NOVEL COMPOUNDS FOR DIAGNOSIS
20230174536 · 2023-06-08
Assignee
Inventors
Cpc classification
A61K51/0455
HUMAN NECESSITIES
C07B2200/05
CHEMISTRY; METALLURGY
A61K51/0463
HUMAN NECESSITIES
A61K51/0459
HUMAN NECESSITIES
International classification
C07B59/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to novel compounds of formula (I), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, that can be employed in the imaging of alpha-synuclein aggregates and determining an amount thereof. Furthermore, the compounds can be used for diagnosing a disease, disorder or abnormality associated with an alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites (such as Parkinson's disease), determining a predisposition to such a disease, disorder or abnormality, prognosing such a disease, disorder or abnormality, monitoring the evolution of the disease in a patient suffering from such a disease, disorder or abnormality, monitoring the progression of such a disease, disorder or abnormality and predicting responsiveness of a patient suffering from such a disease, disorder or abnormality to a treatment thereof.
Claims
1. A compound of formula (I) ##STR00886## or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein ##STR00887## is an aryl or a heteroaryl which is directionally selected from the following: ##STR00888## R.sup.0 is H or C.sub.1-C.sub.4alkyl; R.sup.1 is —CN; or halo; or C.sub.1-C.sub.4alkyl; or C.sub.1-C.sub.4alkoxy; or —N(C.sub.1-C.sub.4alkyl).sub.2; or —NH(C.sub.1-C.sub.4alkyl); or H; or R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo; R.sup.2 is aryl, or 5-membered or 6-membered heteroaryl, wherein R.sup.2 is selected from the following: ##STR00889## wherein R.sup.2a, R.sup.2a′ are independently selected from H, or F; R.sup.2b is independently selected from F, OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, NH.sub.2, CN, or C.sub.1-C.sub.4alkoxy; R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3; R.sup.2d is selected from H, F, or OH; R.sup.2e is selected from H, OH, CH.sub.3, or F; Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S; Z.sup.1 is independently N, NH, O, or S; p is 0, 1 or 2; m is 0 or 1; as valency permits, is a combination of single and double bonds; and * is the position of bonding.
2. The compound according to claim 1, having a formula (IIa), (IIb), (IIIa), (IIIb), or (IIIc) ##STR00890## ##STR00891## or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof.
3. (canceled)
4. The compound according to claim 1, wherein R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo, preferably R.sup.1 is selected from the following: ##STR00892##
5. The compound of formula (I) according to claim 1, wherein the compound is selected from: ##STR00893## ##STR00894## ##STR00895## ##STR00896## ##STR00897## ##STR00898## ##STR00899## ##STR00900## ##STR00901## ##STR00902## ##STR00903## ##STR00904## ##STR00905## ##STR00906## ##STR00907## ##STR00908## ##STR00909## ##STR00910## ##STR00911## ##STR00912## ##STR00913## ##STR00914## ##STR00915## ##STR00916## ##STR00917## ##STR00918## ##STR00919## ##STR00920## ##STR00921## ##STR00922## ##STR00923## ##STR00924## ##STR00925## ##STR00926## ##STR00927## ##STR00928## ##STR00929## ##STR00930## or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof.
6. The compound of formula (I) according to claim 1, wherein the compound is a detectably labelled compound, preferably wherein the detectably labelled compound comprises a detectable label selected from a radioisotope, more preferable wherein the compound is a detectably labelled by .sup.2H, .sup.3H or .sup.18F.
7. (canceled)
8. The compound of formula (I) according to claim 6, wherein R.sup.1 is ##STR00931## or wherein R.sup.1 is ##STR00932## wherein F means .sup.18F; and the compound of formula (I) is detectably labelled at least at one available position by .sup.3H (Tritium); or wherein the detectably labelled compound is ##STR00933## wherein T means .sup.3H (Tritium) and F means .sup.18F; or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
9. (canceled)
10. (canceled)
11. A method of imaging of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, wherein the compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, is employed particularly wherein the imaging is positron emission tomography imaging of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, more particularly wherein the imaging is in vitro imaging, ex vivo imaging, or in vivo imaging, preferably the method is in vivo imaging, more preferably the method is brain imaging.
12. (canceled)
13. (canceled)
14. (canceled)
15. A method according to claim 31 wherein the disease, disorder or abnormality is optionally selected from Parkinson's disease (including sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure or Lewy body dysphagia), SNCA duplication carrier, Lewy Body dementia (LBD), dementia with Lewy bodies (DLB) (including “pure” Lewy body dementia), Parkinson's disease dementia (PDD), diffuse Lewy body disease (DLBD), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, Down syndrome, multiple system atrophy (MSA) (including Shy-Drager syndrome, striatonigral degeneration or olivopontocerebellar atrophy), traumatic brain injury, chronic traumatic encephalopathy, dementia puglistica, tauopathies (including Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Niemann-Pick type C1 disease, frontotemporal dementia with Parkinsonism linked to chromosome 17), Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (including sporadic, familial or ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (including Hallervorden-Spatz syndrome), prion diseases, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder, particularly wherein the disease is Parkinson's disease; multiple system atrophy, dementia with Lewy bodies, Parkinson's disease dementia, SNCA duplication carrier, or Alzheimer's disease.
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
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22. (canceled)
23. A diagnostic composition comprising a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and at least one pharmaceutically acceptable excipient, carrier, diluent or adjuvant.
24. A method selected from (A) a method of imaging a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, in a subject, the method comprising the steps: (a) Administering a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the subject; (b) Allowing the compound to bind to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites; and (c) Detecting the compound bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites, or (B) a method of imaging a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, in a subject, the method comprising the steps: (a) Administering a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the subject; and (b) imaging the brain of the subject; or (C) a method of imaging a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, in a subject according to above method (a) and (B), the method comprising the steps: (a) Administering a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; (b) Allowing the compound to bind to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites; (c) Detecting the compound bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites, and (d) Generating an image representative of the location and/or amount of the compound bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites Or (D) a method of positron emission tomography (PET) imaging of alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, in a tissue of a subject, the method comprising the steps: (a) Administering a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the subject; (b) Allowing the compound to penetrate into the tissue of the subject; and (c) Collecting a positron emission tomography (PET) image of the tissue of the subject; wherein the tissue is tissue of the central nervous system (CNS), of the eye or brain tissue, preferably wherein the tissue is brain tissue; or (E) a method of detecting a neurological disease, disorder or abnormality associated with alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, in a subject, the method comprising the steps: (a) Administering a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the subject; (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and (c) Measuring the radioactive signal of the compound, which is bound to the alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites or (F) a method for the detection and/or quantification a alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, in a tissue of a subject, the method comprising the steps: (a) Contacting the tissue with a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the subject; (b) Allowing the compound to bind to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites, and (c) Detecting and/or quantifying the compound bound to the alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, using positron emission tomography or (G) a method of the diagnostic imaging of the brain of a subject, the method comprising the steps: (a) Administering a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the subject; and (b) Obtaining an image of the brain of the subject using positron emission tomography or (H) a method of collecting data for the diagnosis of a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: (a) Bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area or (I) a method of collecting data for determining a predisposition to a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: (a) Bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area or (J) a method of prognosing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, wherein the method comprises the steps: (a) Bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area, and (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time or (K) method of monitoring the progression of a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in a patient, the method comprising the steps: (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time or (L) method of predicting responsiveness of a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to a treatment of the disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, method comprising the steps: (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound according to claim 6, or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time or (M) The method of above methods (H) to (L), wherein the step of optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; comprises determining in which amount of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy, correlating the amount of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the amount of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and optionally comparing the amount of the amount of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area to a normal control value in a healthy control subject.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. A compound of formula (IV-F) ##STR00934## or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.3 is selected from ##STR00935## R.sup.4 is an aryl or a 5-membered or 6-membered heteroaryl, wherein R.sup.4 is selected from: ##STR00936## wherein R.sup.2a, R.sup.2a′ are independently selected from H, or F; R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy; R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3; R.sup.2d is selected from H, F, or —OH; R.sup.2e is selected from H, OH, CH.sub.3, or F; Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S; Z.sup.1 is independently N, NH, O, or S; p is 0, 1 or 2; m is 0 or 1; as valency permits, is a combination of single and double bonds; and * is the position of bonding; or a compound of formula (IV-H) ##STR00937## or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.6 is selected from ##STR00938## R.sup.6 is an aryl or a 5-membered or 6-membered heteroaryl, wherein R.sup.6 is selected from the following: ##STR00939## wherein R.sup.2a, R.sup.2a′ are independently selected from H, X or F; R.sup.2b is independently selected from X, F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy, and wherein C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, or C.sub.1-C.sub.4alkoxy optionally comprise one or more X; R.sup.2c, R.sup.2c′ are independently selected from X, H, F, OH, OCH.sub.3, or CH.sub.3; R.sup.2d is selected from X, H, F, or —OH; R.sup.2e is selected from X, H, OH, CH.sub.3, or F; Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S; Z.sup.1 is independently N, NH, O, or S; p is 0, 1 or 2; m is 0 or 1; as valency permits,
is a combination of single and double bonds; * is the position of bonding; Fluoro is .sup.19F; X is Bromo, Chloro, or Iodo; and wherein R.sup.6 comprises at least one X; or a compound of formula (IV-J) ##STR00940## or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.7 is selected from ##STR00941## R.sup.8 is selected from the following: ##STR00942## wherein R.sup.2a, R.sup.2a′ are independently selected from H, or F; R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy; p is 0, 1 or 2; R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl; as valency permits,
is a combination of single and double bonds; Fluoro is .sup.19F; and * is the position of bonding.
38. The compound according to claim 37 which is of formula (IV-F), wherein LG is selected from Bromo, Chloro, Iodo, C.sub.1-4 alkyl sulfonate and C.sub.6-10 aryl sulfonate, wherein the C.sub.6-10 aryl can be optionally substituted by —CH.sub.3 or —NO.sub.2.
39. The compound according to claim 37, which is of formula (IV-F), and which is of the following formula ##STR00943## or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein LG is mesylate or nosylate; or which is of formula (IV-H) and which is of the following formula ##STR00944## or a pharmaceutically acceptable salt, hydrate, or solvate thereof or which is of formula (IV-J), and which is of the following formula ##STR00945## or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
40. (canceled)
41. (canceled)
42. (canceled)
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44. A method of preparing the compound according to claim 6 comprising (A) reacting a compound of formula (IV-F) ##STR00946## or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.3 is selected from ##STR00947## R.sup.4 is an aryl or a 5-membered or 6-membered heteroaryl, wherein R.sup.4 is selected from: ##STR00948## wherein R.sup.2a, R.sup.2a′ are independently selected from H, or F; R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy; R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3; R.sup.2d is selected from H, F, or —OH; R.sup.2e is selected from H, OH, CH.sub.3, or F; Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S; Z.sup.1 is independently N, NH, O, or S; p is 0, 1 or 2; m is 0 or 1; as valency permits, is a combination of single and double bonds; and * is the position of bonding; with a .sup.18F-fluorinating agent, so that LG is replaced by .sup.18F; or (B) reacting a compound of formula (IV-H) ##STR00949## or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.5 is selected from ##STR00950## R.sup.6 is an aryl or a 5-membered or 6-membered heteroaryl, wherein R.sup.6 is selected from the following: ##STR00951## wherein R.sup.2a, R.sup.2a′ are independently selected from H, X or F; R.sup.2b is independently selected from X, F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy, and wherein C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, or C.sub.1-C.sub.4alkoxy optionally comprise one or more X; R.sup.2c, R.sup.2c′ are independently selected from X, H, F, OH, OCH.sub.3, or CH.sub.3; R.sup.2d is selected from X, H, F, or —OH; R.sup.2e is selected from X, H, OH, CH.sub.3, or F; Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S; Z.sup.1 is independently N, NH, O, or S; p is 0, 1 or 2; m is 0 or 1; as valency permits,
is a combination of single and double bonds; * is the position of bonding; Fluoro is .sup.19F; X is Bromo, Chloro, or Iodo; and wherein R.sup.6 comprises at least one X; with a .sup.3H radiolabeling agent; or (C) reacting a compound of formula (IV-J) ##STR00952## or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.7 is selected from ##STR00953## R.sup.8 is selected from the following: ##STR00954## wherein R.sup.2a, R.sup.2a′ are independently selected from H, or F; R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy; p is 0, 1 or 2; R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl; as valency permits,
is a combination of single and double bonds; Fluoro is .sup.19F; and * is the position of bonding, with a CT.sub.3 radiolabeling agent, wherein T is .sup.3H.
45. The method according to claim 44, wherein the .sup.18F-fluorinating agent is selected from K.sup.18F, Rb.sup.18F, Cs.sup.18F, Na.sup.18F, Rb.sup.18F, Kryptofix[222]K.sup.18F, tetra(C.sub.1-6 alkyl) ammonium salt of .sup.18F, and tetrabutylammonium [.sup.18F]fluoride.
46. (canceled)
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48. (canceled)
49. A test kit for the detection and/or diagnosis of a disease, disorder or abnormality associated with alpha-synuclein aggregates, wherein the test kit comprises at least one compound as defined in claim 1, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof.
50. A kit for preparing a radiopharmaceutical preparation, wherein the kit comprises a sealed vial containing at least one compound as defined in claim 37.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0256]
[0257]
[0258]
[0259]
[0260]
[0261]
[0262]
[0263]
[0264]
[0265]
[0266]
[0267]
[0268]
[0269]
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[0271]
DETAILED DESCRIPTION OF THE INVENTION
[0272] The compounds of the present invention and their precursors are described in the following. It is to be understood that all possible combinations of the following definitions are also envisaged.
[0273] The present invention relates to a compound of formula (I),
##STR00054##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
##STR00055##
is an aryl or a heteroaryl which is directionally selected from the following:
##STR00056##
R.sup.0 is H or C.sub.1-C.sub.4alkyl;
R.sup.1 is —CN; or halo; or C.sub.1-C.sub.4alkyl; or C.sub.1-C.sub.4alkoxy; or —N(C.sub.1-C.sub.4alkyl).sub.2; or —NH(C.sub.1-C.sub.4alkyl); or H, or
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo;
R.sup.2 is aryl, or 5-membered or 6-membered heteroaryl, wherein R.sup.2 is selected from the following:
##STR00057##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S;
Z.sup.1 is independently N, NH, O, or S;
p is 0, 1 or 2;
m is 0 or 1;
as valency permits, is a combination of single and double bonds; and
* is the position of bonding.
[0274] In another embodiment, the invention provides a compound of formula (I), having a formula (IIa) or (IIb),
##STR00058##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof.
[0275] In another embodiment, the invention provides a compound of formula (I), having a formula (IIIa), (IIIb), or (IIIc),
##STR00059##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof.
R.sup.0 is H or C.sub.1-C.sub.4alkyl. Preferably, R.sup.0 is H or CH.sub.3, more preferably R.sup.0 is H.
[0276] In an embodiment, R.sup.1 is H, —CN, halo, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy, —N(C.sub.1-C.sub.4alkyl).sub.2, or —NH(C.sub.1-C.sub.4alkyl). Preferably, R.sup.1 is —CN, halo, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy, —N(C.sub.1-C.sub.4alkyl).sub.2, or —NH(C.sub.1-C.sub.4alkyl). More preferably, R.sup.1 is —CN, F, C.sub.1-C.sub.3alkyl, C.sub.1-C.sub.3alkoxy, or —N(C.sub.1-C.sub.3alkyl).sub.2. Even more preferably, R.sup.1 is —CN, —CH(CH.sub.3).sub.2, —OCH.sub.3, —OCH(CH.sub.3).sub.2, —N(CH.sub.3).sub.2, or —NH—CH(CH.sub.3).sub.2.
[0277] In an embodiment, R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo. Preferably R.sup.1 is selected from the following:
##STR00060##
wherein R.sup.1′ is independently halo; and s=0, 1, 2 or 3.
[0278] More preferably, R.sup.1 is selected from the following:
##STR00061##
[0279] Even more preferably, R.sup.1 is selected from
##STR00062##
[0280] In a preferred embodiment F is preferably .sup.19F or .sup.18F, more preferably .sup.18F.
[0281] In an embodiment R.sup.2 is selected from the following:
##STR00063##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S;
Z.sup.1 is independently N, NH, O, or S;
p is 0, 1 or 2;
m is 0 or 1;
as valency permits, is a combination of single and double bonds; and
* is the position of bonding.
[0282] Preferably, R.sup.2 is selected from the following:
##STR00064##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0283] Preferably, R.sup.2 is selected from the following:
##STR00065##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0284] More preferably, R.sup.2 is selected from the following:
##STR00066##
wherein R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0285] Even more preferably, R.sup.2 is selected from:
##STR00067##
wherein * is the position of bonding.
[0286] In another embodiment, the invention provides a compound of any one of subformulae (IIa) or (IIb),
##STR00068##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein R.sup.0 is methyl or H; R.sup.1 is CH.sub.3 or H; preferably, R.sup.1 is CH.sub.3; and R.sup.2 comprises at least one fluoro and is preferably selected from the following:
##STR00069##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0287] Most preferably, R.sup.2 is selected from
##STR00070##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2e, R.sup.2c, R.sup.2c′, R.sup.z and p are as defined herein above; and wherein at least one of R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, and R.sup.2e is F. F is preferably .sup.19F or .sup.18F, more preferably .sup.18F.
[0288] In another embodiment, the invention provides a compound of any one of subformulae (IIIa) (IIIb), or (IIIc),
##STR00071##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof,
wherein R.sup.0 is methyl or H, preferably R.sup.0 is H;
R.sup.1 is selected from —CN, halo, C.sub.1-C.sub.4alkyl; or C.sub.1-C.sub.4alkoxy, —N(C.sub.1-C.sub.4alkyl).sub.2, —NH(C.sub.1-C.sub.4alkyl), H; or
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo;
[0289] Preferably, R.sup.1 is selected from the following:
##STR00072##
F is preferably .sup.19F or .sup.18F, more preferably .sup.18F; and
R.sup.2 is preferably selected from the following:
##STR00073##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0290] In another embodiment the present invention relates to a compound of formula (IIIa):
##STR00074##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
R.sup.0 is methyl or H, preferably R.sup.0 is H;
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo, preferably R.sup.1 is selected from the following:
##STR00075##
R.sup.1 is preferably substituted with fluoro as follows
##STR00076##
[0291] More preferably R.sup.1 is
##STR00077##
preferably R.sup.1 is
##STR00078##
R.sup.2 is preferably selected from the following:
##STR00079##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0292] Preferably, R.sup.2 is selected from the following:
##STR00080##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0293] More preferably, R.sup.2 is selected from the following:
##STR00081##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
Preferably, R.SUP.2 .is
[0294] ##STR00082##
[0295] More preferably, R.sup.2 is or N
##STR00083##
[0296] Even more preferably, R.sup.2 is
##STR00084##
[0297] In each of the above embodiments, R.sup.2 can be optionally substituted with one or more substituents as disclosed hereinabove. F is preferably .sup.19F or .sup.18F, more preferably .sup.18F.
[0298] In another embodiment the present invention relates to a compound of formula (IIIb):
##STR00085##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
R.sup.0 is methyl or H, preferably R.sup.0 is H;
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo, preferably R.sup.1 is selected from the following:
##STR00086##
R.sup.1 is preferably substituted with fluoro as follows
##STR00087##
[0299] More preferably R.sup.1 is
##STR00088##
preferably R.sup.1 is
##STR00089##
R.sup.2 is preferably selected from the following:
##STR00090##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0300] Preferably, R.sup.2 is selected from the following:
##STR00091##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0301] More preferably, R.sup.2 is selected from the following:
##STR00092##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0302] Preferably, R.sup.2 is
##STR00093##
[0303] More preferably, R.sup.2 is
##STR00094##
[0304] Even more preferably, R.sup.2 is.
##STR00095##
[0305] In each of the above embodiments, R.sup.2 can be optionally substituted with one or more substituents as disclosed hereinabove. F is preferably .sup.19F or .sup.18F, more preferably .sup.18F.
[0306] In another embodiment the present invention relates to a compound of formula (IIIc):
##STR00096##
or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
R.sup.0 is methyl or H, preferably R.sup.0 is H;
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo, preferably R.sup.1 is selected from the following:
##STR00097##
R.sup.1 is preferably substituted with fluoro as follows
##STR00098##
[0307] More preferably R.sup.1 is
##STR00099##
preferably R.sup.1 is.
##STR00100##
R.sup.2 is preferably selected from the following:
##STR00101##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl;
p is 0, 1 or 2; and
* is the position of bonding.
[0308] Preferably, R.sup.2 is selected from the following:
##STR00102##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0309] More preferably, R.sup.2 is selected from the following:
##STR00103##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0310] Preferably, R.sup.2 is
##STR00104##
[0311] More preferably, R.sup.2 is
##STR00105##
[0312] Even more preferably, R.sup.2 is
##STR00106##
[0313] In each of the above embodiments, R.sup.2 can be optionally substituted with one or more substituents as disclosed hereinabove. F is preferably .sup.19F or .sup.18F, more preferably .sup.18F.
[0314] In another embodiment, the present invention provides a compound of formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the preferred compounds are
##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138##
[0315] More preferably, stereoisomers of preferred compounds are
##STR00139##
[0316] In one embodiment the present invention provides a compound of formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound of formula (I) is a detectably labelled compound.
[0317] One embodiment of the present invention provides a compound of formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound is a detectably labelled compound, wherein the detectable label is a radioisotope, and wherein the compound of formula (I) comprise at least one radioisotope.
[0318] Preferably, the detectable label is a radioisotope selected from .sup.18F, .sup.2H and .sup.3H, most preferably .sup.18F or .sup.3H.
[0319] In one embodiment the present invention provides a compound of formula (I), preferably a compound of subformula (IIIa), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound is a detectably labelled compound of formula (III-F)
##STR00140##
or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
R.sup.1 is substituted with .sup.18F as follows
##STR00141##
R.sup.2 is an aryl, or a 5-membered or 6-membered heteroaryl, wherein R.sup.2 is selected from
##STR00142##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
R.sup.2c, R.sup.2c′ are independently selected from H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from H, F, or —OH;
R.sup.2e is selected from H, OH, CH.sub.3, or F;
Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S;
Z.sup.1 is independently N, NH, O, or S;
p is 0, 1 or 2;
m is 0 or 1;
as valency permits, is a combination of single and double bonds; and
* is the position of bonding.
[0320] Preferably R.sup.2 is selected from
##STR00143##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e and p are as defined hereinabove and R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl.
[0321] More preferably, R.sup.2 is selected from the following:
##STR00144##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0322] More preferably, R.sup.2 is selected from the following:
##STR00145##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0323] Preferably, the detectably labelled compound of formula (III-F), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises at least one .sup.18F. Preferably, the substituents of R.sup.2 (e.g. R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.z, and R.sup.2e) optionally can be .sup.18F. More preferably, the detectably labelled compound of formula (III-F), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises one or two .sup.18F. Even more preferably, one .sup.18F.
[0324] Preferred compounds are selected from:
##STR00146##
or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
[0325] A most preferred compound is
##STR00147##
or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
[0326] In one embodiment the present invention provides a compound of formula (I), preferably a compound of subformula (IIIa), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound is a detectably labelled compound of formula (III-H)
##STR00148##
or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, which is detectably labelled at at least one available position by .sup.2H (deuterium “D”) or .sup.3H (Tritium “T”), preferably .sup.3H,
wherein
R.sup.1 is —CN; or halo; or C.sub.1-C.sub.4alkyl; or C.sub.1-C.sub.4alkoxy; or —N(C.sub.1-C.sub.4alkyl).sub.2; or —NH(C.sub.1-C.sub.4alkyl); or H; or
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo; R.sup.1 is preferably selected from
##STR00149##
R.sup.2 is an aryl, or a 5-membered or 6-membered heteroaryl, wherein R.sup.2 is selected from the following:
##STR00150##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, T or F;
R.sup.2b is independently selected from T, F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, CT.sub.3, or C.sub.1-C.sub.4alkoxy;
R.sup.2a, R.sup.2a′ are independently selected from T, H, F, OH, OCH.sub.3, CT.sub.3, or CH.sub.3;
R.sup.2d is selected from T, H, F, or —OH;
R.sup.2e is selected from T, H, OH, CH.sub.3, CT.sub.3, or F;
Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S;
Z.sup.1 is independently N, NH, O, or S;
p is 0, 1 or 2;
m is 0 or 1;
as valency permits, is a combination of single and double bonds;
Fluoro is .SUP.19.F;
[0327] wherein C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, or C.sub.1-C.sub.4alkoxy optionally comprise one or more T, and
* is the position of bonding.
[0328] Preferably, the detectably labelled compound of formula (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises one, two or three T. Preferably, the detectably labelled compound of formula (III-Ha), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises one T. More preferably, the detectably labelled compound of formula (III-Ha), comprises two T. Even more preferably, the detectably labelled compound of formula (III-Ha), comprises three T.
[0329] Preferably, the detectably labelled compound of formula (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, is a compound of formula (III-Ha)
##STR00151##
or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein
R.sup.1 is —CN; or halo; or C.sub.1-C.sub.4alkyl; or C.sub.1-C.sub.4alkoxy; or —N(C.sub.1-C.sub.4alkyl).sub.2; or —NH(C.sub.1-C.sub.4alkyl); or H; or
R.sup.1 is —NH—C.sub.3-C.sub.6cycloalkyl, C.sub.3-C.sub.6cycloalkyl, or heterocyclyl, each of which is optionally substituted with at least one halo;
R.sup.1 is preferably selected from
##STR00152##
R.sup.2 is an aryl, or a 5-membered or 6-membered heteroaryl, wherein R.sup.2 is selected from the following and wherein R.sup.2 is optionally substituted with at least one T,
##STR00153##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, T or F;
R.sup.2b is independently selected from T, F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, CT.sub.3, or C.sub.1-C.sub.4alkoxy, wherein C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, or C.sub.1-C.sub.4alkoxy optionally comprise one or more T;
R.sup.2c, R.sup.2c′ are independently selected from T, H, F, OH, OCH.sub.3, CT.sub.3, or CH.sub.3;
R.sup.2d is selected from T, H, F, or —OH;
R.sup.2e is selected from T, H, OH, CH.sub.3, CT.sub.3, or F;
Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S;
Z.sup.1 is independently N, NH, O, or S;
p is 0, 1 or 2;
m is 0 or 1;
as valency permits, is a combination of single and double bonds;
T is .SUP.3.H (Tritium);
[0330] n is 0 to 3;
with the proviso that the compound of formula (I-Ha) comprises at least one T;
Fluoro is .SUP.19.F; and
[0331] * is the position of bonding.
[0332] Preferably, the detectably labelled compound of formula (III-Ha), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises one, two or three T. Preferably, n is 1.
[0333] Preferably, the detectably labelled compound of formula (III-Ha), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises one T. More preferably, the detectably labelled compound of formula (III-Ha), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises two T. Even more preferably, the detectably labelled compound of formula (III-Ha), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprises three T.
[0334] In a further embodiment, the present invention provides a detectably labelled compound of formulae (III-H) or (III-Ha), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, as disclosed hereinabove, wherein R.sup.2 is an aryl, or a 5-membered or 6-membered heteroaryl selected from
##STR00154##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e and p are as defined hereinabove, R.sup.z is selected from T, H, C.sub.1-C.sub.4alkyl, CT.sub.3, or haloC.sub.1-C.sub.4alkyl; wherein C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl optionally comprise one or more T.
[0335] Preferably, R.sup.2 is selected from the following:
##STR00155##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0336] More preferably, R.sup.2 is selected from the following:
##STR00156##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2e, R.sup.z and p are as defined hereinabove.
[0337] Preferably, R.sup.2 is
##STR00157##
wherein R.sup.z comprises at least one T.
[0338] More preferably, R.sup.2 is
##STR00158##
[0339] A preferred detectably labelled compound of formula (III-H) or (III-Ha), pharmaceutically acceptable salt, hydrate, or solvate thereof is
##STR00159##
wherein T means .sup.3H (Tritium). Preferably, F means .sup.19F.
[0340] In a preferred embodiment, the invention provides a detectably labelled compound of formula (III-H) or (III-Ha), or stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein .sup.3H Tritium (“T”) can be replaced by .sup.2H Deuterium (“D”).
[0341] Preferably, the detectably labelled compounds of formula (I), or of subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, comprise a detectable label, preferably the detectable label is a radioisotope, in particular selected from .sup.18F, .sup.2H and .sup.3H.
[0342] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and their precursors can be detectably labelled. The type of the label is not specifically limited and will depend on the detection method chosen. Examples of possible labels include isotopes such as radionuclides, positron emitters, and gamma emitters. With respect to the detectably labelled compounds of the present invention, or stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof and their precursors which include a radioisotope, a positron emitter, or a gamma emitter, it is to be understood that the radioisotope, positron emitter, or gamma emitter is to be present in an amount which is not identical to the natural amount of the respective radioisotope, positron emitter, or gamma emitter. Furthermore, the employed amount should allow detection thereof by the chosen detection method.
[0343] Examples of suitable isotopes such as radionuclides, positron emitters and gamma emitters include .sup.2H, .sup.3H, .sup.18F, .sup.11C, .sup.13N, and .sup.15O, preferably .sup.2H, .sup.3H, .sup.11C, .sup.13N, .sup.15O, and .sup.18F, more preferably .sup.2H, .sup.3H and .sup.18F, even more preferably .sup.3H and .sup.18F.
[0344] .sup.18F-labelled compounds are particularly suitable for imaging applications such as PET. The corresponding compounds which include fluorine having a natural .sup.19F isotope are also of particular interest as they can be used as analytical standards and references during manufacturing, quality control, release and clinical use of their .sup.18F-analogs.
[0345] Further, substitution with isotopes such as deuterium, i.e., .sup.2H, may afford certain diagnostic and therapeutic advantages resulting from greater metabolic stability by reducing for example defluorination, increased in vivo half-life or reduced dosage requirements, while keeping or improving the original compound efficacy.
[0346] Isotopic variations of the compounds of the invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and their precursors can generally be prepared by conventional procedures such as by the illustrative methods or by the preparations described in the Examples and Preparative Examples hereafter using appropriate isotopic variations of suitable reagents, which are commercially available or prepared by known synthetic techniques.
[0347] Radionuclides, positron emitters and gamma emitters can be included into the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and their precursors by methods which are usual in the field of organic synthesis. Typically, they will be introduced by using a correspondingly labelled starting material when the desired compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and their precursor is prepared. Illustrative methods of introducing detectable labels are described, for instance, in US 2012/0302755.
[0348] The position at which the detectable label is to be attached to the compounds of the present invention and their precursors is not particularly limited.
[0349] The radionuclides, positron emitters and gamma emitters, for example, can be attached at any position where the corresponding non-emitting atom can also be attached. For instance, .sup.18F can be attached at any position which is suitable for attaching F. The same applies to the other radionuclides, positron emitters and gamma emitters. Due to the ease of synthesis, it is preferred to attach .sup.18F at R.sup.1. .sup.3H can be attached at any available position. Preferably it is attached to the pyridine ring. If .sup.2H is employed as a detectable label it can be attached at any available position. Preferably it is attached to the pyridine ring.
[0350] In another embodiment, the present invention relates further to a compound of formula (IV-F) that is a precursor of the compound of formula (III-F)
##STR00160##
wherein
R.sup.3 is substituted with a Leaving Group (LG) as follows
##STR00161##
R.sup.4 is an aryl, or a 5-membered or 6-membered heteroaryl, wherein R.sup.4 is selected from the same list as R.sup.2 of the compound of formula (III-F) as disclosed hereinabove.
[0351] Preferably, the Leaving Group (LG) is halogen, C.sub.1-4 alkyl sulfonate, C.sub.1-C.sub.4alkyl ammonium, nitro, or C.sub.6-10 aryl sulfonate, wherein the C.sub.6-10 aryl can be optionally substituted by —CH.sub.3 or —NO.sub.2. More preferably, the Leaving Group (LG) is bromo, chloro, iodo, C.sub.1-4 alkyl sulfonate, or C.sub.6-10 aryl sulfonate, wherein the C.sub.6-10 aryl can be optionally substituted by —CH.sub.3 or —NO.sub.2. Even more preferably, the Leaving Group (LG) is mesylate, tosylate or nosylate. Even more preferably, the Leaving Group (LG) is mesylate, or nosylate. Preferably the Leaving Group (LG) is mesylate.
[0352] Preferably, R.sup.4 is
##STR00162##
[0353] More preferably, R.sup.4 is
##STR00163##
[0354] Even more preferably, R.sup.4 is
##STR00164##
[0355] Preferably, R.sup.4 is optionally substituted with a .sup.18F.
[0356] A preferred compound is
##STR00165##
[0357] In another embodiment, the present invention relates further to a compound of formula (IV-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, that is a precursor of the compound of formula (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof
##STR00166##
wherein
R.sup.5 is selected from the same list as R.sup.1 of the compound of formula (III-H) as disclosed hereinabove and is preferably selected from
##STR00167##
R.sup.6 is an aryl, or a 5-membered or 6-membered heteroaryl, wherein R.sup.6 is selected from the following:
##STR00168##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, X or F;
R.sup.2b is independently selected from X, F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy, wherein C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, or C.sub.1-C.sub.4alkoxy optionally comprise one or more X;
R.sup.2c, R.sup.2c′ are independently selected from X, H, F, OH, OCH.sub.3, or CH.sub.3;
R.sup.2d is selected from X, H, F, or —OH;
R.sup.2e is selected from X, H, OH, CH.sub.3, or F;
Z is independently N, NH, N(C.sub.1-C.sub.4alkyl), N(haloC.sub.1-C.sub.4alkyl), O, or S;
Z.sup.1 is independently N, NH, O, or S;
p is 0, 1 or 2;
m is 0 or 1;
as valency permits, is a combination of single and double bonds;
* is the position of bonding.
Fluoro is .SUP.19.F;
X is Bromo, Chloro or Iodo; and
[0358] Wherein R.sup.6 comprises at least one X.
[0359] In a further embodiment, the compound of formula (IV-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, R.sup.6 is preferably an aryl, or a 6-membered heteroaryl optionally substituted with one or more X, selected from:
##STR00169##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, R.sup.2c, R.sup.2c′, R.sup.2d, R.sup.2e and p are as defined hereinabove; as valency permits, is a combination of single and double bonds; Fluoro is .sup.19F; and * is the position of bonding.
[0360] Preferably, R.sup.6 is
##STR00170##
More preferably, R.sup.6 is
##STR00171##
[0361] Even more preferably the compound of formula (IV-H) is
##STR00172##
a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof,
with X being selected from Bromo, Chloro and Iodo.
[0362] Preferably, X is bromine.
[0363] A preferred compound is
##STR00173##
a detectably labelled compound, pharmaceutically acceptable salt, hydrate, or solvate thereof.
[0364] In another embodiment the present invention relates further to a compound of formula (IV-J), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, that is a precursor of the compound of formula (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof
##STR00174##
wherein
R.sup.7 is selected from the same list as R.sup.1 of the compound of formula (III-H) as disclosed hereinabove and is preferably selected from
##STR00175##
[0365] R.sup.8 is selected from the following:
##STR00176##
wherein
R.sup.2a, R.sup.2a′ are independently selected from H, or F;
R.sup.2b is independently selected from F, —OH, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl, —NH.sub.2, —CN, or C.sub.1-C.sub.4alkoxy;
p is 0, 1 or 2;
R.sup.z is selected from H, C.sub.1-C.sub.4alkyl or haloC.sub.1-C.sub.4alkyl,
as valency permits, is a combination of single and double bonds;
Fluoro is .SUP.19.F; and
[0366] * is the position of bonding.
[0367] Preferably, R.sup.z is H.
[0368] In a further embodiment of the compound of formula (IV-J), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, R.sup.8 is preferably selected from
##STR00177##
wherein R.sup.2a, R.sup.2a′, R.sup.2b, and p are as defined hereinabove;
[0369] More preferably, R.sup.8 is selected from:
##STR00178##
[0370] A preferred compound is
##STR00179##
or a detectably labelled compound, pharmaceutically acceptable salt, hydrate, or solvate thereof.
Method of Synthesis of Detectably Labelled Compounds
[0371] The present invention relates further to a method for preparing a compound of formula (I), or of subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and in particular a compound of formula (III-F) or (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof comprising a detectable label.
[0372] In one embodiment, the present invention relates to a method for preparing a compound of formula (III-F), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, by radiolabeling a compound of formula (IV-F) with the radioisotope .sup.18F
##STR00180##
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined herein.
[0373] Suitable solvents for the .sup.18F-fluorination comprise DMF, DMSO, acetonitrile, DMA, or mixtures thereof, preferably acetonitrile or DMSO.
[0374] Suitable agents for the .sup.18F-fluorination are selected from K.sup.18F, Rb.sup.18F, Cs.sup.18F, Na.sup.18F, tetra(C.sub.1-6 alkyl) ammonium salt of .sup.18F, kryptofix[222].sup.18F and tetrabutylammonium [.sup.18F]fluoride.
[0375] In one embodiment, the present invention relates to a method of preparing a compound of formula (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, by radiolabeling a compound of formula (IV-H) with the radioisotope .sup.3H
##STR00181##
wherein R.sup.1, R.sup.2, R.sup.5 and R.sup.6 are as defined herein, and
T is .SUP.3.H (Tritium),
[0376] n is 0 to 3, preferably, n is 1 or 2, more preferably, n is 1;
with the proviso that the compound of formula (III-Ha) comprises at least one T,
Fluoro is .SUP.19.F,
[0377] X is Bromo, Chloro, Iodo or H, preferably, X is bromine.
[0378] The .sup.3H radiolabeling agent can be tritium gas. The method can be conducted in the presence of a catalyst such as palladium on carbon (Pd/C), a solvent such as dimethylformamide (DMF) and a base such as N,N-diisopropylethylamine (DIEA).
[0379] In a preferred embodiment, F (Fluoro) is .sup.19F.
[0380] In one embodiment, the present invention relates to a method for preparing a compound of formula (III-H), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, by radiolabeling a compound of formula (IV-J) with a CT.sub.3 radiolabeling agent, wherein T is .sup.3H.
##STR00182##
[0381] The CT.sub.3 radiolabeling agent can be ICT.sub.3 (derivative of iodomethane with .sup.3H). The method can be conducted in the presence of a solvent such as dimethylformamide (DMF) and a base such cesium carbonate or sodium hydride.
Diagnostic Compositions
[0382] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are particularly suitable for imaging of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. With respect to alpha-synuclein protein, the compounds are particularly suitable for binding to various types of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The imaging can be conducted in mammals, preferably in humans. The imaging is preferably in vitro imaging, ex vivo imaging, or in vivo imaging. More preferably the imaging is in vivo imaging: Even more preferably, the imaging is preferably brain imaging. The imaging can also be eye/retinal imaging. The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are particularly suitable for use in diagnostics.
[0383] The diagnostics can be conducted for mammals, preferably for humans. The tissue of interest on which the diagnostics is conducted can be brain, tissue of the central nervous system, tissue of the eye (such as retinal tissue) or other tissues, or body fluids such as cerebrospinal fluid (CSF). The tissue is preferably brain tissue.
[0384] Due to their design and to the binding characteristics, the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are suitable for use in the diagnosis of diseases, disorders and abnormalities associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are particularly suitable for positron emission tomography imaging of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. Diseases involving alpha-synuclein aggregates are generally listed as synucleinopathies (or α-synucleinopathies). The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are suitable for use in the diagnosis of diseases, disorders or abnormalities including, but not limited to, Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), SNCA duplication carrier, dementia with Lewy bodies (“pure” Lewy body dementia), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease and normal aging in Down syndrome). Synucleinopathies with neuronal and glial aggregates of alpha synuclein include multiple system atrophy (MSA) (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy). Other diseases that may have alpha-synuclein-immunoreactive lesions include traumatic brain injury, chronic traumatic encephalopathy, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick type C1 disease), motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder (Jellinger, Mov Disord 2003, 18 Suppl. 6, S2-12; Galvin et al. JAMA Neurology 2001, 58 (2), 186-190; Kovari et al., Acta Neuropathol. 2007, 114(3), 295-8; Saito et al., J Neuropathol Exp Neurol. 2004, 63(4), 323-328; McKee et al., Brain, 2013, 136(Pt 1), 43-64; Puschmann et al., Parkinsonism Relat Disord 2012, 18S1, S24-S27; Usenovic et al., J Neurosci. 2012, 32(12), 4240-4246; Winder-Rhodes et al., Mov Disord. 2012, 27(2), 312-315; Ferman et al., J Int Neuropsychol Soc. 2002, 8(7), 907-914). Preferably, the compounds of the present invention are suitable for use in the diagnosis of Parkinson's disease, multiple system atrophy, dementia with Lewy bodies, Parkinson's disease dementia, SNCA duplication carrier, or Alzheimer's disease, more preferably Parkinson's disease (PD).
[0385] In the methods of diagnosing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, such as Parkinson's disease, or a predisposition therefor in a subject, the method comprises the steps of: [0386] a) administering to the subject a diagnostically effective amount of a compound of the present invention or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0387] b) allowing the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to distribute into the tissue of interest (such as brain or other tissues, or body fluids such as cerebrospinal fluid (CSF)); and [0388] c) imaging the tissue of interest, wherein an increase in binding of the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the tissue of interest compared to a normal control level of binding indicates that the subject is suffering from or is at risk of developing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites.
[0389] The compounds of the present invention or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can be used for imaging of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in any sample or a specific body part or body area of a patient which is suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The compounds are able to pass the blood-brain barrier. Consequently, they are particularly suitable for imaging of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the brain or peripheral organs such as the gut, as well as in body fluids such as cerebrospinal fluid (CSF).
[0390] In diagnostic applications, the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, preferably compounds of formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), are preferably administered in the form of a diagnostic composition comprising the compound of the invention or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof. A “diagnostic composition” is defined in the present invention as a composition comprising one or more compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, in a form suitable for administration to a patient, e.g., a mammal such as a human, and which is suitable for use in the diagnosis of the specific disease, disorder or abnormality at issue. Preferably a diagnostic composition further comprises a physiologically acceptable excipient, carrier, diluent or adjuvant. Administration is preferably carried out as defined below. More preferably by injection of the composition as an aqueous solution. Such a composition may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g., cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); and pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid). The dose of the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, will vary depending on the exact compound to be administered, the weight of the patient, and other variables as would be apparent to a physician skilled in the art.
[0391] While it is possible for the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to be administered alone, it is preferable to formulate them into a diagnostic composition in accordance with standard pharmaceutical practice. Thus, the invention also provides a diagnostic composition which comprises a diagnostically effective amount of a compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, in admixture with, optionally, at least one pharmaceutically acceptable excipient, carrier, diluent or adjuvant.
[0392] Pharmaceutically acceptable excipients are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 15.sup.th Ed., Mack Publishing Co., New Jersey (1975). The pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof.
[0393] Pharmaceutically useful excipients, carriers, adjuvants and diluents that may be used in the formulation of the diagnostic composition of the present invention may comprise, for example, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate, binders, adjuvants, solubilizers, thickening agents, stabilizers, disintegrants, glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colorants, flavors, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-ß-cyclodextrin, polyvinylpyrrolidone, low melting waxes, and ion exchange resins.
[0394] The routes for administration (delivery) of the compounds of the invention, preferably compounds of formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, include, but are not limited to, one or more of: intravenous, gastrointestinal, intraspinal, intraperitoneal, intramuscular, oral (e. g. as a tablet, capsule, or as an ingestible solution), topical, mucosal (e. g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e. g. by an injectable form), intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, epidural and sublingual. Preferably, the route of administration (delivery) of the compounds of the invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, is intravenous.
[0395] For example, the compounds can be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
[0396] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include starch, a cellulose, milk sugar (lactose) or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
[0397] Preferably, in diagnostic applications, the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are administered parenterally. If the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the compounds; and/or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
[0398] As indicated, the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA134AT) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, e. g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e. g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
[0399] Alternatively, the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, may also be dermally or transdermally administered, for example, by the use of a skin patch.
[0400] They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds can be formulated as micronized suspensions in isotonic, pH was adjusted, sterile saline, or, preferably, as solutions in isotonic, pH was adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
[0401] For application topically to the skin, the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
[0402] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing diagnosis.
[0403] The diagnostic compositions of the invention can be produced in a manner known per se to the skilled person as described, for example, in Remington's Pharmaceutical Sciences, 15.sup.th Ed., Mack Publishing Co., New Jersey (1975).
[0404] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are useful as an in vitro analytical reference or an in vitro screening tool. They are also useful in in vivo diagnostic methods.
[0405] The compounds according to the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can also be provided in the form of a mixture comprising a compound according to the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and at least one compound selected from an imaging agent different from the compound according to the invention, a pharmaceutically acceptable excipient, carrier, diluent or adjuvant. The imaging agent different from the compound according to the invention is preferably present in a diagnostically effective amount. More preferably the imaging agent different from the compound according to the invention is an Abeta or Tau imaging agent.
[0406] Diagnosis of a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites or of a predisposition to a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in a patient may be achieved by detecting the specific binding of a compound according to the invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in a sample or a specific body part or body area, which includes the steps of: [0407] (a) bringing the sample or a specific body part or body area suspected to contain the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of the invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, which binds the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, [0408] (b) allowing the compound of the invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies or Lewy neurites) complex (hereinafter “compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex” will be abbreviated as “compound/protein aggregate complex”), [0409] (c) detecting the formation of the compound/protein aggregate complex, [0410] (d) optionally correlating the presence or absence of the compound/protein aggregate complex with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or area, and [0411] (e) optionally comparing the amount of the compound/protein aggregate complex to a normal control value, wherein an increase in the amount of the compound/protein aggregate complex compared to a normal control value may indicate that the patient is suffering from or is at risk of developing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites.
[0412] The compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can be brought into contact with the sample or the specific body part or body area suspected to contain the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites by a suitable method. In in vitro methods the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and a liquid sample can be simply mixed. In in vivo tests the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, is typically administered to the patient by any suitable means. These routes of administration include, but are not limited to, one or more of: oral (e. g. as a tablet, capsule, or as an ingestible solution), topical, mucosal (e. g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e. g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, epidural and sublingual. In some instances, parenteral administration can be preferred.
[0413] After the sample or a specific body part or body area has been brought into contact with the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, the compound is allowed to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The amount of time required for binding will depend on the type of test (e.g., in vitro or in vivo) and can be determined by a person skilled in the field by routine experiments.
[0414] The compound which has bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, can be subsequently detected by any appropriate method. The specific method chosen will depend on the detectable label which has been chosen. Examples of possible methods include, but are not limited to, a fluorescence imaging technique or a nuclear imaging technique such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and contrast-enhanced magnetic resonance imaging (MRI). These have been described and enable visualization of amyloid biomarkers. The fluorescence imaging technique and/or nuclear imaging technique can be employed for monitoring and/or visualizing the distribution of the detectably labelled compound within the sample or a specific body part or body area.
[0415] The presence or absence of the compound/protein aggregate complex is then optionally correlated with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or area. Finally, the amount of the compound/protein aggregate complex can be compared to a normal control value which has been determined in a sample or a specific body part or body area of a healthy subject, wherein an increase in the amount of the compound/protein aggregate complex compared to a normal control value may indicate that the patient is suffering from or is at risk of developing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites.
[0416] The present invention also relates to a method of determining the amount of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in a tissue and/or a body fluid. This method comprises the steps of: [0417] (a) providing a sample representative of the tissue and/or body fluid under investigation; [0418] (b) testing the sample for the presence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with a compound of the present invention; [0419] (c) determining the amount of compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0420] (d) calculating the amount of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the tissue and/or body fluid.
[0421] The sample can be tested for the presence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with a compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, by bringing the sample into contact with a compound of the invention, allowing the compound of the invention to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/protein aggregate complex and detecting the formation of the compound/protein aggregate complex as explained above.
[0422] Monitoring minimal residual disease, disorder or abnormality in a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites who has been treated with a medicament with a compound according to the invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, may be achieved by [0423] (a) bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0424] (b) allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/protein aggregate complex; [0425] (c) detecting the formation of the compound/protein aggregate complex; [0426] (d) optionally correlating the presence or absence of the compound/protein aggregate complex with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0427] (e) optionally comparing the amount of the compound/protein aggregate complex to a normal control value, wherein an increase in the amount of the aggregate compared to a normal control value may indicate that the patient may still suffer from a minimal residual disease, disorder or abnormality.
[0428] How steps (a) to (e) can be conducted has already been explained above.
[0429] In the method for monitoring minimal residual disease, disorder or abnormality, the method can further comprises steps (i) to (vi) before step (a): [0430] (i) bringing a sample or specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, which compound specifically binds to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0431] (ii) allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex; [0432] (iii) detecting the formation of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex; [0433] (iv) correlating the presence or absence of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; [0434] (v) optionally comparing the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex to a normal control value; and [0435] (vi) treating the patient with the medicament.
[0436] Optionally the method can further comprise step (A) after step (d) or step (e):
(A) comparing the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex determined in step (iv) to the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex determined in step (d).
[0437] In order to monitor minimal residual disease, disorder or abnormality over time, steps (a) to (c) and optionally steps (d) and (e) of the method of monitoring minimal residual disease, disorder or abnormality can be repeated one or more times.
[0438] In the method for monitoring minimal residual disease, disorder or abnormality the amount of the compound/protein aggregate complex can be optionally compared at various points of time during the treatment, for instance, before and after onset of the treatment or at various points of time after the onset of the treatment. A change, especially a decrease, in the amount of the compound/protein aggregate complex may indicate that the residual disease, disorder or abnormality is decreasing.
[0439] Predicting responsiveness of a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites and being treated with a medicament can be achieved by [0440] (a) bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0441] (b) allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/protein aggregate complex; [0442] (c) detecting the formation of the compound/protein aggregate complex; [0443] (d) optionally correlating the presence or absence of the compound/protein aggregate complex with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0444] (e) optionally comparing the amount of the compound/protein aggregate complex to a normal control value.
[0445] How steps (a) to (e) can be conducted has already been explained above.
[0446] In the method for predicting the responsiveness, the method can further comprises steps (i) to (vi) before step (a): [0447] (i) bringing a sample or specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, which compound specifically binds to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0448] (ii) allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex; [0449] (iii) detecting the formation of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex; [0450] (iv) correlating the presence or absence of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; [0451] (v) optionally comparing the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex to a normal control value; and [0452] (vi) treating the patient with the medicament.
[0453] Optionally the method can further comprise step (A) after step (d) or step (e):
(A) comparing the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex determined in step (iv) to the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex determined in step (d).
[0454] In order to determine the responsiveness over time, steps (a) to (c) and optionally steps (d) and (e) of the method of predicting responsiveness can be repeated one or more times.
[0455] In the method for predicting responsiveness the amount of the compound/protein aggregate complex can be optionally compared at various points of time during the treatment, for instance, before and after onset of the treatment or at various points of time after the onset of the treatment. A change, especially a decrease, in the amount of the compound/protein aggregate complex may indicate that the patient has a high potential of being responsive to the respective treatment.
[0456] Optionally, the diagnostic composition can be used before, during and after, surgical procedures (e.g. deep brain stimulation (DBS)) and non-invasive brain stimulation (such as repetitive transcranial magnetic stimulation (rTMS)), for visualizing alpha-synuclein aggregates before, during and after such procedures. Surgical techniques, including DBS, improve advanced symptoms of PD on top of the best currently used medical therapy. During the past 2 decades, rTMS has been closely examined as a possible treatment for PD (Ying-hui Chou et al. JAMA Neurol. 2015 Apr. 1; 72(4): 432-440).
[0457] In a further embodiment of the invention, the diagnostic composition can be used in a method of collecting data for monitoring residual disease, disorder or abnormality in a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites who has been treated with a surgical procedure or non-invasive brain stimulation procedure, wherein the method comprises the steps of: [0458] (a) bringing a sample or specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, which compound specifically binds to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0459] (b) allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex; (c) detecting the formation of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex; [0460] (d) optionally correlating the presence or absence of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex with the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0461] (e) optionally comparing the amount of the compound/(alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites) complex to a normal control value.
[0462] It is understood that the term “monitoring minimal residual disease” as mentioned herein relates to the monitoring of the evolution of the disease. For example, monitoring of the evolution of the disease, disorder or abnormality in a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites.
[0463] A compound according to the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, or its precursor can also be incorporated into a test kit for detecting alpha-synuclein protein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The test kit typically comprises a container holding one or more compounds according to the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, or its precursor(s) and instructions for using the compound for the purpose of binding to alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites to form a compound/protein aggregate complex and detecting the formation of the compound/protein aggregate complex such that presence or absence of the compound/protein aggregate complex correlates with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites.
[0464] The term “test kit” refers in general to any diagnostic kit known in the art. More specifically, the latter term refers to a diagnostic kit as described in Zrein et al., Clin. Diagn. Lab. Immunol., 1998, 5, 45-49.
[0465] The dose of the detectably labelled compounds of the present invention, or stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, preferably compounds of formula (III-F) labelled with .sup.18F, will vary depending on the exact compound to be administered, the weight of the patient, size and type of the sample, and other variables as would be apparent to a physician skilled in the art. Generally, the dose could preferably lie in the range 0.001 μg/kg to 10 μg/kg, preferably 0.01 μg/kg to 1.0 μg/kg. The radioactive dose can be, e.g., 100 to 600 MBq, more preferably 150 to 450 MBq.
[0466] In another embodiment the present invention provides a method of imaging a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in a sample or in a specific body part or body area, in particular in a brain or a sample taken from a patient's brain, the method comprising the steps: [0467] (a) Bringing the sample, the specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0468] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0469] (c) Imaging the sample, the specific body part or the body area with an imaging system.
[0470] In another embodiment the present invention provides a method of determining an amount of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in a sample or a specific body part or body area, the method comprising the steps: [0471] (a) Bringing the sample, the specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0472] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0473] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0474] (d) Determining the amount of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0475] (e) Optionally calculating the amount of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample, the specific body part or body area.
[0476] In another embodiment the present invention provides a method of diagnosing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: [0477] (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0478] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0479] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0480] (d) Correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the disease, disorder or abnormality associated with the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites.
[0481] In another embodiment the present invention provides a method of collecting data for the diagnosis of a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: [0482] (a) Bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0483] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0484] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0485] (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area.
[0486] In another embodiment the present invention provides a method of collecting data for determining a predisposition to a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: [0487] (a) Bringing a sample or a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0488] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0489] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0490] (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area.
[0491] If the amount of the compound bound to the alpha-synuclein aggregates is higher than a normal control value of a healthy/reference subject this indicates that the patient is suffering from or is at risk of developing a disease, disorder or abnormality associated with alpha-synuclein aggregates. In particular, if the amount of the compound bound to the alpha-synuclein aggregates is higher than what expected in a person showing no clinical evidence of neurodegenerative disease, it can be assumed that the patient has a disposition to a disease, disorder or abnormality associated with alpha-synuclein aggregates or a synucleinopathy.
[0492] In another embodiment the present invention provides a method of collecting data for prognosing a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, wherein the method comprises the steps: [0493] (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0494] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0495] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0496] (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0497] (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time.
[0498] The progression of a disease, disorder or abnormality and/or the prospect (e.g., the probability, duration, and/or extent) of recovery can be estimated by a medical practioner based on the presence or absence of the compound bound to the alpha-synuclein aggregates, the amount of the compound bound to the alpha-synuclein aggregates or the like. If desired, steps (a) to (c) and, if present, optional step (d) can be repeated over time to monitor the progression of the disease, disorder or abnormality and to thus allow a more reliable estimate.
[0499] In another embodiment the invention provides a method of collecting data for monitoring the evolution of the disease in a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: [0500] (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0501] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0502] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0503] (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0504] (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time.
[0505] Typically the patient is or has been undergoing treatment of the disease, disorder or abnormality associated with alpha-synuclein aggregates or is/has been undergoing treatment of the synucleinopathy. In particular, the treatment can involve administration of a medicament which is suitable for treating the disease, disorder or abnormality associated with alpha-synuclein aggregates.
[0506] In another embodiment the present invention provides a method of collecting data for monitoring the progression of a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, in a patient, the method comprising the steps: [0507] (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with the compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0508] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0509] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0510] (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0511] (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time.
[0512] Typically, the patient is or has been undergoing treatment of the disease, disorder or abnormality associated with alpha-synuclein aggregates or is or has been undergoing treatment of the synucleinopathy. In particular, the treatment can involve administration of a medicament which is suitable for treating the disease, disorder or abnormality associated with alpha-synuclein aggregates.
[0513] In another embodiment the invention provides a method of collecting data for predicting responsiveness of a patient suffering from a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, to a treatment of the disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, the method comprising the steps: [0514] (a) Bringing a sample, a specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites into contact with a compound of formula (I) or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; [0515] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0516] (c) Detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; [0517] (d) Optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0518] (e) Optionally repeating steps (a) to (c) and, if present, optional step (d) at least one time.
[0519] Typically, the patient is or has been undergoing treatment of the disease, disorder or abnormality associated with alpha-synuclein aggregates or is or has been undergoing treatment of the synucleinopathy. In particular, the treatment can involve administration of a medicament which is suitable for treating the disease, disorder or abnormality associated with alpha-synuclein aggregates.
[0520] If the amount of the compound bound to the alpha-synuclein aggregates decreases over time, it can be assumed that the patient is responsive to the treatment. If the amount of the compound bound to the alpha-synuclein aggregates is essentially constant or increases over time, it can be assumed that the patient is non-responsive to the treatment.
[0521] Alternatively, the responsiveness can be estimated by determining the amount of the compound bound to the alpha-synuclein aggregates. The amount of the compound bound to the alpha-synuclein aggregates can be compared to a control value such as a normal control value, a preclinical control value or a clinical control value. Alternatively, the control value may refer to the control value of subjects known to be responsive to a certain therapy, or the control value may refer to the control value of subjects known to be non-responsive to a certain therapy. The outcome with respect to responsiveness can either be “responsive” to a certain therapy, “non-responsive” to a certain therapy or “response undetermined” to a certain therapy. Response to the therapy may be different for the respective patients.
[0522] In yet another embodiment the present invention provides a method, as defined herein, wherein the step of optionally correlating the presence or absence of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, with the presence or absence of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area comprises [0523] determining in which amount of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy; [0524] correlating the amount of the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites with the amount of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area; and [0525] optionally comparing the amount of the amount of the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the sample or specific body part or body area to a normal control value in a healthy control subject.
[0526] The control value can be, e.g., a normal control value, a preclinical control value and/or a clinical control value.
[0527] A “healthy control subject” or “healthy volunteer (HV) subject” is a person showing no clinical evidence of neurodegenerative disease. The person is selected as defined herein, in section 15 “First in human (FIH) study” of the “biological assay description and corresponding results” paragraph.
[0528] If in any of the above summarized methods the amount of the compound bound with the alpha-synuclein aggregates is higher than the normal control value, then it can be expected that the patient is suffering from or is likely to from a disease, disorder or abnormality associated with alpha-synuclein aggregates or from a synucleinopathy.
[0529] Any of the compounds of the present invention can be used in the above summarized methods. Preferably detectably labeled compounds of the present invention, as disclosed herein, are employed in the above summarized methods.
[0530] The specific body part or body area is preferably of a mammal, more preferably of a human, including the full body or partial body area or body part of the patient suspected to contain alpha-synuclein aggregates.
[0531] The sample can be selected from tissue or body fluids suspected to contain alpha-synuclein aggregates, the sample being obtained from the patient. Preferably, the tissue is selected from brain tissue. Examples of body fluids include cerebrospinal fluid (CSF) or blood. The sample can be obtained from a mammal, more preferably a human. Preferably, the sample is an in vitro sample from a patient.
[0532] In an in vivo method, the specific body part or body area can be brought into contact with a compound of the invention by administering an effective amount of a compound of the invention to the patient. The effective amount of a compound of the invention is an amount which is suitable for allowing the presence or absence of alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites in the specific body part or body area to be determined using the chosen analytical technique.
[0533] The step of allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites includes allowing sufficient time for the compound of the invention to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The amount of time required for binding will depend on the type of test (e.g., in vitro or in vivo) and can be determined by a person skilled in the field by routine experiments. In an in vivo method, the amount of time will depend on the time which is required for the compound to reach the specific body part or body area suspected to contain alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. The amount of time should not be too extended to avoid washout and/or metabolism of the compound of the invention.
[0534] The method of detecting the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites is not particularly limited and depends, among others, on the detectable label, the type of sample, specific body part or body area and whether the method is an in vitro or in vivo method. Possible detection methods include, but are not limited to a fluorescence imaging technique or a nuclear imaging technique such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and contrast-enhanced magnetic resonance imaging (MRI). The fluorescence imaging technique and/or nuclear imaging technique can be employed for monitoring and/or visualizing the distribution of the compound of the invention within the sample or the body. The imaging system is such to provide an image of bound detectable label such as radioisotopes, in particular positron emitters or gamma emitters, as present in the tested sample, the tested specific body part or the tested body area. Preferably, the compound bound to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites is detected by an imaging apparatus such as PET or SPECT scanner.
[0535] The amount of the compound bound with the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites can be determined by the visual or quantitative analysis, for example, using PET scan images.
[0536] In any of the above methods, steps (a) to (c) and, if present, optional step (d) can be repeated at least one time. The repetition of the steps is particularly useful in the method of collecting data for prognosing, the method of collecting data for monitoring the evolution of the disease, the method of collecting data for monitoring the progression and the method of collecting data for predicting responsiveness. In these methods, it may be expedient to monitor the patient over time and to repeat the above steps after a certain period of time has elapsed. The time interval before the above mentioned steps are repeated can be determined by a physician depending on the severity of the disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites or the synucleinopathy.
[0537] In a further aspect, the present invention refers to a method of imaging a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, in a subject, the method comprising the steps: [0538] (a) Administering a compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the subject; [0539] (b) Allowing the compound to bind to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites; and [0540] (c) Detecting the compound bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites.
[0541] In a further aspect, the present invention is directed to a method of imaging a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, in a subject, the method comprising the steps: [0542] (a) Administering a compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the subject; and [0543] (b) Imaging the brain of the subject.
[0544] The brain of the subject should be imaged when the compound has bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites. The compound bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites, can then be imaged in the subject's brain.
[0545] In a further aspect, the present invention refers to a method of positron emission tomography (PET) imaging of alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, in a tissue of a subject, the method comprising the steps: [0546] (a) Administering a compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the subject; [0547] (b) Allowing the compound to penetrate into the tissue of the subject; and [0548] (c) Collecting a positron emission tomography (PET) image of the tissue of the subject; wherein the tissue is tissue of the central nervous system (CNS), of the eye or brain tissue, preferably wherein the tissue is brain tissue.
[0549] The PET imaging should be conducted when the compound has penetrate into the tissue and the compound has bound to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites.
[0550] In a further aspect, the present invention is directed a method of detecting a neurological disease, disorder or abnormality associated with alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, in a subject, the method comprising the steps: [0551] (a) Administering a compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the subject; [0552] (b) Allowing the compound to bind to the alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites; and [0553] (c) Measuring the radioactive signal of the compound, which is bound to the alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites.
[0554] The radioactive signal, as mentioned herein, is observed when a detectably labelled compound of the invention, which comprises at least one radiolabelled atom (e.g. .sup.3H, .sup.2H, or .sup.18F), is bound to the alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites.
[0555] In a further aspect, the present invention is directed to a method (e.g., an in vivo or in vitro method) for the detection and/or quantification of alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, in a tissue of a subject, the method comprising the steps: [0556] (a) Contacting the tissue with a compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the subject; [0557] (b) Allowing the compound to bind to the alpha-synuclein aggregates, including, but not limited to, Lewy bodies and/or Lewy neurites; and [0558] (c) Detecting and/or quantifying the compound bound to the alpha-synuclein aggregates, including but not limited to, Lewy bodies and/or Lewy neurites, using positron emission tomography.
[0559] In yet another aspect, the present invention refers to a method of the diagnostic imaging of the brain of a subject, the method comprising the steps: [0560] (a) Administering a compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the subject; and [0561] (b) Obtaining an image of the brain of the subject using positron emission tomography.
[0562] In the methods of the present invention, the compound of the formula (I), or subformulae thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof is typically administered in a detectable amount, i.e., an amount which can be detected by the device which is employed in for detecting the compound in the respective method. The amount is not particularly limited and will depend on the compound of the formula (I), the type of detectable label, the sensitivity of the respective analytical method and the respective device. The amount can be chosen appropriately by a skilled person.
Radiopharmaceutical Preparations
[0563] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, preferably compounds of formula (I), or of subformulae thereof (e.g (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)), can also be employed in kits for the preparation of radiopharmaceutical preparations. Due to the radioactive decay, the radiopharmaceuticals are usually prepared immediately before use. The kit typically comprises a precursor of the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and an agent which reacts with the precursor to introduce a radioactive label into the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof. The precursor of the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can, for example, be a compound having the formula (IV-F), (IV-H), or (IV-J). The agent can be an agent which introduces a radioactive label such as .sup.18F, or .sup.3H.
Pharmaceutical Compositions
[0564] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, can be employed in treating, preventing or alleviating a disease, disorder or abnormality associated with alpha-synuclein aggregates.
[0565] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, preferably compounds of formula (I), are suitable for treating, preventing or alleviating a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies and/or Lewy neurites. Diseases involving alpha-synuclein aggregates are generally listed as synucleinopathies (or α-synucleinopathies). The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, are suitable for treating, preventing or alleviating diseases, disorders or abnormalities including, but not limited to, Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), SNCA duplication carrier, dementia with Lewy bodies (“pure” Lewy body dementia), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease and normal aging in Down syndrome). Synucleinopathies with neuronal and glial aggregates of alpha synuclein include multiple system atrophy (MSA) (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy). Other diseases that may have alpha-synuclein-immunoreactive lesions include traumatic brain injury, chronic traumatic encephalopathy, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick type C1 disease), motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder. (Jellinger, Mov Disord 2003, 18 Suppl. 6, S2-12; Galvin et al. JAMA Neurology 2001, 58 (2), 186-190; Kovari et al., Acta Neuropathol. 2007, 114(3), 295-8; Saito et al., J Neuropathol Exp Neurol. 2004, 63(4), 323-328; McKee et al., Brain, 2013, 136(Pt 1), 43-64; Puschmann et al., Parkinsonism Relat Disord 2012, 18S1, S24-S27; Usenovic et al., J Neurosci. 2012, 32(12), 4240-424 6; Winder-Rhodes et al., Mov Disord. 2012, 27(2), 312-315; Ferman et al., J Int Neuropsychol Soc. 2002, 8(7), 907-914). Preferably, the compounds of the present invention are suitable for treating, preventing or alleviating Parkinson's disease (PD).
[0566] In pharmaceutical applications, the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, is preferably administered in a pharmaceutical composition comprising the compound of the invention. A “pharmaceutical composition” is defined in the present invention as a composition comprising one or more compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, in a form suitable for administration to a patient, e.g., a mammal such as a human, and which is suitable for treating, alleviating or preventing the specific disease, disorder or abnormality at issue. Preferably a pharmaceutical composition further comprises a physiologically acceptable carrier, diluent, adjuvant or excipient. The dose of the compound of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, will vary depending on the exact compound to be administered, the weight of the patient, and other variables as would be apparent to a physician skilled in the art.
[0567] While it is possible for the compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, to be administered alone, it is preferable to formulate them into a pharmaceutical composition in accordance with standard pharmaceutical practice. Thus, the invention also provides a pharmaceutical composition which comprises a therapeutically effective amount of a compound of formula (I), or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, in admixture with, optionally, at least one pharmaceutically acceptable excipient, carrier, diluent or adjuvant.
[0568] Pharmaceutically acceptable excipients are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 15.sup.th Ed., Mack Publishing Co., New Jersey (1975). The pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof.
[0569] Pharmaceutically useful excipients that may be used in the formulation of the pharmaceutical composition of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, may comprise, for example, carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate, binders, adjuvants, solubilizers, thickening agents, stabilizers, disintegrants, glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colorants, flavors, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-ß-cyclodextrin, polyvinylpyrrolidone, low melting waxes, and ion exchange resins.
[0570] The compounds of the present invention, or a detectably labelled compound, stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and their precursors can be synthesized by one of the general methods shown in the following schemes. These methods are only given for illustrative purposes and should not to be construed as limiting.
TABLE-US-00001 Abbreviation Meaning DMFDMA N,N-dimethylformamide dimethyl acetal SNAr nucleophilic aromatic substitution CsF cesium fluoride DMSO dimethylsulfoxide NBS N-bromosuccinimide LG Leaving Group WFI water for injection HPLC High Performance Liquid Chromatography SPE Solid Phase Extraction
General Synthetic Scheme for the Preparation of Compounds and Precursors of this Invention:
##STR00183## ##STR00184##
Commercially available hydrazine can be condensed with the appropriate ketone to afford the corresponding hydrazone. The crude hydrazone can be subjected to ring cyclization using DMF/DMA to give intermediate A. SNAr can be conducted with a suitable nucleophile in a suitable solvent and base to give intermediate B. Alternatively, thermal conditions can be applied without metal catalyst. Deprotection with suitable conditions can afford intermediate C. Finally, intermediate C can be further functionalized using palladium catalyzed amidation or Ullmann reaction to give compounds of formula (I), or of subformulaes thereof (e.g. (IIa), (IIb), (IIIa), (IIIb), (IIIc), (III-F), (III-H)). In this example the starting materials comprise R.sup.0 is H. The above general scheme applies to starting material wherein R.sup.0 is C.sub.1-C.sub.4alkyl.
##STR00185## ##STR00186##
[0571] An alternative approach (Scheme 1A) comprises deprotecting intermediate A, followed by SNAr reaction with a suitable nucleophile which is preferably conducted in the presence of CsF in DMSO. Intermediates C and D can be further functionalized, preferably using copper (I) (Ullmann reaction) in the presence of a base and solvent, to afford formula (IIIa) and intermediate E. Finally, LG can be introduced into intermediate E to give formula (IV-F). In this example the starting materials comprise R.sup.0 is H. The above general scheme applies to starting material wherein R.sup.0 is C.sub.1-C.sub.4alkyl.
##STR00187##
[0572] A general approach is depicted in scheme 1B following the same preferred conditions as described in the general scheme 1 or 1A
[0573] A .sup.18F-precursor can be obtained by treating intermediate A with hydroxypyrrolidine under heating in a suitable solvent. The R.sup.4 group can be introduced by palladium catalyzed amidation or Ullmann reaction. Ultimately, an alcohol intermediate E can be modified into a leaving group using standard conditions to give a compound of formula (IV-F).
[0574] The .sup.3H-precursor can be obtained by introducing an appropriate R.sup.4 group by palladium catalyzed amidation or Ullmann reaction into an intermediate C. Finally, halogenation of pyridine using, for example, NBS in a suitable solvent can give a compound of formula (IV-H).
General Synthesis of .SUP.18.F-Labelled Compounds of the Present Invention
[0575] Compounds having the formula (I) which are labelled by .sup.18F can be prepared by reacting a precursor compound, as described below, with an .sup.18F-fluorinating agent, so that the LG comprised in the precursor compound is replaced by .sup.18F.
[0576] The reagents, solvents and conditions which can be used for the .sup.18F-fluorination are well-known to a skilled person in the field (L. Cai, S. Lu, V. Pike, Eur. J. Org. Chem 2008, 2853-2873; J. Fluorine Chem., 27 (1985):177-191; Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), in: Schubiger P. A., Friebe M., Lehmann L., (eds), PET-Chemistry—The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 15-50). Preferably, the solvents used in the .sup.18F-fluorination are DMF, DMSO, acetonitrile, DMA, or mixtures thereof, preferably the solvent is acetonitrile or DMSO.
[0577] Any suitable .sup.18F-fluorinating agent can be employed. Typical examples include H.sup.18F, alkali or alkaline earth .sup.18F-fluorides (e.g., K.sup.18F, Rb.sup.18F, Cs.sup.18F, and Na.sup.18F). Optionally, the .sup.18F-fluorination agent can be used in combination with a chelating agent such as a cryptand (e.g.: 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane—Kryptofix®) or a crown ether (e.g.: 18-crown-6). Alternatively, the .sup.18F-fluorinating agent can be a tetraalkylammonium salt of .sup.18F or a tetraalkylphosphonium salt of .sup.18F; e.g., tetra(C.sub.1-6 alkyl)ammonium salt of .sup.18F or a tetra(C.sub.1-6 alkyl)phosphonium salt of .sup.18F. Preferably, the .sup.18F-fluorination agent is K.sup.18F, H.sup.18F, Cs.sup.18F, Na.sup.18F tetra(C.sub.1-6 alkyl) ammonium salt of .sup.18F, kryptofix[222].sup.18F or tetrabutylammonium [.sup.18F]fluoride.
[0578] Although the reaction is shown above with respect to .sup.18F as a radioactive label, other radioactive labels can be introduced following similar procedures.
[0579] The invention is illustrated by the following examples which, however, should not be construed as limiting.
EXAMPLES
[0580] All reagents and solvents were obtained from commercial sources and used without further purification. Proton (.sup.1H) spectra were recorded on a Bruker DRX-400 MHz NMR spectrometer, on a Bruker AV-400 MHz NMR spectrometer or Spinsolve 80 MHz NMR spectrometer in deuterated solvents. Mass spectra (MS) were recorded on an Advion CMS mass spectrometer or an UPLC H-Class Plus with Photodiode Array detector and Qda Mass spectrometer from Waters. Chromatography was performed using silica gel (Fluka: Silica gel 60, 0.063-0.2 mm) and suitable solvents as indicated in the specific examples. Flash purification was conducted with a Biotage Isolera One flash purification system using HP-Sil or KP-NH SNAP cartridges (Biotage) and the solvent gradient indicated in the specific examples. Thin layer chromatography (TLC) was carried out on silica gel plates with UV detection.
Preparative Example 1
[0581] ##STR00188##
Step A:
[0582] A suspension of 2-bromo-5-hydrazinylpyridine (3.21 g, 17.07 mmol) and tert-butyl 2,4-dioxopyrrolidine-1-carboxylate (3.40 g, 17.07 mmol) in ethanol (150 mL) was refluxed for 3 h and monitored by TLC. The crude product was concentrated under reduced pressure and diluted with dichloromethane and water. The layers were separated and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (Silica, 50 g column, 60-80% ethyl acetate in heptane) to afford (E)-tert-butyl 4-(2-(6-bromopyridin-3-yl)hydrazono)-2-oxopyrrolidine-1-carboxylate as a brown solid (4.97 g, 79%). .sup.1H NMR (400 MHz, DMSO-d6) δ=9.22 (s, 1H), 8.41 (s, 1H), 7.89 (d, 1H), 7.40 (d, 1H), 7.11 (dd, 1H), 4.57 (s, 1H), 4.30 (s, 2H), 1.45 (s, 9H). MS: 369.06 [M+H].sup.+
Step B:
[0583] The compound from step A (3.9 g, 10.56 mmol) was stirred in 1,1-dimethoxy-N,N-dimethylmethanamine (80 mL) at 50° C. for 3 h 15 min. The reaction mixture was concentrated to ˜10 mL and ethanol was added. The solid was filtered and washed with small portions of ethanol to afford tert-butyl 2-(6-bromopyridin-3-yl)-4-oxo-4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate as a light brown powder (2.30 g, 57%). .sup.1H NMR (400 MHz, DMSO-d6) δ=9.20 (s, 1H), 9.00 (d, 1H), 8.28 (dd, 1H), 7.89 (d, 1H), 4.84 (s, 2H), 1.53 (s, 9H). MS: 324.83 [M-tBu.sup.+H].sup.+
Preparative Examples 1A to 1H
[0584] Following the procedure as described in preparative example 1, using 1,1-dimethoxy-N,N-dimethylmethanamine or N,N-dimethylacetamide dimethyl acetal and the appropriate hydrazone, the following preparative examples were prepared.
TABLE-US-00002 Preparative 1. .sup.1H-NMR hydrazone SM Example 2. MH.sup.+ (ESI)
Preparative Example A
[0585] ##STR00213##
[0586] Preparative Example 1 (1000 mg, 2.64 mmol) was stirred in 4 M HCl in dioxane (37 mL) at room temperature for 1 h 45 min. The solvent was evaporated under reduced pressure and the solid dissolved in dichloromethane. A solution of saturated NaHCO.sub.3 was added, and the aqueous phase was extracted twice with dichloromethane. The combined organic layers were filtrated to afford 2-(6-bromopyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one as a beige solid. (682 mg, 93%). .sup.1H NMR (80 MHz, DMSO-d6) δ 8.96 (d, 2H), 8.37-8.14 (m, 2H), 7.83 (d, 1H), 4.39 (s, 2H). MS: 280.95 [M+H].sup.+
Preparative Examples A1 to A6
[0587] Following the procedure as described in preparative example A, the following preparative examples were prepared.
TABLE-US-00003 Preparative 1. .sup.1H-NMR SM Example 2. MH.sup.+ (ESI)
Preparative Example 2
[0588] ##STR00226##
[0589] In a flask under argon, palladium (II) acetate (41.4 mg, 0.185 mmol) and xantphos (320 mg, 0.554 mmol) were mixed in 1,4-dioxane (18 mL) and heated at 100° C. for a few seconds on a pre-heated block to form the pd-xantphos complex. (R)-3-Fluoropyrrolidine hydrochloride (348 mg, 2.77 mmol), cesium carbonate (1804 mg, 5.54 mmol) and preparative example 1 (700 mg, 1.846 mmol) were added. The flask was degassed and filled with argon three times and the reaction mixture was heated at 120° C. for 30 min. The reaction mixture was cooled at room temperature and the residue was taken up with ethyl acetate and water. The phases were separated and the aqueous phase was extracted twice. The organic layers were combined, dried over Na.sub.2SO.sub.4 and evaporated. The product was purified by flash chromatography (Silica, Silica 25 g column, 0-60% ethyl acetate in dichloromethane) to afford (R)-tert-butyl 2-(6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-4-oxo-4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate as a white solid (200.5 mg, 28%). .sup.1H NMR (400 MHz, DMSO-d6) δ=8.92 (s, 1H), 8.60 (d, 1H), 8.01 (dd, 1H), 6.67 (d, 1H), 5.46 (d, 1H), 4.80 (s, 2H), 3.86-3.57 (m, 2H), 3.54-3.44 (m, 2H), 2.36-2.12 (m, 2H), 1.53 (s, 9H). MS: 388.15 [M+H].sup.+
Preparative Examples 3 to 3D
[0590] Following the Pd-coupling procedure as described in preparative example 2, using the halogenated starting material and the appropriate amine indicated in Table 1a below, the following preparative example was prepared.
TABLE-US-00004 TABLE 1a 1. Yield Halogenated starting Preparative 2. .sup.1H-NMR material Amine Example 3. MH.sup.+ (ESI)
Preparative Example 4
[0591] ##STR00242##
[0592] In a microwave vial, preparative example 1 (250 mg, 0.659 mmol) and (S)-pyrrolidin-3-ol (172 mg, 1.978 mmol) were mixed in ethanol (10 mL). The vial was irradiated at 150° C. for 30 minutes in the microwave. (S)-Pyrrolidin-3-ol (172 mg, 1.978 mmol) was again added and the reaction mixture was irradiated once again at 150° C. for 45 minutes. The reaction mixture was filtered and washed with ethanol to afford (S)-2-(6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one as a white solid (83.5 mg, 44.4%). .sup.1H NMR (80 MHz, DMSO-d6) δ=8.60 (s, 1H), 8.51 (d, 1H), 8.07 (s, 1H), 7.92 (dd, 1H), 6.56 (d, 1H), 4.97 (d, 1H), 4.34 (s, 3H), 3.69-3.37 (m, 4H), 2.24-1.80 (m, 2H). MS: 286.05 [M+H].sup.+
Preparative Example 5
[0593] ##STR00243##
[0594] Preparative example 2 (160 mg, 0.413 mmol) was stirred in 4 M HCl in dioxane (10 mL) at RT for 3 h30. The solvent was evaporated under reduced pressure and the solid was dissolved in dichloromethane. A solution of saturated NaHCO.sub.3 was added, and the aqueous phase extracted twice with dichloromethane. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness to afford (R)-2-(6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one as a white solid (101.5 mg, 86%). .sup.1H NMR (80 MHz, DMSO-d6) δ=8.62 (s, 1H), 8.55 (d, 1H), 7.96 (dd, 1H), 6.63 (d, 1H), 5.75 (s, 1H), 4.34 (s, 2H), 3.92-3.37 (m, 4H), 2.45-1.78 (m, 2H). MS: 287.80 [M+H]+
Alternative Preparative Example 5
[0595] ##STR00244##
[0596] In a vial under argon, Preparative Example A (400 mg, 0.1.433 mmol), (R)-3-fluoropyrrolidine hydrochloride (720 mg, 5.73 mmol), and cesium fluoride (1306 mg, 8.60 mmol) were mixed in dry DMSO (4 mL). The reaction mixture was flushed with argon and stirred at 120° C. for 6 h 30 min. The reaction mixture was cooled down and poured into cold water (pre-cooled in an ice bath). The resulting solution was filtered, and the solid was rinsed with water. 1 mL of isopropanol was used to triturate the solid directly in the fritte, and the solid was dried to afford the product as a beige solid (287 mg, 0.998 mmol, 70%). .sup.1H NMR (80 MHz, DMSO-d6) 8.63 (s, 1H), 8.55 (d, 1H), 8.15-7.79 (m, 2H), 6.64 (d, 1H), 5.46 (d, 1H), 4.34 (s, 2H), 3.96-3.40 (m, 4H), 2.28-1.56 (m, 2H). MS: 288.11 [M+H].sup.+
Alternative Preparative Examples 4 to 4K
[0597] Following the SNAr procedure as described in alternative preparative example 5, using the appropriate amine indicated in Table 1 b below, the following preparative examples were prepared.
TABLE-US-00005 TABLE 1b 1. Yield Halogenated starting Preparative 2. .sup.1H-NMR material Amine Example 3. MH.sup.+ (ESI)
Preparative Examples 6 to 6D
[0598] Following the deprotection procedure of preparative example 5, the following preparative examples were prepared.
TABLE-US-00006 TABLE 2 1. Yield 2. .sup.1H-NMR Starting material Preparative Example 3. MH.sup.+ (ESI)
Preparative Example 7
[0599] ##STR00291##
[0600] In a vial under argon, palladium (II) acetate (13.14 mg, 0.059 mmol) and xantphos (50.8 mg, 0.088 mmol) were mixed in 1,4-dioxane (3 mL), degassed with argon and heated at 100° C. for a few seconds on a pre-heated block to form the pd-xantphos complex. Then, preparative example 4 (83.5 mg, 0.293 mmol), 3-iodopyridine (66.0 mg, 0.322 mmol) and cesium carbonate (286 mg, 0.878 mmol) were added, the mixture was degassed with argon and heated at 100° C. for 45 min. The reaction mixture was filtered and washed with ethyl acetate. The filtrate was recovered, and evaporated to obtain the product as a yellow gum-solid (134.5 mg, 0.371 mmol, quantitative). .sup.1H NMR (80 MHz, DMSO-d6) δ=9.04 (d, 1H), 8.83 (s, 1H), 8.57 (d, 1H), 8.43-8.13 (m, 2H), 7.96 (dd, 1H), 7.44 (dd, 1H), 6.58 (d, 1H), 5.08 (s, 2H), 4.99 (d, 1H), 4.42 (d, 1H), 3.64-3.40 (m, 4H), 2.17-1.75 (m, 2H). MS: 363.08 [M+H].sup.+
Preparative Example 8
[0601] Following the Pd-coupling procedure as described in preparative example 7, using the amide starting material and the appropriate halogenated heteroaryl indicated in Table 3 below, the following preparative example was prepared.
TABLE-US-00007 TABLE 3 1. Yield halogenated 2. .sup.1H-NMR Amide heteroaryl Preparative Example 3. MH.sup.+ (ESI)
Preparative Example 9
[0602] ##STR00295##
Step A
[0603] Under argon atmosphere to a solution of 2-(6-bromopyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (0.5 g, 1.79 mol) in dioxane (20 ml) was added 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (0.451 g, 2.7 mmol), [(dppf)PdCl2] (146 mg, 0.179 mmol) and Cs.sub.2CO.sub.3 (1.16 g, 3.58 mmol) in H.sub.2O (0.2 ml). The mixture was heated at 80° C. for 2 h. The mixture was cooled, the solvent was evaporated under a high vacuum. The residue was dissolved in ethyl acetate and solid filtered. The filter residue was washed with water, and dried to obtain product 0.450 g. MS: 241.1 [M+H].sup.+
Step B
[0604] To a solution of 2-(6-(prop-1-en-2-yl)pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (1 g, 4.16 mmol) in MeOH (75 mL) was added Pd/C (100 mg, 5%). The mixture was stirred at room temperature for 12 hours under H.sub.2 (15 psi). Upon completion, the reaction slurry was filtered and the filtrate was concentrated to give 2-(6-(propan-2-yl)pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (0.85 g). MS: 243.2 [M+H].sup.+
Preparative Example 10
[0605] Following the procedure as described in alternative preparative example 9, using the appropriate boronic ester indicated in Table 3b below, the following preparative example was prepared.
TABLE-US-00008 TABLE 3b Halogenated starting Boronic material ester Preparative Example MH.sup.+ (ESI)
Preparative Example 11
[0606] ##STR00299##
[0607] 2-Propanol (50 μL, 0.7176 mmol) in 0.4 mL DMF was added to a suspension of sodium hydride (36 mg/60% in mineral oil, 0.9 mmol) in 2 mL DMF at RT. The mixture was stirred for 30 minutes, then added to a stirred solution of 2-(6-bromopyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (100 mg, 0.358 mmol) in 2 mL DMF at 60° C. The reaction mixture was heated at 60° C. for 20 hours. After cooling to RT, water and ethyl acetate were added and the layers were separated. The aqueous layer was extracted with ethyl acetate and the organics were combined, dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to provide 2-(6-isopropoxypyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (0.16 g, 35%): MS: 259.2 [M+H].sup.+
Preparative Example 12
[0608] ##STR00300##
[0609] In a seal tube under nitrogen, (R)-2-(6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (120 mg, 0.417 mmol), 2-bromo-5-((2-(trimethylsilyl)ethoxy)methoxy)pyridine (253 mg, 0.835 mmol), copper(I)-iodide (16 mg, 0.0835 mmol) and potassium carbonate (115 mg, 0.835 mmol) were charged and the system was flushed with nitrogen. 1,4-Dioxane (6 mL) and N,N′-dimethylethylenediamine (0.017 mL, 0.167 mmol) were added and the mixture was stirred at 100° C. for 4 h. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in 10 ml of water, and extracted with DCM/MeOH (9:1, 50 ml×2). The combined organic layers were dried over NaSO.sub.4 (5 g), filtered, and concentrated to obtain 80 mg of a pale yellow solid crude. The crude was purified by column chromatography on basic silica gel (100-200 mesh) using a dichloromethane/methanol gradient (100/0.fwdarw.98/2) to afford the desired product as a pale yellow solid (50 mg, 23% yield). .sup.1H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.60 (d, 1H), 8.42-8.33 (m, 1H), 8.18 (dd, 1H), 8.01 (dd, 1H), 7.58 (dd, 1H), 6.66 (d, 1H), 5.54 (s, 1H), 5.28 (s, 2H), 5.05 (s, 2H), 3.85-3.54 (m, 5H), 3.54-3.42 (m, 1H), 2.39-2.08 (m, 2H), 0.90 (dd, 2H), −0.01 (s, 9H). MS: 511.3 [M+H].sup.+
Preparative Examples 13 to 31
[0610] Following the Cu-coupling procedure as described in preparative example 12, using the amide starting material and the appropriate halogenated heteroaryl indicated in Table 3c below, the following preparative examples were prepared.
TABLE-US-00009 TABLE 3c 1. Yield halogenated 2. .sup.1H-NMR Amide heteroaryl Preparative Example 3. MH.sup.+ (ESI)
Examples 1 to 4
[0611] Following the Pd-coupling procedure as described in preparative example 7, using the amide starting material and the appropriate halogenated heteroaryl indicated in the Table 4 below, the following compounds were prepared.
TABLE-US-00010 TABLE 4 1. Yield halogenated Compound of 2. .sup.1H-NMR Amide heteroaryl example 3. MH.sup.+ (ESI)
Alternative Example 1
[0612] ##STR00370##
[0613] In a flask under argon, Preparative Example 5 (285 mg, 0.992 mmol), 3-bromopyridine (0.191 mL, 1.984 mmol), potassium carbonate (274 mg, 1.984 mmol) and copper(I) iodide (37.8 mg, 0.198 mmol) were mixed and the system was flushed with argon. Dioxane (12 mL) and N1,N2-dimethylethane-1,2-diamine (0.042 mL, 0.397 mmol) were added and the mixture was stirred at 110° C. for 4 h. The crude was concentrated under reduced pressure and dissolved in 20 mL of water. Aqueous ammonia (16.30 mL, 114 mmol) was added until the solution was basic (pH 12). The aqueous layer was extracted twice with a solution of DCM/MeOH (9:1). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The solid was suspended in DCM and stirred at 40° C. for 15 minutes. The mixture was cooled down and filtered to afford the product as a white solid (234.3 mg, 65%). .sup.1H NMR (80 MHz, DMSO-d6) δ 9.03 (d, 1H), 8.86 (s, 1H), 8.60 (d, 1H), 8.42-8.15 (m, 2H), 8.01 (dd, 1H), 7.45 (dd, 1H), 6.67 (d, 1H), 5.41 (d, 1H), 5.09 (s, 2H), 4.00-3.37 (m, 4H), 2.28-1.48 (m, 2H). MS: 365.12 [M+H].sup.+
Examples 5 to 138
[0614] Following the procedures as described in preparative example 7, Alternative Example 1 or using the amide starting material and the appropriate halogenated heteroaryl indicated in the Table 4a below, the following Examples were prepared. Alternatively, Pd.sub.2(dba).sub.3, BINAP and Cs.sub.2CO.sub.3 conditions could be applied.
TABLE-US-00011 TABLE 4a 1. Yield halogenated 2. .sup.1H-NMR Amide heteroaryl Compound of example 3. MH.sup.+ (ESI)
Example 139
[0615] ##STR00773##
[0616] A suspension of 2-(6-(pyrrolidin-1-yl)pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (0.08 g, 0.286 mmol), 2,6-difluoropyrazine (0.199 g, 1.72 mmol) and CsF (0.348 g, 2.293 mmol) in DMSO (4 mL) was heated at 130° C. for 30 minutes under microwave irradiation. Then, the reaction mixture was cooled down and poured into ice cold water (3 mL). The resulting slurry was filtered and the solid was rinsed with water (5 mL). The residue was purified by silica-gel (100-200 mesh) column chromatography using 2 to 5% MeOH in DCM to give the desired product (32 mg, 29%). .sup.1H-NMR (400 MHz, DMSO-d6) δ 9.65-9.59 (m, 1H), 8.96 (s, 1H), 8.60-8.55 (m, 1H), 8.38 (dd, 1H), 7.97 (dd, 1H), 6.59 (d, 1H), 5.03 (s, 2H), 3.46-3.41 (m, 4H), 2.01-1.93 (m, 4H). MS: 366.1 [M+H].sup.+
Examples 140 to 161
[0617] Following the procedures as described in Example 54, using the amide starting material and the appropriate amide and fluoro-heteroaryl indicated in the Table 4b below, the following Examples were prepared.
TABLE-US-00012 TABLE 4b 1. Yield halogenated 2..sup.1H-NMR Amide heteroaryl Compound of example 3. MH.sup.+ (ESI)
Example 162
[0618] ##STR00840##
[0619] (R)-2-(6-(3-Fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(5-((2-(trimethylsilyl)ethoxy)methoxy)pyridin-2-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (50 mg, 0.098 mmol) was dissolved in DCM (1.5 mL) and cooled to 0° C. in an ice-bath with stirring. 4M HCl in 1,4-dioxane (0.2 mL) was added to the solution and stirring was continued at RT for 4 h. After completion of the reaction, the solvent was removed under reduced pressure. The residue obtained was dissolved in ice cold water and basified with aq. sat. sodium bicarbonate solution to pH 8-9. The compound was precipitated, and the solids were removed by filtration. The solid was washed with pentane (3 mL) and further dried on high vacuum for 30 mins to afford the desired compound as pale yellow solid (10 mg, 27%). .sup.1H NMR (500 MHz, CF3COOD) δ 8.85 (s, 1H), 8.72 (d, 1H), 8.57 (dd, 1H), 8.52-8.32 (m, 2H), 7.90 (d, 1H), 7.46 (s, 1H), 5.86-5.62 (m, 1H), 5.53 (s, 2H), 4.52-4.01 (m, 4H), 2.87 (s, 1H), 2.75-2.44 (m, 1H). MS: 381.1 [M+H].sup.+
Examples 163 to 181
[0620] Following the deprotection procedure as described example 162, using the O-protected starting material indicated in the Table 4c below, the following examples were prepared.
TABLE-US-00013 TABLE 4c 1.Yield Compound of 2. .sup.1H-NMR Preparative Example example 3. MH.sup.+ (ESI)
Precursor 1
[0621] ##STR00879##
[0622] In a flask under argon, preparative example 7 (135 mg, 0.373 mmol) was dissolved in dichloromethane. Triethylamine (1.038 ml, 7.45 mmol) was added and the reaction mixture was stirred for 5 minutes. Then methanesulfonyl chloride (0.290 ml, 3.73 mmol) was added dropwise to the reaction mixture. The mixture was stirred at room temperature for 20 min. Methanesulfonyl chloride (0.290 ml, 3.73 mmol) was added and the reaction mixture was stirred for 25 min. The reaction mixture was quenched with a 1N aqueous solution of NaOH and then extracted three times with dichloromethane. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The product was purified by flash chromatography (Silica, Silica 12 g column; 0-10% methanol in dichloromethane) to afford (S)-1-(5-(4-oxo-5-(pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-2(4H)-yl)pyridin-2-yl)pyrrolidin-3-yl methanesulfonate as a white solid (17.4 mg, 11%). .sup.1H NMR (80 MHz, DMSO-d6) δ=9.03 (d, 1H), 8.87 (s, 1H), 8.61 (d, 1H), 8.44-8.15 (m, 2H), 8.03 (dd, 1H), 7.45 (q, 1H), 6.68 (d, 1H), 5.45 (s, 1H), 5.09 (s, 2H), 3.67 (d, 4H), 3.27 (s, 3H), 2.41-2.08 (m, 2H). MS: 441.08 [M+H].sup.+
Alternative Procedure
[0623] In a vial under argon and cooled to 0° C., (S)-2-(6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (100 mg, 0.276 mmol), and 4-dimethylaminopyridine (337 mg, 2.76 mmol) were mixed in pyridine (17 mL). Mesyl-Cl (0.108 mL, 1.380 mmol) was added and the mixture was flushed with argon. The reaction mixture was allowed to warm up to RT and was stirred for 2 h, after this time 4-dimethylaminopyridine (169 mg, 1.380 mmol) and Mesyl-Cl (0.054 mL, 0.690 mmol) were added at 0° C. After 40 min, 0.1 N NaOH in water (20 mL) was added to the mixture to basify it. The solution was poured into cold water and filtered. It was washed with water until the pH of the water was 7. The solid was dried under high vacuum for 30 min to afford the compound as an orange solid (86 mg, 71%). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.03 (d, 1H), 8.87 (s, 1H), 8.61 (d, 1H), 8.35 (d, 1H), 8.26 (d, 1H), 8.02 (dd, 1H), 7.45 (dd, 1H), 6.68 (d, 1H), 5.44 (s, 1H), 5.08 (s, 2H), 3.86-3.42 (m, 4H), 3.27 (s, 3H), 2.40-2.24 (m, 2H). MS: 441.1 [M+H].sup.+
Precursor 2
[0624] ##STR00880##
[0625] N-Bromosuccinimide (22 mg, 0.126 mmol) was added to a solution of preparative example 8 (43 mg, 0.097 mmol) in dimethylformamide (3 mL). After stirring for 1 h at room temperature, the reaction mixture was then diluted with water and ethyl acetate. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was triturated in acetonitrile and the solid was collected by filtration. The crude solid was then purified by flash chromatography (Silica, Silica 12 g column; 2-5% methanol in dichloromethane). The fractions were concentrated under reduced pressure and the residue was triturated in acetonitrile. The solid was collected by filtration to afford (R)-2-(5-bromo-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(5-bromopyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one as a beige solid (16 mg, 32%). .sup.1H-NMR δ 8.88 (d, 1H), 8.83-8.70 (m, 1H), 8.51-8.32 (m, 2H), 8.22-7.83 (m, 2H), 5.75-4.77 (m, 3H), 4.32-3.72 (m, 4H), 0.98-0.67 (m, 2H). MS: 523.10 [M+H].sup.+
Precursor 3
[0626] ##STR00881##
[0627] To a solution of preparative example 7 (70 mg, 0.193 mmol) in DCM (3.5 mL) at RT was added triethylamine (0.08 mL, 0.5797 mmol) under N.sub.2 atm. The reaction mixture was cooled to 0° C. then p-toluenesulfonyl chloride (73 mg, 0.3865 mmol) was added portionwise over a period of 10 mins, followed by DMAP (23 mg, 0.193 mmol). Then, the reaction mixture was warmed to RT and allowed to stir for 12 h, progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with sat aq. NaHCO.sub.3 (5 ml) at RT and extracted with 5% MeOH in DCM twice (2×20 ml). The combined organic layers were dried over with Na.sub.2SO.sub.4. Solvent was distilled off under reduced pressure to get a pale yellow coloured solid. The crude compound was purified by column chromatography on basic silica gel (100-200 mesh) by eluting with a DCM/MeOH gradient (100/0.fwdarw.98/2) to afford the desired compound as an off white solid (20 mg, 20%). .sup.1H NMR (400 MHz, DMSO-D6) δ 9.03 (d, 1H), 8.86 (d, 1H), 8.58 (dd, 1H), 8.35 (dd, 1H), 8.32-8.18 (m, 1H), 7.99 (ddd, 1H), 7.69-7.54 (m, 2H), 7.44 (td, 3H), 6.62 (dd, 1H), 5.25-5.01 (m, 3H), 3.76-3.39 (m, 4H), 2.39 (d, 3H), 2.36-2.01 (m, 2H). MS: [M+H]+ 517.3
Precursor 4
[0628] ##STR00882##
[0629] In a flask under argon, preparative example 7 (700 mg, 1.93 mmol) and 4-DMAP (236 mg, 1.93 mmol) were suspended in 9.4 ml pyridine and cooled to 0° C. 4-Nitrobenzolsulfonyl chloride (2.14 g, 9.66 mmol) was added and the suspension was stirred at room temperature for 4 h. 4-DMAP (118 mg, 0.97 mmol) and 4-nitrobenzolsulfonyl chloride (1.07 g, 4.83 mmol) were added at 0° C. The reaction mixture was stirred overnight. Further 4-DMAP (118 mg, 0.97 mmol) and 4-nitrobenzolsulfonyl chloride (1.07 g, 4.83 mmol) were added at 0° C. and the reaction mixture was stirred for 1 day at room temperature. 40 ml 1M NaOH was added and the resulting mixture was centrifuged for 5 min at 6000 ppm. The centrifugation vial was decanted and the remaining solid was washed 4 times with 40 mL water. Water was removed by centrifugation/decantation after each washing step. The remaining solid was suspended in water, transferred to a flask and evaporated to yield the desired product as a brownish solid (871 mg, 83%). .sup.1H NMR (400 MHz, DMSO-d6) δ=9.04 (s, 1H), 8.85 (s, 1H), 8.68-7.90 (m, 8H), 7.46 (bs, 1H), 6.63 (d, 1H), 5.42 (s, 1H), 5.09 (s, 2H), 3.86-3.39 (m, 4H), 2.36-2.05 (m, 2H). MS: [M+H].sup.+ 547.97
Radioligand Synthesis
Example-1 [.SUP.3.H-1]
[0630] ##STR00883##
[0631] Precursor 2 (0.5 mg) was dissolved in dimethylformamide (DMF) (0.3 mL) and N,N-diisopropylethylamine (DIEA) (5 μL) in a tritium reaction vessel. 10% Pd/C (0.5 mg) was added and the vessel was pressurized to 0.5 atm with tritium gas at −200° C. The solution was stirred for 1 h at room temperature, cooled to −200° C. and excess gas was removed. The reaction flask was rinsed with 4×1 mL CH.sub.3OH, passing each of the CH.sub.3OH washes through a celite pad. The combined methanol was removed under vacuum. The material was purified by HPLC. The mobile phase was removed and the product was redissolved in absolute ethanol. (5 mCi with a radiochemical purity of >99% and a specific activity of 43.6 Ci/mmol). T means Tritium (.sup.3H). MS (ESI): m/z=369 (100%) [M+H].sup.+
Example 1-[.SUP.18.F-1]
[0632] ##STR00884##
[0633] Drying step: In a typical procedure, [.sup.18F]fluoride in a shipping vial (target water obtained from a commercial cyclotron facility) was transferred onto and trapped on an ion exchange cartridge. It was then eluted with a solution of potassium carbonate and Kryptofix 222 into the reaction vessel (RV1) of the TRACERlab® module. The solution was first evaporated by heating at 95° C. for 4 min under vacuum and helium flow. Acetonitrile (1 mL) was added to RV1 and the evaporation was continued under the same conditions for 2 min under vacuum and helium flow. After a second addition of acetonitrile (1 mL), final evaporation was carried out at 95° C. for 2 min under vacuum and helium flow. The reactor was then cooled to 60° C.
[0634] Radiolabeling: A solution of Precursor 1 (1 mg) in anhydrous dimethylsulfoxide (0.7 mL) was added to the reaction vessel and the reaction mixture was heated at 100° C. for 10 min. The reactor was cooled to 40° C., diluted with HPLC mobile phase (1.8 mL) and the contents were transferred into the loop-loading vial (RV2). The reactor was rinsed with water for injection (2.5 mL) and the rinse was transferred into RV2. The contents of RV2 were transferred into the HPLC injector loop for purification.
[0635] HPLC purification: Purification was performed by HPLC using a semi-preparative Phenomenex Synergi C18 column (5 μm, 250×10 mm) and eluted with a mixture of acetonitrile/ammonium acetate solution (20 mM) (35/65, v/v) at a flow rate of 4 mL/min. The product fraction was collected in Flask1, containing 20 mL of sodium ascorbate (5 mg/mL) in WFI. The diluted product mixture was passed through a C18 solid-phase extraction cartridge and the cartridge was rinsed with 10 mL of sodium ascorbate (5 mg/mL) in WFI. The radiolabelled product was eluted from the SPE cartridge with 1.0 mL of 200-proof USP grade ethanol into the formulation flask, pre-loaded with 10 mL of formulation base (sodium ascorbate (4.67 mg/mL) in saline). The cartridge was rinsed with 4.0 mL of formulation base and the rinse was mixed with the contents of the formulation flask. The resulting solution was passed through a sterilizing 0.2 μm membrane filter into a sterile, filter-vented vial (final product vial, FPV), pre-filled with 15 mL of normal saline (27% decay corrected yield).
Example-4 [.SUP.3.H-4]
[0636] ##STR00885##
[0637] Example 4 (1.0 mg) was added to a tritium reaction vessel, followed by cesium carbonate (1.0 mg), then DMF (0.1 mL), and finally iodomethane, [3H] (100 mCi). The vessel was sealed and the solution was stirred for 18 h at room temperature. The reaction mixture was transferred to a larger flask and the reaction vessel was rinsed with 4×2 mL methanol. The combined methanol was removed under vacuum. Crude yield: 38 mCi. The material was purified by silica gel column. Mobile phase was removed under vacuum and the product was re-dissolved in 0.05% TFA in water/acetonitrile. The material was further purified by semi-preparative reverse phase HPLC. Mobile phase was removed under vacuum and the product was re-dissolved in absolute ethanol. (4.8 mCi, purity>99%). The specific activity was determined to be 79.98 Ci/mmol by MS.
[0638] MS (ESI): m/z=374 (100%) [M+H].sup.+
Biological Assay Description and Corresponding Results
[0639] 1. Preparation of Human Parkinson's Disease (PD) Brain-Derived Alpha-Synuclein (a-Syn) Aggregates
[0640] The procedure was adapted from the protocol described in Spillantini et al., 1998. Frozen tissue blocks from PD donors were thawed on ice and homogenized using a glass dounce homogenizer. The homogenate was then centrifuged at 11,000×g (12,700 RPM) in an ultracentrifuge (Beckman, XL100K) for 20 minutes at 4° C. using a pre-cooled 70.1 rotor (Beckman, 342184). Pellets were resuspended in extraction buffer [10 mM Tris-HCl pH 7.4, 10% sucrose, 0.85 mM NaCl, 1% protease inhibitor (Calbiochem 539131), 1 mM EGTA, 1% phosphatase inhibitor (Sigma P5726 and P0044)] and centrifuged at 15,000×g (14,800 RPM, a 70.1 Ti rotor) for 20 minutes at 4° C. Pellets were discarded and sarkosyl (20% stock solution, Sigma L7414) was added to the supernatants to a final concentration of 1% at room temperature for one hour. This solution was then centrifuged at 100,000×g (38,000 RPM, 70.1 Ti rotor) for one hour at 4° C. Pellets containing enriched a-syn aggregates were resuspended in PBS and stored at −80° C. until use.
2. Micro-Radiobinding Competition Assay for the Determination of Binding Affinity
[0641] PD brain-derived a-syn aggregates were spotted onto microarray slides. The slides were incubated with the tritiated reference ligand, [.sup.3H]-a-syn-Ref (as described in WO2017/153601) at 20 nM and the example compounds of this invention (non-radiolabelled) either at 1 μM or at increasing concentrations in the range of 50 pM to 2 μM. After incubation, slides were washed and exposed to a phosphor storage screen (GE healthcare, BAS-IP TR 2025). Following exposure, phosphor storage screens were scanned with a laser imaging system (Typhoon FLA 7000) to readout the signal from the radiobinding experiments described above. Quantification of the signal was performed using the ImageJ software package. Non-specific signal was determined with an excess of non-radiolabelled reference ligand (1 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. Competition was calculated as percent, where 0% was defined as the specific binding in the presence of vehicle and 100% as the values obtained in the presence of excess of the non-radiolabelled reference ligand. All measurements were performed with at least two technical replicates. K.sub.i values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site, specific binding model.
[0642] Example compounds were assessed for their potency to compete with the binding of [3H] radiolabelled reference ligand to PD patient brain-derived a-syn aggregates. Results of the micro-radiobinding competition assay for the example compounds tested are shown in Table 5 as: % competition at 1 μM and K.sub.i value. All measurements were performed on the same PD brain-derived a-syn aggregates. The K.sub.i value of compound 1 reported here is the average of two independent experiments.
TABLE-US-00014 TABLE 5 Micro-radiobinding competition assay Example Competition Compound at 1 μM Ki no. (%) (nM) 1 94 30 2 68 Not determined
[0643] Table 5: Assessment of binding affinity by micro-radiobinding competition assay on human PD brain-derived a-syn aggregates. Left, percent (%) competition over the tritiated reference ligand in the presence of 1 μM of example compounds 1 and 2. Right, the Ki value for example compound 1 is shown. As shown in Table 5, example compounds 1 and 2 of the present invention show good binding to PD brain-derived a-syn aggregates.
3. Assessment of Target Engagement of Example-1 [.sup.3H-1] in a-Synucleinopathies and AD Tissues
3A: By High Resolution Micro-Autoradiography
[0644] The protocol was adapted from Marquie et al., 2015. Sections were incubated with tritiated example compound 1 (Example-1 [3H-1]) or a reference Tau ligand ([3H]-Tau-Ref at 60 nM for one hour at room temperature (RT). Sections were then washed as follows: One time in ice-cold 50 mM Tris-HCl pH 7.4 buffer for one minute, two times in 70% ice-cold ethanol for one minute, one time in ice-cold 50 mM Tris-HCl pH 7.4 buffer for one minute and finally rinsed briefly in ice-cold distilled water. Sections were subsequently dried and then exposed to Ilford Nuclear Emulsion Type K5 (Agar Scientific, AGP9281) in a light-proof slide storage box. After five days, the sections were developed by immersing them successively in the following solutions: 1.) Ilford Phenisol Developer (1:5 dilution in H.sub.2O, Agar Scientific, AGP9106), 2.) Ilfostop solution (1:20 dilution in H.sub.2O, Agar Scientific, AGP9104), 3.) Ilford Hypam Fixer (1:5 dilution in H.sub.2O, Agar Scientific, AGP9183) and finally rinsed with H.sub.2O.
[0645] When indicated, immunostaining was also performed on the same section. For image acquisition, sections were mounted using ProLong Gold Antifade reagent (Invitrogen P36930) and imaged on a Panoramic150 Slide Scanner (3DHistech) with a 20× objective capturing separately brightfield and fluorescent images.
3B. By Staining of Sections Using Antibodies
[0646] Brain sections were immunostained using a commercially available antibody, specific for phosphorylated serine at amino acid 129 a-synuclein (a-syn-pS129, rabbit monoclonal, Abcam 51253) or a mouse conformation-dependent anti-Tau antibody (MC1, kindly provided by Peter Davies, Northwell, US) or a commercially available antibody specific for TDP-43 phosphorylated serine at amino acid 409/410 (anti-pTDP-43 pS409/410, Biolegend 829901). Sections were fixed for 15 minutes at 4° C. with 4% formaldehyde (Sigma, 252549) and washed three times for five minutes with 1×PBS (Dulbecco's phosphate buffered saline, Sigma D1408) at RT. Next, sections were saturated and permeabilized in blocking buffer (PBS, 10% NGS, 0.25% Triton X-100) for one hour at RT and incubated overnight at 4° C. with the primary antibody corresponding to a-syn-pS129 or MC1 (in PBS, 5% NGS, 0.25% Triton X-100). The following day, sections were washed three times for five minutes with 1×PBS before incubation with a secondary, AlexaFluor647-labelled goat-anti-rabbit (Abcam, ab150079) or goat-anti-mouse (115-605-166, Jackson ImmunoResearch) antibody for 45 minutes at RT. Following incubation with secondary antibodies the sections were washed three times in PBS before being processed further. For image acquisition, sections were mounted using ProLong Gold Antifade reagent (Invitrogen P36930) and imaged with a Panoramic150 Slide Scanner (3DHistech; Hungary).
[0647] Results: High-resolution micro-autoradiography with Example-1 [.sup.3H-1] was performed on frozen human brain sections from different a-synucleinopathy cases. Strong autoradiography signal from Example-1 [.sup.3H-1] was detected in the form of accumulating silver grains (
4. Assessment of Specific Binding of Example-1 [.sup.3H-1] in Brain Sections from PD, PDD and Non-Demented Control (NDC) Donors by Autoradiography
[0648] Frozen human brain sections from one familial PD case (a-synuclein [SNCA] gene G51 D missense mutation), labelled as SNCA (G51 D), one PDD case and two non-demented control (NDC) cases were first briefly fixed for 15 minutes at 4° C. with 4% paraformaldehyde (Sigma, 252549) and washed three times for five minutes with PBS (Dulbecco's phosphate buffered saline, Sigma) at RT. All slides were then equilibrated for 20 minutes in 50 mM Tris-HCl pH 7.4 buffer prior to use in the experiment. Each brain section was incubated with a fixed concentration (10 nM) of tritiated example compound 1 (Example-1 [.sup.3H-1]) or a reference a-syn ligand ([3H]-a-syn-Ref), or increasing concentrations of Example-1 [.sup.3H-1] in the range of 1.25 nM to 80 nM of tritiated compound in Tris-HCl buffer for two hours at RT (Total binding, ‘−’). To determine non-specific (NS) binding Example-1 [.sup.3H-1] or [3H]-a-syn-Ref was mixed with 1 μM of non-radiolabelled compound (Example 1 or a-syn-Ref respectively, self-block, ‘+’). The slides were washed and placed under Phosphor imaging screens (GE healthcare, BAS-IP TR 2025) in imaging cassettes. Imaging screens were scanned using a laser imaging system (Typhoon, FLA 7000) and resulting images were analyzed using the ImageJ software package. Specific binding was determined by subtracting the non-specific signal from the total signal. K.sub.d values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site specific binding model.
[0649] Results: Example-1 [.sup.3H-1] displayed a dose-dependent autoradiography signal in different a-synucleinopathy tissues, including a PDD (
TABLE-US-00015 TABLE 6 Example-1 Genetic PD [.sup.3H-1] PDD (SNCA (G51D)) B max 1104 3004 Kd 11 nM 13 nM R.sup.2 0.93 0.88
[0650] Table 6: Assessment of binding affinity of Example-1 [.sup.3H-1] on human brain tissue from an idiopathic PD case (PDD) and a familial PD case (G51 D missense mutation) by autoradiography. The dissociation constant (K.sub.d) and binding site occupancy (B.sub.max) were calculated by applying a nonlinear regression curve fit using a one site, specific binding model in GraphPad Prism7. R.sup.2 is the coefficient of determination.
[0651] Additionally, when compared to a reference a-syn ligand, Example-1 [.sup.3H-1] displayed improved total and excellent specific binding on tissues from different a-synucleinopathy cases, as well as very weak binding in non-diseased tissue (NDC), (
5. Saturation Binding Studies on PD Brain-Derived a-Syn Aggregates by Micro-Radiobinding
[0652] PD brain-derived a-syn aggregates were spotted onto microarray slides. The slides were incubated with Example-1 [.sup.3H-1] or [3H]-a-syn-Ref at increasing concentrations in the range of 300 pM to 150 nM. After incubation, slides were washed and exposed to a phosphor storage screen (GE healthcare, BAS-IP TR 2025). Following exposure, phosphor storage screens were scanned with a laser imaging system (Typhoon FLA 7000) to readout the signal from the radiobinding experiments described above. Quantification of the signal was performed using the ImageJ software package. Non-specific signal was determined with an excess of non-radiolabelled reference ligand (Example-1 or a-syn-Ref, respectively, at 2 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. K.sub.d values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site specific binding model.
[0653] Results: Example-1 [.sup.3H-1] was assessed in saturation binding studies on PD tissue homogenates by micro-radiobinding and compared head-to-head with a reference a-syn binder. As shown in
6. Assessment of Displacement of Example-1 [.sup.3H-1] with a-Syn-Ref on PD Brain-Derived a-Syn Aggregates by Micro-Radiobinding
[0654] PD brain-derived a-syn aggregates were spotted onto microarray slides. The slides were incubated with Example-1 [.sup.3H-1] at 20 nM and either a-syn-Ref or compound of Example 1 (non-radiolabelled) at increasing concentrations in the range of 50 pM to 2 μM. After incubation, slides were washed and exposed to a phosphor storage screen (GE healthcare, BAS-IP TR 2025). Following exposure, phosphor storage screens were scanned with a laser imaging system (Typhoon FLA 7000) to readout the signal from the radiobinding experiments described above. Quantification of the signal was performed using the ImageJ software package. Non-specific signal was determined with an excess of non-radiolabelled example compound 1 (2 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. Competition was calculated as percent, where 0% was defined as the specific binding in the presence of vehicle and 100% as the values obtained in the presence of excess of the non-radiolabelled reference ligand. All measurements were performed with at least two technical replicates.
[0655] Results: It was evaluated whether Example-1 [.sup.3H-1] can be displaced by non-radiolabelled a-syn-Ref compound. The a-syn-Ref compound only partially competed with Example-1 [.sup.3H-1] on brain-derived a-syn aggregates from idiopathic PD cases (
7. Radiobinding Competition Assay for Determination of Inhibitor Constant (K.SUB.i.) of Example Compound 1 on AD Brain Homogenates
Preparation of Human Alzheimer's Disease (AD) Brain Homogenates:
[0656] The procedure was adapted from the protocol described in Bagchi et al., 2013. Frozen tissue blocks from AD donors were thawed on ice and homogenized in high salt buffer (50 mM Tris-HCl pH 7.5, 0.75M NaCl, 5 mM EDTA) supplemented with protease inhibitors (Complete; Roche 11697498001) at 4° C. using a glass Dounce homogenizer. The homogenate was centrifuged at 100,000×g (38,000 RPM) in an ultracentrifuge (Beckman, XL100K) for one hour at 4° C. using a pre-cooled 70.1 rotor (Beckman, 342184). Pellets were resuspended in high salt buffer supplemented with 1% Triton X-100 and homogenized at 4° C. using a glass Dounce homogenizer. The homogenates were centrifuged again at 100,000×g (38,000 RPM, 70.1 rotor) for one hour at 4° C. Pellets were resuspended in high salt buffer supplemented with 1% Triton X-100 and 1 M sucrose and homogenized at 4° C. using a glass Dounce homogenizer. The homogenates were centrifuged at 100,000×g (38,000 RPM, 70.1 rotor) for one hour at 4° C. The resulting pellets containing the insoluble fraction were resuspended in PBS, aliquoted and stored at −80° C. until use.
[0657] A fixed concentration of AD insoluble fraction was incubated with a tritiated reference Abeta ligand ([.sup.3H]-Abeta-Ref) at 10 nM and increasing concentrations of non-radiolabelled example compound 1 in the range of 400 μM to 2 μM for two hours at RT. The samples were then filtered under vacuum in GF/C filter plates (PerkinElmer) to trap the aggregates with the bound radioligand and washed five times with 50 mM Tris pH 7.5. The GF/C filters were then dried and scintillation liquid (UltimateGold, PerkinElmer) was added in each well. The filters were analyzed on a Microbeta2 scintillation counter (PerkinElmer). Non-specific signal was determined with an excess of non-radiolabelled reference ligand (2 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. Competition was calculated as percent, where 0% was defined as the specific binding in the presence of vehicle and 100% as the values obtained in the presence of excess of the non-radiolabelled reference ligand. K.sub.i values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site, specific binding model. Measurements were performed with at least two replicates.
[0658] Results: As shown in
TABLE-US-00016 TABLE 7 Example compound 1 Ki 330 nM R.sup.2 0.97
Table 7: Ki value determination of example compound 1 for the displacement of [3H]-Abeta-Ref with non-radiolabelled example compound 1 on AD brain-derived homogenates. K.sub.i, and R.sup.2 values were calculated by applying a nonlinear regression curve fit using a one site, specific binding model in GraphPad Prism7.
8. PK Studies in a Healthy Monkey
[0659] Non-Human Primate (NHP) was injected intravenously (iv) with the .sup.18F-labelled Example-1 [.sup.18F-1] (6.5 mCi) using 1 mL ethanol and 14 mL ascorbate/saline (ascorbate solution was prepared at a concentration of 9.3 mg/mL). Monkey PET scans were performed using a Siemens Focus 220. PET acquisition started immediately before the radioactive dose was injected. Images were generated as dynamic scans for 120 minutes with head focussed. Example-1 [.sup.18F-1] had a quick uptake (3.5 min post injection) with 2.0 SUVmax whole brain. In addition, Example-1 [.sup.18F-1] had a quick washout with peak to half peak of 14 min (
9. Assessment of Specific Binding of Example-1 [.sup.3H-1] in Brain Sections from PD, PDD, MSA, LBV and Non-Demented Control (NDC) Donors by Autoradiography
[0660] Frozen human brain sections from one PD case, two PDD cases, two MSA cases, one LBV case and three non-demented control (NDC) cases were first briefly fixed for 15 minutes at 4° C. with 4% paraformaldehyde (Sigma, 252549) and washed three times for five minutes with PBS (Dulbecco's phosphate buffered saline, Sigma) at RT. All slides were then equilibrated for 20 minutes in 50 mM Tris-HCl pH 7.4 buffer prior to use in the experiment. Each brain section was incubated with a fixed concentration (10 nM) of tritiated example compound 1 (Example-1 [.sup.3H-1]) in Tris-HCl buffer for two hours at RT (Total binding, ‘Total’). To determine non-specific binding (NSB) Example-1 [.sup.3H-1] was mixed with 5 μM of non-radiolabelled compound Example 1. The slides were washed and then exposed and scanned in a real-time autoradiography system (BeaQuant instrument, ai4R).
[0661] Results: Example-1 [.sup.3H-1] displayed target engagement in various a-synucleinopathy tissues, including two MSA, one LBV and two PDD cases (
10. Micro-Radiobinding Competition Assay for the Determination of Binding Affinity
[0662] PD brain-derived a-syn aggregates were spotted onto microarray slides. The slides were incubated with the Example-1 [.sup.3H-1] at 6 nM or 20 nM and the example compounds (non-radiolabelled) at 1 μM and 100 nM. In some cases, the non-radiolabelled example compounds were further assessed for a range of different concentrations, varying from 0.05 nM to 2 μM. After incubation, slides were washed and scanned by a real-time autoradiography system (BeaQuant, ai4R). Quantification of the signal was performed by using the Beamage image analysis software (ai4R). Non-specific signal was determined with an excess of non-radiolabelled Example-1 (2 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. Competition was calculated as percent, where 0% was defined as the specific binding in the presence of vehicle and 100% as the values obtained in the presence of excess of the non-radiolabelled Example-1. K.sub.1 values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site, specific binding model. All measurements were performed with at least two technical replicates. For compounds tested in more than one experiment, the mean of the replicates or K; values in independent experiments is reported.
[0663] Results: Example compounds were assessed for their potency to compete with the binding of Example-1 [.sup.3H-1] ligand to PD patient brain-derived a-syn aggregates. Results of the micro-radiobinding competition assay for the example compounds tested are shown in Table 8 as: % competition at 1 μM and 100 nM. The Table 8 also shows K.sub.i values.
TABLE-US-00017 TABLE 8 Assessment of binding affinity by micro-radiobinding competition assay on human PD brain-derived a-syn aggregates. Percent (%) competition over the tritiated Example-1 [.sup.3H-1] ligand in the presence of 1 μM and 100 nM of example compounds 2-181. Micro-radiobinding competition assay Competition Competition Example at 1 μM at 100 nM Ki Compound no. (%) (%) (nM) 2 98 72 7 3 89 45 57 4 92 58 50* 5 98 68 26 6 98 87 19 7 101 87 5 8 91 67 9 90 69 10 90 58 61** 11 98 50 12 25 13 31 4 14 77 42 15 91 61 16 70 32 17 100 68 21 18 86 23 111 19 83 23 74 20 90 48 21 84 22 72 23 81 24 97 25 94 64 68 26 108 87 20 27 99 87 13 28 99 89 29 91 30 95 80 31 86 32 25 16 33 75 34 89 80 35 73 36 91 74 37 37 38 70 39 66 43 40 77 50 49 41 93 61 42 89 57 43 88 47 44 90 45 92 78 52** 46 61 47 39 48 77 42 132 49 84 48 50 103 94 8 51 100 80 52 29 26 53 54 27 54 88 71 19 55 38 11 56 93 57 88 58 98 85 59 86 60 71 70 61 80 53 62 27 8 63 96 79 64 95 54 2 65 64 66 92 72 67 71 68 63 69 48 16 70 65 71 51 18 72 79 45 73 66 33 74 39 22 75 73 58 76 93 69 77 94 95 78 60 79 74 80 83 51 56 81 90 56 118 82 84 83 47 84 38 14 85 99 72 86 64 87 92 67 15** 88 75 89 75 29 90 51 18 91 84 44 42 92 73 32 93 47 12 94 70 18 95 68 34 54 96 39 24 97 84 33 84 98 76 38 89 99 92 56 82 100 78 101 75 311 102 68 59 103 70 41 104 98 86 105 83 57 149 106 100 95 107 100 92 108 81 54 182 109 67 110 105 75 111 97 82 112 105 85 113 91 75 40 114 99 92 115 98 92 116 97 71 117 67 17 118 94 61 119 51 12 120 83 45 121 84 40 122 61 34 123 58 32 124 98 61 125 99 80 126 83 75 127 86 71 128 85 55 129 74 25 130 85 74 131 98 72 132 93 63 133 59 134 74 135 96 67 136 75 137 84 35 138 57 32 139 77 58 7 140 67 141 66 51 142 99 92 6 143 91 80 8 144 68 49 145 76 46 146 53 32 147 86 72 148 91 77 40 149 53 150 46 37 151 81 51 226 152 86 39 153 57 31 154 99 81 155 94 51 156 85 80 157 43 21 158 54 19 159 67 46 160 73 51 161 65 42 162 110 92 163 99 49 67 164 87 44 165 107 88 166 96 72 55 167 93 68 56 168 36 30 169 96 73 45 170 102 65 171 48 172 85 44 173 54 12 174 64 46 175 65 32 176 54 13 177 100 76 178 103 77 179 95 80 180 96 77 181 75 67 136 K.sub.i values are also shown for selected example compounds. *mean of Ki values in independent experiments using PD brain-derived homogenates from three different donors. **mean of Ki values in independent experiments using PD brain-derived homogenates from two different donors. As shown in Table 8, example compounds 2-181 of the present invention show potent binding to PD brain-derived a-syn aggregates.
11. Assessment of Target Engagement of Example-4 [.sup.3H-4] in a-Synucleinopathies
11A: By High Resolution Micro-Autoradiography
[0664] The protocol was adapted from Marquie et al., 2015. Sections were incubated with tritiated example compound 4 (Example-4 [.sup.3H-4]) or a reference Tau ligand ([.sup.3H]-Tau-Ref at 20 nM for one hour at RT. Sections were then washed as follows: One time in ice-cold 50 mM Tris-HCl pH 7.4 buffer for one minute, two times in 70% ice-cold ethanol for one minute, one time in ice-cold 50 mM Tris-HCl pH 7.4 buffer for one minute and finally rinsed briefly in ice-cold distilled water. Sections were subsequently dried and then exposed to Ilford Nuclear Emulsion Type K5 (Agar Scientific, AGP9281) in a light-proof slide storage box. After five days, the sections were developed by immersing them successively in the following solutions: 1.) Ilford Phenisol Developer (1:5 dilution in H.sub.2O, Agar Scientific, AGP9106), 2.) Ilfostop solution (1:20 dilution in H.sub.2O, Agar Scientific, AGP9104), 3.) Ilford Hypam Fixer (1:5 dilution in H.sub.2O, Agar Scientific, AGP9183) and finally rinsed with H.sub.2O.
[0665] When indicated, immunostaining was also performed on the same section. For image acquisition, sections were mounted using ProLong Gold Antifade reagent (Invitrogen P36930) and imaged on a Panoramic150 Slide Scanner (3DHistech) with a 20× objective capturing separately brightfield and fluorescent images.
11B. By Staining of Sections Using Antibodies
[0666] Brain sections were immunostained using a commercially available antibody, specific for phosphorylated serine at amino acid 129 a-synuclein (a-syn-pS129, rabbit monoclonal, Abcam 51253). Sections were fixed for 15 minutes at 4° C. with 4% formaldehyde (Sigma, 252549) and washed three times for five minutes with 1×PBS (Dulbecco's phosphate buffered saline, Sigma D1408) at RT. Next, sections were saturated and permeabilized in blocking buffer (PBS, 10% NGS, 0.25% Triton X-100) for one hour at RT and incubated overnight at 4° C. with the primary antibody corresponding to a-syn-pS129. The following day, sections were washed three times for five minutes with 1×PBS before incubation with a secondary, AlexaFluor647-labelled goat-anti-rabbit (Abcam, ab150079) antibody for 45 minutes at RT. Following incubation with secondary antibodies the sections were washed three times in PBS before being processed further. For image acquisition, sections were mounted using ProLong Gold Antifade reagent (Invitrogen P36930) and imaged with a Panoramic150 Slide Scanner (3DHistech; Hungary).
[0667] Results: High-resolution micro-autoradiography with Example-4 [.sup.3H-4] was performed on frozen human brain sections from a PD donor. Strong autoradiography signal from Example-4 [.sup.3H-4] was detected in the form of accumulating silver grains (
12. Assessment of Specific Binding of Example-4 [.sup.3H-4] in Brain Sections from PD, MSA and Non-Demented Control (NDC) Donors by Autoradiography
[0668] Frozen human brain sections from one familial PD case (a-synuclein [SNCA] gene G51 D missense mutation), labelled as SNCA, one idiopathic PD case, one MSA case and two non-demented control (NDC) cases were first briefly fixed for 15 minutes at 4° C. with 4% paraformaldehyde (Sigma, 252549) and washed three times for five minutes with PBS (Dulbecco's phosphate buffered saline, Sigma) at RT. All slides were then equilibrated for 20 minutes in 50 mM Tris-HCl pH 7.4 buffer prior to use in the experiment. Each brain section was incubated with a fixed concentration (10 nM) of tritiated example compound 4 (Example-4 [.sup.3H-4]) in Tris-HCl buffer for two hours at RT (Total binding, ‘Total’). To determine non-specific binding Example-4 [.sup.3H-4] was mixed with 5 μM of non-radiolabelled compound (Example 4, ‘NSB’). The slides were washed and then exposed and scanned in a real-time autoradiography system (BeaQuant instrument, ai4R).
[0669] Results: Example-4 [.sup.3H-4] displayed specific binding in various a-synucleinopathy tissues, including a MSA case, a familial PD case and an idiopathic PD case (
13. Saturation Binding Studies on PD Brain-Derived a-Syn Aggregates by Micro-Radiobinding
[0670] PD brain-derived a-syn aggregates were spotted onto microarray slides. The slides were incubated with Example-4 [.sup.3H-4] at increasing concentrations in the range of 1.56 nM to 80 nM. After incubation, slides were scanned by a real-time autoradiography system (BeaQuant instrument, ai4R). Quantification of the signal was performed by using the Beamage image analysis software (ai4R). Non-specific signal was determined with an excess of non-radiolabelled reference ligand (Example-4 at 2 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. K.sub.d values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site specific binding model.
[0671] Results: Example-4 [.sup.3H-4] was assessed in saturation binding studies on PD tissue homogenates by micro-radiobinding (
TABLE-US-00018 TABLE 9 Example-4 PD [.sup.3H-4] homogenates Kd 21 nM R.sup.2 0.86
[0672] Table 9: Assessment of binding affinity of Example-4 [.sup.3H-4] on human PD brain tissue homegenates by micro-radiobinding. The dissociation constant (K.sub.d) and binding site occupancy (B.sub.max) were calculated by applying a nonlinear regression curve fit using a one site, specific binding model in GraphPad Prism7. R.sup.2 is the coefficient of determination.
14. Radiobinding Competition Assay for Determination of Inhibitor Constant (K.SUB.i.) of Example Compound 4 on AD Brain Homogenates
[0673] Human Alzheimer's disease (AD) brain homogenates were prepared according to the procedure disclosed in Example 7 (see above).
[0674] A fixed concentration of AD insoluble fraction was incubated with a tritiated reference Abeta ligand ([.sup.3H]-Abeta-Ref) at 10 nM and increasing concentrations of non-radiolabelled example compound 1 in the range of 400 pM to 2 μM for two hours at RT. The samples were then filtered under vacuum in GF/C filter plates (PerkinElmer) to trap the aggregates with the bound radioligand and washed five times with 50 mM Tris pH 7.5. The GF/C filters were then dried and scintillation liquid (UltimateGold, PerkinElmer) was added in each well. The filters were analyzed on a Microbeta2 scintillation counter (PerkinElmer). Non-specific signal was determined with an excess of non-radiolabelled reference ligand (2 μM) and specific binding was calculated by subtracting the non-specific signal from the total signal. Competition was calculated as percent, where 0% was defined as the specific binding in the presence of vehicle and 100% as the values obtained in the presence of excess of the non-radiolabelled reference ligand. K.sub.i values were calculated in GraphPad Prism7 by applying a nonlinear regression curve fit using a one site, specific binding model. Measurements were performed in two independent experiments with two technical replicates.
[0675] Results: As shown in
TABLE-US-00019 TABLE 10 Example compound 4 Ki 297 nM R.sup.2 0.97
[0676] Table 10: Ki value determination of example compound 4 for the displacement of [.sup.3H]-Abeta-Ref with non-radiolabelled example compound 4 on AD brain-derived homogenates. K.sub.i, and R.sup.2 values were calculated by applying a nonlinear regression curve fit using a one site, specific binding model in GraphPad Prism7.
15. First in Human (FIH) Study
[0677] A Phase 1 study to evaluate .sup.18F-Example 1 as a potential PET radioligand for imaging a-synuclein deposits in the brain of patients with suspected a-synuclein pathology compared to healthy volunteers (HVs) is ongoing. The study objectives are to characterize safety as well as imaging and pharmacokinetics properties of .sup.18F-Example 1, in individuals with suspected idiopathic Parkinson's Disease (PD) and healthy volunteer (HV) subjects. A total of up to 10 subjects may be enrolled (target of up to 5 HV subjects and up to 5 subjects with idiopathic PD).
Inclusion Criteria for all Subjects:
[0678] Subject is able to provide written informed consent, which must be obtained before any assessment is performed. [0679] Female subjects must not be of childbearing potential, or if they are of childbearing potential to agree to use contraception and not donate eggs. At the discretion of the Investigator, subjects without documentation of non-childbearing potential may receive pregnancy testing. [0680] Male subjects with their partners of childbearing potential must commit to the use of 2 methods of contraception, 1 of which is a barrier method for male subjects for the study duration and 90 days after study completion. [0681] Male subjects must not donate sperm for the study duration and for 90 days after study completion. [0682] For subjects receiving arterial cannulation, adequate circulation to the hand for safe placement of arterial line (as determined by Allen's test) and blood clotting (Prothrombin Time [PT] and Partial Thromboplastin Time [PTT]). [0683] If subject takes bupropion, subject must agree to hold this medication for at least 12 hours prior to DaTscan imaging (if performed).
Additional Inclusion Criteria for HV Subjects:
[0684] Males and females aged ≤21 years. [0685] Healthy with no clinically relevant finding on physical examination at Screening and upon reporting to the clinic for the tracer Imaging Visit. [0686] No family history of α-synucleinopathy, including PD, or other early-onset neurological disease associated with dementia. [0687] No personal history of clinically significant neurologic and/or psychiatric disorders. [0688] No evidence of dopamine transporter deficit on Dopamine active transporter (DaT) scan performed either as part of Screening or on previously acquired DaTscan (within 6 months prior to signing consent). [0689] Have a Montreal Cognitive Assessment (MoCA) score 26. [0690] No cognitive impairment as judged by the person in charge (PI).
Additional Inclusion Criteria for Subjects with α-Synucleinopathy: [0691] Males and females aged ≤40 years. [0692] Subjects diagnosed with any of the following: [0693] Idiopathic PD [0694] PD with genetic risk factor (except leucine-rich repeat kinase 2 [LRRK2] mutation) [0695] A brain magnetic resonance imaging (MRI) consistent with a diagnosis of α-synucleinopathy, with no evidence of focal disease to account for the subject's neurological symptoms. [0696] Evidence of dopamine transporter deficit on DaTscan performed either as part of Screening or on previously acquired DaTscan. [0697] Medications taken for symptomatic treatment of α-synucleinopathy must be maintained on a stable dosage regimen for at least 30 days before Screening Visit. [0698] Ability to tolerate lying in the scanner for up to ˜180 minutes without excessive head or jaw tremor or dyskinesia sufficient to cause significant motion artifact on the PET scans.
[0699] After enrollment, subjects will receive 1 intravenous injection of .sup.18F-Example 1 of no more than 10 mCi. .sup.18F-Example 1 brain uptake and pharmacokinetics in human subjects will be visually and quantitively assessed and safety data acquired. .sup.18F-Example 1 PET signal in suspected idiopathic PD cases will be compared cross-sectionally to HV.
16: Formulation
[0700] .sup.18F trapping and elution: [.sup.18F]-fluoride was transferred onto and trapped on an ion exchange cartridge. It was then eluted with an aqueous acetonitrile solution of potassium carbonate (1.6 mg) and Kryptofix 222 (10 mg) into the reaction vessel (RV1). The solution was first evaporated by heating at 95° C. for 4 min under vacuum and helium flow. Acetonitrile (1 mL) was then added to RV1 and the evaporation was continued under the same conditions for 2 min under vacuum and helium flow. After a second addition of acetonitrile (1 mL), a final evaporation was carried out at 95° C. for 2 min under vacuum and helium flow. Finally, the reactor was cooled to 60° C.
[0701] Radiolabeling reaction: A solution of the precursor (1.0 mg) in anhydrous dimethylsulfoxide was added to the reaction vessel and the reaction mixture was heated at 100° C. for 10 min. The reactor is cooled to 40° C., diluted with HPLC mobile phase (1.8 mL) and the contents are transferred into the loop-loading vial (RV2). The reactor was rinsed with water for injection (2.5 mL) and the rinse was transferred into RV2. The contents of RV2 were transferred into the HPLC injector loop for purification.
[0702] Purification and drug product formulation: Purification was performed by HPLC using a semi-preparative Agilent Eclipse XDB C18 column (5 μm, 250×9.4 mm) and eluted with a mixture of methanol/ammonium acetate solution (20 mM, 50/50, v/v) at a flow rate of 4 mL/min. The product fraction was collected in a flask, containing 20 mL of sodium ascorbate (5 mg/mL) in water for injection (WFI). The diluted product mixture was passed through a C18 solid-phase extraction cartridge and the cartridge was rinsed with 10 mL of sodium ascorbate (5 mg/mL) in WFI. The radiolabeled product was eluted from the SPE cartridge with 1.0 mL of 200-proof USP grade ethanol into the formulation flask, pre-loaded with 10 mL of sodium ascorbate (10 mg/mL) in saline. The cartridge was rinsed with 4.0 mL of sodium ascorbate in saline (10 mg/mL) and the rinse was mixed with the contents of the formulation flask. The resulting solution was passed through a sterilizing 0.2 μm membrane filter into a sterile, filter-vented vial (final product vial, FPV), pre-filled with 15 mL of normal saline.
[0703] The stability of the radiolabelled product over time was studied and validated to remain within specifications for 8 hours after the end of synthesis.
[0704] The batch formula quantities are presented in Table 11:
TABLE-US-00020 Precursor 1 mg .sup.a [18F] Fluoride <4 Ci Normal saline 50 mL Ethanol 1 mL Sodium ascorbate 500 mg .sup.a Removed during processing
[0705] The final formulation of the radiolabelled product developed for this study has a volume of 30 mL, with the intent to achieve the following content based on an injected volume of 10 ml in the final dosage form is shown in Table 12:
TABLE-US-00021 Radioactive Normal Sodium amount Carrier saline Ascorbate Ethanol ≤10 mCi ≤10 μg ≤9.67 ml ≤46.7 mg ≤0.33 ml