BACKGROUND OF THE INVENTION Field of the Invention [0002] This invention relates to novel bioactive compounds, methods of diagnostic imaging using radiolabeled compounds, and methods of making radiolabeled compounds.

Background Art [0003] Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, irreversible memory loss, disorientation, and language impairment. Postmortem examination of AD brain sections reveals abundant senile plaques (SPs) composed of amyloid- (Ap) peptides and numerous neurofibrillary tangles (NFTs) formed by filaments of highly phosphorylated tau proteins (for recent reviews and additional citations see Ginsberg, S. D., et al.,"Molecular Pathology of Alzheimer's Disease and Related Disorders,"in Cerebral Cortex : Neurodegenerative andAge-related Changes ita Structure and Function of Cerebral Cortex, Kluwer Academic/Plenum, NY (1999), pp. 603-654; Vogelsberg-Ragaglia, V., et al.,"Cell Biology of Tau and Cytoskeletal Pathology in Alzheimer's Disease,"Alzheimer's Disease, Lippincot, Williams &Wilkins, Philadelphia, PA (1999), pp. 359-372). Familial AD (FAD) is caused by multiple mutations in the A precursor protein (APP), presenilin 1 (PS 1) and presenilin 2 (PS2) genes (Ginsberg, S. D., et al.,"Molecular Pathology of Alzheimer's Disease and Related Disorders,"in Cerebral Cortex : Neurodegenerative and Age-related Changes ira Structure and Function of Cerebral Cortex, Kluwer Academic/Plenum, NY (1999), pp. 603-654; Vogelsberg-Ragaglia, V., et al.,"Cell Biology of Tau and Cytoskeletal Pathology in Alzheimer's Disease,"Alzheimer's Disease, Lippincot, Williams & Wilkins, Philadelphia, PA (1999), pp. 359-372).

[0004] While the exact mechanisms underlying AD are not fully understood, all pathogenic FAD mutations studied thus far increase production of the more amyloidogenic 42-43 amino-acid long form of the Ap peptide. Thus, at least in FAD, dysregulation of Ap production appears to be sufficient to induce a cascade of events leading to neurodegeneration. Indeed, the amyloid cascade hypothesis suggests that formation of extracellular fibrillar A (3 aggregates in the brain may be a pivotal event in AD pathogenesis (Selkoe, D. J.,"Biology of B-amyloid Precursor Protein and the Mechanism of Alzheimer's Disease,"Alzheimer's Disease, Lippincot Williams & Wilkins, Philadelphia, PA (1999), pp. 293-310; Selkoe, D. J., J. Am. Med. Assoc. 283 : 1615-1617 (2000); Naslund, J., et al., J.

Am. Med. Assoc. 283 : 1571-1577 (2000); Golde, T. E., et al., Biochimica et BiophysicaActa 1502 : 172-187 (2000)).

[0005] Various approaches in trying to inhibit the production and reduce the accumulation of fibrillar Ap in the brain are currently being evaluated as potential therapies for AD (Skovronsky, D. M. and Lee, V. M., Trends Pharmacol. Sci. 21 : 161-163 (2000); Vassar, R., et al., Science 286 : 735-741 (1999); Wolfe, M. S., et al., J. Med. Chem. 41 : 6-9 (1998); Moore, C. L., et al., J. Med. Chem. 43 : 3434-3442 (2000); Findeis, M. A., Biochinaica et Biophysica Acta 1502 : 76-84 (2000); Kuner, P., Bohrmann, et al., J. Biol. Chem. 275 : 1673- 1678 (2000)). It is therefore of great interest to develop ligands that specifically bind fibrillar Ap aggregates. Since extracellular SPs are accessible targets, these new ligands could be used as in vivo diagnostic tools and as probes to visualize the progressive deposition of Ap in studies of AD amyloidogenesis in living patients.

[0006] To this end, several interesting approaches for developing fibrillar Ap aggregate-specific ligands have been reported (Ashburn, T. T., et al., Chem. Biol.

3 : 351-358 (1996); Han, G., et al., J. Am. Chem. Soc. 118 : 4506-4507 (1996); Klunk, W. E., et al., Biol. Psychiatry 35 : 627 (1994); Klunk, W. E., et al., Neurobiol. Aging 16 : 541-548 (1995); Klunk, W. E., et al., Society for Neuroscience Abstract 23 : 1638 (1997); Mathis, C. A., et al., Proc. Xllth Intl.

Symp. Radiopharm. Chem., Uppsala, Sweden : 94-95 (1997); Lorenzo, A. and Yankner, B. A., Proc. Natl. Acad. Sci. U. S. A. 91 : 12243-12247 (1994); Zhen, W., et al., J. Med. Chem. 42: 2805-2815 (1999)). The most attractive approach is based on highly conjugated chrysamine-G (CG) and Congo red (CR), and the latter has been used for fluorescent staining of SPs and NFTs in postmortem AD brain sections (Ashburn, T. T., et al., Chem. Biol. 3: 351-358 (1996); Klunk, W.

E., et al., J. Histochem. Cytochem. 37 : 1273-1281 (1989)). The inhibition constants (O for binding to fibrillar Ap aggregates of CR, CG, and 3'-bromo- and 3'-iodo derivatives of CG are 2,800,370,300 and 250 nM, respectively (Mathis, C. A., et al., Proc. XIIth Intl. Symp. Radiophanm. Chem., Uppsala, Sweden : 94-95 (1997)). These compounds have been shown to bind selectively to Ap (1-40) peptide aggregates in vitro as well as to fibrillar Ap deposits in AD brain sections (Mathis, C. A., et al., Proc. XIIth Intl. Symp. Radiopharm. Chem., Uppsala, Sweden : 94-95 (1997)).

[0007] Amyloidosis is a condition characterized by the accumulation of various insoluble, fibrillar proteins in the tissues of a patient. An amyloid deposit is formed by the aggregation of amyloid proteins, followed by the further combination of aggregates and/or amyloid proteins.

[0008] In addition to the role of amyloid deposits in Alzheimer's disease, the presence of amyloid deposits has been shown in diseases such as Mediterranean fever, Muckle-Wells syndrome, idiopathetic myeloma, amyloid polyneuropathy, amyloid cardiomyopathy, systemic senile amyloidosis, amyloid polyneuropathy, hereditary cerebral hemorrhage with amyloidosis, Down's syndrome, Scrapie, Creutzfeldt-Jacob disease, Kuru, Gerstamnn-Straussler-Scheinker syndrome, medullary carcinoma of the thyroid, Isolated atrial amyloid, ß2-microglobulin amyloid in dialysis patients, inclusion body myositis, 02-amyloid deposits in muscle wasting disease, and Islets of Langerhans diabetes Type I1 insulinoma.

[0009] Thus, a simple, noninvasive method for detecting and quantitating amyloid deposits in a patient has been eagerly sought. Presently, detection of amyloid deposits involves histological analysis of biopsy or autopsy materials.

Both methods have drawbacks. For example, an autopsy can only be used for a postmortem diagnosis.

[0010] The direct imaging of amyloid deposits in vivo is difficult, as the deposits have many of the same physical properties (e. g., density and water content) as normal tissues. Attempts to image amyloid deposits using magnetic resonance imaging (MRI) and computer-assisted tomography (CAT) have been disappointing and have detected amyloid deposits only under certain favorable conditions. In addition, efforts to label amyloid deposits with antibodies, serum amyloid P protein, or other probe molecules have provided some selectivity on the periphery of tissues, but have provided for poor imaging of tissue interiors.

[0011] Potential ligands for detecting Ap aggregates in the living brain must cross the intact blood-brain barrier. Thus brain uptake can be improved by using ligands with relatively smaller molecular size (compared to Congo Red) and increased lipophilicity. Highly conjugated thioflavins (S and T) are commonly used as dyes for staining the Ap aggregates in the AD brain (Elhaddaoui, A., et al., Biospectroscopy 1 : 351-356 (1995)). These compounds are based on benzothiazole, which is relatively small in molecular size. However, thioflavins contain an ionic quarternary amine, which is permanently charged and unfavorable for brain uptake.

[0012] Thus, it would be useful to have a noninvasive technique for imaging and quantitating amyloid deposits in a patient. In addition, it would be useful to have compounds that inhibit the aggregation of amyloid proteins to form amyloid deposits and a method for determining a compound's ability to inhibit amyloid protein aggregation.

BRIEF SUMMARY OF THE INVENTION [0013] The present invention provides novel compounds of Formula I, II, III or m'that bind preferentially to amyloid aggregates.

[0014] The present invention also provides diagnostic compositions comprising a radiolabeled compound of Formula I, n, in or mv, and a pharmaceutically acceptable carrier or diluent.

[0015] The invention further provides a method of imaging amyloid deposits, the method comprising introducing into a patient a detectable quantity of a labeled compound of Formula I, II, m or m or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof.

[0016] The present invention also provides a method for inhibiting the aggregation of amyloid proteins, the method comprising administering to a mammal an amyloid inhibiting amount of a compound Formula I, II, In or III' or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof.

[0017] A further aspect of this invention is directed to methods and intermediates useful for synthesizing the amyloid inhibiting and imaging compounds of Formula I, II, m or IR'described herein.

BRIEF DESCRIPTION OF THE FIGURE [0018] FIG. 1A and FIG. 1B depict representative compounds of the present invention and the binding data for these compounds.

DETAILED DESCRIPTION OF THE INVENTION [0019] A first aspect of the present invention is directed to compounds of the following Formula I : or a pharmaceutically acceptable salt thereof, wherein: Y is CH, NR5, O, S or CH=N, where Rs is hydrogen or a C14 alkyl ; m and n are both zero, or m and n are both 1; R3 is CH3, Br, I, F, 125I, 131I, 123I, 18F, 76Br, 77Br or Sn (alkyl) 3 ; R1 and R2 are independently hydrogen, C1-4 alkyl, C2-4 aminoalkyl, C1-4 haloalkyl, haloarylalkyl,-L-Ch, or R'and R'are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O, S or NR6 in said ring, where R6 is hydrogen or Cl 4 alkyl ; and R4 is Cl 4 alkyl ; and L is a covalent bond or a linking group, such as -(CH2)n-, or -(CH2)n-C (O)- where n is 1-5; and Ch is a tetradentate ligand capable of complexing with a metal, such as a ligand selected from the group consisting of : where R9 is hydrogen or a sulfur protecting group, such as methoxymethyl, methoxyethoxymethyl, p-methoxybenzyl or benzyl, and the other variable groups have the preferred values mentioned herein.

[0020] In this embodiment, compounds having Ch ligands, such as those of Formulae VIII, IX, X and XI are complexed with 99m-pertechnetate, as described herein to form metal chelates where Ch is selected from the group consisting of: [0021] Additionally, a rhenium radioisotope can be complexed with the Ch ligand.

[0022] A preferred group of compounds falling within the scope of the present invention include compounds of Formula I wherein Y is selected from NR', O or S. Especially preferred compounds of Formula I include compounds wherein Y is Nus or S, most preferably Y is S.

[0023] Preferred values of R 5 in compounds of Formula I where Y is Nus are hydrogen and C1-4 alkyl, more preferably Rs is hydrogen or methyl, and most preferably Rs is hydrogen.

[0024] A preferred value of m and n in compounds of Formula I is from zero to one, more preferably zero.

[0025] Suitable values of R3 are Br, I, F, 125I, 131I, 123I, 18F, 76Br, or 77Br.

Especially useful values of R3 are 125I, 131I, 123I, 18F, 76Br, or 77Br. more preferably l23I, 1311, 7Br or 77 Br, and most preferably 1231. Preferred embodiments also include intermediates useful in the preparation of compounds of Formula I wherein R3 is Sn (alkyl) 3.

[0026] Preferred compounds are those of Formula I wherein Rl and R2 are independently one of hydrogen, C1-4 alkyl, C1-4 haloalkyl, halophenyl(C1-4)alkyl, or are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O or NR6 in said ring, where R6 is hydrogen or Cl 4 alkyl. Useful values of Rl and R2 include, independently, hydrogen, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, 3-fluoropropyl, 4-fluorobutyl, or 4-fluorobenzyl, or R'and R'are taken together with the nitrogen to which they are attached to form a piperidinyl ring having NR6 in said ring, where R6 is hydrogen or methyl.

[0027] The present invention is also directed to compounds of Formula II : or a pharmaceutically acceptable salt thereof, wherein : Y is O or NR4 where R4 is hydrogen or C, _a alkyl ; R3 is Br, I, F, 125I, 131I, 123I, 18F, 76Br, 77Br or Sn (alkyl) 3 ; R1 and R2 are independently hydrogen, Cl4 alkyl, C24 aminoalkyl, Cl4 haloalkyl, haloarylalkyl, or R'and Rz are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O, S or NR5 in said ring, where Rs is hydrogen or Cl 4 alkyl.

[0028] A preferred group of compounds include compounds of Formula n where Y is NR4 where R4 is hydrogen or methyl. More preferred compounds include compounds where Y is O.

[0029] Useful values of R3 are Br, I, F, 125I, 131I, 123I, 18F, 76Br, or 77Br. Especially suitable values of R3 are 125I, 131I, 123I, 18F, 76Br, or 77Br, more preferably 123I, 1311, 76Br or 77Br, and most preferably 123I. Preferred embodiments also include intermediates useful in the preparation of compounds of Formula lI wherein R3 is Sn (alkyl) 3.

[0030] Preferred compounds are those of Formula II wherein R'and R'are independently one of hydrogen, C14 alkyl, C14 haloalkyl, halophenyl (C1-4)alkyl, or are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O or NR6 in said ring, where R6 is hydrogen or Cl 4 alkyl. Useful values of R1 and R2 include, independently, hydrogen, methyl, ethyl, propyl, butyl, t-butyl, isobutyl, 3-fluoropropyl, 4- fluorobutyl, or 4-fluorobenzyl, or R1 and R2 are taken together with the nitrogen to which they are attached to form a piperidinyl ring having NR6 in said ring, where R6 is hydrogen or methyl.

[0031] The present invention is also directed to compounds of Formula Ht : or a pharmaceutically acceptable salt thereof, wherein: R3 is Br, I, F, 125I, 131I, 123I, 18F, 76Br, or Sn (alkyl) 3 ; R'and R2 are independently hydrogen, C1-4 alkyl, C2-4 aminoalkyl, C1-4 haloalkyl, haloarylalkyl,-L-Ch, or R'and R2 are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O, S or NR5 in said ring, where R5 is hydrogen or Cl alkyl.

[0032] Useful values of R3 are Br, I, F, 125I, 131I, 123I, 18F, 76Br, or 77Br. Especially suitable values of R3 are 125I, 131I, 123I, 18F, 76Br, or 77Br, more preferably 123I, 131I, 76Br or77Br, and most preferably 123I or 125I. Preferred embodiments also include intermediates useful in the preparation of compounds of Formula m wherein R3 is Sn (alkyl) 3.

[0033] Preferred compounds are those of Formula HI wherein R'and R2 are independently one of hydrogen, Cl. alkyl, C1-4 haloalkyl, halophenyl (C1-4) alkyl, or are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O or NR6 in said ring, where R6 is hydrogen or Cl 1 alkyl. Useful values of R1 and R2 include, independently, hydrogen, methyl, ethyl, propyl, butyl, t-butyl, isobutyl, 3-fluoropropyl, 4- fluorobutyl, or 4-fluorobenzyl, or R'and R2 are taken together with the nitrogen to which they are attached to form a piperidinyl ring having NR6 in said ring, where R6 is hydrogen or methyl. Most preferably R1 and R2 are methyl.

[0034] Another preferred group of compounds are compounds of Formulae I, lI, or HI where R'is-L-Ch, R2 is hydrogen or methyl, and R3 is I or methyl. A preferred Ch is Formula IV. A preferred L is -(CH2)n-where n [0035] is 1,2 or 3.

[0036] In a separate embodiment, compounds of Formula HI have R'and R2 groups as defined above, and R3 is-L-Ch, where L and Ch are as defined above.

[0037] In another embodiment, the invention is directed to compounds of Formula III' : or a pharmaceutically acceptable salt thereof, wherein: A, B and D are CH or N, provided that no more than two of A, B and D is N; R3 is Br, I, F, 125I, 131I, 123I, 18F, 76Br, 77Br, haloalkyl or Sn (alkyl) 3; RI and R 2 are independently hydrogen, C1-4 alkyl, C24 aminoalkyl, C1-4 haloalkyl, haloarylalkyl,-L-Ch, or R'and R2 are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O, S or NR5 in said ring, where Rs is hydrogen or Cl 1 alkyl.

[0038] Useful values of R3 are Br, I, F, 125I, 131I, 123I, 18F, 76Br, 77Br or Fmuoro (C1-5) alkyl. Especially suitable values of R3 are'F/fluoromethyl, 18F/fluoroethyl, 18F/fluoropropyl, 18F/fluorobutyl, or 18F/fluoropentyl. Preferred embodiments also include intermediates useful in the preparation of compounds of Formula III' wherein R3 is Sn (alkyl) 3.

[0039] In a preferred group of compounds, A and B are CH, and D is N. In another preferred group of compounds, A and D are CH, and B is N. In another preferred group of compounds, B and D are CH, and A is N.

[0040] Preferred compounds are those of Formula III' wherein R1 and R2 are independently one of hydrogen, CI-4 alkyl, Cl haloalkyl, halphenyl(C1-4)alkyl, or are taken together with the nitrogen to which they are attached to form a 5-to 7-member heterocyclic ring optionally having O or NR6 in said ring, where R6 is hydrogen or C1-4 alkyl. Useful values of R1 and R2 include, independently, hydrogen, methyl, ethyl, propyl, butyl, t-butyl, isobutyl, 3-fluoropropyl, 4- fluorobutyl, or 4-fluorobenzyl, or R'and R2 are taken together with the nitrogen to which they are attached to form a piperidinyl ring having NR6 in said ring, where R6 is hydrogen or methyl. Most preferably Rl and R2 are methyl.

[0041] Another preferred group of compounds are compounds of Formula I, II, m or III' where R1 is -L-Ch, R2 is hydrogen or methyl, and R3 is I or methyl. A preferred Ch is Formula IV. A preferred L is -(CH2)n- where n is 1,2 or 3.

[0042] In a separate embodiment, compounds of Formula mS have Rl and RI groups as defined above, and R3 is-L-Ch, where L is a covalent bond or linking group, such as -(CH2)n-, or -(CH2)n-C(O)- where n is 0-5, and Ch is a tetradentate ligand capable of complexing with a metal as defined above. Most preferably, L is- (CH2) n-, where n is 0, Ch is Formula XI, and R'and R are independently hydrogen or C14 alkyl. In this embodiment, it is most preferable that R'and R2 are both methyl.

[0043] It is also to be understood that the present invention is considered to include stereoisomers as well as optical isomers, e. g. mixtures of enantiomers as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in selected compounds of the present series.

[0044] The compounds of Formula I, II, in or m'may also be solvated, especially hydrated. Hydration may occur during manufacturing of the compounds or compositions comprising the compounds, or the hydration may occur over time due to the hygroscopic nature of the compounds. In addition, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

[0045] When any variable occurs more than one time in any constituent or in Formula I, II, DI or mX, its definition on each occurrence is independent of its definition at every other occurrence. Also combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

[0046] Another aspect of this invention is related to methods of preparing compounds of Formula I, II, m or m. A first method is characterized by forming a benzothiazole of Formula I wherein Y is S by reacting a 2-aminothiophenol with either: a) a 4-aminobenzaldehyde in DMSO at a temperature in the range of 100°C-220°C, and collecting said benzothiazole; or b) a 4-halobenzoic acid derivative in a solvent in the presence of polyphosphoric acid, collecting the product of this reaction, followed by reacting said product with an amine to form said benzothiazole, and collecting said benzothiazole; and optionally reacting a benzothiazole of Formula I wherein Y is S with (alkyl) 3Sn in a solvent in the presence of palladiumlIoxide to form a trialkylstannyl benzothiazole, and collecting the product of this reaction; and optionally reacting a trialkylstannyl benzothiazole of Formula I wherein Y is S with either: a) iodine in a solvent at ambient temperature, and extracting the product; or b) NaI or Na [1211] I in the presence of hydrogen peroxide, and extracting the product.

[0047] A second method is characterized by forming a benzoxazole of Formula I wherein Y is 0 by reacting a 2-amino-5-nitrophenol with a 4-aminobenzoic acid to form a nitro-substituted benzoxazole intermediate, and collecting said intermediate; followed by catalytic hydrogenation of said nitro group to an amino group, and collecting the product of this reaction; and reacting said product with NaNO2 in the presence of H+ and potassium halide to produce a benzoxazole of Formula I wherein Y is O ; and optionally reacting a benzoxazole of Formula I wherein Y is O with (alkyl) 3Sn in a solvent in the presence of palladiumIIoxide to form a trialkylstannyl benzoxazole, and collecting the product of this reaction; and optionally reacting a trialkylstannyl benzoxazole of Formula I wherein Y is O with either: a) iodine in a solvent at ambient temperature, and extracting the product; or b) NaI or Na [l25I] I in the presence of hydrogen peroxide, and extracting the product.

[0048] A third method is characterized by forming a benzimidazole of Formula I wherein Y is N by reacting a 4-bromo-1, 2-diaminobenzene with either: a) a 4- aminobenzaldehyde to form a benzimidazole of Formula I wherein Y is N, and collecting the product, or b) a 4-halobenzaldehyde to form an intermediate benzimidazole, and reacting said intermediate with a monoalkylamine, dialkylamine, or heterocyclic amine in the presence of palladiumlloxide to form a benzimidazole of Formula I wherein Y is N, and collecting the product; and optionally reacting a benzimidazole of Formula I wherein Y is N with (alkyl) 3Sn in a solvent in the presence of palladiumIloxide to form a trialkylstannyl benzimidazole, and collecting the product of this reaction; and optionally reacting a trialkylstannyl benzimidazole of Formula I wherein Y is N with either: a) iodine in a solvent at ambient temperature, and extracting the product; or b) NaI or Na [l25I] I in the presence of hydrogen peroxide, and extracting the product.

[0049] A fourth method is characterized by forming a compound of Formula I wherein Rl or R2 is-L-Ch. In embodiments where R1 or R2 is -L-Ch, the groups R9 are both hydrogen, or can be any of the variety of protecting groups available for sulfur, including methoxymethyl, methoxyethoxymethyl, p-methoxybenzyl or benzyl. Sulfur protecting groups are described in detail in Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons, Inc., New York (1991). Protecting group R9 can be removed by appropriate methods well known in the art of organic synthesis, such as trifluoroacetic acid, mercuric chloride or sodium in liquid ammonia. In the case of Lewis acid labile groups, including acetamidomethyl and benzamidomethyl, R9 can be left intact. Labeling of the ligand with technetium in this case will cleave the protecting group, rendering the protected diaminedithiol equivalent to the unprotected form.

[0050] Tc-99m complexes can be prepared as follows. A small amount of non- radiolabeled compound (1-2 mg) is dissolved in 100 its EtOH and mixed with 200, uL HC1 (1 N) and 1 mL Sn-glucoheptonate solution (containing 8-32, ug SnCl2 and 80-320 ug Na-glucoheptonate, pH 6.67) and 50, uL EDTA solution (0.1 N). [99mTc] Pertechnetate (100-200, uL ; ranging from 2-20 mCi) saline solution are then added. The reaction is heated for 30 min at 100 ° C, then cooled to room temperature. The reaction mixture is analyzed on TLC (EtOH: conc. NH3 9: 1) for product formation and purity check. The mixture can be neutralized with phosphate buffer to pH 5.0.

[0051] The present invention further relates to a method of preparing a technetium-99m complex according to the present invention by reacting technetium-99m in the form of a pertechnetate in the presence of a reducing agent and optionally a suitable chelator with an appropriate Ch-containing compound.

[0052] The reducing agent serves to reduce the Tc-99m pertechnetate which is eluted from a molybdenum-technetium generator in a physiological saline solution. Suitable reducing agents are, for example, dithionite, formamidine sulphinic acid, diaminoethane disulphinate or suitable metallic reducing agents such as Sn (In, Fe (In, Cu (I), Ti (m) or Sb (E). Sn (II) has proven to be particularly suitable.

[0053] For the above-mentioned complex-forming reaction, technetium-99m is reacted with an appropriate compound of the invention as a salt or in the form of technetium bound to comparatively weak chelators. In the latter case the desired technetium-99m complex is formed by ligand exchange. Examples of suitable chelators for the radionuclide are dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, orthophtalic acid, malic acid, lactic acid, tartaric acid, citric acid, ascorbic acid, salicylic acid or derivatives of these acids; phosphorus compounds such as pyrophosphates; or enolates. Citric acid, tartaric acid, ascorbic acid, glucoheptonic acid or a derivative thereof are particularly suitable chelators for this purpose, because a chelate of technetium-99m with one of these chelators undergoes the desired ligand exchange particularly easily.

[0054] The most commonly used procedure for preparing [TcVO] +3N2S2 complexes is based on stannous (IT) chloride reduction of (99mTcpertechnetate, the common starting material. The labeling procedure normally relies on a Tc-99m ligand exchange reaction between Tc-99m (Sn)-glucoheptonate and the N2S2 ligand. Preparation of stannous (H) chloride and preserving it in a consistent stannous (In form is critically important for the success of the labeling reaction.

To stabilize the air-sensitive stannous ion it is a common practice in nuclear medicine to use a lyophilized kit, in which the stannous ion is in a lyophilized powder form mixed with an excess amount of glucoheptonate under an inert gas like nitrogen or argon. The preparation of the lyophilized stannous chloride/sodium glucoheptonate kits ensures that the labeling reaction is reproducible and predictable. The N2S2 ligands are usually air-sensitive (thiols are easily oxidized by air) and there are subsequent reactions which lead to decomposition of the ligands. The most convenient and predictable method to preserve the ligands is to produce lyophilized kits containing 100-500 yg of the ligands under argon or nitrogen.

[0055] A fifth method is characterized by forming an isoxazole of Formula It wherein Y is O by reacting a 3-halo-2-hydroxy benzaldehyde with a substituted benzamine such as 4- (halomethyl)-benzamine to form a phenoxy benzyl ether intermediate, and collecting the intermediate; followed by reacting said intermediate in a solvent in the presence of NaOMe or NaOEt to form an isoxazole of FormulaII wherein Y is O, and collecting the product; and optionally reacting an isoxazole of Formula I wherein Y is O with (alkyl) 3Sn in a solvent in the presence of palladiumlIoxide to form a trialkylstannyl isoxazole of Formula I wherein Y is O, and collecting the product of this reaction; and optionally reacting a trialkylstannyl isoxazole of Formula I wherein Y is O with either: a) iodine in a solvent at ambient temperature, and extracting the product; or b) NaI or Na ["'Ill in-the presence of hydrogen peroxide, and extracting the product.

[0056] A sixth method is characterized by forming an indole of Formula II wherein Y is NR'by reacting a 2-nitro-4-bromo toluene with N-isopropyl-2,2'- iminodiethanol to form a N, N-dimethyl-styryl-2-nitro-4-bromo benzene intermediate, followed by reacting said intermediate with an acid chloride in the presence of triethylamine to produce an a, p-unsaturated ketone, which undergoes intramolecular annulation by heating in dioxane/water, followed by reacting with sodium hydrosulfite to form an indole of Formula II wherein Y is NR4, and collecting the product; and optionally reacting said indole with methyl iodide in the presence of sodium hydride to produce an indole of Formula It wherein Y is NR4 where R4 is methyl, and collecting the product; and optionally reacting an indole of Formula II wherein Y is NR4 with (alkyl) 3Sn in a solvent in the presence of palladiumIIoxide to form a trialkylstannyl indole of Formula II wherein Y is Nord, and collecting the product of this reaction; and optionally reacting a trialkylstannyl indole of Formula II wherein Y is Nord with either: a) iodine in a solvent at ambient temperature, and extracting the product; or b) NaI or Na [l25IlI in the presence of hydrogen peroxide, and extracting the product.

[0057] A seventh method characterized by forming an imidazo [1, 2a] pyridine of Formula m by reacting 2-amino-5-bromo-pyridine with either: a) a 4'-halo-1- halo-benzophenone in a solvent in the presence of sodium bicarbonate to form an intermediate imidazo [1, 2a] pyridine, and collecting the product of the reaction; followed by reacting said intermediate with a monoalkylamine, dialkylamine or heterocyclic amine in the presence of palladiumBoxide to form an imidazo [1, 2a] pyridine of Formula m, or b) a 4'-amino-l-halo-acetophenone in a solvent in the presence of sodium bicarbonate to form an. imidazol, 2a] pyridine of Formula m, and collecting the product of the reaction; and optionally reacting an imidazo [1, 2a] pyridine of Formula m with (alkyl) 3Sn in a solvent in the presence of palladiumIIoxide to form a trialkylstannyl imidazo [1, 2a] pyridine of Formula HI, and collecting the product of this reaction; and optionally reacting a trialkylstannyl imidazol, 2a] pyridine of Formula m with either : a) iodine in a solvent at ambient temperature, and extracting the product; or b) NaI or Na ['25I] I in the presence of hydrogen peroxide, and extracting the product.

[0058] The term"alkyl"as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 8 carbons, preferably 6 carbons, more preferably 4 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, and isobutyl.

[0059] The term"alkoxy"is used herein to mean a straight or branched chain alkyl radical, as defined above, unless the chain length is limited thereto, bonded to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably the alkoxy chain is 1 to 6 carbon atoms in length, more preferably 1-4 carbon atoms in length.

[0060] The term"monoalkylamine"as employed herein by itself or as part of another group refers to an amino group which is substituted with one alkyl group as defined above.

[0061] The term"dialkylamine"as employed herein by itself or as part of another group refers to an amino group which is substituted with two alkyl groups as defined above.

[0062] The term"halo"employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.

[0063] The term"aryl"as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl or tetrahydronaphthyl.

[0064] The term"heterocycle"or"heterocyclic ring", as used herein except where noted, represents a stable 5-to 7-memebered mono-heterocyclic ring system which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatom may optionally be oxidized. Especially useful are rings contain one nitrogen combined with one oxygen or sulfur, or two nitrogen heteroatoms. Examples of such heterocyclic groups include piperidinyl, pyrrolyl, pyrrolidinyl, imidazolyl, imidazlinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, thiazolyl, thiazolidinyl, isothiazolyl, homopiperidinyl, homopiperazinyl, pyridazinyl, pyrazolyl, and pyrazolidinyl, most preferably thiamorpholinyl, piperazinyl, and morpholinyl.

[0065] The term"heteroatom"is used herein to mean an oxygen atom ("O"), a sulfur atom ("S") or a nitrogen atom ("N"). It will be recognized that when the heteroatom is nitrogen, it may form an NRaRb moiety, wherein Ra and Rb are, independently from one another, hydrogen or Cl alkyl, C2 4 aminoalkyl, Cl4 halo alkyl, halo benzyl, or R'and R2 are taken together to form a 5-to 7-member heterocyclic ring optionally having O, S or NR° in said ring, where Rc is hydrogen or Clu alkyl.

[0066] The present invention is further directed to a methods of preparing compounds of the above Formula I, 1I m or mt. The compounds of this invention can be prepared by reactions described in Schemes 1-13.

[0067] Schemes 1 and 2 depict a synthetic route for forming benzothiazoles of Formula I. Heating 5-bromo-2-amino-benzenethiol (Mital, R. L. and Jain, S. K., J Chem Soc (C) : 2148 (1969); Lin, A.-J. and Kasina, S., J Heterocycl Chem 18 : 759 (1981)) and4-dimethylaminobenzaldehyde or4- (4-methylpiperazin-1-yl) benzaldehyde (Tanaka, A., et al., J. Med. Chem. 41 : 2390 (1998)) in DMSO produced benzothiazoles, 1 and 4. Using the same Pd (0)-catalyzed Br to tributyltin exchange reaction, these two bromo derivatives were successfully converted to the corresponding tributyltin derivatives 2 and 5. They were successfully used in an iododestannylation reaction to produce the corresponding iodinated compounds 3 and 6 (yields were between 25-35%; the reactions were not optimized). Thus, the tributyltin derivatives served two useful purposes, i) they served as intermediates for converting bromo to iodo derivatives ; ii) they are also useful as starting material for preparation of radioiodinated"hot"ligand.

SCHEME 1 NH4SCN N KOH, H Br NH2 NH2 20 NH2 Br2, HAC grS OF-IC N N N (SnBu3) , Pd (0) N/ iN, 4 (SnBU3) 2, Pd (o) N Et3N, dioxane (, \ Bussn N 2 12/CHCI3 N/ S /N I 3 SCHEME2 /-l K2C03, DMF F CHO + HNUN-CH3 ^ OHC<N N-CH3 NHs NH2 Br I gH ( N N N-CH (SnBu3) 2, Pd (0) I| \> \/<N N-CH3 DMSO Br ~S L'~/Et3N, dioxane ber 4 12/CHCI, N N\-i N-CH3 N N-CH3 S ici 5 6 [0068] Scheme 3 depicts a synthetic route in which N-monomethylated amines are prepared, and thereafter employed in the parallel synthesis of disubstituted aminophenyl benzothiazole derivatives.

SCHEME 3 NH2 Polyphosphoric acid N Br Pdcat.. N/==\ (SnBu rN/==\ Br I gH HOOC I Bu Pdcat. N (SnBu3) 2, S, N NH M B S \/NHMe Pd I S NHM r u3Sn Parallei synthesis 12/CHCi3, E, N < 4 N </ --NHMe ri- or Na*I/H202 1 S S 7 E: CH3, C2H5, C3H7, C4Hg, C3H6F, C4H8F or [0069] Schemes 4 through 6 depict synthetic routes for forming benzoxazoles of the present invention.

SCHEME 4 NH2/Boric Acid N/ + HOOC N O N H Xylene p2N O 'I 0, H2 i N/NaN02, H+/N Pd/C, H2, Kl 1 \) <-NX p \ 8 8 SCHEME 5 NH + HOOC---NHMe B°ric Acid i N , NH2 < Xylene o NXN2<NHMe 02N 02N, Pd/C, H2 : ll N H N I O /NHMe \ O NHMe 2 KI I 9 SCHEME 6 Boric Acid N + HOOC NHMe I NHMe 02N H Xylene Pd/C) H2 i N NaN02) H+. N -- 2N w O \/NHMe KBr p \/NHMe bu (SnBu3)2 N 12/CHCi3, 6N '-NHMe NHME BU3Sn or Na*I/H2021 0 10 Parallel synthesis NHMe 1<) : o E : CH3, C2H5, C3H7, C4H9, C3H6F, C4H8F or [0070] Schemes 7,8 and 9 depict synthetic routes for preparing indole derivatives and benzimidazole derivatives of the present invention.

SCHEME 7 Nez EtO, OEt Nome2 O j HCNMea f! ] C! 0/- HCNMe2 ci 0 NMe2 Br N02 DMF Br N02 Et3N, PhH Br N02 /N Dioxane,H20 Na2S204 - H Br \ NO H NaH, Mel 4- (SnBU3) 2 N Br N \ Pd (0) Buts N\ /CHC 3 or Na/HzOg'\ 11 11 SCHEME 8 NH HCHO----NH/Br2 NH/SnC12 NH/ . NO2 NaBH4 NO2 Br NO2 Br) NH2 Ber Br OHC4N\ N (BU3sn) 2 N N Pd (O) nu Xylene gu3sn /a/ or t4 Na [1251] 1, H202 12 Na [S)]), HsOs SCHEME9 i NH Xylene N I, OHC I ., N \/ NH Br Br Pd (0) w N (SnBu3) w N NH N> NHME Pd (O) N-&NHME Bu3Sn Parallel synthesis 12/CHC13" : Z 12/CHCI3/>4-NHMe s > N<N I I 13 E: CH3, C2H5, C3H7, C4H9, C3H6F, C4H8F or nif [0071] Scheme 10 depicts a synthetic route for forming benzofuran derivatives of the present invention. Alternatively, benzofurans can be prepared via an intramolecular Wittig Route (Twyman, et al., Tetrahedron Lett 40 : 9383 (1999)) as set forth in Scheme 11.

SCHEME 10 SCHEME 11 [0072] Scheme 12 provides a synthetic route for parallel synthesis of benzofuran derivatives of the present invention.

SCHEME 12 [0073] Schemes 13 through 17 are directed to imidazo [1, 2, a] pyridine derivatives of the present invention.

SCHEME 13 !') > Ns Br2 Br2, s (EtO) 2POH p3Ns Br) Y Et3N Br bu X, 0) _N_'NH2 i.,/a) 2. ) N (SnBu Pd 0 i W N /N-- N/ ETOH, NaHCO3 dioxane 18 dioxine 16 x = 1 12/CHCI3 17x-Br or Na [1251] 1 H O 125 N/ t2s N / 19 SCHEME 14 Br ETOH, NaHC03.. N Br i I. j EtOH, NaHC03.. N NH2 Br p v $r \ N (SnBu3) 2 'IV Pd0 \ N NHMe pd (0) Bu Sn N /NHMe NH2Me Bu 20 Parallel synthesis E 12/CHCI3 N/=\ N/ or Na*I/H202 I orNa/HsOs) \ jN- /\ E: CH3, C2H5, C3H7, C4H9, C3H6F, C4H8F or SCHEME 15 SCHEME 16 RO (CH2) n ETOH, NaHC03 N e N NH2 RO CH n Br) Br R : p-MeO-benzyl HCI seN/MsCI, N/ ru zon MOs (CH2) n [18F] F Kryptofix K2CO3 Ru F CH n N /N 2 n=1-5 SCHEME 17 N Suzuki Reaction /\/ 16H RS RS (J SR RS"HO Labeling Reaction Na9m Tc] TcO4 Sn (ll)/Glucoheptonate < N\ o/\Np » N~N\ p N ! N HN N1 Ho N [0074] Schemes 18 and 19 depict synthetic routes for forming benzopyrimidines of the present invention.

SCHEME 18 NH NH2 BRJDPrOH BrnNH2 THF Br NH2 0 0 Br i-PrOH Br v T Br (quant.) 90% crude yield I N I N 0 onc Xylene, air Br SCHEME 19 NHME NH NH2 OHC N\ Pd cat. Br'v NH2 e.. N Br NH2Me gr s N , NHMe NHMe (SnBu3) 2 AN+J 12/CHCI3 >, N9 Pd (0) Bu3Sn N or Na*UH02 i N NHMe Parallel synthesis . NHMe N N I E: CH3, C2H5, CSH7, C4H9, C3H6F, C4HsF or [0075] Scheme 20 depicts the synthesis of metal-chelated complexes of the present invention, where R9 is as defined above, and Ar is a bicyclic system selected from the group comprising: benzothiazyl, benzoxazolyl, benzimidazolyl, benzofuranyl, imidazo [1, 2a] pyridyl, and benzopyrimidyl.

SCHEME 20 CSR9 R9SD 1 eq. (Boe) 20 CSR9 R9S Cl\~Br SR9 R9S J J H N\-/N, H H/N\--JN, BOC c N N CSR9 R9Sl J SR N N) /==\ cyoc N SR9 R9S /kr N///\/", \--/-130C Ar--O-N \ \ Hg (OAc) 2 CSH NS. Nassm. . Tc04 TFA,Anisole, N N NTcN J Ar N v V H Sn (II) qr N \ O [BnEt3N][ReOCf4] Rez ^ N N CHClg+ MeOH Aro \ NUN [0076] Shemes 21 through 23 are directed to imidazol, 2a] [1, 3] diazepine derivatives of Formula III'.

SCHEME21 SCHEME 22 SCHEME 23 N N N N N /Suzuki Reaction Labeling N tu N f l \ No /p /N \ Suzuki Reaction Labeling N Tu N Suzuki Reaction Labeling 0 ON N N s c s S S [0077] When the compounds of this invention are to be used as imaging agents, they must be labeled with suitable radioactive halogen isotopes. Although 125I- isotopes are useful for laboratory testing, they will generally not be useful for actual diagnostic purposes because of the relatively long half-life (60 days) and low gamma-emission (30-65 Kev) of 1211. The isotope 123I has a half life of thirteen hours and gamma energy of 159 KeV, and it is therefore expected that labeling of ligands to be used for diagnostic purposes would be with this isotope.

Other isotopes which may be used include 131I (half life of 2 hours). Suitable bromine isotopes include 77Br and 76Br.

[0078] The radiohalogenated compounds of this invention lend themselves easily to formation from materials which could be provided to users in kits. Kits for forming the imaging agents can contain, for example, a vial containing a physiologically suitable solution of an intermediate of Formula I, 1I, or HI in a concentration and at a pH suitable for optimal complexing conditions. The user would add to the vial an appropriate quantity of the radioisotope, e. g., Na'z3I, and an oxidant, such as hydrogen peroxide. The resulting labeled ligand may then be administered intravenously to a patient, and receptors in the brain imaged by means of measuring the gamma ray or photo emissions therefrom.

[0079] Since the radiopharmaceutical composition according to the present invention can be prepared easily and simply, the preparation can be carried out readily by the user. Therefore, the present invention also relates to a kit, comprising: (1) A non-radiolabeled compound of the invention, the compound optionally being in a dry condition; and also optionally having an inert, pharmaceutically acceptable carrier and/or auxiliary substances added thereto; and (2) a reducing agent and optionally a chelator; wherein ingredients (1) and (2) may optionally be combined; and further wherein instructions for use with a prescription for carrying out the above- described method by reacting ingredients (1) and (2) with technetium-99m in the form of a pertechnetate solution may be optionally included.

[0080] Examples of suitable reducing agents and chelators for the above kit have been listed above. The pertechnetate solution can be obtained by the user from a molybdenum-technetium generator. Such generators are available in a number of institutions that perform radiodiagnostic procedures. As noted above the ingredients (1) and (2) may be combined, provided they are compatible. Such a monocomponent kit, in which the combined ingredients are preferably lyophilized, is excellently suitable to be reacted by the user with the pertechnetate solution in a simple manner.

[0081] When desired, the radioactive diagnostic agent may contain any additive such as pH controlling agents (e. g., acids, bases, buffers), stabilizers (e. g., ascorbic acid) or isotonizing agents (e. g., sodium chloride).

[0082] The term"pharmaceutically acceptable salt"as used herein refers to those carboxylate salts or acid addition salts of the compounds of the present invention which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term"salts"refers to the relatively nontoxic, inorganic and organic acid addition salts of compounds of the present invention. Also included are those salts derived from non-toxic organic acids such as aliphatic mono and dicarboxylic acids, for example acetic acid, phenyl- substituted alkanoic acids, hydroxy alkanoic and alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Further representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactiobionate and laurylsulphonate salts, propionate, pivalate, cyclamate, isethionate, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as, nontoxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See, for example, Berge S. M., et al., Pharmaceutical Salts, J. Pharn. Sci. 66 : 1-19 (1977) which is incorporated herein by reference.) [0083] In the first step of the present method of imaging, a labeled compound of Formula I, II, HI or m'is introduced into a tissue or a patient in a detectable quantity. The compound is typically part of a pharmaceutical composition and is administered to the tissue or the patient by methods well known to those skilled in the art.

[0084] For example, the compound can be administered either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray.

[0085] In a preferred embodiment of the invention, the labeled compound is introduced into a patient in a detectable quantity and after sufficient time has passed for the compound to become associated with amyloid deposits, the labeled compound is detected noninvasively inside the patient. In another embodiment of the invention, a labeled compound of Formula I, I1, m or m'is introduced into a patient, sufficient time is allowed for the compound to become associated with amyloid deposits, and then a sample of tissue from the patient is removed and the labeled compound in the tissue is detected apart from the patient. In a third embodiment of the invention, a tissue sample is removed from a patient and a labeled compound of Formula I, II, ni or M'is introduced into the tissue sample.

After a sufficient amount of time for the compound to become bound to amyloid deposits, the compound is detected.

[0086] The administration of the labeled compound to a patient can be by a general or local administration route. For example, the labeled compound may be administered to the patient such that it is delivered throughout the body.

Alternatively, the labeled compound can be administered to a specific organ or tissue of interest. For example, it is desirable to locate and quantitate amyloid deposits in the brain in order to diagnose or track the progress of Alzheimer's disease in a patient.

[0087] The term"tissue"means a part of a patient's body. Examples of tissues include the brain, heart, liver, blood vessels, and arteries. A detectable quantity is a quantity of labeled compound necessary to be detected by the detection method chosen. The amount of a labeled compound to be introduced into a patient in order to provide for detection can readily be determined by those skilled in the art. For example, increasing amounts of the labeled compound can be given to a patient until the compound is detected by the detection method of choice. A label is introduced into the compounds to provide for detection of the compounds.

[0088] The term"patient"means humans and other animals. Those skilled in the art are also familiar with determining the amount of time sufficient for a compound to become associated with amyloid deposits. The amount of time necessary can easily be determined by introducing a detectable amount of a labeled compound of Formulae I-E'into a patient and then detecting the labeled compound at various times after administration.

[0089] The term"associated"means a chemical interaction between the labeled compound and the amyloid deposit. Examples of associations include covalent bonds, ionic bonds, hydrophilic-hydrophilic interactions, hydrophobic- hydrophobic interactions, and complexes.

[0090] Those skilled in the art are familiar with the various ways to detect labeled compounds. For example, magnetic resonance imaging (min, positron emission tomography (PET), or single photon emission computed tomography (SPECT) can be used to detect radiolabeled compounds. The label that is introduced into the compound will depend on the detection method desired. For example, if PET is selected as a detection method, the compound must possess a positron-emitting atom, such as"C or l8F.

[0091] The radioactive diagnostic agent should have sufficient radioactivity and radioactivity concentration which can assure reliable diagnosis. For instance, in case of the radioactive metal being technetium-99m, it may be included usually in an amount of 0.1 to 50 mCi in about 0.5 to 5.0 ml at the time of administration.

The amount of a compound of Formulae I-E'may be such as sufficient to form a stable chelate compound with the radioactive metal.

[0092] The thus formed chelate compound as a radioactive diagnostic agent is sufficiently stable, and therefore it may be immediately administered as such or stored until its use. When desired, the radioactive diagnostic agent may contain any additive such as pH controlling agents (e. g., acids, bases, buffers), stabilizers (e. g., ascorbic acid) or isotonizing agents (e. g., sodium chloride).

[0093] The imaging of amyloid deposits can also be carried out quantitatively so that the amount of amyloid deposits can be determined.

[0094] Preferred compounds for imaging include a radioisotope such as'1,'1, 1311, 18F, 76 Br or 77 Br.

[0095] The present invention is also directed at a method of imaging amyloid deposits. One of the key prerequisites for an in vivo imaging agent of the brain is the ability to cross the intact blood-brain barrier after a bolus iv injection. The compounds of this invention possess a core ring system comprised of various substituted, fused 5-and 6-member aromatic rings. Several compounds of this invention contain a benzothiazole core and are derivatives of thioflavins. These compounds contain no quaternary ammonium ion, therefore, they are relatively small in size, neutral and lipophilic (Partition Coefficient = 70 and 312 for 3 and 6a, respectively).

[00961 To test the permeability through the intact blood-brain barrier several compounds of Formula I or IM were injected into normal mice. Initial brain uptake of 3 and 6a in mice after an iv injection was 0.67 and 1.50 % dose/organ, respectively (see Table 1). The brain uptake peaked at 60 min for both compounds with a maximum brain uptake of 1.57 and 1.89 % dose/organ, respectively. The blood levels are relatively low throughout the time points evaluated. For this series of ligands, specific uptake in the brain is relatively high and the retention in the brain is long.

[0097] Another aspect of the invention is a method of inhibiting amyloid plaque aggregation. The present invention also provides a method of inhibiting the aggregation of amyloid proteins to form amyloid deposits, by administering to a patient an amyloid inhibiting amount of a compound of the above Formula I, lI, Mort.

[0098] Those skilled in the art are readily able to determine an amyloid inhibiting amount by simply administering a compound of Formula I, II, III or HI'to a patient in increasing amounts until the growth of amyloid deposits is decreased or stopped. The rate of growth can be assessed using imaging as described above or by taking a tissue sample from a patient and observing the amyloid deposits therein. The compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is sufficient. The specific dosage used, however, can vary. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to those skilled in the art.

[0099] The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.

EXAMPLE 1 2- (4'-Dimethylaminophenyl)-6-iodobenzothiazole, (3) [0100] 2- (4'-Dimethylaminophenyl)-6-bromobenzothiazole (1) : (Stevens, M. F. G., et al., J. Med. Chem. 37 : 1689-1695 (1994); Stevens, M. F. G., et al., PCT Int. Appl. W019940830 : 47 (1995)) [0101] A mixture of 5-bromo-2-amino-benzenethiol (Mital, R. L. andain, S. K., J. Chem Soc (C): 2148 (1969); Lin, A.-J. and Kasina, S., J Heterocycl Chem 18 : 759 (1981)) (306 mg, 1.5 mmol) and 4-dimethylamino benzaldehyde (224 mg, 1. 5 mmol) in DMSO was heated at 180°C for 15 min. Water (10 mL) was added after the mixture was cooled down. The solid was collected by suction and recrystallized in ethyl acetate to give 340 mg of product (68%).

[0102] lH NE (200 MHz, CDC13) : # 3.06 (s, 6H), 6.74 (d, J=9.0 Hz, 2H), 7.52 (d, d, J=8.7,2.0 Hz, 1H), 7.82 (d, J=8.6 Hz, lH), 7.93 (d, J=8.8 Hz, 2H), 7.95 (s, 1H).

[0103] HRMS : m/z Calcd for ClSHl4BrN2S (MH+) : 333.0061; Found : 333.0072.

[0104] 2- (4'-Dimethylaminophenyl)-6-tribytylstannylbenzothiazole (2) : To a solution of 2- (4'-dimethylaminophenyl)-6-bromobenzothiazole (16a) (60 mg, 0.18 mmol) in 1,4-dioxane (2 mL), toluene (2 mL) and triethylamine (2 mL) was added (Bu3Sn) 2 (0.2 mL) followed by Pd (Ph3P) 4 (20 mg). The mixture was stirred at 90°C overnight. Solvent was removed and the residue was purified by PTLC (Hex: EtOAc, 6: 1) to give 33 mg of product (yield 33.6%).

[0105] 1H NMR (200 MHz, CDCl3) : 8 0.90 (t, J=7. 1 Hz, 9H), 1. 10 (t, J=8.0 Hz, 6H), 1.34 (hex, J=7.3 Hz, 6H), 1.57 (m, 6H), 3.05 (s, 6H), 6.74 (d, J=9.0 Hz, 2H), 7.50 (d, d, J=7.9,0.9 Hz, 1H), 7.93 (s, 1H), 7.95 (d, J=8. 5 Hz, lH), 7.97 (d, J=9.0 Hz, 2H).

[0106] HRMS: m/z Calcd for C27H4lN2SSn (MH+) : 545.2012; Found : 545.2035.

[0107] 2- (4'-Dimethylaminophenyl)-6-iodobenzothiazole, (3): To a solution of 2 (45mg, 0.08mmol) in CHCl3 (10 mL) was added a solution of iodine (1 mL, 1M in CHCl3) dropwise at RT until the color maintaining unchanged. The resulting mixture was stirred at RT for 10 min. NaHSO3 solution (2 mL, 5% in water) and KF (1 mL, 1M in MeOH) were added successively. The mixture was stirred for 5 min and the organic phase was separated. The aqueous phase was extracted with CH2CD12 and the combined organic phases was dried over Na2SO4, filtered and concentrated to give crude product which was purified by PTLC (Hex: EtOAc, 6: 1) to give 9 mg of the desired product (yield 29%). 1H NMR (200 MHz, CDCl3) : # 3.06 (s, 6H), 6.73 (d, J=9.0 Hz, 2H), 7.69 (s, 1H), 7.70 (s, 1H), 7.93 (d, J=9. 0 Hz, 2H), 8.15 (s, 1H).

[0108] HRMS : m/z Calcd for C15H15N2IS(MH+): 380. 9922; Found: 380.9914.

[0109] Anal. (C15H14N3IS) : C, H, N.

EXAMPLE 2 2- [4'- (4"-Methylpiperazin-1-yl)-phenyl]-6-iodobenzothiazole, (6) [0110] 2- [4'- (4"-Methylpiperazin-1-yl)-phenyl]-6-bromobenzothiazole (4): The procedure described above to prepare 1 was employed to give 57.2% of product 4 from 4- (4-methylpiperazin-1-yl) benzaldehyde (Tanaka, A., et al., J.

Med. Chem. 41 : 2390 (1998)) (204 mg, 1 mmol) and 5-bromo-2-amino- benzenethiol (204 mg, 1 mmol).

[0111]'H NMR (200 MHz, CDCl3) : 8 2.38 (s, 3H), 2.60 (t, J=5.0 Hz, 4H), 3.38 (t, J=5. 0 Hz, 4H), 6.96 (d, J=8.9 Hz, 2H), 7.54 (d, d, J=8.5,1.9 Hz, 1H), 7.83 (d, J=8. 5 Hz, lH), 7.95 (d, J=8.9 Hz, 2H), 7.98 (s, lH).

[0112] HRMS: m/z Calcd for C18HIgBrN3S (MH+) : 388.0483; Found: 388.0474. <BR> <BR> <BR> <BR> <BR> <P>[0113] 2-4'- (4"-Methylpiperazin-1-yl)-phenyl]-6-tributylstannyl benzothiazole (5): The procedure described above to prepare 2 was employed, 5 was obtained in 23% yield from 4.

[0114] 1H NMR (200 MHz, CDCl3) : 8 0.89 (t, J=7. 2 Hz, 9H), 1.06 (t, J=8. 2 Hz, 6H), 1.30 (hex, J=7.3 Hz, 6H), 1.57 (pen, J=7.2 Hz, 6H), 2.38 (s, 3H), 2.60 (m, 4H), 3.36 (t, J=5.0 Hz, 4H), 6.96 (d, J=8.9 Hz, 2H), 7.52 (d, J=7.9 Hz, 1H), 7.93 (s, lH), 7.95 (d, J=7.9 Hz, lH), 7.98 (d, J=8.9 Hz, 2H).

[0115] HRMS : m/z Calcd for C30H46N3SSn (MH+) : 600.2434; Found: 600.2449.

[0116] 2- [4'- (4"-Methylpiperazin-1-yl)-phenyl]-6-iodobenzothiazoIe, (6): The same reaction as described above to prepare 3 was employed, 6 was obtained in 36% yield from 5.

[0117] 1H NMR (200 MHz, CDCl3) : 8 2.42 (s, 3H), 263 (t, J=4.8 Hz, 4H), 3.40 (t, J=4.9 Hz, 4H), 6.95 (d, J=9.0 Hz, 2H), 7.71 (s, lH), 7.72 (s, 1H), 7.95 (d, J=8.9 Hz, 2H), 8.17 (t, J=1.0 Hz, IM. HRMS : m/z Calcd for ClgHIgN3IS (MH+) : 436.0344; Found : 436.0364. Anal. (C1gHl8N3SI) : C, H, N.

EXAMPLE 3 Preparation of 6-Tributylstannyl-2- (4'-dimethylamino-) phenyl- imidazo [1, 2a] pyridine (18) 6-Bromo-2- (4'-dimethylamino-) phenyl-imidazo [1, 2-a] pyridine (17) [0118] A mixture of 2-bromo-4'-dimethylaminoacetophenone, (968 mg, 4 mmol) and 2-amino-5-bromo-pyridine (692 mg, 4 mmol) in EtOH (25 mL) was stirred under reflux for 2 hr. NaHC03 (500 mg) was added after the mixture was cooled down. The resulting mixture was stirred under reflux for 4.5hr. The mixture was cooled down, filtered to give 655 mg of product, 17 (52%).

'H NMR (200 MHz, CDCl3, 8) : 3.00 (s, 6H), 6.78 (d, J=8.7 Hz, 2H), 7.17 (d, d, J=9.5,1.7 Hz, 1H), 7.49 (d, J=9.5 Hz, 1H), 7.69 (s, 1H), 7.80 (d, J=8.7 Hz, 2H), 8.21 (d, d, J=1.7,0.8 Hz, 1H). Anal. 3a, (Cl^Hl4BrN3) 6-Tributylstannyl-2- (4'-dimethylamino-) phenyl-imidazo [1, 2-a] pyridine (18).

[0119] To a solution of 6-bromo-2- (4'-dimethylamino-) phenyl-imidazo [1,2-a] pyridine, 17, (80 mg, 0.26 mmol) in 1,4-dioxane (10 mL) and triethylamine (2 mL) was added (Bu3Sn) 2 (0.2 mL) in neat followed by Pd (Ph3P) 4 (20 mg).

The mixture was stirred at 90°C overnight. Solvent was removed and the residue was purified by PTLC (Hex: EtOAc= 1 : 1 as developing solvent) to give 23 mg of product, 18 (17%).

'H NMR (200 MHz, CDCl3,#) : 0.90 (t, J=7.2 Hz, 9H), 1.10 (t, J=8. 0 Hz, 6H), 1.33 (hex, J=7.1 Hz, 6H), 1.54 (pen, J=7.2 Hz, 6H), 3.00 (s, 6H), 6.78 (d, J=8. 9 Hz, 2H), 7.11 (d, J=8. 8 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.71 (s, 1H), 7.84 (d, J=8.8 Hz, 2H), 7.95 (d, J=0.8 Hz, 1H). HRMS: m/z Calcld for C27H42N3Sn (M++H) : 528.2400; Found: 528.2402. Anal. 4, (C27H4lN3Sn. 2H20) 6-Iodo-2- (4'-dimethylamino-) phenyl-imidazo [1, 2-a] pyridine, IMPY, (16) [0120] A mixture of 2-bromo-4'-dimethylaminoacetophenone, (484 mg, 2 mmol) and 2-amino-5-iodo-pyridine (440 mg, 2 mmol) in EtOH (25 mL) was stirred under reflux for 2 hr. NaHC03 (250 mg) was added after the mixture was cooled down. The resulting mixture was stirred under reflux for 4hr. The mixture was cooled down, filtered to give 348 mg of product, 3b (48%).

'H NMR (200 MHz, CDCl3,#) : 3.00 (s, 6H), 6.77 (d, J=8.8 Hz,2H), 7.27 (d, d, J=9.4,1.5 Hz, 1H), 7.38 (d, J=9.5 Hz, 1H), 7.66 (s, 1H), 7.79 (d, J=8.8 Hz, 2H), 8.32 (d, J=0.7 Hz, 1H). Anal. 3b, (ClsHl4IN3).

EXAMPLE 4 Preparation of radioiodinated ligand : [125I]IMPY, [125I]18 [0121] The compound, ['25I] 18, was prepared using iododestannylation reactions with tributyltin precursor 17. Hydrogen peroxide (50 yL, 3% w/v) was added to a mixture of 50 AL of the correspondent tributyltin precursor (1µg/µL EtOH), 50 /iL of IN HCl and [125/123I]NaI (1-5 mCi) in a sealed vial. The reaction was allowed to proceed for 10 min at room temperature and terminated by addition of 100 its of sat. NaHSO3. The reaction mixture was either directly extracted (styrylbenzenes) with ethylacetate (3x 1 mL) or extracted after neutralization with saturated sodium bicarbonate solution (thioflavins). The combined extracts were evaporated to dryness. For styrylbenzenes the residues were dissolved in 100 ßL of EtOH and purified by HPLC using a reverse phase column (Waters ubondpad, 3.9 x 300 mm) with an isocratic solvent of 65 % acetonitrile-35 % trifluoroacetic acid (0.1%) in a flow rate of 0.8 mL/min. Thioflavins were purified on a C4 column (Phenomenex Inc., Torrance, CA) eluted with an isocratic solvent of 80 % acetonitrile-20 % 3,3-dimethyl-glutaric acid (5 mM, pH 7.0) in a flow rate of 0.8 mL/min. The desired fractions containing the product were collected, condensed and re-extracted with ethylacetate. The no-carrier-added products were evaporated to dryness and re-dissolved in 100% EtOH (1µCi/µL), The final 18, with a specific activity of 2,200Ci/mmole and a greater than 95% radiochemical purity, were stored at-20°C up to 6 weeks for in vitro binding and autoradiography studies.

EXAMPLE 5 Partition Coefficient determination [0122] Partition coefficients were measured by mixing the [1211] traCer with 3 g each of 1-octanol and buffer (0.1 M phosphate, pH 7.4) in a test tube. The test tube was vortexed for 3 min at room temperature, followed by centrifugation for 5 min. Two weighed samples (0.5 g each) from the 1-octanol and buffer layers were counted in a well counter. The partition coefficient was determined by calculating the ratio of cpm/g of 1-octanol to that of buffer. Samples from the 1- octanol layer were re-partitioned until consistent partitions of coefficient values were obtained. The. measurement was done in triplicate and repeated three times.

EXAMPLE 6 Binding assays using aggregated Ap (l-40) or Ap 42) peptide in solution [0123] The solid forms of peptides Aß(1-40) and Ap (1-42) were purchased from Bachem (King of Prussia, PA). Aggregation of peptides were carried out by gently dissolving the peptide [0. 5 mg/mL for Ap (1-40) and 0.25 mg/mL for Ap (1-42) in a buffer solution (pH 7.4) containing 10 mM sodium phosphate and 1mM EDTA. The solutions were incubated at 37°C for 36-42 h with gentle and constant shaking. Binding studies were carried out in 12 x 75 mm borosilicate glass tubes according to the procedure described with some modifications (Klunk, W. E., et al., Biol. Psychiatry 35 : 627 (1994)). Aggregated fibrils (10-50 nM in the final assay mixture) were added to the mixture containing 50 ml of radioligands (0.01-0. 5 nM) in 40% EtOH and 10 % EtOH in a final volume of 1 mL for saturation studies. Nonspecific binding was defined in the presence of 2 mM thioflavin T for thioflavins. For inhibition studies, ImL of the reaction mixture contained 40 ml of inhibitors (10-5-10-10 Min 10 % EtOH) and 0. 05 nM radiotracer in 40 % EtOH. The mixture was incubated at room temperature for 3 h and the bound and the free radioactivity were separated by vacuum filtration through Whatman GF/B filters using a Brandel M-24R cell harvester followed by 2 x 3 mL washes of 10% ethanol at room temperature. Filters containing the bound I-125 ligand were counted in a gamma counter (Packard 5000) with 70% counting efficiency. The results of saturation and inhibition experiments were subjected to nonlinear regression analysis using software EBDA52 by which Kd and K ; values were calculated. Additional K ; values for compounds of the invention are provided in Fig. 1A and Fig. 1B.

TABLE 1 Inhibition constants (Ki, nM) of compounds on ligand binding to aggregates of Ap (l-40) and Aß(1-42) at 25°C Aggregates of Ap Aggregates of AD (1-40) (1-42) Compounds-vs ['2sI] 3 vs ['2sI] 3 Chrysamine G >1, 000 >2,000 Thioflavin T 116 + 20 294 + 40 1 1. 9 ~ 0. 3 0.8 ~ 0. 3 4 1. 6 ~ 0. 5 5. 0 ~ 0., 8 3 0. 9 ~ 0. 2 2.2 ~ 0. 4 6a 5. 4 ~ 0. 7 6. 4 0. 7 Values are the mean SEM of three independent experiments, each in duplicates.

EXAMPLE 7 In vivo biodistribution of new probes in normal mice [0124] While under ether anesthesia, 0.15 mL of a saline solution containing labeled agents (5-10 mCi) was injected directly into the tail vein of ICR mice (2-3 month-old, average weight 20-30 g). The mice were sacrificed by cardiac excision at various time points post injection. The organs of interest were removed and weighed, and the radioactivity was counted with an automatic gamma counter (Packard 5000). The percentage dose per organ was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. Total activities of blood and muscle were calculated under the assumption that they were 7% and 40% of the total body weight, respectively.

TABLE 2 ["11 Compound 3 (PC=70) Organ 2 min 30 min 60 min 6 h 24 h Blood 15.74 ~ 6. 06 3.26 ~ 0. 05 3.79 ~ 0. 19 1.44 ~ 0. 05 0.29 : t 0. 09 Heart 1.79 ~ 0. 39 0.20 ~ 0. 01 0.17 ~ 0. 02 0.05 ~ 0. 01 0.01 0. 00 Liver 31.62 ~ 2.38 10.93 ~ 2. 34 9.21 ~ 3. 05 1.52 ~ 0. 30 0.30 ~ 0. 07 Brain 0.67 ~ 0.11 0.97 ~ 0. 29 1. 57 ~ 0. 24 0.65 ~ 0.11 0.04 ~ 0. 01 [125I] Compound 6a (PC= 312) Organ 2 min 30 min 60 min 6 h 24 h Blood 8.02 ~ 0. 82 5.15 ~ 0. 23 4.16 ~ 0. 28 1.49 0. 26 0.41 ~ 0. 09 Heart 2.19 ~ 0. 43 0.69 ~ 0. 02 0.66 ~ 0. 06 0.22 ~ 0. 06 0.08 ~ 0. 01 Liver 28.84 ~ 3. 77 21.22 5. 86 17.20 ~ 2. 49 5.79 ~ 1. 24 3.05 ~ 0. 87 Brain 1.50 ~ 0. 10 1.59 ~ 0. 19 1. 89 ~ 0. 43 1.08 ~ 0. 08 0.91 ~ 0. 08 [125I] Compound 8 (PC= 124) Organ 2 min 30 min 60 niin 6 h 24 h Blood 4.31 ~ 0. 34 2.80 ~ 0. 45 2.94 ~ 0.18 2.23 ~ 0. 53 1.68 ~ 0. 56 Heart 1.20 ~ 0. 18 0.19 ~ 0. 05 0.11 ~ 0. 02 0.05 0. 00 0.02 0. 00 Liver 25.04 : t 2. 45 17.45 2. 01 5.57 ~ 0. 39 1.08 0. 11 0.42 0. 08 Brain 1.43 ~ 0. 23 2.08 ~ 0. 03 1.26 ~ 0. 10 0.12 ~ 0. 02 0.01 ~ 0. 00 [Compound 19 (PC=100) Organ 2 min 30 min 1 hr 2 hr 6 hr 24 hr BLOOD 6.41 ~ 0.77 2.44 ~ 0. 36 2.50 ~ 0. 11 1.82 ~ 0. 21 1. 40 ~ 0. 27 0. 18 + 0. 02 HEART 0.79 ~ 0. 14 0.16 ~ 0. 02 0.12 ~ 0. 02 0.08 ~ 0. 01 0.04 ~ 0. 01 0.01 + 0.00 MUSCLE 13. 81 ~ 3.44 6.08 ~ 0. 59 5.03 ~ 1. 03 2.96 ~ 0. 84 1.46 ~ 0. 42 0.27 0. 11 LUNG 1.56 ~ 0. 33 0.31 ~ 0. 07 0.34 ~ 0. 08 0.20 ~ 0. 05 0.12 ~ 0. 05 0.05 ~ 0. 03 KIDNEY 4.75 + 0.49 1. 51 ~ 0. 27 1.17 ~ 0. 29 0.53 ~ 0. 05 0.25 ~ 0. 05 0.05 ~ 0. 01 SPLEEN 0.40 ~ 0. 06 0.09 ~ 0. 02 0.08 ~ 0. 01 0.05 ~ 0. 01 0.04 ~ 0. 01 0.01 ~ 0. 00 LIVER 20.88 ~ 2. 63 6.32 ~ 0. 55 5.88 ~ 0. 85 2.90 ~ 0. 21 1.54 ~ 0. 08 0. 61 ~ 0. 11 SKIN 5. 72. t 0. 90 4. 69 ~ 1. 06 4. 28 ~ 0. 25 3.14 0.51 2.19 0.63 0.22 0.06 BRAIN 2.88 : E 0. 25 0.26 ~ 0. 00 0.21 ~ 0. 03 0. 14 ~ 0. 03 0.06 ~ 0. 02 0.02 ~ 0. 00 % dose/organ, average of 3 mice SD; Average organ weights are: blood, 2 g; muscle, 12 g; liver, g; brain 0.4 g, from which the % dose/g value for each organ or tissue can be calculated.

* (% dose/organ, avg of 3 or 4 mice SD) [0125] Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications, and publications cited herein are fully incorporated by reference herein in their entirety.