U.S. patent application number 12/138261 was filed with the patent office on 2008-12-11 for methods and compositions for treatment of central and peripheral nervous system disorders and novel compounds useful therefor.
This patent application is currently assigned to Israel Institute for Biological Research. Invention is credited to Nira Bar-Ner, Abraham Fisher, Yishai Karton.
Application Number | 20080306103 12/138261 |
Document ID | / |
Family ID | 29401496 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080306103 |
Kind Code |
A1 |
Fisher; Abraham ; et
al. |
December 11, 2008 |
METHODS AND COMPOSITIONS FOR TREATMENT OF CENTRAL AND PERIPHERAL
NERVOUS SYSTEM DISORDERS AND NOVEL COMPOUNDS USEFUL THEREFOR
Abstract
There are provided methods for the treatment of diseases
involving dysfunction of the peripheral and central nervous system
comprising administering one or more spiro compounds. Also provided
are pharmaceutical compositions useful in such methods, compounds
for use in the preparation of such pharmaceutical compositions,
processes for preparing compounds useful in the practice of such
methods, and some novel such compounds per se.
Inventors: |
Fisher; Abraham; (Holon,
IL) ; Bar-Ner; Nira; (Rishon de Zion, IL) ;
Karton; Yishai; (Ness Ziona, IL) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
Israel Institute for Biological
Research
|
Family ID: |
29401496 |
Appl. No.: |
12/138261 |
Filed: |
June 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10512673 |
Jul 8, 2005 |
7439251 |
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PCT/IL03/00357 |
May 1, 2003 |
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12138261 |
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60377433 |
May 3, 2002 |
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Current U.S.
Class: |
514/278 ;
546/18 |
Current CPC
Class: |
A61P 1/10 20180101; A61P
25/14 20180101; C07D 491/10 20130101; A61P 1/00 20180101; A61P
11/00 20180101; A61P 25/16 20180101; A61P 25/20 20180101; A61P
25/24 20180101; A61P 13/10 20180101; A61P 25/32 20180101; A61P
15/02 20180101; A61P 25/22 20180101; A61P 3/04 20180101; A61P 29/00
20180101; A61P 13/08 20180101; A61P 25/28 20180101; A61P 1/04
20180101; A61P 25/30 20180101; C07D 513/10 20130101; A61P 37/04
20180101; A61P 25/04 20180101; A61P 27/02 20180101; A61P 43/00
20180101; A61P 9/00 20180101; A61P 37/00 20180101; A61P 25/02
20180101; A61P 27/04 20180101; A61P 25/18 20180101; C07D 471/10
20130101; A61P 11/06 20180101; A61P 1/12 20180101; A61P 25/00
20180101; C07B 2200/05 20130101; A61P 17/16 20180101; A61P 19/02
20180101; A61P 27/06 20180101 |
Class at
Publication: |
514/278 ;
546/18 |
International
Class: |
A61K 31/438 20060101
A61K031/438; C07D 495/10 20060101 C07D495/10; A61P 43/00 20060101
A61P043/00 |
Claims
1.-130. (canceled)
131. A method of preventing or treating a disease or disorder
associated with M1 muscarinic receptors, said method comprising
administering to a subject in need thereof an effective amount of a
pharmaceutical composition comprising at least one compound of the
formula (I): ##STR00058## wherein: C denotes a spiro carbon atom
shared by ring A and the ring containing a, b, d and c; A is
selected from the group consisting of: ##STR00059## R is selected
from H, C.sub.1-C.sub.8 straight- or branched-chain alkyl or
--CH.sub.2--P(O)(OH).sub.2; a is --O--, --S-- or --S(O)--; b is
--CR.sup.1R.sup.2-- or --C(R.sup.1).dbd.; d is selected from the
group consisting of .dbd.N--, --C(.dbd.O)--, --C(.dbd.S)-- and
--C.dbd.N(R.sup.3).dbd.O; e is selected from the group consisting
of --CH.sub.2--, --CHR.sup.4--, --NH--, --NR.sup.5--,
--N(SO.sub.2R.sup.6)-- and --N(C(.dbd.O)R.sup.6)--; R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are each independently selected from
the group consisting of H, C.sub.1-6 alkoxy, C.sub.2-6
hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and C.sub.1-6
alkyl optionally substituted by one to three phenyls; R.sup.5 is
selected from the group consisting of H, C.sub.1-6 alkoxy,
C.sub.2-6 hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
optionally substituted phenyl, heteroaryl, and C.sub.1-6 alkyl
optionally substituted by one to three phenyls; or, when the
compound of formula (I) is a dimer, in which both halves of the
dimer share a single R.sup.5, R.sup.5 is selected from the group
consisting of --(CH.sub.2).sub.n-- and --(CH.sub.2O).sub.n--,
wherein n is 1 to 6; and R.sup.6 is selected from the group
consisting of C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.2-6 hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, and C.sub.3-7 cycloalkyl, each optionally substituted by
from 1-6 halogen atoms, hydroxy-C.sub.1-6 alkyl, aryl substituted
with a halogen, nitro, amino, hydroxyl, or CF.sub.3 group,
C.sub.1-6 alkyl substituted by one to three aryl groups, and
C.sub.m alkyl-X, wherein m=0 to 6 and X is selected from the group
consisting of indole, C.sub.1-6 alkyl indole, isoindolyl,
3-pyridinyl, 3-piperidinyl, benzimidazolyl, thienyl, isothiazolyl,
imidazolyl, pyrazinyl, benzofuranyl, quinolyl, isoquinolyl,
benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl,
benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl,
isothiazolyl, 1,2,5-thiadiazolyl, isooxazolyl, pyrrolyl, pyrazolyl,
quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl,
quinoxalinyl, xanthinyl, hypoxanthinyl, and pteridinyl; or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite, prodrug or pharmaceutically acceptable salt
thereof, with the proviso that when A is ##STR00060## R is
--CH.sub.3, a is S, b is --CH(CH.sub.2CH.sub.3)-- and d is
--C(.dbd.O)--, then e is not --NH-- (AF267) or an enantiomer
thereof, and with the further proviso that when A is ##STR00061## R
is --CH.sub.3, a is S, b is --C(CH.sub.3)-- and d is .dbd.N--, then
e is not --CH.sub.2-- (AF150(S)).
132. The method of claim 131, wherein said compound is selected
from the group consisting of:
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292),
thia-4,8-diaza-spiro[4.5]decan-3-one;
4-(2,4-Dimethoxybenzyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF286);
8-Methyl-4-pyrrolidin-1-ylmethyl-1-thia-4,8-diaza-spiro[4.5]deca-
n-3-one (AF287);
2-(1-Hydroxy-ethyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF298);
(S)-2-ethyl-8-methyl-3-oxo-1-thia-4,8-diaza-spiro[4,5]decane
8-oxide (AF299);
4-(2,4-Dimethoxy-benzyl)-2-ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]deca-
n-3-one (AF288);
(S)-2-Ethyl-8-methyl-1-oxo-1.lamda..sup.4-thia-4,8-diaza-spiro[4.5]decan--
3-one (AF300);
2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decane-3-thione
(AF510);
(S)-2-Ethyl-4-(4-fluoro-benzenesulfonyl)-8-methyl-1-thia-4,8-dia-
za-spiro[4.5]decan-3-one (AF700);
2-Ethyl-4-[2-(1H-indol-3-yl)-ethyl]-8-methyl-1-thia-4,8-diaza-spiro[4.5]d-
ecan-3-one (AF703);
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one (AF704);
(S)-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4-
.5]decan-3-one (AF704B);
(R)-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4-
.5]decan-3-one (AF704A);
2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF504),
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292) and
(R)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF291) or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
metabolite, prodrug or a pharmaceutically acceptable salt
thereof.
133. The method of claim 131, wherein said composition further
comprises at least one additional pharmacologically active compound
selected from the group consisting of cholinesterase inhibitors,
nicotinic agonists, cholinergic precursors and cholinergic
enhancers, nootropics, peripheral antimuscarine drugs, M2
muscarinic antagonists, M4 antagonists, benzodiapine inverse
agonists, antidepressants, tricyclic antidepressants or
antimuscarinic drugs used in treatment of Parkinson's disease (PD)
or depression, antipsychotic and antischizophrenic agents,
glutamate antagonists and modulators, NMDA antagonists, AMPA
agonists, acetyl-L-carnitine, MAO-B inhibitors, peptides and growth
factors, cholesterol-lowering agents, antioxidants, GSK-3p
inhibitors, Wnt-ligands, P- or y-secretase inhibitors, beta-amyloid
degrading agents, beta-amyloid anti-aggregation agents, cheating
agents, immunotherapeutic compounds against beta-amyloids,
compounds that bind to amyloids, cyclooxygenase (COX)-2 inhibitors,
non-steroidal antiinflammatory drugs, estrogenic agents, estrogenic
receptor modulators, steroidal neuroprotectants, and spin trapping
pharmaceuticals.
134. The method of claim 131, wherein said composition further
comprises at least one additional compound selected from the group
consisting of 2,8-Dimethyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene (AF
150B) and
(S)-2-ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF267B).
135. The method of claim 131, wherein said disease or disorder is
also associated with impaired cholinergic function or where there
is an imbalance in cholinergic function, or with impaired activity
of acetylcholine receptors, and wherein said disease or disorder is
selected from the group consisting of senile dementia of
Alzheimer's type; Alzheimer's disease (AD); Lewy body dementia;
mixed Alzheimer's and Parkinson's disease; multiifract dementia
(MID); fronto-temporal dementia; vascular dementia;
stroke/ischemia, MID combined with stroke/ischemia/head injury;
combined MID and AD; human head injury; age-associated memory
impairments; mild cognitive impairment (MCI); MCI conducive to AD;
cognitive dysfunction; hallucinatory-paranoid states; emotional and
attention disorders; sleep disorders; postoperative delirium;
adverse effects of tricyclic antidepressants; adverse effects of
certain drugs used in the treatment of schizophrenia and
Parkinson's disease; xerostomia, anomia, memory loss and/or
confusion; psychosis; schizophrenia, schizophrenia comorbit with
AD, late onset schizophrenia, paraphrenia, schizophreniform
disorders; anxiety; bipolar disorders; mania; mood stabilization;
cognitive impairments after removal of certain gliomas; tardive
dyskinesia; oxidative stress during oxygen therapy; aphasia;
postencephalitic amnesic syndrome; AIDS dementia; memory
impairments in autoimmune diseases; memory impairments in atypical
depression or schizophrenia; pain, rheumatism, arthritis and
terminal illness; xerophtalmia, vaginal dryness, skin dryness;
immune dysfunctions; neurocrine disorders and dysregulation of food
intake, including bulimia and anorexia; obesity; congenital
ornithine transcarbamylase deficiency; ollivopontocerebral atrophy;
alcohol withdrawal symptoms; substance abuse; Huntington's chorea;
progressive supranuclear palsy; Pick's disease; Friedrick's ataxia;
Gilles de la Tourette disease; Down's syndrome: glaucoma;
fronto-temporal dementia; vascular dementia; mild cognitive;
anxiety; bipolar disorders; ollivopontocerebral atrophy;
presbyopia; autonomic disorders; urinary urge incontinence, asthma
and COPD.
136. The method of claim 131, wherein said disease or disorder is
associated with dysfunction in one or more of the following: brain,
nervous system, cardiovascular system, immune system, neurocrine
system, gastrointestinal system, endocrine and exocrine glands,
eye, cornea, lungs, prostate, or other organs where the cholinergic
function is mediated by muscarinic receptor subtypes, wherein said
dysfunction involves brain amyloid-mediated disorders; glycogen
synthase kinase (GSK3p)-mediated disorders; tau protein
hyperphosphorylation-mediated damages, dysfunctions or diseases;
CNS and PNS hypercholesterolemia- and/or hyperlipidemia-mediated
damages, dysfunctions or diseases; Wnt-mediated signaling
abnormalities; impairment of neuroplasticity; hyperglycemia;
diabetes; endogenous growth factors-mediated diseases, or
combination of additional risk factors; or disease states that
involve apolipoprotein E; or disturbances in which a cholinergic
dysfunction has been implicated, and wherein said disease or
disorder is selected from the group consisting of senile dementia
of Alzheimer's type; Alzheimer's disease (AD); delay of onset of AD
symptoms in a patient at risk for developing AD; Lewy body
dementia; cerebral amyloid angiopathy (CAA); cerebral amyloidosis;
fronto-temporal dementia; vascular dementia; hyperlipidemia;
hypercholesterolemia; multiifract dementia (MID; stroke ischemia;
MID combined with stroke/ischemia/head injury; combined MID and
Alzheimer's disease; human head injury; age-associated memory
impairments; mild cognitive impairment (MCI); MCI conducive to AD;
bipolar disorder; mania; schizophrenia; nonaffective
sychozophrenia; paraphrenia; immune dysfunctions; neurocrine
disorders and dysregulation of food intake.
137. The method of claim 131 wherein said compound of formula (I)
is AF792 or a pharmaceutically acceptable salt thereof.
138. The method of claim 133 wherein said compound of formula (I)
is AF292 or a pharmaceutically acceptable salt thereof.
139. The method of claim 134 wherein said compound of formula (I)
is AF292 or a pharmaceutically acceptable salt thereof.
140. The method of claim 135 wherein said compound of formula (I)
is AF292 or a pharmaceutically acceptable salt thereof.
141. The method of claim 136 wherein said compound of formula (I)
is AF292 or a pharmaceutically acceptable salt thereof.
142. A method for achieving one or more of the following biological
effects: (1) inhibiting the release or synthesis of beta-amyloid
peptide A.beta. in a mammalian cell, tissue or organism, (2)
elevating the level of secreted form of the non-amyloidogenic
amyloid precursor protein, (3) decreasing the level of A.beta.
peptide in the brain of a mammal having an elevated level of
A.beta. in the brain, (4) decreasing level of or inhibiting the
release or synthesis of Apolipoprotein in a mammalian cell, tissue
or organism, (5) decreasing tau hyperphosphorylation in a mammalian
cell, tissue or organism, (6) stimulating the M1 muscarinic
receptor and activating .alpha.-secretase, (7) stimulating the M1
muscarinic receptor and antagonizing one of more of
.beta.-secretase, .gamma.-secretase, or M3-muscarinic receptor, (8)
decreasing paired helical formation in a mammalian cell, (9)
activating the Wnt signaling pathway in a mammalian cell, tissue or
organism, (10) enhancing the activity of endogenous growth factors,
(11) inhibiting GSK3.beta.-mediated effects in a mammal or (12)
treating or reducing cerebral amyloid angiopathy, said method
comprising administering to a system or subject in need thereof an
effective amount of a pharmaceutical composition comprising a
compound or a mixture of compounds selected from the group
consisting of compounds according to formula (I), AF267B and AF150
(S), or racemates, enantiomers, geometrical isomers, diasteromers,
tautomers and pharmaceutically acceptable salts thereof.
143. The method according to claim 142, wherein said compound of
formula (I) is AF292.
144. A method of preventing or treating xerostomia, said method
comprising administering to a subject in need thereof an effective
amount of a pharmaceutical composition comprising AF267B and at
least one pharmaceutically acceptable carrier, diluent or
excipient.
145. A method of preventing or treating impairment associated with
schizophrenia, said method comprising administering to a subject in
need thereof an effective amount of a pharmaceutical composition
comprising AF267-B and at least one pharmaceutically acceptable
carrier, diluent or excipient.
146. A compound of the formula (II): ##STR00062## wherein: C
denotes a spiro carbon atom shared by ring A and the ring
containing a, b, d and e; A is selected from the group consisting
of: ##STR00063## wherein the bridgehead nitrogen is optionally
oxidized to form an N-oxide, R is selected from H, O,
C.sub.1-C.sub.8 straight- or branched-chain alkyl, or
--CH.sub.2--P(.dbd.O)(OH).sub.2; a is --O--, --S-- or --S(O)--; b
is --CR.sup.1R.sup.2-- or --C(R.sup.1)=; d is selected from the
group consisting of .dbd.N--, --C(.dbd.O)--, --C(.dbd.S)-- and
--C.dbd.N(R.sup.3).dbd.O; e is selected from the group consisting
of --CH.sub.2--, --CHR.sup.4--, --NH--, --NR.sup.5--,
--N(SO.sub.2R.sup.6)-- and --N(C(.dbd.O)R.sup.6)--; R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are each independently selected from
the group consisting of H, C.sub.1-6 alkoxy, C.sub.2-6
hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and C.sub.1-6
alkyl optionally substituted by one to three phenyls; R.sup.5 is
selected from the group consisting of H, C.sub.1-6 alkoxy,
C.sub.2-6 hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
optionally substituted phenyl, heteroaryl, and C.sub.1-6 alkyl
optionally substituted by one to three phenyls; or, when the
compound of formula (II) is a dimer, in which both halves of the
dimer share a single R.sup.1, R.sup.5 is selected from the group
consisting of --CH.sub.2).sub.n-- and --CH.sub.2O).sub.n-- wherein
n is 1 to 6; and R.sup.6 is selected from the group consisting of
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, C.sub.2-6
hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and C.sub.3-7
cycloalkyl, each optionally substituted by from 1-6 halogen atoms,
hydroxy-C.sub.1-6 alkyl, aryl substituted with a halogen, nitro,
amino, hydroxyl, or CF.sub.3 group, C.sub.1-6 alkyl substituted by
one to three aryl groups, and C . . . alkyl-X, wherein m=0 to 6 and
X is selected from the group consisting of indole, C.sub.1-6 alkyl
indole, isoindolyl, 3-pyridinyl, 3-piperidinyl, benzimidazolyl,
thienyl, isothiazolyl, imidazolyl, pyrazinyl, benzofuranyl,
quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl,
indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl,
thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, isooxazolyl, pyrrolyl,
pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl,
phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, and
pteridinyl; or an enantiomer, diastereomer, racemate, tautomer,
geometrical isomer, dimer, metabolite or pharmaceutically
acceptable salt thereof, with the proviso that when A is
##STR00064## R is --H or --CH.sub.3, a is S, b is --CH(CH
CH.sub.3)-- and d is --C(.dbd.O)--, then e is not --NR.sup.5--, and
with the further proviso that when A is ##STR00065## R is
--CH.sub.3, a is S, b is --C(CH.sub.3).dbd. and d is .dbd.N--, then
e is not --CH.sub.2-- (AF150(S)).
147. The compound of claim 146, wherein said compound is selected
from the group consisting of, thia-4,8-diaza-spiro[4.5]decan-3-one;
4-benzyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF282);
4-(2,4-Dimethoxybenzyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF286);
8-Methyl-4-pyrrolidin-1-ylmethyl-1-thia-4,8-diaza-spiro[4.5]deca-
n-3-one (AF287);
2-(1-Hydroxy-ethyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF298);
(S)-2-Ethyl-8-methyl-8-oxy-1-thia-4,8-diaza-spiro[4.5]decan-3-on- e
(AF299);
4-(2,4-Dimethoxy-benzyl)-2-ethyl-8-methyl-1-thia-4,8-diaza-spir-
o[4.5]decan-3-one (AF288);
(S)-2-Ethyl-8-methyl-1-oxo-1.lamda..sup.4-thia-4,8-diaza-spiro[4.5]decan--
3-one (AF300);
2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-thione (AF510);
(S)-2-Ethyl-4-(4-fluoro-benzenesulfonyl)-8-methyl-1-thia-4,8-dia-
za-spiro[4.5]decan-3-one (AF700);
2-Ethyl-4-[2-(1H-indol-3-yl)-ethyl]-8-methyl-1-thia-4,8-diaza-spiro[4.5]d-
ecan-3-one (AF703);
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one (AF704);
(S)-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4-
.5]decan-3-one (AF704B);
4-(tert-Butyloxycarbonyl)-8-methyl-1-thia-4,8-diaza-spiro[4,5]decan-3-one
(AF284); 2-Methyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene (AF400);
2,8-Dimethyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene-8-oxide (AF406);
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]-N-oxi-
de (AF600);
N-[(2-Ethyl-8-methyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]-N-
-oxide (AF601);
N-[(2-Methyl-8-phenyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]--
N-oxide (AF602);
Dihydro-5'-methylspiro[1-azabicyclo[2.2.2]octane-3,5'-(4'H)-3'-ylidene-me-
thylamine]-N-oxide (AF603);
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-benzyl-amine]-N-oxi-
de (AF604);
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-isopropyl-amine]-N--
oxide (AF605) and
(R)-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4-
.5]decan-3-one (AF704A) or an enantiomer, diastereomer, racemate,
tautomer, geometrical isomer, metabolite, or a pharmaceutically
acceptable salt thereof.
148. The compound of claim 146 wherein a is --O--; b is
--CR.sup.1R.sup.2--; d is --C.dbd.N(R.sup.3).dbd.O; e is
--CH.sub.2--; and R.sup.1, R.sup.2 and R.sup.3 are each
independently selected from the group consisting of H C.sub.1-6
alkoxy, C.sub.2-6 hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, and C.sub.1-6 alkyl optionally substituted by one to three
phenyls.
149. The compound of claim 146 wherein a is --S-- or --S(O)--; b is
--CR.sup.1R.sup.2--; d is selected from the group consisting of
--C(.dbd.O)-- and --C(.dbd.S)--; e is selected from the group
consisting of --NH--, --NR.sup.5--, --N(SO.sub.2R.sup.6)-- and
--N(C(.dbd.O)R.sup.6)--; R.sup.1 and R.sup.2 are each independently
selected from the group consisting of H, C.sub.1-6 alkoxy,
C.sub.2-6 hydroxyalkyl, C2-alkenyl, C.sub.2-6 alkynyl, and
C.sub.1-(alkyl optionally substituted by one to three phenyls;
R.sup.5 is selected from the group consisting of H, C.sub.1-6
alkoxy, C.sub.2-6 hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, optionally substituted phenyl, heteroaryl, and C.sub.1-6
alkyl optionally substituted by one, two or three phenyls; or, when
the compound of formula (II) is a dimer, in which both halves of
the dimer share a single R.sup.5R.sup.5 is selected from the group
consisting of --CH.sub.2).sub.n-- and --(CH.sub.2O).sub.n-- wherein
n is 1 to 6; and R.sub.6 is selected from the group consisting of
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, C.sub.2-6
hydroxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and C.sub.3-7
cycloalkyl, each optionally substituted by from 1-6 halogen atoms,
hydroxy-C.sub.1-6 alkyl, aryl substituted with a halogen, nitro,
amino, hydroxyl, or CF.sub.3 group, C.sub.1-6 alkyl substituted by
one to three aryl groups, and C.sub.m alkyl-X, wherein m=0 to 6 and
X is selected from the group consisting of indole, C.sub.1-6 alkyl
indole, isoindolyl, 3-pyridinyl, 3-piperidinyl, benzimidazolyl,
thienyl, isothiazolyl, imidazolyl, pyrazinyl, benzofuranyl,
quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl,
indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl,
thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, isooxazolyl, pyrrolyl,
pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl,
phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, and
pteridinyl, with the proviso that when A is ##STR00066## R is --H
or --CH.sub.3, a is S, b is --CH(CH.sub.2CH.sub.3)-- and d is
--C(.dbd.O)--, then e is not --NR.sup.5--.
150. A pharmaceutical composition comprising a therapeutically
effective amount of a compound according to claim 146, or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof, in
admixture with a pharmaceutically acceptable carrier, diluent or
excipient therefor.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods for treating various
central and peripheral nervous system disorders.
BACKGROUND OF THE INVENTION
[0002] The following documents, the contents of which are
incorporated herein by reference, are believed to be relevant:
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187:142-144, 1995; Refolo et al. Soc. Neurosci. Abst., San Diego,
Calif., USA, 2001; Refolo et al. Neurobiol. Dis. 8:890-899, 2001;
Review: Cedazo-Minguez and Cowburn. J. Cell Mol. Med. 5:254-266,
2001; Bales et al. PNAS 96: 15233, 1999; Buttini et al. Neurosci.
97:207, 2000; Hartmann et al, Exp. Neurol. 170:326, 2001; Mudher
and Lovestone. Trends Neurosci. 25:22-6, 2002; Mudher et al. J.
Neurosci. 21:4987-95, 2001; Zhang et al. Nature 395:698-702, 1998;
De Ferrari et al. Brain Res. Brain Res. Rev 33:1-12, 2000; Garrido
et al. FASEB J. 16:1982-4, 2002; Eldar-Finkelman. Trends Molec.
Med. 8:126-32, 2002; Bhat et al. Neurosignals 11:251-61, 2002;
Gentleman et al. NeuroReport 8:1519-1522, 1997; Roberts et al. J.
Neurol Neurosurg. Psychiat., 57:419-425, 1994; Havlik et al.
Neurobiol. Aging, S140, 587, 1998; Mayeux et al. Neurol, 45:
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al. Behav. Brain Res. 70:125-131, 1995; Pike and Hamm. Expt.
Neurol, 147: 55-65, 1997; Pike and Hamm. Pharmacol Biochem. Behav.,
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2001 Refolo et al., Soc. Neurosci. Abst, 2001, San Diego, Calif.,
USA; Cedazo-Minguez et al., Neurosci., 105: 651-661, 2001; Sparks
et al., Neurosci. Lett. 1995; 187:142-144; Dean et al., Mol
Psychiatry 1996; 1:54-8; Dean et al., J. Mol Psychiatry 2002; 7:
1083-91; Raedler et al., Am. J. Psychiatry 160:118, 2003; Borda et
al., J. Immunol 2002; 168:3667-74; Felder et al., Life Sci. 2001
8:2605-13; Bymaster et al., Current Drug Targets--CNS &
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Neurobiol 2002, 68:209-45; Poeggeler et al., Brain Res.
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(Proceedings of the Chiral Europe 96 Symp, Spring Innovations,
Stockport UK, p 103, 1996; Krise et al., J. Med. Chem. 42:
3094-3100 (1999);
[0005] Fassbeder et al., PNAS, 98:5856, 2001; Sparks et al., Exp.
Neurol 1994, 126:88-94; Sparks et al., Nutr. Metab. Cardiovasc.
Dis. 1997: 7:255-266; Beach et al., Neurosci. Lett. 283: 9-12,
2000; Beach et al., Brain Res. 905: 220-223, 2001; Klausner et al.,
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SUMMARY OF THE INVENTION
[0006] There is provided in accordance with an embodiment of the
invention a compound of the formula (I):
##STR00001##
wherein: C denotes a spiro carbon atom shared by ring A and the
ring containing a, b, d and e; A is selected from the group
consisting of:
##STR00002##
wherein R is selected from H, C.sub.1-C.sub.8 straight- or
branched-chain alkyl, or --CH.sub.2--P(--O)(OH).sub.2;
a is --O-- or --S--;
b is --CR.sup.1R.sup.2-- or --C(R.sub.1).dbd.;
[0007] d is selected from the group consisting of .dbd.N--,
--C(.dbd.O)--, --C(.dbd.S)-- and --N(R.sup.3).dbd.O; e is selected
from the group consisting of --CH.sub.2--, --CHR.sup.4--, --NH--,
--NR.sup.5--, --N(SO.sub.2R.sup.6)-- and --N(C(.dbd.O)R.sup.6)--;
R, R.sup.1, R.sup.2, R.sup.3, R.sup.4 are each independently
selected from H, C.sub.1-6 alkyl optionally substituted by one, two
or three phenyls, C.sub.1-6 alkoxy, C.sub.2-6 hydroxyalkyl,
C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; R.sup.5 is independently
selected from H, C.sub.1-6 alkyl optionally substituted by one, two
or three phenyls, C.sub.1-6 alkoxy, C.sub.2-6 hydroxyalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, substituted phenyl, and
heteroaryl; and R.sup.6 is selected from C.sub.1-6 alkyl, C.sub.1-6
alkoxy, C.sub.1-6 alkylthio, C.sub.2-6 hydroxyalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and C.sub.3-7 cycloalkyl, each
optionally substituted by from 1-6 halogen atoms,
hydroxy-C.sub.1-6-alkyl, aryl substituted with a halogen, nitro,
amino, hydroxyl, or CF.sub.3 group, and C.sub.1-6 alkyl substituted
by one, two or three aryl groups, C.sub.1-6 alkyl indole,
isoindolyl, 3-pyridinyl, 3-piperidinyl, benzimidazolyl, thienyl,
isothiazolyl, imidazolyl, pyrazinyl, benzofuranyl, quinolyl,
isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl,
isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl,
thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, isooxazolyl, pyrrolyl,
pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl,
phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, and
pteridinyl; or an enantiomer, diastereomer, racemate, tautomer,
geometrical isomer, dimer, metabolite or pharmaceutically
acceptable salt thereof, with the proviso that when A is
##STR00003##
R is --CH.sub.3, a is S, b is --CH(CH.sub.2CH.sub.3)-- and d is
--C(.dbd.O)--, then e is not --NH-- (AF267 or an enantiomer
thereof), and with the further proviso that when A is
##STR00004##
R is --CH.sub.3, a is S, b is --C(C.sub.1-3)-- and d is .dbd.N--,
then e is not --CH.sub.2-- (AF150(S)).
[0008] In an embodiment of the invention, R.sup.5 is heteroaryl
selected from the group consisting of indole, pyrrolidinyl,
piperidinyl, piperazinyl, furyl, pyridyl, pyrimidyl, thienyl,
isothiazolyl, imidazolyl, pyrazinyl, benzofuranyl, quinolyl,
isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl,
isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl,
thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, isooxazolyl, pyrrolyl,
pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl,
phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl,
5-azacytidinyl, 5-azauracilyl, triazolopyridinyl,
imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl.
[0009] In an embodiment of the invention, the compound is a dimer
of a compound of formula 1, wherein e is --NR.sup.5-- and the two
formula I moieties share a common group R.sup.5 which is selected
from the group consisting of --(CH.sub.2).sub.n-- and
--(CH.sub.2O).sub.n--, wherein n is 1 to 6, or an enantiomer,
diastereomer, racemate, tautomer, metabolite or pharmaceutically
acceptable salt thereof.
[0010] In an embodiment of the invention, the compound is selected
from the group consisting of:
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-amine]-N-oxide;
N-[(2-Ethyl-5-methyl-1-oxa-8-aza-spiro[4.]dec-3-ylidene)-amine]-N-oxide;
N-[(2-Methyl-8-phenyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-amine]-N-oxide-
; Thia-4,8-diaza-spiro[4.5]decan-3-one;
4-(2,4-Dimethoxy-benzyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF286);
8-Methyl-4-pyrrolidin-1-ylmethyl-1-thia-4,8-diaza-spiro[4.5]deca-
n-3-one (AF287);
2-(1-Hydroxy-ethyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF298);
(S)-2-Ethyl-8-methyl-8-oxy-1-thia-4,8-diaza-spiro[4.5]decan-3-on- e
(AF299);
4-(2,4-Dimethoxy-benzyl)-2-ethyl-8-methyl-1-thia-4,8-diaza-spir-
o[4.5]decan-3-one (AF288):
(S)-2-Ethyl-8-meth-1-oxo-1.lamda..sup.4-thia-4,8-diaza-spiro[4.5]decan-3--
one (AF300);
2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decane-3-thione
(AF510);
(S)-2-Ethyl-4-(4-fluorobenzenesulfonyl)-8-methyl-1-thia-4,8-diaz-
a-spiro[4.5]decan-3-one (AF7100);
2-Ethyl-4-[2-(1H-indol-3-yl)-ethyl]-8-methyl-1-thia-4,8-diaza-spiro[4.5]d-
ecan-3-one (AF703);
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one (AF704);
(S)-Ethyl-4-(3-H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.-
5]decan-3-one (AF704B);
(R)-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4-
.5]decan-3-one (AF704A); and
2-Methyl-8-methyl-d.sub.3-1-thia-3,8-diaza-spiro[4.5]dec-2-ene
(AF402),
or an enantiomer, diastereomer, racemate, tautomer, geometrical
isomer, metabolite, or a pharmaceutically acceptable salt
thereof.
[0011] In an embodiment of the invention, the compound is AF292 or
a pharmaceutically acceptable salt thereof
[0012] In an embodiment of the invention, the compound is a
compound wherein A is
##STR00005##
R is H, a is --S--; b is --CH(CH.sub.2CH.sub.3)--; d is
--(C.dbd.O)--; and e is --NH--, i.e.
2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF504), or an
enantiomer, diastereomer, geometrical isomer, racemate, tautomer,
dimer, metabolite or pharmaceutically acceptable salt thereof.
[0013] In an embodiment of the invention, the compound is
(5)-2-Ethyl-1-thia-4,8-diaza-spiro[4,5]decan-3-one (AF292) or its
HCl salt.
[0014] In an embodiment of the invention, the compound is
(R)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF291).
[0015] In an embodiment of the invention, the compound is a
compound wherein A is
##STR00006##
R is --CH.sub.3, a is --O--, d is .dbd.N(R.sup.3).dbd.O, or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof. In
an embodiment of the invention, b is --CH(CH.sub.3)-- and R.sup.3
is --CH, i.e.
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]-N-oxi-
de (AF600). In an embodiment of the invention, b is
--CH(CH.sub.3)-- and R.sup.3 is benzyl, i.e.
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-benzyl-amine]-N-oxi-
de (AF604). In an embodiment of the invention, b is
--CH(CH.sub.3)-- and R.sup.3 is isopropyl. i.e.
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-isopropyl-amine]-N--
oxide (AF605). In an embodiment of the invention, b is
--CH(CH.sub.2CH.sub.3)-- and R.sup.3 is --CH.sub.3, i.e.
N-[(2-Ethyl-8-methyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]-N-
-oxide (AF7601). In an embodiment of the invention, b is
--CH(CH.sub.3)-- and R.sup.3 is phenyl, i.e.
N-[(2-Methyl-8-phenyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]--
N-oxide (AF602).
[0016] In an embodiment of the invention, the compound is a
compound wherein A is
##STR00007##
R is --CH.sub.3, a is --O--, d is .dbd.N(R.sup.3).dbd.O, or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof. In
an embodiment of the invention, b is --CH(CH.sub.3)-- and R.sup.3
is methyl, i.e. Dihydro-5'-methyl
spiro[1-azabicyclo[2.2.2]octane-3,5'-(4'H)-3'-ylidene-methylamine]-N-oxid-
e (AF603).
[0017] In an embodiment of the invention, the compound is a
compound wherein A is
##STR00008##
R is methyl, a is --S--, b is --CH(CH.sub.2CH.sub.3)--; d is
--C(.dbd.O)--; e is --NR.sup.5-- wherein R.sup.5 is selected from
--(CH.sub.2).sub.3-indolyl and --C(O)--(CH.sub.2).sub.3-indolyl,
i.e.
2-Ethyl-4-[2-(1H-indol-3-yl)-ethyl]-8-methyl-1-thia-4,8-diaza-spiro[4.5]d-
ecan-3-one (AF703) or
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one (AF704), or an enantiomer, diastereomer, geometrical
isomer, racemate, tautomer, dimer, metabolite or pharmaceutically
acceptable salt thereof. In an embodiment of the invention, the is
(S)-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4-
.5]decan-3-one (AF704B).
[0018] There is also provided in accordance with an embodiment of
the invention the use of a compound of formula (I), or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof in
the preparation of a pharmaceutical composition. In an embodiment
of the invention, the compound is AF292 or a prodrug of AF292 or a
pharmaceutically acceptable salt of either AF292 or a prodrug of
AF292. In an embodiment of the invention, the prodrug is AF267B or
a pharmaceutically acceptable salt thereof.
[0019] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising at least one
compound of formula (I), or an enantiomer, diastereomer, racemate,
tautomer, geometrical isomer, dimer, metabolite or pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable carrier,
diluent or excipient therefor. In an embodiment of the invention,
the compound is AF292 or a prodrug of AF292 or a pharmaceutically
acceptable salt thereof. In an embodiment of the invention, the
prodrug is AF267B or a pharmaceutically acceptable salt
thereof.
[0020] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor,
comprising administering to a patient in need thereof an
efficacious amount of a compound selected from the group consisting
of compounds of formula (I), AF267 and AF150(S), or an enantiomer,
diastereomer, racemate, tautomer, geometrical isomer, dimer,
metabolite or pharmaceutically acceptable salt thereof. In an
embodiment of the invention, the compound is AF292 or a prodrug of
AF292 or a pharmaceutically acceptable salt thereof. In an
embodiment of the invention, the prodrug is AF267B or a
pharmaceutically acceptable salt thereof.
[0021] There is also provided in accordance with an embodiment of
the invention a compound of formula (I) wherein A is
##STR00009##
R is --CH.sub.3, a is --O--, d is .dbd.N(R.sup.3).dbd.O, or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof in
the preparation of a pharmaceutical composition for both
stimulating the M1 muscarinic receptor and retarding oxidation in
the vicinity of said M1 muscarinic receptor.
[0022] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor agonistic efficacious amount of a compound of
formula (I) wherein A is
##STR00010##
R is --CH.sub.3, a is --O--, d is N(R.sup.3).dbd.O, or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof, and
at least one pharmaceutically acceptable carrier, diluent or
excipient therefor.
[0023] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor,
comprising administering to a patient in need thereof an
efficacious amount of a compound of formula (I) wherein A is
##STR00011##
R is --CH.sub.3, a is --O--, d is .dbd.N(R.sup.3).dbd.O, or an
enantiomer, diastereomer, racemate, tautomer, geometrical isomer,
dimer, metabolite or pharmaceutically acceptable salt thereof
[0024] There is also provided in an embodiment of the invention the
compound
(S)-2-Ethyl-4-(4-fluoro-benzenesulfonyl)-S-methyl-1-thia-4,8-dia-
za-spiro[4.5]decan-3-one (AF700).
[0025] There is also provided in accordance with an embodiment of
the invention the use of the compound AF700, or an enantiomer,
diastereomer, racemate, tautomer, metabolite or pharmaceutically
acceptable salt thereof, in the preparation of a pharmaceutical
composition for both stimulating the M1 muscarinic receptor and
activating cc secretase.
[0026] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor stimulating and .alpha.-secretase activating
efficacious amount the compound AF700, or an enantiomer,
diastereomer, racemate, tautomer, metabolite or pharmaceutically
acceptable salt thereof, and at least one pharmaceutically
acceptable carrier, diluent or excipient therefor.
[0027] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor
and activating a secretase, comprising administering to a patient
in need thereof an efficacious amount of the compound AF700, or an
enantiomer, diastereomer, racemate, tautomer, metabolite or
pharmaceutically acceptable salt thereof
[0028] There is also provided in accordance with an embodiment of
the invention the use of the compound AF700, or an enantiomer,
diastereomer, racemate, tautomer, metabolite or pharmaceutically
acceptable salt thereof, in the preparation of a pharmaceutical
composition for both stimulating the M1 muscarinic receptor and
antagonizing .beta. secretase.
[0029] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor agonistic and .beta.-secretase antagonistic
efficacious amount of the compound AF700, or an enantiomer,
diastereomer, geometrical isomer, racemate, tautomer, dimer,
metabolite or pharmaceutically acceptable salt thereof, and at
least one pharmaceutically acceptable carrier, diluent or excipient
therefor.
[0030] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor
and antagonizing .beta.-secretase, comprising administering to a
patient in need thereof an efficacious amount of the compound
AF700, or an enantiomer, diastereomer, geometrical isomer,
racemate, tautomer, dimer, metabolite or pharmaceutically
acceptable salt thereof.
[0031] There is also provided in accordance with an embodiment of
the invention the use of the compound AF700, or an enantiomer,
diastereomer, racemate, tautomer, metabolite or pharmaceutically
acceptable salt thereof, in the preparation of a pharmaceutical
composition for both stimulating the M1 muscarinic receptor and
antagonizing .gamma.-secretase.
[0032] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor agonistic and Y-secretase antagonistic
efficacious amount of the compound AF700, or an enantiomer,
diastereomer, racemate, tautomer, metabolite or pharmaceutically
acceptable salt thereof, and at least one pharmaceutically
acceptable carrier, diluent or excipient therefor.
[0033] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor
and antagonizing .gamma.-secretase, comprising administering to a
patient in need thereof an efficacious amount of the compound
AF700, or an enantiomer, diastereomer, racemate, tautomer,
metabolite or pharmaceutically acceptable salt thereof.
[0034] There is also provided in accordance with an embodiment of
the invention the use of the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292), or a
metabolite or pharmaceutically acceptable salt thereof, in the
preparation of a pharmaceutical composition for both stimulating
the M1 muscarinic receptor and antagonizing the M3 muscarinic
receptor.
[0035] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor agonistic and M3 muscarinic receptor
antagonistic efficacious amount of the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292), or a
metabolite or pharmaceutically acceptable salt thereof, and at
least one pharmaceutically acceptable carrier, diluent or excipient
therefor.
[0036] There is also provided in accordance with an embodiment of
the invention human or animal blood containing the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292), or a
metabolite or pharmaceutically acceptable salt thereof. In an
embodiment of the invention, the blood is located in a human or
animal body. In an embodiment of the invention, the blood is not
located in a human or animal body.
[0037] There is also provided in accordance with an embodiment of
the invention human or animal blood plasma containing the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292), or a
metabolite or pharmaceutically acceptable salt thereof. In an
embodiment of the invention, the blood is located in a human or
animal body. In an embodiment of the invention, the blood is not
located in a human or animal body.
[0038] There is also provided in accordance with an embodiment of
the invention the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292)
according to claim 1, or a metabolite or pharmaceutically
acceptable salt thereof, whenever located in a human or animal
body.
[0039] There is also provided in accordance with an embodiment of
the invention the compound
(S)-2-ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF267B), for use as a prodrug of the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292).
[0040] There is also provided in accordance with an embodiment of
the invention the use of the compound
(S)-2-ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF267B) as a prodrug of the compound
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292).
[0041] There is also provided in accordance with an embodiment of
the invention the use of a combination of the compounds
(S)-2-ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF267B) and (S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF292) in the preparation of a pharmaceutical composition for
stimulating the M1 muscarinic receptor and antagonizing the M3
muscarinic receptor.
[0042] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor agonistic and M3 muscarinic receptor
antagonistic amount of a combination of the compounds AF267B and
AF292 and at least one pharmaceutically acceptable carrier, diluent
or excipient therefor.
[0043] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor
and antagonizing the M3 muscarinic receptor in a patient,
comprising administering to a patient an efficacious amount of a
combination of the compounds AF267B and AF292.
[0044] In an embodiment of the invention, AF267B and AF292 are
administered together. In an embodiment of the invention, AF267B
and AF292 are administered separately. In an embodiment of the
invention, AF267B and AF292 are administered at different times. In
an embodiment of the invention, AF267B and AF292 are administered
at the same times.
[0045] There is also provided in accordance with an embodiment of
the invention the use of a combination of a first compound which is
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292) and a
second compound selected from the group consisting of compound of
formula (I), AF267B and AF150(S), including racemates, enantiomers,
diastereomers, tautomers, geometric isomers and pharmaceutically
acceptable salts thereof, in the preparation of a pharmaceutical
composition for stimulating the M1 muscarinic receptor while
minimizing adverse side-effects due to stimulation of other mAChR
subtypes.
[0046] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor agonistic amount of a combination of a first
compound which is AF292 and a second compound selected from the
group consisting of compounds of formula (I), AF267B and AF150(S),
including racemates, enantiomers, diastereomers, geometric isomers,
tautomers and pharmaceutically acceptable salts thereof, and at
least one pharmaceutically acceptable carrier, diluent or excipient
therefor.
[0047] There is also provided in accordance with an embodiment of
the invention a method of stimulating the M1 muscarinic receptor
while minimizing adverse side-effects due to stimulation of other
mAChR subtypes in a patient, comprising administering to a patient
an efficacious amount of a combination of a first compound which is
AF292 and a second compound selected from the group consisting of
compounds of formula (I), AF267B and AF150(S), including racemates,
enantiomers, diastercomers, tautomers, geometric isomers and
pharmaceutically acceptable salts thereof. In an embodiment of the
invention, the first compound and the second compound are
administered together. In an embodiment of the invention, the first
compound and the second compound are administered separately. In an
embodiment of the invention, the first compound and the second
compound are administered at different times. In an embodiment of
the invention, the first compound and the second compound are
administered at the same time.
[0048] There is also provided in accordance with an embodiment of
the invention a method for stimulating the M1 muscarinic receptor
in a patient simultaneously with AF267B and AF292, comprising
administering to a patient an amount of AF267B efficacious to form
in vivo an amount of a mixture of AF267B and AF292 efficacious to
stimulate the M1 muscarinic receptor.
[0049] There is also provided in accordance with an embodiment of
the invention
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-
-spiro[4.5]decan-3-one in racemic form (AF704) or as the
S-enantiomer thereof (AF704B), for use as a prodrug of at least one
of the group of AF267B, AF292 and indole-3-propionic acid.
[0050] There is also provided in accordance with an embodiment of
the invention the use of
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one in racemic form (AF704) or as the S-enantiomer thereof
(AF704B), in the preparation of a pharmaceutical composition for
stimulating the M1 muscarinic receptor, retarding oxidation in the
vicinity of the M1 muscarinic receptor, and providing
neuroprotectant activity.
[0051] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising an M1
muscarinic receptor stimulating, oxidation-retarding and
neuroprotectant activity efficacious amount of the compound
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one in racemic form (AF704) or as the S-enantiomer thereof
(AF704B), and a pharmaceutically acceptable carrier, diluent, or
excipient therefor.
[0052] There is also provided in accordance with an embodiment of
the invention a process for the preparation of
2-ethyl-5-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF267)
comprising reacting 4-ethyl piperidone with 2-mercaptobutyric acid
and ammonia. In an embodiment of the invention, the process further
comprising obtaining the enantiomers AF267A (R-enantiomer) and
AF267B (S-enantiomer) by chiral separation.
[0053] There is also provided in accordance with an embodiment of
the invention a process for the preparation of AF267B, comprising
racemizing AF267A and isolating AF267B from the racemic mixture. In
an embodiment of the invention, the isolating comprising separating
the AF267B from the racemic mixture by chiral separation.
[0054] There is also provided in accordance with an embodiment of
the invention a process for the synthesis of AF267B comprising
contacting (R)-2-mercaptobutyric acid with ammonium acetate and
1-methyl-4-piperidone. In an embodiment of the invention the
(R)-2-mercaptobutyric is obtained by contacting
(R)-2-benzoylthiobutyric acid with ammonium hydroxide. In an
embodiment of the invention the (R)-2-benzoylthiobutyric acid is
obtained by contacting (R)-2-bromobutyric acid with cesium
thiobenzoate. In an embodiment of the invention the
(R)-2-bromobutyric acid is obtained by contacting 2-aminobutyric
acid having the R configuration with sodium nitrite, potassium
bromide and hydrobromic acid.
[0055] There is also provided in accordance with an embodiment of
the invention a process for the preparation of .sup.14C-labelled
AF267B, comprising reacting AF287 with .sup.14C-labelled ethyl
bromide, deprotecting the nitrogen atom at the 4-position of the
AF287- and isolating .sup.14C-labelled AF267B by chiral
chromatography.
[0056] There is also provided in accordance with an embodiment of
the invention a process for the preparation of a mixture of
(S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF292) and
(R)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF291)
comprising reacting AF267 with a demethylating agent.
[0057] There is also provided in accordance with an embodiment of
the invention a crystalline form of
(S)-2-ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decane-3-one
(AF267B) characterized by the following data: P212121 (No 16)
a=10.394 ((10) (.alpha.=90.degree.), b=20.133 (2)
(.beta.=90.degree.) c=5.856 (4) (.gamma.=90.degree.), .ANG., T=110
K. In an embodiment of the invention, the crystalline form is
further characterized by the following data: Volume=1224.2 (9)
.ANG..sup.3, Z=4, Fw=202.32, Calculated density, Dc=1.092
Mg/m.sup.3, Absorption coefficient, .mu.=0.232 mm.sup.-1.
[0058] There is also provided in accordance with an embodiment of
the invention a crystalline form of (AF267B) having the
configuration shown in Structure 1 in Example 2.
[0059] There is also provided in accordance with an embodiment of
the invention a process for the preparation of
2,8-Dimethyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene
[AF150(S)]comprising cyclizing
1-methyl-4-N-thioacetylamino-1,2,9,6-tetrahydropyridine. In an
embodiment of the invention the cyclizing is conducted in the
presence of phosphoric acid. In an embodiment of the invention the
1-methyl-4-N-thioacetylamino-1,2,3,6-tetrahydropyridine is obtained
by reduction of reduction of 1-methyl-4-N-thioacetylaminomethyl
pyridinium with sodium borohydride. In an embodiment of the
invention the 1-methyl-4-N-thioacetylaminomethyl pyridinium is
obtained by reacting 4-(acetaminomethyl)-1-methyl-pyridinium iodide
with Lawesson's reagent.
[0060] There is also provided in accordance with an embodiment of
the invention a pharmaceutical formulation comprising AF150(S) in
paraffin oil.
[0061] There is also provided in accordance with an embodiment of
the invention a method for inhibiting the release or synthesis of
beta-amyloid peptide (A.beta.) in a mammalian cell, tissue or
organism comprising administering to a mammalian cell, tissue or
organism an amount of a compound or a mixture of compounds selected
from the group consisting of compounds of formula (I), AF267B and
AF150(S), or racemates, enantiomers, geometrical isomers,
diasteromers, tautomers and pharmaceutically acceptable salts
thereof effective to inhibit the cellular release or synthesis of
A.beta..
[0062] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267B and AF150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for inhibiting the release or synthesis of beta-amyloid peptide
(A.beta.) in a mammalian cell, tissue or organism.
[0063] There is also provided in accordance with an embodiment of
the invention a method for elevating the level of secreted form of
the non-amyloidogenic amyloid precursor protein (.alpha.-APPs) in a
mammalian cell, tissue or organism comprising administering to a
mammalian cell, tissue or organism an amount of a compound or a
mixture of compounds selected from the group consisting of compound
of formula (I), AF267B and AF150(S), or racemates, enantiomers,
geometrical isomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof effective to elevate the level of the
secreted form of the non-amyloidogenic amyloid precursor protein
(.alpha.-APPs).
[0064] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267B and AF150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts, in the preparation of a pharmaceutical composition for
elevating the level of secreted form of the non-amyloidogenic
amyloid precursor protein (.alpha.-APPs) in a mammalian cell,
tissue or organism.
[0065] There is also provided in accordance with an embodiment of
the invention a method for decreasing the level of A.beta. peptide
in the brain of a mammal having an elevated level of A.beta. in the
brain, comprising administering to a mammal having an elevated
level of A.beta. in the brain an amount of a compound or a mixture
of compounds selected from the group consisting of compounds of
formula (I), AF267B and AF150(S), or racemates, enantiomers,
geometrical isomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof effective to decrease the level of A.beta.
in the brain of said mammal. In an embodiment of the invention the
elevated level of A.beta. in the brain is a result of
hypercholesterolemia. In an embodiment of the invention the
elevated level of A.beta. in the brain is a result of cholinergic
hypofunction.
[0066] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compound of formula (I),
AF267B and AF150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for decreasing the level of A.beta. peptide in the brain of a
mammal having an elevated level of A.beta. in the brain.
[0067] There is also provided in accordance with an embodiment of
the invention a method for inhibiting the synthesis or release of
apolipoprotein (ApoE) in a mammalian cell, tissue or organism
comprising administering to a mammalian cell, tissue or organism an
amount of a compound or a mixture of compounds selected from the
group consisting of compound of formula (I), AF267B and AF150(S),
or racemates, enantiomers, geometrical isomers, diasteromers,
tautomers and pharmaceutically acceptable salts thereof effective
to inhibit the release or synthesis of ApoE in said mammalian cell,
tissue or organism.
[0068] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compound of formula (I),
AF267 and AF 150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for inhibiting the release or synthesis of ApoE in a mammalian
cell, tissue or organism. In an embodiment of the invention the
ApoE is ApoE4.
[0069] There is also provided in accordance with an embodiment of
the invention a method for decreasing levels of apolipoprotein
(ApoE) in a mammalian cell, tissue or organism comprising
administering to a mammalian cell, tissue or organism an amount of
a compound or a mixture of compounds selected from the group
consisting of compound of formula (I), AF267B and AF150(S), or
racemates, geometrical isomers, enantiomers, diasteromers,
tautomers and pharmaceutically acceptable salts thereof effective
to decrease the levels of ApoE in said mammalian cell, tissue or
organism.
[0070] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267B and AF150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for decreasing levels of ApoE in a mammalian cell, tissue or
organism. In an embodiment of the invention the ApoE is ApoE4.
[0071] There is also provided in accordance with an embodiment of
the invention a method for decreasing tan hyperphosphorylation in a
mammalian cell, tissue or organism comprising administering to a
mammalian cell, tissue or organism a compound or a mixture of
compounds selected from the group consisting of compounds of
formula (I), AF26713 and AF150(S), or racemates, enantiomers,
geometrical isomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof effective to inhibit tau
hyperphosphorylation. In an embodiment of the invention the tau
hyperphosphorylation is A.beta.-induced tau
hyperphosphorylation.
[0072] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267B and AF150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for decreasing tau hyperphosphorylation in a mammalian cell, tissue
or organism.
[0073] There is also provided in accordance with an embodiment of
the invention a method for decreasing paired helical formation in a
mammalian cell, tissue or organism comprising administering to a
mammalian cell, tissue or organism a compound or a mixture of
compounds selected from the group consisting of compounds of
formula (I), AF267B and AF150(5), or racemates, enantiomers,
geometrical isomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof effective to inhibit tan
hyperphosphorylation.
[0074] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267 and AF150(5), or racemates, enantiomers, diasteromers,
tautomers, geometrical isomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for decreasing paired helical formation in a mammalian cell.
[0075] There is also provided in accordance with an embodiment of
the invention a method for activating the Wnt signaling pathway in
a mammalian cell, tissue or organism comprising administering to a
mammalian cell, tissue or organism a compound or a mixture of
compounds selected from the group consisting of compounds of
formula (I), AF267B and AF150(5), or racemates, enantiomers,
diasteromers, tautomers, geometrical isomers and pharmaceutically
acceptable salts thereof effective to inhibit Wnt
abnormalities.
[0076] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267 and AF150(S), or racemates, enantiomers, diasteromers,
tautomers, geometrical isomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for activating the Wnt signaling pathway in a mammalian cell,
tissue or organism.
[0077] There is also provided in accordance with an embodiment of
the invention a method for inhibiting GSK3.beta.-mediated effects
in a mammalian cell tissue or organism comprising administering to
a mammalian cell, tissue or organism a compound or a mixture of
compounds selected from the group consisting of compounds of
formula (I), AF267B and AF150(S), or racemates, enantiomers,
diasteromers, tautomers, geometrical isomers and pharmaceutically
acceptable salts thereof effective to inhibit GSK3.beta.-mediated
effects. In an embodiment of the invention the GSK3.beta.-mediated
effects are selected from the group consisting of tau
hyperphosphorylation, apoptosis, .beta.-catenin degradation, and
decrease in Wnt target genes.
[0078] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267 and AF150(S), or racemates, enantiomers, diasteromers,
tautomers, geometrical isomers and pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for inhibiting GSK3.beta.-mediated effects in a mammal. In an
embodiment of the invention the method is used in response to
insults induced by A.beta. peptides or oxidative stress starvation
to Wnt signaling, apoptosis, or cell viability.
[0079] There is also provided in accordance with an embodiment of
the invention a method for enhancing the activity of endogenous
growth factors, i.e. neutrophins, in a cell, comprising
administering to a mammalian cell, tissue or organism an amount of
a compound or a mixture of compounds selected from the group
consisting of compounds of formula (I), AF267B and AF150(S), or
racemates, enantiomers, geometrical isomers, diasteromers,
tautomers and pharmaceutically acceptable salts thereof which alone
is effective as a neurotrophic agent and which acts synergistically
with said neurotrophins.
[0080] There is also provided in accordance with an embodiment of
the invention the use of a compound or a mixture of compounds
selected from the group consisting of compounds of formula (I),
AF267B and AF150(S), or racemates, enantiomers, diasteromers,
tautomers, geometrical isomers and pharmaceutically acceptable
salts thereof which alone is effective as a neurotrophic agent and
which acts synergistically with endogenous growth factors, i.e.
neurotrophins, in the preparation of a pharmaceutical composition
for enhancing the activity of neurotrophins.
[0081] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition for inhibiting the
release or synthesis of beta-amyloid peptide (A.beta.), for
elevating the level of secreted form of the non-amyloidogenic
amyloid precursor protein (.alpha.-APPs), for decreasing the level
of A.beta.peptide in the brain of a mammal having an elevated level
of A.beta. in the brain, for inhibiting the release or synthesis of
ApoE, for decreasing levels of ApoE, for decreasing tart
hyperphosphorylation, for decreasing paired helical formation, for
activating the Wnt signaling pathway, for increasing beta-catenin,
for inhibiting GSK3.beta.-mediated effects or for enhancing the
activity of neurotrophins, comprising a compound or a mixture of
compounds selected from the group consisting of compounds of
formula (I), AF267B and AF150(S), or racemates, enantiomers,
geometrical isomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof, and at least one pharmaceutically
acceptable carrier, diluent or excipient.
[0082] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising at least one
compound selected from the group consisting of compounds of formula
(I), AF267B and AF150(S), or racemates, enantiomers, geometrical
isomers, diasteromers, tautomers and pharmaceutically acceptable
salts thereof and at least one additional pharmacologically active
compound selected from the group consisting of: cholinesterase
inhibitors, nicotinic agonists, cholinergic precursors and
cholinergic enhancers, nootropics, peripheral antimuscarine drugs,
M2 muscarinic antagonists, M4 antagonists, benzodiapine inverse
agonists, antidepressants, tricyclic antidepressants or
antimuscarinic drugs used in treatment of Parkinson's disease (PD)
or depression, antipsychotic and antischizophrenic agents,
glutamate antagonists and modulators, NMDA antagonists, AMPA
agonists, acetyl-L-carnitine, MAO-B inhibitors, peptides and growth
factors, cholesterol-lowering agents, antioxidants, GSK-3.beta.
inhibitors, Wnt-ligands, .beta.- or .gamma.-secretase inhibitors,
beta-amyloid degrading agents, beta-amyloid anti-aggregation
agents, chelating agents, immunotherapeutic compounds against
beta-amyloids, compounds that bind to amyloids, cyclooxygenase
(COX)-2 inhibitors, non-steroidal antiinflammatory drugs,
estrogenic agents, estrogenic receptor modulators, steroidal
neuroprotectants, and spin trapping pharmaceuticals.
[0083] In an embodiment of the invention the compound is AF292 or a
prodrug of AF292 or a pharmaceutically acceptable salt thereof.
[0084] There is also provided in accordance with an embodiment of
the invention a method for treating or reducing cerebral amyloid
angiopathy comprising administering to a patient in need thereof
(a) an efficacious amount of a compound selected from the group
consisting of AF267B, AF292, and AF704B pharmaceutically acceptable
salts thereof or mixtures of such compounds or salts, and (b) an
efficacious amount of a compound selected from an immunotherapeutic
compound against beta-amyloids and compounds that bind to
amyloids.
[0085] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition comprising (a) an
efficacious amount of a compound selected from the group consisting
of AF267B, AF292, and AF704B pharmaceutically acceptable salts
thereof or mixtures of such compounds or salts, and (b) an
efficacious amount of a compound selected from an immunotherapeutic
compound against beta-amyloids and compounds that bind to amyloids,
and a pharmaceutically acceptable carrier, diluent or exicipient
therefor.
[0086] There is also provided in accordance with an embodiment of
the invention the use of a combination of (a) a compound selected
from the group consisting of AF267B, AF292, and AF704B
pharmaceutically acceptable salts thereof or mixtures of such
compounds or salts, and (b) a compound selected from an
immunotherapeutic compound against beta-amyloids and compounds that
bind to amyloids, in the preparation of a pharmaceutical
composition for treating or reducing cerebral amyloid
angiopathy.
[0087] There is also provided in accordance with an embodiment of
the invention a method for treating in a mammal diseases associated
with impaired cholinergic function or diseases where there is an
imbalance in cholinergic function, or diseases with impaired
activity of acetylcholine receptors from the group consisting of,
senile dementia of Alzheimer's type; Alzheimer's disease (AD); Lewy
body dementia; mixed Alzheimer's and Parkinson's disease;
multiifract dementia (MID); fronto-temporal dementia; vascular
dementia; stroke/ischemia, MID combined with stroke/ischemia/head
injury; combined MID and AD; human head injury; age-associated
memory impairments; mild cognitive impairment (MCI); MCI conducive
to AD; cognitive dysfunction (including forgetfulness, acute
confusion disorders, attention-deficit disorders, focus and
concentration disorders); hallucinatory-paranoid states; emotional
and attention disorders; sleep disorders; postoperative delirium;
adverse effects of tricyclic antidepressants; adverse effects of
certain drugs used in the treatment of schizophrenia and
Parkinson's disease; xerostomia, anomia, memory loss and/or
confusion; psychosis; schizophrenia, schizophrenia comorbit with
AD, late onset schizophrenia, paraphrenia, schizophreniform
disorders; anxiety; bipolar disorders; mania; mood stabilization;
cognitive impairments after removal of certain gliomas; tardive
dyskinesia; oxidative stress during oxygen therapy; aphasia;
postencephalitic amnesic syndrome; AIDS dementia; memory
impairments in autoimmune diseases including lupus, multiple
sclerosis, Sjogren's syndrome, chronic fatigue syndrome, and
fibromyalgia; memory impairments in atypical depression or
schizophrenia; pain, rheumatism, arthritis and terminal illness;
xerophtalmia, vaginal dryness, skin dryness; immune dysfunctions;
neurocrine disorders and dysregulation of food intake, including
bulimia and anorexia; obesity; congenital ornithine
transcarbamylase deficiency; ollivopontocerebral atrophy; alcohol
withdrawal symptoms; substance abuse including withdrawal symptoms
and substitution therapy; Huntington's chorea; progressive
supranuclear palsy; Pick's disease; Friedrick's ataxia; Gilles de
la Tourette disease; Down's syndrome; glaucoma; presbyopia;
autonomic disorders including dysfunction of gastrointestinal
motility and function such as inflammatory bowel disease, irritable
bowel syndrome, diarrhea, constipation, gastric acid secretion and
ulcers; urinary urge incontinence, asthma, COPD; comprising
administering to a mammal in need of such treatment a compound a
mixture of compounds selected from the group consisting of
compounds of formula (I), AF267B and AF150(S), or racemates,
enantiomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof in an amount effective to treat one of
said diseases. In an embodiment of the invention the compound is
AF292 or a prodrug of AF292 or a pharmaceutically acceptable salt
thereof.
[0088] There is also provided in accordance with an embodiment of
the invention a method for preventing or treating central or
peripheral nervous system disease states due to dysfunction in one
or more of the following: brain, nervous system, cardiovascular
system, immune system, neurocrine system, gastrointestinal system,
or endocrine and exocrine glands, eye, cornea, lungs, prostate, or
other organs where the cholinergic function is mediated by
muscarinic receptor subtypes, wherein said dysfunction involves:
brain amyloid-mediated disorders; glycogen synthase kinase
(GSK3.beta.)-mediated disorders; tau protein
hyperphosphorylation-mediated damages, dysfunctions or diseases:
CNS and PNS hypercholesterolemia- and/or hyperlipidemia-mediated
damages, dysfunctions or diseases; Wnt-mediated signaling
abnormalities; impairment of neuroplasticity; hyperglycemia;
diabetes; endogenous growth factors-mediated diseases, or
combination of additional risk factors; or disease states that
involve apolipoprotein E; or disturbances in which a cholinergic
dysfunction has been implicated including: senile dementia of
Alzheimer's type, Alzheimer's disease (AD), delay of onset of AD
symptoms in a patient at risk for developing AD, Lewy body
dementia, cerebral amyloid angiopathy (CAA), cerebral amyloidosis,
fronto-temporal dementia, vascular dementia, hyperlipidemia,
hypercholesterolemia, multiifract dementia (MID), stroke ischemia,
MID combined with stroke/ischemia cad injury, combined MID and
Alzheimer's disease, human head injury, age-associated memory
impairments, mild cognitive impairment (MCI), MCI conducive to AD,
bipolar disorder, mania, schizophrenia, nonaffective
sychozophrenia, paraphrenia, immune dysfunctions, neurocrine
disorders and dysregulation of food intake, including bulimia and
anorexia, weight control, obesity, inflammation; comprising
administering to a mammal in need of such treatment a compound or a
mixture of compounds selected from the group consisting of
compounds of formula (I), AF267B and AF150(S), or racemates,
enantiomers, diasteromers, tautomers and pharmaceutically
acceptable salts thereof in an amount effective to treat at least
one of said diseases. In an embodiment of the invention the
compound is AF292 or a prodrug of AF292 or a pharmaceutically
acceptable salt thereof,
[0089] There is also provided in accordance with an embodiment of
the invention a method for treating a patient with AD, MCI, Lewi
Body Dementia, fronto-temporal dementia, vascular dementia, memory
impairment in head injury, AIDS dementia in order to inhibit
further deterioration in the condition of said patient comprising
administering to said patient an efficacious amount of a compound
or a mixture of compounds selected from the group consisting of
compounds of formula (I), AF267B and AF150(S), or racemates,
enantiomers, diasteromers, tautomers, geometric isomers and
pharmaceutically acceptable salts thereof
[0090] In an embodiment of the invention the compound is AF292 or a
prodrug of AF292 or a pharmaceutically acceptable salt thereof.
[0091] There is also provided in accordance with an embodiment of
the invention a method of treating schizophrenia, comprising
administering to a patient in need thereof an efficacious amount of
a compound selected from the group consisting of AF267B, AF292,
pharmaceutically acceptable salts thereof and mixtures of AF267B,
AF292 or salts thereof,
[0092] There is also provided in accordance with an embodiment of
the invention the use of AF267B. AF292, pharmaceutically acceptable
salts thereof or a mixture of AF267B, AF292 or pharmaceutically
acceptable salts thereof in the preparation of a pharmaceutical
composition for the treatment of schizophrenia.
[0093] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition for the treatment of
schizophrenia, comprising AF267B, AF292, pharmaceutically
acceptable salts thereof or a mixture of AF267B, AF292 or
pharmaceutically salts thereof and at least one pharmaceutically
acceptable carrier, diluent or excipient.
[0094] There is also provided in accordance with an embodiment of
the invention a method of ameliorating symptoms of schizophrenia,
comprising administering to a patient in need thereof an
efficacious amount of a compound selected from the group consisting
of AF267B, AF292, pharmaceutically acceptable salts thereof and
mixtures of AF267B, AF292 or pharmaceutically acceptable salts
thereof.
[0095] There is also provided in accordance with an embodiment of
the invention the use of AF267B, AF292, pharmaceutically acceptable
salts or mixtures of AF267B, AF292 or pharmaceutically acceptable
salts thereof in the preparation of a pharmaceutical composition
for amelioration of symptoms of schizophrenia.
[0096] There is also provided in accordance with an embodiment of
the invention a pharmaceutical composition for amelioration of
symptoms of schizophrenia, comprising a compound selected from
AF267B, AF292, pharmaceutically acceptable salts thereof and
mixtures of AF267B. AF292 or pharmaceutically acceptable salts
thereof and at least one pharmaceutically acceptable carrier,
diluent or excipient.
[0097] In this patent application,
[0098] "alkyl" means a linear or branched chain of 1-8 carbon
atoms, e.g. methyl, ethyl, propyl, isopropyl etc.
[0099] "alkoxy" means --O-alkyl, e.g. to methoxy, ethoxy, propoxy,
isopropoxy, etc.
[0100] "alkenyl" means a linear or branched chain of 2-8 carbon
atoms having at least one C--C double bond in the chain.
[0101] "alkynyl" means a linear or branched chain of 2-8 carbon
atoms having at least one C--C triple bond in the chain.
[0102] "alkylthio" means --S-alkyl, e.g. methylthio, ethylthio,
propylthio, isopropylthio etc.
[0103] "cycloalkyl" refers to mono- and bicyclic ring structures
containing 3-12 carbon atoms. Examples of cycloalkyl are
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl, and
norbornyl.
[0104] "aryl" refers to a mono- or bicyclic aromatic ring structure
containing 5-12 carbon atoms. Examples of aryl are phenyl, naphthyl
and benzyl.
[0105] "heterocyclic" refers to mono- and bicyclic ring structures
containing 4-12 carbon atoms and at least one nitrogen, oxygen or
sulfur atom.
[0106] "heteroaryl" refers to a mono- or bicyclic aromatic ring
structure containing 4-12 carbon atoms and at least one nitrogen,
oxygen or sulfur atom. Examples of heterocyclic and heteroaryl are
indolyl, isoindolyl, 3-pyridinyl, 3-piperidinyl, benzimidazolyl,
thienyl, isothiazolyl, imidazolyl, pyrazinyl, benzofuranyl,
quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl,
indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl,
thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, isooxazolyl, pyrrolyl,
pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl,
phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, and
pteridinyl, pyrrolidinyl, piperidinyl, piperazinyl, furyl, pyridyl,
pyrimidyl, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl,
imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl.
[0107] "halogen atom" may be one of fluorine, chloride, bromine and
iodine.
[0108] Unless noted otherwise, the following abbreviations are
used: [0109] A.beta. .beta.-amyloid [0110] AA arachidonic acid
[0111] AAMI age associated memory impairment [0112] ACh
acetylcholine [0113] AchE-Is acetylcholinesterase inhibitors [0114]
AD Alzheimer's disease [0115] AGP Human .alpha.-glycoprotein [0116]
AGP .alpha.-glycoprotein [0117] ApoE apolipoprotein [0118] APP
amyloid precursor protein [0119] AUC area under the curve [0120]
BDNF brain derived growth factor [0121] bFGF basic fibroblast
growth factor [0122] CAA cerebral amyloid angiopathy [0123] CCh
carbachol [0124] CDX methyl-.beta.-cyclodextrin [0125] CE collision
energy [0126] CHI closed head injury [0127] CNS central nervous
system [0128] CSF cerebrospinal fluid [0129] DAPI
4,6-diamidino-2-phenylindole [0130] DCC dicyclohexylcarbodiimide
[0131] DDW double distilled water [0132] DMF N,N-dimethyl formamide
[0133] DMAP 4-dimethylaminopyridine [0134] DMPU
N,N'-dimethyl-N,N'-propylene urea [0135] ECG electrocardiogram
[0136] EGF epidermal growth factor [0137] FACS Fluorescence
activated cell sorter [0138] FCS fetal calf serum [0139] GC gas
chromatography [0140] GSK3.beta. glycogen synthase kinase [0141]
HERG human ether-a-go-go related gene [0142] HPLC high performance
liquid chromatograph [0143] HS horse serum [0144] HSA human serum
albumin [0145] i.c.v., icv intracerebroventricular [0146] i.p., ip
intraperitoneally [0147] i.v., iv intravenous [0148] LBD Lewy Body
disease [0149] LC liquid chromatograph [0150] LDA di-isopropylamine
[0151] Li lithium [0152] Mt mAChR 1 muscarinic receptor [0153]
mAChR muscarinic receptor [0154] mCPBA m-chloroperbenzoic acid
[0155] MCI minimal cognitive impairment [0156] MID multiifract
dementia [0157] MRSA muscarinic receptor selective agonists and
potent antioxidants [0158] MS mass spectrometry [0159] MTBE
Methyl-t-butyl ether [0160] MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
[0161] MWM Morris water maze [0162] nbm nucleus basalis
magnocellularis [0163] NFT neurofibrillary tangles [0164] NGF Nerve
Growth Factor [0165] NMDA N-methyl-D-Aspartate [0166] NMR nuclear
magnetic resonance [0167] NOAEL no-adverse-effect-level [0168] NSS
neurological severity scores [0169] OXO-M oxotremorine-M [0170] PA
passive avoidance [0171] PBS phosphate-buffered saline [0172] PD
Parkinson's disease [0173] PHF paired helical filaments [0174] PI
phosphoinositide [0175] PKC protein kinase C [0176] PMSF
Phenylmethyl sulfonylfuoride [0177] PNS peripheral nervous system
[0178] po per os, oral [0179] PS-1 presenilin-1 [0180] PZ
pirenzepine [0181] QNB quinuclidinyl benzilate [0182] REL ratio of
escape latency [0183] RID Ratio of Investigation Duration [0184]
ROS reactive oxygen species [0185] RPL ratio of path length [0186]
RSA Receptor Selective Antioxidants [0187] s.c., Sc subcutaneously
[0188] SDAT senile dementia of Alzheimer's type [0189] SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis [0190]
SMB Simulated Moving Bed [0191] TBI traumatic brain injury [0192]
Tg transgenic [0193] THF tetrahydrofuran [0194] TUNEL Terminal
deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling
[0195] A1 (human); A2A (human); adenosine receptor subtypes [0196]
A3 (human) [0197] AT1 (Human Recombinant) angiotensin [0198] BZD
(central) benzodiazepine [0199] B2 (Human Recombinant) bradykinin
[0200] CCKA (Human Recombinant) (CCK1) cholecystokinin [0201] D1
(Human Recombinant); dopamine receptor subtypes [0202] D2S (Human
Recombinant) [0203] ETA (Human Recombinant) endothelin [0204] GABA
(non-selective) gamma-aminobutyric acid [0205] GAL2 (h) galanin
[0206] IL-8B (Human Recombinant) (CXCR2) chemokine receptor subtype
[0207] CCR1 (Human Recombinant) chemokine receptor subtype [0208]
H1 (central); H2 histamine receptor subtypes [0209] MC4 (Human
Recombinant) melanocortine [0210] ML1 melatonin [0211] NK2 (Human
Recombinant); tachykinin [0212] NK3 (Human Recombinant) [0213] Y1
(human); neuropeptide, neuropeptide [0214] Y2 (human) [0215] NT1
(Human Recombinant) (NTS1) neurotensin [0216] delta 2 (Human
Recombinant); opiate receptor subtypes [0217] (DOP); kapp (KOP);
opiate, [0218] mu (Human Recombinant) (MOP) [0219] ORL1 (Human
Recombinant) (NOP) orphanin [0220] 5-HT.sub.1A (Human Recombinant);
serotonin subtypes [0221] 5-HT.sub.1B; 5-HT.sub.2A (Human
Recombinant); 5-HT.sub.3 Human Recombinant); 5-HT.sub.5A (Human
Recombinant) (5-ht5A); 5-HT.sub.6 (Human Recombinant); 5-HT.sub.7
(human) [0222] sst (non-selective) somatostatin [0223] VIP1 (human)
(VPAC1) vasoactive intestinal peptide [0224] V1a (Human
Recombinant) vasopressin
[0225] NE transporter (human) Norepinephrine
[0226] The term "geometrical isomers" refers to isomerism across a
double-bond, e.g. cis/trans isomerism and E/Z isomerism, as well as
conformational isomers, e.g. and syn/anti isomerism.
[0227] The term "pharmaceutically acceptable addition salts" refers
to salts known in the art to be acceptable in pharmaceutical
practice, for example acid addition salts such as hydrochloric acid
salts, maleic acid salts, and citric acid salts. Pharmaceutically
acceptable acid addition salts include salts derived form inorganic
acids such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorus, and the like, as well as the
salts derived from organic acids, such as aliphatic mono- and
dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy
alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and
aromatic sulfonic acids, etc. Such salts thus include sulfate,
pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate,
monohydrogenphosphate, dihydrogenphosphate, metaphosphate,
pyrophosphate, chloride, bromide, iodide, acetate, propionate,
caprylate, isobutyrate, oxalate, malonate, succinate, suberate,
sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate,
methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate,
toluenesulfonate, phenylacetate, citrate, lactate, maleate,
tartrate, methanesulfonate, and the like. Also contemplated are the
salts of amino acids such as arginate, gluconate, galacturonate,
and the like; see, for example, Berge et al., "Pharmaceutical
Salts," J. of Pharmaceutical Science, 1977; 66:1-19.
[0228] The acid addition salts of the basic compounds are prepared
by contacting the free base form with a sufficient amount of the
desired acid to produce the salt in the conventional manner. The
free base form may be regenerated by contacting the salt form with
a base and isolating the free base in the conventional manner. The
free base forms differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar solvents,
but otherwise the salts are equivalent to their respective free
base for purposes of the present invention.
[0229] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metal
hydroxides, or of organic amines. Examples of metals used as
cations are sodium, potassium, magnesium, calcium, and the like.
Examples of suitable amines are N,N'-dibenzylethylenediamine,
chloroprocaine, choline, diethanolamine, ethylenediamine,
N-methylglucamine, and procaine; see, for example, Berge et al.,
supra., 1977.
[0230] The base addition salts of acidic compounds are prepared by
contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The
free acid form may be regenerated by contacting the salt form with
an acid and isolating the free acid in a conventional manner. The
free acid forms differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar solvents,
but otherwise the salts are equivalent to their respective free
acid for purposes of the present invention.
[0231] The term "metabolite" refers to a form of a compound
obtained in a human or animal body by action of the body on the
administered form of the compound, for example a de-methylated
analogue of a compound bearing a methyl group which is obtained in
the body after administration of the methylated compound as a
result of action by the body on the methylated compound.
Metabolites may themselves have biological activity.
[0232] The term "prodrug" refers to a form a compound which after
administration to a human or animal body is converted chemically or
biochemically to a different compound in said body having
biological activity. A prodrug form of a compound may itself have
biological activity.
[0233] The novel compounds of embodiments of the present invention,
and compounds which may be used in accordance with embodiments of
the present invention may have at least one chiral center, and may
accordingly exist as enantiomers or as mixtures of enantiomers
(e.g., racemic mixtures). Where the compounds possess two or more
chiral centers, they may additionally exist as
diastereoisomers.
[0234] In some embodiments of the present invention, there are
provided pharmaceutical compositions and the use of certain
compounds in the manufacture of pharmaceutical compositions. Such
compositions may be in a form suitable for oral (e.g. in the form
of capsules, tablets, granules, powders or beads), rectal,
parenteral, intravenous, intradermal, subcutaneous, transdermal or
topical administration, or for administration by insufflation or
nasal spray, iontophoretic, buccal, or sublingual lingual
administration. Such compositions may be in unit dosage form. The
compound of formula (I), or, in those embodiments in which AF267B
or AF150(S) may be employed, may be present in the unit dosage in
an amount in the range of about 0.5 to about 100 mg. In an
embodiment of the invention the compound is present in an amount of
about 5 to about 100 mg. In an embodiment of the invention the
compound is present in an amount of about 10 to about 50 mg. These
amounts may represent a single dose or the total of 2-4 individual
doses for administration from 2 to 4 times per day. In an
embodiment of the invention, the pharmaceutical composition is in
sustained release form.
[0235] Certain of the compounds in some embodiments of the present
invention can exist in unsolvated forms as well as solvated forms,
including hydrated forms. In general, the solvated forms, including
hydrated forms, are equivalent to unsolvated forms and are intended
to be encompassed within the scope of the present invention.
[0236] The compounds AF150(S) and AF267B have been described in
U.S. Pat. No. 5,852,029.
[0237] In some embodiments of the invention, the compounds have
antioxidant activity. Such antioxidant activity may be the result
of such compounds being N-oxides, such as AF600, or it may be the
result of such compounds having an anti-oxidant moiety linked at
the 4-position nitrogen.
[0238] The skilled artisan will appreciate that many factors
influence the selection of any compound for application in clinical
therapy, e.g., effectiveness for the intended purpose, safety,
possible side-effects and therapeutic index. The skilled artisan
will thus appreciate how to interpret the expression
"pharmaceutically acceptable quaternary compounds" which are
structurally derived from the inventive compounds having a tertiary
nitrogen atom, as this expression is used in the present
specification and claims.
[0239] The compounds used in embodiments of the present invention
can be prepared and administered in a wide variety of oral and
parenteral dosage forms. Thus, the compounds used can be
administered by injection, that is, intravenously, intramuscularly,
intracutaneously, subcutaneously, intraduodenally, or
intraperitoneally. Also, the compounds can be administered by
inhalation, for example, intranasally. Additionally, the compounds
can be administered transdermally. It will be appreciated by those
skilled in the art that the following dosage forms may comprise as
the active component, either a compound of Formula (I) or a
corresponding pharmaceutically acceptable salt of a compound of
Formula (I), in accordance with embodiments of the invention
optionally with AF267B or AF150(S) present as well.
[0240] For preparing pharmaceutical compositions from compounds of
formula (I), optionally also including AF267B or AF150(S),
pharmaceutically acceptable carriers can be either solid or liquid.
Solid form preparations include powders, tablets, pills, capsules,
cachets, suppositories, and dispersible granules. A solid carrier
can be one or more substances which may also act as diluents,
flavoring agents, binders, preservatives, tablet disintegrating
agents, or an encapsulating material.
[0241] In powders, the carrier is a finely divided solid which is
in a mixture with the finely divided active component. In tablets,
the active component or components is mixed with the carrier having
the necessary binding properties in suitable proportions and
compacted in the shape and size desired.
[0242] In an embodiment of the invention, the powders and tablets
contain from five or ten to about seventy percent of the active
compound. Suitable carriers include magnesium carbonate, magnesium
stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,
tragacanth, methylcellulose, sodium carboxymethylcellulose, a low
melting wax, cocoa butter, and the like. The term "preparation" is
intended to include the formulation of the active compound with
encapsulating material as a carrier providing a capsule in which
the active component with or without other carriers, is surrounded
by a carrier, which is thus in association with it. Similarly,
cachets and lozenges are included. Tablets, powders, capsules,
pills, cachets, and lozenges can be used as solid dosage forms
suitable for oral administration.
[0243] For preparing suppositories, a low melting wax, such as a
mixture of fatty acid glycerides or cocoa butter, is first melted
and the active component is dispersed homogeneously therein, as by
stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool, and thereby to
solidify.
[0244] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water propylene glycol solutions.
For parenteral injection liquid preparations can be formulated in
solution in aqueous polyethylene glycol solution.
[0245] Aqueous solutions suitable for oral use can be prepared by
dissolving the active component in water and adding suitable
colorants, flavors, stabilizing and thickening agents as
desired.
[0246] Aqueous suspensions suitable for oral use can be made by
dispersing the finely divided active component in water with
viscous material, such as natural or synthetic gums, resins,
methylcellulose, sodium carboxymethylcellulose, and other
well-known suspending agents.
[0247] Also included are solid form preparations which are intended
to be converted, shortly before use, to liquid form preparations
for oral administration. Such liquid forms include solutions,
suspensions, and emulsions. These preparations may contain, in
addition to the active component, colorants, flavors, stabilizers,
buffers, artificial and natural sweeteners, dispersants,
thickeners, solubilizing agents, and the like.
[0248] In an embodiment of the invention the pharmaceutical
preparation is in unit dosage form. In such form the preparation is
subdivided into unit doses containing appropriate quantities of the
active component. The unit dosage form can be a packaged
preparation, the package containing discrete quantities of
preparation, such as packeted tablets, capsules, and powders in
vials or ampoules. Also, the unit dosage form can be a capsule,
table, cachet, or lozenge itself or it can be the appropriate
number of any of these in packaged form.
[0249] The quantity of active component in a unit dose preparation
may be varied or adjusted as recited above, according to the
particular application and the potency of the active component. The
composition can, if desired, also contain other compatible
therapeutic agents.
[0250] In therapeutic use, the compounds utilized in accordance
with embodiments of this invention may be administered at the
initial dosage of about 0.01 mg to about 100 mg/kg daily. In an
embodiment of the invention, a daily dose range of about 0.01 mg to
about 10 mg/kg is used. In another embodiment of the invention, a
daily dose range of 10 to 50 mg/kg is used. The dosages, however,
may be varied depending upon the requirements of the patient, the
severity of the condition being treated, and the compound or
compounds being employed. Determination of the proper dosage for a
particular situation is within the skill of the art. Generally,
treatment is initiated with smaller dosages which are less than the
optimum dose of the compound. Thereafter, the dosage is increased
by small increments until the optimum effect under the
circumstances is reached. For convenience, the total daily dosage
may be divided and administered in portions during the day, if
desired.
[0251] The methods used for preparing compounds of the invention
include methods which are essentially known to organic chemists for
the formation of the five-membered rings, ring-substitution,
changing the degree of ring saturation/unsaturation,
interconvertion of salts and bases, quaternary salt formation, and
so forth. In these synthetic methods, the starting materials may
contain a chiral center and, when a racemic starting material is
employed, the resulting product is a mixture of R, S enantiomers,
Alternatively, a chiral isomer of the starting material may be
employed and, if the reaction protocol employed does not racemize
this starting material, a chiral product is obtained. Such reaction
protocols may involve inversion of the chiral center during
synthesis. Select compounds of formula (I) are capable of existing
in a number of stercoisomeric forms including geometric isomers
such as E and Z (in the nitrones) and enantiomers. The invention
includes each of these stereoisomeric forms, and to mixtures
thereof (including racemates). The different stereoisomeric forms
may be separated one from the other by the usual methods, or any
given isomer may be obtained by stereospecific or asymmetric
synthesis. It will be appreciated, therefore, that while exemplary
methods of preparing certain compounds of the invention will be
described, other methods may also be used to prepare the compounds,
as will be known by skilled person.
[0252] When the five-membered ring is thiazolidine-3'-one ring, for
example, the compounds may be prepared by forming this ring by
reacting the corresponding N-heterocyclic ketone with 2-mercapto
carboxylic acid [R.sup.1CH(SH)CO.sub.2H] and ammonia, and the 4-N
atom in the product may be substituted in known manner. These
reactions may be illustrated as follows in Scheme 1, where the
N-heterocyclic ketone is exemplarily 1-methylpiperidine-4-one.
##STR00012##
[0253] The leaving group in "R.sup.5-(leaving group)" may be e.g.
bromide or chloride. This substitution reaction to obtain structure
(B) may be conducted under essentially known condition, e.g. by
reacting of (A) in presence of an alkali such as lithium
di-isopropylamine (LDA) and using a solvent such as tetrahydrofuran
(THF).
[0254] Compound structure (A) or compound structure (B) can be
separated into two enantiomers by chiral HPLC, e.g. chiral
preparative liquid chromatograph (LC) separation of AF267
(R.sup.1=Et, R.sup.5.dbd.H) gave AF267A [R.sup.1=(R)-Et,
R.sup.5.dbd.H] and the active enantiomer AF267B [R.sup.1=(S)-Et,
R.sup.5.dbd.H]. A large scale cost-effective method was developed
and a maximum 50% yield of each enantiomer can be obtained
(yield>97% of each enantiomer, with ee>99% and HPLC
purity>99%).
[0255] The method can be further expanded for an even more
practical separation for those skilled in the art using a Simulated
Moving-Bed (SMB) technology for chiral separation as defined by
Mazzoti et al., (Proceedings on the Chiral Europe 96 Symp., Spring
Innovations, Stockport UK, p 103, 1996).
[0256] AF267B can also be produced by racemization of the AF267A by
chemical means, e.g. base-catalysis, or by enzyme-catalyzation
followed by chiral HPLC or SMB separation.
[0257] Structure (B) may be prepared by reacting the corresponding
N-heterocyclic ketone with 2-mercapto acid [R.sup.1CH(SH)CO.sub.2H]
and primary amine. This reaction may be illustrated as follows in
Scheme 2, where the N-heterocyclic ketone is exemplarily
1-methylpiperidine-4-one:
##STR00013##
[0258] Structure (B) may also be obtained by reacting (A) under
conditions to obtain amide bond as described below in Scheme 3,
where the reacting acid is exemplarily 3-indolpropionic acid:
##STR00014##
[0259] The R.sup.1 group in structure (A) or in structure (B) may
be obtained by reaction of the 2-unsubstituted compound (A) or (B)
with alkyl halide or alkyl aldehyde under standard conditions to
effect substitution in the 2-position. These reactions may be
illustrated as follows in Scheme 4:
##STR00015##
[0260] This method can be applied to .sup.14C-labeling of AF267.
The introduction of the ethyl moiety by alkylation of the readily
available N-protected AF277 (AF287) with .sup.14C-labeled ethyl
bromide, followed by removal of the protecting group yield
.sup.14C-labeling of AF267. The synthetic pathway, which may be
used analogously to prepare AF267 enriched with .sup.13C, is
described below in Scheme 5:
##STR00016##
[0261] The 1-methyl group in structure (A) and in structure (B) may
be removed by reaction with m-chloroperbenzoic acid/FeCl.sub.2 or
with demethylating agent such as phenylchloroformate as shown in
Scheme 6:
##STR00017##
[0262] AF504 or AF292 may also be prepared by reacting the
corresponding N-heterocyclic ketone with 2-mercaptocarboxylic acid
[R.sup.1CH(SH)CO.sub.2H] and primary amine. This reaction may be
illustrated as follows in Scheme 7, where the N-heterocyclic ketone
is N--BOC-piperidine-4-one (BOC=tert-Butoxycarbonyl):
##STR00018##
[0263] Stereospecific synthesis of structure (A) or (B) may also be
obtained by reacting the corresponding N-heterocyclic ketone with
the appropriate 2-mercaptocarboxylic acid, for example: when
1-methylpiperidine-4-one is reacted with (S)-2-mercaptobutyric acid
and NH.sub.3, AF267B is obtained, as shown in Scheme 8 [the (S)
configuration is based on the x-ray crystallography of AF267B]:
##STR00019##
[0264] (S)-2-Mercaptobutyric acid is commercially available or is
prepared from (R)-bromobutyric acid. This reaction may be
illustrated as follows in Scheme 9:
##STR00020##
[0265] When compound (A) is reacted with oxidizing agent such as
hydrogen peroxide or m-chloroperbenzoic acid, structure (D) or
structure (E) is obtained as shown in Scheme 10:
##STR00021##
[0266] The thio analog of structure (A) or (B) may in general be
obtained by reacting the corresponding thiazolidinone ring in
structure (A) with Lawesson's reagent, for example as shown in
Scheme 11:
##STR00022##
[0267] Bivalent compounds containing essentially two ligands within
the same molecule may be obtained by reacting the corresponding
compound structure (A) with a spacer under the same conditions to
obtained compound structure (B) as described earlier. The spacer
(leaving group) is exemplarily alkaneldihalide, alkanediol,
alkanediacid, poly(ethylene glycol). This reaction may be
illustrated as follows in Scheme 12:
##STR00023##
[0268] N-Phosphonooxymethyl prodrugs were reported in Krise et al.,
J Med. Chem. 42: 3094-3100 (1999). Such moieties of
N-phosphonooxymethyl can be used also for synthesis of prodrug (H)
for improving the water solubility of tertiary amine-containing
compounds (B) as shown below. The tertiary amine in compound
structure (B) undergoes a nucleophilic substitution reaction with
di-tert-butyl chloromethyl phosphate which results in the formation
of the quaternary ammonium phosphate protected prodrug. The free
acid form of the prodrug is obtained after removal of the tertiary
butyl groups as shown in Scheme 13:
##STR00024##
[0269] The nitrones, compounds of type (I) and (J), can be prepared
by reacting the spiro-ketone with alkyl hydroxyl amine or aryl
hydroxyl amine. These reactions may be illustrated as follows in
Schemes 14A and 14B:
##STR00025##
##STR00026##
[0270] Alternatively, structure (I) can be prepared by reacting a
five-membered ring carbonyl with alkyl hydroxylamine or aryl
hydroxylamine. The resulting nitrone is cyclized to form the Spiro
structure.
[0271] The compound of formula
##STR00027##
designated AF150(S), is described in U.S. Pat. No. 5,407,938.
However, the synthesis of this compound has now been improved. The
improved synthesis is described in the following scheme 15:
##STR00028##
[0272] AF150(S) was obtained by reaction of 4-picolylamine with
acetic anhydride/methyl iodide, followed by reaction with
Lawesson's Reagent. The obtained thiopyridinium iodide was reduced
to give thioacetylamino-tetrahydropyridine which was cyclized to
form AF150(S).
[0273] When prepared as a free base, AF150(S) is a colorless
liquid. The free base may be stored cold (-20-0.degree. C.) as a
bulk material in dark storage under dry vacuum. AF150(S) may also
be obtained as a salt. In an embodiment of the invention, citric
acid is used to obtain a stable salt that can be used eventually in
a large scale production. A white crystalline citric acid salt of
AF150(S) was prepared by mixing AF150(S) free base with citric acid
in 2-propanol and tetrahydrofuran solution. In comparison to the
tree base, the salt: (a) lacks color development even at high
temperature (accelerated stability test), and (b) shows high
stability if the bulk is kept under anhydrous conditions even at
high temperature (accelerated stability test).
[0274] In another embodiment of the invention, AF150(S) is provided
in pharmaceutical acceptable paraffin oil. The stability of 10% w/w
AF150(S) in paraffin oil was examined at 40.degree. C., under air
or nitrogen atmosphere and in the presence or absence of
tocopherol. No degradation products above 0.1% were detected. A
slight yellow color was observed in samples without tocopherol but
color was not developed in samples containing 0.5% w/w tocopherol
in AF150(S).
[0275] It may noted that the N-methyl group in AF150(S) may be
removed by reaction with m-chloroperbenzoic acid/FeCl.sub.2 as
shown in Scheme 16:
##STR00029##
[0276] When AF150(S) is reacted with oxidizing agent such as
m-chloroperbenzoic acid, AF406 is obtained.
##STR00030##
[0277] Cold simulation to .sup.14C-labeling of AF150(S), AF402, was
obtained by using d.sub.3-iodomethane instead iodomethane in the
first step of the synthesis, according to the scheme of the
synthesis of AF150(S). The synthesis of AF402 is described in
Scheme 17 below:
##STR00031##
[0278] It is to be understood that whereas in the foregoing
description, the illustrative compounds of the invention have shown
piperidine, and quinuclidine rings, other any nitrogen-containing
heterocyclic rings suitable for spiro-configuration with the
depicted spiro five-membered ring may be substituted therefore.
Such compounds may be made by using the corresponding ketone, in
analogy to the use of 3- or 4-piperidone to obtain compounds shown
above. A similar remark applies to the practical EXAMPLES, which
are merely illustrative and not limitative.
[0279] The invention will now be illustrated by the following
non-limiting EXAMPLES.
EXAMPLE 1
Synthesis of
(S)-2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
##STR00032##
[0280] Step 1
Synthesis of 2-mercaptobutyric acid
[0281] 2-Bromobutyric acid (3.2 kg, 19.16 mol) was introduced in a
cooled (ice-water bath) flask. It was stirred and aqueous potassium
hydroxide (18.2 mol) was added gradually (0.5 h) while the
temperature was maintained at 30-40.degree. C. Cooling was stopped
and potassium O-ethyldithiocarbonate (3.48 kg, 21.7 mol) was added
in portions so that the temperature did not exceed 50.degree. C.
(0.5 h). The reaction mixture was stirred at 50.degree. C. for 1
hour, then cooled to 15-20.degree. C. (ice-water bath).
Ethylenediamine (2.6 l, .about.39 mol) was added during a period of
20 min while the temperature was maintained at 45-55.degree. C.
(external cooling). The resulting suspension was stirred at
50.degree. C. for two hours, cooled to 20.degree. C., filtered and
the solid was washed with 2.times.1.5 liter warm water
(40-50.degree. C.). The aqueous filtrate and washings were
combined, cooled below 20.degree. C. (ice-water bath) and aqueous
sulfuric acid [5.2 l, 50% (w/w)] was added slowly while keeping the
temperature at 45-55.degree. C. (0.5 hour, to pH=2). The solution
was cooled to 30.degree. C. and transferred to a 25 liter container
equipped with a mechanical stirrer. Methyl-t-butyl ether (MTBE, 3
l) was added and the mixture stirred and left overnight at room
temperature. The upper oily phase was separated and the lower
aqueous phase was filtered under reduced pressure to remove a
precipitate which was formed. The aqueous phase was extracted with
MTBE, the extracts were combined and the MTBE was removed. The oily
residue was dissolved in cyclohexane (51) and kept in a
refrigerator overnight. A lower phase was formed. It was separated
and extracted with cyclohexane. The cyclohexane extracts were
combined with the upper phase, cyclohexane was removed and the
2-mercaptobutyric (crude) acid was dried in vacuum (54.degree. C./2
mmHg) to yield 2-mercaptobutyric acid (2.18 kg, 18.16 mol).
Step 2
Synthesis of AF267 (racemate)
[0282] 2-Mercaptobutyric acid (705 g, 5.88 moles) and a mixture of
cyclohexane/tert-butyl alcohol (1:3.5 (w/w), 4.3 l) were introduced
into a flask. The solution obtained was stirred and heated to
40-60.degree. C. Gaseous ammonia was bubbled through the solution
till all or most of the 2-mercaptobutyric acid was converted to its
ammonium salt. The bubbling of the ammonia was stopped, the
reaction mixture was heated to reflux and a solution of
1-Methyl-4-piperidone (496 g, 4.39 mol) in a cyclohexane/tert-butyl
alcohol mixture (500 ml) was added. After 1 hr, the solution become
clear and the bubbling of ammonia was renewed and after 13 hrs
(addition of piperidone and reaction time afterwards) the reaction
mixture was cooled and left overnight at room temperature.
Hydrochloric acid solution prepared by diluting aqueous
concentrated acid (one volume) with water (two volumes) (960 ml)
was added and the mixture stirred for 1 h. The solution obtained
(pH.about.2-3) was cooled to 25.degree. C. and the lower aqueous
phase was separated and made basic with aqueous potassium hydroxide
(pH.about.8.5-9) then left overnight at room temperature. The
product which precipitated was filtered and washed with cold water
(100 ml) to give wet powder. The filtrate was basified to pH 9 and
left overnight at 5.degree. C., filtered and washed with 50 ml cold
water to give 72 g powder. The same procedure was repeated to
synthesize a second batch of AF267 (multiplied by a factor of 1.2).
The corps were combined to give 1.6 kg of wet product. The crude
combined product was dissolved in 4.5 liter of hot water
(95.degree. C.), filtered and the clear solution was left at room
temperature for 10 hrs, filtered and dried for 24 hrs (50.degree.
C., 1 mmHg) to give AF267 (1.048 kg, 50.7% yield). The filtrate was
concentrated, cooled overnight at 5.degree. C., filtered, washed
(100 ml cold water) and dried to give AF267 (215 g, 10.4% yield).
Total AF267 yield: 61%. mp. 142-144.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta.1.02 (t, j=7.3 Hz, CH.sub.3CH.sub.2), 1.7-1.8
(m, CH.sub.3CHH), 1.96-2.07 (m), 2.30 (s, NCH.sub.3), 2.3-2.36 (m,
2H), 2.6-2.7 (bs, 2H), 3.80 (dd, j=8.7, 3.9 Hz, 1H, SCH) ppm. MS me
214 (M.sup.+), 181 (M.sup.+-SH); Anal. (C.sub.10H.sub.18N.sub.2OS)
calcd. C, 56.04; H, 8.47; N, 13.07; S, 14.96. found C, 55.92; H,
8.44; N, 13.23, S 14.81.
Step 3
Chiral Separation of AF267B and AF267A
[0283] Prochom LC 110 High Performance Preparative Liquid
Chromatograph
[0284] Column: CHIRALPAK.RTM. ASV (lot number JG 001)
[0285] Pump flow rate: 500 ml/min
[0286] Pressure: 12.7 bar
[0287] Column Temp 26.degree. C.
[0288] Mobile phase: Acetonitrile/EtOH 85:15
[0289] Concentration: 37 gr/l
[0290] UV Detection: 240/230 nm
[0291] Following elution the eluent was evaporated to dryness.
[0292] First eluting enantiomer (AF267A): ee: 99.7 (687.1 gr;
purity 99.3%; Yield: 97%)
[0293] Second eluting enantiomer (AF267B): ee: 99.8 (694.9 gr;
99.9% Yield: 98%).
[0294] Residual solvent (e.g. acetonitrile) was removed by further
addition of ethanol and evaporation to dryness.
[0295] By analogous syntheses AF292 and other related compounds may
be prepared.
EXAMPLE 2
X-Ray Single Crystal Structure Analysis of
(S-2-Ethyl-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF267B)
[0296] Crystal data: C.sub.10H.sub.19NOS+H.sub.2O (monohydrate),
transparent, light yellow, prisms, crystal size
0.2.times.0.2.times.0.4 mm.sup.3; crystal system, orthorhombic,
space group: P212121 (No 16) a=10.394 (10) (.alpha.=90.degree.),
b=20.133 (2) (.beta.=90.degree.), c=5.856 (4) (.gamma.=90.degree.),
.ANG., from 25 reflection, T=110 K, Volume=1224.2 (9) .ANG..sup.3,
Z=4, Fw=202.32, Calculated density, Dc=1.092 Mg/m.sup.3, Absorption
coefficient, .mu.=0.232 mm.sup.-1.
[0297] Data collection and treatment: Rigaku AFC5R four-circle
diffractometer, MoK.alpha., graphite monochromator (.lamda.=0.71073
.ANG.), 11461 reflections collected, Theta range for data
collection: 2.82.degree..ltoreq..theta..ltoreq.7.53.degree.; Index
ranges: -13.ltoreq.h.ltoreq.13, -26.ltoreq.k.ltoreq.26,
0.ltoreq.l.ltoreq.7, .omega. scan method, scan width=1.2.degree.,
scan speed 2'/min, typical half-height peak width=0.45.degree., 3
standards collected 62 times each, with a 3% change of intensity;
Reflections collected: 6272 measurements, 2833 independent
reflections [R (int)=0.0604, Bijvoet reflections kept
separated].
[0298] Solution and refinement: structure solved by direct methods
(SHELXS-97). Full-matrix least-squares refinement based on F.sup.2
(SHELXL-97). Idealized hydrogens were placed and refined in a
riding mode, water hydrogens found from the difference Fourier map,
148 parameters; final R indices: R.sub.1=0.0671 (based on F.sup.2)
for data with I>2 sigma(I) wR.sup.2=0.1484 and R.sub.1=0.0747
wR.sup.2=0.1553 for all data, goodness-of-fit on F.sup.2=1.13,
largest electron density=0.745 e/.sup.3- around S atom.
[0299] Absolute configuration: The absolute configuration of the
molecule was determined using Flack's parameter approach and the
alternative refinement of the enantiomeric twinning component. Both
methods show unequivocally that the present coordinates belong to
the correct absolute configuration (S enantiomer). The crystals of
this compound were prepared from crystallization in
toluene/petroleum ether/methanol.
##STR00033##
EXAMPLE 3
Chiral Synthesis of (R)-2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro 4.5
decan-3-one, AF267A
##STR00034##
[0301] The asymmetric synthesis of AF267A was performed in order to
model the synthesis of the S enantiomer by using optically active
2-mercaptobutyric acid in the synthesis of AF267. Optically active
2-mercaptobutyric acid was synthesized starting from
L-(+)-2-aminobutyric acid (I) which has the S configuration. The
amino acid was converted to (S)-2-bromobutyric acid (II) with
retention of configuration by treatment with sodium nitrite,
potassium bromide and hydrobromic acid. The enantiomeric purity of
the obtained bromide was checked by proton NMR measured in the
presence of (R)-(+)-N-benzyl-.alpha.methylbenzylamine and compared
to the spectrum of the racemic bromide measured at the same
conditions. The presence of only one enantiomer was detected by
this method. The bromide was converted to (R)-2-benzoylthiobutyric
acid (III) (with inversion of configuration) by treatment with
cesium thiobenzoate in DMF. Debenzoylation of (III) was
accomplished without racemization by aminolysis (1N ammonium
hydroxide at room temperature).
[0302] The obtained (R)-2-mercaptobutyric acid was purified by
distillation and then reacted with ammonium acetate and
1-methyl-4-piperidone in boiling cyclohexane. The crude reaction
mixture was analyzed by GC on a chiral column and found to contain
AF267A accompanied by a small (2-3%) amount of AF267B.
EXAMPLE 4
Chiral synthesis of
(S)-2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
AF267B
[0303] This compound is obtained as in EXAMPLE 3, except that the
starting material used is (R)-2-bromobutyric acid.
EXAMPLE 5
Synthesis of (S)-2-Ethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one,
AF292
##STR00035##
[0305] To a cooled (ice-salt bath) stirred solution of AF267B (6.1
gr, 0.028 mol) in dichloromethane (60 ml) was added
m-chloroperbenzoic acid (mCPBA) in small portions over a period of
15 min mCPBA (70%, 7.02 gr, 0.028 mol total). The mixture was
stirred for 1 hr and then treated with iron (II) chloride (2.14 ml
of 1M solution in water). Stirring and cooling (-10.degree. C.)
were continued for 1 hr and then stirring continued for 2 hrs at
room temperature. Ethylene diamine (1.9 ml, 0.285 mol), sodium
hydroxide (30.5 ml of 2N aqueous solution), and petroleum ether
40-60.degree. C. (60 ml) were added. After vigorous shaking the
layers were separated, the aqueous layer was extracted with mixture
of dichloromethane/petroleum ether 1:1 (600 ml) followed by
dichloromethane (first 600 ml then 300 ml). The combined extracts
were dried (Na.sub.2SO.sub.4), filtered and the solvents were
removed under reduced pressure. Flash chromatography (silica-gel
60, 230-400 mesh, Merck 1.09385, elution with methanol/chloroform
ammonium hydroxide 10:89:1 v/v) of the residue gave AF292. .sup.1H
NMR (CDCl.sub.3) .delta. 1.02 (t, j=7 Hz, 3H), 1.76, 1.92 and 2.07
(3.times.m, 6H), 2.84 (m, 2H), 3.04 (m, 2H), 3.83 (dd, j=8.8, 4 Hz,
1H), 7.66 (NH) ppm; .sup.13C NMR (CDCl.sub.3) .delta. 11.50, 27.24,
42.87, 43.95, 44.10, 48.80, 64.22, 175.22 ppm: MS m/e 200(M.sup.+).
The compound was >99.9%/(purity by HPLC, GC.
[0306] The hydrochloride salt of AF292 was formed by addition of
HCl (4M in methanol) and recrystallised from methanol-diethyl ether
to give a white precipitate that was filtered and dried. .sup.1H
NMR (D.sub.2O) .delta. 0.78 (t, j=7.3 Hz, 3H), 1.58 (m, 1H), 1.76
(m, 1H), 2.05 (m, 4H), 3.07 (m, 2H), 3.30 (m, 2H), 3.92 (dd, j=7.9,
4.0 Hz, 1H) ppm.
EXAMPLE 6
Reactions for Preparation of
[.sup.14C]-2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one;
R and S Isomers
##STR00036##
[0308] In a septum-capped dried flask (10 ml) equipped with a
magnetic bar and nitrogen inlet, a solution of diisopropylamine
(0.078 ml, 0.56 mmol) in dry THF (1.4 ml) is introduced by a
syringe and cooled to 0.degree. C. n-BuLi (0.9 M in hexane, 0.62
ml, 0.56 mmol) is added, the reaction mixture is stirred at
0.degree. C. for 20 min and then cooled to -78.degree. C. A
solution of AF287 (0.1375 gr, 0.51 mmol) in dry THF (0.4 ml) and
dry N,N'-dimethyl-N,N'-propylene urea (DMPU) (0.6 ml) is added
dropwise (30 min) and the reaction mixture is stirred for 20 min at
-78.degree. C. Ethyl bromide (0.043 ml, 0.56 mmol) labeled at the
I-carbon with .sup.14C is added in one portion, the temperature is
allowed to rise to room temperature and the reaction mixture is
stirred for an additional 4 h. The solvents are removed under
reduced pressure, first using a water pump at 25.degree. C. for 20
min and then an oil pump (-4 mm Hg) at .about.60.degree. C. for
.about.30 min (using a needle which introduced a stream of air to
remove the solvent faster). Flash chromatography of the residue
gives racemic AF267 (95 mg, .about.86% yield). Preparative chiral
HPLC may be used to separate the enantiomers. By following the
above procedure using ethyl bromide which was not labelled,
preparative HPLC of 60 mg of the racemate obtained after flash
chromatography afforded non-isotopically labeled AF267B (17.6
mg).
EXAMPLE 7
Synthesis of
(S)-2-Ethyl-8-methyl-8-oxy-1-thia-4,8-diaza-spiro[4.5]deca-3-one,
AF299
##STR00037##
[0310] A solution of mCPBA (70%, 2.62 gr, 10.64 mmol) in
dichloromethane (40 ml) was added slowly (0.5 hr) to a cold
(0.degree. C.) and stirred solution of AF267B (2.07 gr, 9.67 mmol)
in dichloromethane (40 ml). The cooling bath was removed and the
reaction mixture was stirred at room temperature for 2 hrs and then
the solvent was removed under reduced pressure. Flash
chromatography (methanol/chloroform/ammonium hydroxide 10:89:1 v/v)
of the residue and precipitation of the product as a solid from
methanol-acetonitrile gave the N-oxide, AF299; .sup.1H NMR
(CDCl.sub.3) .delta. 0.99 (t, j=7.3 Hz, CH.sub.3CH.sub.2), 1.74 and
2.09 (2m, CH.sub.3CH.sub.2), 1.82 (m, 2H), 3.9 (m, 2H), 3.36 (s,
CH.sub.3N.sup.+), 3.33-3.45 (m, 4H), 3.78 (br NH), 3.83 (dd,
j=3.74, 8.84 Hz, CH.sub.3CH.sub.2CH) ppm; MS m/e 230 (M.sup.+).
EXAMPLE 8
Synthesis of
(S)-2-Ethyl-8-methyl-1-oxo-1.lamda..sup.4-thia-4,8-diaza-spiro[4.5]decan--
3-one, AF300
##STR00038##
[0312] A solution of AF267B (1.72 gr, 0.008 mol) in water (2.5 ml)
was cooled (ice-water bath) and trifluoroacetic acid (3.5 ml) was
added. To the cold stirred obtained mixture was added hydrogen
peroxide (30%, 0.57 ml, 0.008 mol), the cooling bath was removed
and the reaction mixture was stirred at room temperature over
night. Sodium sulfite was added and the pH of the solution was
adjusted to 9 with a saturated solution of sodium carbonate. The
aqueous phase was extracted with dichloromethane (2.times.100 ml)
and then with ethyl acetate (1.times.50 ml). The organic extracts
were combined, dried (MgSO.sub.4) and the solvent was evaporated.
Flash chromatography (silica-gel 60, 230-400 mesh, Merck 1.09385,
elution with methanol/chloroform ammonium hydroxide 10:89:1 v/v)
gave AF300. .sup.1H NMR (CDCl.sub.3) .delta. 1.19 (t, j=7.3 Hz, 3H,
CH.sub.3), 1.85-2.0 (m, 5H), 2.17 (m, 1H), 2.27 (m, 1H), 2.34 (s,
3H, NCH.sub.3), 2.6 (m, 2H), 3.30 (dd, j=3.56, 11.13 Hz,
CH.sub.3CH.sub.2CH), 6.37 (br NH) ppm; MS (EI) m/e 230
(M.sup.+).
EXAMPLE 9
Synthesis of
2-(1-Hydroxy-ethyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
(AF298)
##STR00039##
[0314] To a cold (0.degree. C.) solution of diisopropylamine (0.28
ml, 0.002 mol) in dry THF (12 ml) under argon atmosphere was added
a solution of n-butyllithium (1.4M in hexane, 1.4 ml, 0.002 mol),
the mixture was stirred for 20 min and then cooled to -78.degree.
C. A solution of AF287 (0.41 g, 0.0015 mol) in THF (3 ml) was added
dropwise (10 min) and the resulting mixture was stirred at
-78.degree. C. for additional 10 min. Acetaldehyde (0.85 ml, 0.015
mol) was added in one portion and after ten min at -78.degree. C.
acetic acid (0.11 ml, 0.002 mol) was added in one portion and the
temperature was allowed to raise to room temperature. The reaction
mixture was added to chloroform (200 ml) and the organic phase was
washed with water (2.times.20 ml), separated and dried. The solvent
was evaporated and flash chromatography (silica,
CHCl.sub.3/MeOH/NH.sub.4OH 80/20/1) of the residue gave AF298
(0.132 g). .sup.1H-NMR (CDCl.sub.3) 1.24 (d, j=6.05 Hz, 3H,
CH.sub.3), 1.7-2.2 (m, CH.sub.2), 2.31 (s, 3H, NCH.sub.3),
2.60-2.80 (m, CH.sub.2), 3.71 (d, j=9.41 Hz, 1H, SCH), 3.94-4.04
(m, 1H, CHO), 4.75-4.9 (br OH), 7.1-7.2 (br NH) ppm. MS m/e 230
M(.sup.+).
EXAMPLE 10
Synthesis of
(S)-2-Ethyl-4-(4-fluoro-benzenesulfonyl)-8-methyl-1-thia-4,8-diaza-spiro[-
4.5]decan-3-one, AF700
##STR00040##
[0316] Into a cold (0.degree. C., ice-water bath) solution of
lithium hexamethyldisilazane (5 ml, 1M in THF) was added AF267B
(1.5 g, 0.007 mol) in small portions over a period of 15 min under
argon atmosphere. The cooling bath was removed and the reaction
mixture was stirred at room temperature for 40 min.
4-fluorobenzylsulfonyl chloride (1.37 g, 0.007 mol) was added
(during the addition the temperature was kept below 20.degree. C.,
cooling bath) and the reaction mixture was left at room temperature
under argon atmosphere overnight. Dichloromethane (100 ml) was
added. The reaction mixture was washed with water (20 ml) the
organic phase was separated, dried (MgSO.sub.4) and evaporated.
Flash chromatography (silica, ethyl acetate/methanol/aqueous
ammonia 10/2/0.1) gave AF700 (0.46 gr, 0.0015 mol). .sup.1H-NMR
(CDCl.sub.3, 300 MHz) .delta. 0.94 (t, j=7.14 Hz, 3H, CH.sub.3),
1.64-1.77 (m, 3H, CHH+CH.sub.2), 1.88-2.00 (m, 3H, CHH+CH.sub.2),
2.15-2.29 (br, 2H, CH.sub.2), 2.29 (s, 3H, NCH).sub.3, 2.51-2.60
(br, 2H, CH.sub.2), 4.24 (dd, j=7.99, 3.72 Hz, 1H, SCH), 7.21 (app.
t, j=8.53 Hz, 2H, Ar) 8.07-8.12 (m, 2H, Ar).
EXAMPLE 11
Synthesis of
8-Methyl-4-pyrrolidin-1-ylmethyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one,
AF287
##STR00041##
[0318] A solution of
8-Methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (AF277 5.13 g,
0.0275 mol), formaldehyde (37% solution, 2.75 ml) and pyrrolidine
(2.3 ml, 0.0275 mole) in ethanol (2.3 ml) was refluxed for 4 h,
then left at room temperature over night. Toluene (10 ml) was added
and the solvent was evaporated. Boiling pentane (100 ml) was added
to the residue and the solution was decanted. The trituration with
hot pentane was repeated four times, the pentane layers were
combined, cooled to 0.degree. C. and the precipitate was collected
and identified as AF287 (4.6 gr, 63% yield). .sup.1H-NMR
(CDCl.sub.3) .delta. 1.69-1.77 (m, 5H), 2.20-2.29 (s and m, 5H,
CH.sub.3 and CH.sub.2), 2.43-2.58 (m, 6H), 2.82-2.86 (m, 2H), 3.50
(s, 2H, SCH.sub.2), 4.14 (s, 2H, NCH.sub.2N) ppm. MS (EI) m/le
269(M.sup.+); 198; 84; 70.
EXAMPLE 12
Synthesis of
2-Ethyl-4-(3-1H-indol-3-yl-propionyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5-
]decan-3-one, AF704
##STR00042##
[0320] A solution of AF267 (1.2 gr, 0.0056 mol), 3-indolpropionic
acid (1.37 gr, 0.0072 mol), dicyclohexylcarbodiimide (DCC) (1.57
gr, 0.0076 mol) and 4-dimethylaminopyridine (DMAP) (0.93 gr, 0.0076
mol) in dichloromethane (120 ml) was stirred at room temperature
for 3 days. The reaction mixture was washed with water (2.times.40
ml), the organic phase was dried and evaporated. Flash
chromatography (silica, CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) gave
the title compound which was triturated in acetone. The solution
was filtered to remove the impurities, then the acetone was
evaporated and the obtained thick oil was triturated in ether. The
obtained solid (AF704), 400 mg, was filtered and dried. Mp.
122.5-124.5.degree. C.; .sup.1H-NMR (CDCl.sub.3) .delta. 1.01 (t,
j=7.4 Hz, 3H, CH.sub.3CH.sub.2), 1.50 (m, 1H), 1.59 (m, 1H),
1.61-1.73 (m, 1H, CH.sub.3CHH), 2.1-2.15 (m, 1H, CH.sub.3CHH), 2.18
(dt, j=12.4, 2.4 Hz, 1H), 2.29 (s, 3H, NCH.sub.3), 2.33 (m, 1H),
2.83 (m, 2H), 2.99 (dt, j=12.55, 4.39 Hz, 1H), 3.01 (t, j=7.42 Hz,
2H, CH.sub.2), 3.16 (dt, j=12.7, 4.39 Hz, 1H), 3.28 (m, 2H), 3.67
(dd, j=8.9, 4.18 Hz, 1H, SCH), 7.04 (br s, C.dbd.CH), 7.11 (t,
j=7.8 Hz, ArH), 7.18 (t, j=7.1 Hz, ArH), 7.34 (d, j=8.08 Hz, ArH),
7.64 (d, j=7.58 Hz, ArH), 8.01 (br NH) ppm; MS (EI) m/e 385
M(.sup.+), 214, 181, 171, 143, 130 (100%).
[0321] When the starting material is AF267B, the enantiomer AF704B
is obtained.
EXAMPLE 13
Synthesis of
2-Ethyl-4-[2-(1H-indol-3-yl)-ethyl]-8-methyl-1-thia-4,8-diaza-spiro[4.5]d-
ecan-3-one AF703
##STR00043##
[0323] In a three-necked flask equipped with a magnetic stirrer and
Dean-Stark and dropping funnel a solution of 2-mercaptobutyric acid
(7.38 gr, 0.065 mol) in a mixture of t-butanol/cyclohexane (30/104
gr/gr, 86 ml) was heated to 40.degree. C. Tryptamine (10.8 gr,
0.068 mol) was added in four portions (20 min), the reaction
mixture was stirred for additional 45 min. and then heated to
reflux, A solution of 1-methyl-4-piperidone (6.18 ml, 0.049 mol) in
t-butanol/cyclohexane (30/104 gr/gr, 10 ml) was added dropwise
during 40 min and the reflux continued for 5 hrs (1 ml water was
collected). Mixture of HCl/water (2:2 v/v) was added until pH 2-3,
the aqueous phase was separated, basified to pH 10 with solution of
potassium hydroxide and extracted with dichloromethane. The organic
phase was separated, dried and the solvent was evaporated. Flash
chromatography (silica, CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) of the
residue gave the title compound. Recrystallization from boiling
hexane eave pure AF703. .sup.1H-NMR (CDCl.sub.3) .delta. 1.04 (t,
j=7.35 Hz, 3H, CH.sub.3), 1.65-1.68 (m, 2H), 1.71-1.80 (m, 1H,
CH.sub.3CHH), 2.13-2.34 (m, 5H), 2.31 (s, 3H, NCH), 2.86 (m, 2H),
3.05-3.10 (m, 2H), 3.41-3.49 (m, 1H), 3.51-3.65 (m, 1H), 3.80 (dd,
j=8.8, 3.9 Hz, 1H, SCH), 7.02 (d, j=2.32 Hz, 1H, ArH), 7.14 (ddd,
j=7.35, 7.35, 1.17 Hz, 1H, ArH), 7.20 (ddd, j=7.45, 7.45, 1.39 Hz,
1H, ArH), 7.35 (d, j=7.4 Hz, 1H, ArH), 7.77 (d, j=7.42 Hz, 1H,
ArH), 8.03 (brNH) ppm.
EXAMPLE 14
Synthesis of 2,8-Dimethyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene AF
150(S)
##STR00044##
[0324] Step 1
Synthesis of 4-(acetaminomethyl)-1-methyl-pyridinium iodide
[0325] To a cold (ice-water bath) solution of 4-picolylamine (1070
gr, 9.9 mol) in methanol (3 l) was added dropwise acetic anhydride
(1400 gr, 13.7 mol). During addition the reaction temperature was
kept between 10 and 30.degree. C. When the addition of the reagent
was complete the reaction mixture was left overnight at room
temperature. Iodomethane (800 ml, 24.8 mol) was added to the
reaction mixture which was cooled with water bath, under nitrogen
atmosphere. During addition the reaction temperature was kept below
25.degree. C. When the addition was complete, the reaction mixture
was protected from light and left at room temperature overnight.
The excess iodomethane was evaporated, crystallization was induced,
the reaction mixture was cooled (ice bath) and isopropanol (1.5 l)
was added. The reaction mixture was left at -30.degree. C.
overnight, the precipitate was filtered off, washed with
isopropanol and dried. 4-(Acetaminomethyl)-1-methyl-pyridinium
iodide (2043 gr, 7 mol) was obtained as yellow powder (71% yield).
.sup.1H-NMR (D.sub.2O) .delta. 2.10 (s, CH.sub.3CO), 4.32 (s,
CH.sub.3N'), 4.63 (s, CH.sub.2NHCO), 7.89 (d, j=6.6 Hz, 2H), 8.67
(d, j=6.6 Hz, 2H) ppm.
Step 2
Synthesis of 1-methyl-4-N-thioacetylaminomethylpyridinium
iodide
[0326] A stirred solution of
4-(Acetaminomethyl)-1-methyl-pyridinium iodide (1.92 kg, 6.5 mol)
and Lawesson's reagent (1.87 kg, 4.6 mol) in acetonitrile (6 l) was
warmed to 80.degree. C. for 17 hrs. Then the reaction mixture was
cooled to room temperature and stirred for an additional 4 hrs. The
crude thioamide was filtered off and washed with acetonitrile (2.8
l). To the obtained thioamide was added ethyl acetate (10 l) and
the suspension was refluxed for 1 h. At 72-73.degree. C. a noxious
gas was evolved and trapped with NaOH solution. The suspension was
cooled to 60.degree. C. The thioacetamide was filtered off, washed
with ethyl acetate (2 l) and dried (45.degree. C.). 2 kg of
thioamide was obtained.
Step 3
Synthesis of
1-methyl-4-N-thioacetylamino-1,2,3,6-tetrahydropyridine
[0327] A suspension of 1-methyl-4-N-thioacetylaminomethylpyridinium
iodide (2.07 kg, 6.7 mol) in water (6.2 l) was prepared in 25 l
flask and stirred at room temperature. A solution of sodium
borohydride (383 gr, 10.1 mol) in water (1.2 l) was added dropwise
over a period of 3.5 h so that the temperature was maintained below
32.degree. C. The reaction mixture was stirred for 2 h at room
temperature, ethanol (600 ml) was added and stirring was continued
for 30 min. Sodium carbonate (780 g) was added and the reaction
mixture was stirred overnight. Dichloromethane (4 l) was added, and
stirring was continued for 45 min. The solution was filtered in
order to remove the solid which was washed with chloroform (1 l)
and water (0.5 l). The filtrate was decanted, and the aqueous phase
was extracted with chloroform (2.5 l). The organic phases were
combined and washed with 10% sodium thiosulfate solution (2.2 l).
The aqueous phase was extracted with chloroform (1 l), the organic
phases were combined, dried over magnesium sulfate, filtered and
concentrated. Acetone (1.5 l) was added, the suspension was stirred
for 30 min at room temperature and for 45 min at 0.degree. C. The
thioacetamide was filtered off, washed with acetone (1.5 l) and
dried (50.degree. C.). 860 gr of thioacetamide was obtained. mp.
140.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 2.21 (m, 2H), 2.36
(s, CH.sub.3N), 2.56 (s, CH.sub.3CS), 2.57 (t, 2H), 2.95 (m, 2H),
4.23 (d, CH.sub.2NH), 5.65 (m, CH.dbd.C), 7.41 (bs, NH) ppm. MS m/e
184 (M.sup.+), 151, 150, 149, 141, 140, 126, 114, 109 (100%), 109,
96, 94, 82, 70.
Step 4
Synthesis of 2,8-dimethyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene
AF150(S)
[0328] In three-necked round bottom flask (5 l) equipped with a
mechanical stirrer polyphosphoric acid (2.1 kg) was stirred and
heated to 100.degree. C. The thioacetamide (840 gr) was added in
small portion. At the end of the addition the temperature was
raised to 170.degree. C. and this temperature was maintained for
2-3 hrs. The hot reaction mixture was slowly poured into a stirred
cold aqueous solution of sodium carbonate (25%, 10 l). The basicity
was raised by addition of aqueous sodium hydroxide solution (50%,
350 ml). The reaction mixture was extracted with chloroform
(2.times.3 l), the organic phases were combined, dried
(Na.sub.2SO.sub.4) and evaporated. The residue was dissolved in
petroleum ether (3.5 l) to give a solution and small amount of
insoluble material. Evaporation of the petroleum ether solution
gave crude AF150(S). The described procedure was repeated and the
crude AF150(S) from the combined batches was treated with activated
carbon and was distilled twice under reduced pressure (0.5 mmHg,
61.degree. C.) to give AF150(S) (>1.2 Kg, global yield of 40%).
.sup.1H NMR (CDCl.sub.3) .delta. 1.8-2.0 (m, 4H), 2.18 (t, 3H,
CH.sub.3C), 2.28 (s, 3H, C.sub.1-3N), 3.9 (m, 2H, CH.sub.2) ppm; IR
(C.dbd.O) 1636 cm.sup.-1; MS. M/e 184 (M.sup.+).
EXAMPLE 15
Synthesis of 2,8-Dimethyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene
8-oxide AF406
##STR00045##
[0330] A solution of m-chloroperbenzoic acid, mCPBA, (1.40 gr, 8.11
mmol) in dichloromethane (30 ml) was added gradually to a solution
of AF150(S) (1.45 gr, 7.88 mmol) in dichloromethane (10 ml). The
reaction was stirred at room temperature overnight. Chromatography
of the reaction mixture was on a column of natural aluminum oxide
(Merck)(metanol:chloroform 1/49) gave AF406 (0.75 gr) as a
crystalline solid. A sample was crystallized from ethylacetate. A
very hygroscopic solid was obtained. mp. 130-132.degree. C.
(145-159.degree. C. dec.); .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.89 (m, 2H), 2.2 (t, j=10.5 Hz, CH.sub.3C.dbd.N), 2.82 (m,
2H), 3.24 (m, 2H), 3.25 (s, CH.sub.3N.sup.+O.sup.-), 3.35 (m, 2H),
4.03 (q, j=1.5 Hz, CH.sub.2N.dbd.C) ppm; IR (CHCl.sub.3) 2947,
1636, 1448, 1153, 931, 664 cm.sup.-1; MS (EI) 200 (M.sup.+), 184,
182, 149, 141, 140, 126, 110, 109, 108, 96, 82, 70.
EXAMPLE 16
Synthesis of 2-Methyl-1-thia-3,8-diaza-spiro[4.5]dec-2-ene,
AF400
##STR00046##
[0332] A solution of AF406 (2 gr, 0.01 mol) in chloroform (10 ml)
was cooled to -10.degree. C.-5.degree. C. in an ice-salt bath. A
solution of FeCl.sub.2 (1M, 0.7 ml) was added and the two phase
reaction mixture was stirred for 4.5 h. The color of the reaction
mixture changes with time from dark green to dark orange-brown. To
the cooled reaction mixture was added cautiously a mixture of
petroleum-ether (10 ml), ethylene diamine (600 mg, 0.01 mol) and 2N
sodium hydroxide (10 ml, 0.02 mol). The pH of the water phase was
13. The organic was separated and the aqueous phase was extracted
with chloroform, acidified with 5N HCl to pH=9 and extracted again
with chloroform. All the organic fractions were combined, dried on
potassium carbonate, filtered and evaporated. Chromatography
[silica-gel (RIEDEL DE Haen 31607), CHCl.sub.3:MeOH:NH.sub.4OH
90/9/1] of the residue gave AF400. mp. 40.degree. C.; .sup.1H NMR
(CDCl.sub.3) .delta. 1.74 (m, 2H), 1.92 (m, 2H), 2.19 (t, j=1.8 Hz,
3H), 2.71 (m, 2H), 3.05 (m, 2H), 3.92 (q, j=1.8 Hz, 2H) ppm; MS m/e
171 (M.sup.++1), 170 (M.sup.+).
EXAMPLE 17
Synthesis of
2-Methyl-8-methyl-d.sub.3-1-thia-3,8-diaza-spiro[4.5]dec-2-ene.
AF402
##STR00047##
[0334] To a stirred cold (ice-water bath) solution of
4-picolylamine (50 g, 0.462 mol) in methanol (200 ml) acetic
anhydride (75 g, 0.735 mol) was added slowly (1 hr). The
temperature was kept at 10-15.degree. C. during the addition. The
reaction mixture was left overnight at room temperature. TLC
[silica, chloroform/methanol/ammonia (33%) 90:10:1 (v/v)] showed
one spot at Rf.about.0.4. The product,
N-Pyridin-4-ylmethyl-acetamide was not isolated and was processed
to the next step.
[0335] Part of the reaction mixture (43 ml) was evaporated. The
acetamide salt was obtained as yellow oil (14.5 g). Part of the oil
(1.9 g, .ltoreq.0.06 mol) was dissolved in methanol (40 ml),
stirred under nitrogen atmosphere and protected from light.
Iodomethane-d.sub.3 (10 g, 0.07 mol) was added, maintaining the
temperature of the reaction mixture at 15-25.degree. C. The
reaction mixture was left overnight at room temperature then
triturated twice with ether (2.times.200 ml).
4-(Acetamido-methyl)-1-methyl-d.sub.3-pyridinium iodide was
obtained as yellow solid TLC [silica, chloroform/methanol/ammonia
(33%) 90:10:1 (v/v)] showed one spot at Rf.about.0.05] and was
reduced in the next step without further purification.
[0336] To a cold (ice-water bath) solution of the pyridinum iodode
salt in methanol (40 ml) under nitrogem atmosphere, sodium
borohydride (3.9 gr, 0.2 mol) was gradually added (2 hr) so the
temperature was maintained at 15-30.degree. C., the reaction
mixture was stirred for additional 2 hrs at room temperature and
left overnight without stirring. The solvent was evaporated.
N-(1-methyl-d.sub.3-1,2,3,6-tetrahydropyridine-4-ylmethyl)-acetamide
was obtained as stick yellowish oil (8.5 g). .sup.1H-NMR
(CDCl.sub.3) .delta. 1.98 (s, 3H, CH.sub.3CN), 2.12 (m, 2H), 2.52
(t, 2H), 2.91 (m, 2H), 3.76 (d, CH.sub.2NHCO), 5.52 (m, CH.dbd.C),
6.66 (br. s, NH) ppm. MS in/e 172 (M.sup.+).
[0337] In a three-necked round bottom flask (250 ml) equipped with
an addition funnel and condenser with a calcium chloride tube on
its top, a solution of the tetrahydropyridine acetamide (8.5 gr) in
dry acetonitrile (70 ml) was added. To the stirred solution,
phosphorous pentasulfide (6.7 gr, 0.03 mol) was added followed by
triethylamine (12 gr, 0.12 mol) which was added from the additional
funnel during 10-15 min. The obtained solution was heated under
reflux for 5 hrs and then left at 15.degree. C. for three days. The
solution was evaporated, basified with 10% aqueous potassium
carbonate, then extracted with chloroform. The organic phase was
dried and evaporated. Crude black
N-(1-methyl-d.sub.3-1,2,3,6-tetrahydropyridine-4-ylmethyl)-thioacetamide
(6.62 gr, 0.036 mol) was obtained. .sup.1H-NMR (CDCl.sub.3) .delta.
2.21 (m, 2H), 2.56 (s, CH.sub.3CS), 2.58 (t, j=4.4 Hz, 2H), 2.97
(m, 2H), 4.23 (d, j=4.4 Hz, CH.sub.2NH), 5.65 (m, CH.dbd.C), 7.41
(br s, NH) ppm. MS m/e 187(M.sup.+).
[0338] In a flask (150 ml), polyphosphoric acid (30 g) was added to
the crude thioacetamide (6.5 gr). The reaction was stirred and
heated to 170.degree. C. for 3.5 h. The hot reaction mixture was
slowly poured into a stirred 25% aqueous sodium carbonate (150 ml).
25% Aqueous sodium carbonate (50 ml) was added to the residue in
the reaction flask and the two solutions were combined. The
basicity was raised by addition of an 50% aqueous sodium hydroxide
(6 ml) and the reaction mixture was extracted with chloroform. The
organic phase was separated, dried and evaporated. The residue was
dissolved in petroleum ether (I 00 ml) to give after 12 h at
-20.degree. C. a solution and a small amount of insoluble material.
The petroleum ether solution was evaporated and the obtained oil (5
g) was distilled at reduced pressure (b.p. 50-53.degree. C., 0.2
mmHg) to give AF402 (2.15 g, 0.012 mol). .sup.1H-NMR (CDCl.sub.3)
.delta. 1.87 (m, 2H), 1.94 (m, 2H), 2.1 (m, 2H), 2.2 (t, j=1.7 Hz,
3H), 2.76 (m, 2H), 3.92 (m, 2H) ppm. MS m/e 187 (M.sup.+).
EXAMPLE 18
Synthesis of
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]-N-oxi-
de, AF600
##STR00048##
[0340] To a solution of N-methylhydroxylamine hydrochloride (0.85
gr, 0.01 mol) in ethanol (13.5 ml) was added sodium acetate (0.84
gr, 0.01 mol). A white precipitate was obtained, a solution of
2,8-dimethyl-1-oxa-8-aza-spiro[4.5]decan-3-one (1.62 gr, 0.0088
mol) in ethanol (7 ml) was added and the mixture was stirred at
room temperature for 4.5 hrs. The solvent was evaporated,
dichloromethane (675 ml) was added and the obtained solution was
washed with 20% aqueous sodium carbonate. The organic phase was
dried, the solvent was evaporated and sash chromatography (silica,
CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) gave AF600 (1.5 gr) as a
mixture of two isomers [less polar isomer (A)/more polar isomer (B)
1:4]. .sup.1H-NMR (CDCl.sub.3) .delta. 1.45 [d, j=6.37 Hz, CH.sub.3
(A)], 1.54 [d, j=6.48 Hz, CH.sub.3 (B)], 1.7-1.9 (m), 2.29 [S,
NCH.sub.3 (A)], 2.31 [s, NCH.sub.3 (B)], 2.32-2.5 (m, 3H), 2.62 (s,
CH.sub.2), 3.63 [s, CH.sub.3NO (A)], 3.68 [s, CH.sub.3NO (B)], 4.78
[br CH (A)], 4.85 [br CH (B)] ppm. MS m/e 212 (M.sup.+), 196, 169,
126, 110, 96, 70.
EXAMPLE 19
Synthesis of
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-benzyl-amine]-N-oxi-
de, AF604
##STR00049##
[0342] To a solution of N-benzylhydroxylamine hydrochloride (1.27
gr, 0.008 mol) in ethanol (6 ml) was added sodium acetate (0.65 gr,
0.008 mol). A white precipitate was obtained. A solution of
2,8-dimethyl-1-oxa-8-aza-spiro[4.5]decan-3-one (1.3 gr, 0.0072 mol)
in ethanol (3.5 ml) was added and the mixture was stirred at room
temperature for 4 hrs. The solvent was evaporated, dichloromethane
(450 ml) was added and the obtained solution was washed with 20%
aqueous sodium carbonate. The organic phase was dried, the solvent
was evaporated and flash chromatography (silica.
CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) gave AF604 (1.81 gr) as a
mixture of two isomers [less polar isomer (A)/more polar isomer (B)
1:8]. .sup.1H-NMR (CDCl.sub.3) .delta. 1.42 [d, j=6.35 Hz, CH.sub.3
(A)], 1.53 [d, j=6.45 Hz, CH.sub.3 (B)], 1.7-1.9 (m), 2.29 (S,
NCH.sub.3), 2.32-2.55 (m), 2.62 (m, CH.sub.2), 4.85 (br CH), 4.91
[s CH.sub.3NO (A)], 4.97 [s, CH.sub.3NO (B)] ppm. MS m/e 288
(M.sup.+), 272, 254, 197, 153, 91(100%).
EXAMPLE 20
Synthesis of
N-[(2,8-Dimethyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-isopropyl-amine]-N--
oxide, AF605
##STR00050##
[0344] To a solution of n-isopropylhydroxylamine hydrochloride
(1.84 gr, 0.01 mol) in ethanol (7 ml) was added sodium acetate
(0.91 gr, 0.011 mol). A white precipitate was obtained. A solution
of 2,8-dimethyl-1-oxa-8-aza-spiro[4.5]decan-3-one (1.84 gr, 0.01
mol) in ethanol (4 ml) was added and the mixture was stirred at
room temperature for 4.5 hrs. The solvent was evaporated,
dichloromethane (500 ml) was added and the obtained solution was
washed with 20% aqueous sodium carbonate. The organic phase was
dried, the solvent was evaporated and flash chromatography (silica,
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 90/10/1) gave AF605 (1.5 gr) as a
mixture of two isomers [less polar isomer (A)/more polar isomer (B)
1:4]. .sup.1H-NMR (CDCl.sub.3) .delta. 1.40 (d, j=6.6 Hz,
CH.sub.3CH.sub.2), 1.42 [d, j=6.2 Hz, CH.sub.3 (A)], 1.6-1.8 (m),
2.29 [s, CH.sub.3 (A)], 2.31 [s, CH.sub.3 (B)], 2.35-2.57 (m, 3H),
2.63-2.71 (m, 2H, CH.sub.2), 4.05 [m, CHNO (A)], 4.18 [m, CHNO
(B)], 4.83 (m, 1H, OCH) ppm.
EXAMPLE 21
Synthesis of
N-[(2-Ethyl-8-methyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]-N-
-oxide, AF601
##STR00051##
[0346] To a solution of N-methylhydroxylamine hydrochloride (0.95
gr, 0.01 mmol) in ethanol (14.3 ml) was added sodiumacetate (0.94
gr, 0.011 mol). A white precipitate was obtained, a solution of
2-ethyl-8-methyl-1-oxa-8-aza-spiro[4.5]decan-3-one (2.1 gr, 0.01
mol) in ethanol (7.4 ml) was added and the mixture was stirred at
room temperature for 5.5 hrs. The solvent was evaporated,
dichloromethane (770 ml) was added and the obtained solution was
washed with 20% aqueous sodium carbonate. The organic phase was
dried, the solvent was evaporated and flash chromatography (silica,
CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) gave AF601 (2.4 gr) as a
mixture of two isomers [less polar isomer (A) and more polar isomer
(B)]. .sup.1H-NMR (CDCl.sub.3) 0.94 [t, j=7.4 Hz, CH.sub.3CH.sub.2
(B)], 0.99 [t, j=7.4 Hz, CH.sub.3CH.sub.2 (A)], 1.59 (m, 1H),
1.74-1.86 (m), 1.98-2.05 (m, 2H), 2.29 [s, NCH.sub.3 (A)], 2.30 [s,
NCH.sub.3 (B)], 2.45-2.60 (m, 5H), 3.63 [s, CH.sub.2 (A)], 3.69 [s,
CH.sub.2 (B)], 4.66 [br OCH (A)], 4.78 [br OCH (B)] ppm. MS m/e 226
(M.sup.+), 209, 197, 181, 169, 152, 138, 126, 110, 96, 70.
EXAMPLE 22
Synthesis of
N-[(2-methyl-8-phenyl-1-oxa-8-aza-spiro[4.5]dec-3-ylidene)-methyl-amine]--
N-oxide, AF602
##STR00052##
[0348] To a solution of N-methylhydroxylamine hydrochloride (0.33
g, 0.004 mol) in ethanol (5.4 ml) was added sodiumacetate (0.32 gr,
0.004 mol). A white precipitate was obtained, a solution of
8-methyl-2-phenyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one (0.85 gr,
0.004 mol) in ethanol (3 ml) was added and the mixture was stirred
at room temperature for 4.5 hrs. The solvent was evaporated,
dichloromethane (250 ml) was added and the obtained solution was
washed with 20% aqueous sodium carbonate. The organic phase was
dried, the solvent was evaporated and flash chromatography (silica,
CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) gave AF602 (130 mg) as a
mixture of two isomers [less polar isomer (A)/more polar isomer (B)
1:3]. .sup.1H-NMR (CDCl.sub.3) .delta. 1.24 (m, CH.sub.2), 1.7-1.97
(m), 2.29 [s, NCH.sub.3 (A)], 2.31 [s, NCH.sub.3 (B)], 2.32-2.5
(m), 2.72 (m, CH.sub.2), 3.30 (m), 3.67 [s, CH.sub.3NO (B)], 3.71
[s, CH.sub.3NO (A)], 5.49 [br CH (A)], 4.74 [br CH (B)], 7.28-7.56
(m, ArH) ppm. MS m/e 274 (M.sup.+), 257 (100%), 245, 168, 112, 96,
70.
EXAMPLE 23
Synthesis of
Dihydro-5'-methylspiro[1-azabicyclo[2.2.2]octane-3,5'-(4'H)-3'-ylidene-me-
thylamine-N-oxide, AF603
##STR00053##
[0350] To a solution of N-methylhydroxylamine hydrochloride (1.17
gr, 0.014 mol) in ethanol (15 ml) was added sodium acetate (1.15
gr, 0.014 mol). A white precipitate was obtained, a solution of
dihydro-5'-methylspiro[1-azabicyclo[2.2.2]octane-3,5'-(4'H)-3'-one
(2.1 gr, 0.01 mol) in ethanol (7.4 ml) was added and the mixture
was stirred at room temperature for 4.5 hrs. The solvent was
evaporated, dichloromethane (770 ml) was added and the obtained
solution was washed with 20% aqueous sodium carbonate. The organic
phase was dried, the solvent was evaporated and flash
chromatography (silica, CHCl.sub.3/MeOH/NH.sub.4OH 90/10/1) gave
AF601 (0.45 gr) as a mixture of two isomers [less polar isomer (A)
and more polar isomer (B)]. .sup.1H-NMR (D.sub.2O) .delta. 1.22 [t,
j=7.09 Hz, CH (B)], 1.46 [t, j=7.0 Hz, CH (A)], 1.35 [d, j=6.5 Hz,
(CH3).sub.2CH], 1.41-1.58 (m), 1.57-1.9 (m), 2.59 (m), 2.67-3.01
(m), 3.01-3.26 (m), 3.47 [s, N(O)CH.sub.3 (A)], 3.49 (m), 3.52 [s,
N(O)CH.sub.3 (B)] ppm. MS m/e 224 (M.sup.+), 207, 195, 178,
13&, 124, 96, 83 (100%).
EXAMPLE 24
Synthesis of
2-Ethyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]-decane-3-thione,
AF510
##STR00054##
[0352] A mixture of AF267 (214 mg, 1 mmol) and Lawesson's Reagent
(280 mg, 0.692 mmol) in acetonitrile (5 ml) was heated under reflux
for 17 hrs. The solvent was removed and residue was dissolved in
concentrate aqueous sodium carbonate (0.5 ml) and then extracted
with ethyl acetate. The extract was dried and the solvent
evaporated. The residue (250 mg), recrystallized first from toluene
and then from acetonitrile gave pure AF510. .sup.1H-NMR
(CDCl.sub.3) .delta. 1.03 (t, CH.sub.3CH.sub.2), 1.83 (m),
1.93-2.44 (m), 2.30 (s, CH.sub.3N), 2.80 (m, 2H), 4.21 (dd, SCH),
8.66 (br, NH) ppm. MS m/e 230 (M.sup.+), 197 (M.sup.+-SH), 156,
128, 96 (100%).
EXAMPLE 25
Synthesis of
4-Benzyl-8-methyl-1-thia-4,8-diaza-spiro[4,5]decan-3-one, AF282
##STR00055##
[0354] In a three-necked flask equipped with a magnetic stirrer and
Dean-Stark and two dropping funnels a solution of mercaptoacetic
acid (8 ml, 0.242 mol) in benzene (75 ml) was heated to reflux.
Benzyl bromide (13 ml, 0.242 mol) and 1-methyl-4-piperidone (9.33
ml, 0.08 mol) were added simultaneously dropwise (45 min) and the
reaction mixture was refluxed for additional 1.5 h (2 ml of water
were collected). The reaction mixture was cooled to room
temperature, water (30 ml) was added and the organic phase was
separated, dried and the solvent was evaporated. Flash
chromatography (silica, 10(% methanol in chloroform) of the residue
gave AF282 (3.8 g). .sup.1H-NMR (CDCl.sub.3) .delta. 1.65 (m, 2H),
2-19 (m, 4H), 2.27 (s, 3H, NCH.sub.3), 2.78 (m, 2H), 3.64 (s, 2H,
SCH.sub.2), 4.78 (s, 2H, NCH.sub.2), 7.25 (5H, Ar) ppm.
EXAMPLE 26
Synthesis of
4-(2,4-Dimethoxybenzyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one
AF286
##STR00056##
[0356] In a three-necked flask equipped with a magnetic stirrer and
Dean-Stark a solution of 1-methyl-4-piperidone (0.27 ml, 2.4 mmol),
2,4-dimethoxybenzylamine hydrochloride (0.72 gr, 3.5 mmol) and
mercaptoacetic acid (0.24 ml, 3.5 mmol) in benzene (5 ml) was
refluxed for 3 h. The reaction mixture was cooled to room
temperature, water (10 ml) was added and the organic phase was
separated. The pH of the aqueous phase was adjusted to pH 10 with
2.5N aqueous sodium hydroxide solution and then the aqueous phase
was extracted with chloroform, the organic phases were combined,
dried and the solvent was evaporated. Flash chromatography (silica,
10% methanol in chloroform) of the residue gave AF286 (140 mg, 15%
yield). .sup.1H-NMR (CDCl.sub.3) .delta. 1.66 (m, 2H), 2.21 (m,
4H), 2.27 (s, 3H, NCH.sub.3), 2.77 (m, 2H), 3.64 (s, 2H,
SCH.sub.2), 3.78 (s, 3H, OCH.sub.3), 3.80 (s, 3H, OCH.sub.3), 4.52
(s, 2H, NCH.sub.2),
EXAMPLE 27
Synthesis of
4-(tert-Butyloxycarbonyl)-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one-
, AF284
##STR00057##
[0358] To a solution of AF277 (2.60 gr, 13.97 mmol) in
dichloromethane (60 ml), triethylamine (1.95 ml, 13.99 mmol)
di-tert-butyl dicarbonate (3.85 ml, 16.76 mmol) and
4-dimethylaminopyridine (1.71 gr, 13.99 mmol) were added. The
obtained solution was stirred overnight at room temperature then
the solvent was evaporated. Flash chromatography (silica,
CHCl.sub.3/MeOH/NH.sub.4OH 80/20/1) of the residue gave AF284 (4
gr, >95% yield). 1H-NMR (CDCl.sub.3) .delta. 1.55 (s, 9H,
OC(CH.sub.3).sub.3), 1.70-1.82 (m, 2H), 2.26 (m, 2H), 2.28 (s, 3H,
NCH.sub.3), 2.83-2.87 (m, 4H), 3.53 (s, 2H, SCH.sub.2) ppm.
EXAMPLE 28
Formulation of AF150(S) in Paraffin Oil+Stability Studies
[0359] The stability of 10% w/w AF150(S) in pharmaceutical
acceptable paraffin oil (Paraffin oil Eur. Ph) was examined at
40.degree. C. under air or nitrogen atmosphere, in the presence or
absence tocopherol. Samples were analyzed after two months storage.
TLC, HPLC and GC were used to detect and determine the quantity of
possible degradation product(s). Color changes were determined in
comparison with Paraffin oil with or without tocopherol under the
same conditions. AF150(S) was stable in the paraffin oil
formulation. Degradation products, the thiolamide derivative
(M.sup.+ 202) obtained by hydrolysis of AF150(S), above 0.1% were
not detected in any tested samples. A slight yellow color was
observed in samples without tocopherol but color was not developed
in samples containing 0.5% w/w tocopherol in AF 150(S).
EXAMPLE 29
AF150(S) Citrate Salt+Stability Studies
[0360] To a solution of AF150(S) (30.13 gr, 163.8 mmol) in
2-propanol (60 ml) and tetrahydrofuran (100 ml) was added dropwise
over 45 min a solution of anhydrous citric acid (29.51 gr, 153.5
mmol) in 2-propanol (200 ml). The resulted mixture was stirred at
room temperature under argon atmosphere for additional 2 h, then
the resulted white precipitate was filtered and washed with hexane
under argon atmosphere. The white solid was introduced into a
drying pistol which contained P.sub.2O.sub.5, the drying pistol was
evacuated (0.2 mmHg) temperature and then heated at 55-60.degree.
C. for 6 h. AF150(S) citrate (53.8 g, 87.6% yield) was obtained.
TLC (2% NH.sub.4OH in methanol) Rf 0.57; mp. 146.5-147.5.degree.
C.; .sup.1H NMR (300 MHz, D.sub.2O--Na.sub.2CO.sub.3, pH 12)
.delta. 1.80 (m, 4H), 2.08 (s, 3H, CH.sub.3C.dbd.N), 2.17 (s and m,
5H, CH.sub.2+CH.sub.3N.sup.+), 2.46 (ABq, j=15.2 Hz, 4H,
2CH.sub.2CO.sub.2H), 2.73 (m, 2H), 3.81 (s, 2H, CH.sub.2N.dbd.C)
ppm. .sup.13C NMR (300 MHz, D.sub.2O--Na.sub.2CO.sub.3, pH 12)
19.77, 36.17, 44.16, 45.64, 53.08, 72.71, 75.03, 170.91, 179.19 and
181.86 Hz.
[0361] HPLC analyses of samples of this salt stored under various
conditions (at 60.degree. C. for three months; in air at room
temperature for three months) compared to a reference standard
stored under anhydrous conditions showed the salt to be highly
stable."
EXAMPLE 30
Brain Penetration of Compounds
[0362] 1. pKa of AF267B: The free-base (non-ionized) form of the
compound AF267B crosses the brain blood barrier. Since AF267B has a
pKa of 7.8, at the pH=7.35 of the cerebrospinal fluid (CSF), 26.2%
of the compound are in a free base form, calculated as shown below.
This indicates that AF267B is highly penetrable into the brain
since the free base is the specie that crosses the blood brain
barrier. In comparison, some other known pharmaceutical CNS active
compounds, for example wherein the base is quinuclidinyl, have a
pKa.gtoreq.9 (where the tertiary amine is highly basic). For such
compounds at the relevant pH of 7.35, only 2.2% are in non-ionized
form. This indicates a higher preference for the brain for AF267B
vs. such compounds. These calculations are based on the
following:
BH.sup.+.fwdarw.B+H
% B=100-% BH.sup.+=100-100/[1+10.sup.(pH-pKa)]
pKa(AF267B)=7.8
pH(CSF)=7.3
% B=100-100/1+10.sup.-45]
% B=26.2%
For a base B' having a pKa=9.0
% B'=100-100/1+10.sup.-1.65]
% B'=2.2%
2) Rats were treated with AF267B (2 mg/kg, po) and plasma vs. brain
levels of the drug were analyzed by GC. It was found that AF267B
has a preference for the brain vs plasma: [0363] a) by comparing
the area under the curve extrapolated to infinite time (AUC) both
in whole brain (ng/gr)*hr vs plasma (ng/ml)*hr both iv (1 mg/kg)
and po (2 mg/kg), respectively, it was found that the ratio of AUC
brain/AUC plasma is: for males 1.79 (iv) and 2.43 (po) and for
females 1.32 (iv) and 1.25 (po). Thus greater amounts of the
compound are found in the brain than in plasma; [0364] b) by
comparing the ratio of C.sub.max brain (ng/g)/C.sub.max plasma
(ng/ml) (po) it was found that 25% of the compound in males and 16%
in females is found in the brain. This calculation was based on the
brain weight (2 gr) vs. total plasma volume (14 ml). This also
indicates a high percentage of the compound in the brain. [0365] 3)
ex vivo studies of AF150(S) and AF267B (100 .mu.mole/kg, po) in
mice brain tissue (GC analysis of AF115(S) or AF267B vs a standard
compound (AF261) added to the brain tissue, or by displacement of a
radioactive muscarinic compound such as tritiated-oxotremorine-M
from the brain tissue) also show clearly a high brain penetration
vs. plasma (GC and binding studies). AF150(S): T.sub.max=1-10 min;
T.sub.1/2=21 and 53 min (two phases), iv; MRT (mean retention
time)=50 min. iv, po. AF150(S) has a fast brain penetration (1 min
iv); C.sub.max=40.7 .mu.mole/kg (40.7% in brain from the amount
administered po); AF267B: detected in the brain between 2-240 min
after dosing, a peak at 20-30 min, MRT=128 ml; C.sub.max=36.4
.mu.mole/kg (36.4% in brain from the amount administered po).
EXAMPLE 31
Detection of AF292 Following AF267B Administration to Beagle
Dogs
[0366] The purpose of this study was to determine the levels of
AF267B and AF292 (a metabolite of AF267B) in dog plasma following
13 weeks subchronic singly daily administration of AF267B (1.5, 3
and 6 ml/kg, po to male and female dogs) according to the method
(see below).
[0367] Internal Standard (AF261):
2-Methyl-8-methyl-1-thia-4,8-diaza-spiro[4.5]decan-3-one; MW 200.
The analytical plasma samples originate from the in-life part of
this study. Concentrations of AF267B and AF292 were determined
by:
[0368] Column: Purospher STAR RP18e (4.times.50 mm, 3 .mu.m)
[0369] Mobile Phases Solvent A: 1 g/l (HN.sub.4).sub.2CO.sub.3
(H.sub.2O)
[0370] Solvent B: Methanol
[0371] Loop/Injection Volume: 50 .mu.l/10 .mu.l
[0372] Ionisation Mode: Atmospheric Pressure Chemical Ionisation
(APCI); positive ions
[0373] Sheath Gas Pressure: Nitrogen: 70 psi
[0374] Capillary Temperature: 250.degree. C.
[0375] Spray Voltage: .about.3.6 kV
[0376] Detection Mode: SRM (selected reaction monitoring)
[0377] AF292: m/z: 201.0 [CE (CE=collision energy 30V)].fwdarw.m/z:
70.0 (0.0-5.2 min)]
[0378] AF261: m/z: 201.0 (CE 35V).fwdarw.m/z: 70.0 (5.2-6.2 min);
Internal standard
[0379] AF267B: m/z: 215.0 (CE 30V).fwdarw.m/z: 70.0 (6.2-10.0
min)
[0380] Collision Gas (CID). 2.5 mTorr/Argon
[0381] Results:
[0382] After 13 weeks of repeated daily dosing AF267B has a plasma
half life of 1-2 hrs with T.sub.max=1.5-3 h, C.sub.max (ng/ml)
162-1352 (linearly dose-dependent) and AUC.sub.(0-t)
(ng*h/ml)=712-3947 (linearly dose-dependent). AF292 has an
approximately ten fold longer plasma life (-9-20 hrs) with
T.sub.max=3 hrs, C.sub.max (ng/ml)=136-555 and AUC.sub.(0-t)
(ng*h/ml)=616-2451 (linearly dose-dependent). In comparison to
AF267B, the pharmacokinetic profile of AF292 can be summarized as
follows: AF292 has a T.sub.1/2 in plasma about 3-5 times longer
than AF267B (for example, AF292 T.sub.1/2=10.6 hr for females,
versus T.sub.1/2=AF267B in females). The C.sub.max of AF292 is
50-90% vs. the C.sub.max of AF267B. AF292 shows an apparent shift
to the right of C.sub.max vs. the C.sub.max of AF267B (due to a
delay in the appearance in plasma of AF292 vs. AF267B). On the
basis of this observation, it will be appreciated that AF267B and
AF292 may together form a pharmaceutical combination with a longer
plasma T.sub.1/2 than either compound alone. Such a combination may
be adminstered as such.
EXAMPLE 32
Effects of the Tested Compounds on Secretion of .alpha.-APP.sub.s
in Cell Cultures Stabily Transfected with the M1 mAChR and in Rat
Primary Hippocampal and Cortical Neuronal Cultures
[0383] Cells were plated in 6 well culture plates and used at the
age of 3-5 days after plating. Cells were washed twice in
serum-free medium and incubated for 1 hour at 37.degree. C. with
AF150(S) and AF267B or AF292. The cell cultures were exposed for 1
hr to various concentrations of these tested compounds
(10.sup.-6-10.sup.-3 M), and to carbachol (10.sup.-4 M). Cells
exposed to medium alone are referred as controls. Carbachol,
rivastigmine and deprenyl were used as reference compounds.
[0384] Cell supernatants were collected into Eppendorff tubes
containing a cocktail of protease inhibitors (5 units/ml aprotinin,
5 mg/ml pepstatin A, 5 mg/ml leupeptin and 10.sup.-4 M
Phenylmethylsulfonylfluoride (PMSF, a protease inhibitor); Sigma,
USA). The collected media were concentrated with Centricon tubes
(Amicon, Beverly, Mass., USA) and kept frozen for .alpha.-APP.sub.s
secretion determination. Equal amounts of protein (50-100 .mu.g)
were loaded and separated on 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Followed by
western blotting onto nitrocellulose membrane, blocked by fat-free
milk, and probed for 24 hours at 4.degree. C., with
anti-Alzheimer's precursor protein, A4 monoclonal antibody 22C11
(0.25 .mu.g/ml; Boehringer Mannheim, Germany). The nitrocellulose
membranes were washed and incubated for 2 hours at room temperature
with peroxidase-linked goat anti-mouse IgG antibodies (Jackson
Immunoresearch, USA), followed by extensive washout and staining
with enhanced chemiluminescence detection system (Amersham).
Quantitative evaluation of the immunoreactive bands, on the exposed
films, was performed by video-imaging densitometry (Gel-aid
software; Gialai Co., Israel). APP.sub.s levels were expressed as
x-fold increase over basal levels. In cell cultures stably
transfected with M1 mAChR, .alpha.-APP.sub.s-induced secretion was
also calculated as % of maximal response to 10.sup.-4M
carbachol.
[0385] .alpha.-APP.sub.s secretion in cells expressing the M1 mAChR
increased dose-dependently following agonist stimulation. The
maximal increase was obtained at 10.sup.-4 M AF267B (about 6-fold
increase over control) and was equal to the maximal increase
elicited by carbachol. Addition of the muscarinic antagonist,
atropine at 10.sup.-5 M, inhibited completely the secretion of
APP.sub.s induced by AF267B, indicating that the effect of AF26VB
is mediated via M1 mAChR. In several experiments, the activity of
AF267B was compared to the activity of AF150(S). The results show
that AF267B is more efficacious and potent than AF150(S) (50% of
maximal response of carbachol for AF150(S) vs equal to maximal
response of carbachol for AF267B). Additionally AF267B is more
efficacious and more potent than its racemate (AF267) or AF102B
(50% of maximal response of carbachol) on .alpha.-APP.sub.s
secretion, while the less potent enantiomer AF267A is similar in
potency to AF102B.
[0386] AF292 was as effective as AF267B (EC50=3 .mu.M) and
carbachol in activating elevation of APP.sub.s, while rivastigmine
and deprenyl were not effective in elevating APP, levels. Addition
of atropine (10 .mu.M) inhibited the secretion of APP, induced by
carbachol, AF267B and AF292, indicating that the effect of these
agonists is mediated via M.sub.1 mAChR.
[0387] Taken together, these results show that AF267B is a
selective M1 muscarinic agonist and a "drug-prodrug" for AF292,
which itself is a selective M1 muscarinic agonist and a weak M3
muscarinic antagonist. AF267B and AF292 together form a
pharmaceutical combination with a longer plasma half life and
longer muscarinic activity than either compound alone.
[0388] Using the above tests, AF700 and AF704 were also found to be
effective in increasing APP.sub.s levels in this preparation (at
100 .mu.M 50% of maximal effect of carbachol).
[0389] The effect of various muscarinic agonists on the levels of
secreted APP.sub.s were followed using rat primary cell cultures
prepared from hippocampus, cerebral cortex (both which contain
mainly M1 mAChR) and spinal cord (which contains M2 receptors). In
this study the effects of carbachol (a non-selective muscarinic
agonist), oxotremorine (>M2 selective muscarinic agonist),
physostigmine (a cholinesterase inhibitor) and AF102B, AF150(S) and
AF267B (M1 selective muscarinic agonists) on APP.sub.s secretion
were tested.]
[0390] Primary cell cultures were prepared from embryos of
Sprague-Dawley rats. The experiments were performed with cultures
of hippocampus, cerebral cortex and spinal cord following the
guidelines "Guide for Care and Use of Laboratory animals", National
Research Council, Washington, D.C. 1996.
[0391] Brain tissues, hippocampus, cerebral cortex and spinal cord
were removed from 13-14 or 18-19-day-old rat fetuses, respectively,
by free-hand dissection and transferred into cold Gey's Balanced
salt Solution (Gibco, BRL) containing 6 mg/ml glucose. After
removal of meninges, the dissected tissue was mechanically
dissociated using Pasteur pipettes followed by tripsyn-DNAase
solution to obtain cell suspension. Dissociated cells were
transferred to Dulbeco's Modified Eagle Medium (Biological Ind.
Beit-Haemek, Israel) containing: 6 mg/ml glucose; 2 mM L-glutamate,
1000 IU/ml penicillin. The cell suspension was plated on
poly-L-lysine (1 mg/ml)-pre-coated 12-well culture tissue plates at
a density of 4.times.10.sup.5 and 6.times.10.sup.5 cells/well for
hippocampal and cortical cells, respectively. Cell cultures were
maintained for about 2 weeks in 37.degree. C. incubator (95% air
& 5% CO.sub.2). Cells at 11-14 days in vitro were extensively
washed and then subjected to various treatments as detailed below.
Hippocampal and cortical cells were incubated with the tested
ligands at a concentration of 100 .mu.M for 1 h in magnesium-free
Locke-HEPES buffer consisting of: 154 mM NaCl, 5.6 mM KCl, 3.6 mM
NaHCO.sub.3, 1.3 mM CaCl.sub.2, 5.6 mM glucose and 10 mM HEPES, pH
7.4, containing 0.02% BSA. In the blockade studies the muscarinic
agonists were co-incubated with the antagonist, pirenzepine (10
.mu.M). Cells exposed to buffer alone were referred to as control.
At the end of the incubation period, the conditioned media was
removed and transferred to Eppendorff tubes, which contained a
cocktail of protease inhibitors (as specified above). The
supernatants were concentrated by centrifugation (2,500.times.g for
45 min at 4.degree. C.) using Centricon-30 concentrators (Amicon,
Inc. MA USA) and frozen at -70.degree. C. till APP.sub.s levels
were determined.
[0392] The content of protein in samples was determined in
microplates according to Bio-Rad assay. Equal protein amounts of
each sample (.apprxeq.40 .mu.g/lane) were loaded on 10% SDS-PAGE.
When electrophoresis was completed, gels were blotted onto
nitrocellulose membranes, blocked by fat-free milk and APP.sub.s
bands were probed using the anti-Alzheimer precursor protein A4
(monoclonal 22C11, Boehringer Mannheim) and the secondary probe
peroxidase-linked rabbit anti mouse IgG (Jackson ImmunoResearch,
P). Following extensive washout the bands were stained with TMB
(single solution, Zymed Lab., California) or developed with the
Renaissance Chemiluminescence Reagent (DuPont, NEN) followed by
exposure to an autoradiography film (Hyperfilm-ECL, Amersham).
Quantitative determination of the total APP.sub.s bands was
performed by video-imaging densitometry (Gel-aid software, Galai
Co. Israel). Data obtained for APP.sub.s were expressed as fold
increase over control where the control was cells incubated with
Locke buffer alone.
[0393] Primary rat cortical and hippocampal cultures cell cultures
were exposed to the non-selective agonist carbachol (CCh) and
oxotremorine (>M2 selective), to the M1 muscarinic agonists,
AF150(S) and AF267B and to the cholinesterase inhibitor,
physostigmine, all at 100 .mu.M. The M1 agonists induced a
significant increase in APP.sub.s secretion in both cell systems
used, hippocampus and cortex as compared to levels determined in
control cell cultures. In cortical cell cultures the increase in
APPs levels ranged from 2.5 to 3.1-fold increase over control and
an increase in the range of 1.8-2.8-fold over control was found in
hippocampal cell cultures. AF150(S) and AF267B were more potent
than CCh (2.8-fold and 1.5-fold over control, respectively).
Oxotremorine and physostigmine were inactive. APP.sub.s-induced
secretion by AF150(S), AF267B and CCh was completely blocked by the
M1 selective antagonist, pirenzepine (10 KIM). These agonists did
not activate APP.sub.s secretion in the spinal cord cultures, as
these neurons do not contain M1 mAChR.
EXAMPLE 33
Neurite-Outgrowth Response to Muscarinic Agonists in the Absence or
Presence of Neurotrophins
[0394] Rat pheochromocytoma cells transfected with M1 mAChR cells
were grown as described in Gurwitz et al., (NeuroReport 6, 485,
1995). For determination of neurite outgrowth, cells plated in
six-well plates were used 3-5 days after plating. Growth factors
were added 1 day after plating and muscarinic agonists were added
for the last 24 hrs.
[0395] Cells were observed under an inverted microscope. The
percent of cells with neurite longer than cell diameter were scored
in three random fields of several hundred cells from each well.
Results were expressed as a percent of cells with neurites.
Treatments were performed in triplicate cells. Both NGF (50 ng/ml)
and EGF (10 ng/ml) were added 1 day after plating. Muscarinic
agonists were added 24 hours before scoring.
[0396] The neurotrophic-like effects of AF102B, AF150(S) and AF267B
vs carbachol (CCh) and their interaction with neurotrophins such as
NGF, basic fibroblast growth factor (bFGF) and epidermal growth
factor (EGF) were evaluated. Maximal response to CCh was 80%
compared to 60% for AF267B and 30% for AF150(S). Pretreatment of
rat pheochromocytoma cells transfected with M1 mAChR cells with NGF
synergistically augments the neurotrophic response to all ligands
tested and the efficacy of the agonists tested was increased.
[0397] There is an observable difference between the cellular
response of rat pheochromocytoma cells transfected with M1 mAChR
cells to NGF and bFGF on the one hand (induce differentiation) and
to epidermal growth factor, EGF on the other hand (induces
proliferation). The proliferating profile of EGF changed in the
presence of muscarinic agonists as EGF together with muscarinic
agonists induced an accelerated differentiation.
[0398] Taken together, the above results show that M1 selective
agonists, alone or in combination with either endogenous or
exogenously administered growth factors, may be used to induce
neurotrophic effects beneficial in the treatment of
neurodegenerative disorders, such as AD.
[0399] AF292, AF700 and AF704 were also found to be neurotrophic,
and this effect was blocked totally by atropine, indicating the
mucarinic nature of the effect.
EXAMPLE 34
Effects on A.beta. Levels In Vitro
[0400] Primary mixed rat cortical neurons infected with recombinant
Semliki Forest virus encoding either the human APP695 or APP C99 or
C111 (Fassbeder et al., PNAS, 98: 5856, 2001), were treated with
one of the test compounds (AF102B, AF150 or AF267B, respectively
for 5-8 hours. In these cell cultures APP is cleaved by .gamma.-
and .beta.-secretase to produce A.beta., whereas .alpha.-secretase
destroys A.beta.. However, .alpha.-secretase is not present
intracellularly. Cells were lysed and A.beta. precipitated with W02
(anti A.beta. antibody).
[0401] The mechanism of A.beta. modulation was tested using C99
(and C111) that are truncated constructs generated from APP. Unlike
APP. C99/C111 are direct substrates for .gamma.-secretase. With
both constructs it is possible to directly assay for
.gamma.-secretase activity, while with APP this is not possible.
Both constructs are--as compared to APP--inefficient substrates for
.alpha.-secretase. Synergistic effects were also evaluated with CDX
(methyl-.beta.-cyclodextrin), an agent which extracts cholesterol
from the plasma membrane. CDX inhibits also A.beta. production.
CDX-treated cells were treated in addition to the respective
muscarinic agonist for 5 min to reduce the cholesterol content.
[0402] A.beta. levels were reduced upon treatment with the
muscarinic agonists both in the cell lysate and medium in this
system. AF267B was at least 5-fold more potent than AF150(S) in
decreasing A.beta. levels, being active in the .mu.M range. A
synergistic effect between AF267B and the general cholesterol
lowering agent, CDX (5 mM), in their efficacy to decrease A.beta.
in this system to undetectable A.beta. levels, was observed. It was
also observed in these studies that the present M1 agonists, in
addition to activating .alpha.-secretase, inhibit
.gamma.-secretase. AF267B reduced the release of A.beta.-like
fragments (all fragments being in the 3-4 Kda) range by
approximately 50%. This is equivalent to a .gamma. secretase
activity reduction of 50%. This was also evidenced by a complete
loss of the p3 fragment (a fragment of APP resulting from
.gamma.-secretase cleavage) in the AF267B (1 mM)-treated cells vs
the control. No other compounds have been reported with such a
combined beneficial property on the various secretases (.alpha.-,
.beta.-, and .gamma.-). The results indicate that the combination
of an M1 agonist with a cholesterol lowering agent, such as a
statin, enables the lowering of the dosage of the M1 agonist and
thus reduction of possible side effects of the M1 agonist.
EXAMPLE 35
AF267B Decreases Elevated .beta.-Amyloids in Cortex in
Hypercholesterolemic Rabbits
[0403] Dietary cholesterol induces Alzheimer-like
A.beta.-immunoreactivity in rabbit brain (Sparks et al., Exp.
Neurol 1994; 126:88-94; Sparks et al., Nutr. Metab. Cordiovasc.
Dis. 1997, 7:255-266). New Zealand white male rabbits were allowed
food and water ad libitum. Animals were fed either standard chow or
chow supplemented with 2% cholesterol by weight (Purina) for 10
weeks. One group of animals were injected s.c. once a day with 0.9%
sterile saline and the other group of cholesterol-fed animals were
administered drug (AF267B; 1 mg/kg, s.c. body weight). Following 10
weeks of treatment animals were sacrificed and evaluated for
A.beta. immunohistochemistry when all sections were stained
simultaneously.
[0404] Limited neuronal A.beta. was observed in cortex and hilus of
chow fed rabbits. Among the cholesterol-fed animals injected with
saline there are abundant neurons contained identifiable A.beta..
Such neurons were observably smaller than those occasionally
encountered in a control animal. The number of neurons expressing
A.beta. immunoreactivity was reduced 25-30% in the animals
administered AF267B, and the intensity of the immunoreactivity was
reduced approximately 50%. It was also noted that the neurons
expressing A.beta. after AF267B treatment were similar in size to
those encountered in control brain and therefore larger than those
found in cholesterol-fed saline injected rabbit brain,
[0405] These results show that AF267B is effective in decreasing
elevated A.beta. immunoreactivity in the brain following
hypercholesterolemia, and has a neuroprotective effect on the
neurons that contain these AD peptides.
EXAMPLE 36
AF267B Decreases Elevated .beta.-Amyloids in Cortex in
Hypocholinergic Rabbits (Lesioned Rabbits)
[0406] It is known that experimentally-induced cortical cholinergic
denervation results in biochemical elevations of cortical A.beta.
concentrations and in histologic A.beta. deposition (Beach et al,
Neurosci. Lett. 283: 9-12, 2000), and that administration of
muscarinic M1-selective agents to normal animals decreases CSF
A.beta. concentrations (Beach et al., Brain Res. 905: 220-223,
2001). In the present example, animals with nbm lesions were
treated with AF267B, an M1-selective agonist, to determine whether
the lesion-induced increases in CSF and cortical A.beta. could be
prevented or reduced by chronic M1 receptor activation.
[0407] Twenty-eight female New Zealand White rabbits, about 2.5 kg
each (young adults) were used. Twenty-one received lesions of the
cholinergic nucleus basalis magnocellularis (nbm). The lesion was
accomplished with unilateral i.c.v. injections of an immunotoxin
consisting of the ribosomal toxin saporin conjugated to the
monoclonal antibody ME20.4, which is directed against the
low-affinity neurotrophin receptor, p75. The ME20.4 antibody is
made against monkey p75 and also recognizes rabbit p75. The dose of
immunotoxin was 32.4 .mu.g in 12 .mu.l; this was delivered to the
right lateral ventricle 2 mm lateral to the bregma. Seven animals
received i.c.v. injections of sterile normal saline (sham lesion).
Animals which received the immunotoxin were divided into 3 groups
of 7. One group received twice-daily s.c. injections of AF267B;
each dose was 1 mg/kg for a total daily dose of 2 mg/kg. Another
group received physostigmine hemisalicylate in normal saline by
s.c. osmotic pump at a daily dose of 3 mg/kg. The third group
received twice-daily sterile saline s.c. injections. The animals
which received a sham lesion were implanted with s.c. osmotic pumps
filled with sterile normal saline. Animals were euthanized 4 weeks
after surgery. In the case of animals receiving AF267B injections,
all animals received a final injection approximately 2-3 hours
before sacrifice. Four animals died prematurely [(1 control animal
and 3 physostigmine-treated animals), 1 to post-op hemorrhage, 1
was euthanized after developing uncontrollable seizures, 1 was
euthanized due to hindlimb paralysis induced by i.m. injection of
anesthetic agents prior to surgery and 1 was found dead with no
cause of death found at autopsy)] and were excluded from analysis.
Cerebrospinal fluid was taken from the cisterna magna of all
animals at the time of sacrifice; the brain was removed and sliced
coronally into 0.5 cm slices. One slice, at the level of the
hypothalamus (this slice has hippocampus, as well as cortex), was
fixed in 4% paraformaldehyde and processed for immunohistochemical
staining with an antibody to A.beta.. The other slices were frozen
on sheets of dry ice (the other slices are the non-fixed 0.5 cm
coronal slices of cerebrum, brainstem and cerebellum). Western blot
analysis for A.beta. and sAPP.alpha. was performed on the CSF from
2 of the 4 groups, those with nbm lesion and normal saline
treatment and those with nbm lesion and AF267B.
[0408] Quantification of the 4 kDa band representing CSF A.beta.
showed a noticeable decrease in CSF A.beta. in the AF267B-treated
animals versus the control animals (p=0.05, unpaired, two-tailed
t-test). There was no significant difference in the intensity of
the bands representing sAPP.alpha.. Sections from the same 2 groups
of animals stained immunohistochemically for A.beta. revealed
vascular A.beta. deposition as well as perivascular diffuse
deposits in all animals. The lesion-and-treatment study showed that
both AF267B and physostigmine reduced histologic deposition and
biochemical levels of A.beta.. Histologic A.beta. deposition was
reduced to 6.4% and 12% of the lesioned, untreated group for
physostigmine and AF267B, respectively. Analysis of variance found
that the two treatment groups differed significantly (p=0.01) with
respect to A: deposition (A: deposition was high in the untreated
lesioned animals vs low in the animals which were lesioned and
treated with AB267B) and that both AF267B and physostigmine-treated
groups differed significantly from the lesioned, untreated group
(p<0.05, Fisher's LSD) with respect to A.beta. deposition
(A.beta. deposition was high the untreated lesioned animals vs low
in the lesioned animals treated with AB267B or physostigmine).
[0409] The results show that AF267B treatment of animals with nbm
lesions reduces the increases in CSF A.beta. and brain A.beta.
deposition that are induced by the lesion, and indicates that M1
muscarinic agonists such as AF267B may used as preventative therapy
for AD.
EXAMPLE 37
Prevention of Cytotoxicity and Programmed Cell Death (Apoptosis)
Induced by Various Insults (Deprivation from Growth Factors or
Growth Factor Found in Serum Amyloids Oxidative Stress)
[0410] Confluent rat pheochromocytoma cells transfected with the M1
mAChR cultures were detached with trypsin, washed and plated in
24-well, 6-well, 60-mm or 100-mm plates that were precoated with
rat tail collagen (Sigma, Israel). Several experiments were
performed in serum-free medium, For
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
assay, cells (1.5.times.10.sup.4) were plated in 96-well
collagen-precoated plates, in serum-free medium, with or without
various drugs for 24 hours. For differentiation, cells were grown
in the presence of 1% FCS and 1% HS, with addition of 50 ng/ml NGF
for 7 days, to cause full differentiation. Cells were grown either
on 100-mm plates (5.times.10 cells per plate; MTT, Fluorescence
activated cell sorter (FACS) activities), or on Chamber-Slides
(1.5.times.10.sup.4 cells; TUNEL) that were precoated with
collagen, or on 13-mm glass cover-slip pretreated with
Poly-L-Lysine, in 24-well plates (7500 cells per well; DAP I).
After 7 days, cells were washed and the medium was either replaced
to serum-free, or cells were detached and replated in serum-free
medium. Cells in serum-free medium were treated either with A.beta.
peptides that were previously "aged" or with H.sub.2O.sub.2. Tested
compounds were added together with the insults for the indicated
time, unless otherwise stated.
Cell Viability Assay:
[0411] Cells were plated in 96-well plates in 100 .mu.l medium.
After exposure to various treatments, 10 .mu.l of 5 mg/ml MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;
Sigma, Israel] solution in phosphate-buffered saline (PBS) was
added to each well. Plates were incubated for 2 hrs at 37.degree.
C. followed by hydrocholoric acid-isopropanol addition (100 .mu.l
of 0.04N HCl/isopropanol). Plates were read using ELISA reader at a
wavelength of 570 nm.
Nuclear Staining of DNA:
[0412] Cells were grown and treated on glass coverslips. After
treatments, cells were fixed in cold methanol for 5 min at
-20.degree. C. followed by treatment with cold acetone at 4.degree.
C. for 2 min and washing in PBS. The coverslips were fluorescently
stained with DAPI (4,6-diamidino-2-phenylindole; 5 mg/ml, Sigma,
Israel) an intercalating agent that enables visualization of
chromatin condensation in the cell nuclei, for 15 min at room
temperature. Cells were washed three times with PBS, mounted in a
solution of glycerol containing 22 mM 1,4-diazabicyclo(2,2,2)
octane (Sigma, Israel) to prevent fading, and viewed for nuclear
chromatin morphology with a fluorescence microscope. Apoptotic and
viable cells were counted (200 cells per coverslip, each experiment
was performed in duplicates).
TUNEL Assay:
[0413] This method reveals DNA fragmentation in individual
apoptotic cells. The TUNEL (Terminal deoxynucleotidyl transferase
(TdT) mediated dUTP nick end labeling) method enzymatically labels
DNA fragments at the 3'OH ends (representing the DNA strand breaks)
with fluorescein-dUTP (Boehringer Mannheim, Germany). Briefly,
following treatment, cells grown on Chamber slides were fixed with
paraformaldehyde solution (4%) in PBS, pH 7.4 for 30 min at room
temperature. Following washing with PBS cells were permeabilized
using 0.1% Triton X-100, 0.1% sodium citrate solution, for 2 min on
ice (4.degree. C.), washed with PBS and 50 .mu.l of TUNEL reaction
mixture was added on each sample for 1 hr at 37.degree. C., in a
humidified chamber. Evans Blue reagent (diluted 1:2000 in PBS) was
added for 5 min and the slides were viewed with fluorescence
microscope.
Fluorescence Activated Cell Sorter (FACS) Analysis:
[0414] Cells were differentiated for 7 days and detached from the
plates with Trypsin. 10.sup.6 cells were replated in 50-mm
collagen-coated plates in serum-free medium in presence or absence
of various treatments. 200-g-centrifuged pellets were prepared and
resuspended in 300 .mu.l PBS. 4 ml of cold methanol (-20.degree.
C.) were added to each test tube and the fixation was carried out
for 15 min at -20.degree. C. Cells were washed in PBS, spun and
resuspended in 1 ml PBS. Five microliters of Propidium iodide stock
solution (Sigma; 10 mg/ml) and 5 .mu.l of 20 mg/ml solution of
RNAse A were added for 5-10 min at room temperature. Fluorescence
of individual nuclei was measured using Fluorescence Activated Cell
Sorter (FACScan; Becton Dickinson Corp.) excited at 488 nm
wavelength and collected through 575.+-.21 nm BP filter. The data
were analyzed by Cell Quest software computer system. By this
method we were able to measure DNA content of the cells (apoptotic
cells have less DNA). Cells in G1 phase of cell cycle are after
mitosis and have less DNA than cells in G2/M phase (before and
during mitosis). Apoptotic cells were identified as the pre-G1
phase.
[0415] The MTT assay measures primary early changes within the
cells, reflecting the integrity of the electron transport chain and
provides a readout of cellular redox activity. This test is a
specific, early indicator of the mechanism of 3-amyloid-mediated
cell death and can be used to detect rapid inhibitory response.
Starved, undifferentiated, rat pheochromocytoma cells transfected
with M1 mAChR cells alone reduced cell viability by 10-20% and this
effect was further augmented (up to 50-60% inhibition) by
increasing concentrations of the neurotoxic full-length
.beta.-amyloid (.beta.-A.sub.1-42) peptide and its fragment,
(.beta.-A.sub.25-35) (0.5-20 .mu.M). When such cells were
serum-deprived and treated with .beta.-amyloids, in the absence or
presence of muscarinic agonists, the cell death induced by
.beta.-A.sub.25-35 (1 .mu.M) after 24 hrs was significantly
attenuated by addition of carbachol or AF292, AF150(S) and
AF267B.
[0416] The potential of carbachol, AF150(S), AF267B and AF292 in
protecting cells transfected with M1 mAChR cells from a direct
oxidative stress induced by H.sub.2O.sub.2 was tested. These
agonists were observed to protect the cells from
H.sub.2O.sub.2-induced toxicity.
[0417] Surprisingly, muscarinic agonists were detected in compounds
that have a muscarinic pharmacophore to which an antioxidant moiety
is attached. These include AF604, AF700 and AF704, the structures
of which include a selective M1 agonist moiety linked to an
antioxidant moiety. Notably, AF700, AF703 and in particular AF704
and AF704B are more effective than carbachol and AF267B against
.beta.-A.sub.25-35 (10 & 20 .mu.M)-induced cytotoxicity.
[0418] Using DAPI, an intercalating agent which enables
visualization of chromatin condensation in the cell nuclei,
apoptotic cell death after A.beta. or H.sub.2O.sub.2 treatment was
followed. A.beta.-treated cells showed the morphology of apoptotic
cells that shrank and lost their processes. DAPI staining revealed
nuclear condensation and fragmented chromatin that is indicative of
apoptotic processes. AF150(S) and AF267B were found to protect
these cells from apoptosis and the neuritic-like processes are well
observed.
[0419] A two-fold increase in apoptotic cell death was observed
after starvation for 24 hrs of the cells transfected with the M1
receptor (but not in the untransfected cells). AF150(S) and AF267B
significantly protect the apoptotic cell death induced by
starvation. Atropine (a non-selective muscarinic antagonist) and
pirenzepine (an M1 selective antagonist), reversed the protecting
effect of the muscarinic agonists only in the M1 transfected
cells.
[0420] Treatment of cells transfected with M1 mAChr with
.beta.-A.sub.25-35 (25 .mu.M) or .beta.-A.sub.1-42 (25 .mu.M)
further increased by 1.5-2 fold the apoptotic cell death over
starvation. The selective toxic effect of both peptides was shown
using the reversed peptide (.beta.-A35-25) that did not induce
apoptosis. A.beta.-induced apoptosis was prevented by AF150(S) and
AF267B only in the M1 transfected cells, while muscarinic
antagonists reversed these effects.
[0421] Oxidative insult induced by H.sub.2O.sub.2 (25 and 50 .mu.M)
increased the apoptotic population by 1.5 and 2.5 fold over
starvation, respectively. AF150(S) and AF267B prevented
H.sub.2O.sub.2-induced apoptosis and their effect was selective to
M1 mAChR activation, as atropine reversed the effect.
[0422] The TUNEL method also reveals DNA fragmentation that occurs
following apoptosis in individual apoptotic cells. The number of
TUNEL-positive stained cells increased after 25 .mu.M
.beta.-amyloid 25-35 treatment indicating on apoptotic process,
while AF150(S) (100 .mu.M) was able to block the apoptosis so that
most of the cells were TUNEL-negative stained. The cells retained
their processes, and only the cytoplasm was stained in red.
[0423] Quantification of apoptotic population was performed by
measurement of cell DNA content, after various treatments using
FACS analysis. DNA histograms obtained from serum-deprived neuronal
cultures in the M1 transfected cells revealed the appearance of
apoptotic cell population with degraded (subdiploid) DNA content
(M1=pre-G1 phase). About 20% of total cells underwent apoptotic
death after starvation for 24 hrs. Carbachol, AF150(S) and AF267B,
protected cells from apoptotic death during starvation.
.beta.-Amyloid 25-35 or .beta.-amyloid 1-42 increased the apoptotic
population to 30-35% of the cells. Co-addition of carbachol,
AF150(S) or AF267B reduced the apoptotic population significantly,
even below the values observed after starvation. The effect of the
muscarinic agonists was blocked by 10 .mu.M atropine indicating the
involvement of M1 mAChR activation in the survival effects.
Moreover, in non-transfected cells, the agonists were ineffective
on starvation- and .beta.-amyloid-induced apoptosis.
EXAMPLE 38
Protection Against Cell Death Induced by N-methyl-D-Aspartate
(NMDA)
[0424] Primary rat brain cell cultures derived from 18-19 day old
embryonic (Sprague Dawly rats) rat brains were prepared by
mechano-dissociation. The brains were removed into cold Gey's
Balanced salt Solution (GBSS, Gibco, BRL) containing 6 mg/ml
glucose. The hippocamous and cortex were separated and transferred
to Dulbecco's Modified Eagle Medium (DMEM, Biological Industries,
Bet-Haemek, Israel) containing 6 mg ml glucose, 2 mM L-glutamine
(Biological Industries, Bet-Haemek, Israel), 1000 I.U./ml
Penicillin G sodium and 3% ultrosere G (Gibco, BRL). Following cell
dissociation using a fire polished Pasteur pipette, the resulting
cell suspension was plated on tissue cultures precoated with
poly-L-lysine (30,000-70,000 MW, Sigma) 1 mg/ml in borate buffer.
Cells were plated at a density of 80000 cells/well on a 96 well
culture plate, or at 400000 cells/well (hippocampal cells) and
600000 cell/well (cortical cells) on 12 well culture plates. Cell
cultures were maintained in growth cell medium at 37.degree. C. 5%
CO.sub.2/95% O.sub.2 for about 2 weeks. Glial cell proliferation
was arrested following 3-4 days in culture by addition of
5-fluoro-2'-deoxyuridine/uridine/cytosine arabinoside mixture (5 mM
final concentration).
[0425] For evaluating exposure to NMDA, cortical and hippocampal
cell culture at 10 days in culture plated in 96 well culture plates
were exposed to 100 or 200 .mu.M NMDA (RBI, USA). Neuroprotective
potency of compounds from formula I was compared to that elicited
by 20 .mu.M of MK801 (a non-competitive NMDA receptor antagonist).
Cell cultures were also exposed in parallel to medium alone and
were referred as to controls. In order to produce widespread
neuronal injury, all exposures were carried out for 20-24 hrs prior
assessing neuronal cell death. Neurotoxicity was quantitatively
assessed by measuring the extent of mitochondrial activity in
living cells using
2,3-bis[2-methoxy-4-nitro-5-sulphopheny]-2H-tetrazolium-carboxanilide
inner salt]-based assay (Klausner et al., Biotechnol 5:779-786,
1987; Lipman et al., Cytotechnol. 8:129-176, 1992).
[0426] AF150(S) and AF267B were effective in prevention of cell
death. The neuroprotective potency of 100 M of these agonists
against 100 .mu.M NMDA-induced toxicity was similar to that
elicited by 20 .mu.M MK801, but slightly less against 200 .mu.M of
NMDA.
EXAMPLE 39
1. Effects on Tau Protein Hyperphosphorylation
[0427] Primary cell cultures were grown as described in EXAMPLE 38.
Cell supernatants were removed and the cells were washed once in
medium prior to the addition of cold phosphate buffer saline ph=7.4
(PBS) solution containing 0.2 mM EDTA. Cells were scraped using a
rubber policeman, transferred to Eppendorf tubes, and centrifuged
at 4.degree. C. Cell pellets were resuspended in lysis buffer (EDTA
5 mM, Tris 50 mM, Triton 1%, NaCl 150 mM) containing protease
inhibitors (5 units/ml apronitin, 5 mg/ml pepstatin, 5 mg/ml
leupeptin and 0.1 mM PMSF, Sigma) and subjected to centrifugation
at 4.degree. C. Supernatants were transferred into Eppendorf tubes
and kept at -20.degree. C. till analyzed. The extent of tau-1
immunoreactivity (an antibody that recognizes a non-phosphorylated
tau at Ser.sup.199) was determined by western blots.
[0428] AF 150(S) and AF267B were effective in elevating tau
immunoreactivity both in cortical and hippocampal cell cultures in
a dose range of 1-100 .mu.M.
2. Effects on Tau Phosphorylation and Antagonism of A.beta.-Induced
Effects on Tau Phosphorylation
[0429] Following the experimental design of Sadot et al., J.
Neurochem. 66:877-880, 1996, including immunoblotting with AT8
antibody, an antibody that recognizes phosphorylated tau at
Ser.sup.199/202, it was found that AF267B, AF292 AF704, and AF704B
(100 .mu.M) induce dephosphorylation of tau proteins in these cell
cultures to the control level. By linking an M1 agonist to all
antioxidant moiety tau phosphorylation may be decreased, providing
a new therapeutic strategy in a variety of CNS disease states due
to combined damage due to oxidative stress and tau
hyperphosphorylation.
EXAMPLE 40
Effects on ApoE Synthesis and Secretion in Rat Primary Type 1
Astrocyte Cultures
[0430] Primary cultures of type 1 astrocytes were derived from the
cortex of Sprague Dawley rats. The compounds tested were dissolved
in the culture media and added to cells for 24, 48, 72 and 96 hrs.
Media was then removed and kept frozen until ApoE protein levels
were evaluated by immunoblot analysis as described in Poirier et
al., Neuroscience 55: 81-90 (1993). Alternatively the methods of
Cedazo-Minuez et al. (Neurosci. 105: 651-661, 2001) may be
employed. AF102B at 96 hrs was inactive toward ApoE metabolism
(synthesis and secretion). The compound AF267B, and to a lesser
extent AF150(S), inhibits the production of ApoE over time with a
maximal effect at 96 hrs. The effects observed (60-80% inhibition)
occur at 0.1 nM, a very low concentration of the compound. These
results indicate that this compound inhibits apoE production,
including ApoE4 in rat astrocytes, and thus may be used in
therapies in which inhibition of ApoE production is indicated.
EXAMPLE 41
Competition Binding Assay for Muscarinic Receptor with an Agonist
as the Labeled Probe
[0431] Oxotremorine-M (OXO-M) is an agonist that binds to all
muscarinic receptor subtypes with similar affinities. The ability
of a test compound to displace [.sup.3H]OXO-M binding provides a
measure for the affinity of the test compound to the receptor
agonist binding site. The competition of [.sup.3H]OXO-M binding
with AF292 and its enantiomer, AF291, have K.sub.i values of 0.27
and 6.56 .mu.M respectively as compared to the lull agonist
carbachol which shows a K.sub.i of 0.05 .mu.M.
[0432] Pirenzepine (PZ), a muscarinic antagonist, binds
preferentially to M1 receptors while OXO-M binds to all mAChR
subtypes non-selectively; the ratio between the K.sub.i values for
PZ versus that of OXO-M may be indicative of the selectivity of the
tested compound. The smaller the ratio, the more M1 selective is
the tested compound. Competition of [.sup.3H]PZ binding to rat
cortical membranes with AF292 (K.sub.i=1.39 .mu.M) and its
enantiomer AF291 (K.sub.i=10.7 .mu.M) shows that AF292 is the
active enantiomer. The OXO-M/PZ ratio is .about.0.36 showing a
moderately high selectivity for the M1 receptor.
EXAMPLE 42
Muscarinic Receptor Selectivity
1. Functional Studies in Cell Cultures Transfected with Human
Muscarinic Receptor Subtypes
[0433] 1.1 AF292 was tested for its agonistic or antagonistic
properties, its potency, and its selectivity towards the human M1
vs. M3 and M5 receptors in activating phosphoinositide (PI)
hydrolysis according to the method of Gurwitz et al., Eur. J.
Pharmacol 267, 21, 1993. The effects of the compounds were
atropine-sensitive in activating PI hydrolysis demonstrating, their
muscarinic nature. AF292 and AF267B were found to be partial
agonists at the M1 receptor, showing .about.35% and .about.66%
activity, respectively, versus carbachol with respect to PI
turnover measured in this paradigm No activity was seen at the M3
and M5 receptor with AF292, as compared with AF267B (.about.30% vs.
carbachol at the M3). AF292 was both a partial agonist at the M1
receptor and a weak antagonist at the M3 receptor (pK.sub.b=0.66
.mu.M), with no agonistic activity at M2 or M5 mAChR. The effects
on the M2 receptor were measured in modified cell cultures that
show an increase in intracellular Ca ions following activation with
carbachol. In spite of an 8-fold increase in activity induced by
carbachol on the M2 mAChR, AF292 was inactive as an agonist at all
tested concentrations (10.sup.-9-10.sup.-3M).
[0434] 1.2. AF292 was tested for its agonistic or antagonistic
properties, its potency, and its selectivity towards the human M1
vs. M3 and M5 receptors in activating arachidonic acid (AA)
hydrolysis according to the method of Gurwitz et al., Eur. J.
Pharmacol. 267, 21, 1993. AF292 was more potent on AA release
induced by M1 mAChR (80%) than on PI turnover (35%), but still
inactive as an agonist (AA release) on M3 and M5 mAChR. Thus AF292
is a more efficacious agonist on M1 mAChR-mediated AA release
(mediated via phospholipase A2) than on P1 (mediated via
phospholipase C). In summary, not only is AF292 highly selective
for the M1 mAChR as an agonist, but it also exhibits distinct
activation of select G-proteins (e.g. not all the G-proteins are
activated to the same extent by AF292, unlike carbachol, which acts
as a non-selective mAChR agonist that activates all these receptors
to the same extent.
2. Binding Studies to Muscarinic Receptors and Other Systems
[0435] 2.1 In competitive binding studies against the following
ligands for mAChR receptors, AF267B was found to be highly
selective for the following M1 mAChR subtypes: QNB [Muscarinic
Antagonist in Rat Cortical (CTX) Membranes] (K.sub.i=49.6.+-.9
.mu.M); QNB [Muscarinic Antagonist in Rat Cerebellar Membranes]
(K.sub.i=45.2.+-.10.8 .mu.M); Pirenzepine (M.sub.1 selective
Antagonist in CTX) (K.sub.i=3.74.+-.0.59 .mu.M); Oxotremorine-M
(Muscarinic Agonist in CTX) (K.sub.i1.62.+-.0.34 .mu.M)]; vs.
Serotonin, 5HT.sub.3 51.3% inhibition at 10.sup.-4 M;
Opiods/Opiate, Non-Selective 52.5% inhibition at 10.sup.-4 M; with
no binding at all to Adrenergic (A), .alpha.1A, Adrenergic;
.alpha.1B, Adrenergic; .alpha.2A (Human Recombinant); Adrenergic,
.alpha.2B; Adrenergic, .alpha.2C (Human Recombinant); Adrenergic,
.beta.1; Adrenergic, .beta.2; Benzodiazepine (BZD), Peripheral;
Clozapine; Dopamine, D1; Dopamine, D2 (Human Recombinant);
Dopamine, D3 (Rat Recombinant); GABA A, Agonist Site; GABA A,
Benzodiazepine, Central; Glutamate, AMPA Site; Glutamate, Kainate
Site; Glutamate, NMDA Agonist Site; Glutamate, NMDA, Glycine;
Glycine, Strychnine-Sensitive; Histamine, H1; Histamine, H2;
Histamine, H3; Nicotinic; Ganglionic site; Nicotinic, Neuronal
site; Serotonin, 5HT1A (Human Recombinant); Serotonin, 5HT.sub.1B;
Serotonin, 5HT.sub.4: Serotonin, 5HT.sub.6 (Rat Recombinant);
Serotonin, 5HT.sub.7 (Rat Recombinant); Choline Acetyltransferase;
Glutamic Acid Decarboxylase; Monoamine Oxides A, MAO-A; Monamine
Oxidase B, MAO-B.
[0436] 2.2. In binding studies, AF292 (10 .mu.M) was found to be
highly selective for the mAChR subtypes {M1 (human) (55%), M2
(human) (61%), M3 (human) (55%)} with no binding at all to:
adenosine A1 (human); A2A (human); adenosine A3 (human); alpha 1
adrenergic (non-selective); alpha 2 (non-selective); beta 1
(human); angiotensin, AT1 (human recombinant); benzodiazepine (BZD)
(central); bradykinin, B2 (human recombinant); cholecystokinin
(CCKA) (human recombinant) (CCK1); dopamine D1 (human recombinant);
D2S (human recombinant), endothelin, ETA (human recombinant); GABA
(non-selective); galanin, GAL2 (human); chemokine, IL-8B (human
recombinant) (CXCR2); chemokine, CCR1 (human recombinant);
histamine, H1 (central); histamine, H2: melanocortine, MC4 (human
recombinant); melatonin, ML1; tachykinin, NK2 (human recombinant);
NK3 (human recombinant); neuropeptide, Y1 (human); neuropeptide, Y2
(human); neurotensin, NT1 (human recombinant) (NTS1); opiate, delta
2 (human recombinant) (DOP): opiate, kappa (KOP); opiate mu (human
recombinant) (MOP); orphanin, ORL1 (human recombinant) (NOP);
serotonin, 5-HT.sub.1A (human recombinant); serotonin, 5-HT.sub.1B;
serotonin, 5-HT.sub.2A (human recombinant); serotonin, 5-HT.sub.3
(human recombinant); serotonin, 5-HT.sub.5A (human recombinant)
(5-ht5A); 5-HT.sub.6 (human recombinant); 5-HT.sub.7 (human);
somatostatin, sst (non-selective); vasoactive intestinal peptide,
VIP1 (human) (VPAC1); vasopressin V1a (human recombinant);
Ca.sup.2+, channel (L, verapamil site); K+V channel; SK+Ca channel;
Na.sup.+ channel (site 2); Cl.sup.- channel; norepinephrine NE
transporter (human).
EXAMPLE 43
Effects in Aging Microcebes
[0437] Aging microcebes show similar cognitive deficits and
cerebral lesions to those observed in aging humans and in AD
patients. Thus this is a good animal model for AD, mimicking the
three major hallmarks in AD (plaques (A.beta.), paired helical
filaments (hyperphosphorylated and aggregated .tau.) and cognitive
dysfunction). This model may also be used to mimic MCI conducive to
AD
[0438] In this model AF150(S) [chronic treatment for 18 months]: i)
improved the cognitive and behavioral impairments ii) decreased
hyperphosphorylated .tau. proteins and the number of neurons
containing aggregated .tau. protein (e.g. indicative of diseased
brains) and the number of paired helical filaments; and iii)
decreased astrogliosis and inflammation. This indicates AF150(S)
may be used as a drug to treat or modify the effects of AD but does
not produce tolerance following prolonged treatment.
EXAMPLE 44
M1 Agonists Reduce Neurobehavioral Impairments Following Closed
Head Injury in Mice
[0439] Closed head injury (CHI) was induced in mice as described in
Chen et al., J. Neurotrauma, 15: 231-237. 1998. Neurological
severity scores (NSS) were assessed using a battery of 10
parameters (10=worst outcome, 0=normal function). The compounds
tested (1 mg compound/kg body weight) vs. placebo-treated animals
were injected ip 5 min after CHI vs. placebo-treated animals.
Treated mice were evaluated at 1 h, to determine the severity of
injury, and at 24 and 48 h to determine recovery. The NSS was as
follows: 1. Control (N=10): 7.80+/-0.25 (1 h); 5.30+/-0.33 (24 h);
4.20+/-0.47 (48 h). 2. AF150(S) (N=10): 8.00+/-0.21 (1 h);
4.30+/-0.26** (24 h); 2.90+/-0.23.sup.b (48 h); AF267B (N=9):
7.89+/-0.26 (1 h); 3.67+/-0.24* (24 h); 2.89+/-0.26.sup.c (48 h)
[*p=0.03; **p=0.03; .sup.ap=0.009; .sup.bp=0.005;
.sup.cp=0.004].
[0440] All the compounds tested showed a highly significant
improvement on the motor functions. Recovery was faster in the
AF267B treated animals in two balance tests (beam walk) [22% vs 80%
in control at 24 h (3 cm) or 33% vs. 80% in control at 48 h (2
cm)].
EXAMPLE 45
AF150(S), AF267B in Social Memory in Rats
[0441] Social olfactory recognition in rodents has been shown to
assess short term memory and to be sensitive to cholinergic drugs
(Dantzer et al., Psychopharmacol. 91:363-368, 1987; Perio et al.,
Psychopharmacol. 97: 262-268, 1989). In this example the effect of
AF150(S) and AF267B on investigatory behavior of naive rats was
tested.
[0442] 12 Male Wistar rats, 400-530 gr (4-5 months old) were used.
Rats were housed individually 14 days before testing. Juvenile
Wistar rats 40-50 gr (at arrival) were kept in groups of 6 and
served as social stimuli for the adult rats. Animals were kept at
21.degree. C..+-.1, with an inverse light-dark cycle (light on from
2:00 P.M. to 2:00 A.M.). The sessions were conducted 7 hr into the
dark part of the light/dark cycle, under red illumination.
[0443] Adult rats were placed in a dim illumination room 1.5 h
before the beginning of the social test. All juveniles were
isolated in cages for 30 min prior to the beginning of the
experiment. At the beginning of testing, an unfamiliar juvenile rat
was placed in the home cage of an adult rat for 5 min. The time
spent by the adult rat in investigating the juvenile rat was
recorded. The adult rat was then immediately (1-2 min) treated with
vehicle or test compound. Two hours later, the same juvenile was
presented to the same adult rat for another 5 min period, a time
when normally the stimulus juvenile is no longer identified (i.e.,
the adult rat investigates the juvenile for the same amount of time
as during the first presentation). Thus under the influence of a
purported memory enhancing drug, the time spent in investigating
the same juvenile is expected to be reduced. Two days later, a
juvenile, different from the one used for the first exposure, was
presented to the adult rat, 2 h after drug or vehicle
administration. Any reduction in social exploration of this
different juvenile is thus considered as reflecting a nonspecific
effect of the drug (i.e., not memory related). On no occasion was a
subject tested twice with the same juvenile stimulus animal, nor
was a juvenile used more than once in a 48-hr period.
[0444] AF150(S) or AF267B (0.5, 1 and 5 mg/kg, p.o.) or vehicle,
phosphate buffer saline (PBS) were administered to the adult rats
immediately after the first exposure to the juvenile rat.
[0445] Time spent in social investigation of stimulus juvenile rat
was measured (in see) and then expressed for each animal as the
ratio of the second exposure to the first exposure (Ratio of
Investigation Duration (RID)). This transformation to RIDs was used
in order to minimize possible individual as well as day-to-day
variations in baseline performance (Perio et al., Psychopharmacol.
97: 262-268, 1989). Therefore any reduction in investigation time
during the second exposure will lead to a RID which is less than 1,
indicating that the animal recognizes the juvenile rat. Analysis
for repeated measurement was made by a 3-way ANOVA and post hoc
comparisons were made by simple main effects contrasts
analysis.
[0446] AF150(S) and AF267B decreased the investigation time of the
same juvenile compared to placebo group in a dose-dependent manner.
This improvement of memory cannot be attributed to non-specific
effects, since it was not observed when a different juvenile was
used for the second exposure. Both compounds thus appear to
facilitate social memory in naive rats.
[0447] No significant difference was found between the total RIDs
of the two compounds, but the interaction between similar/different
juvenile x doses of both drugs was found statistically significant,
[F(3/33)=14.9, p<0.0001]. Specifically, both compounds
significantly reduced the investigatory time of the same juvenile
at all three doses tested (p<0.001) relative to placebo.
Furthermore, a significant difference was found between the RIDs of
the 0.5 mg/kg and the two other doses (p<0.05); The RIDs
observed for the 1 and 5 mg/kg were significantly lower than those
observed for the 0.5 mg/kg.
[0448] It should be noted that a significant difference was found
between the RIDs of the same juvenile group and a different
juvenile group at all three doses (p<0.01-p<0.001).
EXAMPLE 46
Effects of AF267B and AF292 on Passive Avoidance (PA) in
Cholinotoxin (AF64A)-Treated Rats
[0449] The general procedure followed is described in Fisher et
al., J. Pharmacol. Exptl. Therap., 257: 392, 1991. AF64A (10 mM)
was prepared by alkaline hydrolysis of acetylethylcholine mustard
HCl. Rats, anesthesized with Equithesin (0.3 ml/100 g IP) were
injected bilaterally by stereotaxic application of AF64A (3 nmol 2
.mu.l/side) or saline (2 .mu.l) into the lateral cerebral
ventricles (AP=-0.8; L=.+-.1.5 mm from bregma; and DV=-4.8 mm from
skull surface). Infusions were made via a CMA 100 microinjection
pump, through a 30-gauge injection cannula, at a constant rate of
0.25 .mu.l/min. The cannula was left in place for 4 min after
injection to allow diffusion of the solution into the ventricles.
Compounds or phosphate-buffer-saline (PBS) were administrated once,
p.o., immediately after shock. Retention was tested 72 h after
training.
[0450] A significant difference was found in the initial latency
between all AF64A-injected rats (29.02.+-.3.1 s) and all saline
injected rats (20.55.+-.1.95 s), F(1/72)=5.13, p<0.05. A
statistically significant interaction was found between
AF64A-injection.times.drug treatment, F(3/72)=11.99, p<0.001, in
the retention latency. The retention latency of AF64A-injected rats
treated with PBS (67.1.+-.18.9 s) was significantly shorter (poorer
memory) than that of saline-injected rats treated with PBS
(455.3.+-.55.1) (p<0.001, by simple main effects contrasts
analysis).
[0451] The retention latencies of AF64A-injected rats treated with
AF267B, 0.1 mg/kg (440.7.+-.46.4 s), and AF292, 1 mg kg
(447.+-.46.8 s), were significantly longer (better memory) than
that of AF64A-injected rats treated with PBS (p<0.001, by simple
main effects contrasts analysis). No significant difference was
found between the retention latency of AF64A-rats treated with
AF267B, 0.03 mg/kg (105.7.+-.31.9 s), and that of AF64A-rats
treated with PBS. No significant differences were found between the
latencies of any of the saline-injected groups.
[0452] AF64A-injected rats demonstrated a clear impairment in
retention of the PA task. The minimal effective dose of AF267B in
attenuating AF64A-induced retention deficiencies is less than 0.1
mg/kg, p.o. Both AF267B, 0.1 mg/kg, and AF292, 1 mg/kg, in the PA
task, are efficacious in improving AF64A-induced retention
deficiencies, compared to AF64A-injected rats treated with PBS. The
minimal effective dose of AF292 may be below 1 mg/kg, po.
EXAMPLE 47
Effects of AF267B Cognitive Impairments Induced by AF64A in Rats in
the MWM Test
[0453] AF64A or saline-injected (6 months old) Sprague-Dawley rats
were tested in the Morris Water Maze (MWM) task. The paradigm used
assesses spatial learning abilities in a reference memory regimen,
and involves training (days 1-4), transfer test (Probe trial--day
4, 3 min following the last training trial) and reversal test (day
5).
[0454] At 4 months post-operation, each of the AF64A and saline
groups of rats was randomly subdivided into four treatment
subgroups (n=9): subgroups 1-3 were treated with AF267B in doses of
0.3, 1, and 3 mg/kg, po, in a volume of 10 ml/kg, whereas subgroup
4 (control group) was treated with the vehicle, phosphate-buffer
saline, 10 mM (PBS) in the same volume. Drugs and PBS were
administered once a day for 5 days before starting the behavioral
testing, and then for the duration of the 5-day experiment, 30 min
before testing.
[0455] The three measures, escape latency, path length and swimming
speed were analyzed by MANOVA, followed by simple main effects
contrast analysis.
[0456] Results: AF64A-injected rats showed a significantly longer
escape latency than saline-injected rats, F(1/64)=10.56,
p<0.005. In terms of path length, AF64A-injected rats showed a
significantly slower learning curve than saline-injected rats,
F(3/192)=4.01, p<0.11. AF267B had no significant effect on
learning; however, AF64A-injected rats treated with AF267B-1 mg/kg
showed a tendency for improvement, in escape latency only, while
AF267B-3 mg/kg tended to impair performance in these rats. No
correlation was found between the cognitive measures (escape
latency and path length) and the nonspecific, motor measure
(swimming speed).
[0457] All saline-injected rats showed a spatial bias in the probe
trial, in both parameters, F(3/192)=7.86, p<0.001, and
F(3/192)=7.44, p<0.001, for escape latency and path length,
respectively. On the other hand, AF64A-injected rats treated with
PBS showed only a partial spatial bias on this test. However,
AF64A-injected rats treated with AF267B showed a complete spatial
bias, similar to that of saline-injected rats, as presented in
escape latency only, F(9/192)-2.3, p<0.025. No significant
differences were found between the various doses of AF267B, in
their beneficial effect on memory.
[0458] No significant differences were found between any of the
groups tested in the reversal test. However, AF64A injection tended
to deteriorate cognitive performance, in both measures.
Additionally, AF64A-injected rats treated with AF267B-1 mg/kg
showed a tendency for improvement, while AF64A-injected rats
treated with AF267B-3 mp/kg showed a tendency for impairment on
this test.
EXAMPLE 48
The Effects of AF150(S), AF267B, Rivastigmine and Nicotine on MWM
Performance of C57BL/10SnJ vs. C57BL6J Mice
[0459] C57BL/10SnJ (B10) mice were selected due to their small
hippocampi and decreased number of hippocampal pyramidal neurons;
the cell loss seemed to be associated with poor spatial learning.
Deficiencies in spatial memory tasks observed in these animals were
reported to be responsive to cholinergic manipulation (scopolamine)
(Simons et al., Life Sci. 42, 375-383, 1988), and both AChE
inhibitors (physostigmine) and muscarinic agonists (AF102B,
PD151832) (Simons et al., Life Sci. 42, 375-383, 1988; Vincent et
al. Brain Res., 597, 264-268, 1992; Schwarz et al. Drug Dev. Res.,
40, 133-143, 1997) have shown positive effects in this model, using
the MWM.
[0460] Each group of mice was randomly divided into 7 treatment
groups (n=12-14/group). Groups 1-2 were treated with AF150(S) at
doses of 0.5 and 1 mg/kg, i.p., in a volume of 10 ml/kg, groups 3-4
were treated with AF267B at the same doses and volume, groups 5-6
were treated with rivastigmine and nicotine, respectively, at the
dose of 1 mg kg, i.p., and group 7 was treated with the solvent,
saline 0.9%. All tested compounds and saline were administered once
a day for 4 days before starting the behavioral testing, and then
for the duration of the 5-day experiment, 30 minutes before
testing.
[0461] Training: Each mouse was trained for four consecutive days,
four trials (one block) per day, in which the platform position
remained constant and was located in the center of the southeast
quadrant of the pool. Within each block of four trials, each mouse
started at each of the starting locations, but the sequence of
locations was randomly selected. A trial consisted of placing a
mouse by hand into the water facing the wall of the pool at one of
four starting locations, north, south, east or west, around the
pool's perimeter. Escape latency (the time to find the platform),
path length (the distance traveled by the mouse) and speed (the
swimming rate of the mouse) were recorded on each trial by the
monitoring system.
[0462] For each mouse, the path length, escape latency, and
swimming speed of the four trials on each of the 4 training days
were grouped into blocks (one block for each day). The scores of
all three measures were analyzed by a three-way MANOVA
(2.times.7.times.4) with one repeated variable (days) and two non
repeated variables [mice strain--C57BL/10SnJ or C57BL6J, and
treatment--two doses of each, AF150(S) and AF267B, rivastigmine,
nicotine (one dose for each) and saline]. Specific comparisons were
performed, using the simple main effects contrasts analysis, which
is specifically suited for testing significant interactions.
[0463] Escape latency. Small-hippocampi mice showed significantly
longer escape latencies (indicating a worse RM performance) than
normal hippocampus rats. AF150(S) and AF267B, and rivastigmine,
positively affected the training performance of small hippocampus
mice, F(6/161) 6.39, p<0.0001. Specifically, both doses of each
of the muscarinic compounds improved the escape latencies of small
hippocampus mice, compared to control group (p<0.01-p<0.001);
Furthermore, AF267B showed a dose-response curve in its effect on
performance (p<0.02) while AF 150(S) affected performance
equally by both doses. Both AF150(S) and AF267B affected
performance more effectively than rivastigmine
(p<0.05-p<0.001, respectively). AF150(S) increased the escape
latencies of normal hippocampus mice, by both doses
(p<0.05-p<0.01) whereas AF267B did not significantly affect
the escape latencies of these mice during training. While nicotine
had no improving effect of memory deficits shown by small
hippocampus mice, it degraded the performance of normal hippocampus
mice (p<0.02). The results also indicated a significant general
effect of training, F(3/483)=90.49, p<0.0001; the escape
latencies of all groups decreased linearly during the four training
days (p<0.0001, by a polynomial contrast).
[0464] Path length. Small hippocampi mice showed significantly
longer path lengths than normal hippocampus mice, F(6,161)=2.35.
p<0.033. AF150(S) (both doses), AF267B (the higher dose) and
rivastigmine positively affected the performance of small
hippocampus mice (p<0.05-p<0.01). AF150(S) (only the higher
dose) significantly (p<0.05) impaired the path length of normal
hippocampus mice whereas neither AF267B nor rivastigmine had any
effect on the path length of these mice. Nicotine had no
significant effect on the performance of any of the mice strains
tested. The results also indicated a significant general effect of
training, F(31483)=86.98, p<0.0001; the path lengths of all
groups decreased linearly during the four training days
(p<0.0001, by a polynomial contrast).
[0465] Swimming speed. Motor activity differences were observed
between the two strains of mice treated with saline: The swimming
speed of small hippocampus mice was significantly lower than that
of normal hippocampus mice, F(6/161)-14.32, p<0.0001.
Furthermore, both muscarinic drugs significantly increased the
swimming speed of small hippocampus mice. Specifically, AF267B
enhanced the swimming speed in a dose dependent manner (p<0.001,
relative to control; p<0.02, between doses) while the enhancing
effect of AF150(S) was equal in both doses (p<0.001). Moreover,
AF267B-1 mg/kg significantly enhanced the swimming speed more
strongly (p<0.001) than AF150(S)-1 mg/kg. Neither rivastigmine
nor nicotine had any significant effect on the swimming speed of
small hippocampus mice. AF150(S) significantly
(p<0.01-p<0.001) impaired the swimming speed of normal
hippocampus mice while AF267B had no such effect, likewise,
rivastigmine (p<0.01) and nicotine (p<0.001) significantly
decreased the swimming speed of these mice. The results also
indicated a significant general effect of training, F(3/483) 15.34,
p<0.0001; the swimming speeds of all groups increased linearly
during the four training days (p<0.0001), by a polinomial
contrast).
[0466] Transfer test. During trial No. 17, on the fourth day, the
platform was entirely removed from the pool (a probe trial). In
this trial, the mouse was placed into the water for a limited
period (30 s), and its spatial bias was measured by recording the
relative distribution of escape latency and path length over the
four quadrants of the pool. The path length and escape latency for
the transfer trial (trial No. 17) were analyzed by a three-way
MANOVA (2.times.7.times.4) with one repeated variable (quadrant in
the pool) and two non repeated variables [mice strain--C57BL/10SnJ
or C57BL6J, and treatment--two doses of each, AF150(S) and AF267B,
rivastigmine and nicotine (one dose for each) and saline].
[0467] The three-way interaction for the escape latency measure was
found statistically significant, F(18/483)=1.62, p<0.05, while
the interaction for the path length measure was close to
significance, F(18/483)=-1.5, p<0.08. Normal hippocampus mice
treated with saline showed a complete spatial bias in the transfer
test. They spent significantly more time in the training quadrant
(p<0.001) relative to the three other quadrants of the pool. On
the other hand, small hippocampus mice treated with saline showed
only a partial spatial bias in this test; They spent significantly
more time in quadrant No. 1 relative to quadrants No. 3
(p<0.001) and 4 (p<0.05) but not relative to quadrant No. 2.
However, small hippocampus mice treated with AF150(S) or AF267B (by
both doses), or rivastigmine, showed a complete spatial bias, like
normal hippocampus mice. In contrast, small hippocampus mice
treated with nicotine showed only a partial spatial bias, like
small hippocampus mice treated with saline. Normal hippocampus mice
treated with AF150(S)-1 mg/kg showed only a partial spatial bias in
the transfer test while all other normal hippocampus mice treated
with the other drugs showed a complete spatial bias in this test.
The results of the path length measure were very similar to those
of the escape latency measure.
[0468] Reversal test. During trials 18-21, on the fifth day, the
platform position was changed to the northwest quadrant, opposite
to the training quadrant. Thus, during reversal learning, the
platform location was moved relative to the configuration of
objects within the room, but the pool occupied the same place
within the room throughout the entire experiment. Testing of the
rats and measures taken were the same as in training.
[0469] For each mouse, the escape latency, path length and swimming
speed of the reversal test (trials No. 18-21) were grouped into one
block. All three measures were analyzed by a two-way MANOVA
(2.times.7) with two variables (mice strain C57BL/10SnJ or C57BL6J,
and treatment--two doses of each, AF150(S) and AF267B,
rivastigmine, nicotine (one dose for each) and saline).
[0470] Escape latency. Small hippocampi mice showed significantly
longer escape latencies during reversal learning than normal
hippocampus mice, F(6/161)=3.26, p<0.005. Both muscarinic drugs,
AF150(S) and AF267B (by both doses), significantly
(p<0.05-p<0.01) improved the escape latency of small
hippocampus mice while AF150(S)-0.5 mg/kg significantly (p<0.05)
impaired the escape latency of normal hippocampus mice. Both
rivastigmine and nicotine had no significant effect on either
C57BL/10SnJ or C57BL6J mice.
[0471] Path length. The only significant effect (p<0.05) shown
in this measure was the impairment of reversal learning by
AF150(S)-1 mg/kg in normal hippocampus mice (F(6/161)=2.85,
p<0.011, for the two-way interaction).
[0472] Swimming speed. No significant differences were obtained
between the saline-treated, two strains of mice in motor activity.
However, both muscarinic drugs, by both doses, significantly
(p<0.01-0.001) increased the swimming speeds of small hippocampi
mice, F(6/161) 8.71, p<0.0001. The swimming speeds of normal
hippocampi mice were significantly decreased by AF150(S)-0.5 mg/kg
(p<0.02), AF267B-1 mg/kg (p<0.05), rivastigmine (p<0.01)
and nicotine (p<0.05).
[0473] AF150(S), AF267B, and the AChE inhibitor, rivastigmine,
significantly attenuated these impairments in mice with small
hippocampus. The improvement of cognitive functioning was more
pronounced during acquisition and retention, although a similar
improvement was shown by both muscarinic compounds in reversal
learning. In contrast, nicotine had no beneficial effect on the
cognitive performance of small hippocampi mice. A dose-response
effect of AF267B was demonstrated in acquisition, by the
differential improvement of the cognitive deficits shown in escape
latency measure. The beneficial effect of the 1 mg/kg dose was
significantly stronger than that of the 0.5 mg/kg dose. During
transfer trial, non-treated, small hippocampi mice showed only
partial memory deficits concerning the platform location. A
significant improvement of these deficits was demonstrated equally
well by both muscarinic drugs, at the two doses tested, as well as
by rivastigmine, but not by nicotine. The contribution of AF267B
and AF150(S) to the improvement of learning and memory processes is
emphasized by two findings: the dose-response effect shown by
AF267B in acquisition, and the beneficial effect of both drugs
demonstrated in the probe (transfer) trial. In this respect it
should be noted that the probe trial is the foremost procedure in
the MWM task, providing measures that quantity the strength and
accuracy of the original learning.
EXAMPLE 49
AF150(S) is Effective in Restoration of Cognitive Impairments in
Ischemic Rats
[0474] Transient ischemia in rats was induced by a modification of
the ischemia model (Volpe et al., Stroke 20: 1700-1706, 1989). This
was done by a bilateral carotid artery occlusion in Sprague Dawley
rats: (42 male, 3 months old, weighing 270-340 g) combined with
reduction in blood pressure induced by sodium nitropruside.
Ischemia was induced in 21 rats whereas the other 21 rats served as
sham controls. Under pentobarbital anasthesia (30 mg/kg, ip),
sodium nitropruside 4.8 mg/kg/hr) was infused through a cannula
implanted in the tail vein, for a period of 25 min. Five min after
the initiation of the infusion, at the time when mean blood
pressure was maintained at 30-60 mmHg (initial levels A110 mm Hg)
both carotid arteries were clamped for 20 min. Immediately
afterwards 1.8 mEq sodium bicarbonate solution was administered ip
in order to minimize systemic acidosis. The sham operated rats were
anesthetized as the ischemic rats and were infused with saline.
Their carotid arteries were exposed but were not subject to carotid
clamping. In rats subjected to ischemia, a mortality of about 30%
was recorded within 24 hr after surgery. Animals were allowed to
recover for 3 weeks prior to behavioral testing. Rats were randomly
assigned to one of four groups: ischemic and sham-operated rats
which were treated with AF150(S) (0.5 mg/kg, po) and ischemic and
sham-operated control rats treated with double distilled water
(DDW) (10 ml/kg, po). Each groups comprised of 10-11 rats. AF 50(S)
was administered immediately following operation, once a day (6
days/week) for weeks before starting the behavioral testing, and
then for the duration of the three-weeks experiment, 60 min before
testing. The evaluation of the animals was done using the
working-memory matching-to-sample paradigm in the MWM.
[0475] The ratio of escape latency (REL) and the ratio of path
length (RPL) were calculated by the ratio of block no 2/block no 1
for each parameter. REL and RPL reflect the relative saving in
performance from trial no 1 to trial no 2. REL and RPL were
analyzed by a 3-way ANOVA (2.times.2.times.3), with one repeated
variable (weeks) and two non-repeated variables (Operation-ischemia
sham-operated and Treatment-AF150(S)/DDW).
[0476] For both REL and RPL the interaction between operation x
treatment was found statistically significant (F(1/32)=8.08;
p<0.01 and F(1/32)=6.75; p<0.025, for REL and RPL,
respectively). Simple main effects contrasts analysis showed that
both REL and RPL of ischemic rats treated with DDW were higher than
those of control rats treated with DDW (p<0.01 and P<0.02 for
REL and RPL, respectively). This result indicates a deficit in
working memory processes of ischemic rats compared to control
rats.
[0477] AF150(S) significantly improved working memory performance
of ischemic rats, compared with DDW-treatment; both REL and RPL of
ischemic rats treated with AF150(S) were significantly lower
(p<0.05) than those of ischemic rats treated with DDW. Control
rats treated with AF 150(S) did not show any significant change in
performance. No differences in swimming speed were found in any of
the tested groups. In conclusion, chronic administration of AF
150(S), 0.5 mg/kg, po, showed a clear improvement of working memory
performance in ischemic rats during the three weeks of the
experiment (following 3-6 weeks of drug administration).
Nonspecific, motor coordination effects could explain neither the
behavioral effects of the ischemic rats, nor the improving effects
of AF 150(S), because no significant effects were demonstrated in
the swimming ability of the rats in the Norris water maze
testing.
EXAMPLE 50
Effects in Trihexyphenidyl Treated Rats--AF150(S), AF267B, AF292,
AF704
[0478] Trihexyphenidyl is a selective M1 muscarinic antagonist that
crosses the blood brain barrier and induces memory and learning
impairments (Bymaster et al., J Pharmacol Exp. Ther. 267: 16-24,
1993; Roldan et al., Neurosci. Lett. 230: 93-96, 1997; Kimura et
al., Brain Res. 834: 6-12, 1999).
[0479] Naive Wistar rats were used in the experiments below. The
passive avoidance (PA) task is comprised of training (acquisition)
phase and a retention phase. In the training procedure each rat was
individually placed in the small illuminated compartment and after
60 sec. of familiarization/adaptation, the door to the large
compartment was opened and the latency to enter was measured
(Initial Latency). Immediately following entry into the dark
compartment, the door was closed and inescapable foot shock (0.6 mA
for 3 sec) was delivered through the grid floor. A cutoff point of
180 sec was used for initial latency. Animals that failed to enter
(step-through) within 180 sec were excluded from the experiment.
After the acquisition trial the rat was returned to its home cage.
Retention of the passive avoidance task was measured 24 h later, by
again placing the rat in the light compartment and after a 60 sec
adaptation interval, the door was opened and the latency to
re-enter the dark compartment was measured. A cutoff point of 300
sec was used for retention latency. Animals that failed to step
through within 300 sec were removed from the apparatus and a 300
sec latency was recorded for them.
[0480] The tested compounds include: AF150(S) (0.5, 1 and 5 mg/kg,
p.o.), AF267B (0.5, 1 and 5 mg kg, p.o.), AF102B 1 mg/kg, p.o.).
The retention latency of trihexyphenidyl rats treated with
AF150(S)-5 mg/kg (222.+-.25.6), AF267B--0.5 mg/kg (181.1.+-.35.4),
AF267B--1 mg/kg (290.1.+-.8.5) and AF102B--1 mg/kg (234.4.+-.35.3)
was significantly longer than that of trihexyphenidyl rats treated
with double distilled water (DDW) (82.9.+-.19.55)
(p<0.01-0.001). Furthermore, the retention latency of the
trihexyphenidyl rats treated with AF267B--1 mg/kg was significantly
longer than that of trihexyphenidyl rats treated with AF267B--0.5
mg/kg (p<0.01) or trihexyphenidyl rats treated with AF150(S)-5
mg/kg (p<0.05). No difference was found in the retention latency
between control groups treated with various drugs or DDW. AF704 was
also significantly effective in this test. Thus retention latency
of trihexyphenidyl rats treated with DDW (116.25.+-.36.36) was
significantly shorter than that of control (DDW) rats treated with
DDW (300+0) (p<0.001). However, the retention latency of
trihexyphenidyl rats treated with AF704-0.1 mg/kg, po
(214.70.+-.36.63), 0.5 mg/kg, po (283.50.+-.17.39) and 1 mg/kg, po
(274.44.+-.26.97) was significantly longer than that of
trihexyphenidyl rats treated with DDW (p<0.01-0.001).
[0481] AF292 was the most potent compound among the tested
agonists. AF292 was significantly effective at a dose of 0.1-0.05
mg/kg, po. When the lowest dose of AF292 0.03 mg/kg, po was tested
after 24 and 72 hrs, only the 72 hrs delay showed a significant
effect on PA in retention latency (225.9.+-.36 vs.
DDW--93.1.+-.29.0; p<0.01 compared to trihexyphenidyl rats
treated with DDW). No effects were found in the initial latency.
These results show AF292 to be a highly potent agonist (e.g. more
potent than AF150(S) by two orders of magnitudes), despite the
higher potency of AF 50(S) in binding studies against pirenzepine
(high affinity & low affinity). This effect of AF292 cannot be
attributed only to a higher bioavailability of AF292 vs AF ISO(S)
in rats (49% vs. 31%).
EXAMPLE 51
Effects of AF267B, AF292 on Cognitive Function in Aged Rats
[0482] Old (22-24 months old) and young (three months old)
Sprague-Dawley rats had been tested in the MWM. Old rats showed a
significantly slower learning curve than young rats, F(3/267)=6.74,
p<0.0001, and F(3/267)=4.66, p<0.003, for escape latency and
path length, respectively. No significant effect was found for any
of the test compounds on learning; however, aged rats treated with
AF267B-1 mg/kg showed a tendency for improvement. All young rats
showed a spatial bias in the probe trial, in both parameters,
relative to old rats, F(3/267)=34.91, p<0.0001, and
F(31267)=9.06, p<0.0001, for escape latency and path length,
respectively. No significant effect was found for any of the test
compounds on memory; however, relative to old rats treated with
DDW, aged rats treated with AF292 in both doses, showed a tendency
for partial spatial bias in this test.
[0483] Old rats showed significantly worse performance during
reversal learning than young rats. AF267B-1 mg/kg improved
significantly the reversal learning of aged rats, F(4/88)=2.62,
p<0.04, and F(41/88)-2.58, p<0.04, for escape latency and
path length, respectively. The beneficial effects of AF267B on
reversal learning of old rats could not be attributed to
nonspecific, motor coordination effects, since AF267B had no
significant effect on the swimming ability of these rats. AF292 did
not reach significance in the reversal learning of aged rats, yet
from the shape of the curves there is a tendency of improvement at
both doses tested, 1 and 0.5 mg/kg, po.
EXAMPLE 52
CNS Safety Profile of AF292 (Table 1)
[0484] AF292 was evaluated in rodents for possible effects on
general behavior and other CNS related pharmacological effects. No
significant physical or behavioral signs were observed in rats
administered AF292 at 1, 10, 30, 60, or 100 mg/kg orally, as
compared to the vehicle control group. No behavioral or physical
signs were observed 24 hours after administration. All rats were
retained for 14 days, and throughout this retention period all rats
appeared normal. In comparison, the compound AF267B begins to show
some effects (salivation and lacrimation at about 40 mg/kg po)
already in the first hour after administration.
EXAMPLE 53
Cardiovascular Safety of AF292
[0485] 1. Astemizole (human ether-a-go-go related gene (HERG)
Channel) Binding Assay. AF292 was inactive in this binding assay as
it failed to inhibit [.sup.3H]-Astemizole binding to the
hERG-encoded channel.
[0486] 2. Isolated Guinea Pig Right Atrium (atrial fibrillation).
AF292 had no significant effect on contractile force, but tended to
slightly reduce the contractile rate beyond what is seen in the
vehicle group.
[0487] 3. Effects of AF292 (dose 5 mg/kg orally) on cardiac
electrophysiological, cardio-haemodynamic in instrumented, awake
dogs. Healthy trained and chronically instrumented female Beagle
dogs of varying age and ranging in body weight from 9.4 to 12 kg,
were used for recording of the cardiovascular parameters: heart
rate, diastolic and systolic blood pressure, pressure rate product,
LV dp/dt max, LV dp/dt max/pd, LV dp/dt min, cardiac output, stroke
volume, systemic vascular resistance and the ECG parameters (PQ-,
QRS-, QT-, QTcBazett--(QTcB), QTcFridericia--(QTcF) and QTcVan de
Water--(QTcVdW)) interval duration and QT-dispersion. During the
last 18 hours prior to the experiments, the dogs had no access to
food. Water was available ad libitum. At the beginning of each
experiment, control values of the various parameters were recorded
for at least 30 min. Thereafter, 5 mg/kg of AF292 (n=4) or the
corresponding volume of the solvent (n=4) was administered orally
by gavage. The various haemodynamic parameters were recorded
continuously for 4 hours thereafter.
[0488] AF292 orally administered at a dose of 5 mg/kg has no
statistically significant and relevant effect on hemodynamic or ECG
parameters: e.g. blood pressure, cardiac contractility (LV dp/dt
max; LV dp/dt max/pd) and relaxation (LV dp/dt min), stroke volume,
systemic vascular resistance the duration of the PQ-, QRS-, QT-,
QTcB-, QTcF- and QTcVdW-interval, QT-dispersion and on
ECG-morphology.
EXAMPLE 54
Effects of AF292 on Cytochrome P450 Isoform Inhibition
[0489] Cytochrome P450 activity can be an indicator for potential
drug-drug interactions. AF292 was evaluated in a microtiter plate
assay for P450 inhibition. AF292, at a concentration up to 10
.mu.M, did not induce significant inhibition of CYP1A2, CYP2C9,
CYP2C.sub.19, CYP3A4 and CPY2D6 isoforms, five major human P450
enzymes responsible for drug metabolism and associated drug-drug
interactions.
EXAMPLE 55
In Vitro Metabolizm of AF292
[0490] The metabolic stability of AF292 was evaluated by monitoring
its disappearance while incubated with rat, rog, monkey, and human
hepatic microsomes. Testosterone, and propranolol were run as assay
controls. The results are shown in Table 1.
EXAMPLE 56
Human Colon Adenocarcinoma (Caco-2) Cell Permeability Studies of
AF292
[0491] This test is used to determine intestinal permeability of
tested compounds. Caco-2 cells, when grown on semipermeable
filters, spontaneously differentiate in culture to form confluent
monolayers which both structurally and functionally resemble the
small intestinal epithelium. Because of this property they are
useful as an in vitro model for the study of drug absorption and
metabolism during absorption in the intestinal mucosa. Caco-2
monolayers were grown to confluence on collagen-coated,
microporous, polycarbonate membranes in 12 well plates and
permeability of the test material was determined. The average
permeability coefficient (P.sub.app) of AF292 was
17.4.times.10.sup.6 cm sec ranking it as having a high absorption
potential (bi-directional assay performed).
EXAMPLE 57
Protein Binding
[0492] Protein binding studies were carried out in human plasma,
.alpha..sub.1-glycoprotein, human serum albumin (HSA), and
Dulbecco's Phosphate Buffered Saline (PBS). AF292 was added to a
final concentration of 10 .mu.M. The results showed 0% protein
binding in PBS buffer dose conc (.mu.M) and in human
.alpha.-glycoprotein (AGP), (see Table 1).
EXAMPLE 58
Pharmacokinetic Profile of AF292 in Rats and Dogs
[0493] The results of the PK profile of AF292 in rats and Beagle
dogs (overnight fasting; drug administered in water solution;
gavage) are listed in Table 1.
EXAMPLE 59
Toxicology Profile of AF267B
[0494] AF267B has been extensively tested in chronic toxicity
studies for up to 13 weeks in the Wistar rat and the Beagle dog, In
the dog, the no-adverse-effect-level (NOAEL) is considered to be in
the range of 6-9 mg/kg/day, po. Effects seen in the dog (>9
mg/kg) and rat (>40 mg/kg) are consistent with the profile of
AF267B as a muscarinic agonist, without toxic cardiovascular
effects (tested in awake dogs). AF267B was evaluated extensively in
beagle dogs for up to 13 weeks in oral toxicity studies and
electrocardiograms (ECG) were routinely recorded pre- and
post-administration of the test agent. Heart rate, P wave duration
& amplitude, P-Q, QRS, and QT intervals were measured and no
changes in the ECG considered to be related to the administration
of the test agent were observed.
EXAMPLE 60
Effects of AF267B on Rat Hippocampal Neurons Exposed to A.beta.
Fibrils as Followed by Survival and Apoptosis; GSK-3 Activity;
Cytoplasmic and Nuclear Stabilization of .beta.-Catenin; Cyclin D1
Expression
[0495] This study was performed in rat hippocampal primary cell
cultures using the methods described by Garrido J L et al. (FASEB
J. 2002, 16:1982).
[0496] AF267B does not affect the survival and morphology of
hippocampal neurons at concentrations of 0.5-50 .mu.M. However
AF267B (10 .mu.M) protects>90% the hippocampal neurons from
A.beta.1-40 (5 .mu.M) that alone caused a 45% decrease in survival
and in morphology. These protective effects of AF267B are mediated
by M1 muscarinic receptors since these are blocked by pirenzepine
(10 nM), an M1 antagonist. AF267B (100 .mu.M) decreased GSK-3.beta.
activity by 60% in cultures of rat hippocampal neurons. In such a
preparation 10 .mu.M A.beta. fibrils increased GSK-3.beta. activity
to 370% vs control (100%). Furthermore AF267B (10 .mu.M)
antagonized the effects of A.beta. fibrils (10 .mu.M) decreasing
GSK-3.beta. activity to the same of 150% vs. control. AF267B (10
.mu.M) prevented A.beta.1-40 (5 .mu.M)-induced apotosis to the
control level. When cultured hippocampal neurons were exposed to
A.beta.1-40 (5 .mu.M), soluble .beta.-catenin was degraded and this
degradation was prevented by AF267B (100 .mu.M). In fact, AF267B
increased soluble .beta.-catenin level above control in a
concentration dependent manner (e.g. 1 .mu.M (100%) 10 .mu.M
(300%), 100 .mu.M (350%)). A.beta.1-40 (5 .mu.M) decreased nuclear
.beta.-catenin (by 60%), an effect blocked by AF267B (1 .mu.M (140%
vs control)). These protective effects of AF267B are mediated by M1
mAChR since these are blocked by pirenzepine (10 nM). The
destabilizing effect of A.beta.1-40 (5 .mu.M) in rat hippocampal
neurons was shown by dendritic shrinkage detected by
immunofluorescent stain. The position of the nucleus was shown by
c-jun antibody. AF267B protected the neurons and the cells have
healthy neurites when treated with this M1 agonist. The effect of
AF267B is M1 mAChR mediated since it is blocked by pirenzepine (10
nM). Finally AF267B has a protective effect (50% increase vs.
control) against A.beta.1-40 (5 .mu.M)-induced decrease (40% vs.
control) of cyclin D1, a target gene of the Wnt pathway. Again this
effect of AF267B is blocked by pirenzepine (10 nM).
[0497] The results shown here indicate that AF267B protects
neuronal cells as evaluated by MTT reduction, immunofluorescence of
neurofilaments and apoptotic analysis.
[0498] As shown above, compounds used in embodiments of the present
invention are low molecular compounds that are capable of crossing
the blood brain barrier. Many of these compounds have additional
beneficial effects including, inter ala, improvement of memory and
learning in a variety of animal models that mimic various aspects
of AD and other related disorders with an excellent safety
margin.
[0499] Table 1 compares some of the results of tests on AF292 and
AF267B.
TABLE-US-00001 TABLE 1 EFFECT AF292 AF267B Trihexyphenidyl- 0.03,
0.05, 0.1, 0.3, 0.5 mg/kg, p.o. 0.5, 1, 5 positive effects
rats-Passive positive effects; MED</0.03 mg/kg, MED 0.1-0.5
mg/kg, p.o. avoidance p.o. LONG DURATION OF ACTION CNS (rat): IRWIN
TEST.sup.@ Mydriasis: 25 mg/kg, p.o. General 1, 10, 30, 60, 100
mg/kg, p.o. Salivation: 40 mg/kg, p.o. Observation No effect on:
Lacrimation: 50 mg/kg, p.o. motor activity {open field; vertical
& Hypothermia: 50 mg/kg, p.o. horizontal screen; rotarod;
locomotor Gnawing; 50 mg/kg, p.o. ataxia; body posture & tone;
tremors; Convulsion: 50 mg/kg, p.o. twiches; paralysis; catalepsy}
Sedation: 100 mg/kg, p.o. reflexes {righting; corneal; pinnal;
Chromodacryorrhea: 100 mg/kg, extension; limb tone; flexor p.o.
withdrawal; startle} IRWIN TEST: 1, 10, 100 mg/kg, excitation or
sedation {convulsion p.o. clonic/tonic; opisthotonus; 1 & 10 no
effect on gross vocalization; C-tail; Straub tail; behavior &
physiological state. circling; stereotypies; sedation; 100 mg/kg:
abnormalities of hypnosis (sleep} carriage, apathy, decreased eye
condition {palpebral ptosis; corneal reflex, diarrhea, reduced
lacrimation; chromodacryorrhea; grooming, increased urination,
enophtalamus; exophtalamus} salivation, lacrimation & skin
condition {skin plasticity, chromodacryorrhea, reduced
piloerection, blanching (ear); locomotor activity, passivity,
hyperemia, cyanosis} decreased respiration, reduced various effects
{salivation; diarrhea; startle response (onset: 0.5 hr; diuresis;
response to duration 3-6 hrs.). handling; abdominal constriction;
rectal temp.; death} [.sup.3PZ]; rat 1.64 +/- 0.13 3.74 +/- 0.59
cortex; Ki, .mu.M [.sup.3OXO-M]; rat 0.57 +/- 0.15 1.62 +/- 0.34
cortex, Ki, .mu.M .alpha.-APPs 100% Equipotent with AF267B &
100% secretion carbachol.cndot.(CCh), EC50 = 3 .mu.M; Even in cell
cultures though less efficacious on PI transfected with rat M1
mAChR (% of max CCh) MTT assay Equipotent with AF267B & CCh at
100% against 100 .mu.M; Even though less efficacious A.beta. 25-35
& on PI H.sub.2O.sub.2 in cell cultures transfected with rat M1
mAChR PI turnover PI, AA: M1 mAChR: 35%, >88% PI, AA: M1 mAChR:
66%, 100% vs. CCh (as PI., AA: M3 mAChR: Not active as PI: M3
mAChR: 30% 100%) agonist PI, rat M1 mAChR: 75% in cell cultures An
M3 antagonist (pKb = 660 nM) transfected M2 mAChR - no effects as
agonist with the human PI, AA: M5 mAChR - no effect as mAChR
agonist subtype PI, rat M1 mAChR; 50% Metabolic Rat Liver
Microsomes: T1/2 = 92 min Rat Liver Microsomes: T1/2 = 88 min
Stability Dog Liver Microsomes: T1/2 > 100 min Dog Liver
Microsomes: T1/2 > 100 min Monkey Liver Microsomes: T1/2 = 74
min Monkey Liver Microsomes: T1/2 = 27 min Human Liver Microsomes:
T1/2 > 100 min Human Liver Microsomes: T1/2 > 100 min {BETTER
THAN AF267B} Protein Binding PBS = 0% PBS = 0% Human
.alpha.-glycoprotein (AGP) = 0% AGP = 5.7% Human serum albumin
(HSA) = 22% HSA = 32% Human Plasma = 35% Human Plasma = 25%
Pharmacokinetic Rats, 10 mg/kg, Dogs, 5 mg/kg, Rats, 5 mg/kg,
Dogs., 1 mg/kg, study{circumflex over ( )} p.o. p.o. p.o. p.o.
T1/2, hr 0.99 2.04 0.64 1.33 Tmax, hr 0.5 0.8 0.25 0.58-0.75 MRT,
hr 1.5 4.85 1 1.96 Cmax, ng/ml 1906 1193 852, 1704** 257, 1085#
AUC.sub.(0-inf), 2146 4648 773, 1546** 552, 2760# (ng*hr/ml)
Bioavailability 49 70 30*** 53 (% F) Toxicokinetic F(1x): outlier,
3901-4840; M F (1x): 985-3179; M (1x): 800-3691 study: 13 w* (1x):
1142-10932 F(13 w): 1033-4164; M(13 w): AUC(0-inf) F (13 w):
1866-12723; M(13 w): 1609- 1041-4471 ng*h/ml 4170 F(1x): 4.7-5.3;
M(1x): 3.8-5 MRT (area) [h] F(1x): outlier, 8.7-18.7; M(1x): 5-27.3
F(13 w): 3.6-5.2; M(13 w): 3.0-4.2 Elimination half F(13 w):
5.1-33.7; M(13 w): 5-7.8 Mean T1/2 (M) = 2.1 +/- 0.6 life [h] Mean
T1/2 (M) = 6.3 +/- 5.9; Mean T1/2 (F) = 3.3 +/- 1.5 Mean T1/2 (F) =
10.6 +/- 7.6 *Dogs: AF267B (1.5, 3, 6 mg/kg, p.o.; single =
1.times. & for 13 weeks = 13 w); (AF292 obtained from AF267B,
in vivo); treatment by gavage 1/2 h after feeding; solid drug in
capsule. Plasma levels of the drug are analyzed at various time
points by LC-MS; **extrapolated for 10 mg/kg; ***calculated for 2
mg/kg, po; #(extrapolated for 5 mg/kg). Oral and intravenous area
under the concentration vs. time curve (AUC) were compared to
determine the % biovailability (% F) by the following formula: Dose
(IV) .times. AUC (oral)/Dose (oral) .times. AUC(IV). A % F of over
30% generally suggests good bioavailability. Cmax levels are
equally important to determine if sufficient plasma levels are
attained to produce the desired pharmacological effect and a value
>1 .mu.M is usually sufficient. AUMC is the first statistical
moment of the AUC and is used to calculate the mean residence time
(MRT = AUMC/AUC) which is the average time the compound is in the
animal. The Cmax represents the maximum concentration observed, the
Tmax is the time to reach that maximum concentration and the T1/2
is the calculated half-life of the compound in plasma (ln2 .times.
MRT). Clearance is the volume of fluid (containing compound) from
which compound is removed completely per unit time. {circumflex
over ( )}Rat and dogs were dosed intravenously (iv) and by oral
gavage. Plasma levels of the drug are analyzed at various time
points by LC-MS-MS. .sup.@Irwin, S. PSYCHOPHARM 13: 222-257,
1968.
[0500] The results shown in Table 1 indicate that AF267B and AF292
have the same affinity, but differing efficacy for mAChR
subtypes.
[0501] It will be appreciated by persons skilled in the art that
the present invention is not limited by what has been particularly
shown and described hereinabove. Rather the scope of the present
invention includes both combinations and subcombinations of the
features described hereinabove as well as modifications and
variations thereof which would occur to a person of skill in the
art upon reading the foregoing description and which are not in the
prior art.
* * * * *