CN1189816A - Azocompounds for the antemortem diagnosis of alzheimer's disease and in vivo imaging and prevention of amyloid depositon - Google Patents

Abstract

Amyloid binding compounds which are non-azo derivatives of Chrysamine G, pharmaceutical compositions containing, and methods using such compounds to identify Alzheimer's brain in vivo and to diagnose other pathological conditions characterized by amyloidosis, such as Down's Syndrome are described. Pharmaceutical compositions containing non-azo derivatives of Chrysamine G and methods using such compositions to prevent cell degeneration and amyloid-induced toxicity in amyloidosis associated conditions are also described. Methods using non-azo Chrysamine G derivatives to stain or detect amyloid deposits in biopsy or post-mortem tissue are also described. Methods using non-azo Chrysamine G derivatives to quantify amyloid deposits in homogenates of biopsy and post-mortem tissue are also described.

Description

Be used for the preceding diagnosis of Alzheimer death and in-vivo imaging and the azo-compound that prevents amyloid beta deposition

The present invention uses the National Institute of Ageing fund, grant number AG-05443, and AG-05133 and AG-08974, and finish.The application is the further part application of the U.S. Patent application serial number 08/432,019 of submission on May 1 nineteen ninety-five, and the latter is the further part application of the U.S. Patent application serial number 08/282,289 of submission on July 29th, 1994.

Background of invention

The present invention relates to be applicable to the evaluation that in vivo shows the compound of amyloid beta deposition the patient.More particularly, the present invention relates to a kind ofly, in the live body brain, make the method for diagnosing before the death of amyloid beta deposition imaging Alzheimer.The present invention also relates to the purposes of this compounds in treatment.

Alzheimer (" AD ") is a kind of neurodegenerative disease, it is characterized in that the damaged of loss of memory and other cognitive abilitys.Mckhann etc., neurological (Neurology) 34:939 (1984).It is a kind of reason of most common dementia in the U.S..AD may attack the younger person in 40-50 year, still, owing to be difficult to not have the big biosy of brain tissue of harm, and thereby be difficult to know disease time.The popular of AD increased with the age, estimated to have among the crowd in 85-90 year influenced by it up to 45-50%.Evans etc., JAMA 262:2551 (1989); Katzman, neurological (Neurology) 43:13 (1993).

According to definition, AD is diagnosed by the inspection of cerebral tissue when postmortem usually clearly.Khachaturian, neurological introduction (Arch.Neurol.) 42:1097 (1985); Mckhann etc., neurological (Neurology) 34:939 (1984).According to neuropathology, there is neuritis spot (NP) in being characterized as of this disease, neurofibrillary tangles (NFT), and neurone loss, and various other discoveries.Mann, old and feeble genesis mechanism (Mech, Ageing Dev.) 31:213 (1985).Die from the amyloid in the proteinaceous cell outer core that has neuritis spot in patient's the necrotomy section of cerebral tissue of Alzheimer with AD feature.

The amyloid of these neuritis spots nuclear is made up of the albumen that is called amyloid-beta (A β), and this albumen is arranged in the structure that the beta sheet leaf is dominant.Mori etc., journal of biological chemistry (Journal of Biological Chemistry) 267:17082 (1992); Kirschner etc., PNAS 83:503 (1986).The neuritis spot is the early stage constant feature of this disease.Mann etc., neurological magazine (J.Neurol.Sci.) 89:169:Mann etc., old and feeble genesis mechanism (Mech.Ageing Dev.) 31:213 (1985); Terry etc., neuropathology experiment magazine (J.Neuropathol.Exp.Neurol.) 46:262 (1 987).

The deposition at the initial stage of A β may occur in a very long time before clinical symptom occurs.Be generally used for diagnosing AD " minimum (micro-) microscopy standard " number based on the neuritis spot of in brain, finding.Khachaturian, neurological introduction (Arch.Neurol.), Supra (1985).Unfortunately, the quantity of neuritis spot must after death can measured.

The proteic neuritis spot of starch-containing sample is AD and Downs syndromes patient and the apolipoproteins E that develops into AD probably ₄The key character of allelotrope homozygote brain selective area.Corder etc., science (Science) 261:921 (1993); Divry, P., neuropathy and psychiatry magazine (J.Neurol, Psych.27:643-657 (1927); Wisniewski etc., inZimmerman, H. M. (ed): the development of neuropathology (PROGRESS INNEUROPATHOLOGY), (Grune and Stratton, N.Y.1973) pp.1-26.By with thioflavine S or Congo redly can easily detect amyloid in the brain to brain section dyeing.Puchtler etc., histological chemistry and cytochemistry magazine (J.Histochem.Cytochem.), 10:35 (1962).In showing, dichroism shown as yellow-green colour polarization look by the amyloid of congo red staining.Dichroism is in conjunction with the result who is the foliation structure of amyloid beta-folding.Glenner, medical journal (N.Eng.J.Med.) 302:1283 (1980).Biological chemistry and histochemical going through to amyloid can be consulted Glenner, medical journal (N.Eng.J.Med.) 302:1333 (1980).

So far, the diagnosis of AD is mainly by clinical criteria assessment, biosy of brain tissue and become celestial fabric study and realize.Improve the research of alzheimer's disease diagnosis method in vivo and comprise (1) genetics test, (2) immunoassay and (3) imaging technique.

Can infer that according to the catastrophe point of the A β precursor protein of finding in the rare family that has AD autosome dominant form at several A β metabolic disturbance is necessity and the sufficient condition that AD forms.Hardy, natural genetics (Nature Genetics) 1:233 (1992); Hardy etc., science (Science) 256:184 (1992).These sudden changes occur in and produce the necessary N-of A β and C-end from its precursor protein and split near the branch.St.George-Hyslop etc., science (Science) 235:885 (1987); Kang etc., nature (Nature) 325:733 (1987); Potter WO 92/17152.Yet the genetic analysis of a large amount of AD families has proved that AD is heterogeneous.St.George-Hyslop etc., nature (Nature) 347:194 (1990).Chain only in the family of some AD early onset thereofs, appearance on karyomit(e) 21 marks and in the family of AD late onset, not occurring.Recently, its product prediction contains karyomit(e) 14 genes of the regional also similar integral membrane proteins of multiple transfer film by evaluations such as Sherrington, nature (Nature) 375:754-760 (1995).This gene can reach and account for 70% of early onset thereof autosome dominance AD.The increase that preliminary data presentation karyomit(e) 14 sudden changes cause A β to produce.Scheuner etc., summary (Soc.Neurosci.Abstr.) 21:1500 (1995) of association of neurology department.The sudden change of closely similar with it gene is identified on the karyomit(e) 1 of the Volga German clan with early onset thereof AD.Levy-Lahad etc., science (Science) 269:973-977 (1995).

The auxiliary of AD diagnosis has been proposed as in genotypic screening to apolipoproteins E.Scott, nature (Nature) 366:502 (1993); Roses, neuroscience annual report (Ann.Neurol.) 38:6-14 (1995).But this technology is difficult to carry out, and this is because apolipoproteins E4 allelotrope only is the risk factor of AD, rather than the sign of disease.It does not exist in a lot of AD patients, and exists in a lot of not dull-witted old men.Bird, neuroscience annual report (AnnNeurol.) 38:2-4 (1995).

Immunoassay has been applied to the inspection of AD patient's neurochemistry mark, is used for checking the AD amyloid related with cerebrospinal fluid.Warner, analytical chemistry (Anal, Chem.) 59:1203A (1987); Potter, WO 92/17152; Glenner etc., U.S. Patent No. 4,666,829.The method of these diagnosis AD also fails to find all AD patients, early stage in disease particularly, and be invasive, need the backbone puncture.And, developed monoclonal antibody, be used for the probe of A β imaging.Majocha etc., Journal of Nuclear Medicine (J.Nucl.Med.), 33:2184 (1992); Najocha etc., WO 89/06242 and Majocha etc., United States Patent (USP) 5,231,000.The main drawback of antibody probe is to be difficult to make these macromole to pass through hemato encephalic barrier.Use antibody to diagnose AD to need significantly unusual hemato encephalic barrier in vivo, so that it is entered in the brain.Evidence proof AD patient hemato encephalic barrier on the function of not making us believing exists unusual really.Kalaria, the cerebrovascular and brain metabolism comment (Cerebrovascular ﹠amp; BrainMetabolism Review 4:226 (1992).

A β antibody is used for the AD diagnosis also shortcoming, and this is because they typically make the sedimentary A β dyeing of diffusion outside the neuritis spot, and this diffusion spot is usually preponderated.Yamaguchi etc., neuropathology (Acta Neuropathol.), 77:314 (1989).The diffusion spot can be independent class damage, not necessarily is included in the dull-witted process of AD, and this can find out by find the diffusion spot in normal control group of the same clan and geriatric dog.Moran etc., clinical medicine (MedicinaClinica) 98:19 (1992); Shimada etc., veterinary science magazine (Journal of VeterinaryMedical Science) 54:137 (1992); Ishihara etc., brain research (Brain Res.) 548:196 (1991); Giaccone etc., neurological (Neurosci.Lett) 144:178 (1990).If even the diffusion spot be the omen of neuritis spot, the pathology activity of the key of AD may be converted into the process of the neuritis spot relevant with the halogen sex change for obvious benign diffusion spot.Therefore, needing proof neuritis spot and NETs is the AD physiopathology mark that has more feature than the equally also antibody of mark diffusion spot.

Recently, radio-labeling A β peptide has been used to the diffusion in the mark AD brain section, reaches the spot of the types of nerve closely.Maggio etc., WO93/04194.These peptides have all shortcomings of antibody.Particularly peptide can not normally be popped one's head in and the reaction of diffusion spot by hemato encephalic barrier with because of these with the required amount of imaging, and they may not be narrow spectrum to AD.

The Congo red diagnosis that can be used for amyloidosis in the in vivo non-brain parenchymal tissue.Congo redly may be unsuitable in vivo the sedimentary diagnosis of A β in the brain, this is that the iodate of injected dose O.03% is Congo red can to enter the brain entity because have only.Tubis etc., U.S.'s pharmaceutical journal (J.Amer.Pharm.Assn.) 49:422 (1960).With Congo red relevant radioiodinated BDB compounds,, the existence that is used for amyloid beta deposition in external and intravital patient's organ and the inspection at position, place have been disclosed as Benzo Orange R and Direct Blue 4.Quay etc., United States Patent(USP) Nos. 5,039,511 and 4,933,156.But to Congo red similar, disclosed all compounds of Quay all contain the strongly-acid sulfonic group, and it has strictly limited these compounds and has entered in the brain, make it be difficult to reach imaging significant quantity or detection significant quantity in the brain entity.

Can not measure the research that the AD amyloid beta deposition has hindered this fatal disease before dead.The method of quantitative assay amyloid beta deposition before dead, not only as the diagnostic tool under a kind of slight or clinical clouding of consciousness situation but also as with prevent A β be deposited as purpose result of treatment detection means and be required.Therefore, at present most important be still seek a kind of by to the amyloid imaging in the live body brain entity and in the safe specific method of antemortem diagnosis AD.Though carried out the various trials of in vivo diagnosing AD at present, still do not had the dead preceding method of surveying amyloid in the brain.Do not have a kind of method to use hypotoxicity, can pass through hemato encephalic barrier, and than normal brain easier with the AD brain detection thing at amyloid of bonded high-affinity effectively, be beneficial to before the patient is dead the deposition of AD amyloid in the mensuration brain.Therefore, still do not have the method proof of in vivo diagnosing AD and reached these discrimination standards.

Recent data presentation amyloid binding compounds has the possibility of treatment AD and type 2 diabetes.As mentioned above, in the AD brain, there is the spot of two kinds of wide types, diffusion spot and neuritis spot (typically).The diffusion spot seems not induce the morphology reaction as reactive astrocyte, malnutritive aixs cylinder, microgliacyte, cynapse forfeiture, and whole complement activations of finding in the neuritis spot.Joachim etc., American Journal of Pathology (Am.J.Patho1.) 135:309 (1989); Masliah etc., loc.cit.137:1293 (1990); Lue and Rogers, dull-witted (Dementia) 3:308 (1992).The reaction of these morphology all means at contiguous neuritis spot and neurotoxic and cytopathy reaction has taken place the sedimentary zone of A β closely.A β-inductive neurotoxic and cytopathy are reported in some cell in vitro models to some extent.Yankner etc., science (Science) 250:279 1990); Roher etc., BBRC 174:572 (1991); Frautschy etc., Proc.Natl.Acad.Sci.88:83362 (1991); Shearman etc., loc.cit.91:1470 (1994).The aggegation that has now found that A β peptide must cause external neurotoxic.Yankner, neurophysiology annual report (Neurobiol.Aging) 13:615 (1992).The difference of A β state of aggregation can illustrate that the toxic reaction of diffusion spot peripheral nerve is less in diffusion spot and the neuritis spot.Lorenzo and Yankner, Proc.Natl.Acad.Sci., 91:12243 (1994).Recently, the result of Congo red vitro inhibition A β-inductive neurotoxic and cytometaplasia has all been reported in three laboratories.Burgevin etc., neuroscience report (NeuroReport) 5:2429 (1994); Lorenzo and Yankner, Proc.Natl, Acad.Sci.91:12243 (1994); Pollack etc., neuroscience wall bulletin (Neuroscience Letters) 184:113 (1995); Pollack etc., neuroscience wall bulletin (Neuroscience Letters) 197:211 (1995).As if its mechanism comprise and suppress fibriilar formation and prevent to form fibriilar neurotoxic character.Lorenzo and Yankner, Proc.Natl.Acad.Sci.91:12243 (1994).The Congo red protection islet cells that also shown is avoided the neurotoxic effect that dextrin (amylin) causes.Lorenzo and Yankner, Proc.Natl.Acad.Sci.91:12243 (1994).Dextrin is a kind of Fibrinopeptide similar to A β, and it is in diabetes B patient's pancreas accumulation.

Known some azoic dyestuff of those skilled in the art can cause cancer.Morgan etc., environmental health perspective (Environmental Health Perspectives, 102 (supp.) 2:63-78 (1994).This potential carcinogenicity is owing to such reason to a great extent.Azoic dyestuff by intestinal bacterium in large quantities metabolism in free human body amine.Cerniglia etc., and biochemical physiological Study (Biochem, Biophys.Res.Com.), 107:1224-1229 (1982).When using some benzidine dye (and much the p-diaminodiphenyl of other replacements), unhindered amina is a carcinogen.These facts are slightly enlightening to the amyloid imaging research that the indivisible radiolabeled dyestuff of high specific activity is injected directly in the blood.In this case, using dosage is insignificant, and dyestuff has been avoided intestinal bacterium.

In therepic use, these factors are extremely important.It is unacceptable discharging a kind of known carcinogen from the treatment compound.Second problem that the azoic dyestuff metabolism brings be a large amount of medicines of taking before absorption by the metabolism of intestinal bacterium institute.Its shortcoming is that even without discharging metabolic substd, it has also reduced bioavailability.

Therefore, need to seek amyloid bonded compound, it is to Congo red similar, but can enter in the brain (Congo red can not).Such compound can be used for preventing forming relevant cytopathy with protofibril, thereby can treat the pathological state with amyloid related disease, and treatment diabetes B islet cells is poisoned.

Further need to seek nontoxic, bioavailability is high, thereby can be used for treating with amyloid bonded compound.

Brief summary of the invention

Therefore, the purpose of this invention is to provide a kind of by in vivo to the amyloid imaging in the brain entity, thereby in the method for the safe special efficacy of antemortem diagnosis AD.It is hypotoxic that another object of the present invention provides a kind of use, to the amyloid high-affinity, can pass through hemato encephalic barrier, and can distinguish the probe of AD brain and normal brain activity, identifies the method for AD amyloid beta deposition in the brain before patient is dead.Another purpose provides a kind of A of preventing β deposition or toxic AD methods of treatment.Another purpose provides a technology that in living tissue or after death amyloid beta deposition is dyeed and detects in the tissue samples.Another purpose provide a kind of in biological tissue or tissue samples homogenate after death the method for quantitative assay amyloid beta deposition.

In order to reach these purposes or other purpose, according to an aspect of the present invention, provide here and amyloid bonded formula I compound or its water soluble non-toxic salt: Wherein: Be N=N, C ≡ C, or CR '=CR ' (wherein R ' expression H or low alkyl group); X is C (R ") ₂

(wherein R " be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

Wherein R ' is H or low alkyl group) or X be CH=CH, N=N, C=O, O, NR ' (wherein R ' expression H or low alkyl group), S, or SO ₂R ₁And R ₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or group as follows: Q is selected from down one of array structure independently of one another, respectively contains a carboxylic acid or sour sample functionality: IA, IB, and IC, ID, IE, IF and IG, wherein

IA has structure as follows:

R wherein ₃, R ₄, R ₅, R ₆, or R ₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or group as follows:

On two Q ', all be its R wherein ₃, R ₄, R ₅, R ₆, or R ₇At least one is arranged is hydroxyl, tetrazolium ， oxadiazole, NO ₂, or carboxyl; And work as R ₅Be hydroxyl R ₆R during for carboxyl or its lower alkyl esters ₁, R ₂, R ₃, R ₄, or R ₇Have at least one not to be H; Work as R ₅Be amino, R ₆Be carboxyl or its lower alkyl esters, and R ₇During for hydroxyl, R ₁, R ₂, R ₃, or R ₄At least one is not H; Work as R ₂Be CH ₃, R ₅Be hydroxyl, and R ₆During for carboxyl or its lower alkyl esters, R ₁, R ₃, R ₄, or R ₇At least one is not H; Work as R ₃, R ₄, or R ₇Be CH ₃, R ₅Be hydroxyl R ₆During for carboxyl or its lower alkyl esters, R ₁Or R ₂At least one is not H or CH ₃

IB has structure as follows:

Wherein: R ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or

On two Q ', all be its R wherein ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆Have at least one to be hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

IC has structure as described below: Wherein: R ₁₇, R ₁₈, R ₁₉, R ₂₀, or R ₂₁In at least one is H, F, Cl, Br, I low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or the triazene that is shown below:

(R wherein ₈And R ₉Be low alkyl group) or

ID has structure as follows:

Wherein: R ₂₂, R ₂₃, or R ₂₄Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or

The heterocyclic radical of one of representing in following six formulas:

IE has structure as follows:

Wherein: R ₂₅, R ₂₆, or R ₂₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or

The heterocyclic radical of one of representing in following six formulas:

IF has structure as follows:

Wherein: R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂One of be above-mentioned formula I definition

Connect base, remaining R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or

All be its R on two Q ' wherein ₂₈, R ₂₉, R ₃₀, R ₃₁Or R ₃₂Have at least one to be hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

IG has structure as follows:

R wherein ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉One of be as above-mentioned formula I definition

Connect base, remaining R ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or

All be R on two Q ' wherein ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉Have at least one to be hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

Another object of the present invention provide definition as above with amyloid bonded formula I compound, or its water soluble non-toxic salt, wherein substituent R ₁-R ₇And R ₁₀-R ₃₉In have at least one be selected from by ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F, ¹²⁵I, CH ₂-CH ₂- ¹⁸F, O-CH ₂-CH ₂- ¹⁸F, CH ₂-CH ₂-CH ₂- ¹⁸F, O-CH ₂-CH ₂-CH ₂- ¹⁸(wherein at least one carbon is the carbon containing substituting group that limits among F and the formula I ¹¹C or ¹³C) one group of formation.

Another object of the present invention provide definition as above with amyloid bonded formula I compound, or its water soluble non-toxic salt, wherein when by combine when measuring compound and A β bonded dissociation constant (K with synthetic A β peptide or Alzheimer cerebral tissue _D) between 0.0001 to 10.0 μ M.

Other purpose of the present invention provides a kind of synthetic definition as above, and at least one substituent R wherein ₁-R ₇And R ₁₀-R ₃₉Be selected from by ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F and ¹⁹One group of constituting of F, can with the method for amyloid bonded formula I compound or its water soluble non-toxic salt, comprising making wherein at least one substituent R ₁-R ₇And R ₁₀-R ₃₉For the definition of trialkyltin as above with amyloid bonded formula I compound or its water soluble non-toxic salt with contain ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹The step of the halide reagent reaction of F.

Another object of the present invention provides a kind of in vivo pharmaceutical composition of amyloid beta deposition imaging that is used for, comprising (a) substituent R ₁-R ₇And R ₁₀-R ₃₉In have at least one be selected from by ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹(wherein at least one carbon is the carbon-containing group that limits among F and the formula I ¹¹C or ¹³C) constitute one group, or its water soluble non-toxic salt and (b) pharmaceutically acceptable carrier.

Another object of the present invention provides a kind of method that in vivo detects amyloid beta deposition, may further comprise the steps: but (a) use the aforementioned pharmaceutical compositions of detection limit, and (b) in this live body, detect this compound and deposit combining of amyloid.One object of the present invention also provides a kind of method that detects amyloid beta deposition in vivo, and wherein amyloid beta deposition betides in detected person's brain.Method of the present invention can be used for suspecting to suffer from the Alzheimer of being selected from, Down syndrome, and apolipoproteins E ₄The patient of this class of allelotrope homozygote and amyloidosis diseases associated and syndromes.

Another object of the present invention relates to cytopathy and toxic pharmaceutical composition and the method that forms association in the prevention symptom relevant with amyloidosis such as AD and the diabetes B with protofibril.Such pharmaceutical composition comprises chrysamine G (Chrysamine G) or its said derivative and pharmaceutically acceptable carrier.Such compound is nontoxic.

Another object of the present invention relates to as to living tissue or amyloid beta deposition radiography and dyeing and the application of the detection thing measured in the tissue samples after death.

Another object of the present invention relate to living tissue or after death in the tissue samples radio-labeling of amyloid beta deposition quantitative assay survey the application of thing.

Another object of the present invention relates to a kind of method of distinguishing Alzheimer brain and normal brain activity.

Other purposes of the present invention, feature and advantage will become more obvious from following detailed description.But, should be understood that what detailed description that these represent preferred version of the present invention and embodiment just were presented in illustrational mode, therefore from this detailed description, various variations within essence spirit of the present invention and the scope and modification all are conspicuous for a person skilled in the art.

Brief description of the drawings

Figure 1A represents chrysamine G (Chrysamine G) and several chrysamine G analogue or derivatives of being synthesized and having tested, comprises 3-iodine derivative (3-JCG), 3-iodine dimethoxy derivative (3-JCG (OM) ₂), dimethyl esters derivative (CG (COOMe) ₂), amphyl, Whitfield's ointment (SA), anils (1/2 CG), Congo red, 3,3 '-the diiodo-derivative (3,3 '-I ₂CG), 3,3 '-two br-derivatives (3,3 '- Br ₂3,3 CG), '-dichloro-derivatives (3,3 '-Cl ₂CG), the chemical structure of 3-br-derivatives (3-bromine BrCG) and 5-fluorosalicylic acid derivatives ((5-FSA) CG).Among the figure numeral refer to each compound suppress [ ¹⁴C] chrysamine G and synthetic peptide, A β (10-43) bonded K _i(in μ M).

Figure 1B represents chrysamine G analogue or the derivative that several have been synthesized and have tested, comprise 3,3 '-dicarboxylic acid derivatives (3,3 '-(COOH) ₂2,2 of chrysamine G CG), '-disulfonic acid derivatives (2,2 '-(SO ₃) ₂CG), 3-bromine, 3-sec.-propyl salicyclic acid derivatives (3-Br-(3-iPrSA) CG), 3-sec.-propyl salicyclic acid derivatives ((3-iPrSA) CG), 2,4-biphenol derivative (2,4-Diphenol), γ-resorcylic acid derivative ((6-OHSA) CG), 3,3 ', 5,5 ' tetramethyl benzidine derivative (3,3 ', 5,5 '-(CH ₃) ₄3,3 CG), '-the dimethyl derivative (3,3 '-(CH ₃) ₂2,2 CG), '-the dimethyl derivative (2,2 '-(CH ₃) ₂CG), benzisoxazole derivatives (CGBenzisoxazole) and the 3-carboxyl alkyne derivatives (chemical structure of 3-(COOH)-C3C).Among the figure numeral refer to each compound suppress [ ¹⁴C] chrysamine G and synthetic peptide, A β (10-43) bonded K _i(in μ M).

Fig. 2 A-2K represents chrysamine G and chrysamine G analogue, the chemical structure of the trialkyltin derivative of special heterocyclic analogs.Notice that these structural formulas represent half of a molecule, this molecule is that the center is symmetric with top-right wavy key, unless the trialkyltin part is only on one side of xenyl.The trialkyltin derivative for the stable intermediate of preparation high specific activity halo radioactivity derivative and moment precursor.Heterocyclic analogs is illustrated in the selectivity mode that replaces the slightly acidic part on the same structure position of moderate acid hydroxy-acid group of chrysamine G.These trialkyltin precursor compounds are represented with its protonated form, also comprise their non-protonization form and tautomeric form but those skilled in the art understand these figure.2A) chrysamine G.2B) chrysamine G trialkyltin derivative.2C) 3-hydroxyl-1, the similar thing trialkyltin of 2-benzoisoxazole derivative.2D) phthalic imidine or isoindole-1,3 (2H)-two keto analog trialkyltin derivative.2E) Phthalocyclohydrazide or 2,3-benzo pyridazine-1,4 (2H, 3H)-two keto analog trialkyltin derivative.2F) 2,3-benzoxazine-1,4 (3H)-two keto analog trialkyltin derivative.2G) (2H) 1,3-benzoxazine-2,4 (3H)-two keto analog trialkyltin derivative.2H) (3H) 2-isoquinoline 99.9 (benzazine)-1,3 (2H)-two keto analog trialkyltin derivative.2I) 1,8-naphthalimide analogue trialkyltin derivative.2J) tetrazolium analogue trialkyltin derivative.2K) oxadiazole analogue trialkyltin derivative.

Fig. 3 by the analog of several chrysamine Gs make [ ¹⁴C] chrysamine G and A β (10-43) bonded replacement curve.Be abbreviated as among Fig. 1 used.Fig. 3 A) chrysamine G (empty triangle); (5-FSA) CG (real rhombus); 3,3 '-(COOH) ₂CG (real square); 2,2 '-(SO ₃) ₂CG (real circular).Fig. 3 B) chrysamine G (empty triangle); Congo red (empty circular); Anils (empty inverted triangle); Phenol derivatives (empty square); Whitfield's ointment (X).Show in high density and to show in conjunction with the enhanced curve and to have formed micelle.Bedaux, F etc., pharmacy weekly (Pharm, Weekblad) 98:189 (1963).

Fig. 4 A chrysamine G and A β (10-43) bonded Scatchard curve.This curve representation is to two kinds of nonlinear least squares of binding site model independently.Straight line is represented one-component.

Fig. 4 B be [ ¹⁴C] CG and typical contrast (rhombus) and AD brain sample (square) bonded Scatchard analysis.The dotted line slope is identical with the AD line so that with the contrast slope ratio.AD brain sample is 48NP/ * 200 multiplying powers, 0.35 μ M K _DAnd 790fmol/ μ g B _MaxAlbumen.Contrast is 0.48 μ M K _DAnd 614fmol/ μ gB _MaxAlbumen.

Fig. 5 is the curve of expression combination mensuration with respect to the linear relationship of peptide concentration.About 0.9 μ gA β (10-43) is used for typical mensuration.

Fig. 6 A is the curve of expression chrysamine G and A β (10-43) bonded time course.

Fig. 6 B is in conjunction with velocity constant (K ₁) the mensuration graphic representation.

Fig. 6 C is that chrysamine G is gone up dissociated time course curve from A β (10-43).

Fig. 7 is an interactional molecular model synoptic diagram between chrysamine G and the A β.

Fig. 8 A for expression combined [ ¹⁴C] the synoptic diagram of relation in the amount of chrysamine G and the AD brain sample between neuritis spot (NP) number.

Fig. 8 B for expression combined [ ¹⁴C] the synoptic diagram of relation in the amount of chrysamine G and the AD brain sample between neurofibrillary tangles (NFT) number.In Fig. 8 A and 8B, the x-axle is illustrated in/middle volume (n=10) or go up in the Bielschowsky stained of temporo cortex (n=10) average N P or NFT number with * 200 enlargement ratio, filled symbols and solid line represent not have the brain of amyloid blood vessel disease, and open symbols and dotted line represent to have the brain of amyloid blood vessel disease.Y-axis is illustrated in the homogenate that is used for the adjacent brain sample of painted brain total, pure bonded [ ¹⁴C] chrysamine G (fmol/ μ g albumen).75 μ g albumen and 150nM[have approximately been used ¹⁴C] chrysamine.

Fig. 9 A, 9B and 9C Fig. 9 A show the AD brain sample that has above the 20NPs/x200 enlargement ratio, i.e. " high spot AD brain " sample, in be attached to the chrysamine G in each brain zone.Fig. 9 B shows to have the AD brain sample that is lower than the 20NPs/x200 enlargement ratio, i.e. " low spot AD brain " sample, in be attached to the chrysamine G in each brain zone.Data point represent to be combined in specified brain zone [ ¹⁴C] chrysamine G be combined in the cerebellum of same brain [ ¹⁴C] ratio of chrysamine G.Horizontal strip chart shows mode, and the error strip chart shows standard deviation in contrast (annulus) and the AD brain (rhombus among 9A and the 9B).The brain zone comprises antinion (FP), head of caudate nucleus (CAU), on/middle volume (SMF), last temporo (ST), lower wall (IP) and look portion (OC) cortex.Asterisk refer to the contrast ratio have notable difference ( ^*P＜0.05; ^*P＜0.001).Fig. 9 C represents two Down ' s syndromes brain samples.Rhombus represents not find 23 years old Down ' s syndromes patient's of AD symptom brain among the 9C.The 9C intermediate cam is represented 51 years old Down ' s syndromes patient's brain.This patient develops into AD 40 years old the time as most of Down ' s syndromes patients.

Figure 10 for will [ ¹⁴C] curve of the level of chrysamine G the chrysamine G mouse tissue of putting to death from outside tail vein injection and time shown in the figure.Absolute activity in hollow symbol and the fine rule representation unit cpm/g tissue (left side axle).Solid symbol and solid line are represented the ratio of brain radioactivity and kidney (last figure) or blood (middle figure).Ratio is marked on right axle.

The last figure of Figure 11: use Stokes and Triokey, the method for clinical pathology magazine (J.Clin.Pathol.) 26:241-242 (1973) is cut into slices with temporal lobe under the painted AD brain of chrysamine G.Two neuritis spots are high-visible.Figure below: with the AD patient's of amyloid blood vessel disease adjacent temporal lobe section.Left figure: with the painted brain section of congo red method of Puchtler.Puchtler etc., histological chemistry and cytochemistry magazine (J.Histochem.Cytochem.) 10:35 (1962).Flagpole pattern is represented 20 microns.Right figure: the adjacent brain section that replaces congo red staining in the Puchtler method with chrysamine G.Two sections have all easily shown the vascular that identical amyloid is full of.The a small amount of background autofluorescence of red corpuscle and lipofuscin is also high-visible.All Photomicrographs all use fluorescein isothiocyanate (FITC) filter to obtain.

Figure 12 shows that having or not chrysamine G to exist increases the bar graph of A β (25-35) concentration to the influence of the redox active of rabbit pheochromocytoma (PC-12) cell down, it is by bromination 3-[4,5-dimethylthiazole-2-yl]-2,5-phenylbenzene tetrazolium, MTT, reductive action is measured.The absorption of MTT reduzate at the 560nm place is marked in vertical axle.Have only the effect of A β (25-35) to be shown in solid bar graph, show dose is decided by that the MTT reductive reduces among the figure.Be shown in blank space in the solid bar graph with the significant difference of control group (no A β (25-35), no chrysamine G).The provide protection of 20 μ M chrysamine Gs is shown in the hollow bar graph.MTT reductive significant difference is shown in the hollow bar graph with surplus when having or not chrysamine G to exist.

Figure 13.Show the bar graph of the concentration of increase chrysamine G to the protection effect of the reductive action of A β (25-35) inductive rabbit pheochromocytoma (PC-12) cellular oxidation reducing activity; it is by bromination 3-[4,5-dimethylthiazole-2-yl]-2,5-phenylbenzene tetrazolium; MTT, reductive action is measured.The absorption of MTT reduzate at the 560nm place is marked in vertical axle.The effect of chrysamine G is shown in the solid bar graph during no A β (25-35).Control group (no A β (25-35), no chrysamine G) is at no significant difference in the presence of the no A β (25-35) and between the chrysamine G of any concentration.MTT reductive action when the chrysamine G that exists 1 μ MA β (25-35) to increase with concentration is shown in the hollow bar graph.Under each concentration chrysamine G condition, be shown in the white place of solid bar graph in the significant difference that has or not the MTT reductive action of A β (25-35) between existing, the significant difference that adds the MTT reductive action between the chrysamine G that increases concentration at A β (25-35) control group (no chrysamine G) and A β (25-35) is shown in the black place of hollow bar graph.

Figure 14 chrysamine G amphyl nonactive with it is to the comparison by A β (25-35) inductive toxic action.Except that control group, the concentration of A β (25-35) is 1 μ M in all experiments.Concentration is that the chrysamine G of 0.1 and 1 μ M has shown provide protection, but amphyl does not show provide protection, has perhaps also increased the toxicity of A β.

The detailed description of preferred version

The present invention developed chrysamine G and radiolabeled derivative thereof by intravital hemato encephalic barrier and with neuritis (but non-diffusion) spot in sedimentary A β, with sedimentary A β in the cerebrovascular amyloid, and with the amyloid bonded ability that constitutes by sedimentary albumen among the NFT.Chrysamine G is Congo red derivative, and its important textural difference is that Congo red sulfonic acid part is replaced by hydroxy-acid group in chrysamine G (Fig. 1).This structural modification makes chrysamine G than Congo red and macromole such as antibody is easier enters in the brain.Tubis etc., American Pharmaceutical Association's will (J.Am.Pharmaceut.Assn.) 49:422 (1960).And chrysamine G may be the AD physiopathology sign more special than antibody, and the latter is mark diffustivity spot also, and this is because chrysamine G has tangible specificity to neuritis spot and NFTs.

Chrysamine G derivative of the present invention has following feature: (1) combines with the specificity of synthetic A β in vivo, and (2) combine with the non-diffusion spot of neuritis in brain section, the ability of the hemato encephalic barrier that (3) are in vivo gone beyond by difficulty.

Method of the present invention can be measured the generation and the position of amyloid beta deposition in patient's organ or the body district, particularly brain.This method comprises uses the formula I compound that contains defined above being called as " mensuration compound " of mensuration significant quantity or the pharmaceutical composition of its pharmaceutically acceptable water-soluble salt to the patient.After referring to use, " mensuration significant quantity " can satisfy the combine amount of measuring required mensuration compound of compound with amyloid.After referring to use, " imaging significant quantity " can satisfy the compound amount that picture is measured needs that is combined into to compound and amyloid.

The present invention uses the amyloid probe, this probe and impassivity damage imaging technique such as nuclear magnetic resonance spectroscopy (MRS) or nuclear magnetic resonance art (MRI), or γ imaging art, be used from the in vivo quantitative assay of amyloid beta deposition as positron radiation x-ray tomography gamma radiography (PET) or single photon emission x-ray ct gamma radiography (SPECT).Term " in vivo imaging " refers to measure the chrysamine G of mark, the method of the chrysamine G derivative of mark or the formula I compound of the present invention of aforesaid mark, with regard to the γ imaging, measurement is from checked organ or the regional ray of launching, and it is expressed the ratio of the total binding in described ratio is undertaken during the same in-vivo imaging method by the total binding in the standardized tissue and (for example being divided by) in another tissue of same measured with total binding value or a kind of ratio.In vivo total combination is with total signal definition of measuring in a tissue by the living imaging technology, and this technology does not need by injecting equivalent tagged compound and excessive unmarked for the second time, but the identical compound of other chemical property is proofreaied and correct." testee " is Mammals, and preferred people most preferably suspects the people who suffers from dementia.

In order to realize the purpose of living imaging, the type of available determining instrument is to select the important factor of given mark.For example, radio isotope and ¹⁹F is specially adapted to the in vivo imaging in the inventive method.The type of institute's use instrument will determine radionuclide or stable isotopic selection.For example, selected radionuclide must have can by the decay form measured of given type instrument.Another factor that needs to consider is the transformation period of nucleic.Transformation period should be long enough to still can measure under measured's absorption time reaches the situation of maximum value, is short to be unlikely to the measured is produced persistent harmful radioactive.Radio-labeled compound of the present invention can use γ imaging art to measure, and wherein the suitable wavelength gamma-rays of emission is measured.The γ imaging method includes, but is not limited to SPECT and PET.Measure for SPECT, the preferential radio-labeling of selecting lacks the particulate emission, and produces a large amount of photons in the 140-200keV scope.Measure for PET, radio-labeling will be the radionuclide of emission positron, as ¹⁹F, it will eliminate the gamma-rays of two 511keV, and this can measure by the PET photographic apparatus.

Use of the present invention and amyloid bonded compound/probe carry out imaging and quantitative assay to amyloid beta deposition in vivo.These compounds can with not damaged neuroimaging technology such as nuclear magnetic resonance spectroscopy (MRS) or nuclear magnetic resonance (MRI), positron radiation x-ray tomography art (PET) and single photon emission x-ray ct gamma radiography (SPECT) are collaborative to be used.According to the present invention, can the chrysamine G derivative be used by common technique of organic chemistry known in the art ¹⁹F or ¹³C mark and be used for MRS/MRI.See, for example, March, J. " Advanced Organic Chemistry: reaction, mechanism, and structure (ADVANCED ORGANIC CHEMISTSY:REACTIONS, MECHANISMS, AND STRUCTURE) (third edition, 1985) " fits into reference with in it hereby.Also can be for PET by technology known in the art and Fowler.J. and Wolf. A. at " positron radiation x-ray tomography gamma radiography and radioautograph " (POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY) (Phelps, M, Mazziota, J., and Schelbert, H.eds.) 391-450 (Raven Press, NY1986) use by the method described in ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br is chrysamine G derivative mark, hereby with the content of above-mentioned document as a reference.Also can be for SPECT by any usefulness in several technology of this area ¹²³I mark chrysamine G derivative.Referring to, for example, Kulkarni, radioactivity is used and principle magazine (Int.J.Rad.Appl.﹠amp; Inst.) (Part B) 18:647 (1991), hereby as a reference with its content.In addition, available any suitable radioiodine isotropic substance for example, but is not limited to ¹³¹I, ¹²⁵I, or ¹²³I, by direct iodate diazotization aminoderivative, through iodate diazonium substance markers chrysamine G derivative, referring to Greenbaum, F. U.S.'s pharmaceutical journal (Am.J.Pharm.) 108:17 (1936), or any in the use means known in the art by unsettled diazotization amine is changed into stable triazene, or by inactive halogenated precursors being changed into stable trialkyltin derivative, convert it into iodide then and mark chrysamine G derivative, referring to, Satyamurthy and Barrio organic chemistry magazine (J.Org.Chem.) 48:4394 (1983), Goodman etc., organic chemistry magazine (J.Org.Chem.) 49:2322 (1984), Mathis etc., tagged compound and radiopharmaceuticals magazine (J.Labell.Comp.and Radiopharm.) 1994:905; Chumpradit etc., medicochemistry magazine (J.Med.Chem.) 34:877 (1991); Zhuang etc., medicochemistry magazine (J.Med.Chem.) 37:1406 (1994); Chumpradit etc., medicochemistry magazine (J.Med.Chem.) 37:4245 (1994).For example the stable triazene of chrysamine G or trialkyltin derivative or its analogue with contain ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ³⁸F or ¹⁹The halogenating agent reaction of F.Therefore, stable trialkyltin derivative and the analogue thereof of chrysamine G is the new precursor that is used for synthetic a lot of radiolabeled compounds of the present invention.Therefore, these trialkyltin derivatives are a preferred version of the present invention.

Also in known metal radioactively labelled substance, as technetium-99m ( ^99mTc) mark chrysamine G derivative.Radio-labeling field those of ordinary skill need not too many experiment just can modify the ligand of substituting group introducing in conjunction with such metal ion.Then, can use the chrysamine G derivative of metal mark to measure amyloid beta deposition.

Method of the present invention can be used can be by the isotropic substance of nuclear magnetic resonance spectrometry, thereby realizes in vivo imaging and spectroscopic analysis.Useful especially element comprises in the nuclear magnetic resonance spectroscopy ¹⁹F and ¹³C.

The radio isotope that is applicable to the object of the invention comprises beta emitter, gamma emitter, positron emitter and X-gamma ray emission body.These radio isotope comprise ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br and ⁷⁶Br.According to the present invention, be applicable to that the suitable stable isotope of nuclear magnetic resonance (MRI) or spectrum (MRS) comprises ¹⁹F and ¹³C.Be applicable to living tissue homogenate or the radio isotope of the external quantitative analysis of amyloid in the tissue homogenate after death, comprise ¹²⁵I, ¹⁴C and ³H.The preferred radioactively labelled substance that is used in vivo PET imaging is ¹⁸F, what be used for the SPECT imaging is ¹²³I, what be used for MRS/MRI is ¹⁹F, and be used in vitro study be ³H or ¹⁴C.But according to the present invention, the ordinary method of any diagnostic exploration that is used to develop all can be used.

This method can be used to diagnose the AD under slight or the clinical confusion situation.This technology also will be opened up amyloid beta deposition high risk population such as Down ' s syndromes, the AD of family, and the longitudinal research of the amyloid beta deposition in the apolipoproteins E4 allelotrope homozygote.Corder etc., science (Science) 261:921 (1993).Whether the method for following the amyloid beta deposition time sequence can be measured deposition and take place for a long time before dementia begins, and perhaps whether deposition is irrelevant with dementia.This method can be used for monitoring to prevent that amyloid beta deposition from being the result of treatment of purpose.

In general, great changes have taken place for the dosage general of mensuration mark chrysamine G derivative, can be by the experienced doctor in this area according to various factors such as age, situation, sex, and patient's the state of an illness, taboo, any treatment of carrying out possible time the and other variable factors are regulated.Dosage can be from 0.001mg/kg to 1000mg/kg, and preferred 0.1mg/kg changes in 100mg/kg.

Administration to the patient can be a topical, also can be to be administered systemically, and the mode of finishing administration has intravenously administrable, artery administration, intrathecal drug delivery (through cerebrospinal fluid) etc.Position during according to inspection also can be administration in intradermal administration or the body cavity.Compound combine with amyloid the back sufficiently long for some time, for example 30 minutes after 48 hours.By conventional imaging technique MRS/MRI for example, SPECT, the plane scintillography, PET and outgoing imaging technique, etc., check experimenter's specific region.Accurate source recording must depend on aforesaid patient's special factor, depends on the position when checking, the type of medication and employed marker; Concrete for a person skilled in the art medication is conspicuous.For the brain imaging, preferably measure the radiolabeled chrysamine G of bonded or chrysamine G derivative or analogue amount (total or specific combination) and with its with the amount that is attached to supracerebellar mark chrysamine G of patient or chrysamine G derivative relatively.The same ratio of then that this ratio is suitable with age normal brain activity relatively.

Pharmaceutical composition of the present invention is preferably with the form administration of injectable composition.Be used for the typical composition of this purpose and comprise pharmaceutically acceptable carrier.For example, said composition can contain chrysamine G or the chrysamine G derivative that contains about 10mg human serum albumin and be labeled from about 0.5 to 500 microgram in the phosphate buffered saline buffer of NaCl at every milliliter.Other pharmaceutically acceptable carriers comprise the aqueous solution, non-toxic excipients, comprise salt, sanitas, buffer reagent and as at REMINGTON ' S pharmaceutical science (REMINGTON ' S PHARMACEUTICAL SCIENCES), 15thEd.Easton:Mack Pubishing Co.PP.1405-1412 and 1461-1497 (1975) and National Formulary 14 editions (THE NATIONAL FORMULARY XIV.), 14thEd.Washington: the vehicle described in the American Pharmaceutical Association (American Pharmaceutical Association) (1975), hereby as a reference with the content of above-mentioned document.

The example of non-aqueous solvent has propylene glycol, ethylene glycol, vegetables oil and injection organic ester such as ethyl oleate.Aqueous carrier comprises water, alcohol/aqueous solution, and salt brine solution, parenteral carrier such as sodium-chlor, Ringer ' s dextrose, etc.Intravenous vehicles comprises the replenisher of mobile and nutrition.Sanitas comprises biocide, oxidation inhibitor, sequestrant and rare gas element.The pH value of pharmaceutical composition and the definite concentration of each component can be regulated according to this area routine techniques.Referring to, the pharmacological basis (THE PHARMACOLOGICALBASIS FOR THERAPEUTICS) (7th Ed) of Goodman and Gilman ' s treatment.

Particularly preferred pharmaceutical composition of the present invention is except in vivo can combining with the amyloid specificity and can be by the hemato encephalic barrier, and under suitable dosage level nontoxic and pharmaceutical composition with gratifying validity period.Molecular model

Molecular model is made according to Evans and Sutherland PS-300 computer graphics system, operation computer model program MacroModel (version 2 .5 can (ColumbiaUniversity) C.Still obtains from the Columbia University).Produce A β peptide chain with antiparallel beta sheet form.Kirschner etc., Proc.Natl.Acad.Sci. U.S is (1986) A.83:503.Employed amyloid peptide need not further structural modification.The arrangement of A β peptide should make 4.67  that are spaced apart of alternate chain, and this is the fibriilar feature of beta sheet.Kirschner，supra。Arrange with the chrysamine G energy minimization and with the protofibril model, it is contacted with-20 districts with hydrophobicity phenylalanine-19 with A β (10-43) Methionin-16 to greatest extent.

Synthesize peptide specific bonded characterized with A β: affinity, kinetics

Feature, farthest combination

At first use and be called the synthetic A β peptide analysis chrysamine G of A β (10-43) and the feature of bonded chrysamine G derivative.Select the 10-43 peptide to be because existing report shows that this Toplink provides a model system that comprises A β peptide entire infrastructure feature.Hilbich etc., molecular biology magazine (J.Mol.Biol.) 218:149 (1991).10-43 amino acid fragment with A β of 9-fluorene methyl chloro-formic ester (FMOC) chemical feature is synthesized by University of Pittsburgh peptide compound experiment chamber.This peptide is identified by mass spectrum, the M of main ingredient _RBe 3600g/mole (calculated value 3598).The method of use Hiblich etc. is further purified this peptide, in brief, this method is made up of following step: with 70% formic acid at Biogel P10 post (2 * 180cm, the 200-400 order, Biorad, Richmond, CA) wash-out, then carry out the order size exclusion chromatography at Biogel P4 post (2 * 180cm, 200-400 order) the wash-out second time with 1M acetate.Hiblich etc., molecular biology magazine (J.Mol.Biol.) 218:149 (1991).With this peptide-80 ℃ of freeze-drying and be stored to and be used in conjunction with measuring.The aminoacid sequence of A β (10-43) is as follows:

??10	??11	??12	??13	??14	??15	??16	??17	??18	??19	??20	??21
												??Tyr	??Glu	??Val	??His	??His	??Gln	??Lys	??Leu	??Val	??Phe	??Phe	??Ala
												??22	??23	??24	??25	??26	??27	??28	??29	??30	??3?1	??32	??33
??Glu	??Asp	??Val	??Gly	??Ser	??Asn	??Lys	??Gly	??Ala	??Ile	??Ile	??Gly
??34	??35	??36	??37	??38	??39	??40	??41	??42	??43
												??Leu	??Met	??Val	??Gly	??Gly	??Val	??Val	??Ile	??Ala	??Thr

Combination to synthetic A β (10-43) is measured

In conjunction with carrying out in the borosilicate glass tube that is determined at 12 * 75mm.The cold chrysamine G derivative that in 10% ethanol/water, adds various concentration.Adding ethanol is in order to prevent the formation of micelle.This situation often betides in the azoic dyestuff derivative, and these micelles even under the situation of no peptide, all can be intercepted and captured by filter.In above-mentioned solution, the A β (10-43) that adds 25 μ l 0.36mg/ml is at H ₂Suspension among the O, and add 10% ethanol and make volume reach 950 μ l.Incubation is after 10 minutes under the room temperature, add 50 μ l be present in 40% ethanol [ ¹⁴C] chrysamine G, reach use [ ¹⁴C] the desired 100-125nM of chrysamine G preparation final [ ¹⁴C] chrysamine G concentration.With binding mixture incubation 30 minutes at room temperature.At room temperature (Gaithersburg, Whatman GF/B filter MD) carries out isolated by vacuum filtration bonded and free radioactive substance, follows every part with twice of 10% washing with alcohol of 3ml by using BrandelM-24R Cell Harvester.With filter at the 4ml Cytoscint of 7.0ml plastics flickers bottle that packs into ^-ES scintillator (ICN Biomedicals, Inc.Irvine, CA) middle equilibrate overnight.Said determination and all incubation liquid is prepared three parts at least in conjunction with in measuring, its result with on average ± standard deviation represents.

Dynamics research

With A β (10-43) bonded [ ¹⁴C] dynamics research of chrysamine G undertakies by above-mentioned filtration assay method in 13 * 100mm borosilicate glass test tube.For bonded kinetics, with 25 μ l0.36mg/ml A β (10-43) be dissolved in 475 μ l10% ethanol and 0 time with 4.5ml 125nM[ ¹⁴C] chrysamine G adds this solution.With the at room temperature rapid vortex of this mixture and 5,10,20,30,45,60,75,135,240, with time of 300 seconds by use Brandel M-24R CellHarvester (Gaithersbug, Whatman GF/B filter vacuum filtration MD) to be to stop association reaction, then with twice of 10% washing with alcohol of every part of 3ml; The bonded radioactivity is pressed said determination.

For the kinetics of dissociating, 25 μ l 0.36mg/mlA β (10-43) are dissolved in 450 μ l10% ethanol, then add in 40% ethanol of 25 μ l2.5 μ M [ ¹⁴C] chrysamine G.With this mixture vortex and room temperature incubation 30 minutes.In 0 time this mixture is diluted with the cold chrysamine G in 10% ethanol of 45ml 10 μ M, with the rapid vortex of this mixture and 0.5,1.5,3,5 and 15 minutes time stops to dissociate by above-mentioned filter method, presses said determination bonded radioactivity.Specificity bonded characterized with the Alzheimer brain

Chrysamine G combines with AD and contrast brain homogenate

Postmortem brain sample obtains from Alzheimer research centre neuropathology basis teaching and research room of University of Pittsburgh (Neuropathology Core of the Alzheimer ' s Disease ResearchCenter of the University of Pittsburgh).According to disclosed NIA meeting report specified standards, control sample is restricted to the sample of the neuropathology discrimination standard (enough NPs or NFTs number) that does not reach AD, Khachaturian, neurological outline (Arch.Neurol.) 42:1097 (1985).The brain sample that is studied is eight control samples (age 58-75).11 AD (age 61-84) and two Down syndromes brains (age 23 and 51).Have six to be high spot number (＞20 NPs/ * 200 enlargement ratios) in the AD brain, five are low spot number (＜20 NPs/ * 200 enlargement ratios).Two control samples are that clinical manifestation is dull-witted but does not have NPs or NFTs and be diagnosed as the brain sample of " dementia that lacks tangible histologic characteristics ".See Knopman dementia (Dementia) 4:132 (1993).Another control sample has the symptom of dull-witted and olivopontocerebellar atrophy.Clinical or the histological sign of other control sample impassivity diseases.The postmortem sample is freezing and store under this temperature up to by homogenate at-70 ℃ rapidly.To five parts be studied brain on and in before return and to separate between in the cortex with the tie point of last temporo siocortex 2 and 4 layers but NPs and NFTs counting in the section of adjacent areas (* 200 ratio of enlargement).In the qualitative evaluation/the amyloid blood vessel disease that exists in the middle volume cortex.Use Bielschowsky silver impregnation method to identify NPs and NFTs, use the congo red staining method to identify brain amyloid blood vessel disease.These methods are by open in detail.See Moossy etc., neurological outline (Arch.Neurol.) 45:251 (1988).Be used for CG/volume or on temporo cortex bonded sample adjacent with the cortex that is used for NP and NFT counting substantially.

It is taking from of 10-20mg brain/ml and middle volume cortex tie point that about 100mg is present in concentration in 10% ethanol, last temporo cortex, antinion, head of caudate nucleus, lower wall cortex, occipitalia cortex, or cerebellum Polytron ^Tissue homogenizer (PT10/35, Brinkman InstrumentsInc.Westbury, NY) 6 homogenate 30 seconds in the position.Not every zone all can obtain from each brain.With 25-150 μ l aliquot of tissue (pressing Lowry etc., the method for journal of biological chemistry (J.Biol.Chem.) 193:265 (1951), about 25-300 μ g) with at final volume be 10-750nM[in 10% ethanol of 1.0ml ¹⁴C] CG (26.8 Ci/mole) places 12 * 75mm borosilicate glass test tube incubation 30 minutes at room temperature together.Under the normal circumstances, approximately use 150 μ g albumen and 75nM[for the research of cerebellum ratio ¹⁴C] CG, for NPs, NFTs approximately uses 75 μ g albumen and 150nM[with the sick relevant research of amyloid blood vessel ¹⁴C] CG.In order to prevent that forming micelle with the disazo dyes derivative need use ethanol because even under amorphous situation this sizing material all intercepted and captured by filter.Under the room temperature by use BrandelM-24R Cell Harvester (Gaithersburg, Whatman GF/B filter vacuum filtration MD) and separation and combination with the free radioactive substance, follow every part with twice of 3ml 10% washing with alcohol.With filter at the 4ml Cytosint of 7.0ml plastics flickers bottle that packs into ^-ES scintillator (ICN Biomedicals, Inc., Irvine, CA) middle equilibrate overnight is counted then.Saturated (distinctive) is in conjunction with being defined as the combination that in the presence of the unlabelled CG of 20 μ M total binding deducts residue (unsaturated).Except as otherwise noted, in conjunction with in measuring, incubation liquid is prepared three parts at least at all, and its result represents with ± standard deviation.The result both can the institute to fmol bonded in the brain district [ ¹⁴C] the proteic absolute value representation of CG/ μ g, also can this brain district in the proteic expression recently of fmol/ μ g in the cerebellum of fmol/ μ g albumen and same brain.Octanol/water is distributed

The chrysamine G of the about 75 μ M of preparation or the solution of its analogue in 5.0ml 1-octanol.(0.15M NaCl, the 5mM potassiumphosphate is pH7.4) and with its rapid vortex mixed to add the salt solution of 5ml phosphate buffered.1,000g is centrifugal, and this mixture that impels forms tangible two-phase then.Separate each layer with separating funnel, in each layer of each 600 μ l,, measure the optical density of 389nm place chrysamine G and the λ max of each analogue with 400 μ l alcohol dilutions.After proofreading and correct the molar absorption coefficient difference in two kinds of solvents, measure concentration, partition ratio is illustrated in concentration in the octanol layer divided by the concentration in water layer.Experiment divides three parts and carries out.Chrysamine G and the imaging of Alzheimer brain amyloid precipitation bonded

In order to be illustrated more clearly in combining of CG and tissue, according to Puchtler etc., the alkaline Congo red method of histological chemistry's cytochemistry magazine (J.Histochem.Cytochem.) 10:355 (1962) and Stokes and Trickey, the method of clinical pathology magazine (J.Clin.Pathol.) 26:241-242 (1973) is with CG and Congo red 8 microns adjacent paraffin section dyeing to the serious sedimentary AD brain of cerebrovascular amyloid.In the CG dyeing course, except that Congo red with the CG replacement, other programs are identical.The slide glass that uses the inspection of fluorescein isothiocyanate (FITC) filter to be colored.Compound is by the mensuration of hemato encephalic barrier

Mice study is given about 0.03 μ Ci/g[in the female Swiss-Webster injected in mice 0.9%NaCl solution by lateral tail vein ¹⁴C] chrysamine G.Injection back is with 15 minutes, and 35 minutes, 1 hour, mouse was put to death by the neck dislocation in the interval of 4 hours and 24 hours.Collect rapidly carotid artery blood, brain, liver, and kidney are weighed and use the obscure glass homogenizer at distillation/deionization H ₂Homogenate among the O, get aliquots containig weigh pack into a 18.0ml plastics flickers bottle (Beckman Poly-Q-Vial) and adding 10.0ml flicker mixture (Cytoscint ^-ES (ICN)) and after the equilibrate overnight count.In the tissue [ ¹⁴C] chrysamine G content is with cpm/mg tissue expression.

Remove injection 0.05 μ Ci/g[ ¹⁴C] chrysamine G and put to death outside the mouse at 60 minutes, by above-mentioned experiment of carrying out from tissue, extracting radioactive substance.Then with brain with liver shifts out and extract with the method for Folch.Folch etc., journal of biological chemistry (J.Biol.Chem.) 226:447 (1957).In two kinds of tissues, the radioactive substance that is extracted more than 95% is contained in organic layer.With the organic layer evaporate to dryness, resuspending and is expelled to (Prep Nova Pak HR Silica on the silicagel column in a small amount of 10% methyl alcohol/90%ACN, 7.8 * 300mm, Waters, Milford MA) also carries out same solvent (isocratically) wash-out with identical solvent.Under these conditions, 99% radioactive substance wash-out in solvent fraction early comes out, but most of lipid retention time is longer, and this can be expelled in the above-mentioned anti-phase C4 column system fraction early suitably.All solvent fractions are early collected, drying, and resuspending in the 10%ACN/90% sodium phosphate buffer (5mM, pH6) in, it is expelled on the C4 post with the cold chrysamine G of identifying.Collect one minute fraction, add 10mlCytoscint ^Count behind-the ES.

At another preferred version, the present invention relates to prevent with certain " with amyloidosis " situation under protofibril form pharmaceutical composition and the method that relevant cytopathy becomes to poison, above-mentioned " with amyloidosis " situation comprises as Alzheimer, Down ' s syndromes and diabetes B, the courageous and upright amyloidosis (Dutch) of heredity cerebral hemorrhage, amyloid A (reactivity), secondary amyloidosis, family's Mediterranean fruit fly, with urticaria and deaf family's amyloid ephrosis (Muckle-Wells syndromes), amyloid λ L-chain or amyloid KL-chain are (spontaneous, relevant with myelomatosis or macroglobulinemia) A β 2M (chronic hemodialysis), (family's amyloid polyneuropathy is (Portuguese for ATTR, Japan, Sweden), family's amyloid myocardosis (Denmark), isolating heart amyloid, (systemic senile amyloidosis), AIAPP or amylopectin nesidioblastoma, atrial natriuretic peptide (atrial naturetic factor) (isolating atrium amyloid), procalcitonin (the plain medullary carcinoma of first shape), gelsolin (family's amyloidosis (France)), cystatin c (heredity cerebral hemorrhage (Iceland)) with amyloidosis, AApo-A-I (family amyloid neuropathy-Iowa), AApo-A-II (quickening the mouse aging), with amyloid related celloglobulin, and Asor or PrP-27 (scrapie, Creutzfeld Jacob disease, the Gertsmann-Straussler-Scheinker syndromes, bovine spongiform encephalitis) or as the homozygous people of apolipoproteins E4 equipotential group.This method comprises suffering from or very likely developing into above-mentioned treatment target with the amyloidosis diseases associated and use the pharmaceutical composition that comprises chrysamine G or its a kind of aforesaid derivative.

Because some two azo-compound may be carcinogens, therefore treatment compound of the present invention include only nontoxic, the compound of no carcinogenesis.That is, the present invention proposes passes through only to use based on 3,3 ', 5,5 '-problem of two azo-compounds (solution) the potential carinogenicity of tetramethyl benzidine.A lot of researchs have proved 3,3 ', 5,5 '-tetramethyl benzidine is no mutagenicity, the p-diaminodiphenyl of the replacement of non-carcinogenesis.Holland etc., tetrahedron (Tetrahedron), 30:3299-3302 (1974).De Serres and Ashby (eds.) carcinogen short-time test assessment (Evaluation of Short-Term Test for Carcinogens), Elsevier North-Holland, 1981.3,3 ', 5,5 '-tetramethyl benzidine has been used as the p-diaminodiphenyl substitution product in azoic dyestuff.Josephy and Weerasooriya, chemicobiology interacts (Chem.-Biol.Interactions), 1984:375-382, (1984).Ashby etc., the generation of cancer (Carcinogenesis) 3:1277-1282 (1982).The inventor has synthesized the tetramethyl-analogue (4 of chrysamine G, 4 '-two (3-carboxyls-4-hydroxyphenyl azo-group)-3,3 ', 5,5 '-tetramethyl biphenyl) and found that it and synthetic A β (10-43) bonded Ki value are 0.75 ± .09 μ M, this value is about the twice of chrysamine G.

Use the alkene and the alkyne derivatives of azo-compound can avoid the low potential problems of bioavailability.These compounds are not bacterium or Mammals azo-group reductase enzyme reductive substrate.

In fact, it is because they had both comprised no mutagenicity that the The compounds of this invention that is used for the treatment of purpose is better than known compound, and the benzidine derivative of non-carcinogenesis comprises the alkenyl of non-bacterium azo reductase substrate or the benzidine derivative that alkynyl connects again.

In vitro study has proved that the fibriilar formation of A β neurotoxic sexual needs and it can be by Congo red inhibition.Especially, proved that now Congo red protofibril forms or suppress fibriilar A β neurotoxicity by combining with the protofibril that forms by suppressing with amyloid protofibril bonded.Lorenzo etc., Proc.Natl.Acad.Sci.USA91:12243-12247 (1994).Proved the Congo red amylopectin relevant that suppress, the fibriilar islet cells toxicity of another kind of amyloid with diabetes.Lorenzo etc., supra. also can referring to, Burgevin etc., neuroscience communication (NeuroReport) 5:2429 (1994); Pollack etc., neuroscience wall bulletin (Neurosci.Letters) 184:113-116 (1995); Pollack etc., neuroscience wall bulletin (Neuroscience Letters) 197:211 (1995).These documents show to amyloid bonded compound such as chrysamine G and derivative thereof to Congo red similar, can prevent cytopathy relevant in the amyloidosis symptom and poisoning effectively with protofibril, different is that these compounds are easier to be entered in the brain.

Embodiment 8 and Figure 12 and 13 show that Congo red extremely similar the prevent rat relevant with dosage that has with reporting previously have a liking for the effect of coughing up glucagonoma.Therefore, these external tests provide a kind of selection to be used to prevent form with protofibril the method for compound of the pharmaceutical composition of relevant cytopathy and poisoning.

According to the present invention, at animal model, as " geriatric animals " model of cerebral amyloidosis, Wisniewski etc., europathology and neuroscience experiment magazine (J.Neuropathol.﹠amp; Exp.Neurol.) 32:566 (1973), family's Mediterranean fruit fly mouse model (neurochemistry (Neurochem.), Inc.Kingston, Ontario, Canada) and Alzheimer one type neuropathology mutant mice model, Games etc., among nature (Nature) 373:523-527 (1995), form by measuring the dystrophic aixs cylinder, the loss of cynapse, neurofibrillary tangles forms and gliosis, and the compound of testing chrysamine G for example and said derivative thereof prevents in vivo that the amyloid protofibril from forming or the effect of relevant therewith cytopathy.In family's Mediterranean fruit fly model, the whole body albuminoid degeneration has taken place in animal.In in vivo mensuration according to the present invention, the animal of using and do not handle with The compounds of this invention is carried out the restraining effect that a series of necrotomys form with the comparative assessment amyloid.In the animal model that the brain amyloid forms, except the generation of sequential testing amyloid, also measuring the dystrophic aixs cylinder by the postmortem of a series of usefulness and the animal of not handling with The compounds of this invention forms, the cynapse loss, neurofibrillary tangles forms and gliosis, thereby measures and amyloid related neurodegeneration.

According to the present invention, amyloid or the formation of amyloid protofibril are appearred in expection, the curee of cytopathy and poisoning uses the pharmaceutical composition that contains the chrysamine G or derivatives thereof.In preferred version, the curee comprises, for example, produces the people of brain amyloid most probably, comprises in early days, does not show as dull-witted colony or has patient with amyloidosis diseases associated and diabetes B.The meaning that term " prevents " comprises with protofibril and forms the relevant cytopathy and the improvement of poisoning." improvement " refers to prevent occur in early days toxicity symptom, as the patient of dementia more serious cytopathy and poisoning takes place.

Be used for preventing that the pharmaceutical composition that amyloidogenic disease and protofibril form relevant cytopathy and poisoning usefulness from comprising chrysamine G or its said derivative and pharmaceutically acceptable carrier.In a preferred version, such pharmaceutical composition comprises serum albumin, chrysamine G or chrysamine G derivative and the phosphate buffered saline buffer that contains NaCl.Other pharmaceutically acceptable carriers comprise the aqueous solution, non-toxic excipients, comprise for example REMINGTON ' s PHARMACEUTTCALSCIENCES, 15th Ed., Easton:Mack Publishing Co., pp.1405-1412 and 1461-1487 (1975) and National Formulary 14 editions (THE NATIONALFORMULARY XIV.), 14th Ed.Washington:American PharmaceuticalAssociation (1975), and 18 editions (the UNITED STATESPHARMACOPEIA XVIII of American Pharmacopeia,), 18th Ed.Washington:AmericanPharmaceutical Association (1995), described in salt, sanitas, damping fluid etc. are incorporated the content of above-mentioned document in this into own forces hereby as a reference.

The example of non-aqueous solvent has propylene glycol, polyoxyethylene glycol, vegetables oil and injection organic ester such as ethyl oleate.Aqueous carrier comprises water, alcohol/aqueous solution, and salt brine solution, non-enteron aisle carrier such as sodium-chlor, Ringer ' s dextrose, etc.Intravenous vehicles comprises the development regenerator of mobile and nutrition.Sanitas comprises biocide, oxidation inhibitor, and sequestrant and rare gas element, the pH of pharmaceutical composition and the precise concentrations of each component can be regulated according to this area routine techniques.Referring to, Goodman and Gilman ' s THE PHARMACOLOGICAL BASIS FORTHERAPEUTICS (7th Ed.).

According to the present invention, the form oral administration that such pharmaceutical composition can liquid or solid is with the form intravenous injection or the intramuscular injection of suspension or solution.Term " pharmacy effective dose " is meant and prevents to form the relevant cytopathy and the amount of poisoning with protofibril that this is measured with patient's age, and body weight and situation and change can be regulated according to known ordinary method by those of ordinary skills.In the preferred version, dosage is every day 0.1 to 100mg/kg, perhaps is divided into littler dosage and uses two to four times every day.Such dosage regimen can continue until every day of patient's life.Optionally, can arrive the dosage of 100mg/kg with these six weeks of pharmaceutical composition administered intramuscular every day 1.

On the other hand, the present invention relates in living tissue or after death measure the method for amyloid beta deposition in the tissue.This method comprises the tissue with the solution incubation formalin fixed of above-mentioned formula I compound.Preferred this solution can be used the saturated 25-100% ethanolic soln of formula I compound, (all the other are water).In the incubation process, sedimentary amyloid dyeing or mark during compound will be organized are colored or the settling of mark can be measured or development by any standard method.Such measuring method comprises microtechnique such as bright field, fluorescent, laser-confocal point and cross polarization microscope.

On the other hand, the present invention relates to a kind of quantitative analysis living tissue or the method for amyloid in the tissue after death.This method comprises the chrysamine G derivative of incubation mark, and preferred formula I compound or its water soluble non-toxic salt are with living tissue or the homogenate of tissue after death.This tissue obtains and homogenate by methods known in the art.Although the technician is familiar with various mark substances such as enzyme, chemoluminescence and immunofluorescence compound, preferred mark substance is a radioactively labelled substance.Preferred radioactively labelled substance is ¹²⁵I, ¹⁴C or ³H, the preferred mark substituting group of formula I compound is at least R ₁-R ₇, R ₁₀-R ₂₇In one.The tissue of starch-containing sample proteinosis thing will combine with the chrysamine G derivative of mark.By any method known to the skilled,, incite somebody to action bonded tissue and unconjugated separate tissue then as filtering.Then by any method quantitative analysis known to the skilled bonded tissue.See embodiment 3.Then by will become with the unit conversion of the chrysamine G derivative bonded tissue that is labeled with the typical curve comparison of making by the amyloid of incubation known quantity and radiolabeled chrysamine G derivative every 100mg organize in the micrograms of amyloid.

On the other hand, the present invention relates to a kind of method of distinguishing Alzheimer brain and normal brain activity, this method comprises from (i) cerebellum and (ii) uses other zones outside the cerebellum the brain, from the normal subjects with suspect that the experimenter who suffers from Alzheimer obtains cerebral tissue.See embodiment 3.Use method known to those skilled in the art that this tissue is made homogenate respectively, then with radiolabeled chrysamine G derivative incubation.Calculate each types of organization (cerebellum for example then, non-cerebellum, normally, abnormal) in the amount of radiolabeled chrysamine G derivative bonded tissue, and calculate in the healthy tissues and suspect non-cerebellum part and cerebellum portion of tissue bonded ratio in the experimenter's who suffers from Alzheimer the tissue.Compare these ratios then.If suspect that the ratio of the brain suffer from Alzheimer is higher by 90% than normal brain activity ratio, suffers from Alzheimer really with regard to diagnosable.Synthesizing of embodiment 1. chrysamine Gs and derivative thereof

Synthesizing of chrysamine G

The following reactions steps of synthetic needs of chrysamine G (i.e. 4,4 '-two (3-carboxyl-4-hydroxyphenyl azo)-biphenyl).These reactions steps will be as the general method of " chrysamine G is synthetic ".With p-diaminodiphenyl ● and 2HCl (28.9mg, 0.11mmole, Sigma Chemical Company, St.Louis, MO) 1: 1 DMSO of 1.5ml is equipped with in adding: distillation/deionization H ₂In the 50cc round-bottomed flask of O.Unless otherwise noted, each step reaction is all carried out at 0 ℃.Add 29 μ l concentrated hydrochloric acids, stir and obtain clear solution.In p-diaminodiphenyl solution, drip 15.5mg (0.22mmole) NaNO ₂At 300 μ l1: 1 DMSO/H ₂Solution among the O, the gained pH value of solution is about 2-3.Reaction mixture was stirred 45 minutes; , in this four nitrogen p-diaminodiphenyl mixture, in 10 minutes time, dropping is dissolved in 2.0ml and contains 250mg/ml Na then ₂ CO ₃100%DMSO in 24.8mg (0.18mmole) wintergreen oil (Aldrich), the pH that keeps this suspension is about 10.5.The gained mixture was stirred 1 hour at 0 ℃, at room temperature stir then and spend the night.

Afterwards, with pH regulator to about 7, with the chloroform of every part of 50ml with this mixture extraction three times.With the chloroform extraction liquid that merges H with every part of 50ml ₂O washing three times is concentrated into driedly then, obtains chrysamine G dimethyl ester (i.e. 4,4 '-two (3-methoxycarbonyl-4-hydroxyphenyl azo)-biphenyl), and it is further purified by chloroform/hexane recrystallization.Then by at the about 100ml1 that contains four equivalent NaOH: 1 ethanol: H ₂Refluxed three hours and this ester of hydrolysis among the O.After the ethanol evaporation, with H ₂The O lyophilize obtains the tetra-na salt of chrysamine G.By this tetra-na salt is water-soluble, once remove unhydrolysed dimethyl ester with chloroform washing, pH value is reduced to about 2 and formation chrysamine G free acid, with the ethyl acetate extraction of every part of 50ml three times.With the acetic acid ethyl acetate extract that merges H with every part of 50ml ₂O washing three times also is concentrated into dried.

In the product that obtains under these conditions, there has not been wintergreen oil, Whitfield's ointment or p-diaminodiphenyl, and only micro-mono-substituted product, 4-hydroxyl-4 '-(3-carboxyl-4-hydroxyphenyl azo-biphenyl, this can be by using the anti-phase MPLC of C4 post (Vydac 214-TP510), with 3.5ml/ minute flow velocity, at first use sodium phosphate buffer (5mM, pH6): 90: 10 solvent systems of acetonitrile (ACN), wash-out 10 minutes is increased to acetonitrile 50% then wash-out 20 minutes and identifying then.With the elutriant of this post 290nm 365nm dual wavelength, with diode matrix detector (Perkin Elmer, 235C) monitoring.Under these conditions, chrysamine G at 17.6 minutes by wash-out.

The structure of chrysamine G and derivative thereof is by mark DMSO-d in doing with TMS ₆The 500MHz proton N MR that makes solvent determines.Chrysamine G tetra-na salt peak position ownership is as follows, and wherein SA refers to the proton of the specific ring position of Whitfield's ointment part, and BZ refers to the proton of the specific ring position of p-diaminodiphenyl part: SA-3, locate doublet, J=8.73Hz in 6.75 100 ten thousand/units (ppm), SA-4, dual doublet, J=8.73 and 2.72Hz; 7.82ppm; BZ-2/6, doublet, J=8.44Hz, 7.91ppm; BZ-3/5, doublet, J=8.44Hz, 7.95ppm; And SA-6, doublet J=2.72Hz, 8.28ppm.The λ of the UV/ visible spectrum in 40% ethanol _MxaAt the 389nm place.By with NMR in mark comparison peak area calculate the concentration of chrysamine G and rapidly aliquots containig dilution metering UV/ visible spectrum in 40% ethanol of NMR sample obtained the molar absorption coefficient of chrysamine G.The molar absorption coefficient that is in 40% ethanol at 389nm is 5.5 * 10 ⁴AU/cmM.

By improving one's methods of aforesaid method can synthesize [ ¹⁴C] chrysamine G.Remove and use 100%H ₂Outside the O, by the above-mentioned tetrazotization that carries out p-diaminodiphenyl.Na with 50 μ l 2.5M ₂CO ₃The aqueous solution add 50 μ Ci crystallization Whitfield's ointment-carboxyls in the 0.5ml cone-shaped glass bottle of packing into- ¹⁴Among the C (Sigma).The p-diaminodiphenyl mixture that adds 60 μ l tetrazotizations in this Erlenmeyer flask, vortex also kept 1 hour at 0 ℃.For preventing to form mono-substituted p-diaminodiphenyl by product, in reaction mixture, add and be present in 2.5M Na ₂CO ₃In the 12.5 cold Whitfield's ointments of μ l 250mM (Sigma), and kept 1 hour down at 0 ℃.This Erlenmeyer flask at room temperature placed spend the night.Whole mixtures are dissolved in a small amount of 35%ACN, and it is injected on the C4 post by above-mentioned.Collect peak and the freeze-drying corresponding with chrysamine G standard crystalline phase.By measuring the absorption at 389nm place, then to the radiocounting of the aliquots containig of same sample, and the specific activity at calculating 26.8Ci/mole place.Will [ ¹⁴C] chrysamine G is stored in 40% ethanol.Then with purifying [ ¹⁴C] chrysamine G is expelled on the C4 post with 3.5ml/ minute speed 21%ACN wash-out again, obtained at 10.4 minutes with the common wash-out of standard chrysamine G＞98% radioactive substance.Except that following explanation exception, use the general method of above-mentioned " chrysamine G is synthetic " can synthesize a lot of chrysamine G derivatives.The structure of these derivatives can be examined and determine by NMR.Fig. 1 shows the chemical structure of chrysamine G and several derivatives.

Synthesizing of the 3-sec.-propyl salicyclic acid derivatives of chrysamine G

By replacing p-diaminodiphenyl with 3-bromo biphenyl amine (seeing below) and replacing wintergreen oil, can synthesize 4,4 '-two (3-carboxyl-4-hydroxyl-5-cumyl azo-group)-3-iodine biphenyl by the method for above-mentioned synthetic chrysamine G with 3-sec.-propyl wintergreen oil.Resulting br-derivatives is converted into the trialkyltin derivative, is converted into iodo derivative again by hereinafter detailed description then.

Synthesizing of the pyridine of chrysamine G and diazine derivatives

By replacing p-diaminodiphenyl with 3-bromo biphenyl amine (seeing below) and using 2-hydroxy niacin (or relevant derivative, the 6-hydroxy niacin, the 3-hydroxy-picolinic acid, or hydroxyl diazine carboxylic acid such as 4-hydroxy pyrimidine-6-carboxylic acid) the replacement wintergreen oil, the pyridone carboxylic acid or the hydroxyl diazine carboxylic acid derivative that can synthesize 4,4 '-two (5-carboxyl-6-hydroxyl-3-pyridylazo base)-3-iodo biphenyl and relevant chrysamine G by the method for above-mentioned synthetic chrysamine G.Resulting bromo derivative is converted into the trialkyltin derivative, is converted into iodo derivative again by hereinafter detailed description then.

The naphthalene of chrysamine G, quinoline and benzodiazine derivative synthetic

By replacing p-diaminodiphenyl with 3-bromo biphenyl amine (seeing below) and using 1-hydroxyl-2-naphthoic acid (or relevant derivative, other hydroxynaphthoic acid isomer, hydroxy quinoline carboxylic acid such as kynurenic acid, or the different diazine carboxylic acid of hydroxybenzene such as 5-hydroxy quinoxaline-6-carboxylic acid) the replacement wintergreen oil, method by above-mentioned synthetic chrysamine G can synthesize 4, the hydroxynaphthoic acid of 4 '-two (3-carboxyl-4-hydroxyl-naphthyl azo base)-3-iodo biphenyl and relevant chrysamine G, hydroxy quinoline carboxylic acid, or hydroxy benzo diazine carboxylic acid derivative.Resulting bromo derivative is converted into the trialkyltin derivative, is converted into iodo derivative again by hereinafter detailed description then.

Synthesizing of the nitrophenol derivative of chrysamine G

By replacing p-diaminodiphenyl with 3-bromo biphenyl amine (seeing below) and replacing wintergreen oil with 2-nitrophenols (or other nitrophenols isomer relevant) with derivative, can synthesize the nitrophenol derivative that 4,4 '-two (3-nitro-4-hydroxyl-phenylazo)-3-iodo biphenyl reach relevant chrysamine G by the method for above-mentioned synthetic chrysamine G.Resulting bromo derivative is converted into the trialkyltin derivative, is converted into iodo derivative again by hereinafter detailed description then.

The process for selective of synthetic two azo-based compound

As a kind of process for selective that forms azo-compound, nitroso compound is reacted and the amine coupling in Glacial acetic acid by Mills.Badger, G etc., Australian The Chemicals (Aust.J.Chem.) 17:1036 (1964).Nitroso compound can be by Coleman etc., the method preparation of organic synthesis compilation (Organic Synthesis Collective) Vol.III:p668.For example, with 3-nitrobenzoic acid (Aldrich Chem.Co., Milwaukee, WI) (2.44mmoles) and 150mg NH ₄The solution of Cl in 5ml water merges, and adds in the round-bottomed flask of a 50ml and vigorous stirring.In 5 minutes time, add zinc powder (372mg) with aliquot.After adding zinc powder, temperature begins to raise, but uses ice bath that temperature is remained between 50 and 55 ℃.。This mixture was stirred 20 minutes under this temperature.Filter residual zinc then and wash with boiling water.Place beaker ice-cooled to 0 ℃ the filtrate and the washing lotion that merge by adding.When stirring, in above-mentioned refrigerative azanol mixture, add refrigerative sulphuric acid soln (the 750 μ l vitriol oils add capacity ice makes temperature be cooled to-5 ℃).When stirring, the solution of ice-cooled 170mg sodium dichromate 99 dihydrate in 750 μ l water is all once added.With the p-diaminodiphenyl of gained 3-nitrosobenzoic acid and two/monovalent, 2,7 diamin of luorene or other benzidine derivative in Glacial acetic acid, merge and 70-80 ℃ warm 7 hours, at room temperature place then and spend the night.This mixture dilute with water is also used ethyl acetate extraction, obtain 4,4 '-two (3-carboxyl-phenylazo) biphenyl, 2,7-two (3-carboxyl-phenylazo)-fluorenes, or use specific corresponding two (3-carboxyl-phenylazo) derivative that benzidine derivative produced.

Synthesizing of methoxy derivatives

The methoxy derivatives of all phenolic compounds can be synthetic by following method.Be dissolved in acetone at monovalent, concentration be approximately add in the chrysamine G ester derivative of 5mg/ml with phenolic group 10 normal methyl iodide (Aldrich Chemical Company, Milwaukee, WI) and 10 normal K ₂CO ₃With this suspension returning 6-24 hour, be concentrated into driedly with rotatory evaporator, and use chloroform extraction.Also at room temperature stir 24-27 hour by the concentration that adds 1: 1 extremely about 1mg/ml of THF/0.1 N NaOH with the ester hydrolysis.By remove unhydrolysed methoxyl group-ester with chloroform extraction, the pH value is adjusted to～2, and ethyl acetate is gone in methoxyl group-acid extraction.

Synthesizing of the benzidine derivative that replaces

For the synthetic benzidine compound that replaces, except with 3,3 '-dichlorobenzidine (Pfaltz﹠amp; Bauer, Waterbury, CT), 4,4 '-diamino octafluoro biphenyl, 3,3 ', 5,5 '-tetramethyl benzidine (Aldrich Chemical Company, Milwaukee, WI), 3,3 '-tolidine (Aldrich Chemical Company, Milwaukee, WI), 3,3 '-dimethoxy benzidine (Aldrich Chemical Company, Milwaukee, WI), p-diaminodiphenyl-3,3 '-dicarboxylic acid (Pfaltz ﹠amp; Bauer, Waterbury, CT) or its lower alkyl esters, or 3, (Fluka Chemical Corp., Ronkonkoma NY) replace outside the unsubstituted p-diaminodiphenyl 3-dinitrobenzene p-diaminodiphenyl, react by the general method of above-mentioned synthetic chrysamine G.Also can be used for replacing the p-diaminodiphenyl of other replacements of unsubstituted p-diaminodiphenyl to include, but are not limited to by reference method is synthetic: 3,3 '-'-dibromobiphenyl amine and 3,3 '-diiodobiphenyl amine (Snyder, H. etc., JACS (J.Am.Chem.Soc.) 71:289-291,1994); 3-bromo biphenyl amine and 3-iodine p-diaminodiphenyl (Badger, G. etc., Australian The Chemicals (Aust.J.Chem.) 17:1036-1049,1964; Holland.V. etc., tetrahedron (Tetrahedron) 30:3299-33201974).

Luxuriant and rich with fragrance (Phenanthracene), benzo (c) cinnolines, fluorenes, Fluorenone, carbazole,

Dibenzofuran, dibenzothiophen and dibenzothiophen-9,9-dioxide derivative synthetic

In the tetrazotization step, use 2,7 diamin of luorene (Aldrich Chemical Company, Milwaukee, WI), replace p-diaminodiphenyl, use the same step coupling that is used for tetrazotization p-diaminodiphenyl to make the fluorenes of chrysamine G (2,2 '-methylene radical xenyl) derivative then.Equally, also can be with 2 in the tetrazotization of standard and coupling step, 7-diamino phenanthrene, 2,7-diamino-3,6-dimethylbiphenyl [c] cinnolines, 2,7 diamin of luorene ketone, 9-methyl-2,7-diamino carbazole, 3,7-diamino dibenzofuran (is different from other this polycyclic compounds, the oxo bridge atom of dibenzofuran is 5 rather than 9-position in the conventional ring system, therefore, amino substituting group is 3,7-position rather than as 2 in other compounds, the 7-position, although the position is suitable, 2,7-diamino dibenzothiophen, with 2,7-diamino dibenzothiophen-9,9-dioxide (" benzidine sulfone ") replaces p-diaminodiphenyl.Unsubstituted if desired carbazole can make 9-methyl-2 after coupling, 7-diamino carbazole compound N-demethyl.Press Ullmann and Mallet Ber.31:1694-1696,1898 and Nunn.A. etc., the method that chemistry can will (J.Chem.Soc.) 1952:2797-2803, by reason 2-amino-3 also ', 2 of 5-dinitrobenzene-cis-stilbene preparation, 7-dinitrobenzene phenanthrene and Synthetic 2,7-diamino phenanthrene.Press Snyder etc., the method of JACS (J.Amer.Chem.Soc.) 71:289 (1949), by reducing 3 with the zinc in the NaOH aqueous solution with the NaOBr oxidation, 3 '-dimethyl-6,6 '-dinitrobenzene p-diaminodiphenyl (Aldrich ChemicalCompany, Milwaukee, WI), and the corresponding hydrazono-compound that forms can prepare 2,7-diamino-3,6-dimethylbiphenyl [c] cinnolines.Optionally, can be at first with 3,3 '-dimethyl-6,6 '-dinitrobenzene p-diaminodiphenyl replaces p-diaminodiphenyl tetrazotization and coupling, handles with Zn/NaOH and NaOBr by above-mentioned then.Press Ullmann and Mallet Ber.31:1694-1698,1898 and Nunn, A. etc., chemistry can will (J.Chem.Soc.) method of 1952:2797-2803, by reason 2-amino-3 also ', 2 of 5-dinitrobenzene benzophenone preparation, but 7-dinitrobenzene Fluorenone Synthetic 2,7-diamino Fluorenone.Press Saunders, K.H. and Allen, described in the R.L.M.AROMATIC DIAZO COMPOUNDS 625-640 (Edward Arnold, London, 1985), by reason N-also methylate from 2-amino-3 ', 2 of 5-dinitrobenzene p-diaminodiphenyl preparation, 7-dinitrobenzene carbazole and the 9-methyl-2 for preparing, 7-dinitrobenzene carbazole can synthesize 9-methyl-2,7-diamino carbazole, the full content of the document are incorporated into own forces hereby and are reference.Press Saunders, K.H. and Allen, R.L.M.AROMATIC DIAZOCOMPOUNDS 625-640 (Edward Arnold, London, 1985), described in, by reason 2-amino-3 also ', 3 of 5-dinitrobenzene biphenyl ether preparation, 7-dinitrobenzene dibenzofuran can synthesize 3,7-diamino dibenzofuran, the full content of the document are incorporated into own forces hereby and are reference.According to Courtot and Evain Bull, Soc.Chim.49 (iv): 528,1931 and the method for Cullinane and DaviesRec.Trav.Chim.55:881-886 (1936) can prepare 2,7-diamino dibenzothiophen and 2,7-diamino dibenzothiophen-9,9-dioxide.

Synthesizing of chrysamine G alkynyl (C ≡ C) and thiazolinyl (CH=CH) derivative

Pass through at K by above-mentioned ₂CO ₃Exist down and iodomethane reaction and (Aldrich Chemical Copany, MilwauKee WI) change into methyl ester/methyl ether with 5 bromosalicylic acid.Under existing, palladium makes resulting 2-methoxyl group-5-bromine methyl-formiate and (trimethyl silyl) acetylene (Aldrich Chemical Copany, MilwauKee, WI) reaction.Remove trimethyl silyl, by above-mentioned in the presence of palladium, make two equivalent gained 2-methoxyl group-5-acetylenylbenzene methyl-formiates and 4,4 '-'-dibromobiphenyl (Aldrich Chemical Company, MilwauKee, WI) reaction.Prepare chrysamine G alkynyl analogue by above-mentioned by this ester of hydrolysis, 4,4 '-two (3-carboxyl-4-methoxyphenyl ethynyl)-biphenyl.By ordinary method the alkynyl analogue is reduced into chrysamine G thiazolinyl analogue.

Synthesizing of chrysamine G diiodosalicylic acid derivative

By 5-iodo-salicylic acid (Aldrich Chemical Company, MilwauKee, WI) then take off the synthetic chrysamine G 3-iodo-salicylic acid derivative of iodate, 4,4 '-two (3-carboxyl-4-hydroxyl-5-iodophenyl azo-group)-biphenyl in the iodate of 3-position in the selectivity of 5-position.After forming methyl ester and recrystallization, with light brown, wax shape 3-iodine derivative replaces with wintergreen oil by the step of above-mentioned synthetic chrysamine G.By using 75%CHCl ₃/ 25% hexane wash-out and separate required product from the silicagel column.Then by at 1: 1 ethanol that contains four equivalent NaOH: H ₂O dissolving refluxed three hours and with this ester hydrolysis.

Synthesizing of difluoro chrysamine G

(Aldrich Chemical Company, Milwaukee WI) replace Whitfield's ointment can synthesize the 5-fluorine derivative, 4,4 '-two (2-hydroxyl-3-carboxyl-5-fluorophenyl azo-group)-biphenyl with the 5-fluorosalicylic acid.In the Schiemann reaction, by using ¹⁸The precursor of F mark as [ ¹⁸F] the LiBF4 replacement, through Cs[ ¹⁸F] triazene decomposes, or warp ¹⁸F is to the nucleophilic substitution of X, X=tosyl group wherein, trifyl, NO ₂, ⁺N (CH ₃), or halogen, can prepare chrysamine G [ ¹⁸F] the aryl fluoride derivative.Referring to Fowler, J. and Wolf, A is at POSITRONEMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H.eds.) 391-450 (Raven Press, NY, 1986) and Kilbourn, M. fluoro-18 mark radiopharmaceuticals.(Fluorine-18?labeling?ofradiopharmaceuticals)。(Natl，Acad.Press，Washington，D.C.)(1990)。

Synthesizing of aromatics fluoroalkyl and Fluoroalkyloxy derivative

Use Bishop etc., medicochemistry magazine (J.Med.Chem.) 34:1612 (1991) but method synthesis of aromatic fluoroalkyl derivative, wherein suitable O-allyl group chain Claisen resets and generates the aromatics allyl deriv, it can be obtained fluoro ethyl or fluoropropyl derivative by further functionalized.Optionally, use Sonogashira etc., the palladium catalyzed coupling method of tetrahedron communication (TetrahedronLetters) 4467-4470 (1975) can easily change into the aromatics iodide length by two to five aromatic alkynes that carbon atom is formed.Change into fluoroalkyl derivative by this alkynes subsequently.Press Chumpradit etc., the method for medicochemistry magazine (J.Med.Chem.) 36:21 (1993) can prepare the Fluoroalkyloxy derivative, wherein uses 1-bromine (or iodine or sulphonyl oxygen)-ω-fluothane that suitable phenol alkylation is obtained corresponding fluoroalkyl derivative.

Aralkyl sulphonyl oxygen and alcoxyl sulphonyl oxygen derivative Radiofluorinated

Press Mathis etc., the method for nuclear medicine biology (Nucl.Med.Biol.) 19:571 (1992) is carried out Radiofluorinated, wherein use [ ¹⁸F] fluorochemical substituted aralkyl-or alcoxyl sulphonyl oxygen (for example (alkoxymethyl)-2 benzenesulfonyl) derivative, obtain virtue [ ¹⁸F] fluoroalkyl and [ ¹⁸F] the Fluoroalkyloxy derivative.

The radioiodination and the radiobromination of the chrysamine G derivative by the trialkyltin approach

Synthesizing of chrysamine G trialkyltin derivative

Fig. 2 B has shown the general structure of the trialkyltin derivative of chrysamine G.In general, the trialkyl tinbase will replace in xenyl part 3-position on one side, but other positions comprise that Whitfield's ointment or heterocyclic radical part also is the potential target.These trialkyltin derivatives are used for the stable direct precursor of people's radioiodinated and radiobrominating compound for preparation.More particularly, these trialkyltin derivatives can be used for preparing the halo radioactive compound to using in the amyloid imaging in vivo.

The general method of synthetic trialkyltin derivative

Press disclosed method, comprise Kosugi, M., etc., chemistry circular (Chem.Lett.) 1981:829; Heck.R. pure chemistry and applied chemistry (Pure and Appl Chem.) 1978:691; Echararren, A. and Stille, J. JACS (J.Am.Chem Sco.) 1987:5478; Mitchell, T. Organometallic Chemistry (J.Organometallic Chem.) 1986:1; And Stille, J. pure chemistry and applied chemistry (J.Pure and Applied Chem.) 1985:1771 can be by suitable aryl halide, [(C ₆H ₅) ₃P] ₃Pd (O) and six alkyl, two tin prepare the trialkyltin derivative.Also can be by Mathis etc., the method for tagged compound and radiopharmaceuticals (J.Labell.Comp.and Radiopharm.) 1994:905 uses n-BuLi and chlorination trialkyltin to obtain these derivatives.

3-three alkane of 4,4 '-two (3-methoxycarbonyl-4-hydroxyphenyl azo-group) biphenyl

The base tin derivative is synthetic

By synthetic 3-bromine or 3-iodine p-diaminodiphenyl (seeing above), reach and the wintergreen oil coupling as synthetic chrysamine G tetrazotization, and when needs methoxylation compound, by above-mentioned phenol is methylated, and preparation 3-bromine or 3-iodo-4,4 '-two (3-methoxycarbonyl-4-hydroxyphenyl azo-group) biphenyl or it is by dimethyl ether.Under the argon atmospher, with 1mmol phenolic ester or methoxyl group ester, [(C ₆H ₅) ₃P] ₃Pd (0) (0.1 to 0.2mmol), six dibutyltin dilaurates or hexa methyl ditin (1.25mmol) are with diox (25ml) was 70 ℃ of heating 16 hours.With the reaction mixture cooling and with solvent evaporation.Remove trialkyltin halogenide with the KF aqueous solution.Use the ethyl acetate extraction organism, use dried over mgso, filter, remove solvent under reduced pressure.By the silica gel purification residue, obtain 3-trialkyltin-4,4 '-two (3-methoxycarbonyl-4-hydroxyphenyl azo-group) biphenyl.

The radioiodination of trialkyltin derivative or radiobromination

By disclosed method, as Mathis etc., tagged compound and radiopharmaceuticals magazine (J.Labell.Comp.and Radiopharm.) 1994:905; Chumpradit etc., medicochemistry magazine (J.Med.Chem.) 34:877 (1991); Zhuang etc.: medicochemistry magazine (J.MedChem.) 37:1406 (1994); Chumpradit etc., the medicochemistry magazine (J.Med, Chem.) the described method of 37:4245 (1994), with tributyl or tin trimethyl derivative by Na[ ¹²⁵I] or Na[ ¹²³I] radioiodination or by Na[ ⁷⁵Br] or Na[ ⁷⁶Br] radioiodination.In general, with the 0.5mg trialkyl tin compound, 0.2ml anhydrous acetonitrile, 10 μ l 2M H ₃PO ₄, 2-100 μ l high specific (＞2000 Ci/mmol) Na[ ¹²⁵I] or [ ¹²³I] (or Na[ ⁷⁵Br] or Na[ ⁷⁶Br] solution in the NaOH of pH9-12, and dichloramine-T (DCT) (DCT in the 20 μ l2.5mg/ml acetonitriles) places the 1ml reaction flask.Cover this bottle, in the dark stir this mixture under the room temperature.By the HPLC monitoring reaction, and after 30 minutes with 50 μ l2M Na ₂S ₂O ₃With its quencher.By general chromatographic technique purified product.Mathis etc., tagged compound and radiopharmaceuticals (J.Labell.Comp.and Radiopharm.) 1994:905.Equally, can prepare low specificity by similar approach ¹⁸The F derivative.

Prepare "dead" I, Br, Cl, F and-general method of SH derivative

In general, with Sodium Nitrite and HCl or H ₂SO ₄Can be with Whitfield's ointment 3-or the 4-aminoderivative that shows among Fig. 2, or the corresponding derivative of salicylic heterocyclic analogs changes into corresponding two azo-compounds.Convert it into aryl iodide then by forming iodate two azos; Or can directly prepare the iodine derivative by the method for triazene intermediate.See, for example, Greenbaum, F. U.S.'s pharmaceutical journal (Am.J.Pharm.) 108:17 (1936), satyamurthy, N. and Barrio, J., organic chemistry magazine (J.Org.Chem.) 48:4394 (1983) and Goodman, M. etc., organic chemistry magazine (J.Org.Chem.) 49:2322 (1984).According to the triazene of Sandmeyer reaction, can prepare aromatic bromide and muriate from two azo-compounds with CuCl or CuBr processing or process as preparation iodine derivative.According to Schiemann reaction NaBF ₄, HBF ₄, or NH ₄BF ₄Handle two azo-compounds or can prepare the aryl fluorochemical through the triazene decomposition similar to the iodine derivative.Nucleophilic reagent such as HS with sulfur-bearing ^-, E _tO-CSS ^-, and S ₂ ^2-, handle two azo-compounds and can prepare the aryl thiophenol.Optionally, also can prepare the aryl thiophenol with the nucleophilic reagent substituted aryl halogenide of sulfur-bearing, referring to March, J.ADVANCED ORGANIC CHEMYSTRY:REACTIONS, MECHANISMS., AND STRUCTURE (3rd Edition, 1985).

Preparation radioactivity C, the general method of F and Tc derivative

Outside last method, according to literature method known in the art, can be with SPECT's ^99mTc or use the positron radiation radionuclide ¹¹C, ¹⁸F, ⁷⁵Br and ⁷⁶Br carries out the high specific radio-labeling.Some potential adhoc approach hereinafter will be described, but also have the conspicuous currently known methods of some those skilled in the art that description is arranged in the literature, as Fowler, J. and Wolf, A. positron radiation tagged compound (Positron emitter-labeled compounds) POSITRONEMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H.eds.) P391-450 (Raven Press, NY) (1986), Coenen, H. etc., Radiochimica Acta 34:47 (1983), and Kulkarni, Int J.Rad.Appl.﹠amp; Inst (PartB) 18:647 (1991) as a reference its content incorporates into own forces this.

By preparing with the coordination of aryl thiophenol ^99mThe Tc derivative.Use ¹¹The radio-labeling of C can by above-mentioned to suitable chrysamine G analogue with [ ¹¹C] the methyl iodide replacement, [ ¹¹C] alkylation, or [ ¹¹C] carboxylated method easily methylates by N-, and O-methylates and carries out.[ ¹⁸F] the aryl fluoride compound derivative can be by using ¹⁸In the precursor of F mark such as the above-mentioned Schiemann reaction [ ¹⁸F] LiBF ₄Replace, through Cs[ ¹⁸F] triazene decomposes, or warp ¹⁸F is to the nucleophilic substitution of X, X=tosyl group wherein, trifyl (triflate), NO ₂, ⁺N (CH ₃) ₃, or halogen, and prepare.Use ⁷⁵Br and ⁷⁶The radiobromination of Br can use parent's electricity (Br of similar radioiodination technology ⁺) or nucleophilic (Br ^-) replacement technique finishes, and sees Coehen, H., Supra.

3-hydroxyl-1,2-benzisoxazole derivatives and relevant derivative

Synthesizing of (seeing Fig. 2 C)

According to B shagen (Chem.Ber 100:954-960; 1967) method, by using hydroxylamine hydrochloride, make 2, (TCIAmerica, Portland OR) change into hydroxamic acid to 6-resorcylic acid (γ-resorcylic acid) methyl esters.Still according to the method for B shagen (Chem.Ber 100:954-960,1967), use SOCl earlier ₂, use triethylamine that hydroxamic acid is changed into corresponding 3-hydroxyl-1,2-benzoisoxazole then.Then through the step identical with being used for salicyclic acid derivatives with this compound and p-diaminodiphenyl, 3-bromo biphenyl amine, or other benzidine derivative couplings.Can brominated derivative be changed into trialkyltin and iodinated derivatives by mentioned above then.

Optionally, can make wintergreen oil and p-diaminodiphenyl coupling routinely and by the method for above-mentioned B shagen with gained 4,4 '-two (3-methoxycarbonyl-4-hydroxyphenyl azo-group) biphenyl (or chrysamine G dimethyl ester) changes into hydroxamic acid, changes into benzoisoxazole then.The 3rd class 3-hydroxyl-1, the 2-benzoisoxazole comprises 4 by the dihydroxy-benzene dicarboxylic acid of several isomeries, 6-dihydroxyl-1,3-phthalic acid, 3,6-dihydroxyl phthalic acid and 2,5-dihydric para-phthalic acid (Aldrich Chem, Co., Milwaukee, WI) synthetic.By after standard method and the coupling of p-diaminodiphenyl or derivatives thereof, press the method for above-mentioned B shagen, by dihydroxyl/diester being changed into dihydroxyl/two hydroxamic acid with azanol reaction.Also press the method for above-mentioned B shagen, by using SOCl ₂Change into two benzoisoxazoles with the triethylamine processing.

Phthalic imidine or isoindole-1,3 (2H)-derovatives

Synthesizing of (seeing Fig. 2 D)

Make 3-hydroxyl phthalic anhydride (Aldrich Chemical Company Milwaukee by the method identical with the method that is used for salicyclic acid derivatives, WI) prepare the 3-hydroxyphthalimide with the coupling of p-diaminodiphenyl or derivatives thereof, then by above-mentioned trialkyltin and the iodo-derivative of changing into.

Phthalocyclohydrazide or 2, and 3-benzodiazine-1,4 (2H, 3H)

Synthesizing of-derovatives (seeing Fig. 2 E)

The method identical with being used for salicyclic acid derivatives makes 3-hydroxyl phthalic anhydride (Aldrich Chemical Company Milwaukee, WI) the preparation 3-hydroxyl Phthalocyclohydrazide with the reaction of p-diaminodiphenyl or derivatives thereof is then by above-mentioned trialkyltin and the iodo-derivative of changing into.

2,3-benzoxazine-1,4 (3H)-derovatives (seeing Fig. 2 F) synthetic

(Aldrich Chemical CompanyMilwaukee WI) changes into 2, the 3-benzoxazine with the 3-hydroxyl phthalic anhydride with azanol.Make benzo-oxazine derivative and the coupling of p-diaminodiphenyl or derivatives thereof by the method identical then, then by above-mentioned trialkyltin and the iodo-derivative of being converted into being used for salicyclic acid derivatives.

(2H) 1,3-benzoxazine-2,4 (3H)-derovatives (seeing Fig. 2 G) synthetic

This compound is pressed the method (Chem.Ber.105:1926-1942 of Effenberger etc.; 1972) synthetic.In brief, make phenol and the coupling of p-diaminodiphenyl or derivatives thereof by the method identical with being used for salicyclic acid derivatives.Then by in the presence of triethylamine, this affixture being changed into carbamate with ethoxycarbonyl isocyanic ester (O=C=N-CO-O-Et) reaction.The carbamate (or N-ethoxycarbonyl-phenyl carbamate) that will replace by heating in diphenyl ether changes into the benzoxazine diketone.By above-mentioned the benzoxazine diketone is changed into trialkyltin and iodo-derivative then.

(3H) 2-benzo azine (benzazine)-1,3 (2H)-diketone

Synthesizing of derivative (seeing Fig. 2 H)

With the 3-hydroxyl phenylacetic acid (Aldrich Chemical Company MilWaukee WI) changes into acid amides, then by with make itself and the coupling of p-diaminodiphenyl or derivatives thereof with the identical method of salicyclic acid derivatives.Then by this adducts being changed into N-(3-hydroxyphenyl oxyethyl group)-urethane ester derivative with the Vinyl chloroformate reaction.Make the carbamate of replacement change into the p-diaminodiphenyl diketone by heating in diphenyl ether.By above-mentioned the p-diaminodiphenyl diketone is changed into trialkyltin and iodo-derivative then.

Synthesizing of 1,8-naphthalimide derivative (seeing Fig. 2 I)

Press the standard method of arylamine two azo couplings, as Saunders, K.H. and Allen, R.L. M.AROMATIC DIAZO COMPOUNDS (Edward Arnold, London, 1985) described, hereby its content is incorporated into own forces as a reference, make 4-amino-1,8-naphthalimide and the coupling of p-diaminodiphenyl or derivatives thereof.By above-mentioned two azo-naphthalene dicarboximides are converted into trialkyltin and iodo-derivative then.

Tetrazolium is with oxadiazole derivative (seeing Fig. 2 J and 2K) synthetic

U.S. Patent No. 3 according to Holland and Pereira medicochemistry magazine (J.Med.Chem.) 10:149 (1967) and Holland, 448, method in 107, by with the reaction of sodiumazide or aluminium azide with 2-cyanophenol (Aldrich Chemical Company, Milwaukee WI) changes into tetrazolium.In brief, the 2-cyano group phenol or the cyano group amphyl (1mmol) of handling the chrysamine G among the 40ml DMF with sodiumazide (10mmol) and triethylamine hydrochloride (10mmol) under the argon atmospher.This mixture was stirred 2 hours at 120 ℃, handle with this mixture cooling and by handling similar mode then with above-mentioned chrysamine G.

The He Cheng oxadiazole by the tetrazolium of handling above preparation with acid anhydrides (as diacetyl oxide).A kind of process for selective is Bamford etc., method among medicochemistry magazine (J.Med.Chem.) 38:3502 (1995), in the method, in the presence of dicyclohexyl carbodiimide, handle chrysamine G hydrazine derivative or Whitfield's ointment hydrazine derivative (handle each ester respectively and obtain) with hydrazine with Trapex.

Synthetic as the chrysamine G derivative of contrast

By (Fisher Chemical Co., Fair Lawn NJ) replace every normal p-diaminodiphenyl can synthesize anils with two normal aniline.By with p-diaminodiphenyl-2,2 '-disulfonic acid (Pfaltz﹠amp; Bauer, Inc., Waterbury, but CT) replace the p-diaminodiphenyl Synthetic 2,2 '-the disulfonic acid acid derivative.But by replace every normal Whitfield's ointment synthesizing phenol derivative with monovalent phenol.Congo red (Aldrich acceptable quality) can buy from market.Embodiment 2. chrysamine Gs and chrysamine G derivative combine with the specificity of A β

With combining of synthetic A β (10-43)

Chrysamine G can combine with synthetic A β (10-43) well external.Fig. 4 A has shown that chrysamine G and A β (10-43) bonded Scatchard analyze.K than the component of high-affinity _DBe 0.257 μ M, B _MaxBe 3.18nmoles chrysamine G/mg A β (10-43).The component of low affinity can not be limited by these data well, but it seems K _DBe 4.01 μ M and B _MaxBe 18.7nmoles chrysamine G/mg A β (10-43).Low affinity component show chrysamine G in high density with unique, the combination at low affinity position does not combine with impurity in the preparation.In vivo the amount of Zhu She chrysamine G is so low, so that do not have any combination with low affinity component.Under extremely low concentration, high and low affinity bonded ratio is very big.

As shown in Figure 5, the concentration of the amount of bonded chrysamine G and peptide is linear in the use range.

The bonded dynamic characteristic

Dynamics research has shown obviously combination (Fig. 6 A) fast, when the μ M of [chrysamine G]=112, and t _1/2Be 89 ± 1.8 seconds, finish and some dissociate slowly (Fig. 6 C) t in 1 minute substantially _1/2=55 ± 9.4 seconds [velocity constant of dissociating (k _-1)=1.26 * 10 ^-2Second ^-1].Fig. 6 B shows according to Bennett and Yamamura, Bennett, and J.P. and Yamamura, H.I. is in the conversion of the binding kinetics data of the method for NEUROTRANSMITTER RECEPTOR BINDING (N.Y.:Raven Press1985) pp.61-89.The linear portion of the binding curve among Fig. 6 A is moved in the straight line of Fig. 6 B, wherein ln[B _Eq/ (B _Eq-B _t)] corresponding with the time, B wherein _EqThe amount B of (4 minutes) bonded chrysamine G during for balance _tFor time=bonded amount during t.The slope of this line equals k _ObservedAnd k _-1=(k _Observed-k _-1)/[chrysamine G], k wherein _-1Dissociation rate constant for data determination among Fig. 6 C.Curve is followed following equation among Fig. 6 C: $A_t=A_0{e}^{-k_{-1}t}$ A wherein _tFor time=during t, keep the amount of bonded chrysamine G, A ₀Be the amount of some bonded chrysamine G in time=0, and k _-1Be dissociation rate constant.Association rate constant (the k that from this is analyzed, calculates ₁) be 3.75 * 10 ⁴M ^-1Second ^-1, gained K _D=k _-1/ k ₁=0.34 μ M analyzes in full accord with Scatchard.

Can suppress chrysamine G and A β bonded chrysamine G derivative

Chrysamine G analogue with the chemical structure that shows among Fig. 1 suppress [ ¹⁴C] chrysamine G and A β (10-43) bonded K _iValue and several displacement curves are as shown in Figure 3.K _iBe defined as IC ₅₀/ (1+[L]/K _D), wherein [L] in measuring [ ¹⁴C] concentration (.100-.125 μ M) of chrysamine G, K _DBe 0.26 μ M, the K of chrysamine G _DValue is by above-mentioned Scatchard assay determination.The K of chrysamine G itself _iValue is 0.37 ± 0.04 μ M, with very consistent by Scatchard and dynamic analysis gained result.Congo red K _iValue is 2.82 ± .84 μ M.The difluoro derivatives of chrysamine G, (5-FSA) effect of CG (Fig. 1) is 1/3rd (K of chrysamine G itself _i=1.16 ± 0.19 μ M).The activity prompting of difluoro chrysamine G derivative ¹⁸F difluoro chrysamine G derivative is suitable for the PET imaging, and ¹⁹F difluoro chrysamine G derivative is suitable for the MRS/MRI imaging of brain.

The effect of 3-ICG is slightly stronger than chrysamine G.The activity prompting of 3-ICG derivative ¹²⁵I two fluoro chrysamine G derivatives are suitable for the SPECT imaging.3-ICG (OMe) ₂In, the affinity of methylated 3-ICG phenol reduces by 10 times.CG (COOMe) ₂In because carboxyl is methylated, make more (about 200 times) that affinity reduces.Remove the acidic group part fully, as amphyl, then completely destroy binding affinity.

The acidic group of these results suggest chrysamine G analogues part with play an important role during A β combines, and phenolic group partly plays a driving role.The effect of phenolic group part may be that the acidic group part-structure is localized to be stablized by helping with sour bonded hydrogen.The phenolic group that exists at the ortho position both may be by hydrogen in conjunction with the charge distribution that also may change the acidic group part by the charge distribution that changes whole aroma system.Optionally, phenolic group may be connected with two-dentate of amyloid binding site face by phenolic group and acidic group and participates in directly and the combining of amyloid.As among resorcylic acid derivative (6-OHSA) CG, add second phenolic group and just produced the strongest compound of affinity in this series, its K at the ortho position of carboxylate salt _iValue is 0.094 ± .02 μ M.

It seems that the lipotropy of raising xenyl main chain can increase some affinities.The dihalo derivative, 3,3 '-I ₂CG, 3,3 '-Br ₂CG and 3,3 '-Cl ₂The K of CG _iBe worth all very closely, be about half of chrysamine G.

The distortion of dihedral angle has reduced affinity significantly between the xenyl phenyl ring that is caused by 2 replacements.2,2 of chrysamine G '-disulfonic acid derivatives, 2,2 '-(SO ₃) ₂CG does not have the provable this point of affinity.Because 3,3 '-the dicarboxyl derivative, 3,3 '-(COOH) ₂CG only shows the activity doubly than the low 7-of chrysamine G, therefore increase the acidic group part and may not be to cause 2,2 '-sole cause of disulfonic acid loss of activity.This 2,2 '-derivative is very unique, wherein the sulfonic group that 2-bit space steric hindrance is very big makes xenyl depart from the plane.Molecular model studies show that 2,2 '-disulfonic acid derivatives in dihedral angle between two xenyl phenyl ring be 83 °.In chrysamine G and every other reactive derivative, this angle is approximately 35-40 °.

In order to explore the critical nature of chrysamine G functional group bidentate, we have studied half the combination (Fig. 1) of anils of expression chrysamine G molecule.Bound energy can calculate approx by following equation:

Δ G=-RT ln K _EqWherein Δ G is the energy of association reaction, and R is molecular gas constant [8.31441J/ (mole ° of K)], and T is a ° K temperature, and K _EqBe following equilibrium constant of reaction.

[probe]+[peptide]  [probe peptide] and K _Eg=1/K _D≈ 1/K _iUse K _DBe the chrysamine G of 0.26 μ M, bound energy is approximately 38KJ/mole.If aniline is with half combination of this energy, Qi Wang bound energy is about 19KJ/mole so.To K _iValue is the anils of 73 μ M, and bound energy is 23KJ/mole, and it is quite consistent with desired value.The importance of the hydrophobic region of chrysamine G and anils can be by Whitfield's ointment itself fully in conjunction with active this true proof.

Chrysamine G seems than the high several times of Congo red affinity to this peptide to the affinity of A β.No matter analyze by Scatchard, kinetics methodology is still measured in conjunction with the inhibition method, and the combination that dissociation constant is about 250-400nM all is a reversible.Because the amyloid protofibril is noncrystalline, insoluble character, the composite structure of Congo red or chrysamine G and amyloid still can not be definite by accurate structure technology such as x-ray crystal diffraction or multidimensional NMR.Congo red and the interactional model of amyloid is suggested.Cooper，Lab.Invest.31∶232(1974)；Romhanyi，Virchows?Arch.354∶209(1971)。It is not to combine with independent amyloid peptide molecule that the document thinks Congo red, but crosses because beta sheet protofibril and several directed A beta molecule bonded.Klunk etc., J. Histochem.Cytochem.37:1273 (1989).

Fig. 7 has shown the diagram of this model that uses the MacroModel2.5 making, and wherein chrysamine G is across 5 peptide chains in the antiparallel beta sheet configuration.The not further structural modification of employed peptide.These peptides distance that makes the alternative chain that is in line is 4.76 , and this is the fibriilar feature of beta sheet.The alternative peptide chain is plotted black in white.Chrysamine G (deceiving) is in line for the energy minimum state and with the protofibril model and realizes farthest contacting with hydrophobic phenylaniline 19/20 (figure below) with Methionin 16 (light gray ellipse among the last figure) with realization.Two figure represent that mutual angle is approximately 90 ° same model.The direction that white arrow indicates another diagrammatic sketch to observe.

The distance of 19.1 just in time matches with distance across 5 chain 19.0 (Kirschner etc., Proc.Natl.Acad.Sci.U.S.A.83:503 (1986) show that the distance between the adjacent chain is 4 * 4.76 ) between the chrysamine G carboxylic moiety.If the natural structure of A β contains the (Hilbich etc. just like propositions such as Hilbich, molecular biology magazine (J.Mol.Biol.) 218:149 (1991)) hairpin ring structure, chain 1 and 2 so, 3 and 4,5 and 6, Deng, will with second half doubling in a part, unless this model is identical in other respects.Should be noted that amino-acid residue positively charged in this model, as the Methionin among the A β-16, is essential.Formerly document has shown that amino acid whose quantity positively charged in Congo red combination and the amyloid fibrils sample is relative.Klunk etc., histological chemistry and cytochemistry magazine (J.Histochem.Cytochem.37:1273 (1989).The interactional importance of the character of the bidentate of Fig. 7 model and hydrophobic group can be by chrysamine G monodentate aniline analogue affinity reduction and salicylic non-activity and prove in the stronger stronger fact of compound (see figure 1) activity of lipotropy that p-diaminodiphenyl partly has two halogen atoms.Xenyl near planar importance can be by 2,2 '-this is true and prove for the disulfonic acid derivatives non-activity.Embodiment 3. usefulness chrysamine Gs are distinguished Alzheimer brain and normal brain activity

Chrysamine G and AD brain bonded feature

Carry out chrysamine G and chrysamine G derivative and AD brain sample bonded Scatchard analyzes in order to understand chrysamine G to combine with AD brain enhanced.(Fig. 4 B, table 1).Under employed condition, contrast and AD brain show single in conjunction with component.K in the AD brain _DThan contrast low 16% but difference and not obvious (p=.29).B in the AD brain _MaxComparison is high by 36% according to brain, but difference not obvious equally (p=.09).Therefore, the combination that increases in the AD brain as if mainly be since exist more with contrast brain in identical in conjunction with component, rather than have certain unique component.

Table 1.AD and the comparison that contrasts the brain incorporating parametric

	??K D(μM)	??B max(pmol/μg?prot)
			Contrast (n=6)	????0.47±.049	????0.576±.092
??AD(n＝5)	????0.39±.048	????0.784±.061

CG is obviously relevant with the NPs number in the relevant cortex with the combination of AD brain.Fig. 8 A demonstration [ ¹⁴C] CG in conjunction with the AD brain on/relation of middle volume and last temporo cortex NPs number.No matter comprise contrast (r=0.69; P=0.001) still consider AD brain (r=0.59 separately; P=0.007), the relation with NPs is fairly obvious.Fig. 8 has shown the similar relation to the NFT number.As NPs, no matter comprise contrast (r=0.60; P=0.001) still consider AD brain (r=0.50 separately; P=0.026), the relation with NFTs is fairly obvious.CG relevant with NFTs the people is taken aback, because can make the painted Congo red derivative of NFTs.Right relevant (the r=0.82 of NPs digital display with the NFTs number; P=.0001).

Concerning the brain that is used for this research, only can be had or do not had the qualitative data of amyloid blood vessel disease, thus can not study CG in conjunction with to the similar relation of cerebrovascular amyloid level.As if in the relation between CG combination and NP number, the existence of amyloid blood vessel disease is made us catching wondering.Fig. 8 A shows the brain (r=0.79 of no amyloid blood vessel disease; P=0.01) than having cerebrovascular amyloid precipitation (r=0.49; P=0.15) have closer CG in conjunction with the relation of NP number.In the relation of NFT number, also found similar phenomena.

[ ¹⁴C] chrysamine G and AD brain bonded K _DIn value and the experiment in vitro [ ¹⁴C] chrysamine G and synthetic A β bonded K _DBe worth similarly, illustrate that combination in brain homogenate also can represent the interaction with A β.Chrysamine G is in conjunction with also combining with these structures with relation explanation chrysamine G in brain homogenate of NFTs.Optionally, because NFTs number and NPs number are closely related, so [ ¹⁴C] chrysamine G be in conjunction with just in time may being that chrysamine G combines the epiphenomenon with NPs with the relation of NFTs.

By measuring with the bonded of synthetic A β peptide or Alzheimer cerebral tissue, useful chrysamine G derivative or analogue provided by the invention have K _DAt least at the binding affinity of 0.01 to 10.0 μ M scope; K _DScope is that the high-affinity compound of 0.0001 to 0.01 μ M also can be used for method of the present invention.

From above-mentioned consideration, the chrysamine G combination may not be specific to A β.In other words, chrysamine G comprises A β settlings all in the neuritis spot and the amyloid in the cerebrovascular in conjunction with " load " that may reflect the total amyloid that exists in the amine.Among the NFTs phosphinylidyne T shape proteinosis also may participate in chrysamine G combine Goedert, M. etc., PNAS85:4051 (1988).NFTs also is made up of the quaternary structure antiparallel protofibril similar to A β protofibril.Kirschner etc., Proc.Natl.Acad. Sci.U.S.A.83:503 (1986).

Total and the relevant chrysamine G combination of difference AD and normal brain activity

As mentioned and hereinafter described external in conjunction with measuring method can be used as screening in vivo with brain bonded compound and indication subsequently in vivo the model of imaging research be widely used in this area.Referring to, Young, A. etc., receptor determination: interior (Receptor Assays:In Vitro and In Vivo.) the in POSITRON EMISSION TOMOGRAPHY ANDAUTORADIOGRAPHY (Phelps of external and body, M., Mazziota, J., and Schelbert, H.eds) PP.73-111 (1986).The chrysamine G of mark of the present invention and chrysamine G derivative also can be used for described quantificational expression living tissue of context or amyloid beta deposition external in conjunction with measuring in the tissue after death.

All can be observed in AD brain and the contrast brain homogenate [ ¹⁴C] saturable (specific) combination of chrysamine G, account for the 60-80% of total binding in this combination of AD brain.Non-saturable in AD brain and the contrast brain in conjunction with closely similar.Big according in the brain all compared in saturable or total combination in the AD brain.Although the sensitivity that the use total binding must be lower, this parameter more can indicate the in vivo success of research, and the latter is a final purpose of the present invention.And in order to expand in vivo research, can be with the combining standardized chrysamine G in AD and normal brain activity of the chrysamine G in the cortical area in conjunction with closely similar brain district.Do the calculating of having avoided being difficult in vivo the bonded absolute magnitude of carrying out like this.We can check as the combination in the cerebellum of potential check plot, because typical NPs is extremely rare in this brain district.(Joachim etc., U.S.'s pharmacology magazine (Am.J.Pathol.) 135:309 (1989)).

[ ¹⁴C] chrysamine G is attached to the supracerebellar mean vol of contrast and is attached to the supracerebellar amount of AD (table 2) no better than, and this makes cerebellum usable as internal contrast.Therefore, cerebellum binding ratio (CBR) accurately reflected bonded [ ¹⁴C] chrysamine G absolute magnitude and an individual internal reference thing is provided for each brain.No matter with the fmol/ μ proteic absolute magnitude of g (table 2) expression, still with the cerebellum of same brain in the bonded ratio represent (table 3), the binding capacity in the AD brain is all bigger.CBR is the higher take off data of sensitivity, can show variation very little between the various brains.The use of summing up content and CBRs has promoted the expansion that these in vitro results are studied greatly in body.Following result is all used these unit representations when correspondingly, suitable.

Total bonded compares * in table 2.AD and the contrast brain

The brain district	Contrast (fmol/ μ g albumen)	AD (fmol/ μ g albumen)	The p value
				Cerebellum	??75±13(n＝8)	??73±9(n＝11)	??p＝0.91
Antinion	??58±8(n＝6)	??124±16(n＝10)	??p＜0.006
				On/middle volume	??54±10(n＝8)	??130±21(n＝1?1)	??p＜0.005
Last temporo	??66±17(n＝8)	??121±14(n＝11)	??p＜0.02
				Head of caudate nucleus	??73±11(n＝4)	??123±22(n＝7)	??P＝0.14
Lower wall	??76±13(n＝8)	??137±19(n＝11)	??p＜0.03
				Occipitalia	??64±16(n＝8)	??95±12(n＝11)	??p＝0.15

^*High-as to count the AD brain with low-spot to have concurrently.Table 3. is in bonded total in AD that represents with the ratio of cerebellum and contrast brain * relatively

The brain district	Contrast (CBR)	????AD(CBR)	The p value
				Antinion	??0.87±.04(n＝6)	??1.87±.25(n＝10)	??p＜0.004
On/middle volume	??0.73±.02(n＝8)	??1.84±.18(n＝11)	??p＜0.001
				Last temporo	??0.86±.08(n＝8)	??1.63±.17(n＝11)	??p＜0.002
Head of caudate nucleus	??0.95±.04(n＝4)	??1.76±.31(n＝7)	??P＜0.004
				Lower wall	??0.90±.08(n＝8)	??1.93±.20(n＝11)	??p＜0.001
Occipitalia	??0.77±.13(n＝8)	??1.44±.20(n＝11)	??p＜0.02

* the CBR value of each sample by in this sample ¹⁴C] chrysamine G bonded absolute value divided by with

In the cerebellum sample of one brain [ ¹⁴C] chrysamine G bonded absolute value and obtaining.Numerical value in the table

For the average CBRs in each brain district (± SEM).High-as to count the AD brain with low-spot to have concurrently.

Fig. 9 A and 9B show by standardized six the brain districts of cerebellum of same brain with ¹⁴C] combination of chrysamine G.Fig. 9 A shows chrysamine G and the AD brain that has above the 20NPs/x200 magnification, " high spot is counted the AD brain ", the combination in middle AD brain district.Fig. 9 B shows chrysamine G and has the AD brain that is lower than the 20NPs/x200 magnification, " low spot is counted the AD brain ", the combination in middle AD brain district.In all brain districts, with the AD brain combine obviously more than with combine (the seeing Table 3) of contrast.At last/middle volume cortex, do not have between contrast and any one AD brain sample and overlap.In all the brain districts except that the occipitalia cortex, contrast overlaps with having＞do not have between the AD sample of 20NPs/ * 200 magnifications.In the minimum brain district of typical NPs precipitation, in occipitalia cortex (or cerebellum), the maximum that can be observed between AD and the contrast overlaps.

Fig. 9 C shows from two Down ' s syndromes patient's data.Down ' s syndromes patient can produce A β deposition in 10 years at its 4th, and a lot of people can develop into AD.Wisniewski etc., neurological (Neurology) 35:957 (1985); Schapiro etc., neurobiology aging (Neurobiol.Aging) 13,723 (1992).These patients all show surpass to illumination range [ ¹⁴C] the chrysamine G combination.Because the existing amyloid beta deposition of younger patient (23 years old) but yet there are no the clinical dementia symptom, so Fig. 9 C explanation chrysamine G can occur in the clinical symptom of dementia long ago measuring dull-witted but will develop into the patient of AD and the difference of control group certainly.

Compounds and methods for of the present invention provides the method for two kinds of useful measurement AD brains and normal brain activity difference; (1) total chrysamine G in conjunction with the chrysamine G in (table 2) or (2) given brain district in conjunction with the cerebellum of same brain in the chrysamine G bonded than (table 3).These in vivo quantitative analyses of measuring AD neuritis spot have two big advantages.At first, measure total A β combination by providing a kind of, rather than specificity A β bonded method, the present invention can need not make the patient face quantitative analysis A β deposition under the situation of injecting radioactive substance for the second time in order to measure non-specific binding.For this reason, data are only represented total combination.In all experiments of the present invention, even under the AD situation widely different, do not provide specificity bonded data with contrasting brain yet.

The second, the variation that the chrysamine G derivative absorbs in brain will influence the absolute concentration of chrysamine G in the brain.Therefore, need to find to explain the mechanism of these variations between the experimenter.Seldom show the sedimentary brain of A β district (that is, " blank " in the experiment) by seeking, every patient can be used as his contrast.Because typical NPs extremely rare (Joachim etc., American Journal of Pathology (Am.J.Pathol.) 135:309 (1989)) in the cerebellum, chrysamine G can be used as the contrast that each is studied brain with combining of cerebellum.The result with the chrysamine G in the given brain district in conjunction with the cerebellum of same brain in the chrysamine G bonded recently represent (Fig. 9 and table 3).

For the amyloid among the quantitative assay AD in vivo, should consider the influence of encephalatrophy.Therefore, when we use chrysamine G and chrysamine G derivative in vivo during the quantitative assay amyloid, can proofread and correct encephalatrophy according to the MRI cubing.Be similar to these class methods that this area routine is carried out with the collaborative MRI cubing of carrying out of the inventive method.Referring to, Pearlson, G. and Marsh, L.MAGNETIC RESONANCE IMAGING IN PSYCHIATRYin Annual Review of Psychiatry (Vol.12) Oldham, eds.P.347-381 such as J. (1993).Therefore measuring the total radioactive method of every volume brain Qu will use following equation:

Signal/brain district " A " volume or S/VA are appointed as in this measurement mean that the cerebellum ratio will represent by following formula: S/V wherein _CBBe signal/volume in same experimenter's cerebellum during the same imaging research.Suspection can be had AD or other then is that similar ratio in this ratio in any brain district beyond the patients of cerebellar of pathological state of feature normal control group experimenter's suitable with the age the normal range in same brain district is compared with the amyloid beta deposition.The bonded ratio of high sedimentary brain district of neuritis spot and cerebellum can be used as difference Alzheimer patient and control group experimenter's parameter.Embodiment 4 chrysamine Gs, chrysamine G derivative and Congo red octanol-water partition coefficient

Octanol-water partition coefficient is the tolerance relevant with the compound lipotropy.The lipotropy of compound is high more, the easy more hemato encephalic barrier that passes through.Referring to, Goodman and Gilman ' s THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).The octanol/water partition ratio of chrysamine G is 60.22 ± 3.97 and this Congo red coefficient is 0.665 ± 0.037 (P＜0.001).The lipotropy of this explanation chrysamine G is stronger 90 times than Congo red, and is therefore easier in theory mammiferous hemato encephalic barrier.The 3-iodine and 3,3 of chrysamine G '-the octanol/water partition ratio of diiodo-derivative (Fig. 1) is respectively 72.53 ± .74 and 112.9 ± 7.3.These octanol/water partition ratios show that the lipotropy of on-radiation analogue of some radiolabeled chrysamine G derivatives that these are used in vivo studying is stronger 170 times than Congo red, than the strong twice of chrysamine G.This illustrates that they are than Congo red or chrysamine G is easier enters in the brain.Embodiment 5 chrysamine Gs and chrysamine G derivative are by the ability of hemato encephalic barrier and the metabolism of chrysamine G

They can be by hemato encephalic barrier and near the amyloid beta deposition entity to use the amyloid probe in vivo to diagnose the AD requirement.

Chrysamine G is by the ability of hemato encephalic barrier Swiss-Webster mice study.I.v. injecting the brain/blood ratio that recorded in back 15 minutes surpasses 10: 1, reaches 20: 1 in the time of 35 minutes (Figure 10).From now on, radioactivity almost keeps constant in the brain, but the reduction in the blood and increase in the liver.Brain/kidney ratio is the highest when 15 minutes (sampling back), reaches 0.5.I.v. injection [ ¹⁴C] extract brain and liver 60 minutes the time behind the chrysamine G, on reversed-phase HPLC with chrysamine G reliably jointly by wash-out, obtain again＞95% radioactive substance, illustrate that tangible metabolism does not take place chrysamine G during this period.

Chrysamine G enters in the normal mouse brain, and brain/blood is higher than very.Radioactivity keeps relative constant in first 30 minutes hindbrains.And the reduction in the blood, the rising in the liver.This explanation is compared with further accumulating in brain, and high brain/blood is than more causing chrysamine G to be removed effectively from blood by liver.At 60 minutes, all radioactivity proof chrysamine Gs that detect from brain and liver did not change basically.Congo redly be difficult for passing through hemato encephalic barrier.Tubis etc., J.Amer.Pharm.Assn.49:422 (1960).Most of Congo red by liver and spleen removing, the brain that reaches in the guinea pig/kidney ratio is about 0.07.Tubis etc., supra.Chrysamine G also can be removed by liver, but more enters brain.

The living animal experiment provides the foundation of further mensuration dosage range, by the transport efficacy and the binding ability of hemato encephalic barrier.Preferred especially Games etc. for achieving the above object, the mutant mice model of (natural Nature373:523 (1995)), and cerebral amyloidosis " geriatric animals " model; That is, the animal of the scorching spot of Alzheimer type cerebral nerve that known generation quantity is variable sees Wisniewski etc. as the mouse of sudden change and old dog or monkey.Neuropathology and experiment neurological magazine (J.Neuropathol.﹠amp; Exp.Neurol.) 32:566 (1973), Selkoe etc., science (Science) 235:873 (1987) is used to test combination and detection efficiency.The living tissue of above-mentioned in vivo mensuration requirement monitoring contrast or autopsy tissue are to confirm also existing of quantitative analysis amyloid beta deposition.

Other animal models that are applicable to the test present composition and method can produce with the method for sudden change.For example, Quon etc., nature (Nature), 352:239-241 (1991) use rabbit neuro-specific endolase promotor inhibitor district to make mutant mice.Also referring to, Wirak etc., science (Science), 253:323-325 (1991).Also have other models manufactured by the direct encephalic of animal is used β/A4 peptide (Tate etc., Bull.Clin.Neurosci.), 56:131-139 (1991).

It should be noted that still that not animal model in vivo can be proved to be the fabulous model of AD neuropathology.In other words, they are more suitable for doing the model of the amyloid beta deposition of normal age.All the more so concerning the geriatric animals model.All these models have all shown the advantage of the diffusion spot of being discussed as above-mentioned geriatric dog model.But except that the mutant mice model of Games etc., still have some cerebrovascular amyloids seldom to produce the neuritis spot.Other mutant mice models usually only show the diffusion spot.Therefore, when these models can be used for studying in the brain probe and distribute, these models but were difficult to be presented at according to the desired identical quantitative difference of seeing in the AD brain of above-mentioned chrysamine G and the in vitro study of AD brain bonded significantly.

The chrysamine G derivative is by the assessment of human blood brain barrier ability

To have quilt that about 500Ci/mole specific activity or higher dosage is approximately 10mCi suitably the mark chrysamine G derivative intravenous injection of crossing suffer from the subject of AD to normal subjects or suspection, and by this derivative in SPECT or the PET imaging monitoring analysis brain with respect to the detectability of other organs and be determined at the time course of detectability in the brain.The dosage that can easily detect is defined as " imaging effective dose ".

Chrysamine G and chrysamine G derivative are distinguished the human AD control group suitable with the age

The assessment of ability

The radiolabeled suitably chrysamine G derivative of imaging effective dose is expelled to suspects to have in the subject of the brain amyloid beta deposition that produces owing to pathological condition as AD.After 15 minutes to 24 hours, detect the emission signal that spreads out of in the brain by SPECT or PET.Detect the radioactivity in all the brain districts in the detector sensing range simultaneously.Sensing range will be set to and comprise cerebellum, last temporo cortex, on/major part in middle volume cortex and middle diencephalon district.Before the research, will carry out MRI scanning so that proofread and correct the atrophy of tested brain district cerebral tissue by the method for embodiment 3.The S/V that discusses among the embodiment 3 _A, S/V _CBAnd ratio A variable will be calculated and with the previous similar standard proportional that has obtained from suitable normal control experimenter of age relatively.Embodiment 6 chrysamine Gs and cerebrovascular amyloid bonded histology location

The last figure expression of Figure 11 is by painted two the neuritis spots of chrysamine G.Dyeing process such as Stokes and Trickey, the method for clinical pathology magazine (J.Clin.Pathol.) 26:241-242 (1973) just replaces Congo red with chrysamine G.Except intensity is hanged down sometimes, the identical (not shown) of these settlings and usefulness congo red staining.Following two Photomicrographs of Figure 11 show the adjacent section of the temporal lobe of the AD patient with amyloid blood vessel disease.Cut into slices with the Congo red Puchtler etc. that presses in the lower-left., the method dyeing of histological chemistry and cytochemistry magazine (J.Histochem.Cytochem.) 10:35 (1962) Puchtler.The section of Figure 11 bottom right replaces Congo red method dyeing of pressing Puchtler with chrysamine G.The blood vessel that same amyloid is full of is easily represented in two sections.Also can be observed a small amount of background autofluorescence from red corpuscle and lipofuscin.Two Photomicrographs all use FIC (FITC) filter with laser with the focus microscope photographing.Bright wisp is represented 20 microns.Embodiment 7 chrysamine Gs and the toxic mensuration of chrysamine G derivative

With dosage is 10 and the "dead" chrysamine G intraperitoneal administration of 100mg/kg, does not find significant behavior effect and toxicity on one's body mouse in 72 hours.[ ¹⁴C] the chrysamine G dosage observes the doctor's advice of 1mg/kg.

According to setting up LD ₅₀Test, it seems that chrysamine G seldom shows anxious toxicity.Even the chrysamine G saturated solution that may be injected into the intravital maximum volume of mouse (about 0.025ml/g) of the murder by poisoning that causes with the influence of no fluid volume does not all have tangible behavior variation during to injected in mice (100mg/kg) at least 72 hours the longest trial period.Measure the dosage regimen that the required dosage of radiolabeled derivative is lower than execution this dosage by SPECT or PET.

Congo red with much larger than the amount that is used for radioactivity chrysamine G derivative, safety injection in human body, disclosed Congo red LD ₅₀Be 190mg/kg mouse (Tubis etc., American Pharmaceutical Association's will (J.Amer.Pharm.Assoc.) 49:422 (1960).It and chrysamine G show＞LD of 100mg/kg ₅₀Be worth similar.Therefore, these two kinds of chemically similar compounds have similar hypotoxicity to mouse.

Equally, by injecting with the very wide concentration of scope and can testing other chrysamine G derivatives to mouse or other higher mammiferous toxicity by the various poisoning signs that methods known in the art are zoologizeed.See Goodman and Gilman ' s; THE PHARMACOLOGICAL BASISFOR THERAPEUTICS (7th Ed.).Embodiment 8 chrysamine Gs prevent the mensuration of the toxic ability of A β (25-35)-bring out

Prevent the toxicity of A β (25-35)-bring out in the PC-12 cell

Rat pheochromoblast oncocyte (PC-12) is grown in containing the RPMI1640 medium of 10% foetal calf serum.Is in the 96-hole flat board of 100 μ l with the cell inoculation of about 5000 exponential growths at the medium volume, and 37 ℃ of overnight incubation.To add 20 μ l at A β (25-35) and chrysamine G (CG) or the allied compound that 37 ℃ of quilts condensed 7 days in advance in advance reaches in the aqueous solution of ultimate density (0.01 to 10 μ M A β (25-35) and 0.03 to 20 μ MCG) and cultivates in advance.The cell cultures 24 hours of 5mg/ml MTT (3, (4,5-dimethylthiazole-2-yl) 2,5-phenylbenzene tetrazolium bromide) in the 13.3 μ l sterilization phosphate buffered saline (PBS) will be added in advance.At 37 ℃ after 4.5 hours, add 100 μ l and extract the damping fluid (20%w/vSDS in 50%DMF/ water; 80% acetate with 2.5% and 2.5%1N HCl) the pH value is transferred to 4.7 and with dull and stereotyped overnight incubation.Hansen etc., immunological method magazine (J.Immunol.Methods) 119:203 (1989).Measure the colour-change at 560nm place then.Only add 20 μ l distillation, deionization H ₂The specific absorption of the control wells of O is defined as maximum keep alive power.Add in the hole of 0.1% (ultimate density) trotyl X-100, cell is dissolved, is interpreted as maximum toxicity.

Cultivate the PC12 cell with A β (25-35) and cause of the dependence reduction (Figure 12) of the activity of these cell reduction MTT concentration.Figure 12 shows by what the MTT reduction was measured and is not having the influence of the concentration of increase A β (25-35) in the presence of the chrysamine G to PC12 cellular oxidation reducing activity.The MTT reduzate is marked on the longitudinal axis in the absorption of 560nm.The independent effect of A β (25-35) is shown in the solid bar graph, shows the minimizing MTT reductive effect relevant with dosage.White numeral in the solid bar graph shows the notable difference with contrast (no A β, no chrysamine G).Hollow bar graph shows the provide protection of 20 μ M chrysamine Gs.Black numbers demonstration in the hollow bar graph has or not chrysamine G to have reductive notable difference between the MTT down.

Figure 13 illustrates increases the provide protection that chrysamine G concentration reduces antagonism A β (25-35) inductive PC12 cellular oxidation reducing activity.Chrysamine G effect during no A β (25-35) is shown in the solid bar graph.No A β (25-35) does not have evident difference between the chrysamine G of any concentration and the contrast (no A β, no chrysamine G) when existing.The existence MTT down that hollow bar graph shows 1 μ M A β (25-35) and increases the chrysamine G of concentration reduces.Each concentration chrysamine G of white numeral in the solid bar graph exists down, has or not the MTT reductive notable difference between the A β (25-35).Black numbers in the hollow bar graph shows the MTT reductive notable difference that A β (25-35) contrast (no chrysamine G) and A β (25-35) add to be increased between the chrysamine G concentration.

Formerly technology is reported, and Congo red concentration begins to resist A β-inductive toxicity when surpassing 2 μ M, reaches protection completely during 20 μ M.Burgevin etc., neuroscience report (NeuroReport) 5:2429 (1994); Lorenzo and Yankner, Proc.Natl.Acad.Sci.91:12243 (1994); Pollack etc., neuroscience circular (Neuroscience Letters) 184:113 (1995); Pollack etc., neuroscience circular (Neuroscience Letters) 197:211 (1995).The provide protection of chrysamine G had both depended on that the concentration (Figure 12) of A β (25-35) also depended on the concentration (Figure 13) of chrysamine G.The provide protection of chrysamine G begins when 0.2 μ M obviously, and this concentration is very near chrysamine G and synthetic A β bonded Ki value, 0.37 μ M (Fig. 1).Seem that the effect of chrysamine G is stronger than Congo red, i.e. demonstration effect in 0.1 to 1.0 scope.This is consistent with them and synthetic A β bonded Ki valency, wherein chrysamine G be 0.37 μ M and Congo red be 2.8 μ M (Fig. 1).

In another experiment (Figure 14), by with 1 μ M A β (25-35) cultured cells inspection chrysamine G and its not with the effect of A β bonded amphyl (see figure 1).Chrysamine G shows provide protection at 0.1 and 1 μ M, and amphyl does not show provide protection, has perhaps also increased the toxicity of A β.

The Congo red lipophilic derivatives of these presentation of results, chrysamine G prevent combine the closely similar concentration of A β with it under in the cell cultures by the beta induced toxicity of A.This protection has also shown structure specificity, because can not prevent A β-inductive toxicity with synthetic A β bonded acid derivative.Because chrysamine G can distribute in brain well, because these results prove chrysamine G and have the possibility of treatment AD with A β bonded chrysamine G derivative.

The mechanism of chrysamine G provide protection it be unclear that.There are two kinds of main possibilities.At first, chrysamine G can hinder the cohesion of A β.The second, chrysamine G can hinder the effect (direct or non-direct) of A β to target cell.The cohesion of Congo red inhibition A β, and the A β toxic action of protection antagonism cohesion.Lorenzo and Yankner, Proc.Natl, Acad.Sci.91:12243 (1994).In the above-mentioned experiment to the obstruction of cohesion may not because A β is condensed in advance before cultivating with chrysamine G.Therefore, suppress cohesion and can show it is the important therapeutic action of chrysamine G, but can not explain the provide protection of the A β of chrysamine G antagonism cohesion, in chrysamine G shown in Figure 7 and A β bonded model, how showed chrysamine G " lid " on A β surface.How this may change by cell-surface receptor or other macromole such as the sedimentary viewpoint of complement proteins recognition protofibril, and with may conflict by the viewpoint of the toxic action of the A β of these macromole mediations.Chrysamine G and Congo red may be before A β cohesion and the multiple influence of back generation.The patient sees that from the therapeutics viewpoint this is favourable, because may go to a doctor when having A β cohesion and producing amyloid beta deposition.

Claims

Modification according to the 19th of treaty

Shown in the formula I can with amyloid bonded compound or its water soluble non-toxic salt: Wherein:

Be N=N, C ≡ C, or CR '=CR ' (wherein R ' expression H or low alkyl group); X is C (R ") ₂

(wherein R " be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

Wherein R ' is H or low alkyl group) or X be CH=CH, N=N, C=O, O, NR ' (wherein R ' expression H or low alkyl group), S, or SO ₂R ₁And R ₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo Evil two as follows

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or group as follows:

When formula I compound is two azobenzene compounds, at least one R ₂Be not H, OCH ₃, CH ₃, or halogen; Q is selected from down array structure independently of one another, respectively contains a carboxylic acid or sour sample functional group: IA, IB, and IC, ID, IE, IF and IG, wherein

IA has structure as follows:

R wherein ₃, R ₄, R ₅, R ₆, or R ₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or group as follows:

The R among two Q ' wherein ₃, R ₄, R ₅, R ₆, or R ₇In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂, or carboxyl; And work as R ₅Be hydroxyl R ₆R during for carboxyl or its lower alkyl esters ₁, R ₂, R ₃, R ₄, or R ₇Have at least one not to be H; Work as R ₅Be amino, R ₆Be carboxyl or its lower alkyl esters, and R ₇During for hydroxyl, R ₁, R ₂, R ₃, or R ₄At least one is not H; As two R ₂Just in time be CH ₃, or OCH ₃, R ₅Be hydroxyl, and R ₆During for carboxyl or its lower alkyl esters, R ₁, R ₃, R ₄, or R ₇At least one is not H; Work as R ₃, R ₄, or R ₇Be CH ₃, R ₅Be hydroxyl R ₆During for carboxyl or its lower alkyl esters, R ₁Or R ₂At least one is not H or CH ₃Work as R ₄During for F, R ₇It is not hydroxyl; Work as R ₅Be hydroxyl R ₆During for carboxyl or its lower alkyl esters, R ₂Be not COOH; Work as R ₅Be hydroxyl R ₆During for H, R ₁, R ₂, R ₃, R ₄, or R ₇In at least one is not H; Work as R ₆Be COOH and R ₇R when being hydroxyl ₄It or not sulfo group; Work as R ₂During for carboxyl, R ₃And R ₇Be not NH ₂Or hydroxyl; Work as R ₅Be hydroxyl, R ₆Be carboxyl or its lower alkyl esters, and the X among the formula I is CR '=CR ' time, R ₁, R ₂, R ₃, R ₄, or R ₇Have at least one not to be H; When the X of formula I is SO ₂The time R ₁Or R ₂In have at least one not to be H, perhaps R ₃, R ₅, or R ₇In have one at least for H, hydroxyl, or CH ₃

IB has structure as follows:

Wherein: R ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂, O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

(wherein R ' is H or low alkyl group), or triazene as follows:

(R wherein ₈And R ₉Be low alkyl group) or

The R of two Q ' wherein ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl, work as R ₁₀Be hydroxyl R ₁₆Be (C=O) N (R ') ₂Or when carboxyl or its lower alkyl esters, R ₁Or R ₂In have one at least for Br or I or R ₁₁-R ₁₅In have one at least for H; Work as R ₁₅Be hydroxyl R ₁₆During for carboxyl or its lower alkyl esters, R ₁Or R ₂In have one at least for Br or I or R ₁₀-R ₁₄In have one at least for H; X is SO in formula I ₂The time, R ₁Or R ₂In have one at least for H or R ₁₁-R ₁₄In have one at least for H;

IC has structure as described below:

Wherein: R ₁₇, R ₁₈, R ₁₉, R ₂₀, or R ₂₁In at least one is H, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows: the statement of relevant 19 modifications

According to the international search report of mailing on September 27th, 1996, the applicant replaces former claim 1-4 page or leaf with the replacement page or leaf claim 1-4a page or leaf of submitting herein, so that claim is modified.These are replaced page or leaf and claim 1 are revised to some extent claim 2 to 25 and former submit identical.The modification of described claim 1 is relevant R among the formula I ₂Definition, and revise structural formula IA, the last R group of IB in conjunction with defining, get rid of in the search report thus those compounds that disclosed in the document of being quoted.

The document that the applicant studied the international search report carefully and quoted is noticed and has only been pointed out that claim 1 lacks novelty and inventive measure.Therefore the applicant has revised this claim, so that this citing document is or else as for destroying the novelty and the inventive measure of replacing the claimed compound of page or leaf.

The applicant thinks and replaces contained claim among the page or leaf 1-4a, has novelty and inventive measure each document of quoting in search report.

Claims (25)

1. Shown in the formula I can with amyloid bonded compound or its water soluble non-toxic salt:

   

   Wherein:

   

   Be N=N, C ≡ C, or CR '=CR ' (wherein R ' expression H or low alkyl group); X is C (R ") ₂

   (wherein R " be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   Wherein R ' is H or low alkyl group) or X be CH=CH, N=N, C=O, O, NR ' (wherein R ' expression H or low alkyl group), S, or SO ₂R ₁And R ₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   (wherein R ' is H or low alkyl group), or triazene as follows:

   

   (R wherein ₈And R ₉Be low alkyl group) or group as follows:

   

   Q is selected from down array structure independently of one another, respectively contains a carboxylic acid or sour sample functional group: IA, IB, and IC, ID, IE, IF and IG, wherein

   IA has structure as follows:

   

   R wherein ₃, R ₄, R ₅, R ₆, or R ₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   (wherein R ' is H or low alkyl group), or triazene as follows:

   

   (R wherein ₈And R ₉Be low alkyl group) or group as follows:

   

   The R of two Q ' wherein ₃, R ₄, R ₅, R ₆, or R ₇In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂, or carboxyl; And work as R ₅Be hydroxyl R ₆R during for carboxyl or its lower alkyl esters ₁, R ₂, R ₃, R ₄, or R ₇Have at least one not to be H; Work as R ₅Be amino, R ₆Be carboxyl or its lower alkyl esters, and R ₇During for hydroxyl, R ₁, R ₂, R ₃, or R ₄At least one is not H; Work as R ₂Be CH ₃, R ₅Be hydroxyl, and R ₆During for carboxyl or its lower alkyl esters, R ₁, R ₃, R ₄, or R ₇At least one is not H; Work as R ₃, R ₄, or R ₇Be CH ₃, R ₅Be hydroxyl R ₆During for carboxyl or its lower alkyl esters, R ₁Or R ₂At least one is not H or CH ₃

   IB has structure as follows:

   

   Wherein: R ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   (wherein R ' is H or low alkyl group), or triazene as follows:

   (R wherein ₈And R ₉Be low alkyl group) or The R of two Q ' wherein ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

   IC has structure as described below:

   

   Wherein: R ₁₇, R ₁₈, R ₁₉, R ₂₀, or R ₂₁In at least one is H, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   (wherein R ' is H or low alkyl group), or the triazene that is shown below:

   (R wherein ₈And R ₉Be low alkyl group) or

   ID has structure as follows:

   

   Wherein: R ₂₂, R ₂₃, or R ₂₄Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   (wherein R ' is H or low alkyl group), or triazene as follows:

   

   (R wherein ₈And R ₉Be low alkyl group) or

   

   The heterocyclic radical of one of representing in following six formulas:

   

   IE has structure as follows:

   

   Wherein: R ₂₅, R ₂₆, or R ₂₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   

   (wherein R ' is H or low alkyl group), or triazene as follows:

   

   (R wherein ₈And R ₉Be low alkyl group) or

   The heterocyclic radical of one of representing in following six formulas:

   

   IF has structure as follows:

   

   Wherein: R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂One of be as above-mentioned formula I definition Connect base, remaining R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

   

   (wherein R ' is H or low alkyl group), or triazene as follows:

   (R wherein ₈And R ₉Be low alkyl group) or

   

   The R of two Q ' wherein ₂₈, R ₂₉, R ₃₀, R ₃₁Or R ₃₂In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

   IG has structure as follows:

   

   R wherein ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉One of be as above-mentioned formula I definition

   

   Connect base, remaining R ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, the tetrazolium Huo oxadiazole that is shown below:

   (wherein R ' is H or low alkyl group), or triazene as follows:

   (R wherein ₈And R ₉Be low alkyl group) or The R of two Q ' wherein ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl.
2. 2. the compound of claim 1, wherein substituent R ₁-R ₇And R ₁₀-R ₃₉At least one is selected from ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH ₂-CH ₂- ¹⁸F, O-CH ₂-CH ₂- ¹⁸F, CH ₂-CH ₂-CH ₂- ¹⁸F, O-CH ₂-CH ₂-CH ₂- ¹⁸F, ¹⁹F, ¹²⁵I and wherein at least one carbon be ¹¹C or ¹³The carbon containing substituting group of the formula I definition of C.
3. 3. the compound of claim 1, wherein by to synthetic A β peptide or Alzheimer cerebral tissue when measuring, described compound and A β bonded dissociation constant (K _D) between 0.0001 to 10.0 μ M.
4. 4. one kind is synthesized wherein substituent R ₁-R ₇And R ₁₀-R ₃₉Have at least one to be selected from ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F and ¹⁹The method of claim 1 compound of F comprises making wherein substituent R ₁-R ₇And R ₁₀-R ₃₉Have at least one for the compound of the claim 1 of trialkyltin with contain ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁵F or ¹⁹The halogenating agent of F reacts this step.
5. 5. pharmaceutical composition that is used in vivo amyloid being precipitated into picture comprises compound of (a) claim 2 and (b) pharmaceutically acceptable carrier.
6. 6. method of in vivo measuring amyloid beta deposition those who are investigated may further comprise the steps:

   (a) pharmaceutical composition of the claim 5 of significant quantity is measured in use, and

   (b) measure combining of above-claimed cpd and amyloid beta deposition in this those who are investigated's body.
7. 7. the method for claim 6, wherein said amyloid beta deposition is positioned at those who are investigated's brain.
8. 8. the method for claim 6, wherein said those who are investigated are under a cloud to be suffered from and is selected from following disease or syndromes: Alzheimer, family's Alzheimer, Down ' s syndromes and apolipoproteins E4 allelotrope homozygote.
9. 9. the method for claim 6, wherein said mensuration is selected from the γ imaging, nuclear magnetic resonance and nuclear magnetic resonance spectroscopy.
10. 10. the method for claim 9, wherein said γ imaging both can be PET and also can be SPECT.
11. 11. the method for claim 6, wherein said pharmaceutical composition is through the intravenous injection administration.
12. 12. the method for claim 7 is comprising with this ratio in the bonded ratio of this compound in the combination of this compound in (i) non-cerebellum brain district in this those who are investigated's body and the (ii) cerebellum and the normal those who are investigated's body relatively.
13. 13. form the relevant cytopathy and the method for poisoning with protofibril in the disease that a kind is suppressed relevant amyloidosis, this method comprises suffering from or suspecting the chrysamine G or derivatives thereof that is used pharmacy effective dose by the curer of suffering from above-mentioned disease.
14. 14. the method for claim 13, wherein said chrysamine G derivative can be in conjunction with compound or its water soluble non-toxic salt of amyloid for formula I: Wherein:

    

    Be N=N, C ≡ C, or CR '=CR ' (wherein R ' expression H or low alkyl group); X is C (R ") ₂

    (wherein R " be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    Wherein R ' is H or low alkyl group) or X be CH=CH, N=N, C=O, O, NR ' (wherein R ' expression H or low alkyl group), S, or SO ₂R ₁And R ₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or group as follows: Q is selected from down one of array structure independently of one another, respectively contains a carboxylic acid or sour sample functional group: IA, IB, and IC, ID, IE, IF and IG, wherein

    IA has structure as follows:

    

    R wherein ₃, R ₄, R ₅, R ₆, or R ₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or group as follows:

    

    The R of two Q ' wherein ₃, R ₄, R ₅, R ₆, or R ₇In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂, or carboxyl; And work as R ₅Be hydroxyl R ₆R during for carboxyl or its lower alkyl esters ₁, R ₂, R ₃, R ₄, or R ₇Have at least one not to be H; Work as R ₅Be amino, R ₆Be carboxyl or its lower alkyl esters, and R ₇During for hydroxyl, R ₁, R ₂, R ₃, or R ₄At least one is not H; Work as R ₂Be CH ₃, R ₅Be hydroxyl, and R ₆During for carboxyl or its lower alkyl esters, R ₁, R ₃, R ₄, or R ₇At least one is not H; Work as R ₃, R ₄, or R ₇Be CH ₃, R ₅Be hydroxyl R ₆During for carboxyl or its lower alkyl esters, R ₁Or R ₂At least one is not H or CH ₃

    IB has structure as follows: Wherein: R ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or The R of two Q ' wherein ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

    IC has structure as described below: Wherein: R ₁₇, R ₁₈, R ₁₉, R ₂₀, or R ₂₁In at least one is H, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or the triazene that is shown below:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    ID has structure as follows:

    

    Wherein: R ₂₂, R ₂₃, or R ₂₄Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    The heterocyclic radical of one of representing in following six formulas:

    

    IE has structure as follows:

    Wherein: R ₂₅, R ₂₆, or R ₂₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    The heterocyclic radical of one of representing in following six formulas:

    

    IF has structure as follows:

    Wherein: R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂One of be as above-mentioned formula I definition

    

    Connect base, remaining R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or

    

    The R of two Q ' wherein ₂₈, R ₂₉, R ₃₀, R ₃₁Or R ₃₂In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

    IG has structure as follows:

    

    R wherein ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉One of be as above-mentioned formula I definition

    

    Connect base, remaining R ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    Wherein at the R of two Q ' ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl.
15. 15. the method for claim 14, wherein when Z=Z is N=N, each R ₂Be CH ₃
16. 16. the method for claim 13, wherein be selected from Alzheimer with the amyloidosis diseases associated, Down ' s syndromes, diabetes B, heredity cerebral hemorrhage amyloidosis (Dutch), amyloid A (reactivity), secondary amyloidosis, family's Mediterranean fruit fly, have urticaria and deaf family's amyloid ephrosis (Muckle-wells syndromes), amyloid λ-L-chain or amyloid κ-L-chain are (with idiopathy, myelomatosis or macroglobulinemia are relevant) A β 2M (chronic hemodialysis), ATTR (family's amyloid polyneuropathy) (Portuguese, Japanese, Swedish), family's amyloid myocardosis (Danish), isolating heart amyloid, (general senile amyloidosis), AIAPP or amylopectin nesidioblastoma, anterior chamber's natural factor (isolating anterior chamber's amyloid), preceding thyrocalcitonin (Tiroidina medullary substance knurl), gelsolin (family's amyloidosis (Finnish)), cysteine proteinase inhibitor C (heredity cerebral hemorrhage (Icelandic)) with amyloidosis, AApo-A-I (family's amyloid neuropathy-Iowa), AApo-A-II (quickening old and feeble in the mouse), the amyloid that Fibrinogen is relevant; With Asor or PrP-27 (scrapie, Creutzfeld Jacob disease, Gertsmann-Straussler-Scheinker syndromes, bovine spongiform encephalitis) or be the homozygous people's of apolipoproteins E4 allelotrope case, and with apolipoproteins E4 allelotrope homozygote diseases associated.
17. 17. one kind prevents to form the relevant cytopathy and the pharmaceutical composition of poisoning with the protofibril of relevant amyloidogenic disease, comprising chrysamine G and pharmaceutically acceptable carrier.
18. 18. one kind prevents to form the relevant cytopathy and the pharmaceutical composition of poisoning with the protofibril of relevant amyloidogenic disease, comprising the chrysamine G derivative and the pharmaceutically acceptable carrier of claim 1.
19. 19. measure living tissue or the method for amyloid beta deposition in the tissue after death, may further comprise the steps for one kind:

    (a) water culture of the compound of the tissue of formalin fixed and claim 1 is formed the precipitation of mark, then,

    (b) measure the precipitation of this mark.
20. 20. the method for claim 19, wherein said solution is by forming with the saturated 25-100% ethanol of the compound of claim 1 (all the other are water).
21. 21. the method for claim 19, wherein said mensuration be by being selected from bright-field microscope, fluorescence, and the microscopical microtechnique of confocal laser and cross polarization is carried out.
22. 22. quantitative assay living tissue or the method for amyloid amount in the tissue after death may further comprise the steps:

    A) with radiolabeled chrysamine G derivative and living tissue or tissue homogenate cultivation after death,

    B) will be bonded tissue and not with radiolabeled chrysamine G derivative bonded separate tissue,

    C) quantitative analysis and radiolabeled chrysamine G derivative bonded tissue, and

    D), will become the unit of amyloid micrograms in every 100mg tissue with the unit conversion of radiolabeled chrysamine G derivative bonded tissue by comparing with standard.
23. 23. the method for claim 22, wherein said radiolabeled chrysamine G derivative can be in conjunction with compound or its water soluble non-toxic salt of amyloid for formula I: Wherein:

    

    Be N=N, C ≡ C, or CR '=CR ' (wherein R ' expression H or low alkyl group); X is C (R ") ₂

    (wherein R " be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    Wherein R ' is H or low alkyl group) or X be CH=CH, N=N, C=O, O, NR ' (wherein R ' expression H or low alkyl group), S, or SO ₂R ₁And R ₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or group as follows:

    

    Q is selected from down one of array structure independently of one another, respectively contains a carboxylic acid or sour sample functional group: IA, IB, and IC, ID, IE, IF and IG, wherein

    IA has structure as follows:

    R wherein ₃, R ₄, R ₅, R ₆, or R ₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or group as follows: Wherein at the R of two Q ' ₃, R ₄, R ₅, R ₆, or R ₇In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂, or carboxyl; And work as R ₅Be hydroxyl R ₆R during for carboxyl or its lower alkyl esters ₁, R ₂, R ₃, R ₄, or R ₇Have at least one not to be H; Work as R ₅Be amino, R ₆Be carboxyl or its lower alkyl esters, and R ₇During for hydroxyl, R ₁, R ₂, R ₃, or R ₄At least one is not H; Work as R ₂Be CH ₃, R ₅Be hydroxyl, and R ₆During for carboxyl or its lower alkyl esters, R ₁, R ₃, R ₄, or R ₇At least one is not H; Work as R ₃, R ₄, or R ₇Be CH ₃, R ₅Be hydroxyl R ₆During for carboxyl or its lower alkyl esters, R ₁Or R ₂At least one is not H or CH ₃

    IB has structure as follows:

    

    Wherein: R ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R, N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or Wherein at the R of two Q ' ₁₀, R ₁₁, R ₁₂, R ₁₃, R ₁₄, R ₁₅, or R ₁₆In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

    IC has structure as described below:

    

    Wherein: R ₁₇, R ₁₈, R ₁₉, R ₂₀, or R ₂₁In at least one is H, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or the triazene that is shown below:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    ID has structure as follows:

    

    Wherein: R ₂₂, R ₂₃, or R ₂₄Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    (R wherein ₈And R ₉Be low alkyl group) or

    

    The heterocyclic radical of one of representing in following six formulas:

    

    IE has structure as follows:

    Wherein: R ₂₅, R ₂₆, or R ₂₇Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    The heterocyclic radical of one of representing in following six formulas:

    

    IF has structure as follows:

    

    Wherein: R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂One of be as above-mentioned formula I definition Connect base, remaining R ₂₈, R ₂₉, R ₃₀, R ₃₁, or R ₃₂Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    The R of two Q ' wherein ₂₈, R ₂₉, R ₃₀, R ₃₁Or R ₃₂In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl,

    IG has structure as follows:

    

    R wherein ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉One of be as above-mentioned formula I definition Connect base, remaining R ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉Be H independently of one another, F, Cl, Br, I, low alkyl group, CF ₃, CH ₂-CH ₂F, O-CH ₂-CH ₂F, CH ₂-CH ₂-CH ₂F, O-CH ₂-CH ₂-CH ₂F, CN, (C=O)-and R ', N (R ') ₂, NO ₂, (C=O) N (R ') ₂O (CO) R ', OR ', COOR ', trialkyltin, tetrazolium Huo oxadiazole as follows:

    (wherein R ' is H or low alkyl group), or triazene as follows:

    

    (R wherein ₈And R ₉Be low alkyl group) or

    

    Wherein at the R of two Q ' ₃₃, R ₃₄, R ₃₅, R ₃₆, R ₃₇, R ₃₈, or R ₃₉In have one at least for hydroxyl, tetrazolium ， oxadiazole, NO ₂Or carboxyl.
24. 24. the method for claim 23, wherein substituent R ₁-R ₇And R ₁₀-R ₃₉In at least one is selected from ¹²⁵I, ³H, and wherein have at least a carbon to be ¹⁴The carbon containing substituting group radio-labeling substance markers that defines among the formula I of C.
25. 25. a method of distinguishing Alzheimer brain and normal brain activity, comprising following steps:

    A) from normal those who are investigated with suspect that cerebellum other brain districts in addition obtain tissue the those who are investigated's suffer from Alzheimer (i) cerebellum and the (ii) same brain;

    B) this tissue is cultivated with radiolabeled chrysamine G derivative, and amyloid combines with above-mentioned radiolabeled chrysamine G derivative in this tissue so that make;

    C) according to the method for claim 21, the amount of quantitative analysis and above-mentioned radiolabeled chrysamine G derivative bonded amyloid;

    D) calculate the ratio of cerebellum with the amount of amyloid in the amount of amyloid in the external-brain district and the cerebellum.

    E) ratio with amyloid amount in above-mentioned normal those who are investigated's tissue compares with suspecting the ratio of suffering from amyloid amount in the Alzheimer those who are investigated tissue; And

    F) if the ratio that above-mentioned suspection suffers from those who are investigated's brain of Alzheimer is higher by 90% than the ratio in normal those who are investigated's brain, then can judge to have Alzheimer.

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