U.S. patent application number 13/030239 was filed with the patent office on 2011-06-16 for acetylcholinesterase dual inhibitors.
This patent application is currently assigned to NOSCIRA, S.A.. Invention is credited to Laura Rubio Arrieta, Celia De Austria, Isabel Dorronsoro Diaz, Paola Usan Egea, Ana Martinez Gil, Diana Alonso Gordillo, Ana Fuertes Huerta, Maria Del Monte Millan, Susana Morales-Alcelay, Miguel Medina Padilla, Esther Garcia Palomero, Pilar Munoz Ruiz.
Application Number | 20110144148 13/030239 |
Document ID | / |
Family ID | 27741882 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110144148 |
Kind Code |
A1 |
Gil; Ana Martinez ; et
al. |
June 16, 2011 |
ACETYLCHOLINESTERASE DUAL INHIBITORS
Abstract
The invention provides compounds of formula: ##STR00001## which
have a tacrine moiety connected to an heterocyclic moiety through a
linker. Through careful selection of the substituents and the
linker, the activity and selectivity towards acetylcholinesterase
can be modulated. The compounds show potent AChE inhibition
activities together with modifications in the .beta.-amyloid
aggregation properties by binding simultaneously to the catalytic
and peripheral AChE sites. They are useful in the treatment of AChE
mediated diseases, such as the Alzheimer's disease.
Inventors: |
Gil; Ana Martinez; (Madrid,
ES) ; Diaz; Isabel Dorronsoro; (Madrid, ES) ;
Arrieta; Laura Rubio; (Madrid, ES) ; Gordillo; Diana
Alonso; (Madrid, ES) ; Huerta; Ana Fuertes;
(Madrid, ES) ; Morales-Alcelay; Susana; (Madrid,
ES) ; Millan; Maria Del Monte; (Madrid, ES) ;
Palomero; Esther Garcia; (Madrid, ES) ; Egea; Paola
Usan; (Madrid, ES) ; Austria; Celia De;
(Madrid, ES) ; Padilla; Miguel Medina; (Madrid,
ES) ; Ruiz; Pilar Munoz; (Madrid, ES) |
Assignee: |
NOSCIRA, S.A.
Madrid
ES
|
Family ID: |
27741882 |
Appl. No.: |
13/030239 |
Filed: |
February 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10887974 |
Jul 9, 2004 |
7910739 |
|
|
13030239 |
|
|
|
|
Current U.S.
Class: |
514/297 ;
435/7.8 |
Current CPC
Class: |
C07D 401/12 20130101;
A61P 9/00 20180101; A61P 21/00 20180101; A61P 25/16 20180101; A61P
25/28 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/297 ;
435/7.8 |
International
Class: |
A61K 31/473 20060101
A61K031/473; A61P 25/28 20060101 A61P025/28; A61P 9/00 20060101
A61P009/00; A61P 25/16 20060101 A61P025/16; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 2003 |
GB |
0316094.2 |
Claims
1. A method of treatment of a patient with an AChE-mediated
disease, which comprises administration of an effective amount of a
compound of formula (I): ##STR00063## wherein A, B are
independently selected from CH and N; D is selected from CH, O, S
and N; provided that at least one of A, B or D is an heteroatom;
when D is CH or N, then Z is selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
aryl, substituted or unsubstituted heterocyclyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryloxy; when D
is O or S, then Z is absent; each L is independently selected from
--CR.sub.aR.sub.b--, --CR.sub.a.dbd., --CO--, --O--, --S-- and
--NR.sub.a--; k, m, n, q, x and w are each an integer independently
selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, with the proviso
that k+m+n+q+x+w is at least 4; R.sub.1 to R.sub.6 are
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted aryl,
substituted or unsubstituted heterocyclyl, --COR.sub.a,
--C(O)OR.sub.a, --C(O)NR.sub.aR.sub.b, --C.dbd.NR.sub.a, --CN,
--OR.sub.a, --OC(O)R.sub.a, --S(O).sub.t--R.sub.a,
--NR.sub.aR.sub.b, --NR.sub.aC(O)R.sub.b, --NO.sub.2,
--N.dbd.CR.sub.aR.sub.b and halogen; R.sub.a and R.sub.b are each
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted aryl,
substituted or unsubstituted heterocyclyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryloxy and
halogen; with the proviso that they are not halogen when linked to
a N; t is 0, 1 or 2; or a tautomer, a pharmaceutically acceptable
salt, a prodrug or a solvate thereof.
2. The method of claim 1, for the treatment of a condition selected
from the group consisting of cognitive disorders such as senile
dementia, cerebrovascular dementia, mild recognition impairment,
attention deficit disorder, and/or neurodegenerative dementing
disease with aberrant protein aggregations such as specially
Alzheimers's disease or condition, or prion disease as
Creutzfeld-Jacob disease or Gerstmann-Straussler-Scheinher disease,
or Parkinson's disease or condition, or Polyglutamine disease, or
tauopathies such as Pick's disease, frontotemporal dementia,
supranuclear progressive palsy, or familial amyotrophic lateral
sclerosis or systemic amyloidosis or condition.
3. A method according to claim 2, for the treatment of Alzheimers's
disease.
4. A method of biological assay which employs as reactive a
compound of formula (I): ##STR00064## wherein A, B are
independently selected from CH and N; D is selected from CH, O, S
and N; provided that at least one of A, B or D is an heteroatom;
when D is CH or N, then Z is selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
aryl, substituted or unsubstituted heterocyclyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryloxy; when D
is O or S, then Z is absent; each L is independently selected from
--CR.sub.aR.sub.b--, --CR.sub.a.dbd., --CO--, --O--, --S-- and
--NR.sub.a--; k, m, n, q, x and w are each an integer independently
selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, with the proviso
that k+m+n+q+x+w is at least 4; R.sub.1 to R.sub.6 are
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted aryl,
substituted or unsubstituted heterocyclyl, --COR.sub.a,
--C(O)OR.sub.a, --C(O)NR.sub.aR.sub.b, --C.dbd.NR.sub.a, --CN,
--OR.sub.a, --OC(O)R.sub.a, --S(O).sub.t--R.sub.a,
--NR.sub.aR.sub.b, --NR.sub.aC(O)R.sub.b, --NO.sub.2,
--N.dbd.CR.sub.aR.sub.b and halogen; R.sub.a and R.sub.b are each
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted aryl,
substituted or unsubstituted heterocyclyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryloxy and
halogen; with the proviso that they are not halogen when linked to
a N; t is 0, 1 or 2; or a tautomer, a pharmaceutically acceptable
salt, a prodrug or a solvate thereof.
Description
[0001] This application is a divisional of application Ser. No.
10/887,974 filed Jul. 9, 2004, which claims priority to British
Application No. GB0316094.2, filed Jul. 9, 2003, the contents of
each of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to a series of tacrine derivatives,
to processes for their preparation, to pharmaceutical compositions
containing them, and to their medical use. In particular it relates
to compounds and compositions which show dual site
acetylcholinesterase inhibition, specially to be useful for the
treatment of Alzheimer's disease.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is a progressive neurodegenerative
disorder which is one of the most common causes of mental
deterioration in elderly people, accounting for about 50-60% of the
overall cases of dementia among persons over 65 years of age.
Demographic data indicate that the percentage of elderly in the
population is increasing.
[0004] Brain regions that are associated with higher mental
functions, particularly the neocortex and hippocampus, are those
most affected by the characteristic pathology of AD. This includes
the extracellular deposits of .beta.-amyloid (derived from amyloid
precursor protein, APP) in senile plaques, intracellular formation
of neurofibrillary tangles (containing an abnormally phosphorylated
form of a microtubule associated protein, tau), and the loss of
neuronal synapsis and pyramidal neurons.
[0005] Current treatment approaches in this disease continue being
primarily symptomatic, with the major therapeutic strategy being
based on the cholinergic hypothesis and specifically on
acetylcholinesterase (AChE) inhibition. Over last decade, the
cholinergic hypothesis of AD has launched on the market various
cholinergic drugs primarily AChE inhibitors as tacrine, donepezil
or rivastigmine, and more recently galanthamine, with modest
improvement in the cognitive function of Alzheimer's patients.
These compounds still present some undesired side effects such as
nausea and vomiting.
##STR00002##
[0006] The three dimensional structure of AChE, as determined by
X-ray crystallography, revealed that its active site can apparently
be reached only through a deep and narrow catalytic gorge
Inhibitors of AChE act on two target sites on the enzyme, the
active site and the peripheral site. Inhibitors directed to the
active site prevent the binding of a substrate molecule, or its
hydrolysis, either by occupying the site with a high affinity
molecule (tacrine) or by reacting irreversibly with the catalytic
serine (organophosphates and carbamates). The peripheral site
consists of a less well-defined area, located at the entrance of
the catalytic gorge. Inhibitors that bind to that site include
small molecules, such as propidium and peptide toxins as
fasciculins. Bis-quaternary inhibitors as decamethonium,
simultaneously bind to the active and peripheral sites, thus
occupying the entire catalytic gorge.
[0007] Parallel to the development of antidementia drugs, research
efforts have been focused, among others, on the therapeutic
potential of AChE inhibitors to slow the disorder progression. This
fact was based on a range of evidence, which showed that AChE has
secondary non-cholinergic functions.
[0008] New evidence shows that AChE may have a direct role in
neuronal differentiation. Additionally, the role of AChE in cell
adhesion have been studied. The results indicate that AChE promotes
neurite outgrowth in neuroblastoma cell line through a cell
adhesive role.
[0009] Moreover, recent studies have shown that the peripheral
anionic site of the AChE is involved in the neurotrophic activity
of the enzyme and conclude that the adhesion function of AChE is
located at the peripheral anionic site. This finding has
implications, not only for our understanding of neural development
and its disorders, but also for the treatment of neuroblastoma, the
leukemias, and especially for Alzheimer's disease.
[0010] As it has been previously mentioned, senile plaques are one
of pathological hallmarks in AD in which their main component is PA
peptide. This is found as an aggregated poorly soluble form. In
contrast soluble PA is identified normally circulating in human
body fluids. Structural studies of PA showed that synthetic
peptides containing the sequences 1-40 and 1-42 of .beta.A can
adopt two major conformational states in solution: an amyloidogenic
conformer (.beta.A ac) with a high content of .beta.-sheet and
partly resistant to proteases and a non-amyloidogenic conformer
(.beta.A nac) with a random coil conformation or .beta.-helix and
protease-sensitive. AChE colocalizes with .beta.A peptide deposits
present in the brain of Alzheimer's patients. It is postulated that
AChE binds to a .beta.A nac form acting as a pathological chaperone
and inducing a conformational transition from .beta.A nac into
.beta.A ac in vitro and therefore to amyloid fibrils. AChE directly
promotes the assembly of .beta.A peptide into amyloid fibrils
forming stable .beta.A-AChE complexes.
[0011] Considering the non-cholinergic aspects of the cholinergic
enzyme AChE, their relationship to Alzheimer's hallmarks and the
role of the peripheral site of AChE in all these functions, an
attractive target for the design of new antidementia drugs emerged.
Peripheral or dual site inhibitors of AChE may simultaneously
alleviate the cognitive deficit in Alzheimer's patients and what it
is more important, avoid the assembly of beta-amyloid which
represents a new way to delay the neurodegenerative process.
[0012] Thus, ligands able to interact simultaneously with active
and peripheral sites could implicate several advantages over the
known inhibitors. On one hand, they should improve greatly the
inhibitory potency and on the other hand they should be involved in
neurotrophic activity.
[0013] Very recently some compounds have been reported with both
activities, see Piazzi L. et al., J. Med. Chem., 2003, 46,
2279-2282.
[0014] WO 03033489 describes piperidine derivatives having an
effect of inhibiting acetylcholinesterase and aggregation of
beta-amyloid.
[0015] WO 0117529 discloses halogen substituted tacrine or
bistacrine derivatives for treating Alzheimer's disease. One of the
subgroups presents an indole moiety connected to the tacrine
through a short linker. For example, it describes the preparation
of N-[2-(3-indolyl)ethyl]-6-chlorotacrine of formula:
##STR00003##
[0016] WO 0117529 does not suggest a site of inhibition and does
not give any activity data.
[0017] Castro, A.; Martinez, A. Mini Rev. Med. Chem., 2001, 1,
267-272 describe several families of peripheral and dual binding
site AChE inhibitors, including some tacrine derivatives.
[0018] WO 04032929 discloses dual binding site AChE inhibitors
containing a tacrine moiety connected through a linker to specified
heterocycles such as a tacrine, an indanone or a thiadiazolidinone
moiety.
SUMMARY OF THE INVENTION
[0019] After careful investigation we have designed a structurally
distinct class of dual AChE inhibitors, i.e. compounds that,
following the above ideas, show potent AChE inhibition activities
together with modifications in the .beta.-amyloid aggregation
properties by binding simultaneously to the catalytic and
peripheral AChE sites. In addition, their selectivity can be
modulated and they present low toxicity which makes them candidates
for drug development.
[0020] The compounds of the invention are characterised by the
presence of two main heterocyclic units: a tacrine moiety and a
[6+5] heteroaromatic moiety, connected through an appropriate
linker. We have found that selectivity and activity can be
modulated with the nature and length of the linker, and the nature
and substituents of the above mentioned moieties. As the examples
show, such compounds present high AChE inhibition, low toxicity,
high binding to the peripheral site and inhibition of
.beta.-amyloid aggregation and .beta.-amyloid-AChE aggregation
complex and, if desired, high selectivity.
[0021] In one aspect the invention is directed to a compound of
formula (I):
##STR00004##
wherein A, B are independently selected from CH or N; D is selected
from CH, O, S, N; provided that at least one of A, B or D is an
heteroatom; when D is CH or N, then Z is selected from hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted aryl, substituted or unsubstituted heterocyclyl,
substituted or unsubstituted alkoxy, substituted or unsubstituted
aryloxy; when D is O or S, then Z is absent; each L is
independently selected from --CR.sub.aR.sub.b--, --CR.sub.a.dbd.,
--CO--, --O--, --S-- or --NR.sub.a--; k, m, n, q, x and w are each
an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10, with the proviso that k+m+n+q+x+w is at least 4; R.sub.1 to
R.sub.6 are independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
aryl, substituted or unsubstituted heterocyclyl, --COR.sub.a,
--C(O)OR.sub.a, --C(O)NR.sub.aR.sub.b, --C.dbd.NR.sub.a, --CN,
--OR.sub.a, --OC(O)R.sub.a, --S(O).sub.t--R.sub.a,
--NR.sub.aR.sub.b, --NR.sub.aC(O)R.sub.b, --NO.sub.2,
--N.dbd.CR.sub.aR.sub.b or halogen; R.sub.a and R.sub.b are each
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted aryl,
substituted or unsubstituted heterocyclyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryloxy or
halogen; with the proviso that they are not halogen when linked to
a N; t is 0, 1 or 2; or a tautomer, a pharmaceutically acceptable
salt, a prodrug or a solvate thereof.
[0022] The side chain of formula:
##STR00005##
is suitably a substituent on A or B when A or B is carbon, which
then becomes C in place of CH.
[0023] In another aspect the invention is directed to
pharmaceutical compositions which comprise a compound according to
formula (I) or a pharmaceutically acceptable salt, prodrug or
solvate thereof, and a pharmaceutically acceptable carrier,
adjuvant or vehicle. In a preferred embodiment the formulation is
oral.
[0024] The present invention is also directed to the use of the
above defined compounds in the manufacture of a medicament,
preferably for the treatment of cognitive disorders such as senile
dementia, cerebrovascular dementia, mild recognition impairment,
attention deficit disorder, and/or neurodegenerative dementing
disease with aberrant protein aggregations such as especially
Alzheimers's disease or condition, or prion disease as
Creutzfeld-Jacob disease or Gerstmann-Straussler-Scheinher disease,
or Parkinson's disease or condition, or Polyglutamine disease, or
tauopathies such as Pick's disease, frontotemporal dementia,
supranuclear progressive palsy, or familial amyotrophic lateral
sclerosis or systemic amyloidosis or condition.
[0025] In another embodiment the invention is directed to the use
of the above defined compound in a method of treatment of these
diseases or conditions.
[0026] In another aspect, the invention is directed to the use of
the above defined compounds as reactives for biological assays.
[0027] In another aspect the invention is directed to a process for
preparing a compound of formula I above by coupling the two
heterocyclic moieties through the linker.
DETAILED DESCRIPTION OF THE INVENTION
[0028] In the above definition of compounds of formula (I) the
following terms have the preferred meanings indicated:
[0029] "Alkyl" refers to a straight or branched hydrocarbon chain
radical consisting of carbon and hydrogen atoms, containing no
saturation, having one to eight carbon atoms, and which is attached
to the rest of the molecule by a single bond, e.g., methyl, ethyl,
n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc. Alkyl radicals
may be optionally substituted by one or more substituents such as a
halo, hydroxy, alkoxy, carboxy, cyano, carbonyl, acyl,
alkoxycarbonyl, amino, nitro, mercapto and alkylthio.
[0030] "Amino" refers to a radical of the formula --NH.sub.2,
--NHRa or --NRaRb, wherein Ra and Rb are as defined above.
[0031] "Aryl" refers to a phenyl, naphthyl, indenyl, phenanthryl or
anthracyl radical, preferably phenyl or naphthyl radical. The aryl
radical may be optionally substituted by one or more substituents
such as hydroxy, mercapto, halo, alkyl, phenyl, alkoxy, haloalkyl,
nitro, cyano, dialkylamino, aminoalkyl, acyl and
alkoxycarbonyl.
[0032] "Aralkyl" refers to an aryl group linked to an alkyl group.
Preferred examples include benzyl and phenethyl.
[0033] "Acyl" refers to a radical of the formula --C(O)--Rc or
--C(O)--Rd where Rc is an alkyl radical and Rd is an aryl radical,
e.g., acetyl, propionyl, benzoyl, and the like. Other acyl groups
are possible.
[0034] "Cycloalkyl" refers to a 3- to 10-membered monocyclic or
bicyclic radical which is saturated or partially saturated, and
which consist of carbon and hydrogen atoms. Unless otherwise stated
specifically in the specification, the term "cycloalkyl" is meant
to include cycloalkyl radicals which are optionally substituted by
one or more substituents such as alkyl, halo, hydroxy, amino,
cyano, nitro, alkoxy, carboxy and alkoxycarbonyl.
[0035] "Fused aryl" refers to an aryl group, especially a phenyl or
heteroaryl group, fused to another ring.
[0036] "Alkoxy" refers to a radical of the formula --ORa where Ra
is an alkyl radical as defined above, e.g., methoxy, ethoxy,
propoxy, etc.
[0037] "Hal-" or "Halo" refers to bromo, chloro, iodo or
fluoro.
[0038] "Heterocyclyl" refers to a 3- to 15 membered ring radical
which consists of carbon atoms and from one to five heteroatoms
selected from the group consisting of nitrogen, oxygen, and sulfur,
preferably a 4- to 8-membered ring with one or more heteroatoms,
more preferably a 5- or 6-membered ring with one or more
heteroatoms. For the purposes of this invention, the heterocycle
may be a monocyclic, bicyclic or tricyclic ring system, which may
include fused ring systems; and the nitrogen, carbon or sulfur
atoms in the heterocyclyl radical may be optionally oxidised; the
nitrogen atom may be optionally quaternized; and the heterocyclyl
radical may be partially or fully saturated or aromatic. Examples
of such heterocycles include, but are not limited to, azepines,
benzimidazole, benzothiazole, furan, isothiazole, imidazole,
indole, piperidine, piperazine, purine, quinoline, thiadiazole,
tetrahydrofuran.
[0039] References herein to substituted groups in the compounds of
the present invention refer to the specified moiety that may be
substituted at one or more available positions by one or more
suitable groups, e.g., halogen such as fluoro, chloro, bromo and
iodo; cyano; hydroxyl; nitro; azido; alkanoyl such as a C1-6
alkanoyl group such as acyl and the like; carboxamido; alkyl groups
including those groups having 1 to about 12 carbon atoms or from 1
to about 6 carbon atoms and more preferably 1-3 carbon atoms;
alkenyl and alkynyl groups including groups having one or more
unsaturated linkages and from 2 to about 12 carbon or from 2 to
about 6 carbon atom; alkoxy groups having one or more oxygen
linkages and from 1 to about 12 carbon atoms or 1 to about 6 carbon
atoms; aryloxy such as phenoxy; alkylthio groups including those
moieties having one or more thioether linkages and from 1 to about
12 carbon atoms or from 1 to about 6 carbon atoms; alkylsulfinyl
groups including those moieties having one or more sulfinyl
linkages and from 1 to about 12 carbon atoms or from 1 to about 6
carbon atoms; alkylsulfonyl groups including those moieties having
one or more sulfonyl linkages and from 1 to about 12 carbon atoms
or from 1 to about 6 carbon atoms; aminoalkyl groups such as groups
having one or more N atoms and from 1 to about 12 carbon atoms or
from 1 to about 6 carbon atoms; carbocyclic aryl having 6 or more
carbons, particularly phenyl or naphthyl and aralkyl such as
benzyl. Unless otherwise indicated, an optionally substituted group
may have a substituent at each substitutable position of the group,
and each substitution is independent of the other.
[0040] In the compounds of the invention the [6+5] heteroaromatic
moiety can be selected among others from indole, isoindole,
benzimidazole, indazole, benzothiophene, benzotriazole,
benzoisoxazole, benzofurane, isobenzofurane.
[0041] A preferred class of compounds of formula (I) is that in
which A and preferably also B are CH. D is preferably N.
[0042] In a preferred embodiment the [6+5] heteroaromatic moiety is
an indole or indazole unit, preferably a substituted or
unsubstituted indole. In this case good results are obtained when
the linker is connected to the position 2 or 3 of the indole unit,
more preferably to the position 3. More generally, in one variation
of the present invention, the linker is on a carbon of the hetero
ring at a position adjacent to the benzene ring, being for example
the 3 position of the indole. Such compounds are of the
formula:
##STR00006##
[0043] Another preferred class of compounds of formula (I) is that
in which the tacrine moiety is substituted. More preferably it has
a halogen substituent. In a preferred embodiment the tacrine moiety
has a chloro substituent at position 6. This gives improved
activity and selectivity towards AChE.
[0044] We have found that the linker between the two units plays an
important role in the activity and selectivity. Indeed, a linker
containing a number of L units in the range between 4 and 18, more
preferably between 7 and 13 gives good results. Most preferred are
values between 8 and 12, and specially of about 10 or 11.
[0045] In a preferred embodiment the linker
-(L).sub.k-(L).sub.m-(L).sub.n-(L).sub.q-(L).sub.x-(L).sub.w- is
selected from the formulae
--(CH.sub.2).sub.k--CO--NR.sub.a--(CH.sub.2).sub.w--,
--(CH.sub.2).sub.k--NR.sub.a--CO--(CH.sub.2).sub.w--,
--(CH.sub.2).sub.k--CO--NR.sub.a--(CH.sub.2).sub.q--NR.sub.a--(CH.sub.2).-
sub.w--,
--(CH.sub.2).sub.k--NR.sub.a--CO--(CH.sub.2).sub.q--NR.sub.a--(CH-
.sub.2).sub.w--,
--(CH.sub.2).sub.k--O--CO--NR.sub.a--(CH.sub.2).sub.w-- wherein k,
q, w and R.sub.a are as defined above. More preferably, the linker
-(L).sub.k-(L).sub.m-(L).sub.n-(L).sub.q-(L).sub.x-(L).sub.w- has
the formulae --(CH.sub.2).sub.k--CO--NR.sub.a--(CH.sub.2).sub.w--
or --(CH.sub.2).sub.k--O--CO--NR.sub.a--(CH.sub.2).sub.n--. R.sub.a
is usually H. The integer k is preferably 1 or 2, especially 2. The
integer w is suitably from 6 to 9, especially 6 or 7.
[0046] It is preferred that the linker contains one or more amide
units, they can be at any position in the linker.
[0047] It is to be understood that the present invention includes
all combinations of the mentioned particular and preferred
groups.
[0048] In one aspect, preferred compounds of this invention are of
the formula (II):
##STR00007##
[0049] In a related aspect, more preferred compounds are of the
formula (III):
##STR00008##
[0050] In the compounds of the various formulae of this invention,
Z is preferably selected from H and CH.sub.3, especially H.
R.sub.1, R.sub.3 and R.sub.4 are preferably H. R.sub.2 is
preferably selected from H, -Hal, and --CN, especially H.
Preferably R.sub.5 is halogen and R.sub.6 is hydrogen.
[0051] In particular, we prefer that R.sub.1, R.sub.2, R.sub.3,
R.sub.4, Z are hydrogen; R.sub.5 is halogen especially chloro; and
the linker includes an amido function, preferably an amido function
flanked by oligomethylene groups. Thus, the linker is preferably of
formula --(CH.sub.2).sub.k--CONH--(CH.sub.2).sub.n--, where the sum
of k and w is preferably in the range 6 to 10, especially 7 to 9.
Suitably k is less than w, with k being 1, 2 or 3.
[0052] As variants, R.sub.1 to R.sub.5 are independently selected
from the group consisting of hydrogen, --CN, and halogen; and/or
each L is independently selected from the group consisting of
--CR.sub.aR.sub.b--, --CO-- and --NR.sub.a.
[0053] As a further aspect, the present invention extends to
compounds of the formula (A):
##STR00009##
[0054] Where:
[0055] L is independently selected from --C(R')(R'')--, --CO--,
--O-- or --NR'--
[0056] n is zero, one, two, three, four, five, six, seven, eight,
nine or ten
[0057] R' and R'' are independently selected from hydrogen, alkyl,
aryl, heteroaryl, halo, haloalkyl, alkoxy, alkylthio
[0058] A is independently selected from --CO--, --C(R')(R'')--,
.dbd.C(R')--, --N(R')--, .dbd.N--, --O--, --S(O).sub.t--
[0059] B is independently selected from --C(R')--, .dbd.C--,
--N--,
[0060] C is independently selected from --C(R')(R'')--,
.dbd.C(R')--, --N(R')--, .dbd.N--, R.sub.1, R.sub.2, R.sub.3,
R.sub.4 and R.sub.5 are independently selected from hydrogen,
alkyl, alkoxy, alkylthio, cycloaklyl, haloalkyl, halo, aryl,
--(Z).sub.n-aryl, heteroaryl, --OR.sub.3, --C(O)R.sub.3,
--C(O)OR.sub.3, --S(O).sub.t, cyano, nitro, mercapto
[0061] T is zero, one or two
[0062] Z is independently selected from C(R.sub.3)(R.sub.4)--,
--C(O)--, --O--, --C(.dbd.NR.sub.3)--, --S(O).sub.t--,
N(R.sub.3)--.
[0063] In a related aspect, the compounds of this invention are in
agreement with both the formula (I) and the formula (A). Such
compounds are of the formula (B):
##STR00010##
where the respective definitions are selected to overlap with those
of both formula (I) and formula (A).
[0064] Preferably for the formula (B), the following definitions
will apply:
A is CH or N;
B is CH or N;
D is CH, O, S or N;
[0065] at least one of A, B and D is a heteroatom; Z is hydrogen
alkyl, alkoxy, cycloalkyl, haloalkyl, aryl, aralkyl, heteroaryl;
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 are hydrogen,
alkyl, alkoxy, alkylthio, cycloalkyl, haloalkyl, halo, aralkyl,
heteraryl, OR.sub.3, COR.sub.S, COOR.sub.S, SO.sub.tR.sub.3 where t
is 0, 1 or 2; at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4 is
hydrogen; L is CR.sub.aR.sub.r; CO, O, NR.sub.a where R.sub.a and
R.sub.r are hydrogen, alkyl, aryl, heteroaryl, halo, haloalkyl,
alkoxy; k, m, n, q, x and w are 0 to 10, provided that the total is
not more than 40 and preferably x and w are 0.
[0066] Unless otherwise stated, the compounds of the invention are
also meant to include compounds which differ only in the presence
of one or more isotopically enriched atoms. For example, compounds
having the present structures except for the replacement of a
hydrogen by a deuterium or tritium, or the replacement of a carbon
by a .sup.13C-- or .sup.14C-enriched carbon or .sup.15N-enriched
nitrogen are within the scope of this invention.
[0067] The term "pharmaceutically acceptable salts, derivatives,
solvates, prodrugs" refers to any pharmaceutically acceptable salt,
ester, solvate, or any other compound which, upon administration to
the recipient is capable of providing (directly or indirectly) a
compound as described herein. However, it will be appreciated that
non-pharmaceutically acceptable salts also fall within the scope of
the invention since those may be useful in the preparation of
pharmaceutically acceptable salts. The preparation of salts,
prodrugs and derivatives can be carried out by methods known in the
art.
[0068] For instance, pharmaceutically acceptable salts of compounds
provided herein are synthesized from the parent compound which
contains a basic or acidic moiety by conventional chemical methods.
Generally, such salts are, for example, prepared by reacting the
free acid or base forms of these compounds with a stoichiometric
amount of the appropriate base or acid in water or in an organic
solvent or in a mixture of the two. Generally, nonaqueous media
like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are
preferred. Examples of the acid addition salts include mineral acid
addition salts such as, for example, hydrochloride, hydrobromide,
hydroiodide, sulphate, nitrate, phosphate, and organic acid
addition salts such as, for example, acetate, maleate, fumarate,
citrate, oxalate, succinate, tartrate, malate, mandelate,
methanesulphonate and p-toluenesulphonate. Examples of the alkali
addition salts include inorganic salts such as, for example,
sodium, potassium, calcium, ammonium, magnesium, aluminium and
lithium salts, and organic alkali salts such as, for example,
ethylenediamine, ethanolamine, N,N-dialkylenethanolamine,
triethanolamine, glucamine and basic aminoacids salts.
[0069] Particularly favoured derivatives or prodrugs are those that
increase the bioavailability of the compounds of this invention
when such compounds are administered to a patient (e.g., by
allowing an orally administered compound to be more readily
absorbed into the blood) or which enhance delivery of the parent
compound to a biological compartment (e.g., the brain or lymphatic
system) relative to the parent species.
[0070] Any compound that is a prodrug of a compound of formula (I)
is within the scope of the invention. The term "prodrug" is used in
its broadest sense and encompasses those derivatives that are
converted in vivo to the compounds of the invention. Such
derivatives would readily occur to those skilled in the art, and
include, depending on the functional groups present in the molecule
and without limitation, the following derivatives of the present
compounds: esters, amino acid esters, phosphate esters, metal salts
sulfonate esters, carbamates, and amides.
[0071] The compounds of the invention may be in crystalline form
either as free compounds or as solvates and it is intended that
both forms are within the scope of the present invention. Methods
of solvation are generally known within the art. Suitable solvates
are pharmaceutically acceptable solvates. In a particular
embodiment the solvate is a hydrate.
[0072] The compounds of formula (I) or their salts or solvates are
preferably in pharmaceutically acceptable or substantially pure
form. By pharmaceutically acceptable form is meant, inter alia,
having a pharmaceutically acceptable level of purity excluding
normal pharmaceutical additives such as diluents and carriers, and
including no material considered toxic at normal dosage levels.
Purity levels for the drug substance are preferably above 50%, more
preferably above 70%, most preferably above 90%. In a preferred
embodiment it is above 95% of the compound of formula (I), or of
its salts, solvates or prodrugs.
[0073] The compounds of the present invention represented by the
above described formula (I) may include enantiomers depending on
the presence of chiral centres or isomers depending on the presence
of multiple bonds (e.g. Z, E). The single isomers, enantiomers or
diastereoisomers and mixtures thereof fall within the scope of the
present invention.
[0074] The compounds of formula (I) defined above can be obtained
by a convergent pathway strategy by coupling the two heterocyclic
moieties which contain part of the linker. Synthetic procedures to
obtain the intermediates containing the tacrine moiety or the
benzofused 5 ring heterocyclic system are available in the
literature and involve standard organic synthesis procedures. The
person skilled in the art of organic synthesis will readily design
the process for each compound depending on the desired
functionality of the heterocycles and the nature of the linker to
be obtained. See for example WO 0117529 and WO04032929. Other
intermediates are reported in the literature.
[0075] 9-alkylaminotetrahydroacridines can be synthesized following
the previously reported procedures [Carlier, P. R.; Chow. E. S.-H;
Han, Y.; Liu, J.; El Yazal, J.; Pang Y.-P. J. Med. Chem., 1999, 42,
4225-4231]. The general method for the synthesis of indole
derivatives was previously described in Padwa. A. et al, Synthesis,
1994, 9, 993-1004. 5-Cyanoindole-3-propionic acid can be
synthesized according to the method reported in the literature
[Agarwal, A.; Jalluri, R. K.; Dewitt Blanton, C.; and Will Taylor,
E., Synthetic communications, 1993, 23, 8, 1101-1110]. Alternative
heterocycles can be prepared and used. General methods of organic
synthesis are available for example in "March's Advanced Organic
Chemistry: Reactions, Mechanisms, and Structure" 5th Edition Wiley;
Wiley series "The Chemistry of Heterocyclic Compounds"; Wiley
Series "Compendium of Organic Synthetic methods", etc.
[0076] Schemes 1 and 2 exemplify processes for the preparation of
compounds of the invention when the linker contains an amide or a
carbamate bond:
##STR00011##
##STR00012##
[0077] Alternative processes for compounds with amine, ether, ester
or other units in the linker will be readily apparent to the
skilled person.
[0078] For example to a solution of the corresponding indole
derivative in anhydrous THF was added 1,1'-carbonyldiimidazole
under N.sub.2, and the resulting mixture was stirred for 4 hours at
room temperature. A solution of the corresponding
9-alkylaminotetrahydroacridine in THF was added and the stirring
was continued for further 20 hours. After evaporation of the
solvent under reduced pressure, water was added and the resulting
mixture was extracted with dichloromethane. The combined organic
extracts were washed with saturated NaCl solution and dried with
Na.sub.2SO.sub.4. Evaporation of the solvent under reduced pressure
gave a residue which was purified by silica gel flash-column
chromatography as indicated bellow for each case.
[0079] The reaction products may, if desired, be purified by
conventional methods, such as crystallisation or chromatography.
Where the above described processes for the preparation of
compounds of the invention give rise to mixtures of stereoisomers,
these isomers may be separated by conventional techniques such as
preparative chromatography. If there are chiral centers the
compounds may be prepared in racemic form, or individual
enantiomers may be prepared either by enantiospecific synthesis or
by resolution.
[0080] One preferred pharmaceutically acceptable form is the
crystalline form, including such form in a pharmaceutical
composition. In the case of salts and solvates the additional ionic
and solvent moieties must also be non-toxic. The compounds of the
invention may present different polymorphic forms, it is intended
that the invention encompasses all such forms.
[0081] The typical compounds represented by the above-mentioned
formula (I) of the present invention, a salt thereof, a solvate or
a prodrug of them exhibit a superior acetylcholinesterase
inhibitory action. Therefore, another aspect of this invention
relates to a method of treating, improving or preventing an AChE
related disease or condition which method comprises administering
to a patient in need of such a treatment a therapeutically
effective amount of a compound of formula (I) or a pharmaceutical
composition thereof. Among the diseases that can be treated are
cognitive disorders as senile dementia, cerebrovascular dementia,
mild recognition impairment, attention deficit disorder, and/or
neurodegenerative dementing disease with aberrant protein
aggregations as specially Alzheimers's disease or condition, or
prion disease as Creutzfeld-Jacob disease or
Gerstmann-Straussler-Scheinher disease, or Parkinson's disease or
condition, or Polyglutamine disease, or tauopathies as Pick's
disease, frontotemporal dementia, supranuclear progressive palsy,
or familial amyotrophic lateral sclerosis or systemic amyloidosis
or condition.
[0082] The present invention further provides pharmaceutical
compositions comprising a compound of this invention, or a
pharmaceutically acceptable salt, derivative, prodrug or
stereoisomers thereof together with a pharmaceutically acceptable
carrier, adjuvant, or vehicle, for administration to a patient.
[0083] Examples of pharmaceutical compositions include any solid
(tablets, pills, capsules, granules etc.) or liquid (solutions,
suspensions or emulsions) composition for oral, topical or
parenteral administration.
[0084] In a preferred embodiment the pharmaceutical compositions
are in oral form, either solid or liquid. Suitable dose forms for
oral administration may be tablets, capsules, syrups or solutions
and may contain conventional excipients known in the art such as
binding agents, for example syrup, acacia, gelatin, sorbitol,
tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,
sugar, maize starch, calcium phosphate, sorbitol or glycine;
tabletting lubricants, for example magnesium stearate;
disintegrants, for example starch, polyvinylpyrrolidone, sodium
starch glycollate or microcrystalline cellulose; or
pharmaceutically acceptable wetting agents such as sodium lauryl
sulfate.
[0085] The solid oral compositions may be prepared by conventional
methods of blending, filling or tabletting. Repeated blending
operations may be used to distribute the active agent throughout
those compositions employing large quantities of fillers. Such
operations are conventional in the art. The tablets may for example
be prepared by wet or dry granulation and optionally coated
according to methods well known in normal pharmaceutical practice,
in particular with an enteric coating.
[0086] The pharmaceutical compositions may also be adapted for
parenteral administration, such as sterile solutions, suspensions
or lyophilized products in the appropriate unit dosage form.
Adequate excipients can be used, such as bulking agents, buffering
agents or surfactants.
[0087] The mentioned formulations will be prepared using standard
methods such as those described or referred to in the Spanish and
US Pharmacopoeias and similar reference texts.
[0088] Administration of the compounds or compositions of the
present invention may be by any suitable method, such as
intravenous infusion, oral preparations, and intraperitoneal and
intravenous administration. Oral administration is preferred
because of the convenience for the patient and the chronic
character of the diseases to be treated.
[0089] Generally an effective administered amount of a compound of
the invention will depend on the relative efficacy of the compound
chosen, the severity of the disorder being treated and the weight
of the sufferer. However, active compounds will typically be
administered once or more times a day for example 1, 2, 3 or 4
times daily, with typical total daily doses in the range of from
0.1 to 1000 mg/kg/day. It will be appreciated that it may be
necessary to make routine variations to the dosage, depending on
the age and condition of the patient, and the route of
administration.
[0090] The compounds and compositions of this invention may be used
with other drugs to provide a combination therapy. The other drugs
may form part of the same composition, or be provided as a separate
composition for administration at the same time or at different
time.
[0091] The following examples are given as further illustration of
the invention, they should not be taken as a definition of the
limits of the invention.
EXAMPLES
[0092] The general procedures for the preparation of compounds of
the invention have been described above.
Example 1
##STR00013##
[0093]
N-[5-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl]-3-(1H--
indol-3-yl)-propionamide
[0094] Reagents: Indole-3-propionic acid (57 mg, 0.3 mmol), THF
anhydrous (3 ml), 1,1'-carbonyldiimidazol (51 mg, 0.32 mmol), and
6-chloro-9-(5-aminopentylamino)-1,2,3,4-tetrahydroacridine (100 mg,
0.32 mmol).
[0095] Purification: silica gel column chromatography using
DCM/MeOH (7:1). Yellow solid, yield: 121 mg (83%).
[0096] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta.ppm): 8.53 (brs,
1H), 7.87 (d, 1H, J=8.8 Hz), 7.88 (d, 1H, J=2.4 Hz), 7.55 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.30 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.21 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.13 (td, 1H, J=8 Hz, J=1.2 Hz), 7.06 (td,
1H, J=8.0 Hz, J=0.8 Hz), 6.90 (m, 1H), 5.61 (m, 1H), 4.24 (brs,
1H), 3.43 (t, 2H, J=6.4 Hz), 3.16 (c, 2H, J=6.4 Hz), 3.10 (t, 2H,
J=7.2 Hz), 3.01 (m, 2H), 2.52 (m, 2H), 2.56 (t, 2H, J=7.2 Hz), 1.84
(m, 4H), 1.60 (2H, m), 1.38 (m, 2H), 1.25 (m, 2H).
[0097] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.9,
159.0, 151.4, 147.9, 136.5, 134.6, 127.2, 126.6, 124.9, 124.5,
122.0, 121.9, 119.4, 118.7, 118.2, 115.4, 114.8, 111.4, 49.5, 39.4,
37.7, 33.6, 31.5, 29.6, 24.9, 24.3, 23.1, 22.7, 21.8.
ESI-MS[M+H.sup.+].sup.+ 489.
Example 2
##STR00014##
[0098]
3-(1H-Indol-3-yl)-N-[5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-penty-
l]-propionamide
[0099] Reagents: Indole-3-propionic acid (63 mg, 0.33 mmol), THF
anhydrous (3 ml), 1,1'-carbonyldiimidazol (57 mg, 0.35 mmol), and
9-(5-aminopentylamino)-1,2,3,4-tetrahydroacridine (100 mg, 0.35
mmol).
[0100] Purification: silica gel column chromatography using
DCM/MeOH (3:1). Yellow solid. Yield: 147 mg (97%).
[0101] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.53 (brs,
1H), 7.90 (t, 2H, J=8.4 Hz), 7.55 (t, 1H, J=8.4 Hz), 7.52 (dd, 1H,
J=8.0 Hz, J=0.8 Hz), 7.35 (t, 1H, J=8.4 Hz), 7.30 (dd, 1H, J=8.0
Hz, J=0.8 Hz), 7.13 (td, 1H, J=8 Hz, J=1.2 Hz), 7.05 (td, 1H, J=8.0
Hz, J=0.8 Hz), 6.90 (m, 1H), 5.61 (m, 1H), 3.90 (brs, 1H), 3.40 (m,
2H), 3.19 (c, 2H, J=6.4 Hz), 3.10 (t, 2H, J=7.2 Hz), 3.01 (m, 2H),
2.62 (m, 2H), 2.56 (t, 2H, J=7.2 Hz), 1.80-2.00 (m, 4H), 1.60 (m,
2H), 1.38 (m, 2H), 1.25 (m, 2H).
[0102] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.9,
158.6, 150.8, 147.0, 136.5, 128.9, 128.8, 127.0, 123.8, 123.0,
122.1, 122.0, 120.1, 119.4, 118.8, 116.2, 114.8, 111.4, 49.6, 39.5,
37.8, 34.4, 31.7, 29.7, 25.2, 24.5, 23.5, 23.2, 21.8.
ESI-MS[M+H.sup.+].sup.+ 455.
Example 3
##STR00015##
[0103]
N-[5-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-hexyl]-3-(1H-i-
ndol-3-yl)-propionamide
[0104] Reagents: Indole-3-propionic acid (70 mg, 0.37 mmol), THF
anhydrous (3 ml), 1,1'carbonyldiimidazol (63 mg, 0.39 mmol), and
6-chloro-9-(6-aminohexylamino)-1,2,3,4-tetrahydroacridine (131 mg,
0.39 mmol).
[0105] Purification: silica gel column chromatography using
DCM/MeOH (50:1, 25:1, 20:1). Yellow solid. Yield: 143 mg (77%).
[0106] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.40 (brs,
1H), 7.85 (d, 1H, J=2.4 Hz), 7.84 (d, 1H, J=8.8 Hz), 7.55 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.29 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.23 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.15 (td, 1H, J=8.0 Hz, J=1.2 Hz), 7.07
(td, 1H, J=8.0 Hz, J=0.8 Hz), 6.97 (m, 1H), 5.41 (m, 1H), 4.42
(brs, 1H), 3.41 (t, 2H, J=6.4 Hz), 3.13 (c, 2H, J=6.4 Hz), 3.09 (t,
2H, J=7.2 Hz), 3.01 (m, 2H), 2.64 (m, 2H), 2.54 (t, 2H, J=7.2 Hz),
1.91-1.88 (m, 4H), 1.59-1.53 (2H, m), 1.36-1.27 (m, 4H), 1.22-1.16
(m, 2H).
[0107] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.7,
159.4, 150.7, 148.0, 136.4, 134.0, 127.3, 127.1, 124.6, 124.2,
121.8, 119.1, 118.6, 118.4, 115.7, 114.7, 111.3, 49.5, 39.4, 37.6,
34.1, 31.8, 29.6, 26.6, 26.6, 24.8, 23.1, 22.8, 21.7. ESI-MS: m/z
[M+H.sup.+].sup.+ 503.
Example 4
##STR00016##
[0108]
N-[7-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-heptyl]-3-(1H--
indol-3-yl)-propionamide
[0109] Reagents: Indole-3-propionic acid (70 mg, 0.37 mmol), THF
anhydrous (3 ml), 1,1'-carbonyldiimidazol (63 mg, 0.39 mmol), and
6-chloro-9-(7-aminoheptylamino)-1,2,3,4-tetrahydroacridine (135 mg,
0.39 mmol).
[0110] Purification: silica gel column chromatography using
AcOEt/MeOH (50:1). Yellow solid. Yield: 151 mg (79%).
[0111] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.10 (brs,
1H), 7.87 (d, 1H, J=8.8 Hz), 7.85 (d, 1H, J=2.4 Hz), 7.57 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.32 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.24 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.16 (td, 1H, J=8 Hz, J=1.2 Hz), 7.09 (td,
1H, J=8.0 Hz, J=0.8 Hz), 6.99 (m, 1H), 5.32 (m, 1H), 3.91 (brs,
1H), 3.45 (t, 2H, J=6.4 Hz), 3.13 (c, 2H, J=6.4 Hz), 3.11 (t, 2H,
J=7.2 Hz), 3.02 (m, 2H), 2.65 (m, 2H), 2.55 (t, 2H, J=7.2 Hz),
1.92-1.88 (m, 4H), 1.64-1.57 (m, 2H), 1.36-1.14 (m, 8H).
[0112] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.4,
159.3, 150.6, 147.9, 136.1, 133.8, 127.3, 126.9, 124.4, 124.0,
121.8, 121.6, 119.0, 118.5, 118.2, 115.6, 114.7, 111.0, 49.5, 39.3,
37.5, 34.0, 31.7, 29.4, 28.9, 26.7, 26.6, 24.6, 22.9, 22.7, 21.5.
ESI-MS: m/z [M+H.sup.+].sup.+ 517.
Example 5
##STR00017##
[0113]
N-[8-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-octyl]-3-(1H-i-
ndol-3-yl)-propionamide
[0114] Reagents: Indole-3-propionic acid (70 mg, 0.37 mmol), THF
anhydrous (3 ml), 1,1'-carbonyldiimidazol (63 mg, 0.39 mmol), and
6-chloro-9-(8-aminooctylamino)-1,2,3,4-tetrahydroacridine (140 mg,
0.39 mmol).
[0115] Purification: silica gel column chromatography using.
AcOEt/MeOH (50:1). Yellow solid. Yield: 104 mg (53%)
[0116] .sup.1H-NMR (CDCl.sub.3, 400 MHz .delta. ppm): 8.21 (brs,
1H), 7.86 (d, 1H, J=8.8 Hz), 7.85 (d, 1H, J=2.4 Hz), 7.57 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.24 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.24 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.16 (td, 1H, J=8.0 Hz, J=1.2 Hz), 7.09
(td, 1H, J=8.0 Hz, J=0.8 Hz), 6.99 (m, 1H), 5.35 (m, 1H), 3.91
(brs, 1H), 3.46 (t, 2H, J=6.4 Hz), 3.14 (c, 2H, J=6.4 Hz), 3.10 (t,
2H, J=7.2 Hz), 3.01 (m, 2H), 2.65 (m, 2H), 2.55 (t, 2H, J=7.2 Hz),
1.92-1.89 (m, 4H), 1.64-1.58 (m, 2H), 1.36-1.31 (m, 4H), 1.28-1.14
(m, 6H).
[0117] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.6,
159.4, 150.8, 148.1, 136.4, 133.9, 127.4, 127.1, 124.6, 124.1,
121.9, 121.8, 119.1, 118.6, 118.4, 115.7, 114.8, 111.3, 49.7, 39.6,
37.7, 34.2, 31.9, 29.7, 29.3, 27.0, 26.8, 24.8, 23.1, 22.9, 21.
ESI-MS: m/z [M+H.sup.+].sup.+ 531.
Example 6
##STR00018##
[0118]
N-[9-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-nonyl]-3-(1H-i-
ndol-3-yl)-propionamide
[0119] Reagents: indole-3-propionic acid (28 mg, 0.15 mmol), THF
anhydrous (3 ml), 1,1'-carbonyldiimidazol (25 mg, 0.15 mmol), and
6-chloro-9-(9-aminononylamino)-1,2,3,4-tetrahydroacridine (57 mg,
0.15 mmol).
[0120] Purification: silica gel column chromatography using.
DCM/MeOH (7:1). Yellow solid. Yield: 10 mg (14%).
[0121] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.53 (brs,
1H), 7.87 (d, 1H, J=8.8 Hz), 7.88 (d, 1H, J=2.4 Hz), 7.57 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.30 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.21 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.13 (td, 1H, J=8 Hz, J=1.2 Hz), 7.06 (td,
1H, J=8.0 Hz, J=0.8 Hz), 6.90 (m, 1H), 5.61 (m, 1H), 4.24 (brs,
1H), 3.50 (m, 2H), 3.19 (c, 2H, J=6.4 Hz), 3.10 (t, 2H, J=7.2 Hz),
3.01 (m, 2H), 2.62 (m, 2H), 2.56 (t, 2H, J=7.2 Hz), 1.81 (m, 4H),
1.52 (m, 2H), 1.01-1.40 (m, 13H).
[0122] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.7,
159.0, 151.2, 147.8, 136.5, 134.3, 127.3, 127.2, 125.0, 124.4,
122.1, 122.0, 119.3, 118.8, 118.2, 115.3, 115.0, 111.4, 49.8, 39.8,
37.8, 34.0, 32.0, 29.8, 29.7, 29.5, 29.5, 27.2, 27.1, 25.0, 23.4,
23.0, 22.0. ESI-MS[M+H.sup.+].sup.+ 545.
Example 7
##STR00019##
[0123]
N-[10-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-decyl]-3-(1H--
indol-3-yl)-propionamide
[0124] Reagents: indole-3-propionic acid (47 mg, 0.25 mmol), THF
anhydrous (4 ml), 1,1'-carbonyldiimidazol (44 mg, 0.27 mmol), and
6-chloro-9-(10-aminodecylamino)-1,2,3,4-tetrahydroacridine (105 mg,
0.27 mmol).
[0125] Purification: silica gel column chromatography using
DCM/MeOH (10:1). Yellow solid. Yield: 21 mg (19%).
[0126] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.63 (brs,
1H), 7.89 (d, 1H, J=8.8 Hz), 7.87 (d, 1H, J=2.4 Hz), 7.56 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.32 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.21 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.14 (td, 1H, J=8 Hz, J=1.2 Hz), 7.08 (td,
1H, J=8.0 Hz, J=0.8 Hz), 6.97 (m, 1H), 5.61 (m, 1H), 4.24 (brs,
1H), 3.50 (m, 2H), 3.19 (c, 2H, J=6.4 Hz), 3.10 (t, 2H, J=7.2 Hz),
3.01 (m, 2H), 2.63 (m, 2H), 2.56 (t, 2H, J=7.2 Hz), 1.80-2.00 (m,
4H), 1.51 (m, 2H), 1.01-1.40 (m, 14H).
[0127] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.7,
159.0, 151.2, 147.8, 136.5, 1343, 127.3, 127.2, 125.0, 124.4,
122.1, 122.0, 119.3, 118.8, 118.2, 115.3, 115.0, 111.4, 49.8, 39.8,
37.8, 34.0, 32.0, 29.8, 29.7, 29.6, 29.5, 29.5, 27.2, 27.1, 25.0,
23.4, 23.0, 22.0. ESI-MS[M+H.sup.+].sup.+ 559.
Example 8
##STR00020##
[0128]
N-(3-{[3-(1,2,3,4-tetrahydro-acridin-9-ylamino)-propyl]-methyl-amin-
o}-propyl)-3-(1H-indol-3-yl)-propionamide
[0129] Reagents: Indole-3-propionic acid (56 mg, 0.29 mmol), THF
anhydrous (4 ml), 1,1'carbonyldiimidazol (50 mg, 0.31 mmol), and
N.sup.1-[3-(1,2,3,4-tetrahydro-acridin-9-ylamino)-propyl]-N.sup.1-methyl--
propane-1,3-diamine (100 mg, 0.31 mmol).
[0130] Purification: silica gel column chromatography using
DCM/MeOH (20:1+0.1% NH.sub.3, 10:1+0.2% NH.sub.3, 10:1+0.4%
NH.sub.3. Yellow solid. Yield: 70 mg (46%)
[0131] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.80 (brs,
1H), 7.86 (t, 2H, J=8.4 Hz), 7.51 (t, 1H, J=8.4 Hz), 7.46 (d, 1H,
J=8.4 Hz), 7.27 (td, 1H, J=7.0 Hz, J=2.0 Hz), 7.25 (d, 1H, J=7.0
Hz), 7.10 (td, 1H, J=8.0 Hz, J=1.2 Hz), 7.03 (td, 1H, J=8.0 Hz,
J=1.2 Hz), 6.85 (d, 1H, J=2.4 Hz), 6.37 (t, 1H, J=4.5 Hz), 5.00
(brs, 1H), 3.46 (m, 2H), 3.19 (c, 2H, J=6.3 Hz), 3.07-3.01 (m, 4H),
2.63 (m, 2H), 2.47 (t, 2H, J=7.0), 2.36 (t, 2H, J=6.4 Hz), 2.24 (t,
2H, J=6.8 Hz), 1.91 (s, 3H), 1.86-1.84 (m, 4H), 1.70-1.67 (m, 2H),
1.54-1.50 (m, 2H). .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm):
172.0, 158.3, 150.8, 147.2, 136.3, 128.4, 128.3, 127.1, 123.6,
122.8, 121.8, 121.8 120.2, 119.0, 118.5, 115.9, 114.7, 111.3, 56.6,
56.1, 48.7, 42.2, 38.5, 37.7, 34.0, 28.5, 26.6, 25.3, 23.2, 22.9,
21.7. ESI-MS: m/z [M+H.sup.+].sup.+ 498.
Example 9
##STR00021##
[0132]
N-(3-{[3-(6-chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-propyl]-me-
thyl-amino}-propyl)-3-(1H-indol-3-yl)-propionamide
[0133] Reagents: indole-3-propionic acid (56 mg, 0.29 mmol), THF
anhydrous (4 ml), 1,1'-carbonyldiimidazol (50 mg, 0.3 .mu.mol), and
N.sup.1-[3-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-propyl]-N.sup.-
1-methyl-propane-1,3-diamine (100 mg, 0.31 mmol).
[0134] Purification: silica gel column chromatography using
DCM/MeOH (20:1+0.1% NH.sub.3, 10:1+0.2% NH.sub.3, 10:1+0.4%
NH.sub.3). Yellow solid. Yield: 70 mg (46%)
[0135] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.79 (br,
1H), 7.83 (d, 1H, J=8.8 Hz), 7.79 (d, 1H, J=2.4 Hz), 7.30 (dd, 1H,
J=8.0 Hz, J=1.2 Hz), 7.24 (dd, 1H, J=8.0 Hz, J=0.8 Hz), 7.14 (dd,
1H, J=8.8 Hz, J=2.4 Hz), 7.08 (td, 1H, J=8 Hz, J=1.2 Hz), 7.01 (td,
1H, J=8.0 Hz, J=0.8 Hz), 6.86 (m, 1H), 6.30 (m, 1H), 3.91 (brs,
1H), 3.45 (m, 2H), 3.17 (c, 2H, J=7.2 Hz), 3.03 (t, 2H, J=6.4 Hz),
2.94 (m, 2H), 2.50 (m, 2H), 2.46 (t, 2H, J=7.2 Hz), 2.41-2.23 (m,
2H), 2.22-2.18 (m, 2H), 2.10 (s, 3H), 1.18 (m, 4H), 1.73-1.60 (m,
2H), 1.59-1.49 (m, 2H).
[0136] .sup.13C-NMR (CDCl.sub.3, 100 MHz, .delta. ppm): 172.7,
159.5, 151.0, 148.0, 136.4, 134.0, 127.3, 127.2, 124.7, 124.1,
121.9, 122.0, 119.2, 118.6, 118.4, 115.7, 114.8, 111.4, 56.7,
56.21, 49.0, 42.3, 38.6, 37.8, 34.2, 28.5, 26.8, 25.2, 23.2, 23.0,
21.8. ESI-MS: m/z [M+H.sup.+].sup.+ 531.
Example 10
##STR00022##
[0137] 1H-Indole-3-carboxylic acid
[5-(6-chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl]-amide
[0138] Reagents: Indole-3-carboxylic acid (151 mg, 0.94 mmol), THF
anhydrous (4 ml), 1,1'-carbonyldiimidazol (153 mg, 0.94 mmol), and
6-chloro-9-(8-aminopentylamino)-1,2,3,4-tetrahydroacridine (276 mg,
0.90 mmol).
[0139] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 198 mg (52%)
[0140] .sup.1H-NMR (CD.sub.3OD, 400 MHz, .delta. ppm): 8.07 (d, 1H,
J=9.0 Hz), 8.06 (dt, 1H, J=8.0 Hz and J=0.8 Hz), 7.80 (s, 1H), 7.69
(d, 1H, J=2.0 Hz), 7.41 (d, 1H, J=8.0 Hz), 7.22 (dd, 1H, J=9.0 Hz
and J=2.0 Hz), 7.17 (td, 1H, J=8.0 Hz and J=1.2 Hz), 7.12 (td, 1H,
J=8.0 Hz and J=1.2 Hz), 3.58 (t, 2H, J=7.0 Hz), 3.37 (t, 2H, J=7.0
Hz), 2.88 (t, 2H, J=6.2 Hz), 2.65 (t, 2H, J=6.2 Hz), 1.79 (m, 4H),
1.71 (m, 2H), 1.63 (m, 2H), 1.45 (m, 2H).
[0141] .sup.13C-NMR (CD.sub.3OD, 100 MHz, .delta. ppm): 168.6,
160.3, 153.4, 148.6, 138.2, 135.6, 128.9, 127.2, 126.7, 126.6,
125.1, 123.5, 122.0, 121.8, 119.5, 116.7, 112.9, 112.0, 40.1, 34.3,
32.0, 30.7, 26.1, 25.4, 24.0, 23.6. ESI-MS[M].sup.+ 461.07.
Example 11
##STR00023##
[0142] 1H-Indole-3-carboxylic acid
[6-(6-chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-hexyl]-amide
[0143] Reagents: Indole-3-carboxylic acid (153 mg, 0.95 mmol), THF
anhydrous (10 ml), 1,1'-carbonyldiimidazol (154 mg, 0.95 mmol), and
6-chloro-9-(8-aminohexylamino)-1,2,3,4-tetrahydroacridine (300 mg,
0.90 mmol).
[0144] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 120 mg (28%)
[0145] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 10.24 (brs,
1H), 7.89 (m, 1H), 7.85 (d, 1H, J=9.4 Hz), 7.83 (d, 1H, J=2.0 Hz),
7.71 (d, 1H, J=2.8 Hz), 7.39 (m, 1H), 7.18-7.22 (m, 3H), 6.13 (t,
1H, J=5.8 Hz), 4.0 (brs, 1H), 3.46 (m, 4H), 2.97 (m, 2H), 2.60 (m,
2H), 1.84 (m, 4H), 1.60 (m, 4H), 1.39 (m, 4H).
[0146] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 166.0,
159.6, 151.0, 148.1, 136.7, 134.2, 128.4, 127.2, 124.8, 124.7,
124.3, 122.8, 121.5, 119.7, 118.3, 115.7, 112.4, 112.1, 49.5, 39.4,
33.9, 31.7, 30.0, 26.7, 26.6, 24.6, 22.9, 22.6. ESI-MS[M+H].sup.+
476.
Example 12
##STR00024##
[0147] 1H-Indole-3-carboxylic acid
[7-(6-chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-heptyl]-amide
[0148] Reagents: Indole-3-carboxylic acid (147 mg, 0.91 mmol), THF
anhydrous (10 ml), 1,1'-carbonyldiimidazol (147 mg, 0.91 mmol), and
6-chloro-9-(8-aminoheptylamino)-1,2,3,4-tetrahydroacridine (300 mg,
0.87 mmol).
[0149] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 226 mg (51%)
[0150] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 9.88 (brs,
1H), 7.90 (dd, 1H, J=6.3 Hz, J=3.0 Hz), 7.87 (d, 1H, J=9 Hz), 7.84
(d, 1H, J=2.0 Hz), 7.73 (d, 1H, J=2.7 Hz), 7.41 (dd, 1H, J=6.3 Hz,
J=3.0 Hz), 7.23 (d, 1H, J=9 Hz), 7.23-7.19 (m, 2H), 6.06 (t, 1H,
J=5.5 Hz), 4.0 (brs, 1H), 3.46 (m, 4H), 2.98 (m, 2H), 2.61 (m, 2H),
1.85 (m, 4H), 1.59 (m, 4H), 1.34 (m, 6H).
[0151] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 165.7,
159.6, 151.1, 148.2, 136.6, 134.2, 128.3, 127.4, 124.8, 124.7,
124.3, 122.8, 121.6, 119.8, 118.4, 115.8, 112.4, 112.3, 49.6, 39.5,
34.0, 31.8, 29.9, 29.1, 27.0, 26.9, 24.7, 22.9, 22.7.
ESI-MS[M].sup.+ 489.
Example 13
##STR00025##
[0152] 1H-Indole-3-carboxylic acid
[8-(6-chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-octyl]-amide
[0153] Reagents: Indole-3-carboxylic acid (92 mg, 0.57 mmol), THF
anhydrous (3 ml), 1,1'-carbonyldiimidazol (92 mg, 0.57 mmol), and
6-chloro-9-(8-aminooctylamino)-1,2,3,4-tetrahydroacridine (196 mg,
0.54 mmol).
[0154] Purification: silica gel column chromatography (eluent:
Hexane/EtOAc 1:2+0.1% NH.sub.3, 1:3+0.2% NH.sub.3). Yellow solid.
Yield: 110 mg (40%).
[0155] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 10.51 (brs,
1H), 7.85 (m, 1H), 7.79 (d, 1H, J=9.0 Hz), 7.76 (d, 1H, J=2.0 Hz),
7.65 (d, 1H, J=1.6 Hz), 7.31 (m, 1H), 7.15 (dd, 1H, J=9.0 Hz, J=2.0
Hz), 7.10 (m, 2H), 6.09 (m, 1H), 3.9 (brs, 1H), 3.36 (c, 4H, J=7.3
Hz), 2.91 (m, 2H), 2.53 (m, 2H), 1.78 (m, 4H), 1.52 (m, 4H),
1.26-1.19 (m, 8H)
[0156] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 165.4,
158.9, 150.4, 147.5, 136.1, 133.5, 127.7, 126.6, 124.2, 123.6,
122.1, 122.0, 120.7, 119.1, 117.7, 115.0, 111.7, 111.4, 48.9, 38.9,
33.2, 31.1, 29.3, 29.1, 28.5, 26.3, 26.1, 23.9, 22.3, 22.0.
ESI-MS[M].sup.+ 503.
Example 14
##STR00026##
[0157]
N-[7-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-heptyl]-2-(1H-i-
ndol-3-yl)-acetamide
[0158] Reagents: Indole-3-acetic acid (1.10 g, 6.3 mmol), THF
anhydrous (50 ml), 1,1'-carbonyldiimidazol (1.07 g, 6.6 mmol), and
6-chloro-9-(8-aminoheptylamino)-1,2,3,4-tetrahydroacridine (2.29 g,
6.6 mmol).
[0159] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 2.48 g (80%)
[0160] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 9.25 (brs,
1H), 7.79 (d, 1H, J=9 Hz), 7.79 (d, 1H, J=2.0 Hz), 7.45 (d, 1H,
J=8.0 Hz), 7.29 (d, 1H, J=8.0 Hz), 7.6 (dd, 1H, J=9 Hz, J=2.0 Hz),
7.11 (t, 1H, J=8.0 Hz), 7.03 (t, 1H, J=8.0 Hz), 7.03 (s, 1H), 5.71
(t, 1H, J=5.5 Hz), 3.82 (brs, 1H), 3.65 (s, 2H), 3.34 (t, 2H, J=7.0
Hz), 3.08 (c, 2H, J=6.6 Hz), 2.93 (brs, 2H), 2.56 (brs, 2H), 1.81
(m, 4H), 1.48 (m, 2H), 1.28-1.06 (m, 8H).
[0161] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 171.4,
159.4, 150.6, 148.0, 136.4, 133.7, 127.2, 126.8, 124.4, 123.9,
123.7, 122.2, 119.6, 118.4, 118.2, 115.5, 111.3, 108.5, 49.3, 39.2,
33.7, 33.3, 31.4, 29.1, 28.6, 26.5, 26.3, 24.3, 22.7, 22.4.
ESI-MS[M].sup.+ 503.
Example 15
##STR00027##
[0162]
N-[5-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl]-4-(1H--
indol-3-yl)-butyramide
[0163] Reagents: Indole-3-butyric acid (134 mg, 0.66 mmol), THF
anhydrous (10 ml), 1,1'-carbonyldiimidazol (107 mg, 0.66 mmol), and
6-chloro-9-(5-aminopentylamino)-1,2,3,4-tetrahydroacridine (200 mg,
0.63 mmol).
[0164] Purification: silica gel column chromatography using
EtOAc/MeOH (100:1). Yellow solid. Yield: 220 mg (44%).
[0165] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.47 (brs,
1H), 7.84 (d, 1H, J=1.9 Hz), 7.83 (d, 1H, J=10.0 Hz), 7.54 (d, 1H,
J=7.4 Hz), 7.30 (d, 1H, J=7.4 Hz), 7.21 (dd, 1H, J=9.0 Hz, J=1.9
Hz), 7.13 (td, 1H, J=7.4 Hz, J=1.2 Hz), 7.05 (td, 1H, J=7.4 Hz,
J=1.2 Hz), 6.10 (d, 1H, J=2.4 Hz), 5.52 (t, 1H, J=5.4 Hz), 3.91
(brs, 1H), 3.40 (m, 2H), 3.18 (c, 2H, J=6.4 Hz), 2.98 (brs, 2H),
2.76 (t, 2H, J=7.0 Hz), 2.60 (brs, 2H), 2.18 (t, 2H, J=7.0 Hz),
2.02 (m, 2H), 1.85 (m, 4H), 1.61 (m, 2H), 1.45 (m, 2H), 1.36-1.31
(m, 2H).
[0166] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 173.3,
159.8, 150.8, 148.3, 136.5, 134.1, 127.6, 127.6, 124.7, 124.4,
122.0, 122.7, 119.2, 118.9, 118.6, 116.0, 115.6, 111.3, 49.5, 39.2,
36.4, 34.2, 31.4, 29.6, 26.3, 24.7, 24.7, 24.3, 23.0, 22.8.
ESI-MS[M].sup.+ 503.
Example 16
##STR00028##
[0167]
N-[6-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-hexyl]-4-(1H-i-
ndol-3-yl)-butyramide
[0168] Reagents: Indole-3-butyric acid (134 mg, 0.66 mmol), THF
anhydrous (8 ml), 1,1'-carbonyldiimidazol (107 mg, 0.66 mmol), and
6-chloro-9-(5-aminohexylamino)-1,2,3,4-tetrahydroacridine (200 mg,
0.63 mmol).
[0169] Purification: silica gel column chromatography using
DCM/MeOH (20:1, 20:1+0.01% NH.sub.3). Yellow solid. Yield: 163 mg
(48%).
[0170] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.93 (brs,
1H), 7.85 (d, 1H, J=1.9 Hz), 7.84 (d, 1H, J=9.0 Hz), 7.51 (d, 1H,
J=8.0 Hz), 7.29 (d, 1H, J=8.0 Hz), 7.20 (dd, 1H, J=9.0 Hz, J=2.0
Hz), 7.10 (t, 1H, J=7.5 Hz), 7.02 (t, 1H, J=7.5 Hz), 6.87 (d, 1H,
J=1.4 Hz), 5.80 (brs, 1H), 4.12 (brs, 1H), 3.41 (t, 2H, J=7.4 Hz),
3.16 (c, 2H, J=6.6 Hz), 2.97 (brs, 2H), 2.72 (t, 2H, J=7.4 Hz),
2.56 (brs, 2H), 2.17 (t, 2H, J=7.4 Hz), 1.99 (m, 2H), 1.83 (m, 4H),
1.56 (m, 2H), 1.41 (m, 2H), 1.35-1.21 (m, 4H).
[0171] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 173.6,
159.2, 151.4, 147.7, 136.7, 134.5, 127.6, 126.9, 125.1, 124.5,
122.0, 121.9, 120.0, 118.9, 118.3, 115.6, 115.4, 111.5, 49.5, 39.4,
36.5, 33.7, 31.8, 29.8, 26.7, 26.6, 26.4, 24.8, 24.7, 23.0, 22.7.
ESI-MS[M].sup.+ 517.
Example 17
##STR00029##
[0172]
N-[6-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-hexyl]-3-(1H-i-
ndol-3-yl)-acrylamide
[0173] Reagents: Indole-3-acrylic acid (88 mg, 0.47 mmol), THF
anhydrous (6 ml), 1,1'-carbonyldiimidazol (76 mg, 0.47 mmol), and
6-chloro-9-(5-aminohexylamino)-1,2,3,4-tetrahydroacridine (150 mg,
0.45 mmol).
[0174] Purification: silica gel column chromatography using
EtOAc/MeOH (100:1, 100:1+0.1% NH.sub.3). Yellow solid. Yield: 20 mg
(8%).
[0175] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.93 (brs,
1H), 7.83-7.76 (m, 4H), 7.33-732 (m, 2H), 7.19-7.08 (m, 3H), 6.35
(d, 1H, J=15.0 Hz), 5.79 (t, 1H, J=5.9 Hz), 3.92 (brs, 1H), 3.38
(t, 2H, J=7.0 Hz), 3.33 (c, 2H, J=6.6 Hz), 2.94 (brs, 2H), 2.56
(brs, 2H), 1.81 (m, 4H), 1.61-1.36 (m, 4H), 1.32 (m, 4H).
[0176] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 167.8,
159.4, 151.0, 147.9, 137.5, 134.8, 134.2, 129.0, 127.0, 125.4,
124.9, 124.3, 122.8, 120.9, 120.2, 118.3, 115.6, 113.1, 112.2,
49.4, 39.6, 33.8, 31.7, 29.8, 26.7, 26.6, 24.6, 22.9, 22.6.
ESI-MS[M].sup.+ 501.
Example 18
##STR00030##
[0177]
2-(5-Bromo-1H-indol-3-yl)-N-[7-(6-chloro-1,2,3,4-tetrahydro-acridin-
-9-ylamino)-heptyl]-acetamide
[0178] Reagents: 5-Bromoindole-3-acetic acid (155 mg, 0.61 mmol),
THF anhydrous (10 ml), 1,1'-carbonyldiimidazol (99 mg, 0.61 mmol),
and 6-chloro-9-(8-aminoheptylamino)-1,2,3,4-tetrahydroacridine (200
mg, 0.58 mmol).
[0179] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 185 mg (54%)
[0180] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 9.36 (brs,
1H), 7.86 (d, 1H, J=9.0 Hz), 7.83 (d, 1H, J=2.3 Hz), 7.63 (t, 1H,
J=0.8 Hz), 7.23 (m, 1H), 7.21 (m, 2H), 7.09 (d, 1H, J=2.3 Hz), 5.72
(t, 1H, J=5.8 Hz), 3.95 (brs, 1H), 3.64 (s, 2H), 3.42 (t, 2H, J=7.2
Hz), 3.15 (c, 2H, J=6.6 Hz), 2.98 (brs, 2H), 2.61 (brs, 2H), 1.86
(m, 4H), 1.56 (m, 2H), 1.34 (m, 2H), 1.30-1.17 (m, 4H), 1.14 (m,
2H).
[0181] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 171.2,
159.5, 151.09, 148.1, 135.2, 134.2, 128.9, 127.3, 125.4, 125.3,
124.9, 124.3, 121.4, 118.4, 115.7, 113.2, 113.1, 108.6, 49.6, 39.6,
34.0, 33.5, 31.8, 29.5, 29.0, 26.8, 26.7, 24.6, 23.0, 22.7.
ESI-MS[M+1, .sup.79Br].sup.+ 581, [M+1, .sup.81Br].sup.+ 583.
Example 19
##STR00031##
[0182]
N-[6-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-hexyl]-3-(5-is-
ocyano-1H-indol-3-yl)-propionamide
[0183] Reagents: 5-Cyanoindole-3-propionic acid (111 mg, 0.52
mmol), THF anhydrous (10 ml), 1,1'-carbonyldiimidazol (84 mg, 0.52
mmol), and
6-chloro-9-(8-aminoheptylamino)-1,2,3,4-tetrahydroacridine (164 mg,
0.49 mmol).
[0184] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 60 mg (22%).
[0185] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 9.48 (brs,
1H), 7.89 (s, 1H), 7.87 (d, 1H, J=9.0 Hz), 7.82 (d, 1H, J=2.1 Hz),
7.33 (brs, 2H), 7.23 (dd, 1H, J=9.0 Hz, J=2.1 Hz), 7.10 (brs, 1H),
5.70 (t, 1H, J=5.6 Hz), 4.00 (brs, 1H), 3.44 (t, 2H, J=7.2 Hz),
3.18 (c, 2H, J=6.6 Hz), 3.08 (t, 2H, J=7.2 Hz), 2.98 (brs, 2H),
2.62 (brs, 2H), 2.52 (t, 2H, J=7.4 Hz), 1.88 (m, 4H), 1.58 (m, 2H),
1.42-1.24 (m, 4H), 1.40-1.24 (m, 4H), 1.23-1.18 (m, 2H).
[0186] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 172.5,
159.7, 151.2, 148.3, 138.3, 135.3, 134.42, 127.6, 127.4, 125.0,
124.9, 124.7, 124.6, 124.4, 121.1, 118.7, 116.2, 116.1, 112.4,
102.5, 49.6, 39.6, 37.6, 34.2, 31.9, 29.8, 26.7, 24.9, 23.2, 22.9,
21.3. ESI-MS[M].sup.+ 528.
Example 20
##STR00032##
[0187] 1H-Methylindole-3-carboxylic acid
[7-(6-chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-heptyl]-amide
[0188] Reagents: 1-Methylindole-3-carboxylic acid (212 mg, 1.21
mmol), THF anhydrous (10 ml), 1,1'-carbonyldiimidazol (197 mg, 1.21
mmol), and
6-chloro-9-(8-aminoheptylamino)-1,2,3,4-tetrahydroacridine (400 mg,
1.16 mmol).
[0189] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 45 mg (8%)
[0190] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 7.88 (d, 1H,
J=7.0 Hz), 7.86 (d, 1H, J=9 Hz), 7.84 (d, 1H, J=2.3 Hz), 7.63 (s,
1H), 7.33 (d, 1H, J=7.0 Hz), 7.28-7.20 (m, 3H, 5.97 (t, 1H, J=5.5
Hz), 3.9 (brs, 1H), 3.44 (m, 4H), 2.99 (m, 2H), 2.62 (m, 2H), 1.87
(m, 4H), 1.59 (m, 4H), 1.36 (m, 6H).
[0191] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 165.3,
159.7, 150.9, 148.3, 137.3, 134.0, 132.4, 127.7, 125.3, 124.7,
124.3, 122.6, 121.5, 120.0, 118.5, 115.9, 111.1, 110.2, 49.7, 39.5,
34.2, 33.4, 31.8, 30.0, 29.1, 27.0, 26.9, 24.7, 23.0, 22.8.
ESI-MS[M].sup.+ 503.
Example 21
##STR00033##
[0192] 1H-Indazole-3-carboxylic acid
[7-(6-chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-heptyl]-amide
[0193] Reagents: Indazole-3-carboxylic acid (162 mg, 1.00 mmol),
THF anhydrous (5 ml), 1,1'-carbonyldiimidazol (170 mg, 1.05 mmol),
and 6-chloro-9-(8-aminoheptylamino)-1,2,3,4-tetrahydroacridine (364
mg, 1.05 mmol).
[0194] Purification: silica gel column chromatography (eluent:
EtOAc/MeOH 50:1). Yellow solid. Yield: 6 mg (1%)
[0195] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 11.26 (brs,
1H), 8.34 (d, 1H, J=8.0 Hz), 7.84 (d, 1H, J=2.0 Hz), 7.82 (d, 1H,
J=9.0 Hz), 7.43 (dd, 1H, J=8.0 Hz), 7.5 (t, 1H, J=7 Hz), 7.23-7.17
(m, 2H), 7.03 (t, 1H, J=5.5 Hz), 3.9 (brs, 1H), 3.42 (m, 4H), 2.96
(brs, 2H), 2.58 (brs, 2H), 1.82 (m, 4H), 1.57 (m, 4H), 1.32 (m,
6H)
[0196] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 162.9,
159.6, 151.1, 148.2, 141.5, 139.6, 134.2, 127.5, 127.4, 124.8,
124.4, 122.9, 122.8, 122.1, 118.5, 115.8, 109.9, 49.7, 39.0, 34.1,
31.8, 29.8, 29.1, 26.9, 26.8, 24.7, 23.0, 22.7. ESI-MS[M].sup.+
490.
Example 22
[0197] The indole-tacrine carbamate derivatives were synthesized
following a similar method to that reported in the literature:
Bruce, A.; Spangle, L. A.; Kaldor, S. W.; Tetrahedron Letters,
1996, 7, 937-940. The synthetic strategy is summarized in scheme
2.
Synthesis of intermediate carbonic acid 2-(1H-indol-3-yl)-ethyl
ester 4-nitro-phenyl ester
[0198] To a solution of 2-(1H-indol-3-yl)-ethanol (1600 mg, 9.92
mmol), in N-methyl morpholine (2000 mg, 19.84 mmol), was added
p-nitrophenyl chloroformate (4000 mg, 19.84 mmol), and the mixture
was stirred for 24 hours at room temperature. Water was added and
the mixture was extracted with dichloromethane. Evaporation of the
solvent gave a residue which was purified by silica gel column
chromatography using a mixture of DCM/Hx (3:1) as eluent to produce
1034 mg (32%) of the title compound as a yellow solid.
[0199] General Synthesis of Carbamate Derivatives:
[0200] To a solution of the carbonic acid 2-(1H-indol-3-yl)-ethyl
ester 4-nitro-phenyl ester, was added a solution of the
corresponding alkylaminotetrahydroacridine in DMF, in presence of
DMAP, and the resulting mixture was stirred for 24 hours at room
temperature. After evaporation of the solvent under reduced
pressure water was added and the mixture extracted with
dichloromethane. Evaporation of the solvent gave a residue which
was purified by silica gel column chromatography as indicated
bellow for each case.
Example 23
##STR00034##
[0201]
[5-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl]-carbamic
acid 2-(1H-indol-3-yl)-ethyl ester
[0202] Reagents:
N1-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-yl)-pentane-1,5-diamine
(500 mg, 1.58 mmol), carbonic acid 2-(1H-indol-3-yl)-ethyl ester
4-nitro-phenyl ester (260 mg, 0.79 mmol), DMAP (1930 mg, 1.58
mmol).
[0203] Purification: silica gel chromatography using DCM/MeOH
(7:0.5) as eluent. Yield: 126 mg (32%).
[0204] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.51 (brs,
1H), 7.86 (m, 2H), 7.57 (m, 1H, J=8.0 Hz), 7.28 (m, 1H, J=8.0 Hz),
7.21-7.23 (m, 1H), 7.14-7.10 (m, 1H), 7.07-7.03 (m, 1H), 6.97-6.82
(m, 1H), 4.77 (brs, 1H), 4.33-4.30 (m, 2H), 4.04 (m, 2H), 3.14-3.13
(m, 2H), 3.05-2.98 (m, 4H), 2.58 (brs, 2H), 1.87-1.83 (m, 4H),
1.62-1.61 (m, 2H), 1.49-1.47 (m, 2H), 1.37-1.35 (m, 2H).
[0205] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 165.7,
159.4, 151.3, 147.7, 136.5, 134.6, 127.7, 127.0, 124.9, 124.6,
122.3, 122.1, 119.4, 118.9, 118.3, 116.3, 115.8, 112.2, 49.5, 40.8,
33.7, 31.5, 30.0, 25.4, 24.7, 24.2, 23.0, 22.7. ESI-MS[M+H].sup.+
505.1
Example 24
##STR00035##
[0206]
[6-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-hexyl]-carbamic
acid 2-(1H-indol-3-yl)-ethyl ester
[0207] Reagents:
N1-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-yl)-hexane-1,6-diamine
(610 mg, 1.84 mmol), carbonic acid 2-(1H-indol-3-yl)-ethyl ester
4-nitro-phenyl ester (300 mg, 0.92 mmol), DMAP (225 mg, 1.84
mmol).
[0208] Purification: DCM/MeOH (7:0.5). Yield: 100 mg (21%).
[0209] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.25 (brs,
1H), 7.90-7.87 (m, 2H), 7.62 (d, 1H, J=8.0 Hz), 7.34 (d, 1H, J=8.0
Hz), 7.30-7.25 (m, 2H), 7.20-7.16 (m, 1H), 7.13-7.08 (m, 1H),
7.03-7.02 (m, 1H), 4.67 (brs, 1H), 4.36-4.33 (m, 2H), 3.46 (brs,
2H), 3.17-3.03 (m, 6H), 2.66 (brs, 2H), 1.92-1.90 (m, 4H),
1.66-1.33 (m, 6H).
[0210] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 159.5,
156.7, 150.7, 148.0, 136.1, 134.0, 127.5, 124.5, 124.2, 122.0,
119.3, 118.7, 118.3, 115.7, 112.1, 111.1, 65.0, 49.4, 40.6, 34.0,
31.6, 30.0, 26.4, 26.3, 25.2, 24.5, 23.0, 22.6. ESI-MS[M+H].sup.+
519.1.
Example 25
##STR00036##
[0211]
[7-(3-Chloro-1,2,3,4-tetrahydro-acridin-9-ylamino)-heptyl]-carbamic
acid 2-(1H-indol-3-yl)-ethyl ester
[0212] Reagents:
N1-(6-Chloro-1,2,3,4-tetrahydro-acridin-9-yl)-heptane-1,7-diamine
(344 mg, 1.0 mmol), carbonic acid 2-(1H-indol-3-yl)-ethyl ester
4-nitro-phenyl ester (166 mg, 0.5 mmol), DMAP (122 mg, 1.0
mmol).
[0213] Purification: chromatography purification using DCM/MeOH
(7:0.5) as eluent. Yield: 70 mg (40%) crystalline solid, colour
amber.
[0214] .sup.1H-NMR (CDCl.sub.3, 400 MHz, .delta. ppm): 8.25 (brs,
1H), 7.90-7.88 (m, 2H), 7.62 (d, 1H, J=8.0 Hz), 7.34 (d, 1H, J=8.0
Hz), 7.30-7.25 (m, 2H), 7.20-7.15 (m, 1H), 7.13-7.08, (m, 1H),
7.03-7.02 (m, 1H), 4.71 (brs, 1H), 4.35-4.32 (m, 2H), 3.5-3.45 (m,
2H), 3.17-3.12 (m, 2H), 3.09-3.06 (m, 2H), 3.03 (brs, 2H), 2.64
(brs, 2H), 1.91-1.88 (m, 4H), 1.64-1.60 (m, 2H), 1.47-1.44 (m, 2H),
1.31-1.25 (m, 6H).
[0215] .sup.13C-NMR (CD.sub.3Cl, 100 MHz, .delta. ppm): 159.6,
156.9, 151.2, 148.2, 136.4, 134.3, 127.7, 127.5, 124.9, 124.5,
122.3, 122.2, 119.0, 118.5, 115.8, 112.3, 111.4, 65.1, 49.8, 41.1,
34.0, 31.9, 30.2, 29.2, 27.0, 26.7, 25.4, 24.7, 23.1, 22.8.
ESI-MS[M+H].sup.+ 533.10.
Example 26
Comparative Example
[0216] The N-[2-3(Indolyl)ethyl]-6-chlorotacrine having a short
linker was synthesized following the reported method (Ming-Kuan, H.
U. and Jiajiu, S. WO 01/17529) and its characterization by .sup.1H
NMR and .sup.13C NMR was in agreement with that in the literature
(same reference).
##STR00037##
Example 27
Biological Evaluation
Acetylcholinesterase (AChE) Inhibition (from Bovine
Erythrocytes)
[0217] AChE inhibitory activity was evaluated at 30.degree. C. by
the colorimetric method reported by Ellman [Ellman, G. L.;
Courtney, K. D.; Andres, B.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88-95]. The assay solution consisted of 0.1 M
phosphate buffer pH 8, 0.3 mM 5,5'-dithiobis (2-nitrobenzoic acid)
(DTNB, Ellman's reagent), 0.02 unit AChE (Sigma Chemical Co. from
bovine erythrocytes), and 0.5 mM acetylthiocholine iodide as the
substrate of the enzymatic reaction. The compounds tested were
added to the assay solution and pre incubated with the enzyme for 5
min at 30.degree. C. After that period, the substrate was added.
The absorbance changes at 405 nm were recorded for 5 min with a
microplate reader Digiscan 340T, the reaction rates were compared,
and the percent inhibition due to the presence of test compounds
was calculated. The reaction rate was calculated with, at least,
triplicate measurements, and the percent inhibition due to the
presence of test compound was calculated relative to the
compound-free control. The compound concentration producing 50% of
AChE inhibition (IC.sub.50) was determined. The results are shown
in table 1.
[0218] Butyrylcholinesterase (BuCHe) Inhibition (from Human
Serum)
[0219] BuChE inhibitory activity was evaluated at 30.degree. C. by
the colorimetric method reported by Ellman. The assay solution
consisted of 0.01 unit BuChE from human serum, 0.1 M sodium
phosphate buffer pH 8, 0.3 mM 5,5'-dithiobis (2-nitrobenzoic acid)
(DTNB, Ellman's reagent), and 0.5 mM butyrylthiocholine iodide as
the substrate of the enzymatic reaction. Enzyme activity was
determined by measuring the absorbance at 405 nm during 5 minutes
with a microplate reader Digiscan 340T. The tested compounds were
preincubated with the enzyme for 10 minutes at 30.degree. C. The
reaction rate was calculated with, at least, triplicate
measurements. The IC.sub.50 is defined as the concentration of each
compound that reduces a 50% the enzymatic activity with respect to
that without inhibitors. The results are shown in table 1.
[0220] Toxicity Measurement
[0221] The cytotoxicity effect of the molecules was tested in the
human neuroblastoma cell line SH-SYSY. These cells were cultured in
96-well plates in DULBECCO'S MOD EAGLE medium, supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin, and grown in
a 5% CO.sub.2 humidified incubator at 37.degree. C.
[0222] The cells were plated at 10.sup.4 cells for each well, at
least, 48 hours before the toxicity measure. Cells were exposed for
24 hours to the compounds at different concentrations (from
10.sup.-5 to 10.sup.-9), quantitative assessment of cell death was
made by measurement of the intracellular enzyme lactate
dehydrogenase (LDH) (cytotoxicity detection kit, Roche). The
quantity of LDH was evaluated in a microplate reader Anthos 2010,
at 492 and 620 nm. Controls were taken as 100% viability. The
results are shown in table 1.
[0223] Propidium Competition
[0224] Propidium exhibits an increase in fluorescence on binding to
AChE peripheral site, making it a useful probe for competitive
ligand binding to the enzyme.
[0225] Fluorescence was measured in a Fluostar optima plate reader
(BMG). Measurements were carried out in 100 .mu.l solution volume,
in 96-well plates. The buffer used was 1 mM Tris/HCl, pH 8.0. 10
.mu.M AchE was incubated, at least 6 hours, with the molecules at
different concentrations. 20 .mu.M propidium iodide was added 10
min before fluorescence measurement. The excitation wavelength was
485 nm, and that of emission, 620 nm. The results are shown in
table 1.
TABLE-US-00001 TABLE 1 IC.sub.50 IC.sub.50 Propidium AChE BuChE
Toxicity competition Compound Structure (nM) (nM) (.mu.M) (.mu.M) 1
##STR00038## 4 100 >100 >100 2 ##STR00039## 70 1 >100 10 3
##STR00040## 0.02 2.9 100 1000 4 ##STR00041## 0.06 0.1 10 10 5
##STR00042## 0.5 5.7 10 10 6 ##STR00043## 4.4 9.6 >100 >100 7
##STR00044## 21.9 54 >100 10 8 ##STR00045## 147 0.03 >100
1000 9 ##STR00046## 2.88 7.57 >100 10 10 ##STR00047## 180 9.5
>10 100 11 ##STR00048## 33 1.7 >10 100 12 ##STR00049## 36 19
>10 100 13 ##STR00050## 46 22.4 >10 10 14 ##STR00051## 0.18
11.7 >10 10 15 ##STR00052## 0.34 3.2 >10 10 16 ##STR00053##
0.48 5.6 >10 100 17 ##STR00054## 18 77 >10 10 18 ##STR00055##
0.63 1.7 >10 100 19 ##STR00056## 0.72 11.7 >10 100 20
##STR00057## 10.9 206 >10 100 21 ##STR00058## 95 79 >10 100
23 ##STR00059## 1.5 13.6 >10 100 24 ##STR00060## 0.7 3.2 >10
100 25 ##STR00061## 3 59 >10 10 Comparative Example ##STR00062##
537
Example 27
Inhibition of .beta.-amyloid aggregation
[0226] The generation of AChE-A.beta. complexes were carried out as
described previously [Alvarez, A et al. J. Neurosci., 1998, 18,
3213-3223; Munoz, F. J.; Inestrosa, N. C. FEBS Lett., 1999, 450,
205-209]. Stock solutions of A.beta..sub.1-40 (rPeptide, Georgia
USA) at 3.5 mM were dissolved in PBS (pH 7.4) after HFIP treatment
to obtain monomeric starting material, according to manufacture's
recommendations. For co-incubation experiments, 0.1 mM of peptide
was mixed with human recombinant acetylcholinesterase (huAchE,
Sigma-Aldrich) in the same buffer at molar ratio A.beta.:huAChE
200:1, and stirred for 48 hours in a microtiter plate at room
temperature. The fibrils obtained were characterized by Congo Red
(CR) binding.
[0227] For the inhibition of .beta.-amyloid aggregation, the
compounds tested were used at the IC.sub.50 defined in the previous
paragraph of the biological evaluation. 50 nM propidium iodide was
used as reference [Inestrosa, N. C et al., J. Neuron, 1996, 16,
881-891].
[0228] To quantify the amount of fibrils aggregated, the binding to
CR was done as described [Klunk, W E.; Pettegrew, J W.; Abraham, D
J. J. Hystochem Cytochem., 1989, 8, 1293-1297]. Briefly, 5.5 nl
aliquot of the aggregation mixture was added to 132 nl of a 25 nM
CR solution (100 mM phosphate buffer pH 7.4, 150 mM NaCl), and
incubated for 30 minutes at room temperature. Absorbance was
measured at 480 and 540 nm and the molarity of aggregates
calculated by CR (M)=(A.sub.540/25295)-(A.sub.480/46306).
[0229] In the conditions above described, the indol compound
derivative's 3 and 8 showed a 15% and 17% reduction respectively,
in the .beta.-amyloid-huAChE aggregation complex. The peripheral
inhibitor propidium iodide used as reference did show a 10%
reduction.
* * * * *