U.S. patent application number 14/465773 was filed with the patent office on 2014-12-11 for compound suitable for the treatment of synucleopathies.
The applicant listed for this patent is Neuropore Therapies, Inc.. Invention is credited to Eliezer MASLIAH, Edward M. ROCKENSTEIN, Igor Flint TSIGELNY, Wolfgang WRASIDLO.
Application Number | 20140364610 14/465773 |
Document ID | / |
Family ID | 43629477 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140364610 |
Kind Code |
A1 |
MASLIAH; Eliezer ; et
al. |
December 11, 2014 |
COMPOUND SUITABLE FOR THE TREATMENT OF SYNUCLEOPATHIES
Abstract
The present invention relates to certain heteroaromatic
compounds of Formula (Ia), or pharmaceutically acceptable salts
thereof, and uses of such compounds in the treatment of
synucleopathies.
Inventors: |
MASLIAH; Eliezer; (San
Diego, CA) ; ROCKENSTEIN; Edward M.; (Spring Valley,
CA) ; WRASIDLO; Wolfgang; (San Diego, CA) ;
TSIGELNY; Igor Flint; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Neuropore Therapies, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
43629477 |
Appl. No.: |
14/465773 |
Filed: |
August 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13516543 |
Aug 28, 2012 |
8846682 |
|
|
PCT/US2010/060862 |
Dec 16, 2010 |
|
|
|
14465773 |
|
|
|
|
61287082 |
Dec 16, 2009 |
|
|
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Current U.S.
Class: |
544/369 ;
544/366; 544/373; 546/201 |
Current CPC
Class: |
C07D 413/12 20130101;
C07D 473/00 20130101; A61P 25/28 20180101; C07D 403/14 20130101;
C07D 487/04 20130101; C07D 417/12 20130101; A61P 25/16 20180101;
A61P 25/00 20180101; A61P 25/14 20180101; C07D 403/12 20130101;
C07D 413/14 20130101; A61P 21/02 20180101; C07D 417/14
20130101 |
Class at
Publication: |
544/369 ;
546/201; 544/366; 544/373 |
International
Class: |
C07D 417/14 20060101
C07D417/14; C07D 403/14 20060101 C07D403/14; C07D 413/14 20060101
C07D413/14 |
Claims
1-16. (canceled)
17. A compound of Formula (Ia): ##STR00359## wherein R.sub.1 is a
hydrophilic aromatic or heterocyclic group; R.sub.2 is an aliphatic
hydrocarbon or alicyclic hydrophobic group or nil; R.sub.3 is an
aliphatic or alicyclic group with basic character; X is NH, O, S,
or CH.sub.2; Y is C.dbd.O, C.dbd.S, or C.dbd.NH; each Z is
independently C, CH, C.dbd.O, O, S, --N--, or NH; and L is
CH.sub.2, CO.sub.2, CH.sub.2--S, CH.sub.2--SO, CH.sub.2--SO.sub.2,
O, S, or C.dbd.O, or nil; or a pharmaceutically acceptable salt
thereof or a pharmaceutically acceptable solvate of said compound
or salt.
18. The compound of claim 17, wherein Z.sub.1 is C or N; Z.sub.2 is
C, N, C.dbd.O, or NH; Z.sub.3 is C.dbd.O, N, or C; Z.sub.4 is C;
and Z.sub.5 is N, S, NH, O, C.dbd.O, or S.
19. The compound of claim 17, wherein R.sub.1 is a bicyclic
heteroaromatic group.
20. The compound of claim 19, wherein R.sub.1 is selected from the
group consisting of: ##STR00360##
21. The compound of claim 17, wherein R.sub.2 is an aliphatic
hydrocarbon or alicyclic hydrophic group.
22. The compound of claim 17, wherein R.sub.2 is an alkyl group
with 1 to 8 carbon atoms.
23. The compound of claim 17, wherein R.sub.2 is an alkyl group
with 1 to 5 carbon atoms.
24. The compound of claim 17, wherein R.sub.2 is selected from the
group consisting of: ##STR00361##
25. The compound of claim 17, wherein R.sub.3 is: ##STR00362##
26. The compound of claim 17, wherein ##STR00363## is selected from
the group consisting of: ##STR00364##
27. The compound according to claim 17, selected from the group
consisting of: ##STR00365## ##STR00366## and pharmaceutically
acceptable salts thereof.
28. A pharmaceutical composition comprising a compound of claim 17
or a pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable excipients.
29. A method of treating a synucleopathy comprising administering
to a patient in need of such treatment an effective amount of a
compound of claim 17 or a pharmaceutically acceptable salt
thereof.
30. The method of claim 29, wherein the synucleopathy is
Parkinson's disease, Parkinson's disease with dementia, dementia
with Lewy bodies, Pick's disease, Down syndrome, multiple system
atrophy, amyotrophic lateral sclerosis (ALS), or Hallervorden-Spatz
syndrome.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/516,543, filed Aug. 28, 2012, which is a U.S. National Phase
Application of International Application No. PCT/US2010/060862,
filed Dec. 16, 2010, which claims priority to U.S. Provisional
Application No. 61/287,082, filed Dec. 16, 2009, the entire
contents of each of which is incorporated herein by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to compounds suited to be used
to treat and/or prevent synucleopathies.
[0003] Protein misfolding and aggregation into toxic oligomers has
been linked with the neurodegenerative process in Alzheimer's
Disease (AD), Parkinson's Disease (PD) and other age-associated
neurological disorders. Together, AD and PD affect over 10 million
people in the US and Europe alone. In PD and related conditions
such as Dementia with Lewy bodies (DLB), Parkinson's Disease
Dementia (PDD), and Multiple System Atrophy (MSA), the damage of
nerve terminals has been linked to abnormal accumulation of
alpha-synuclein (SYN), a synaptic protein that, under physiological
conditions, is involved in synaptic vesicle recruitment and
plasticity. Jointly, PD, PDD and DLB are denominated Lewy body
disease (LBD). In patients with PD, motor deficits have been linked
to the degeneration of dopaminergic neurons. However, patients with
PD also develop non-motor symptoms such as memory and olfactory
deficits that result from the degeneration of other neuronal
populations in the CNS.
[0004] Previous studies have considered SYN as an unstructured
molecule; however, studies in biological membranes and molecular
dynamics studies over prolonged periods of time have shown that SYN
can adopt complex structures with two-alpha helixes at the
N-terminus and a movable C-terminus tail. Based on these studies,
it was recently discovered that SYN could form propagating and
non-propagating dimers. The propagating dimers arrange in a tail to
tail conformation (N-term of one SYN with the N-term of the other
SYN) that allows for the incorporation of additional SYN molecules.
The non-propagating dimers (N-term of one SYN with the C-term of
the other SYN) arrange in a head to tail orientation and do not
allow further aggregation. Molecular dynamics simulations and in
vitro studies demonstrated that propagating dimers might constitute
the nidus for the formation of toxic oligomers (pentamers,
hexamers, heptamers) that are centrally involved in the
pathogenesis of PD and related conditions.
[0005] Most compounds currently under testing for PD are designed
to improve dopaminergic neurotransmission. A few new experimental
compounds have been developed to target SYN aggregation by blocking
fibril formation rather than oligomers. The role of fibril
formation in PD is controversial, and most recent studies consider
that fibrilization might play a role at isolating more toxic
oligomers.
[0006] A number of relatively specific and non-specific SYN
inhibitors are currently under development. Most of these molecules
such as curcumin, rifampicin and flavinoids display anti-oxidant
properties. However, none of the compounds known specifically
target SYN arrays involved in the formation of toxic oligomers.
[0007] It is an object of the present invention to provide
compounds which specifically block the formation of propagating
dimers and toxic SYN oligomers. Consequently, these compounds can
be used to treat individuals suffering from synucleopathies, slow
down the progress and prevent the outbreak of said diseases.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention relates to a compound of formula
(I):
##STR00001##
wherein
[0009] R.sub.1 is a substituted or unsubstituted aromatic hetero-
or homocyclic or a substituted or unsubstituted alicyclic hetero-
or homocyclic group,
[0010] R.sub.2 is an alkyl group with 1 to 18 carbon atoms or a
substituted or unsubstituted cycloalkyl or aryl group,
[0011] R.sub.3 is a substituted or unsubstituted aromatic hetero-
or homocyclic or a substituted or unsubstituted alicyclic hetero-
or homocyclic group,
[0012] L is a single bond, an alkyl group having 1 to 6, preferably
1 to 5, more preferably 1 to 4, even more preferably 1 to 3, carbon
atoms, NHCO, O, S, NHCONH or NHCOO,
[0013] X, Y and Z are independently O, N, NH, S or CH,
[0014] W is a single bond or an alkyl group having from 1 to 6
carbon atoms,
[0015] or a pharmaceutically acceptable salt thereof or a
pharmaceutically acceptable solvate of said compound or salt.
[0016] It turned out that the organic heteroaromatic compounds
according to the present invention having formula (I) specifically
block the formation of propagating dimers and toxic SYN oligomers.
These compounds bind selectively misfolded SYN and prevent
aggregation of oligomers into toxic species. These compounds
consequently block the formation of propagating dimers.
[0017] It was found that organic compounds in the molecular weight
range from 150 to 600, preferably 200 to 500, having a central
heteroaromatic ring structure linked to three different types of
moieties R.sub.1, R.sub.2 and R.sub.3 shown in the general formula
above, are suitable to block SYN aggregation. The central scaffold
of the compound according to formula (I) is composed of --NH--CO--
and a heteroaromatic ring structure. This scaffold is linked via
linkage moieties designated as L and W to R.sub.1, R.sub.2 and
R.sub.3 (formula I). R.sub.1, R.sub.2 and R.sub.3 are diversity
inputs for affecting the affinity of the compound to the SYN
target. R.sub.1 is a substituted or unsubstituted aromatic hetero-
or homocyclic or a substituted or unsubstituted alicyclic hetero-
or homocyclic group. R.sub.1 is preferably a substituted or
unsubstituted aromatic or heterocyclic group, preferably a fused
heteroaromatic ring incorporating at least one basic nitrogen atom,
and R.sub.2 is preferably either a linear aliphatic moiety or a
short chain aliphatic moiety connected to a alicyclic ring
structure. It is noted that substituent R.sub.2 of formula (I) is
hydrophobic. This property of R.sub.2 is important for the
biological activity of the compounds of the present invention.
R.sub.3 is a substituted or unsubstituted aromatic hetero- or
homocyclic or a substituted or unsubstituted alicyclic hetero- or
homocyclic group. R.sub.3 is preferably composed of a linear
alicyclic or linear chain structure with basic character including
basic nitrogen atoms.
[0018] The central scaffold composed of a heterocyclic 5-membered
ring of a variety of structures including triazoles, imidazoles,
imides, oxazoles, thiazoles and any combination of heteroatoms
independently having nitrogen, oxygen or sulfur atoms in the rings.
The linker fragment L can be either a hydrocarbon chain, an ester
group, a thioether, methylene sulfoxide, methylene sulfone or a
simple oxygen, sulfur or carbonyl bridge, preferably NHCO, O, S,
NHCONH or NHCOO. Linker W can be nil (i.e. single bond), resulting
in a compound lacking substituent R.sub.3 or being bound directly
to a carbon atom of the heterocyclic 5-membered ring according to
formula (I), or an alkyl group comprising or consisting of 1 to 6
or 1 to 15, preferably 1 to 10, more preferably 1 to 8, even more
preferably 1 to 5 carbon atoms.
[0019] The heteroaromatic compounds described herein are designed
to bind to pathological forms of SYN, which, based on previous
studies, are usually located in the membranes. In contrast, the
physiological SYN is usually found in the cytoplasmic fraction.
This shows that the compounds of the present invention have access
to the abnormal SYN, while the native molecule is affected by said
compounds.
[0020] Substituents R.sub.1 and R.sub.3 may be identical or
different.
[0021] According to a particularly preferred embodiment of the
present invention, substituent L is NHCONH. It turned out that a
compound of formula (I) or (Ia) having an urea group at this
position is more stable than compounds wherein L is another
substituent.
[0022] The term "pharmaceutically acceptable salt", as used herein,
relates to salts which are toxicologically safe for human and
animal administration. For example, suitable pharmaceutically
acceptable salts include, but are not limited to, salts of
pharmaceutically acceptable inorganic acids such as hydrochloric,
sulphuric, phosphoric, nitric, carbonic, boric and sulfamic acids,
or salts of pharmaceutically acceptable organic acids such as
acetic, propionic, butyric, tartaric, hydroxymaleic, fumaric,
maleic, citric, lactic, gluconic, benzoic, succinic,
methanesulphonic, oxalic, phenylacetic, toluenesulphonic,
benezenesulphonic, salicyclic, sulphanilic, aspartic, glutamic,
edelic, stearic, palmitic, oleic, lauric, pantothenic, tannic,
ascorbic and valeric acids.
[0023] An alternative aspect of the present invention relates to a
compound of formula (Ia):
##STR00002##
wherein
[0024] R.sub.1 is a hydrophilic aromatic or heterocyclic group,
[0025] R.sub.2 is an aliphatic hydrocarbon or alicyclic hydrophobic
group or nil,
[0026] R.sub.3 is an aliphatic or alicyclic group with basic
character,
[0027] X is NH, O, S, or CH.sub.2,
[0028] Y is C.dbd.O, C.dbd.S or C.dbd.NH,
[0029] Z is independently CH, C.dbd.O, O, S, --N-- or NH, and
[0030] L is CH.sub.2, CO.sub.2, CH.sub.2--S, CH.sub.2--SO,
CH.sub.2--SO.sub.2, O, S or C.dbd.O, or nil,
[0031] or a pharmaceutically acceptable salt thereof or a
pharmaceutically acceptable solvate of said compound or salt.
[0032] Z.sub.1 of formula (Ia) may be C or N, Z.sub.2 may be C, N,
C.dbd.O or NH, Z.sub.3 may be C.dbd.O, N or C, Z.sub.4 may be C and
Z.sub.5 may be N, S, NH, O, C.dbd.O or S.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 shows patterns of alpha-synuclein accumulation in the
brains of patients with DLB and PD. (A) Immunoblot analysis showing
increased alpha-synuclein oligomers accumulation in the membrane
fractions of LBD cases compared to controls and AD. (B-E) By
immunocytochemistry, alpha-synuclein accumulates in synapses,
neuronal cell bodies and axons. (F) Molecular dynamics studies
illustrating alpha-synuclein docking to the membranes.
[0034] FIG. 2 shows chemical structure and synthesis of
heteroaromatic organic compounds that inhibit synuclein.
[0035] FIG. 3 shows examples of chemical compositions and formulas
of heteroaromatic organic compounds that inhibit synuclein.
[0036] FIG. 4 shows cell free immunoblot analysis of the effects of
the heteroaromatic organic compounds at blocking synuclein
aggregation.
[0037] FIG. 5 shows immunoblot analysis of the effects of the
heteroaromatic organic compounds at reducing alpha-synuclein
aggregation in a neuronal cell based assay.
[0038] FIG. 6 shows confocal analysis of the effects of the
heteroaromatic organic compounds at ameliorating neuronal
pathology. (A-E) Analysis of levels of neuronal alpha-synuclein
accumulation. (F-J) analysis of the neurite length and
extension.
[0039] FIG. 7 shows an analysis of effects of the heteroaromatic
organic compounds in calcium levels in neuronal cells expressing
alpha-synuclein.
[0040] FIG. 8 shows an overview of the chemical synthesis of a
compound according to the present invention.
[0041] FIG. 9 shows a mass spectrum of the product obtained in
example 1.
DETAILED DESCRIPTION OF THE INVENTION
[0042] R.sub.1, R.sub.2 and R.sub.3 of formula (Ia) are preferably
hydrophilic aromatic or heterocyclic groups, aliphatic hydrocarbon
or alicyclic hydrophobic groups or aliphatic or alicyclic groups
with basic character, respectively.
[0043] According to a preferred embodiment of the present
invention, R.sub.1 of formula (I) is selected from the group
consisting of a phenyl, naphthyl, pyridinyl, pyrimidinyl,
quinolinyl, benzothienyl, indolyl, pyrazinyl, isoindolyl,
isoquinolyl, quinazolinyl, imidazolinyl, benzofuranyl, thienyl,
pyrrolyl and thiazolyl group, or a substituted heteroring structure
comprising alkoxy substituents or halo substituents selected from
the group consisting of fluoro, chloro, bromo or iodo groups.
[0044] According to a further preferred embodiment of the present
invention, R.sub.2 of formula (I) is a substituted or unsubstituted
cycloalkyl group selected from the group consisting of cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, or
a substituted or unsubstituted aryl group selected from the group
consisting of phenyl, alkoxyphenyl and halide substituted phenyl
groups, said halide substituted phenyl groups comprising fluoro,
chloro, bromo or iodo groups.
[0045] According to a preferred embodiment of the present
invention, R.sub.3 of formula (I) is selected from the group
consisting of piperadine, piperazine, morpholino, thiomorpholine,
imidazolo, pyrrolidonyl, pyrrolyl, pyrazolyl, imidazolyl,
imidazolidinyl and substituted N-substituted piperazine comprising
methyl, ethyl, propyl, butyl, pentyl, hexanyl, heptyl or octyl
substituents.
[0046] According to a particularly preferred embodiment of the
present invention, R.sub.1 is a bicyclic heteroaromatic group,
preferably selected from the group consisting of
##STR00003##
[0047] According to a preferred embodiment of the present
invention, R.sub.2 is an alkyl group with 1 to 15, preferably 1 to
10, more preferably 1 to 8, even more preferably 1 to 5, carbon
atoms.
[0048] According to a further preferred embodiment of the present
invention, R.sub.2 is selected from the group consisting of
##STR00004##
[0049] R.sub.3 is preferably a hetero-alicyclic group preferably
selected from the group consisting of
##STR00005##
[0050] According to a preferred embodiment of the present
invention,
##STR00006##
is preferably selected from the group consisting of
##STR00007##
[0051] According to a particularly preferred embodiment of the
present invention the compounds of the present invention having
general structure of formula (I) have the following
substituents:
##STR00008##
TABLE-US-00001 TABLE A No. R1 R2 R3 L W X Y Z 1 ##STR00009##
##STR00010## ##STR00011## NHCONH -- S CH N 2 ##STR00012##
##STR00013## ##STR00014## NHCO -- S CH N 3 ##STR00015##
##STR00016## ##STR00017## NHCOO -- S CH N 4 ##STR00018##
##STR00019## ##STR00020## NHCO -- S N CH 5 ##STR00021##
##STR00022## ##STR00023## NHCO -- NH N N 6 ##STR00024##
##STR00025## ##STR00026## NHCONH -- S N CH 7 ##STR00027##
##STR00028## ##STR00029## NHCONH -- NH N N 8 ##STR00030##
##STR00031## ##STR00032## NHCONH -- S CH N 9 ##STR00033##
##STR00034## ##STR00035## NHCO -- S CH N 10 ##STR00036##
##STR00037## ##STR00038## NHCOO -- S CH N 11 ##STR00039##
##STR00040## ##STR00041## NHCO -- S N CH 12 ##STR00042##
##STR00043## ##STR00044## NHCO -- NH N N 13 ##STR00045##
##STR00046## ##STR00047## NHCONH -- S N CH 14 ##STR00048##
##STR00049## ##STR00050## NHCONH -- NH N N 15 ##STR00051##
##STR00052## ##STR00053## NHCONH -- S CH N 16 ##STR00054##
##STR00055## ##STR00056## NHCO -- S CH N 17 ##STR00057##
##STR00058## ##STR00059## NHCOO -- S CH N 18 ##STR00060##
##STR00061## ##STR00062## NHCO -- S N CH 19 ##STR00063##
##STR00064## ##STR00065## NHCO -- NH N N 20 ##STR00066##
##STR00067## ##STR00068## NHCONH -- S N CH 21 ##STR00069##
##STR00070## ##STR00071## NHCONH -- NH N N 22 ##STR00072##
##STR00073## ##STR00074## NHCONH -- S CH N 23 ##STR00075##
##STR00076## ##STR00077## NHCO -- S CH N 24 ##STR00078##
##STR00079## ##STR00080## NHCOO -- S CH N 25 ##STR00081##
##STR00082## ##STR00083## NHCO -- S N CH 26 ##STR00084##
##STR00085## ##STR00086## NHCO -- NH N N 27 ##STR00087##
##STR00088## ##STR00089## NHCONH -- S N CH 28 ##STR00090##
##STR00091## ##STR00092## NHCONH -- NH N N 29 ##STR00093##
##STR00094## ##STR00095## NHCONH -- S CH N 30 ##STR00096##
##STR00097## ##STR00098## NHCO -- S CH N 31 ##STR00099##
##STR00100## ##STR00101## NHCOO -- S CH N 32 ##STR00102##
##STR00103## ##STR00104## NHCO -- S N CH 33 ##STR00105##
##STR00106## ##STR00107## NHCO -- NH N N 34 ##STR00108##
##STR00109## ##STR00110## NHCONH -- S N CH 35 ##STR00111##
##STR00112## ##STR00113## NHCONH -- NH N N 36 ##STR00114##
##STR00115## ##STR00116## NHCONH -- S CH N 37 ##STR00117##
##STR00118## ##STR00119## NHCO -- S CH N 38 ##STR00120##
##STR00121## ##STR00122## NHCOO -- S CH N 39 ##STR00123##
##STR00124## ##STR00125## NHCO -- S N CH 40 ##STR00126##
##STR00127## ##STR00128## NHCO -- NH N N 41 ##STR00129##
##STR00130## ##STR00131## NHCONH -- S N CH 42 ##STR00132##
##STR00133## ##STR00134## NHCONH -- NH N N 43 ##STR00135##
##STR00136## ##STR00137## NHCONH -- S CH N 44 ##STR00138##
##STR00139## ##STR00140## NHCO -- S CH N 45 ##STR00141##
##STR00142## ##STR00143## NHCOO -- S CH N 46 ##STR00144##
##STR00145## ##STR00146## NHCO -- S N CH 47 ##STR00147##
##STR00148## ##STR00149## NHCO -- NH N N 48 ##STR00150##
##STR00151## ##STR00152## NHCONH -- S N CH 49 ##STR00153##
##STR00154## ##STR00155## NHCONH -- NH N N 50 ##STR00156##
##STR00157## ##STR00158## NHCONH -- S CH N 51 ##STR00159##
##STR00160## ##STR00161## NHCO -- S CH N 52 ##STR00162##
##STR00163## ##STR00164## NHCOO -- S CH N 53 ##STR00165##
##STR00166## ##STR00167## NHCO -- S N CH 54 ##STR00168##
##STR00169## ##STR00170## NHCO -- NH N N 55 ##STR00171##
##STR00172## ##STR00173## NHCONH -- S N CH 56 ##STR00174##
##STR00175## ##STR00176## NHCONH -- NH N N 57 ##STR00177##
##STR00178## ##STR00179## NHCONH -- S CH N 58 ##STR00180##
##STR00181## ##STR00182## NHCO -- S CH N 59 ##STR00183##
##STR00184## ##STR00185## NHCOO -- S CH N 60 ##STR00186##
##STR00187## ##STR00188## NHCO -- S N CH 61 ##STR00189##
##STR00190## ##STR00191## NHCO -- NH N N 62 ##STR00192##
##STR00193## ##STR00194## NHCONH -- S N CH 63 ##STR00195##
##STR00196## ##STR00197## NHCONH -- NH N N 64 ##STR00198##
##STR00199## ##STR00200## NHCONH -- S CH N 65 ##STR00201##
##STR00202## ##STR00203## NHCO -- S CH N 66 ##STR00204##
##STR00205## ##STR00206## NHCOO -- S CH N 67 ##STR00207##
##STR00208## ##STR00209## NHCO -- S N CH 68 ##STR00210##
##STR00211## ##STR00212## NHCO -- NH N N 69 ##STR00213##
##STR00214## ##STR00215## NHCONH -- S N CH 70 ##STR00216##
##STR00217## ##STR00218## NHCONH -- NH N N 71 ##STR00219##
##STR00220## ##STR00221## NHCONH -- S CH N 72 ##STR00222##
##STR00223## ##STR00224## NHCO -- S CH N 73 ##STR00225##
##STR00226## ##STR00227## NHCOO -- S CH N 74 ##STR00228##
##STR00229## ##STR00230## NHCO -- S N CH 75 ##STR00231##
##STR00232## ##STR00233## NHCO -- NH N N 76 ##STR00234##
##STR00235## ##STR00236## NHCONH -- S N CH 77 ##STR00237##
##STR00238## ##STR00239## NHCONH -- NH N N 78 ##STR00240##
##STR00241## ##STR00242## NHCONH -- S CH N 79 ##STR00243##
##STR00244## ##STR00245## NHCO -- S CH N 80 ##STR00246##
##STR00247## ##STR00248## NHCOO -- S CH N 81 ##STR00249##
##STR00250## ##STR00251## NHCO -- S N CH 82 ##STR00252##
##STR00253## ##STR00254## NHCO -- NH N N 83 ##STR00255##
##STR00256## ##STR00257## NHCONH -- S N CH 84 ##STR00258##
##STR00259## ##STR00260## NHCONH -- NH N N 85 ##STR00261##
##STR00262## ##STR00263## NHCONH -- S CH N 86 ##STR00264##
##STR00265## ##STR00266## NHCO -- S CH N 87 ##STR00267##
##STR00268## ##STR00269## NHCOO -- S CH N 88 ##STR00270##
##STR00271## ##STR00272## NHCO -- S N CH 89 ##STR00273##
##STR00274## ##STR00275## NHCO -- NH N N 90 ##STR00276##
##STR00277## ##STR00278## NHCONH -- S N CH 91 ##STR00279##
##STR00280## ##STR00281## NHCONH -- NH N N 92 ##STR00282##
##STR00283## ##STR00284## NHCONH -- S CH N 93 ##STR00285##
##STR00286## ##STR00287## NHCO -- S CH N 94 ##STR00288##
##STR00289## ##STR00290## NHCOO -- S CH N 95 ##STR00291##
##STR00292## ##STR00293## NHCO -- S N CH 96 ##STR00294##
##STR00295## ##STR00296## NHCO -- NH N N 97 ##STR00297##
##STR00298## ##STR00299## NHCONH -- S N CH 98 ##STR00300##
##STR00301## ##STR00302## NHCONH -- NH N N 99 ##STR00303##
##STR00304## ##STR00305## NHCONH -- S CH N 100 ##STR00306##
##STR00307## ##STR00308## NHCO -- S CH N 101 ##STR00309##
##STR00310## ##STR00311## NHCOO -- S CH N 102 ##STR00312##
##STR00313## ##STR00314## NHCO -- S N CH 103 ##STR00315##
##STR00316## ##STR00317## NHCO -- NH N N 104 ##STR00318##
##STR00319## ##STR00320## NHCONH -- S N CH 105 ##STR00321##
##STR00322## ##STR00323## NHCONH -- NH N N 106 ##STR00324##
##STR00325## ##STR00326## NHCONH -- S CH N 107 ##STR00327##
##STR00328## ##STR00329## NHCO -- S CH N 108 ##STR00330##
##STR00331## ##STR00332## NHCOO -- S CH N 109 ##STR00333##
##STR00334## ##STR00335## NHCO -- S N CH 110 ##STR00336##
##STR00337## ##STR00338## NHCO -- NH N N 111 ##STR00339##
##STR00340## ##STR00341## NHCONH -- S N CH 112 ##STR00342##
##STR00343## ##STR00344## NHCONH -- NH N N -- is a single bond
[0052] The compound according to the present invention is
preferably selected from the group consisting of:
##STR00345##
[0053] Compounds which can be summarized under formula (Ia) may
have further the following substituents and structures:
##STR00346##
may be selected from the group consisting of:
##STR00347##
[0054] Formula (Ia) may additionally comprise the following
structures:
##STR00348## ##STR00349##
[0055] The compounds of the present invention can be used for
treating, ameliorating and/or preventing synucleopathies.
[0056] The synucleopathies are preferably selected from the group
consisting of Parkinson's Disease, Parkinson's Disease with
Dementia, Dementia with Lewy bodies, Pick's Disease, Down's
Syndrome, Multiple System Atrophy, Amyotrophic Lateral Sclerosis
(ALS) and Hallervorden-Spatz Syndrome.
[0057] Another aspect of the present invention relates to a
pharmaceutical preparation comprising an effective amount of a
compound according to the present invention or a pharmaceutically
acceptable salt thereof or a pharmaceutically acceptable solvate of
said compound or salt, and one or more pharmaceutically acceptable
excipients.
[0058] The compounds of the present invention or a pharmaceutically
acceptable salt thereof may be formulated by following any number
of techniques known in the art of drug delivery. The compounds or
pharmaceutically acceptable salts thereof may of course be
administered by a number of means keeping in mind that all
formulations are not suitable for every route of administration.
They can be administered in solid or liquid form. The application
may be oral, rectal, nasal, topical (including buccal and
sublingual) or by inhalation. The compounds of the invention or a
pharmaceutically acceptable salt thereof may be administered
together with conventional pharmaceutically acceptable adjuvants,
carriers and/or diluents. The solid dosage forms comprise tablets,
capsules, powders, pills, pastilles, suppositories, gels and
granular forms of administration. They may also include carriers or
additives, such as flavors, dyes, diluents, softeners, binders,
preservatives, lasting agents and/or enclosing materials. Liquid
forms of administration include solutions, suspensions and
emulsions. These may also be offered together with the
above-mentioned additives.
[0059] Solutions and suspensions of the compounds of the invention
or pharmaceutically acceptable salts thereof (provided of course
that these solutions and suspensions have a suitable viscosity) may
be injected. If the suspension is too viscous for injection the
pharmaceutical preparation may be implanted using devices designed
for such purposes. Sustained release forms are generally
administered via parenteral or enteric means. Parenteral
administration is another route of administration of the compounds
of the present invention or pharmaceutically acceptable salts
thereof.
[0060] The administration of the compounds of the present invention
may involve an oral dosage form. Oral dose formulations are
preferably administered once or twice daily, three times daily in
the form of a capsule or tablet, for instance, or alternatively as
an aqueous based solution. If the compounds of the present
invention are administered intravenously, the administration may
occur either daily, continuously, once a week or three times a
week.
[0061] It is also possible to provide pharmaceutical compositions
which, in addition to the compounds of the present invention,
comprise other substances which are suited for treating, preventing
or relieving the symptoms of synucleopathies and Parkinson's-like
disorders. These combinations may be administered in solid or
liquid form in a single formulation or composition or in separate
formulations or compositions.
[0062] According to a preferred embodiment of the present
invention, the pharmaceutical compositions contain from about 0.01
mg to about 5.0 g, preferably from about 0.05 mg to 2 g, more
preferably from about 0.5 mg to 1 g, even more preferably from
about 1 mg to 500 mg, of the compound of the present invention. The
compounds of the present invention can be administered to a patient
in an amount of about 0.01 mg to about 5 g, preferably of about
0.05 mg to 2 g, more preferably from about 0.5 mg to 1 g, even more
preferably from about 1 mg to about 500 mg per kg body ledge
weight.
[0063] The compounds of the present invention may also be provided
as sustained release oral formulations. These formulations
generally comprise the compounds of the invention having decreased
solubility in order to delay absorption into the bloodstream. In
addition, these formulations may include other components, agents,
carriers, etc., which may also serve to delay absorption of the
compounds. Microencapsulation, polymeric entrapment systems, and
osmotic pumps, which may or may not be bioerodible, may also be
used to allow delayed or controlled diffusion of the compounds from
a capsule or matrix.
[0064] As used herein, the term "effective amount" in the context
of treating or preventing alpha-synucleopathies or Parkinson's-like
disorders, especially PD, relates to the administration or addition
of an amount of the compound of the present invention that is
effective for the prevention and/or treatment of existing
synucleopathies or Parkinson's-like disorder. The effective amount
will vary depending on the health and physical condition of the
individual to be treated, the taxonomic group of the individual to
be treated, the formulation of the composition, the assessment of
the medical situations and other relevant factors.
[0065] Another aspect of the present invention relates to the use
of a compound according to the present invention or a
pharmaceutically acceptable salt thereof or a pharmaceutically
acceptable solvate of said compound or salt for the manufacture of
a medicament for treating, ameliorating and/or preventing
synucleopathies.
[0066] The synucleopathies are preferably selected from the group
consisting of Parkinson's Disease, Parkinson's Disease with
Dementia, Dementia with Lewy bodies, Pick's Disease, Down's
Syndrome, Multiple System Atrophy, Amyotrophic Lateral Sclerosis
(ALS) and Hallervorden-Spatz Syndrome.
[0067] A further aspect of the present invention relates to a
method for producing the compounds of the present invention.
[0068] The compounds of formula (I) as well as of formula (Ia) may
be prepared by the methods of known chemical reactions and
procedures, some from starting materials which are well known in
the art. General preparative methods described in "Introduction to
Organic Chemistry" by Streitwieser et al. (Macmillan Publishers,
4th Edition, 1992) can be followed to aid one skilled in the art in
synthesizing these compounds, with more detailed examples being
provided in the reaction schemes below and in the examples.
[0069] Substituted and unsubstituted oxadiazoles, thiazoles,
triazoles, imidazoles, thiatriazoles, thiophenes, pyrroles,
pyrrolines, pyrazoles may be prepared by using standard methods
(see, for example, A R Katritzky, Comprehensive Heterocyclic
Chemistry II, Vol. 5. M H Palmer. Heterocyclic Compounds, Arnold
Ltd, London (1967)).
[0070] Overall reaction schemes for the synthesis of specific
compounds of formula I are shown below.
[0071] The synthesis of oxazoles as exemplified in the example
leading to compound A can be accomplished in the condensation
reaction of two fragments, one containing the indole ring (compound
7) and one containing the oxazole scaffold (compound 13), followed
by deblocking of the piperidine moiety as shown in compound 11.
[0072] Compound 7 is a general intermediate from which other
analogs can be prepared. For example, the triazole compound B can
be synthesized via the compound 7 intermediate via amidation of
compound 18 following deblocking reactions as shown for compound
A.
[0073] Alternatively, an analog containing the thiazole scaffold
can be made via the compound 7 intermediate and intermediate
compound 23 having the thiazole ring.
[0074] A reaction route leading an imide ring scaffold and having
an isobutene side chain in place of the butyl ester group can be
prepared as shown in the reaction scheme involving intermediate
compounds 25-34.
[0075] Still other variations are shown in the scheme involving the
synthesis of compounds E and F.
[0076] Reaction schemes for the synthesis of inhibitors:
##STR00350## ##STR00351## ##STR00352##
[0077] A modification of the above scheme starting with tryptophan
is shown below. This modification significantly reduces the number
of steps needed to obtain the final products.
[0078] The reaction of equimolar quantities of tryptophan (compound
1) with butanol yields compound 2. The amino group in piperazine
carboxylic acid can be blocked by standard methods described in T W
Greene et. al. (Protective Groups in Organic Synthesis, Third
edition, Wiley Interscience (1999)), which can be further reacted
with compound 9 in the general scheme to obtain the oxazole
intermediate. Deesterification of 12 with sodium hydroxide yields
the carboxylic acid 13. Reaction of intermediate 13 with compound 2
yields the intermediate 14, and deblocking of 14 gives the final
product (compound A1).
##STR00353##
[0079] A variation of the above synthetic procedure as shown below
yields the triazole intermediate compound 18, which can be further
reacted similarly to compound A1 using the intermediate compound 2
to give the final triazole analog compound B1.
##STR00354##
[0080] Alternatively, the procedures can be adapted to obtain a
thiazole analog as shown in the example of the reaction scheme
below.
##STR00355##
[0081] All reactions can be performed in flame-dried or oven dried
glassware under positive pressure of argon or nitrogen. The
reaction vessels were stirred magnetically. Sensitive liquids and
solutions were transferred via syringe and introduced into the
reaction vessels through rubber septa.
[0082] Another aspect of the present invention relates to a method
for treating, ameliorating and/or preventing synucleopathies and/or
their symptoms by administering to an individual suffering or being
at risk to suffer from said synucleopathies an effective amount of
a compound or a pharmaceutical preparation according to the present
invention.
[0083] A further aspect of the present invention relates to the use
of the compounds of the present invention as biomarkers. The
compounds of the present invention can be used, when labelled
accordingly (e.g., radioactively), in positron emission tomography
(PET) for determining whether a patient comprises .alpha.-synuclein
or any other plaques to which the compounds of the present
invention are able to bind. This allows the localization of the
plaques within the body and also allows identification of the
amount of said plaques. This allows the medical doctor to treat or
prevent conditions associated with synucleopathy. Methods to label
compounds are accordingly well known in the art.
[0084] The present invention is further illustrated by the
following figures and examples without being restricted
thereto.
EXAMPLES
Example 1
[0085] The chemical synthesis of the compounds having formula (I)
as defined above is exemplified by the synthesis of the compound
having the following structure:
##STR00356##
[0086] The synthesis scheme is depicted in FIG. 8.
1. Synthesis of 2-piperazinyl-thiazole-5-carboxylic acid 2
[0087] 2-Bromothiazole carboxylic acid 1 (1 g; 4.8 mmol) and
piperazine (6.2 g; 72 mmol; 24 equiv.) were dissolved in dioxane,
K.sub.2CO.sub.3 (3.32 g; 24 mmol; 5 equiv.) was added and the
suspension was refluxed overnight. The solvent was removed at the
rotary evaporator, the residue dissolved in ethanol, filtered and
recrystallized. The crude product was dried at the oil pump and
directly used in the following step.
2. Protection of 2 with Boc
[0088] Crude 1, triethylamine (24 mmol; 3.4 ml) and
di-tert-butyl-dicarbonate (14.4 mmol; 3.14 g) were dissolved in
methanol and refluxed overnight. The solvents were removed at the
rotary evaporator and the residue was chromatographed over a short
silica gel column.
[0089] Yield of 3: 1.18 g (72% over two steps).
3. Synthesis of tryptophan butylester 5
[0090] L-Tryptophan (5 g; 24.5 mmol) and thionyl chloride (73.5
mmol; 5.3 ml) were dissolved in n-butanol (80 ml) and stirred at
100.degree. C. overnight. The product 5 precipitated after cooling
to room temperature and was filtered off and washed with ice cold
butanol and petrol ether. The product was dried at the oil pump,
yielding 5.8 g of 5 (91%).
4. Coupling of 3 and 5
[0091] a.) Formation of the Succinimidyl Ester
[0092] 2-(1-Boc-piperazin-4-yl)-thiazole-5-carboxylic acid 3 (250
mg; 0.8 mmol), N-hydroxysuccinimide (110 mg; 0.95 mmol),
diisopropylcarbodiimide (0.15 ml; 0.95 mmol) and DMAP (5 mg; 0.04
mmol) were dissolved in dry dichloromethane and stirred at room
temperature overnight (reaction monitoring by thin layer
chromatography). A 1 M aqueous solution of KHSO.sub.4 was added,
the precipitate was removed by filtration and the organic phase was
extracted with water.
[0093] b.) Coupling with 5
[0094] The crude succinimidyl ester, tryptophan butyl ester 5 (250
mg; 0.96 mmol) and triethylamine (0.65 ml) were dissolved in dry
THF (5 ml) and stirred at 50.degree. C. for 24 h. THF was removed
in vacuo. The residue was taken up in dichloromethane and extracted
with aqueous 1 M KHSO.sub.4. Crude 6 was used in the following step
without purification.
5. Formation of n-butyl amide 7
[0095] Crude 6 was dissolved in 10 ml n-butylamine and refluxed
overnight (reaction monitoring by HPLC-MS). The solvent
n-butylamine was removed at the rotary evaporator, the residue was
dried at the oil pump, recrystallized from methanol and purified by
column chromatography (DCM.fwdarw.MeOH). Yield: 340 mg (0.61 mmol;
64% over two steps).
6. Deprotection of 7
[0096] The Boc-protected 6 was dissolved in 45% THF, 45%
trifluoroacetic acid and 10% water, and the THF and TFA were slowly
removed at the rotary evaporator. The residue was lyophilized,
precipitated in diethyl ether and purified by chromatography on
silica gel (DCM.fwdarw.MeOH). Yield: 223 mg (0.49 mmol; 80%). The
product obtained was subjected to mass spectrometry (see FIG.
9).
[0097] Thin-layer chromatography was performed using Whatman
pre-coated glass-backed silica gel plates. Visualization of the
gels was effected by either ultraviolet illumination of exposure to
iodine vapour. Column chromatography was performed using 230-400
mesh EM Science silica gel.
[0098] NMR spectra were measured using a Varian 500
spectrophotometer. NMR spectra were measured with deuterated
chloroform, methanol or DMSO, as standard.
[0099] LC/Mass spectra were obtained on an Agilent 1100 series
instrument equipped with a quaternary pump, a variable length
detector and a C-18 column.
Example 2
The Compounds of Formula Ia
##STR00357##
[0100] may be prepared by the methods of known chemical reactions
and procedures, some from starting materials which are well known
in the art.
Example 3
[0101] To screen the effectiveness and ideal doses for the HAOC
(Heteroaromatic organic compounds) of the present invention at
blocking SYN aggregation two sets of assays can be utilized. The
first set involves in vitro assays in cell free and cell based
systems and the second includes in vivo studies in transgenic mouse
models of PD.
[0102] In the present example the following HAOC, denominated as
NPT200-5, was used:
##STR00358##
[0103] The objective is to identify with the in vitro assays a
positive response by demonstrating a 50% effect in 2 out of the 3
assays at a 1 .mu.M dose.
[0104] 1. In Vitro Assays of SYN Aggregation and Toxicity
[0105] The in vitro studies include the following: i) effects on
SYN oligomers in a cell free immunoblot assay of SYN aggregation;
ii) effects on SYN accumulation and neurite outgrowth in neuronal
cultures infected with a LV-SYN construct; and iii) effects on SYN
oligomers in neuronal cultures infected with a LV-SYN
construct.
[0106] For this purpose, recombinant SYN (1 .mu.M, Calbiochem, USA)
will be incubated at 37 and then at 56.degree. C. for 16 hrs. After
1 hour of incubation, the NPT200-5 and analogs will be added to the
mix at concentrations ranging from 1 nM to 100 .mu.M. Samples will
be subjected to immunoblot analysis with the rabbit polyclonal SYN
antibody (Millipore) and mouse monoclonal antibody against SYN
(SYN211, 1:1000, Sigma) and analyzed in the VersaDoc imaging system
using the Quantity One software (BioRad, Hercules, Calif.,
USA).
[0107] For the neuronal cell based assays, the neuroblastoma line
B103 will be used. Cells will be infected with LV-SYN vector for 24
hrs, treated with the NPT200-5 at 0, 0.1, 1 and 10 .mu.M for 24 hrs
in serum free media. To infect the neural cells with LV vectors,
multiples of infections (MOI) of 0.1, 1 or 5 (based on TU/ml on
293T cells) will be used. After 4-5 days in vitro, the % of
transgene-expressing cells will be analyzed. The B103 cells will be
maintained at 37.degree. C., 5% CO.sub.2 in Dulbecco's modified
eagle medium (DMEM, high glucose) supplemented with 10% fetal
bovine serum (Irvine Scientific, Irvine, Calif.) and 1% v/v
penicillin/streptomycin. For analysis of SYN aggregation cell
homogenates will be analyzed by immunoblot with antibodies against
SYN and in coverslips by immunocytochemistry. For evaluation of
neurite outgrowth, coverslips will be immunostained with an
antibody against MAP2 and analyzed with a digital Olympus
microscope and the Image Quant System.
[0108] The LDH release assay (CytoTox 96 assay, Promega) will be
performed to measure levels of toxicity (if any). Additional
confirmation of cell viability will be obtained utilizing Hoechst
staining and calcein AM/ethidium homodimer staining (Live/Dead
assay, Molecular Probes). All assays will be performed in
triplicate in 96-well plates according to the manufacturers'
instructions.
[0109] 2. In Vivo Studies in Transgenic Models of SYN
Accumulation.
[0110] Once a series of compounds are identified to be most active
the next step will be to inject NPT200-5 and controls into our SYN
transgenic (tg) mice, to test the in vivo effects. The first set of
experiments will be daily injections for 2 weeks with compounds at
1, 10 and 100 nM. Blood, CSF, brain and liver will be analyzed for
levels of SYN and compound. After preliminary data is obtained,
more extensive studies with groups of 20 mice will be performed
with daily injections in 3 and 6 month old mice for 3 and 6 month
durations of treatment. Mice will be analyzed behaviorally,
neuropathologically and biochemically for SYN aggregation and
neurodegeneration. Blood and CSF will be analyzed for levels of SYN
and NPT200-5 by mass spectrometry and NMR. The compounds will be
further refined and modified to increase permeability, access into
the brain and bio-availability. The selected compounds will be
first tested for toxicity in non tg mice. SYN knockout mice are
viable and neurologically intact. This suggests that using a
compound that blocks SYN will have low or no toxicity when tested
in the SYN tg mice.
[0111] The lead compounds screened from these in vivo experiments
will then be submitted for toxicological studies and prepared for a
phase I clinical trial. The long term objective is to obtain
funding and develop this compound for a phase II clinical trial in
patients with PD.
[0112] The compounds of the present invention lead to a novel
therapy for PD, LBD, AD and MSA based on blocking neurotoxic SYN
oligomerization in the cell membrane.
[0113] Computer simulations and calculations to pre-screen for the
compounds that most likely might block SYN aggregation were
performed. One (FIG. 4) of these heteroaromatic organic compounds
with the appropriate controls was tested in a cell-free system. For
this purpose recombinant SYN (10 .mu.M) was incubated at 37.degree.
C. for 0, 8, 16, and 24 hours with the peptides at 0, 0.1, 1 and 10
.mu.M. Control experiments were performed with compounds that did
not recognize the aggregated SYN molecules (control-1), with beta
and gamma-synuclein as well as with a mutant SYN molecule that
could not bind the peptide. The mixture was run in a gel, followed
by immunoblot testing with SYN antibodies. This study showed that
NPT200-5 (FIG. 2) was capable of completely blocking SYN
aggregation at early and later time points of the oligomerization
process (FIG. 4). For this assay, SYN was used at 5 .mu.M. The
NPT200-5 reduced SYN aggregation by 50% at the 0.1 .mu.M
concentration.
[0114] To test the activity of the NPT200-5 in vivo, the B103
neuronal cell line was infected with a lentivirus expressing SYN
(wildtype) or an empty vector (control), and cells expressing SYN
were exposed to the NPT200-5 at 0, 0.1, 1 and 10 .mu.M for 24 hrs.
Cells were analyzed for SYN aggregation by immunoblot, confocal
microscopy, neurite outgrowth and survival assays. By immunoblot,
compared to controls, neuronal cells infected LV-SYN displayed the
presence of high level expression of SYN monomer (14 kDa) as well
as oligomers consistent with dimers, trimers and tetramers in the
soluble and insoluble fractions (FIG. 5). After treatment with
NPT200-5, there was a 50-60% reduction in the levels of aggregates
(but also the monomers) in the various fractions (FIG. 5).
Treatment with vehicle or with a control inactive compound had no
effects in the levels of SYN. The NPT200-5 reduced SYN levels by
50% at the 0.1 .mu.M concentration.
[0115] Similarly, neuronal cells were plated in coverslips,
infected with the LV-SYN vector for 24 hrs, treated with the
NPT200-5 at 0, 0.1, 1 and 10 .mu.M for 24 hrs in serum free media
and analyzed by immunocytochemistry, confocal microscopy and image
analysis. Compared to LV-empty vector control, neuronal cells
infected with LV-SYN showed high levels of SYN accumulation
(similar to what may be observed in the brains of SYN tg mice and
patients with PD) (FIG. 6). After treatment with NPT200-5, there
was a 60-65% reduction in the levels of aggregates in the neuronal
cell bodies and neurites (FIG. 6). Treatment with vehicle or with a
control inactive compound had no effects on the levels of SYN. The
NPT200-5 reduced SYN levels by 50% at the 0.1 .mu.M concentration.
Neuronal cells expressing high levels of SYN displayed reduced
neurite outgrowth when analyzed with an antibody against the
cytoskeletal protein MAP2. The NPT200-5 treatment (0.1 .mu.M)
ameliorated the deleterious effects on neurite length extension and
improved cellular morphology (FIG. 6). Treatment with vehicle or
with a control inactive compound had no protective effects.
[0116] Next, to ascertain the effects on neuronal activity, cells
were infected with the LV-SYN vector for 24 hrs, treated with the
NPT200-5 at 0, 0.1, 1 and 10 .mu.M for 24 hrs in serum free media,
loaded with Flou-4 and analyzed by FLIPR assay to determine
Ca++levels. Compared to the LV-empty vector control, neuronal cells
infected with LV-SYN showed 25-30% higher levels of Ca++flow (FIG.
7). After treatment with NPT200-5, levels of Ca++were back to
baseline (FIG. 7). Treatment with the vehicle or with a control
inactive compound was unable to re-establish Ca++levels. The
NPT200-5 improved Ca++levels by 50% at the 0.1 .mu.M concentration.
Finally, to examine the effects on neuronal survival, the MTT, LDH
and BrDu assays were performed. This study showed no toxic effects
of the NPT200-5 compounds at doses ranging from 0.1-10 .mu.M (FIG.
7). All in vitro and cell-based assays were repeated at least 4
times and experiments were performed blind.
[0117] The next step was to inject NPT200-5 and controls into SYN
transgenic (tg) mice, to test the in vivo effects. The first set of
experiments was daily injections for 2 weeks with compounds at 1,
and 100 nM. Blood, CSF, brain and liver were analyzed for levels of
SYN and compound. After preliminary data had been obtained, more
extensive studies with groups of 20 mice were performed with daily
injections in 3 and 6 month old mice for 3 and 6 month durations of
treatment. Mice were analyzed behaviorally, neuropathologically and
biochemically for SYN aggregation and neurodegeneration. Blood and
CSF were analyzed for levels of SYN and NPT200-5 by mass
spectrometer and NMR. The compounds were further refined and
modified to increase permeability, access into the brain and
bio-availability. The selected compounds were tested for toxicity
in non tg mice. SYN knockout mice are viable and neurologically
intact. This suggests that using a compound that blocks SYN will
have low or no toxicity when tested in the SYN tg mice.
* * * * *