cell 87:493-506 (1996)).Phenotype worsens to hypokinesia gradually.The brain of these transgenic mices also show neuro chemistry with histological unusual; neurotransmitter receptor (glutamic acid for example; dopaminergic) variation, reduce the N-acetyl group aspartic acid (a kind of labelling of neuron integrity) of concentration and striatum and the brain size that reduces.Therefore, evaluation can comprise evaluation and neurotransmitter levels, neurotransmitter receptor level, the brain size parameter relevant with the striatum size.In addition, the unusual aggregation that contains total length human Huntington protein or its transgenic part is present in the cerebral tissue of these animals (for example, R6/2 transgenic mice strain).Referring to, for example, Mangiarini et al.Cell 87:493-506 (1996), Davies et al.Cell90:537-548 (1997), Brouillet, Functional Neurology 15 (4): 239-251 (2000) and Cha et al.Proc.Natl.Acad.Sci.USA 95:6480-6485 (1998).
For the test experiments chemical compound (for example, chemical compound as herein described or be present in chemical compound in the library as herein described) effect in animal model, the experimental compound of variable concentrations is administered to transgenic animal, for example by experimental compound is injected in the circulation of animal.In one embodiment, the hungtington's chorea-sample symptom in the evaluation animal.Monitor for example progress of hungtington's chorea-sample symptom then, for example as top about as described in the mouse model, whether can cause alleviating of symptom or postpone with the treatment of determining experimental compound.In another embodiment, monitor the depolymerization of Huntington protein aggregation in these animals.Can put to death animal then, and obtain brain section.Analyze then and whether have the aggregation that contains transgenic human Huntington protein, its part or comprise human Huntington protein or its a part of fusion rotein in the brain section.This analysis can comprise, for example, with anti--Huntington protein antibody brain tissue slice is dyeed, add identification anti--Huntington protein antibody with FITC put together two anti-(for example, anti--Huntington protein antibody is mice Anti-Human antibody, two is anti-to people's antibody specificity), and by the fluorescence microscope protein aggregate.Perhaps, anti--Huntington protein antibody can directly be puted together FITC.Pass through the level of fluorescence microscope Huntington protein aggregation then.
Also can obtain the Drosophila melanogaster model system of hungtington's chorea.Referring to, for example, Steffan et al., Nature, 413:739-743 (2001) and Marsh et al., HumanMolecular Genetics 9:13-25 (2000).For example, genetically modified fruit bat can be transformed into expressing human Huntington protein, its part (for example exons 1) or comprise human Huntington protein or its a part of fusion rotein, have in the fusion rotein for example comprise at least 36 glutamine polyglutamyl amine district (for example, repeat (preferably 51 repetitions or more a plurality of) codings (perhaps, the CAG of arbitrary number repetition can be CAA) by CAG).Polyglutamyl amine district can repeat the section coding by the CAG of the exons 1 of coding poly Q section.Also these transgenic flies can be transformed in neuron (for example, in the fruit bat eye) expressing human Huntington protein, its part (for example exons 1) or comprise human Huntington protein or its a part of fusion rotein.
Can give transgenic fly with experimental compound (for example, the experimental compound of variable concentrations) or compound administration as herein described, for example, by the pharmaceutical composition of inclusion compound being used into the animal or the part of chemical compound of feeding as food.Using of chemical compound can occur in the fruit bat life cycle stage.Animal be can monitor, the minimizing or the delay of hungtington's chorea-sample symptom, the depolymerization of Huntington protein aggregation, or the fatality rate that reduces, and/or the neuronic degeneration of monitoring light receptor whether can be caused with the treatment of determining chemical compound.
In drosophila compound eye, can easily observe because human Huntington protein, its part (for example exons 1) or comprise human Huntington protein or neural degeneration that its a part of Expression of Fusion Protein causes regular trapezoidal the rearranging of 7 visible sense bars (subcellular sheet feeding type) that described compound eye is generated by the light receptor neuron of each fruit bat ommatidium.Human Huntington protein, its part (for example exons 1) or comprise human Huntington protein or its a part of Expression of Fusion Protein can cause feeling the carrying out property forfeiture of bar.Thereby, can estimate the neuronal degeneration of the animal of using experimental compound.
Morely et al. (2002) Proc.Nat.Acad.USA Vol.99:10417 has described the beautiful nematicide system that is used to estimate the relevant protein aggregation of hungtington's chorea.
Estimate hungtington's chorea
Chemical compound as herein described can be used to improve at least a symptom of the hungtington's chorea of object.
Can be estimated and/or be monitored many kinds of methods of hungtington's chorea.Many kinds of clinical symptoms of known this disease and indication.Hungtington's chorea can cause the dyskinesia, psychiatry difficulty and cognitive the variation.The degree of these symptoms, age of onset and performance can change.The dyskinesia can comprise fast, carelessly, dancing-sample motion, be called chorea.
Estimate a kind of method of hungtington's chorea and use unified hungtington's chorea rating scale (UNDRS).Also can be individually or use one test in combination, to estimate at least a symptom of whether having improved hungtington's chorea.UNDRS is documented in MovementDisorders (vol.11:136-142,1996) and Marder et al.Neurology (54:452-458,2000).The seriousness of the quantitative hungtington's chorea of UNDRS meeting.It is divided into a plurality of subdivisions: motion, cognition, behavior, function.In one embodiment, use one subdivision to come evaluation object.By different problem summations, can calculate these marks with each part.Some part (for example chorea and myodystonia) can comprise grades each limbs, face, cheek-oral cavity-tongue and trunk dividually.
Exemplary motion evaluation comprises: eye tracking, and saccade causes, saccade speed, dysphonia, tongue stretches out, and refers to the tip-tap ability, and is prostrate/as to lie on the back, fist-hands-palm order, arm is stiff, and is bradykinesia, maximum tension obstacle (trunk, upper limb and lower limb), maximum chorea is (for example, trunk, face, upper limb and lower limb), gait, front and back walking, and backward progression.A kind of exemplary treatment can cause the variation of total motion scoring 4 (TMS-4), and this is the sub-scale of UHDRS, for example through 1 year.
Diabetes
The invention provides the method for treatment and prevent diabetes.The example of diabetes comprises insulin dependent diabetes mellitus (IDDM) and non--insulin dependent diabetes mellitus (IDDM).For example this method comprises, uses chemical compound as herein described for the patient who suffers from diabetes or be in the risk of diabetes.In some example, reduce (IGT) or empty stomach hyperglycemia by having glucose tolerance, the patient can be differentiated to being in the danger that diabetes take place.
For example, chemical compound as herein described can be administered to object with the treatment effective dose,, improve glycemic control (promptly lower fasting glucose), or make insulin sensitivity normalization to reduce gluconeogenesis.Can give the object of suffering from diabetes or obesity with compound administration.
Insulin dependent diabetes mellitus (IDDM) (type i diabetes) is a kind of autoimmune disease, and wherein insulitis causes the destruction of pancreas J-cell.When the clinical episodes of type i diabetes, the b cell of mass production insulin is destroyed, only 15% to 40% can also produce insulin (McCulloch etal. (1991) Diabetes 40:673-679).B-cell fault causes depending on throughout one's life the injection of insulin of every day and to the exposure of the acute and late complication of this disease.
Type ii diabetes is the impaired metabolic disease of glucose homeostasis, it is characterized in that hyperglycemia or hyperglycemia, as defective insulin action (it shows as insulin resistant, defective insulin secretion, or the two) the result.The type ii diabetes patient has and insulin resistant and/or impaired relevant unusual carbohydrate, lipid and the Proteometabolism of insulin secretion.This disease can cause pancreatic beta cell to be destroyed, and finally causes absolute insulin deficit.Do not have insulin, keep high glucose level in the blood.That the long term of hyperglycemia comprises is blind, renal failure and to the relatively poor blood circulation in these zones, and this can cause foot and ankle amputation.Reach aspect this seriousness the prevention patient, earlier detection is crucial.Most of glycosuria patients have the diabetes of non--insulin dependency form, are called type ii diabetes now.
The present invention comprises that also treatment is relevant with diabetes or is derived from the method for the disease of diabetes that described disease for example end-organ is damaged diabetic gastroparesis, diabetic neuropathy, arrhythmia etc.
The example molecule model of type ii diabetes comprises: the transgenic mice (US 6,127,598) with deficiency Nkx-2.2 or Nkx-6.1; Fat fa/fa (ZDF) rat (US 6569832) of Zucker diabetes; And macaque, it spontaneously forms obesity, and often develop into tangible type ii diabetes subsequently (Hotta et al., Diabetes, 50:1126-33 (2001); And transgenic mice, it has the IGF-I receptor (KR-IGF-IR) of dominance negative, and has type ii diabetes-sample insulin resistant.
Metabolism syndrome
The invention provides a kind of method for the treatment of metabolism syndrome, comprise the chemical compound described herein of using effective dose to object.
Metabolism syndrome (for example, X syndrome) is characterised in that one group of metabolic risk factors of a philtrum.They comprise: central obesity (excess fat tissue around abdominal part and abdominal part), atherogenic dyslipidemia (dyslipidemia-mainly be high triglyceride and low HDL cholesterol-their promote the speckle assembly in arterial wall); Insulin resistant or glucose intolerance (human body can not suitably utilize insulin or blood glucose); Short thrombosis state (for example, high fibrinogen in the blood or plasminogen activator inhibitor [1]); Hypertension (that is hypertension) (130/85mmHg or higher); With short scorching state (for example, hypersensitivity C-reactive protein raises in the blood).
This syndromic potential cause is overweight, health inertia and inherited genetic factors.The people who suffers from metabolism syndrome suffer from coronary heart disease, with arterial wall in speckle assemble the danger increase of relevant other disease (for example, apoplexy and peripheral vascular disease) and type 2 diabetes mellitus.Metabolism syndrome is closely related with the generalized metabolic disorder that is known as insulin resistant, and wherein human body can not use insulin effectively.
The adipose cell relevant disease
The invention provides the adipogenic method of enhancing, comprise to object and use chemical compound as herein described.For example, object can be a underweight, has the fat content of minimizing, or needs extra adipose cell, no matter is that (for example, at regional area, skin of face for example) still capapie partly.
Chemical compound also can be used to regulate adipose cell, for example, and lipocyte, for example, the differentiation of adipose cell.For example, can use chemical compound as herein described to effectively prevent the fatty amount of gathering with the state of normal or pathology.Comprise obesity with the adipose cell diseases associated." obesity " refers to that object has the situation of the Body Mass Index (BMI) more than or equal to 30." overweight " refers to that object has the situation more than or equal to 25.0 Body Mass Indexs.Body Mass Index and other definition are with reference to " NIH Clinical Guidelines on the Identification andEvaluation, and Treatment of Overweight and Obesity in Adults " (1998).More specifically, obesity can cause the type ii diabetes of successive stages.Clinically, these stages can be characterized by normal glucose tolerance, glucose tolerance reduces, hyperinsulinemia diabetes and hypoinsulinemia diabetes.It is relevant that carrying out property glucose like this stores impaired rising with basic blood glucose.
The example of other adipose cell relevant disease comprises dyslipidemia, and hyperlipemia (comprising high triglyceride, high LDL, high fatty acid level).
The fat exemplary model of treatment comprises two kinds of main animal model systems: the 1) calorie intake by about 60% fat content of feeding to rodent, the obesity that the diet that causes brings out (DIO).Handle the animal that reaches 12-16 week with such diet and obtain significant body weight (＞50% increases), accumulation excess fats amount becomes hyperglycemia, hyperinsulinemia and insulin resistant.In this model, random time that can be before the beginning diet or in forming the process of obesity, test compounds.2) db/db saltant mice (leptin receptor spontaneous mutant).These animals can show and the similar phenotype of DIO animal, but more serious aspect different reading.Can handle animal with like the DIO model class.Read as the alternative of SirT1 inhibitor activity, can put to death animal born of the same parents along with therapeutic scheme, and biochemical the acetylation state that FoxO1 albumen increases in the different tissues, for example liver, muscle and the white adipose tissue of assessing.
Chemical compound as herein described can be used for the treatment of AMD.Degeneration of macula comprises and being characterised in that and many kinds of diseases of unusual relevant the carrying out property central vision forfeiture of Bruch's membrane and retinal pigment epithelium (referring to, for example, U. S. application 20030138798).AMD occurs among 1.2% 52 to 64 years old the crowd and 20% surpass among 75 years old the patient (referring to, for example, U. S. application 20030087889).Degeneration of macula takes place with two kinds of forms: " atrophic " (" non-exudative " or " do " form) and " exudative " (" is wet " form).The more uncommon form of AMD is " atrophic AMD ", and it is because due to the dead RPE cell (U. S. application 20030093064).
The symptom of AMD comprises: the straight line in the visual field shows as wavy; Literal on books, magazine and the newspaper thickens; Block the optic centre with dark or white space (referring to, for example, U. S. application 20030065020).
The example molecule labelling that can be used to estimate the AMD state comprises: the nucleotide sequence or the proteic aminoacid sequence of FBNL of the gene of coding FBNL: 345Arg＞Trp and 362Arg＞Gln; (referring to, for example, U. S. application 20030138798); The increase of the yellow ethylethanolamine of pigment A2E, N-retinyl-N-Asian TV Station finally causes cytochrome C to be released into Cytoplasm (U. S. application 20030050283); At various degeneration of macula-correlation molecules self-antibody, described molecule comprises fibulin-3, vitronectin, β-crystalline protein A2, β-crystalline protein A3, β-crystalline protein A4, β-crystalline protein S, calreticulin, 14-3-3 albumen ε, serotransferrin, albumin, keratin, pyruvate carboxylase, or villin 2 (referring to, for example, U. S. application 20030017501); The abnormal activity of complement pathway molecule (comprising clusterin, C6 or C5b-9 complex) or level (referring to, for example, U. S. application 20020015957); With the accumulation (Suter et al., J Biol Chem.275:39625-30 (2000)) of pigment lipofuscin in the lysosome of retinal pigment epithelium (RPE) cell.
Tissue repair
Chemical compound as herein described also can be used to regulate tissue repair or structural state.The exemplary enforcement of tissue repair comprises wound healing, burn, ulcer (for example, the ulcer of diabetics, for example, diabetic ulcer of foot), surgical wound, skin ulcer and scratch.This method can reduce at least a symptom of this tissue.For example, this method comprises, uses the chemical compound as herein described of (for example, partly or capapie) effective dose.
Chemical compound can be used for the disease or the disease of dermatological.
Skeletal muscle atrophy
Amyotrophy comprises many nervimuscular, metabolic, immune and obstacle nerve and diseases, and hunger, malnutrition, metabolic stress, diabetes, aging, muscular dystrophy or myopathy.Amyotrophy occurs in the ageing process.Amyotrophy also is derived from the minimizing of muscle and uses or useless using.Symptom comprises the minimizing of skeletal muscle tissue amount.In the man, reduce 1/3 at 50 to 80 years old muscle quantities.
Amyotrophic some characterization of molecules comprises the rise of ubiquitin ligase and the forfeiture of parapeptone (Furuno et al., J.Biol.Chem., 265:8550-8557,1990).These proteic disintegrates can followingly be followed the trail of, for example, and by measuring 3-methyl-histidine production, it is the particular components of actin, and in some muscle, be particular components (Goodman, the Biochem.J of myosin, 241:121-12,1987 and Lowell, et al., Metabolism, 35:1121-112,1986; Stein and Schluter, Am.J.Physiol.Endocrinol.Metab.272:E688-E696,1997).The release of creatine kinase (a kind of cell injury labelling) (Jackson, et al., Neurology, 41:101 104,1991) also can be indication.
Multiple sclerosis
Multiple sclerosis (MS) is a kind of focal inflammation of cerebral white matter and neuromuscular disease of autoimmune degeneration of being characterised in that.White matter becomes inflammation, and the inflammation heel (form " the sick damage ", its sign is the infiltration of many immunocytes, especially T-cell lymphocyte and macrophage along with myelinic destruction.MS can cause slowing down of neural impulse transmission or blocking-up fully, causes somatic function to weaken thus or loses.MS patient can have one of many ranks of MS (for example, recurrence-remission form MS, former progress type MS, secondary progress type and MarburgShi modification MS).
Symptom can comprise for example fuzzy or diplopia of visual problem, red-green distortion, or even eye blind, the limbs myasthenia is coordinated and equilibrium problem, muscular spasticity, muscle fatigue, paraesthesia, of short duration abnormal sensory is numbness, twinge or " pin and point " sensation for example, and in the worst case, the part or completely the paralysis.About half MS patient also can experience cognitive impairment, for example, relatively poor concentrate, attention, memory and/or judgement (referring to, for example, US2003-0130357 and 2003-0092089).
The molecular marker of MS comprises many inherited genetic factorss, for example, Caucasian's haplotype DRB*1501-DQA1*0102-DQB1*0602 (U. S. application 20030113752), point mutation in Protein-tyrosine-phosphatase receptor-C type (U. S. application 20030113752), the proteic disappearance of wild type SARG-1-, the SARG-1-albumen that has sudden change, or the disappearance of the nucleic acid of encoding wild type SARG-1 or sudden change (referring to, for example, U. S. application 20030113752) and the albumen indicant (for example, myelin basic protein autoantibody in the cerebrospinal fluid) (referring to, for example, U. S. application 20030092089).
The cell of MS and animal model comprise the transgene mouse model (experimental autoimmune encephalomyelitis (EAE)) of chronic MS, for example, as Goverman et al., Cell.72:551-60 (1993) is described, with as Brok et al., Immunol.Rev., the primate model summarized of 183:173-85 (2001).
Amyotrophic lateral sclerosis (ALS; Lou GehrigShi disease)
Chemical compound as herein described can be used to regulate ALS.ALS refers to a class disease, and it comprises upper and lower motor neuron.The sickness rate of ALS significantly increases in the old people.These diseases are characterised in that significant pathological abnormalities, comprise the selectivity and the degeneration of carrying out property of lower motor neuron in the spinal cord that causes motor neuron death and the upper motor neuron in the cerebral cortex, this causes the reduction of the muscle under they control and becomes thin, and causes paralysis.The example of ALS disease comprises classical ALS (typically influencing lower and upper motor neuron), primary lateral sclerosis (PLS, typically only influence upper motor neuron), PBP (PBP or oblongata morbidity, typically from swallowing, chew the ALS version with parathria beginning), progressive myatrophy (PMA, typically only influence lower motor neuron) or familial AL (the hereditary version of ALS), or the combination of these situations (referring to, for example, U. S. application 20020198236 and U. S. application 20030130357).
By neurologic examination or alternate manner, MRI for example, FVC, MUNE etc. (referring to, for example, U. S. application 20030130357), can estimate individual ALS situation.Symptom comprises the myasthenia of hands, arm, lower limb; Swallow or dyspnea; Ballism (fasciculation) and muscle spasm; Use with the minimizing of limbs.The present invention includes amount, use the reagent of regulating the IGF-1/GH axle with one or more ALS symptoms of effective alleviation, for example, in suffering from ALS or being in individuality in the danger of ALS.
The method of estimating individual ALS situation can comprise evaluation " excitability amino acid transporter 2 types " (EAAT2) albumen or gene, copper-Cu/Zn SOD (SOD1) albumen or gene, mitochondrion composite I activity, polyamines (putraceine for example, spermine and spermidine) level, the gene of the guanosine triphosphatase regulator that ornithine decarboxylase activity and coding are inferred (is seen Nat.Genet., 29 (2): 166-73 (2001)).
Be used for the mice that assessing compound comprises the sod gene with change to the cell and the animal of the effect of ALS state, for example, the SOD1-G93A transgenic mice, its people G93A SOD that is driven by endogenesis promoter that carries variable copy number suddenlys change, SOD1-G37R transgenic mice (Wong et al., Neuron, 14 (6): 1105-16 (1995)); SOD1-G85R transgenic mice (Bruijn et al., Neuron, 18 (2): 327-38 (1997)); Express the beautiful nematicide strain (Oeda et al., Hum Mol Genet., 10:2013-23 (2001)) of mutant human SOD1; With fruit bat (the Phillips et al. that expresses the sudden change in the Cu/Zn superoxide dismutase (SOD), Proc.Natl.Acad.Sci.U.S.A., 92:8574-78 (1995) and McCabe, Proc.Natl.Acad.Sci.U.S.A., 92:8533-34 (1995)).
Neuropathy
Chemical compound as herein described can be used to regulate neuropathy.Neuropathy can comprise the maincenter that caused by the general disease that influences motion, sensation, sensorimotor or autonomic nerve, hereditary situation or toxic agents and/or peripheral nervous dysfunction (referring to, for example, U. S. application 20030013771).
Symptom can be with the particular type of the reason of nerve injury and affected nerve and is different.For example, the symptom of motor neuron comprises clumsiness or the myasthenia of carrying out the health task, tired out behind the labour in a small amount, the difficulty of standing or walking and the reduction of neuromuscular reflex or disappearance (U. S. application 20030013771).The symptom of autonomic neuropathy comprises constipation, the reduction (U. S. application 20030013771) of arrhythmia and the reflection of position hypotension.The symptom of esthesioneurosis comprises pain and numbness; The tingling of hands, lower limb or foot; With extreme sensitivity to contacting.The symptom of retinopathy comprises blurred vision, the unexpected forfeiture of vision, black speck, and flash of light.
The Guillain-Barr syndrome is a type games neuropathy, and it usually occurs in influenza-sample disease or other metainfective 2 to 3 weeks.Symptom comprises the unable of rising, and is wherein unable from lower limb, rises to upper limb.The rising of protein level in the spinal fluid and leukocyte number do not increase yet, and (U. S. application 20030083242) can take place.
Exemplary disease
Other exemplary disease that chemical compound described herein is suitable for and definition comprise following:
" disease that the age is relevant " or " with the age diseases associated " are such disease or diseases, when submitting the application to, and surpassing among 100,000 people's the selected crowd, its sickness rate in surpassing 60 years old human individual is than at least 1.5 times of the human individual's height between 30-40 year.Preferred crowd is an American population.Can pass through sex and/or ethnic restriction crowd.
" infirmities of age " is such disease or disease, when submitting the application to, and surpassing among 100,000 people's the selected crowd, and its sickness rate is at least 70% in surpassing 70 years old human individual.In one embodiment, infirmities of age is the disease beyond cancer or the heart and lung diseases.Preferred crowd is an American population.Can pass through sex and/or ethnic restriction crowd.
Disease with " susceptibility factor that the age is relevant " refers to such disease or disease, the outside mediation of its former reason, but when submitting the application to, and in American population, its seriousness or symptom significantly increase than the human individual between 30-40 year in surpassing 60 years old human individual.For example, pneumonia is to be caused by pathogen, but the seriousness of this disease in surpassing 60 years old people is higher than the human individual between 30-40 year.
" neoplastic disease " is such disease or disease, and the cell that it is characterized in that having autonomous growth or replication capacity for example, is characterised in that the abnormality or the situation of proliferative cell growth." neoplastic disease that the age is relevant " is also to be the neoplastic disease of relevant disease of age.
" non--neoplastic disease " is such disease or disease, and its feature does not lie in the cell with autonomous growth or replication capacity." non--neoplastic disease that the age is relevant " is also to be the non-neoplastic disease of relevant disease of age.
" neurological disorder " is such disease or disease, it is characterized in that the unusual or dysfunction of neuronal cell or neuron sustenticular cell (for example, neuroglia or muscle).This disease or disease can influence maincenter and/or peripheral nervous system.Exemplary neurological disorder comprises neuropathy, skeletal muscle atrophy, and neurodegenerative disease, for example, assemble the neurodegenerative disease cause by polyglutamyl amine at least in part or remove at least in part by the gathering of polyglutamyl amine cause neurodegenerative disease.Exemplary neurodegenerative disease comprises: Alzheimer, amyotrophic lateral sclerosis (ALS), and parkinson." neurological disorder that the age is relevant " is also to be the neurological disorder of relevant disease of age.
" cardiovascular disease " is such disease or disease, it is characterized in that the unusual or dysfunction of cardiovascular system (for example, heart, lung or blood vessel).Exemplary cardiovascular disease comprises: arrhythmia, chronic heart failure, cerebral infarction, coronary artery disease, hypertension (being hypertension), and cardiomyopathy." cardiovascular disease that the age is relevant " is also to be the cardiovascular disease of relevant disease of age.
" metabolism disorder " is such disease or disease, it is characterized in that metabolic unusual or dysfunction.One kind of metabolism disorder is the disorder of glucose or insulin metabolism." relevant metabolism disorder of age is also to be the metabolism disorder of relevant disease of age.
" dermatological diseases " is such disease or disease, it is characterized in that the unusual or dysfunction of skin." organization factors of dermatological " refers to help skin and any basic organization (for example, supporting tissue) of skin function and/or outward appearance (for example, beauty treatment outward appearance).
Implementing relevant exemplary disease and disease with some comprises: cancer (for example, breast carcinoma, colorectal carcinoma, CCL, CML, carcinoma of prostate); Skeletal muscle atrophy; Adult morbidity type diabetes; Diabetic nephropathy, neuropathy (for example, esthesioneurosis, autonomic neuropathy, motor neuron, retinopathy); Obesity; Bone resorption; The degeneration of macula that age is relevant, ALS, Alzheimer, Bell's palsy, atherosclerosis, cardiovascular disease are (for example, arrhythmia, chronic heart failure, cerebral infarction, coronary artery disease, hypertension (being hypertension), and cardiomyopathy), chronic renal failure, type ii diabetes, ulcer, cataract, hypermetropia, glomerulonephritis, the Guillan-Barre syndrome, hemorrhagic apoplexy, short-term and longterm memory forfeiture, rheumatoid arthritis, inflammatory bowel, multiple sclerosis, SLE, Crohn disease, osteoarthritis, parkinson, pneumonia, and urinary incontinence.In addition, many neurodegenerative diseases and with protein aggregation (for example, except that polyglutamyl amine assemble) or protein misfolding diseases associated also can be that the age is relevant.The symptom of disease and diagnosis are that the medical science practitioner knows.Also compositions can be administered to just individuality by other method of this disease is treated, for example, with the individuality of chemotherapy (for example, and having neutropenia, atrophy, cachexia, nephropathy, neuropathy) or elective surgery treatment.
Test kit
Chemical compound described herein can provide in test kit.This test kit comprises: (a) chemical compound described herein, for example comprise compound compositions described herein and, (b) information material randomly.This information material can be descriptive material, guiding material, marketing material or other material, and it is relevant with methods described herein and/or the application of chemical compound described herein in methods described herein.
To the form of the information material of test kit without limits.In one embodiment, information material can comprise production about chemical compound, chemical compound molecular weight, concentration, Expiration Date, batch or the information of production site information etc.In one embodiment, information material relates to the method for administered compound.
In one embodiment, information material can comprise with suitable method uses chemical compound described herein to carry out the explanation of methods described herein, for example, and with proper dosage, dosage form or mode of administration (for example, dosage as herein described, dosage form or mode of administration).In another embodiment, information material can comprise the explanation of compound administration described herein being given suitable object, and described object is the people for example, for example suffers from disease described herein or is in people in the disease danger described herein.
To the form of the information material of test kit without limits.In many cases, information material, for example description provides with printed article, for example, the text of printing, accompanying drawing and/or photo, for example, label or printed leaves.Yet, can also provide information material by other form, for example braille, computer readable-material, videograph or audio recording.In another embodiment, the information material of test kit is contact details, for example, actual address, e-mail address, network address or telephone number, the user of test kit can therefrom obtain the essential information about chemical compound described herein and/or its application in methods described herein.Certainly, information material can also provide in any combination.
Except chemical compound described herein, the compositions of this test kit also can comprise other composition, as solvent or buffer agent, stabilizing agent, antiseptic, correctives (for example, bitterness antagonist or sweetener), aromatic or other cosmetic composition and/or be used for the treatment of second kind of reagent of situation described herein or disease.Optionally, other composition can be included in the test kit, but in the compositions or container different with chemical compound described herein.In this class embodiment, test kit can comprise about becoming phase-splitting to mix with other chemical compound described herein or about using the explanation of chemical compound described herein with other composition.
Chemical compound described herein can provide in any form, for example, and liquid, exsiccant or freeze-dried.Preferably, chemical compound described herein is pure basically and/or aseptic.When providing chemical compound described herein in liquid solution, liquid solution is aqueous solution preferably, preferred aseptic aqueous solution.When providing chemical compound described herein, carry out reprovision by adding suitable solvent usually with dried forms.Solvent, for example, aseptic water or buffer can be chosen wantonly and be provided in the test kit.
This test kit can comprise one or more containers, is used to hold contain compound compositions described herein.In some embodiments, test kit comprises the container that separates, separator or the compartment that is used for compositions and information material.For example, compositions can be contained in bottle, bottle or the syringe, and information material can be contained in plastics sleeve or the parcel.In other embodiments, the assembly that separates with test kit is contained in the single undivided container.For example, compositions is contained in bottle, bottle or the syringe, it is the information material of label form.In some embodiments, that test kit comprises is a plurality of (for example, a bag) one container, each container contains the chemical compound described herein of one or more unit dosage forms (for example, dosage form described herein).For example, test kit comprises a plurality of syringes, ampoule, paper tinsel bag or blister package, and each contains the chemical compound described herein of single unit dose.The container of test kit can be gastight a, waterproof (being not saturating for the variation of moisture or evaporation for example) and/or lighttight.
Test kit randomly comprises the device that is suitable for using compositions, for example, and syringe, inhaler, pipette, tweezers, measuring spoon, dropper (for example, eye dropper), swab (for example, cotton swab or peg wood) or any this class delivery apparatus.In a preferred embodiment, this device is medical embedded device, and for example packing is inserted for operation.
Genetics information
Can obtain SIRT1 hereditary information, for example, by estimating hereditary material (for example, DNA or RNA) from object (for example, as described below).Hereditary information refers to about any indication at the nucleotide sequence content at one or more nucleotide place.Hereditary information can comprise, for example, and about the existence whether indication of specific polymorphism (for example, one or more nucleotide diversities).Exemplary polymorphism comprises single nucleotide polymorphism (SNP), and restriction site or limited fragment length insert, inversion, and disappearance repeats (for example, trinucleotide repeats, and retrovirus repeats), etc.
In table 2, listed exemplary SIRT1 SNP.
Table 2: exemplary SIRT1 SNP
| Initial | Stop | dbSNP rs# | Local locus | transID | avg.het | s.e.het |
| 69520160 69520607 69530733 69531621 69535743 69536360 69536618 69536736 69536742 69539733 69540006 69540390 69540762 69540970 69541621 69544136 69547213 69549191 69551326 69557788 69558999 | 69520160 69520607 69530733 69531621 69535743 69536360 69536618 69536736 69536742 69539733 69540006 69540390 69540762 69540970 69541621 69544136 69547213 69549191 69551326 69557788 69558999 | rs730821 rs3084650 rs4746715 rs4745944 rs3758391 rs3740051 rs932658 rs3740053 rs2394443 rs932657 rs737477 rs911738 rs4351720 rs2236318 rs2236319 rs768471 rs1885472 rs2894057 rs4746717 rs2224573 rs2273773 | SIRT1: locus; SIRT1: locus; SIRT1: locus; SIRT1: locus; SIRT1: locus; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1; | NM_012238 | 0.267438 0.424806 0.118187 0.222189 0.455538 0 0.430062 | 0 0 0 0 0.153425 0.114325 0 0 0 0 0.201473 0 0 0.135429 0.102018 0.01 0 0 0 0 0.135492 |
| 69559302 69564725 69564728 69564741 69564744 69565400 69566230 69566318 69567559 69567728 69568961 69568962 69569231 69569461 69570479 69570580 69570983 69572334 69573968 69574252 69575032 | 69559302 69564725 69564728 69564741 69564744 69565400 69566237 69566318 69567559 69567728 69568961 69568962 69569231 69569461 69570479 69570580 69570983 69572334 69573968 69574252 69575032 | rs3818292 rs1063111 rs1063112 rs1063113 rs1063114 rs3818291 rs5785840 rs2394444 rs1467568 rs1966188 rs2394445 rs2394446 rs4746720 rs752578 rs2234975 rs1022764 rs1570290 rs2025162 rs4141919 rs14819 rs14840 | SIRT1: intron; SIRT1; SIRT1; SIRT1; SIRT1; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1: intron; SIRT1; SIRT1; SIRT1; SIRT1; SIRT1; SIRT1: locus; SIRT1: locus; DKFZP564G092: locus; DKFZP564G092: locus; DKFZP564G092: locus; | ； NM_012238 ； NM_012238 ； NM_012238 ； NM_012238 ； NM_012238 ：UTR； NM_012238 ：UTR； NM_012238 ：UTR； NM_012238 ：UTR； NM_012238 ：UTR； | 0.456782 0.179039 0.0392 | 0.10598 0 0 0 0 0.132983 0 0 0 0 0 0 0 0 0 0 0.167405 0 0 0 |
Can digitally write down or exchange hereditary information in many ways.Typical expression comprises one or more, or text-string.For example, use 2, can describe biallelic marker.In one embodiment, whether first bit representation exists first allele (for example, less important allele), and whether second bit representation exists another allele (for example, main allele).With regard to multiallelic labelling, for example, wherein may exist to surpass 2 allele, can use extra order and other form coding (for example, two-symbol, hexadecimal text, for example, ASCII or Unicode, etc.).In some embodiment, hereditary information is described haplotype, for example, and a plurality of polymorphisms on same chromosome.But, in many embodiments, (unphased) of hereditary information right and wrong phase.
According to hereditary information, can make decision about whether using chemical compound described herein about SIRT1.For example, the method for using chemical compound described herein can comprise, estimates the nucleic acid from object, obtains the hereditary information about SIRT1 or another kind of sirtuin, and uses chemical compound described herein.
The data base
The present invention also provides the data base, its will about or the information of differentiating one or more chemical compounds described herein and parameter correlation connection about the patient, for example, the patient of this paper disease to be treated.Parameter can be a General Parameters, for example, and blood pressure, central body temperature etc., or the parameter relevant (for example, as described herein) with specified disease or disease.
Embodiment
Among all embodiment below, mention chemical compound according to their appointments (being the chemical compound of example) in table 1.
Embodiment 1:HeLa apoptosis is measured
Use detects ELISA plus test kit from the cell death of Roche Applied Science, estimates the effect that following exemplary compounds is measured the apoptosis of HeLa cell.
| Chemical compound | Dosage | On | SD | |
| 8 8 8 8 8 5 | 0 0.5 2.5 10 25 0 | 1.12 1.23 1.85 2.11 2.27 0.92 | 0.15 0.04 0.24 0.25 0.20 0.07 |
| 5555 resveratrol resveratrol resveratrol resveratrol resveratrol DMS0 DMSO DMSO DMSO DMSO | 0.5 2.5 10 25 0 0.5 2.5 10 25 0 0.5 2.5 10 25 | 1.00 0.97 1.07 0.91 0.73 0.83 0.84 1.01 0.56 0.72 0.79 0.91 0.76 1.18 | 0.08 0.11 0.02 0.07 0.08 0.05 0.02 0.07 0.08 0.09 0.12 0.13 0.09 0.20 |
The reagent tabulation:
| Reagent name | Supply status | The source | Catalog number (Cat.No.) | | |
| 1 2 3 4 5 6 7 8 9 10 11 | People SirT1 Fluor de Lys substrate Fluor de Lys developer NAD niacinamide Trizma-HCl sodium chloride magnesium chloride potassium chloride polyoxyethylene sorbitan monolaurate (Tween-20) Fluor de Lys deacetylation standard items | 2.5 or 3.5U/ul 50mM; 20x concentrate solid solid solid solid solid solid 100% 10mM in DMSO is in DMSO | Biomol Biomol Biomol Sigma Calbiochem Sigma Sigma Sigma Sigma Sigma Biomol | SE-239 KI-104 KI-105 N-1636 481907 T-5941 S-9888 M-2393 P-3911 P-7949 KI-142 | -20C-20C-20C-greenhouse, greenhouse, 20C Room greenhouse greenhouse temperature-20C |
List of devices:
| The instrument title | The instrument source | Catalog number (Cat.No.) |
| 1 2 3 | The fluorescence plate reader is worked in coordination with | BIO-TEK Apogent Discoveries VWR | SIAFR 2069 1540 |
The disposable product tabulation:
| Disposable product | The source | Catalog number (Cat.No.) | |
| 1 2 3 4 | The dull and stereotyped sealing film of the reagent storage that the tip 25ml of the dull and | Greiner/Bellco Apogent Discoveries Apogent Discoveries Apogent Discoveries | 4507-84075 7421 8095 4418 |
The standard reagent preparation:
| The reagent name of preparation | The component title | Molecular weight | Group component (in water) | Final concentration of component | Store | |
| 1 2 3 4 5 6 7 8 | Tris-HCl, pH 8.0 sodium chloride magnesium chloride potassium chloride polyoxyethylene sorbitan monolaurate NAD niacinamide are measured buffer solution | Trizma-HCl HCl NaCl MgCl 2KCl Tween-20 NAD niacinamide Tris-HCl, pH 8.0 NaCl KCl MgCl2Tween 20 | 157.6 58.44 203.3 74.55 717 122 | 157.6g/L to 10% stoste of the 100mM stoste of the 270mM stoste of the 5M stoste of the 1M stoste of pH 8.0 292g/L 20.33g/L 20.13g/L 1ml/10ml 0.0717g/ml 0.0061g/ml 25ml/L 27.4ml/L 10ml/L 10ml/L 5ml/L | 1M pH 8.0 5M 100mM 270mM 10％ 100mM 50mM 25mM 137mM 2.7mM 1mM 0.05％ | Greenhouse, greenhouse, Room greenhouse temperature-20 ℃-20 ℃ 4 ℃ |
| * prepares that following work stock solution is following to be prepared in measuring buffer before be about to using | ||||||
| 9 10 11 | 2x substrates enzymes mixture developer/termination reagent | Flour de Lys substrate NAD Biomol SirT1 20x developer concentrate niacinamide | The 100mM stoste of 6ul/ml 20ul/ml * * is according to the specific activity of lot.Ex: 3.5U/ul, 50mM stoste/ml of 35.71ul/ml 50ul/ml 20ul | 300uM 2mM 0.125U/ul (0.5U/ hole) 1x, 1mM in measuring buffer solution | Ice ice ice | |
Embodiment 3:
In order to determine whether chemical compound 8 can suppress mammalian enzyme, with construct transfection 293T cell, described construct is designed to express the people SIRT1 that merges mutually with glutathione-S-transferase, to allow from the cell extract fast purifying.After cracking, with glutathion-agarose pearl incubation, washing is washed once in SIRT1 enzymatic determination buffer several times at last in lysis buffer then with cell extract.In the presence of the chemical compound 8 that a series of concentration are arranged, the pearl that combines GST-SIRT1 is added to Fleur-de-lys measure (Biomol).From Fig. 2 a as can be seen, the EC of 8 couples of mammal SIRT1 of chemical compound
50The value that value can obtain with the people's enzyme to recombinant bacteria production is compared.
As can be seen from Figure 2B, after etoposide was handled, chemical compound 8 entered cell and increases p53 acetylation (at lysine 382 places).In the described experiment of Fig. 2 B; be with or without in the presence of the SIRT1 inhibitor (chemical compound 8 or nicotiamide); handle the NCI-H460 cell with 20uM etoposide (a kind of DNA damage agent), and by Western blot, the amount of observing acetylizad p53 (at lysine 382 places).Compare with independent DMSO, chemical compound 8 can significantly increase the p53 acetylation, and 1uM and 10uM are effective equally.
Embodiment 4:
Whether tested the enantiomer of chemical compound 8, wherein every kind of enantiomer has the purity greater than 90% enantiomeric excess, more effective than the mixture of enantiomer to determine single enantiomer.Having in the presence of the 20uM etoposide, handling the NCI-H460 cell 6 hours with chemical compound 8 (+) and 8 (-), cracking subsequently, and use Ab-6 (Oncogene Science) to carry out the immunoprecipitation of p53.With the antibody (Cell Signaling) of the acetylizad lysine 382 of discerning p53, survey extract.Fig. 3 shows that chemical compound 8 has active and enantiomer non-activity.More specifically, having in the presence of the etoposide, the enantiomeric compounds 8 (+) of non-activity can not cause enhanced p53 acetylation, and chemical compound 8 (-) can cause proteic acetylizad remarkable increase of p53 and stabilisation.
Embodiment 5:
In the result of experiment, we have confirmed that the acetylizad ability of enhancing p53 of chemical compound is relevant to the vitro efficacy of SIRT1 with it below shown in Figure 4.With 1uM concentration, similar compounds on a series of structure is added to cell.Only with IC less than 1uM
50Those chemical compounds that suppress SIRT1 have strengthened the p53 acetylation, and have the IC greater than 1uM
50Chemical compound can not.
Embodiment 6:
The described experiment confirm of Fig. 5, in depending on the reticent mensuration of the active yeast of SIRT1, non-activity enantiomeric compounds 8 (+) cell growth of chemical compound 8 is effect not, and activated enantiomer 8 (-) can suppress SIRT1, and allowing to express URA3, it can the blocking-up growth in the presence of 5-fluorouracil.(URA at the telomere place) is used for coming SCREENED COMPOUND based on zymic mensuration with the SL8c strain.Cell is grown in-URA culture medium, select to go reticent cell.Next day, in containing the fresh YPD of 2% glucose, 1: 20 diluting cells was grown 5 hours then.Then, in SD and SD+0.1%5FOA culture medium, with cell dilution to OD=0.01.Then, in 10ul SD or SD+0.1%5FOA culture medium, the serial dilution chemical compound.140 μ l cell suctions are measured in the 96 hole flat boards, and 30 ℃ of growths 18-24 hour.
Embodiment 7:
Embodiment 8:
In order to assess the variation whether 8 pairs of acetylizad influences of p53 of chemical compound cause the p53 function, experimentize the cell survival of measuring after DNA damage.Be with or without in the presence of the SIRT1 inhibitor, with the etoposide damage NCI-H460 cell of variable concentrations.As shown in Figure 7, in this was measured, chemical compound 8 self was not significantly regulated the p53 function.
Embodiment 9:
In the presence of etoposide that a series of concentration are arranged and 1uM chemical compound 8, with the density in 800/hole, with the cell plating in 96 hole cytostar flat boards.With 24 hours intervals, measure thymidine and mix.As shown in Figure 9, this experiment confirm, when etoposide is handled, before and add afterwards under the condition of chemical compound, the growth characteristics to the NCI-H460 cell between etoposide and the chemical compound 8 does not have synergism.
Embodiment 10:
Be with or without in the presence of the chemical compound 8, making HEK293 cell serum starvation 24 hours, cracking then, and immunoblotting assay p27 albumen.As can be seen from Figure 9, handle cells, can cause abolishment the rise of the cell cycle inhibitor p27 of serum starvation-mediation with chemical compound 8.The explanation to this result that proposes is, the inactivation that the deacetylation of SIRT1-mediation causes the p27 of FOXO1-mediation to transcribe, and the interpolation of chemical compound 8 reverses this effect.
Embodiment 11
With GFP-hSIRT2 isotype 1 (green) transfection HeLa cell.After transfection 36 hours, add 1 μ M TSA and DMSO or 50 μ M chemical compounds 8.In morning next day, fixed cell is changed processing thoroughly, and to acetylizad tubulin dyeing (redness).In the cell of handling with DMSO, in the cell of expressing SIRT2, there is considerably less acetylizad tubulin, in the cell of handling with chemical compound 8, tubulin is shown the effect of having blocked SIRT2 by acetylation more to heavens.
Use western blot analysis, also can observe the effect of chemical compound.With eGFP (contrast) or mice SIRT2 isotype 1 (mSIRT2) transfection 293T cell.Add TSA, to increase the amount of acetylizad tubulin, chemical compound to the 10 μ M that adds DMSO simultaneously or list below.
Operation is described:
Step is described
The 2x substrate of the hole count aequum that 1 preparation is to be determined.Every hole needs 5ul
2 distribute 5ul 2x substrate in experimental port
3 distribute the 1ul experimental compound in experimental port
Distribute 1ul chemical compound solvents/diluents in the positive control hole
Distribute Iul 1mM nicotiamide to 50% to suppress in the hole
Distribute 1ul 10mM nicotiamide to 100% to suppress in the hole
4 distribute 4ul to measure buffer in negative control hole (no enzyme contrast)
The enzyme of the hole count aequum that 5 preparations are to be determined.Every hole needs the 4ul enzymatic mixture
6 distribute the 4ul enzymatic mixture in experimental port and positive control hole
7 build and 37 ℃ of incubations 45 minutes
8 in 30 minutes before use, the 1x developer/termination reagent of the hole count aequum that preparation is measured
9 distribute 10ul 1x developer/terminations reagent to porose in
10 room temperature incubation at least 15 minutes
11 in the fluorescence plate reader reading, excite=350-380nm emission=440-460
Fluor de Lys in 12 substrates has intrinsic fluorescence, before data are carried out any calculating, it need be deducted as a setting.From negative control hole, can find these values.
Appendix 1: use the standard substance of Fluor de Lys deacetylation, the preparation standard curve
The 1 1uM diluent by the preparation standard product is determined the concentration range of the standard substance of the deacetylation that will be used in combination with said determination.The 1uM diluent and the 10ul developer that mix 10ul carry out reading in wavelength and the sensitivity setting identical with reading mensuration.Use the estimated value of AFU (flat fluorescent arbitrarily)/uM, determine the concentration range that in standard curve, to test.
2 in measuring buffer, the serial dilutions of the standard substance of preparation Fluor de Lys deacetylation, and it strides target concentration range
3 suction amount 10ul measure buffer to ' 0 ' hole
4 suction amount 10ul standard substance diluents are in the hole
5 suction amount 10ul developers are in the hole, and room temperature incubation 15 minutes
6 read flat board at above-mentioned wavelength
7 draw the graph of a relation of fluorescence signal (y) to the concentration (x) of the standard substance of Fluor de Lys deacetylation, and definite slope is as AFU/uM
The scheme of the inhibitor of test developer reaction
1 from standard curve, is chosen in the concentration (for example 5uM) of the standard substance of the deacetylation that produces the fluorescence signal that is equivalent to positive control in the mensuration
2 distribute the standard substance (for example 10uM) of 5ul 2x deacetylation
3 distribute the 1ul chemical compound, and 4ul measures buffer
4 distribute the 10ul developer
5 in room temperature incubation 15 minutes (or with screening in identical time), and at the be provided with reading identical with screening
Embodiment 12: Synthetic 2-chloro-5,6,7,8,9,10-six hydrogen cyclohepta [b] indole-6-Methanamides
Preparation 3-bromo-2-oxo cycloheptane methyl formate: (50g 294mmol) is dissolved in carbon tetrachloride (200mL), and is cooled to 0 ℃ with 2-oxo-1-cycloheptane methyl formate.Through about 30 minutes, by addition funnel add bromine (46.8g, 294mmol).Remove cooling bath then, make reaction be warmed up to room temperature.Under nitrogen, stirring at room reaction 4 days.After 4 days, solution is poured into the separatory funnel of moisture (1L).Wash organic facies with water, and through dried over sodium sulfate.Vacuum is removed solvent, obtains (72.4g, 291mmol, 99% thick productive rate).Without being further purified, further use this material.
Preparation 2-chloro-5,6,7,8,9,10-six hydrogen cyclohepta [b] indole-6-methyl formates: with 3-bromo-2-oxo cycloheptane methyl formate (16.8g, 67.4mmol) and 4-chloroaniline (18.4g, 98%, 2.13 equivalent 143.6mmol) adds the flask with thermometer, nitrogen inlet and machine mixer.Along with the temperature of mixture surpasses 140 ℃, heat release fast relatively and violent gas take place emit.React with water cooling immediately.Reactant mixture is dissolved in DCM (200mL).Change material over to separatory funnel, and water (2 * 50mL), 3HCl (3 * 50mL), water (2 * 50mL), the salt water washing, through Na
2SO
4Drying, and vacuum is removed solvent.Thick residue is applied to Biotage, and with 9/1 heptane/eluent ethyl acetate, obtains the product 10g (53%) of pale solid shape,, it is used for next reaction without being further purified.
Preparation 2-chloro-5,6,7,8,9,10-six hydrogen cyclohepta [b] indole-6-Methanamides: with 2-chloro-5,6,7,8,9, (10g 36mmol) is dissolved in 7N ammonia in methanol (350mL), and changes the Parr pressure reactor over to 10-six hydrogen cyclohepta [b] indole-6-methyl formates.The purge reaction vessel substitutes all air with nitrogen simply.Then 90 ℃ of reacting by heating 48 hours.Reaction is cooled to room temperature, and vacuum is removed solvent, and thick residue is applied to Biotage, and gradient elution (1/1 heptane/ethyl acetate to 0/1 heptane/ethyl acetate), obtain the foamed product of canescence, it is ground with DCM, obtain the pure products 2g (21%) of pale solid shape.
Multiple embodiments of the present invention has been described.Yet, should be appreciated that and can carry out multiple improvement, and do not break away from the spirit and scope of the present invention.Other embodiment is in claims.
Sequence table
<110>Elixir Pharmaceuticals，Inc.
＜120〉method of treatment disease
<130>13407-092WO1
<140>US 11/077,664
<141>2005-03-11
<150>US 10/940,269
<151>2004-09-13
<160>7
<170>FastSEQ for Windows Version 4.0
<210>1
<211>747
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>1
Met Ala Asp Glu Ala Ala Leu Ala Leu Gln Pro Gly Gly Ser Pro Ser
1 5 10 15
Ala Ala Gly Ala Asp Arg Glu Ala Ala Ser Ser Pro Ala Gly Glu Pro
20 25 30
Leu Arg Lys Arg Pro Arg Arg Asp Gly Pro Gly Leu Glu Arg Ser Pro
35 40 45
Gly Glu Pro Gly Gly Ala Ala Pro Glu Arg Glu Val Pro Ala Ala Ala
50 55 60
Arg Gly Cys Pro Gly Ala Ala Ala Ala Ala Leu Trp Arg Glu Ala Glu
65 70 75 80
Ala Glu Ala Ala Ala Ala Gly Gly Glu Gln Glu Ala Gln Ala Thr Ala
85 90 95
Ala Ala Gly Glu Gly AsP Asn Gly Pro Gly Leu Gln Gly Pro Ser Arg
100 105 110
Glu Pro Pro Leu Ala Asp Asn Leu Tyr Asp Glu Asp Asp Asp Asp Glu
115 120 125
Gly Glu Glu Glu Glu Glu Ala Ala Ala Ala Ala Ile Gly Tyr Arg Asp
130 135 140
Asn Leu Leu Phe Gly Asp Glu Ile Ile Thr Asn Gly Phe His Ser Cys
145 150 155 160
Glu Ser Asp Glu Glu Asp Arg Ala Ser His Ala Ser Ser Ser Asp Trp
165 170 175
Thr Pro Arg Pro Arg Ile Gly Pro Tyr Thr Phe Val Gln Gln His Leu
180 185 190
Met Ile Gly Thr Asp Pro Arg Thr Ile Leu Lys Asp Leu Leu Pro Glu
195 200 205
Thr Ile Pro Pro Pro Glu Leu Asp Asp Met Thr Leu Trp Gln Ile Val
210 215 220
Ile Asn Ile Leu Ser Glu Pro Pro Lys Arg Lys Lys Arg Lys Asp Ile
225 230 235 240
Asn Thr Ile Glu Asp Ala Val Lys Leu Leu Gln Glu Cys Lys Lys Ile
245 250 255
Ile Val Leu Thr Gly Ala Gly Val Ser Val Ser Cys Gly Ile Pro Asp
260 265 270
Phe Arg Ser Arg Asp Gly Ile Tyr Ala Arg Leu Ala Val Asp Phe Pro
275 280 285
Asp Leu Pro Asp Pro Gln Ala Met Phe Asp Ile Glu Tyr Phe Arg Lys
290 295 300
Asp Pro Arg Pro Phe Phe Lys Phe Ala Lys Glu Ile Tyr Pro Gly Gln
305 310 315 320
Phe Gln Pro Ser Leu Cys His Lys Phe Ile Ala Leu Ser Asp Lys Glu
325 330 335
Gly Lys Leu Leu Arg Asn Tyr Thr Gln Asn Ile Asp Thr Leu Glu Gln
340 345 350
Val Ala Gly Ile Gln Arg Ile Ile Gln Cys His Gly Ser Phe Ala Thr
355 360 365
Ala Ser Cys Leu Ile Cys Lys Tyr Lys Val Asp Cys Glu Ala Val Arg
370 375 380
Gly Asp Ile Phe Asn Gln Val Val Pro Arg Cys Pro Arg Cys Pro Ala
385 390 395 400
Asp Glu Pro Leu Ala Ile Met Lys Pro Glu Ile Val Phe Phe Gly Glu
405 410 415
Asn Leu Pro Glu Gln Phe His Arg Ala Met Lys Tyr Asp Lys Asp Glu
420 425 430
Val Asp Leu Leu Ile Val Ile Gly Ser Ser Leu Lys Val Arg Pro Val
435 440 445
Ala Leu Ile Pro Ser Ser Ile Pro His Glu Val Pro Gln Ile Leu Ile
450 455 460
Asn Arg Glu Pro Leu Pro His Leu His Phe Asp Val Glu Leu Leu Gly
465 470 475 480
Asp Cys Asp Val Ile Ile Asn Glu Leu Cys His Arg Leu Gly Gly Glu
485 490 495
Tyr Ala Lys Leu Cys Cys Asn Pro Val Lys Leu Ser Glu Ile Thr Glu
500 505 510
Lys Pro Pro Arg Thr Gln Lys Glu Leu Ala Tyr Leu Ser Glu Leu Pro
515 520 525
Pro Thr Pro Leu His Val Ser Glu Asp Ser Ser Ser Pro Glu Arg Thr
530 535 540
Ser Pro Pro Asp Ser Ser Val Ile Val Thr Leu Leu Asp Gln Ala Ala
545 550 555 560
Lys Ser Asn Asp Asp Leu Asp Val Ser Glu Ser Lys Gly Cys Met Glu
565 570 575
Glu Lys Pro Gln Glu Val Gln Thr Ser Arg Asn Val Glu Ser Ile Ala
580 585 590
Glu Gln Met Glu Asn Pro Asp Leu Lys Asn Val Gly Ser Ser Thr Gly
595 600 605
Glu Lys Asn Glu Arg Thr Ser Val Ala Gly Thr Val Arg Lys Cys Trp
610 615 620
Pro Asn Arg Val Ala Lys Glu Gln Ile Ser Arg Arg Leu Asp Gly Asn
625 630 635 640
Gln Tyr Leu Phe Leu Pro Pro Asn Arg Tyr Ile Phe His Gly Ala Glu
645 650 655
Val Tyr Ser Asp Ser Glu Asp Asp Val Leu Ser Ser Ser Ser Cys Gly
660 665 670
Ser Asn Ser Asp Ser Gly Thr Cys Gln Ser Pro Ser Leu Glu Glu Pro
675 680 685
Met Glu Asp Glu Ser Glu Ile Glu Glu Phe Tyr Asn Gly Leu Glu Asp
690 695 700
Glu Pro Asp Val Pro Glu Arg Ala Gly Gly Ala Gly Phe Gly Thr Asp
705 710 715 720
Gly Asp Asp Gln Glu Ala Ile Asn Glu Ala Ile Ser Val Lys Gln Glu
725 730 735
Val Thr Asp Met Asn Tyr Pro Ser Asn Lys Ser
740 745
<211>389
<212>PRT
＜213〉homo sapiens
<400>2
Met Ala Glu Pro Asp Pro Ser His Pro Leu Glu Thr Gln Ala Gly Lys
1 5 10 15
Val Gln Glu Ala Gln Asp Ser Asp Ser Asp Ser Glu Gly Gly Ala Ala
20 25 30
Gly Gly Glu Ala Asp Met Asp Phe Leu Arg Asn Leu Phe Ser Gln Thr
35 40 45
Leu Ser Leu Gly Ser Gln Lys Glu Arg Leu Leu Asp Glu Leu Thr Leu
50 55 60
Glu Gly Val Ala Arg Tyr Met Gln Ser Glu Arg Cys Arg Arg Val Ile
65 70 75 80
Cys Leu Val Gly Ala Gly Ile Ser Thr Ser Ala Gly Ile Pro Asp Phe
85 90 95
Arg Ser Pro Ser Thr Gly Leu Tyr Asp Asn Leu Glu Lys Tyr His Leu
100 105 110
Pro Tyr Pro Glu Ala Ile Phe Glu Ile Ser Tyr Phe Lys Lys His Pro
115 120 125
Glu Pro Phe Phe Ala Leu Ala Lys Glu Leu Tyr Pro Gly Gln Phe Lys
130 135 140
Pro Thr Ile Cys His Tyr Phe Met Arg Leu Leu Lys Asp Lys Gly Leu
145 150 155 160
Leu Leu Arg Cys Tyr Thr Gln Asn Ile Asp Thr Leu Glu Arg Ile Ala
165 170 175
Gly Leu Glu Gln Glu Asp Leu Val Glu Ala His Gly Thr Phe Tyr Thr
180 185 190
Ser His Cys Val Ser Ala Ser Cys Arg His Glu Tyr Pro Leu Ser Trp
195 200 205
Met Lys Glu Lys Ile Phe Ser Glu Val Thr Pro Lys Cys Glu Asp Cys
210 215 220
Gln Ser Leu Val Lys Pro Asp Ile Val Phe Phe Gly Glu Ser Leu Pro
225 230 235 240
Ala Arg Phe Phe Ser Cys Met Gln Ser Asp Phe Leu Lys Val Asp Leu
245 250 255
Leu Leu Val Met Gly Thr Ser Leu Gln Val Gln Pro Phe Ala Ser Leu
260 265 270
Ile Ser Lys Ala Pro Leu Ser Thr Pro Arg Leu Leu Ile Asn Lys Glu
275 280 285
Lys Ala Gly Gln Ser Asp Pro Phe Leu Gly Met Ile Met Gly Leu Gly
290 295 300
Gly Gly Met Asp Phe Asp Ser Lys Lys Ala Tyr Arg Asp Val Ala Trp
305 310 315 320
Leu Gly Glu Cys Asp Gln Gly Cys Leu Ala Leu Ala Glu Leu Leu Gly
325 330 335
Trp Lys Lys Glu Leu Glu Asp Leu Val Arg Arg Glu His Ala Ser Ile
340 345 350
Asp Ala Gln Ser Gly Ala Gly Val Pro Asn Pro Ser Thr Ser Ala Ser
355 360 365
Pro Lys Lys Ser Pro Pro Pro Ala Lys Asp Glu Ala Arg Thr Thr Glu
370 375 380
Arg Glu Lys Pro Gln
385
<210>3
<211>399
<212>PRT
＜213〉homo sapiens
<400>3
Met Ala Phe Trp Gly Trp Arg Ala Ala Ala Ala Leu Arg Leu Trp Gly
1 5 10 15
Arg Val Val Glu Arg Val Glu Ala Gly Gly Gly Val Gly Pro Phe Gln
20 25 30
Ala Cys Gly Cys Arg Leu Val Leu Gly Gly Arg Asp Asp Val Ser Ala
35 40 45
Gly Leu Arg Gly Ser His Gly Ala Arg Gly Glu Pro Leu Asp Pro Ala
50 55 60
Arg Pro Leu Gln Arg Pro Pro Arg Pro Glu Val Pro Arg Ala Phe Arg
65 70 75 80
Arg Gln Pro Arg Ala Ala Ala Pro Ser Phe Phe Phe Ser Ser Ile Lys
85 90 95
Gly Gly Arg Arg Ser Ile Ser Phe Ser Val Gly Ala Ser Ser Val Val
100 105 110
Gly Ser Gly Gly Ser Ser Asp Lys Gly Lys Leu Ser Leu Gln Asp Val
115 120 125
Ala Glu Leu Ile Arg Ala Arg Ala Cys Gln Arg Val Val Val Met Val
130 135 140
Gly Ala Gly Ile Ser Thr Pro Ser Gly Ile Pro Asp Phe Arg Ser Pro
145 150 155 160
Gly Ser Gly Leu Tyr Ser Asn Leu Gln Gln Tyr Asp Leu Pro Tyr Pro
165 170 175
Glu Ala Ile Phe Glu Leu Pro Phe Phe Phe His Asn Pro Lys Pro Phe
180 185 190
Phe Thr Leu Ala Lys Glu Leu Tyr Pro Gly Asn Tyr Lys Pro Asn Val
195 200 205
Thr His Tyr Phe Leu Arg Leu Leu His Asp Lys Gly Leu Leu Leu Arg
210 215 220
Leu Tyr Thr Gln Asn Ile Asp Gly Leu Glu Arg Val Ser Gly Ile Pro
225 230 235 240
Ala Ser Lys Leu Val Glu Ala His Gly Thr Phe Ala Ser Ala Thr Cys
245 250 255
Thr Val Cys Gln Arg Pro Phe Pro Gly Glu Asp Ile Arg Ala Asp Val
260 265 270
Met Ala Asp Arg Val Pro Arg Cys Pro Val Cys Thr Gly Val Val Lys
275 280 285
Pro Asp Ile Val Phe Phe Gly Glu Pro Leu Pro Gln Arg Phe Leu Leu
290 295 300
His Val Val Asp Phe Pro Met Ala Asp Leu Leu Leu Ile Leu Gly Thr
305 310 315 320
Ser Leu Glu Val Glu Pro Phe Ala Ser Leu Thr Glu Ala Val Arg Ser
325 330 335
Ser Val Pro Arg Leu Leu Ile Asn Arg Asp Leu Val Gly Pro Leu Ala
340 345 350
Trp His Pro Arg Ser Arg Asp Val Ala Gln Leu Gly Asp Val Val His
355 360 365
Gly Val Glu Ser Leu Val Glu Leu Leu Gly Trp Thr Glu Glu Met Arg
370 375 380
Asp Leu Val Gln Arg Glu Thr Gly Lys Leu Asp Gly Pro Asp Lys
385 390 395
<210>4
<211>314
<212>PRT
＜213〉homo sapiens
<400>4
Met Lys Met Ser Phe Ala Leu Thr Phe Arg Ser Ala Lys Gly Arg Trp
1 5 10 15
Ile Ala Asn Pro Ser Gln Pro Cys Ser Lys Ala Ser Ile Gly Leu Phe
20 25 30
Val Pro Ala Ser Pro Pro Leu Asp Pro Glu Lys Val Lys Glu Leu Gln
35 40 45
Arg Phe Ile Thr Leu Ser Lys Arg Leu Leu Val Met Thr Gly Ala Gly
50 55 60
Ile Ser Thr Glu Ser Gly Ile Pro Asp Tyr Arg Ser Glu Lys Val Gly
65 70 75 80
Leu Tyr Ala Arg Thr Asp Arg Arg Pro Ile Gln His Gly Asp Phe Val
85 90 95
Arg Ser Ala Pro Ile Arg Gln Arg Tyr Trp Ala Arg Asn Phe Val Gly
100 105 110
Trp Pro Gln Phe Ser Ser His Gln Pro Asn Pro Ala His Trp Ala Leu
115 120 125
Ser Thr Trp Glu Lys Leu Gly Lys Leu Tyr Trp Leu Val Thr Gln Asn
130 135 140
Val Asp Ala Leu His Thr Lys Ala Gly Ser Arg Arg Leu Thr Glu Leu
145 150 155 160
His Gly Cys Met Asp Arg Val Leu Cys Leu Asp Cys Gly Glu Gln Thr
165 170 175
Pro Arg Gly Val Leu Gln Glu Arg Phe Gln Val Leu Asn Pro Thr Trp
180 185 190
Ser Ala Glu Ala His Gly Leu Ala Pro Asp Gly Asp Val Phe Leu Ser
195 200 205
Glu Glu Gln Val Arg Ser Phe Gln Val Pro Thr Cys Val Gln Cys Gly
210 215 220
Gly His Leu Lys Pro Asp Val Val Phe Phe Gly Asp Thr Val Asn Pro
225 230 235 240
Asp Lys Val Asp Phe Val His Lys Arg Val Lys Glu Ala Asp Ser Leu
245 250 255
Leu Val Val Gly Ser Ser Leu Gln Val Tyr Ser Gly Tyr Arg Phe Ile
260 265 270
Leu Thr Ala Trp Glu Lys Lys Leu Pro Ile Ala Ile Leu Asn Ile Gly
275 280 285
Pro Thr Arg Ser Asp Asp Leu Ala Cys Leu Lys Leu Asn Ser Arg Cys
290 295 300
Gly Glu Leu Leu Pro Leu Ile Asp Pro Cys
305 310
<210>5
<211>310
<212>PRT
＜213〉homo sapiens
<400>5
Met Arg Pro Leu Gln Ile Val Pro Ser Arg Leu Ile Ser Gln Leu Tyr
1 5 10 15
Cys Gly Leu Lys Pro Pro Ala Ser Thr Arg Asn Gln Ile Cys Leu Lys
20 25 30
Met Ala Arg Pro Ser Ser Ser Met Ala Asp Phe Arg Lys Phe Phe Ala
35 40 45
Lys Ala Lys His Ile Val Ile Ile Ser Gly Ala Gly Val Ser Ala Glu
50 55 60
Ser Gly Val Pro Thr Phe Arg Gly Ala Gly Gly Tyr Trp Arg Lys Trp
65 70 75 80
Gln Ala Gln Asp Leu Ala Thr Pro Leu Ala Phe Ala His Asn Pro Ser
85 90 95
Arg Val Trp Glu Phe Tyr His Tyr Arg Arg Glu Val Met Gly Ser Lys
100 105 110
Glu Pro Asn Ala Gly His Arg Ala Ile Ala Glu Cys Glu Thr Arg Leu
115 120 125
Gly Lys Gln Gly Arg Arg Val Val Val Ile Thr Gln Asn Ile Asp Glu
130 135 140
Leu His Arg Lys Ala Gly Thr Lys Asn Leu Leu Glu Ile His Gly Ser
145 150 155 160
Leu Phe Lys Thr Arg Cys Thr Ser Cys Gly Val Val Ala Glu Asn Tyr
165 170 175
Lys Ser Pro Ile Cys Pro Ala Leu Ser Gly Lys Gly Ala Pro Glu Pro
180 185 190
Gly Thr Gln Asp Ala Ser Ile Pro Val Glu Lys Leu Pro Arg Cys Glu
195 200 205
Glu Ala Gly Cys Gly Gly Leu Leu Arg Pro His Val Val Trp Phe Gly
210 215 220
Glu Asn Leu Asp Pro Ala Ile Leu Glu Glu Val Asp Arg Glu Leu Ala
225 230 235 240
His Cys Asp Leu Cys Leu Val Val Gly Thr Ser Ser Val Val Tyr Pro
245 250 255
Ala Ala Met Phe Ala Pro Gln Val Ala Ala Arg Gly Val Pro Val Ala
260 265 270
Glu Phe Asn Thr Glu Thr Thr Pro Ala Thr Asn Arg Phe Arg Phe His
275 280 285
Phe Gln Gly Pro Cys Gly Thr Thr Leu Pro Glu Ala Leu Ala Cys His
290 295 300
Glu Asn Glu Thr Val Ser
305 310
<210>6
<211>355
<212>PRT
＜213〉homo sapiens
<400>6
Met Ser Val Asn Tyr Ala Ala Gly Leu Ser Pro Tyr Ala Asp Lys Gly
1 5 10 15
Lys Cys Gly Leu Pro Glu Ile Phe Asp Pro Pro Glu Glu Leu Glu Arg
20 25 30
Lys Val Trp Glu Leu Ala Arg Leu Val Trp Gln Ser Ser Ser Val Val
35 40 45
Phe His Thr Gly Ala Gly Ile Ser Thr Ala Ser Gly Ile Pro Asp Phe
50 55 60
Arg Gly Pro His Gly Val Trp Thr Met Glu Glu Arg Gly Leu Ala Pro
65 70 75 80
Lys Phe Asp Thr Thr Phe Glu Ser Ala Arg Pro Thr Gln Thr His Met
85 90 95
Ala Leu Val Gln Leu Glu Arg Val Gly Leu Leu Arg Phe Leu Val Ser
100 105 110
Gln Asn Val Asp Gly Leu His Val Arg Ser Gly Phe Pro Arg Asp Lys
115 120 125
Leu Ala Glu Leu His Gly Asn Met Phe Val Glu Glu Cys Ala Lys Cys
130 135 140
Lys Thr Gln Tyr Val Arg Asp Thr Val Val Gly Thr Met Gly Leu Lys
145 150 155 160
Ala Thr Gly Arg Leu Cys Thr Val Ala Lys Ala Arg Gly Leu Arg Ala
165 170 175
Cys Arg Gly Glu Leu Arg Asp Thr Ile Leu Asp Trp Glu Asp Ser Leu
180 185 190
Pro Asp Arg Asp Leu Ala Leu Ala Asp Glu Ala Ser Arg Asn Ala Asp
195 200 205
Leu Ser Ile Thr Leu Gly Thr Ser Leu Gln Ile Arg Pro Ser Gly Asn
210 215 220
Leu Pro Leu Ala Thr Lys Arg Arg Gly Gly Arg Leu Val Ile Val Asn
225 230 235 240
Leu Gln Pro Thr Lys His Asp Arg His Ala Asp Leu Arg Ile His Gly
245 250 255
Tyr Val Asp Glu Val Met Thr Arg Leu Met Lys His Leu Gly Leu Glu
260 265 270
Ile Pro Ala Trp Asp Gly Pro Arg Val Leu Glu Arg Ala Leu Pro Pro
275 280 285
Leu Pro Arg Pro Pro Thr Pro Lys Leu Glu Pro Lys Glu Glu Ser Pro
290 295 300
Thr Arg Ile Asn Gly Ser Ile Pro Ala Gly Pro Lys Gln Glu Pro Cys
305 310 315 320
Ala Gln His Asn Gly Ser Glu Pro Ala Ser Pro Lys Arg Glu Arg Pro
325 330 335
Thr Ser Pro Ala Pro His Arg Pro Pro Lys Arg Val Lys Ala Lys Ala
340 345 350
Val Pro Ser
355
<210>7
<211>400
<212>PRT
＜213〉homo sapiens
<400>7
Met Ala Ala Gly Gly Leu Ser Arg Ser Glu Arg Lys Ala Ala Glu Arg
1 5 10 15
Val Arg Arg Leu Arg Glu Glu Gln Gln Arg Glu Arg Leu Arg Gln Val
20 25 30
Ser Arg Ile Leu Arg Lys Ala Ala Ala Glu Arg Ser Ala Glu Glu Gly
35 40 45
Arg Leu Leu Ala Glu Ser Ala Asp Leu Val Thr Glu Leu Gln Gly Arg
50 55 60
Ser Arg Arg Arg Glu Gly Leu Lys Arg Arg Gln Glu Glu Val Cys Asp
65 70 75 80
Asp Pro Glu Glu Leu Arg Gly Lys Val Arg Glu Leu Ala Ser Ala Val
85 90 95
Arg Asn Ala Lys Tyr Leu Val Val Tyr Thr Gly Ala Gly Ile Ser Thr
100 105 110
Ala Ala Ser Ile Pro Asp Tyr Arg Gly Pro Asn Gly Val Trp Thr Leu
115 120 125
Leu Gln Lys Gly Arg Ser Val Ser Ala Ala Asp Leu Ser Glu Ala Glu
130 135 140
Pro Thr Leu Thr His Met Ser Ile Thr Arg Leu His Glu Gln Lys Leu
145 150 155 160
Val Gln His Val Val Ser Gln Asn Cys Asp Gly Leu His Leu Arg Ser
165 170 175
Gly Leu Pro Arg Thr Ala Ile Ser Glu Leu His Gly Asn Met Tyr Ile
180 185 190
Glu Val Cys Thr Ser Cys Val Pro Asn Arg Glu Tyr Val Arg Val Phe
195 200 205
Asp Val Thr Glu Arg Thr Ala Leu His Arg His Gln Thr Gly Arg Thr
210 215 220
Cys His Lys Cys Gly Thr Gln Leu Arg Asp Thr Ile Val His Phe Gly
225 230 235 240
Glu Arg Gly Thr Leu Gly Gln Pro Leu Asn Trp Glu Ala Ala Thr Glu
245 250 255
Ala Ala Ser Arg Ala Asp Thr Ile Leu Cys Leu Gly Ser Ser Leu Lys
260 265 270
Val Leu Lys Lys Tyr Pro Arg Leu Trp Cys Met Thr Lys Pro Pro Ser
275 280 285
Arg Arg Pro Lys Leu Tyr Ile Val Asn Leu Gln Trp Thr Pro Lys Asp
290 295 300
Asp Trp Ala Ala Leu Lys Leu His Gly Lys Cys Asp Asp Val Met Arg
305 310 315 320
Leu Leu Met Ala Glu Leu Gly Leu Glu Ile Pro Ala Tyr Ser Arg Trp
325 330 335
Gln Asp Pro Ile Phe Ser Leu Ala Thr Pro Leu Arg Ala Gly Glu Glu
340 345 350
Gly Ser His Ser Arg Lys Ser Leu Cys Arg Ser Arg Glu Glu Ala Pro
355 360 365
Pro Gly Asp Arg Gly Ala Pro Leu Ser Ser Ala Pro Ile Leu Gly Gly
370 375 380
Trp Phe Gly Arg Gly Cys Thr Lys Arg Thr Lys Arg Lys Lys Val Thr
385 390 395 400
Claims (46)
1. treat or prophylactic method, this method comprises the chemical compound with formula (I) from effective dose to object that use:
Wherein,
R
1And R
2With they bonded carbon, form C
5-C
10Cycloalkyl, C
5-C
10Heterocyclic radical, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, C
6-C
10Aryl, or C
6-C
10Heteroaryl, each in them can be randomly by 1-5 R
5Replace; Or R
1Be H, S-alkyl, or S-aryl, and R
2Be amidoalkyl, wherein nitrogen is replaced by alkyl, aryl or aryl alkyl, and each in them is randomly further replaced by alkyl, halogen, hydroxyl or alkoxyl;
R
3And R
4With they bonded carbon, form C
5-C
10Cycloalkyl, C
5-C
10Heterocyclic radical, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, C
6-C
10Aryl, or C
6-C
10Heteroaryl, each in them are randomly by 1-5 R
6Replace;
Each R
5And R
6Be halogen independently, hydroxyl, C
1-C
10Alkyl, C
1-C
6Haloalkyl, C
1-C
10Alkoxyl, C
1-C
6Halogenated alkoxy, C
6-C
10Aryl, C
5-C
10Heteroaryl, C
7-C
12Aralkyl, C
7-C
12Heteroarylalkyl, C
3-C
8Heterocyclic radical, C
2-C
12Alkenyl, C
2-C
12Alkynyl, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, carboxyl, carboxylate (ester), cyano group, nitro, amino, C
1-C
6Alkyl amino, C
1-C
6Dialkyl amido, sulfydryl, SO
3H, sulfate (ester), S (O) NH
2, S (O)
2NH
2, phosphate (ester), C
1-C
4Alkylene dioxo base, oxo, acyl group, amino carbonyl, C
1-C
6Alkyl amino-carbonyl, C
1-C
6Dialkyl amino carbonyl, C
1-C
10Alkoxy carbonyl, C
1-C
10The thio alkoxy carbonyl, diazanyl carbonyl, C
1-C
6Alkyl hydrazine carbonyl, C
1-C
6Dialkyl group diazanyl carbonyl, the hydroxyl amino carbonyl; The alkoxy amino carbonyl; Or R
5Or R
6One of and R
7Formation contains the cyclic group of 4-6 carbon, a 1-3 nitrogen, a 0-2 oxygen and 0-2 sulfur, and it is randomly by oxygen or C
1-C
6Alkyl replaces;
X is NR
7, O, or S; Y is NR
7 ', O or S;
The optional two keys of----representative;
R
7And R
7 'Be hydrogen independently of one another, C
1-C
6Alkyl, C
7-C
12Aryl alkyl, C
7-C
12Heteroaryl alkyl; Or R
7With R
5Or R
6One of form the cyclic group contain 4-6 carbon, a 1-3 nitrogen, a 0-2 oxygen and 0-2 sulfur, it is randomly by oxygen or C
1-C
6Alkyl replaces; With
N is 0 or 1.
2. the process of claim 1 wherein R
1And R
2With they bonded carbon, form C
5-C
10Cycloalkyl, C
5-C
10Heterocyclic radical, C
5-C
10Cycloalkenyl group, C
5-C
10Heterocycloalkenyl, C
6-C
10Aryl, or C
6-C
10Heteroaryl, each in them can be randomly by 1-5 R
5Replace.
3. the process of claim 1 wherein R
1And R
2With they bonded carbon, form C
5-C
10Cycloalkenyl group.
4. the method for claim 3, wherein R
1And R
2By R
5Replace.
5. the method for claim 4, wherein R
5Be to be substituted the C that base replaces
1-C
6Alkyl or be substituted the amino carbonyl that base replaces.
6. the method for claim 5, wherein substituent group is amino substituent group or amino carbonyl.
7. the process of claim 1 wherein R
3And R
4With they bonded carbon, form C
6-C
10Aryl.
8. the method for claim 5, wherein R
3And R
4By R
6Replace.
9. the method for claim 6, wherein R
6Be halogen or C
1-C
6Alkyl.
10. the process of claim 1 wherein that n is 0.
11. the process of claim 1 wherein that X is NR
7
12. the process of claim 1 wherein that n is 0, and X is NR
7
13. the method for claim 1, it has following formula (X):
Formula (X).
14. the method for claim 13, wherein R
6Be halogen or C
1-C
6Alkyl.
15. the method for claim 13, wherein R
5It is amino carbonyl.
16. the method for claim 13, it has following formula (XI):
Formula (XI).
17. the method for claim 16, wherein R
6Be halogen or alkyl.
18. the method for claim 16, wherein R
5It is amino carbonyl.
19. the method for claim 16, wherein R
6Be halogen or alkyl, and R wherein
5It is amino carbonyl.
20. the method for claim 13, wherein chemical compound is a 6-chloro-2,3,4,9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides.
21. the method for claim 20, wherein chemical compound comprises the enantiomer with-14.1 optical rotations (c=0.33 DCM) that surpasses 60% enantiomeric excess.
22. the method for claim 21, wherein chemical compound comprises the enantiomer with-14.1 optical rotations (c=0.33 DCM) that surpasses 90% enantiomeric excess.
23. the chemical compound of claim 1, wherein with respect to non--SirT1 sirtuin, chemical compound preferentially suppresses SirT1.
24. the chemical compound of claim 1, wherein chemical compound has at least 5 times preferentially to SirT1.
25. the chemical compound of claim 1, wherein chemical compound has K less than about 1 μ M to SirT1
i
26. the process of claim 1 wherein that disease is a neoplastic disease.
27. the method for claim 26, wherein neoplastic disease is a cancer.
28. the process of claim 1 wherein that disease is a neurodegenerative disease.
29. the method for claim 28, wherein neurodegenerative disease is Alzheimer or parkinson.
30. the process of claim 1 wherein that disease is the relevant disease of adipose cell.
31. the method for claim 30, wherein using of chemical compound understood the lipogenesis that strengthens in the object.
32. the process of claim 1 wherein that disease is diabetes.
33. the method for claim 32, wherein object has type i diabetes.
34. the method for claim 32, wherein object has type ii diabetes.
35. the process of claim 1 wherein that object is differentiated in the danger that is in diabetes.
36. the method for claim 35 wherein reduces by glucose tolerance, and the patient is differentiated to be in the danger of diabetes.
37. the method for claim 35 wherein by the empty stomach hyperglycemia, is differentiated the patient for to be in the danger of diabetes.
38. the process of claim 1 wherein that disease is a metabolism syndrome.
39. the method for claim 38, wherein object has atherogenic dyslipidemia.
40. the method for claim 38, wherein to as if fat.
41. the method for claim 38, wherein object has insulin resistant or impaired glucose intolerance.
42. the method for claim 38, wherein object has hypertension.
43. have the chemical compound of formula (XI),
R wherein
6Be halogen or C
1-C
6Alkyl, and
P is 0,1, or 2; And
Wherein, chemical compound has and the 6-chloro-2,3,4 with optical rotation (c=0.33 DCM) of-14.1, the identical relative spatial chemistry of stereoisomer of 9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides.
44. the chemical compound of claim 43, wherein R
6Be chlorine or methyl.
45. the chemical compound of claim 43, wherein p is 1.
46. have the 6-chloro-2,3,4 of-14.1 optical rotation (c=0.33 DCM), the stereoisomer of 9-tetrahydrochysene-1H-carbazole-1-benzoic acid amides.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10/940,269 US20050209300A1 (en) | 2003-09-12 | 2004-09-13 | Methods of treating a disorder |
| US10/940,269 | 2004-09-13 | ||
| US11/077,664 | 2005-03-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101035527A true CN101035527A (en) | 2007-09-12 |
Family
ID=38731658
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200580033718 Pending CN101035527A (en) | 2004-09-13 | 2005-09-13 | Methods of treating a disorder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101035527A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636451A (en) * | 2017-03-10 | 2017-05-10 | 河北医科大学 | Biomarker for detecting occlusion or stenosis of coronary artery and preparation method thereof, and reagent kit containing biomarker |
| CN107077854A (en) * | 2014-07-28 | 2017-08-18 | 弗劳恩霍夫应用研究促进协会 | For processor, method and the computer program handled using truncation analysis or synthesis window lap audio signal |
| CN114371289A (en) * | 2020-10-15 | 2022-04-19 | 山东秉泰生物科技有限公司 | Marker for predicting tumor cell chemotherapy drug resistance and application thereof |
| CN114507290A (en) * | 2020-11-16 | 2022-05-17 | 上海市第一人民医院 | Fusion protein derived from truncated human SirT2 protein, and preparation method and application thereof |
| CN115029347A (en) * | 2022-05-11 | 2022-09-09 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating liver and kidney cell fibrosis, recombinant plasmid and virus inhibition |
-
2005
- 2005-09-13 CN CN 200580033718 patent/CN101035527A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107077854A (en) * | 2014-07-28 | 2017-08-18 | 弗劳恩霍夫应用研究促进协会 | For processor, method and the computer program handled using truncation analysis or synthesis window lap audio signal |
| US11664036B2 (en) | 2014-07-28 | 2023-05-30 | Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. | Processor and method for processing an audio signal using truncated analysis or synthesis window overlap portions |
| CN106636451A (en) * | 2017-03-10 | 2017-05-10 | 河北医科大学 | Biomarker for detecting occlusion or stenosis of coronary artery and preparation method thereof, and reagent kit containing biomarker |
| CN106636451B (en) * | 2017-03-10 | 2020-07-24 | 河北医科大学 | Biomarker for detecting coronary artery occlusion stenosis, preparation method thereof and kit containing biomarker |
| CN114371289A (en) * | 2020-10-15 | 2022-04-19 | 山东秉泰生物科技有限公司 | Marker for predicting tumor cell chemotherapy drug resistance and application thereof |
| CN114371289B (en) * | 2020-10-15 | 2024-02-23 | 山东秉泰生物科技有限公司 | Marker for predicting tumor cell chemotherapy resistance and application thereof |
| CN114507290A (en) * | 2020-11-16 | 2022-05-17 | 上海市第一人民医院 | Fusion protein derived from truncated human SirT2 protein, and preparation method and application thereof |
| CN115029347A (en) * | 2022-05-11 | 2022-09-09 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating liver and kidney cell fibrosis, recombinant plasmid and virus inhibition |
| CN115029347B (en) * | 2022-05-11 | 2024-02-20 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating hepatic and renal cell fibrosis, recombinant plasmid and virus inhibition |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8486990B2 (en) | SirT inhibitors that bind to NAD | |
| CN1305850C (en) | Sulfonylamino-derivatives as novel inhibitors of histone deacetylase | |
| CN1149204C (en) | 1-heterocycle substd. diarylamines | |
| CN1149085C (en) | Inhibition of RAF kinase using substituted heterocyclic ureas | |
| CN1960966A (en) | Hydrazide-containing CFTR inhibitor compounds and uses thereof | |
| CN1061658C (en) | Tricyclic benzazepine vasopression antagonists | |
| CA2538759C (en) | Substituted heterocyclic compounds as sirtuin inhitibitors | |
| JP2007515429A (en) | How to treat a disorder | |
| CN1798733A (en) | Carbonyl compounds as inhibitors of histone deacetylase for the treatment of disease | |
| CN1913880A (en) | Method for treating ADAMTS-5-associated disease | |
| CN101061111A (en) | DNA-PK inhibitors | |
| CN1791412A (en) | Novel compounds and their use in therapy | |
| CN1694703A (en) | Use of indolyl derivatives for the manufacture of a medicament for the treatment allergic rhinitis | |
| CN1901908A (en) | Therapeutic compounds and uses thereof | |
| CN1845915A (en) | Bipyridyl amines and ethers as modulators of metabotropic glutamate receptor-5 | |
| CN101035527A (en) | Methods of treating a disorder | |
| CN101035758A (en) | Phenyl derivatives and methods of use | |
| CN1768036A (en) | Indolone-acetamide derivatives, processes for preparing them and their uses | |
| CN1714076A (en) | Novel aryl ureido benzoic acid derivatives and their use | |
| CN1547472A (en) | Fused pyrimidines as d-alanyl-d-alanine ligase inhibitors | |
| CN1117092C (en) | Azablicyclic 5-HT, receptor ligands | |
| CN1812992A (en) | HIV integrase inhibitors | |
| CN1921841A (en) | Amines and amides for the treatment of diseases | |
| CN1505509A (en) | Method for treating allergies using substituted pyrazoles | |
| CN1268949A (en) | Fused thiophene compounds and medical use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20070912 |