U.S. patent application number 17/020318 was filed with the patent office on 2020-12-31 for novel 1,4-bis(3-aminopropyl)piperazine derivative and its use.
The applicant listed for this patent is CENTRE HOSPITALIER REGIONAL ET UNIVERSITAIRE DE LILLE (CHRU), INSERM (Institute National de la Sante et de la Recherche Medicale), UNIVERSITE DE LILLE 2 DROIT ET SANTE. Invention is credited to Luc BUEE, Marion GAY, Nicolas LE FUR, Patricia MELNYK, Nicolas SERGEANT.
Application Number | 20200407325 17/020318 |
Document ID | / |
Family ID | 1000005086921 |
Filed Date | 2020-12-31 |
United States Patent
Application |
20200407325 |
Kind Code |
A1 |
SERGEANT; Nicolas ; et
al. |
December 31, 2020 |
NOVEL 1,4-BIS(3-AMINOPROPYL)PIPERAZINE DERIVATIVE AND ITS USE
Abstract
The present invention relates to the compound having Formula (I)
or pharmaceutically acceptable salts or solvates thereof, and its
use in treating and/or preventing a Tauopathy. ##STR00001##
Inventors: |
SERGEANT; Nicolas; (Lille
Cedex, FR) ; BUEE; Luc; (Lille Cedex, FR) ;
MELNYK; Patricia; (Lille, FR) ; GAY; Marion;
(Lille, FR) ; LE FUR; Nicolas; (Roubaix,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (Institute National de la Sante et de la Recherche
Medicale)
CENTRE HOSPITALIER REGIONAL ET UNIVERSITAIRE DE LILLE (CHRU)
UNIVERSITE DE LILLE 2 DROIT ET SANTE |
Paris Cedex 16
Lille
Lille |
|
FR
FR
FR |
|
|
Family ID: |
1000005086921 |
Appl. No.: |
17/020318 |
Filed: |
September 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16009979 |
Jun 15, 2018 |
10807958 |
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17020318 |
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15558921 |
Sep 15, 2017 |
10017476 |
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PCT/EP2016/055633 |
Mar 16, 2016 |
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16009979 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 219/12
20130101 |
International
Class: |
C07D 219/12 20060101
C07D219/12 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2015 |
EP |
15305384.8 |
Claims
1-7. (canceled)
8. A method of treating and/or preventing a disease selected from
Amyotrophic lateral sclerosis and parkinsonism-dementia complex,
Chronic traumatic encephalopathy, cerebral amyloid angiopathy,
Dementia pugilistica, Familial British Dementia, Familial Danish
Dementia, Gerstmann-Straussler-Scheinker disease, Guadeloupean
parkinsonism, Hallervorden-Spatz disease, inclusion body myositis,
multiple system atrophy, Steinert Myotonic dystrophy, Myotonic
dystrophy type II, Neurodegeneration with brain iron accumulation,
Niemann-Pick disease, Non-Guamanian motor neuron disease with
neurofibrillary tangles, Postencephalitic parkinsonism, Subacute
sclerosing panencephalitis, multi-infarct dementia, chronic
traumatic encephalopathy (CTE), traumatic brain injury (TBI),
comprising administering to a patient in need thereof a
pharmaceutically effective amount of a compound of Formula I
##STR00005## or a pharmaceutically acceptable salt thereof.
9. A method according to claim 8, wherein the disease is Steinert
Myotonic dystrophy and Myotonic dystrophy type II.
10. A method according to claim 8, wherein the disease is Steinert
Myotonic dystrophy.
11. A method according to claim 8, wherein the compound of Formula
I is administered as a composition of the compound or a
pharmaceutically acceptable salt thereof and at least one
pharmaceutically acceptable carrier, diluent, excipient and/or
adjuvant.
12. A method for decreasing pathological Tau protein
phosphorylation in a patient suffering from Amyotrophic lateral
sclerosis and parkinsonism-dementia complex, Chronic traumatic
encephalopathy, cerebral amyloid angiopathy, Dementia pugilistica,
Familial British Dementia, Familial Danish Dementia,
Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism,
Hallervorden-Spatz disease, inclusion body myositis, multiple
system atrophy, Steinert Myotonic dystrophy, Myotonic dystrophy
type II, Neurodegeneration with brain iron accumulation,
Niemann-Pick disease, Non-Guamanian motor neuron disease with
neurofibrillary tangles, Postencephalitic parkinsonism, Subacute
sclerosing panencephalitis, multi-infarct dementia, chronic
traumatic encephalopathy (CTE) or traumatic brain injury (TBI), in
need thereof, comprising administering to said patient an effective
amount of a compound of Formula I ##STR00006## or a
pharmaceutically acceptable salt thereof.
13. A method according to claim 12, wherein the disease is Steinert
Myotonic dystrophy and Myotonic dystrophy type II.
14. A method according to claim 12, wherein the disease is Steinert
Myotonic dystrophy.
Description
[0001] The present invention relates to a novel compound,
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl
Jpiperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine, and
pharmaceutically acceptable salts or solvates thereof, their
preparation, pharmaceutical compositions containing them and use of
the same in the treatment and/or prevention of Tauopathies.
BACKGROUND OF THE INVENTION
[0002]
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]--
1,2,3,4-tetrahydroacridin-9-amine which has the structure of
Formula I:
##STR00002##
[0003] belongs to a family of 1,4-bis(3-aminopropyl)piperazine
derivatives previously disclosed in WO 2006/051489 and which are
useful for the treatment and/or prevention of neurodegenerative
diseases with APP dysfunction.
[0004] A majority of neurodegenerative diseases have common
cellular and molecular mechanisms including protein aggregation and
inclusion body formation.
[0005] The definition of the different types of lesions is made
regarding the molecular compound which constitutes them.
Neurofibrillary degeneration (NFD) is one of the most widely
observed brain lesions which results from a cascade of molecular
events combining abnormal modifications of the
microtubule-associated Tau protein isoforms, their progressive
aggregation and accumulation into fibrillar material inside
neurons.
[0006] Tau protein is mainly expressed in neurons of the central
nervous system where it interacts with tubulin to stabilize
microtubules and promotes tubulin assembly into microtubules. This
stabilizing property is controlled by isoforms and phosphorylation.
The Tau isoforms are the product of alternative splicing from
transcript of a single gene MAPT (microtubule-associated protein
Tau) located on chromosome 17.
[0007] Six Tau isoforms exist in human brain tissue, and they are
distinguished by their number of binding domains (three or four
binding domains). The isoforms are a result of alternative splicing
in exons 2, 3, and 10 of the Tau gene. The binding domains are
located in the half carboxy-terminal region of the protein and are
positively-charged (allowing it to bind to the negatively-charged
microtubule). The isoforms with four binding domains containing
exon 10 have a better affinity with microtubules, thus allowing a
better stabilization of these microtubules, than those with three
binding domains.
[0008] Tau is a phosphoprotein, the phosphorylation sites of which
are essentially distributed on both sides of the microtubule
binding domain. The phosphorylation of Tau results in disruption of
microtubule organization.
[0009] In several neurodegenerative diseases termed "Tauopathies",
abnormally and hyperphosphorylated Tau protein isoforms aggregate
into fibrillar structures within neurons to form the so-called
neurofibrillary tangles (NFTs). These Tauopathies mainly differ
from each other in both Tau isoform phosphorylation and content.
Hyperphosphorylation, abnormal phosphorylation and aggregation are
constantly present in neurofibrillary degeneration.
[0010] The quality, intensity and/or spatial location of
neurofibrillary degeneration is extremely correlated with the
impairment of cognitive functions. These impairments are
particularly incapacitating for the patient who is not autonomous
any more.
[0011] As a consequence, there is a need for treatments able to
avoid the pathological aggregation of Tau protein in the human
brain and the consequences of this aggregation.
[0012] Some treatments have focused on kinase inhibitors,
phosphatase activators or anti-Tau antibodies (see for example WO
2014028777). However, none of these treatments have successfully
led to a commercial treatment of Tauopathies up to the Applicant's
knowledge, especially in the case of rare neurodegenerative
diseases such as Pick's disease, Progressive supranuclear palsy
(PSP), Corticobasal degeneration or Frontotemporal dementia with
Parkinsonism linked to chromosome 17 (FTDP-17) mutations of
MAPT.
[0013] There is thus still a need in the art for a treatment for
Tauopathies able to successfully reestablish a normal Tau
metabolism.
SUMMARY OF THE INVENTION
[0014] The present invention is based on the unexpected findings
that the compound of Formula I,
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,-
4-tetrahydroacridin-9-amine, is useful in rectifying the metabolism
of the Tau protein in particular by altering pathological Tau
protein phosphorylation and by increasing Tau protein
proteolysis.
[0015] The invention is thus directed to the compound of Formula I
or pharmaceutically acceptable salts or solvates thereof, as well
as compositions comprising such compounds and their use in treating
and/or preventing a Tauopathy.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The invention concerns the compound having Formula I
##STR00003##
[0017] or pharmaceutically acceptable salts or solvates
thereof.
[0018] The compound of Formula I as well as its pharmaceutically
acceptable salts or solvates may be isotopically labelled,
especially deuterated. The deuterated form is useful for the dosage
of the compound in a biological environment.
[0019] The compound of formula I or pharmaceutically acceptable
salts or solvates thereof are useful for rectifying the metabolism
of the Tau protein on the following essential points:
[0020] 1) decreasing the phosphorylation of Tau protein;
[0021] 2) increasing the catabolite products of Tau protein.
[0022] By "Tauopathy" is meant a disorder characterized by an
abnormal metabolism of Tau protein, in particular by an abnormal
phosphorylation and/or hyperphosphorylation of Tau protein and/or
the presence of elevated (higher than a normal control level, e.g.
higher than a normal, control level for an individual or population
of individuals of the same age group) levels of Tau or Tau isoform
or Tau polypeptides and/or pathological forms of Tau in a cell, a
tissue, or a fluid, preferably in brain tissue and/or cerebrospinal
fluid. More specifically, a Tauopathy refers to a disorder wherein
intracellular aggregates of abnormally modified Tau proteins are
observed such as neurofibrillary tangles (NFT), Pick bodies,
astrocytic tufted and/or muscle inclusion bodies, preferably at
least neurofibrillary tangles (NFT).
[0023] Tauopathies include, but are not limited to, Alzheimer's
disease, Amyotrophic lateral sclerosis and parkinsonism-dementia
complex of Guam, Argyrophilic grain disease, Chronic traumatic
encephalopathy, cerebral amyloid angiopathy, Corticobasal
degeneration, Creutzfeldt-Jakob disease, Dementia pugilistica,
Diffuse neurofibrillary tangles with calcification, Down's
syndrome, Familial British Dementia, Familial Danish Dementia,
Frontotemporal dementia (FTD), Frontotemporal dementia with
Parkinsonism linked to chromosome 17 (FTDP-17),
Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism,
Hallervorden-Spatz disease, inclusion body myositis, multiple
system atrophy, Steinert Myotonic dystrophy, Myotonic dystrophy
type II, Huntington disease Neurodegeneration with brain iron
accumulation, Niemann-Pick disease of type C, Non-Guamanian motor
neuron disease with neurofibrillary tangles, Paget's disease,
Pick's disease, Postencephalitic parkinsonism, Prion protein
cerebral amyloid angiopathy, Progressive subcortical gliosis,
Progressive supranuclear palsy (PSP), SLC9A6-related mental
retardation, Subacute sclerosing panencephalitis, Tangle-only
dementia, multi-infarct dementia, ischemic stroke, chronic
traumatic encephalopathy (CTE), traumatic brain injury (TBI),
stroke and White matter Tauopathy with globular glial
inclusions.
[0024] Due to its ability to rectify the metabolism of Tau protein
as described above, the compound of Formula I or a pharmaceutically
salt or solvate thereof is useful as a medicament, in particular
for use in treating or preventing a Tauopathy.
[0025] Hence, the invention also concerns the compound of Formula I
or a pharmaceutically acceptable salt or solvate thereof as defined
herein for use in treating and/or preventing a Tauopathy selected
from Alzheimer's disease, Amyotrophic lateral sclerosis and
parkinsonism-dementia complex, Argyrophilic grain disease, Chronic
traumatic encephalopathy, cerebral amyloid angiopathy, Corticobasal
degeneration, Creutzfeldt-Jakob disease, Dementia pugilistica,
Diffuse neurofibrillary tangles with calcification, Down's
syndrome, Familial British Dementia, Familial Danish Dementia,
Frontotemporal dementia (FTD), Frontotemporal dementia with
Parkinsonism linked to chromosome 17 (FTDP-17), Frontotemporal
lobar degeneration, Gerstmann-Straussler-Scheinker disease,
Guadeloupean parkinsonism, Hallervorden-Spatz disease, inclusion
body myositis, multiple system atrophy, Myotonic dystrophy,
Neurodegeneration with brain iron accumulation, Niemann-Pick
disease, Non-Guamanian motor neuron disease with neurofibrillary
tangles, Pick's disease, Postencephalitic parkinsonism, Prion
protein cerebral amyloid angiopathy, Progressive subcortical
gliosis, Progressive supranuclear palsy (PSP), SLC9A6-related
mental retardation, Subacute sclerosing panencephalitis,
Tangle-only dementia, multi-infarct dementia, ischemic stroke,
chronic traumatic encephalopathy (CTE), traumatic brain injury
(TBI), stroke and White matter Tauopathy with globular glial
inclusions. More preferably, the Tauopathy is selected from
Alzheimer's disease, Frontotemporal dementia with Parkinsonism
linked to chromosome 17 (FTDP-17), Progressive supranuclear palsy
(PSP), Corticobasal degeneration, Pick's disease and Frontotemporal
Dementia (FTD). More preferably, the Tauopathy is selected from
Frontotemporal dementia with Parkinsonism linked to chromosome 17
(FTDP-17), Progressive supranuclear palsy (PSP), Corticobasal
degeneration, Pick's disease and Frontotemporal Dementia (FTD).
[0026] In other terms, the invention also provides for a method of
treating and/or preventing a Tauopathy, in particular those cited
above as well as embodiments thereof, comprising administering to a
patient in need thereof a pharmaceutically effective amount of the
compound of Formula I or a pharmaceutically acceptable salt or
solvate thereof as described herein.
[0027] In one particular embodiment, the invention also concerns a
compound of Formula I or a pharmaceutically acceptable salt or
solvate thereof as defined herein for use in delaying in a patient
the onset of a Tauopathy as described above.
[0028] In other terms, the invention provides a method for delaying
in a patient the onset of a Tauopathy, comprising administering to
a patient in need thereof a pharmaceutically effective amount of
the compound of Formula I or a pharmaceutically acceptable salt or
solvate thereof.
[0029] According to a further feature of the present invention
there is provided a method for modulating pathological Tau
metabolism, in a patient, preferably a warm blooded animal, and
even more preferably a human, in need of such treatment, which
comprises administering to said patient an effective amount of the
compound of Formula I, or a pharmaceutically acceptable salt or
solvate thereof.
[0030] More preferably, there is provided a method for altering
pathological Tau protein phosphorylation in a patient, preferably a
warm blooded animal, and even more preferably a human, in need of
such treatment, which comprises administering to said patient an
effective amount of the compound of Formula I or a pharmaceutically
acceptable salt or solvate thereof.
[0031] Generally, for pharmaceutical use, the compound of Formula I
or a pharmaceutically acceptable salt or solvate thereof may be
formulated as a pharmaceutical composition comprising at least the
compound of the invention or a pharmaceutically acceptable salt or
solvate thereof and at least one pharmaceutically acceptable
carrier, diluent, excipient and/or adjuvant, and optionally one or
more additional therapeutic agents and/or active ingredients.
[0032] By means of non-limiting examples, the pharmaceutical
composition may be in a dosage form suitable for oral
administration, for parenteral administration (such as by
intravenous, intramuscular or subcutaneous injection or intravenous
infusion), for topical administration (including ocular), for
administration by inhalation, by a skin patch, by an implant, by a
suppository, etc. Such suitable administration forms--which may be
solid, semi-solid or liquid, depending on the manner of
administration--as well as methods and carriers, diluents and
excipients for use in the preparation thereof, will be clear to the
skilled person; reference is made to the latest edition of
Remington's Pharmaceutical Sciences. The pharmaceutical
compositions may be formulated in solid form and re-dissolved or
suspended prior to use.
[0033] Some preferred, but non-limiting examples of dosage forms
include tablets, pills, powders, lozenges, sachets, cachets,
elixirs, suspensions, emulsions, solutions, syrups, aerosols,
ointments, cremes, lotions, soft and hard gelatin capsules,
suppositories, drops, sterile injectable solutions and sterile
packaged powders (which are usually reconstituted prior to use) for
administration as a bolus and/or for continuous administration,
which may be formulated with carriers, excipients, and diluents
that are suitable per se for such formulations, such as lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
polyethylene glycol, cellulose, (sterile) water, methylcellulose,
methyl- and propylhydroxybenzoates, talc, magnesium stearate,
edible oils, vegetable oils and mineral oils or suitable mixtures
thereof. The pharmaceutical compositions can optionally contain
other substances that are commonly used in pharmaceutical
formulations, such as lubricating agents, wetting agents,
emulsifying and suspending agents, dispersing agents,
disintegrating agents, stabilizing agents, isotonic agents, bulking
agents, fillers, preserving agents, sweetening agents, flavoring
agents, perfuming agents, coloring agents, antibacterial agents
and/or antifungal agents such as parabens, chlorobutanol, phenol,
sorbic acid, dispensing agents, flow regulators, release agents,
etc. The compositions may also be formulated so as to provide
rapid, sustained or delayed release of the active compound(s)
contained therein.
[0034] The pharmaceutical compositions of the invention are
preferably in a unit dosage form, and may be suitably packaged, for
example in a box, blister, vial, bottle, sachet, ampoule or in any
other suitable single-dose or multi-dose holder or container (which
may be properly labeled); optionally with one or more leaflets
containing product information and/or instructions for use.
Generally, such unit dosages will contain between 0.05 and 1000 mg,
and usually between 1 and 500 mg, of the at least one compound of
the invention, e.g. about 10, 25, 50, 100, 200, 300 or 400 mg per
unit dosage.
[0035] Usually, depending on the condition to be prevented or
treated and the route of administration, the active compound of the
invention will usually be administered between 0.01 to 100 mg per
kilogram, more often between 0.1 and 50 mg, such as between 1 and
25 mg, for example about 0.5, 1, 5, 10, 15, 20 or 25 mg, per
kilogram body weight of the patient per day, which may be
administered as a single daily dose, divided over one or more daily
doses, or essentially continuously, e.g. using a drip infusion.
[0036] The compound of Formula I of the invention and its
pharmaceutically acceptable salts and solvates can be prepared by
different ways with reactions known by the person skilled in the
art. Reaction schemes as described in the example section
illustrate by way of example different possible approaches.
[0037] The salts and solvates thereof can be prepared according to
techniques known in the art such as those involving precipitation,
crystallization, recrystallization, lyophilisation, phase transfer
or ion exchange resins.
[0038] Generally, pharmaceutically acceptable salts of compounds of
Formula I may for example be prepared as follows:
[0039] (i) by reacting the compound of Formula I with the desired
acid;
[0040] (ii) by converting one salt of the compound of Formula I to
another by reaction with an appropriate acid or by means of a
suitable ion exchange column.
[0041] All these reactions are typically carried out in solution.
The salt, may precipitate from solution and be collected by
filtration or may be recovered by evaporation of the solvent. The
degree of ionization in the salt may vary from completely ionized
to almost non-ionized.
Definitions
[0042] The definitions and explanations below are for the terms as
used throughout the entire application, including both the
specification and the claims.
[0043] Unless otherwise stated any reference to compounds of the
invention herein, means the compound of Formula I as such as well
as their pharmaceutically acceptable salts and/or solvates.
[0044] When describing the compounds of the invention, the terms
used are to be construed in accordance with the following
definitions, unless indicated otherwise.
[0045] The terms "salt" refers to any acid addition salt obtained
from the compound of the invention, said salt having an essentially
similar biological activity compared to the biological activity of
the compound of the invention. Suitable pharmaceutically acceptable
acid addition salts are formed from acids which form non-toxic
salts. Non-limiting examples include the acetate, adipate,
aspartate, benzoate, besylate, bicarbonate, carbonate, bisulphate,
sulphate, borate, camsylate, citrate, cyclamate, edisylate,
esylate, formate, fumarate, gluceptate, gluconate, glucuronate,
hexafluorophosphate, hibenzate, hydrochloride/chloride,
hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate,
malate, maleate, malonate, mesylate, methylsulphate, naphthylate,
2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate,
pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,
pyroglutamate, saccharate, stearate, succinate, tannate, tartrate,
tosylate, trifluoroacetate and xinofoate salts. Hemisalts of acids
may also be formed, for example, hemisulphate salts. Preferably,
pharmaceutically acceptable salts include the hydrochloride,
hydrobromide, bisulphate, sulphate, nitrate, citrate, acetate, and
phosphate salts. The hydrochloride is particularly preferred.
[0046] The term "solvate" is used herein to describe a molecular
complex comprising the compound of the invention and one or more
pharmaceutically acceptable solvent molecules, for example, ethanol
or water. The term "hydrate" is employed when said solvent is
water.
[0047] All references to compounds of Formula I include references
to pharmaceutically acceptable salts and solvates thereof.
[0048] The compounds of the invention include compounds of Formula
I as hereinbefore defined, including all polymorphs and crystal
habits thereof, prodrugs and isomers thereof (including optical,
geometric and tautomeric isomers) and isotopically labeled
compounds of Formula I.
[0049] The term "patient" refers to a warm-blooded animal, more
preferably a human, who/which is awaiting the receipt of, or is
receiving medical care or is/will be the object of a medical
procedure.
[0050] The term "human" refers to a subject of both genders and at
any stage of development (i.e. neonate, infant, juvenile,
adolescent, adult). In one embodiment, the human is an adolescent
or adult, preferably an adult.
[0051] The terms "treat", "treating" and "treatment, as used
herein, are meant to include alleviating, attenuating or abrogating
a condition or disease and/or its attendant symptoms.
[0052] The terms "prevent", "preventing" and "prevention", as used
herein, refer to a method of delaying or precluding the onset of a
condition or disease and/or its attendant symptoms, barring a
patient from acquiring a condition or disease, or reducing a
patient's risk of acquiring a condition or disease.
[0053] The term "therapeutically effective amount" (or more simply
an "effective amount") as used herein means the amount of active
agent or active ingredient (e.g.
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl
Jpiperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine) that is
sufficient to achieve the desired therapeutic or prophylactic
effect in the patient to which/whom it is administered.
[0054] The term "administration", or a variant thereof
(e.g./`administering"), means providing the active agent or active
ingredient (e.g.
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,-
4-tetrahydroacridin-9-amine), alone or as part of a
pharmaceutically acceptable composition, to the patient in
whom/which the condition, symptom, or disease is to be treated or
prevented.
[0055] By "pharmaceutically acceptable" is meant that the
ingredients of a pharmaceutical composition are compatible with
each other and not deleterious to the patient thereof.
[0056] The term "pharmaceutical vehicle" as used herein means a
carrier or inert medium used as solvent or diluent in which the
pharmaceutically active agent is formulated and/or administered.
Non-limiting examples of pharmaceutical vehicles include creams,
gels, lotions, solutions, and liposomes.
[0057] The present invention will be better understood with
reference to the following examples. These examples are intended to
representative of specific embodiments of the invention, and are
not intended as limiting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1: Determination by ID gel electrophoresis of the
expression of the Tau protein by using Pan-Tau antibodies
(Tau-Nter, Tau-Cter) and of the phosphorylation of the Tau protein
by using phospho-dependent Tau antibodies (S199P, S396) and
non-phosphorylated epitote comprised between 198-205 (Tau-1), in
non-induced conditions (NI), induced but non-treated conditions
(NT) and with administration of Compound I.HQ.
[0059] FIG. 2: Determination by 2D gel electrophoresis of the
expression of the Tau protein by using Pan-Tau antibodies
(Tau-Nter, Tau-Cter) and of the phosphorylation of the Tau protein
by using phospho-dependent Tau antibody (S396), with an induced but
non-treated condition (Ctrl) and with administration of Compound
I.HCl.
[0060] FIG. 3: Increase Tau C-terminal catabolites and reduce
phosphorylation at serine 199. The 2D pattern of Tau proteins from
the brain of WT animals treated with 0.5 mg/Kg or 1 mg/Kg was
revealed a pan-Tau antibody (Tau-Cter) and a phospho-dependent
antibody against the phosphorylated serine 199.
[0061] FIG. 4: Morris water maze evaluation of the spatial memory
of Wild-type C57BL/6J animal (WT) and Thy-Tau22 mice, treated or
not with 0.5 mg/Kg of Compound I-HQ.
[0062] A: Representation of the distance to reach the platform
(path length) in function of time (days).
[0063] B: Representation of the percent of time spent in the target
quadrant ("T") (where the platform is positioned during the
learning phase) versus the others ("O").
[0064] FIG. 5: Immunohistochemical measurement of tau pathology in
Thy-Tau22 mice untreated (0 mg/kg) or treated with Compound I-HQ
(0.5 mg/kg Compound I-HQ).
[0065] FIG. 6: Biochemical analysis of Tau expression, catabolites
and phosphorylation by ID SDS-PAGE and Western-blot.
CHEMISTRY EXAMPLES
[0066] The following abbreviations are used throughout the present
application: .degree. C.: Celsius degrees, BINAP:
2,2'-bis(diphenylphosphino)-1,1'-binaphtyl, dba:
dibenzylideneacetone, DCE: dichloroethane, DCM: dichloromethane,
.delta.: NMR chemical shifts expressed in ppm, eq: equivalent(s),
Et: ethyl, g: gram(s), HPLC: high performance liquid
chromatography, L: liter(s), LCMS: HPLC coupled to a mass
spectrometer, Me: methyl, mg: milligram(s), min: minute(s), mL:
milliliter(s), mol: mole(s), mmol: millimole(s), .mu..eta.o:
micromole(s), MS: Mass Spectrometry, NMR: nuclear magnetic
resonance, ppm: party per million, RT: room temperature (ca
15-25.degree. C.), Xantphos:
4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene, rt: retention
time.
[0067] All reported temperatures are expressed in degrees Celsius
(.degree. C.); all reactions were carried out at room temperature
(RT) unless otherwise stated.
[0068] Material and Methods
[0069] Chemicals and solvents were obtained from commercial
sources, and used without further purification unless otherwise
precised. Reactions were monitored by TLC performed on
Macherey-Nagel Alugram.RTM. Sil 60/UV.sub.254 sheets (thickness 0.2
mm). Purification of products was carried out by either column
chromatography using Macherey-Nagel silica gel (230-400 mesh).
[0070] NMR spectra were recorded on a Bruker DRX 300 spectrometer
(operating at 300 MHz for .sup.1H and 75 MHz for .sup.13C).
Chemical shifts are expressed in ppm relative to either
tetramethylsilane (TMS) or to residual proton signal in deuterated
solvents. Chemical shifts are reported as position (6 in ppm),
multiplicity (s=singlet, d=doublet, t=triplet, sept=septuplet,
br=broad and m=massif), coupling constant (J in Hz), relative
integral and assignment. The attributions of protons and carbons
were achieved by analysis of ID and 2D experiments (.sup.1H,
.sup.13C).
[0071] Mass spectra were recorded on a Varian triple quadrupole
1200 W mass spectrometer equipped with a non-polar C18 TSK-gel
Super ODS (4.6.times.50 mm) column, using electro spray ionization
and a UV detector (diode array).
[0072] The compound of formula I can be prepared according to the
three methods set out in Scheme 1 below.
##STR00004##
Example 1: synthesis of tert-Butyl
N-[3-(4-{3-[(1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}piperazin-1-yl)p-
ropyl] carbamate (1.2)
[0073] To an oven-dried flask and under nitrogen atmosphere, were
placed 9-chloro-1,2,3,4-tetrahydroacridine (0.50 g, 2.3 mmol),
Cs.sub.2C0 3 (1.05 g, 3.22 mmol) in dioxane (7.45 mL). The mixture
was deoxygenated by passing a stream of nitrogen through it.
Xantphos (0.20 g, 0.35 mmol) and Pd.sub.2(dba).sub.3 (0.11 g, 0.12
mmol) were added. Then tert-butyl
N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}carbamate 1.1 prepared
according to J. Med. Chem. 2003, 46, 542-557 (0.83 mg, 2.76 mmol)
dissolved in dioxane (7.45 mL) was added to the mixture. The
reaction mixture was stirred for 24 hours. The solution was
filtered through a celite pad and evaporated. The residue was
purified by flash chromatography (eluent DCM/MeOH 0 to 10% then
DCM/MeOH sat NH3 0 to 4%. The title compound (1.00 g, 90% yield)
was obtained as yellow oil.
[0074] H NMR (300 MHz), .delta. (ppm, CDCl.sub.3): 7.95 (dd,
.sup.3J=1.1 Hz, .sup.3J=8.5 Hz, 1H); 7.82 (dd, .sup.3J=0.9 Hz,
.sup.3J=8.6 Hz, 1H); 7.45 (ddd, .sup.4J=1.2 Hz, .sup.3J=6.8 Hz,
.sup.3J=8.2 Hz, 1H); 7.24 (ddd, .sup.4J=1.2 Hz, .sup.3J=6.8 Hz,
.sup.3J=8.3 Hz, 1H); 5.53 (br t, 3=5.2 Hz, 1H); 5.06 (br t, 31=5.4
Hz, 1H); 3.48 (m, 2H); 3.12 (m, 2H); 2.98 (t, .sup.3J=6.0 Hz, 2H);
2.66 (t, .sup.3J=6.0 Hz, 2H); 2.55-2.32 (M, 12H); 1.85-1.72 (M,
6H); 1.59 (m, 2H), 1.35 (s, 9H).
[0075] .sup.13C NMR (75 MHz), .delta. (ppm, CDCl.sub.3): 158.4;
156.0; 151.0; 147.5; 128.7; 128.0; 123.3; 123.0; 120.3; 115.9;
78.7; 57.0; 53.2; 48.9; 39.8; 34.0; 28.4; 27.2; 26.4; 25.7;
22.9.
[0076] LC-MS (ESI) mlz Calculated: 482.3, Found: 482.4 [M+H].sup.+,
382.2 [M+H-Boc].sup.+; rt: 1.8 min
Example 2:
N-{3-[4-(3-Aminopropyl)piperazin-1-yl]propyl}-1,2,3,4-tetrahydr-
oacridin-9-amine (1.3)
[0077] tert-Butyl
N-[3-(4-{3-[(1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}piperazin-1-yl)p-
ropyl] carbamate 1.2 (90 mg, 0.19 mmol) was dissolved in dioxane
saturated with HCl (5 mL). The reaction mixture was stirred for 15
hours. The solvent was evaporated. The residue was dissolved in
MeOH(NH.sub.3) and stirred for 2 hours. The solvent was evaporated.
The residue was dissolved in 15 mL of DCM and washed with saturated
NaHCO.sub.3 solution (3.times.10 mL). The organic layer was dried
over MgSO.sub.4, filtered and evaporated. The residue was purified
by column chromatography (eluent DCM/MeOH(NH.sub.3) 9.4:0.6 (v/v)).
The title compound (70 mg, 98% yield) was obtained as yellow
oil.
[0078] H NMR (300 MHz), .delta. (ppm, CDCl.sub.3): 8.01 (dd,
.sup.4J=0.9 Hz, .sup.3J=8.5 Hz, 1H); 7.87 (dd, .sup.4J=0.9 Hz,
.sup.3J=8.5 Hz, 1H); 7.51 (ddd, .sup.4J=1.4 Hz, .sup.3J=6.8 Hz,
.sup.3J=8.3 Hz, 1H); 7.30 (ddd, V=1.3 Hz, .sup.3J=6.8 Hz,
.sup.3J=8.3 Hz, 1H); 5.13 (br s, 1H); 3.55 (t, 3=6.0 Hz, 2H); 3.04
(t, .sup.3J=6.4 Hz, 2H); 2.71 (M, 4H); 2.65-2.37 (M, 12H);
1.91-1.79 (M, 8H); 1.64 (m, 2H).
[0079] .sup.13C NMR (75 MHz), .delta. (ppm, CDCl.sub.3): 158.4;
151.1; 147.5; 128.7; 128.1; 123.4; 123.0; 120.3; 115.9; 57.0; 53.4;
49.0; 40.8; 34.0; 30.5; 27.2; 25.7; 22.9.
[0080] LC-MS (ESI) mlz Calculated: 382.3, Found: 382.2 [M+H].sup.+;
rt: 1.3 min;
Example 3:
N-[3-(4-{3-[(2-Methylpropyl)amino]propyl}piperazin-1-yl)propyl]-
-1,2,3,4-tetrahydroacridin-9-amine (1.4)
[0081] To a stirred solution of
N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}-1,2,3,4-tetrahydroacridin-9-
-amine 1.3 (100 mg, 0.26 mmol) in toluene (5 mL) was added
isobutyraldehyde (0.04 mL, 0.4 mmol). The reaction mixture was
refluxed with a Dean-Stark apparatus for 1 hour (until no more
drops of water appeared). The toluene was evaporated and the
residue was dissolved in DCE (5 mL). NaBH 4 (15 mg, 0.4 mmol) was
added and the mixture was stirred for 15 hours. 10 mL of saturated
NaHCO 3 solution was added. The mixture was stirred for 1 hour. 5
mL of DCM was added and the layers were separated. The organic
layer was washed with saturated NaHCO 3 solution (2.times.10 mL).
The organic layer was dried over MgS0.sub.4, filtered and
evaporated. The compound was used without purification for the next
step.
[0082] H NMR (300 MHz), .delta. (ppm, CDCl.sub.3): 8.03 (dd,
.sup.4J=1.0 Hz, .sup.3J=8.5 Hz, 1H); 7.90 (dd, .sup.4J=0.9 Hz,
.sup.3J=8.5 Hz, 1H); 7.54 (ddd, .sup.4J=1.3 Hz, .sup.3J=6.8 Hz,
.sup.3J=8.3 Hz, 1H); 7.33 (ddd, .sup.4J=1.3 Hz, .sup.3J=6.8 Hz,
.sup.3J=8.3 Hz, 1H); 5.14 (br s, 1H); 3.57 (m, 2H); 3.05 (t,
.sup.3J=6.2 Hz, 2H); 2.74 (t, .sup.3J=5.5 Hz, 2H); 2.65 (t,
.sup.3J=6.9 Hz, 2H); 2.56-2.40 (M, 14H); 1.94-1.66 (M, 11H); 0.92
(d, .sup.3J=6.6 Hz, 6H).
[0083] .sup.13C NMR (75 MHz), .delta. (ppm, CDCl.sub.3): 158.5;
151.1; 147.6; 128.8; 128.1; 123.4; 123.0; 120.3; 116.0; 58.2; 57.4;
53.5; 49.0; 34.1; 28.5; 27.2; 25.7; 23.0; 20.7.
[0084] LC-MS (ESI) mlz Calculated: 438.3, Found: 438.3 [M+H].sup.+;
rt: 1.5 min.
Example 4: Isobutyraldehyde-</7:
2-(.sup.2H.sub.3)methyl(2,3,3,3-.sup.2H.sub.4)propanal
N-Methoxy-2-(.sup.2Hg)methyl-N-methyl(.sup.2H.sub.4)propanamide
[0085] To a stirred solution of
2-(2H.sub.3)methyl(.sup.2H.sub.4)propanoic acid (93 .mu.L, 1 mmol)
in DCM (3.8 mL) was added N-methylmorpholine (0.715 mL, 6.5 mmol),
EDC 1HC1 (249 mg, 1.3 mmol), HOBt (175 mg, 1.3 mmol) and
N,O-dimethylhydroxylamine hydrochloride (204 mg, 2.1 mmol). The
mixture was stirred for 15 hours. 5 mL of DCM were added to the
reaction mixture and the organic layer was washed with saturated
NaHCO 3 solution (2.times.5 mL), HCl 1M solution (2.times.5 mL) and
brine (1.times.5 mL).
[0086] The organic layer was used without evaporation or
purification for the next step.
[0087] H NMR (300 MHz), .delta. (ppm, CDCl.sub.3): 3.70 (s, 3H,
H.sub.b); 3.19 (s, 3H, H.sub.a).
[0088] .sup.13C NMR (75 MHz), .delta. (ppm, CDCl.sub.3): 178.3;
61.4; 32.3; 29.1; 18.3.
[0089] LC-MS (ESI) mlz Calculated: 139.2, Found: 139.8 [M+H]+
Isobutyraldehvde-<j7:
2-(.sup.2H.sub.3)methyl(2,3,3,3-.sup.2H.sub.4)propanal
[0090] To a solution of
N-Methoxy-2-(.sup.2H.sub.3)methyl-N-methyl(.sup.2H.sub.4)propanamide
in DCM (5 mL, 1 mmol) was added LiAlH.sub.4 (0.9 mL, 0.9 mmol). The
mixture was stirred for 1 hour. Saturated KHS0.sub.4 solution (4.2
mL) was added dropwise. 5 mL of DCM were added and the organic
layer was washed with saturated NaHCO 3 solution (2.times.5 mL),
HCl 1M solution (2.times.5 mL) and brine (1.times.5 mL). The
organic layer was dried, filtered and used as it is.
Example 5:
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)prop-
yl]-1,2,3,4-tetrahydroacridin-9-amine (I)
[0091] First Protocol (iv)
[0092] To a stirred solution of
N-[3-(4-{3-[(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,4-t-
etrahydroacridin-9-amine 1.4 (230 mg, 0.52 mmol) in toluene (10 mL)
was added isobutyraldehyde (0.07 mL, 0.8 mmol). The reaction
mixture was heated with a Dean-Stark apparatus for 1 hour (until no
more drops of water appeared). The toluene was evaporated and the
residue was dissolved in DCE (10 mL). NaBH.sub.4 (30 mg, 0.8 mmol)
was added and the mixture was stirred for 15 hours. 15 mL of
saturated NaHCO 3 solution was added. The mixture was stirred for 1
hour. 10 mL of DCM was added and the layers were separated. The
organic layer was washed with saturated NaHCO 3 solution
(2.times.15 mL). The organic layer was dried over MgSO 4, filtrated
and evaporated. The residue was purified by column chromatography
on alumina (eluent DCM/MeOH(NH.sub.3) 9.8:0.2 (v/v)).
[0093] The title compound (30 mg, 12% yield) was obtained as
colorless oil.
[0094] Second Protocol (v)
[0095] To the organic layer containing isobutyralehyde-<i7
(Example 4) was added
N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}-1,2,3,4-tetrahydr-
oacridin-9-amine 1.3 (80 mg, 0.21 mmol). The mixture was stirred
for 1 hour and STAB (190 mg, 0.899 mmol) was added. The mixture was
stirred for 15 hours. 5 mL of saturated NaHCO 3 solution was added
and the mixture was stirred for 1 hour. The layers were separated
and the organic layer was washed with saturated NaHCO 3 solution
(2.times.5 mL). The organic layer was dried, filtrated and
evaporated. The residue was purified by flash chromatography
(DCM/MeOH(NH.sub.3), 10:0 to 9.5:0.5 (v/v)).
[0096] The title compound (36 mg, 34% yield) was obtained as
colorless oil.
[0097] Third Protocol (vii)
[0098] In a oven-dried flask and under a nitrogen atmosphere, were
placed 9-chloro-1,2,3,4-tetrahydroacridine (257 mg, 1.12 mmol),
(+/-) BINAP (167 mg, 0.15eq), Cs.sub.2CO 3 (546 mg, 1.4eq),
Pd.sub.2(dba).sub.3 (162 mg, 0.15eq) and 3 mL of dry 1,4-dioxane.
N-[3-[4-(3-aminopropyl)piperazin-1-yl]propyl]-N-isobutyl-2-methyl-propan--
1-amine 1.5 (349 mg, 2.07 mmol) prepared according to Ryckebusch A
et al. J. Med. Chem. 2003, 46, 542-557 in dry dioxane (3 mL) was
added and the mixture was stirred at 90.degree. C. for 12 h. The
solution was filtered through a celite pad and evaporated. The
residue was purified by flash chromatography on silica gel
(CH.sub.2Cl.sub.2/MeOH//95/5).
[0099] The title compound (419 mg, 12% yield) was obtained as
colorless oil.
Characterization of
N-r3-r4-(3-aminopropyl)piperazin-1-yllpropyll-N-isobutyl-2-methyl-propan--
1-amine 1.5
[0100] .sup.1H NMR (300 MHz), .delta. (ppm, CD.sub.3OD): 3.05 (t,
.sup.3J=1.2 Hz, 2H); 3.00-2.60 (M, 18H,); 2.10-1.80 (M, 6H); 1.02
(d, .sup.3J=6.5 Hz, 12H).
[0101] .sup.13C NMR (75 MHz), .delta. (ppm, CD.sub.3OD): 61.1;
53.8; 53.5; 50.7; 37.6; 24.2; 22.5; 21.3; 19.5.
[0102] LC-MS (ESI) m/z calculated: 313.3, found: 313.2 [M+H].sup.+;
rt: 1.15 min
Characterization of
N43-(4-134Bis(2-methylpropyl)aininolpropyl}piperazin-1-yl)propyll-1,2,3,4-
-tetrahydroacridin-9-amine I
.sup.1H-Compound
[0103] H NMR (300 MHz), .delta. (ppm, CDCl.sub.3): 8.45 (d,
.sup.3J=8.6 Hz, 1H); 7.8-7.9 (M, 2H); 7.64 (ddd, .sup.3J=8.6 Hz,
.sup.3J=6.7 Hz, .sup.4J=1.1 Hz, 1H); 4.14 (t, .sup.3J=6.6 Hz, 2H);
3.6-4.1 (M, 8H); 3.35-3.55 (M, 6H); 3.11 (d, .sup.3J=6.8 Hz, 4H);
3.0-3.1 (M, 2H); 2.79 (m, 2H); 2.5-2.3 (M, 4H); 2.20 (sept,
.sup.3J=6.6 Hz, 2H); 1.9-2.0 (M, 4H); 1.09 (d, .sup.3J=6.5 Hz,
12H).
[0104] .sup.13C NMR (75 MHz), .delta. (ppm, CDCl.sub.3): 156.5;
150.9; 138.2; 132.8; 125.5; 124.9; 118.8; 115.9; 112.2; 61.1; 51.2;
44.3; 28.1; 24.7; 24.0; 21.6; 20.4; 19.5; 19.4; 18.1.
[0105] LC-MS (ESI) m/z Calculated: 494.4, Found: 494.4 [M+H].sup.+;
rt: 1.4 min;
<W4-Compound
[0106] H NMR (300 MHz), .delta. (ppm, CDCl.sub.3): 8.04 (dd,
.sup.4J=0.9 Hz, .sup.3J=8.6 Hz, 1H, H.sub.5); 7.90 (dd, .sup.4J=0.7
Hz, .sup.3J=7.7 Hz, 1H, .sup.34); 7.54 (ddd, .sup.4J=1.3 Hz,
.sup.3J=6.8 Hz, .sup.3J=8.2 Hz, 1H, H.sub.7); 7.32 (ddd,
.sup.4J=1.2 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H, H.sub.6); 5.27
(br s, 1H, NH); 3.59 (m, 2H, H.sub.d); 3.07 (t, .sup.3J=5.4 Hz, 2H,
H.sub.4); 2.73 (t, .sup.3J=5.8 Hz, 2H, H.sub.1); 2.57-2.40 (M, 14H,
H.sub.a, 3/4, H.sub.b, 3/4, H.sub.d); 2.04 (s, 4H, 3/4); 1.96-1.82
(M, 6H, H.sub.2, H.sub.3, H.sub.c); 0.92 (m, 2H, 3/4).
[0107] .sup.13C NMR (75 MHz), .delta. (ppm, CDCl.sub.3): 158.3;
151.2; 147.3; 128.6; 128.2; 123.4; 123.1; 120.2; 115.8; 63.7; 57.4;
53.5; 49.2; 33.9; 27.2; 25.8; 22.9; 20.4; 19.4.
[0108] LC-MS (ESI) m/z Calculated: 508.5, Found: 508.4 [M+H].sup.+;
rt: 1.5 min;
Example 6:
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)prop-
yl]-1,2,3,4-tetrahydroacridin-9-amine hydrochloride
[0109] The hydrochloride of the 1 compound of Example 5 obtained
according to the third protocol of Example 5 was formed after
treatment with MeOH/HCl. Compound of example 5 was solubilized in
MeOH, treated with HCl 1M until pH=1 and then lyophilized.
Biological Examples
[0110] Materials and Methods
[0111] Antibodies
[0112] Pan-Tau and phospho-dependent Tau antibodies included
Tau-Nter (M19G, 1/10 000, Sergeant et al., 1999), Tau-Cter (1/10
000, Sergeant et al., 1999, Le Freche et al., 2012), Tau-1 (1/5000,
Merck Millipore), S199P (directed against the phospho-serine 199,
1/4000, Sergeant et al., 1999), Tau-Phospho S396 (directed against
the phosphor-serine 396, 1/10000, Lifetechnologies), Tau-Phospho
404 (1/10 000, Lifetechnologies), 422 (clone 2H9, a homemade
monoclonal antibody developed against a phospho-peptide containing
the phosphorylated serine 422 residue of Tau, numbering according
to the longest human brain Tau-441 isoform), AT8 (1/1000, Thermo
Scientific), AT270 (1/2000, Thermo Scientific), AT100 (1/1000,
Thermo Scientific). Neuron specific enolase (1/10 000),
.beta.-actin (1/5000) and .beta.-tubulin (1/10 000) were from
Sigma-Aldrich.
[0113] Cell Culture and Transfection
[0114] Human neuroblastoma cell line SKNSH-SY5Y cells expressing
the human Tau isoform 412 (with the exon 2 and 10 encoding
sequences) (or SY5Y-Tau46) was as previously described in
Bretteville et al., 2009.
[0115] Drug Treatment
[0116] Neuroblastoma SKNSH-SY5Y cells expressing the human Tau
isoform 412 (with the exon 2 and 10 encoding sequences) were
treated with 10 .mu.M of the hydrochloride salt of
N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,-
4-tetrahydroacridin-9-amine (Example 6, hereinafter named "Compound
I.HQ") which was diluted in the culture medium as described in
Bretteville et al., 2009.
[0117] Animal Treatment
[0118] Wild-type C57BL/6J animals of 3 months were purchased at
Charles Rivers Laboratories (France) and were allow to acclimate in
the animal facility for at least one week before any treatment.
Thy-Tau22 transgenic colonies (C57BL/6J genetic background) were
obtained by crossing heterozygous Thy-Tau22 males with C57BL/6 WT
females. Thy-Tau22 mouse transgenic line exhibits progressive
neuron-specific Alzheimer Disease-like Tau pathology devoid of
motor deficits. In this model, a progressive development of NFT is
observed in the hippocampus and amygdala, which parallels
behavioral impairments (Schindowski et al., 2006). Furthermorever,
in the hippocampus, hyper- and abnormally phosphorylated Tau
species accumulate within the somato-dendritic area (Schindowski et
al., 2006).
[0119] All animals were housed in a pathogen-free facility at 5 to
6 animals per cage (Techniplast Cages 1284L). They had ad libitum
access to food and water in a 12/12-hour light-dark cycle and
maintained in constant temperature of 22.degree. C.
[0120] For animal treatment, animals were randomly distributed and
the compound of Formula I was given in the drinking water at final
concentration of 0.5 or 1 mg/kg. Drinking bottles were changed once
per week, volume consumption was measured along the treatment
period. Food consumption and body weight were assessed. In
wild-type animals the pilot study of drug treatment was performed
for one month to establish the innocuousness of the treatment with
the compound of Formula I. This was based on the physiological,
social and behavioural assessments. Thy-Tau22 males were treated
for 4 months starting at 3 month old. All protocols were approved
by the local ethics committee (n.degree. 342012, CEEA).
[0121] Spatial Memory Assessment Using Morris Water Maze
[0122] Before any behavioral test, exploratory and locomotion of
treated and untreated animal were evaluated in a Open field (OF) 25
cm.times.25 cm arena. Four acquisitions in joined arena were
performed simultaneously. Each mouse was placed in the arena and
allowed to explore for at least 5 min. Parameters including
distance, speed, velocity were acquired by video recording using
the EthoVision video tracking equipment and software (Noldus,
Information Technology, Paris, France) in a dedicated room. The
anxiety, which could interfere with memory test, was evaluated by
the elevated plus maze (EPM). Mice were placed in the centre of a
plus-shaped maze consisting of two 10 cm wide open arms and two
enclosed arms elevated at 50 cm from the floor. Locomotion,
distance, speed and velocity were measured as well as the number of
arm entries, time spent in the open versus closed arms, percentage
of open arm entries and time per minute spent in the open arms on a
total of 5 min test.
[0123] Morris Water Maze
[0124] Spatial learning and memory abilities were assessed in a
standard hidden-platform acquisition and retention version of the
Morris water maze task (Van der Jeugd et al., 2013). A 90-cm
circular pool was filled with water opacified with nontoxic white
paint and kept at 21.degree. C. A 10-cm round rescue platform was
hidden 1 cm beneath the surface of the water at a fixed position.
Four positions around the edge of the tank were arbitrarily
designated in order to divide it into four cardinal points (North,
East, West and South) delineating four quadrants. This target
quadrant contains the rescue platform, surrounded by to adjacent
quadrant and a opposite quadrant. In the acquisition trial, each
mouse was given four swimming trials per day with at least 10 min
of intertrial interval, for four consecutive training days. The
start position was pseudo-randomized across trials. Mice that
failed to find the hidden platform within 2 min were manually
positioned on the platform. They were allowed to remain on it for
15 s before putting them back to their cages. The time required to
find the hidden rescue platform (escape latency) was used as a
spatial learning index and was recorded using the Ethovision XT
video tracking system (Noldus France). Swimming speed and total
distance were also measured. 72 h after the acquisition phase, the
hidden platform was removed and spatial memory was evaluated in a
60 s probe trial. The proportion of time spent in the target
quadrant vs. the other quadrants was considered as a spatial memory
index.
[0125] Mouse Brain Samples
[0126] After behavioral assessment, blood was collected in 1.5 mL
polypropylene tubes (Eppendorf, France), mice were sacrificed by
cervical dislocation to avoid any modifications instrumental to
anesthesia (Lefreche et al. 2012). Hippocampi and cortices were
dissected and snap frozen in 1.5 mL tubes (Eppendorf). Tissue was
homogenized in Tris-HCl buffer (10 mM pH 7.4) containing 320 mM
sucrose, protease inhibitors (Complete mini EDTA-free, Roche)
sonicated and spun at 12,000.times.g for 10 min at 4.degree. C. The
supernatant was recovered, protein concentration was achieved using
the BCA Protein quantification assay kit (Pierce) according to the
manufacturer instruction. The supernatant was further processed for
biochemical analyses or kept at -80.degree. C. For ID or 2D gels,
the same lysis buffer was used. Thus, equal amount protein in Tris
buffer were added with a solution containing 7M urea, 2M thiouree
with 2% SDS and sonicated at 60 Hz 30 pulses.
[0127] ID SDS-PAGE and Western-Blotting
[0128] For the western-blot analysis of Tau proteins, SDS-PAGE was
performed according to the manufacturer's instructions using
Bis-Tris 4-12% gradient acrylamide gel (NuPage.RTM. Bis-Tris
PreCast 12 wells, Life Technologies). The apparent molecular
weights were calibrated using the molecular weight markers (Novex
and Magic Marks, Life Technologies). Electrophoresis was performed
under a continuous tension of lOOV/per gel for 1 h. Proteins were
reversibly stained with Ponceau Red to check the quality of the
efficacy of protein transfer. For western-blotting, the membranes
were blocked in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20
(v/v) (TBS-T) and 5% (w/v) of skimmed milk (TBS-M) for 30 min and
they were incubated overnight at 4.degree. C. the primary antibody.
The nitrocellulose membrane is rinsed three times by gentle shaking
in TBS-T during 10 min each. The membrane was then incubated during
1 h at room temperature with secondary antibody (Peroxydase Labeled
Anti-Rabbit IgG (H+L chains), Vector Laboratories). The
immunoreactive complexes were revealed using the ECL.TM. Western
Blotting kit and image acquisition was performed with the LAS-3000
Luminescence Image Analyser (FujiFilm Lifesciences) or exposed to
Amersham Hyperfilm ECL.TM. (G&E healthcare). Digitized images
were processed with ImageJ Software (NIH).
[0129] Two-Dimensional Gel Electrophoresis
[0130] Two-dimensional gel electrophoresis was performed according
to the recently published procedure (Fernandez-Gomez et al., 2014).
Briefly, cells were rinsed once with PBS and lysed in UTS buffer
(7M urea, 2M thiourea, 2% SDS) and sonicated at 60 Hz 30 pulses.
Lysate was precipitated with methanol/chloroform. 50 g of proteins
were precipitate using the chloroform/methanol procedure and the
pellet was dissolved in 2D buffer [urea 7M, thiourea 2M, 4% CHAPS,
and 0.6% Pharmalytes 3-10 (G&E Healthcare)]. Immobilized pH
gradient strip 3-11 Immobiline.TM. DryStrip of 11 cm (G&E
Healthcare) were rehydrated with 200 .mu.g containing a final
amount of 50 .mu.g of protein and isoelectrofocusing was achieved
with an IPGPhor III (G&E Healthcare) according to the
manufacturer's instructions. The strips were layered onto a 4-12%
Bis-Tris Poly-acrylamide Gel XT.TM. Precast Criterion (BioRad) and
run with a constant tension of 100V as described for ID SDS-PAGE.
The Western blot was performed as above described for ID SDS-PAGE
and Western-blotting.
[0131] Quantification of Tau in Mouse Plasma
[0132] hTau dosage was achieved using the total Tau and phospho-181
hTau Innotest.RTM. ELISA kits (Fujirebio Europe) according to the
manufacturer's instructions. hTau or phospho-Tau dosage was
performed using 25 or 75 L of plasma.
[0133] Immunofluorescence
[0134] Neuroblastoma SY5Y-APPWT cells were plated in cell culture
chamber slides (Labtek) and allowed to grow in DMEM supplemented
with 10% Fetal calf serum. Treatment with compounds were performed
following the same protocol described in the drug treatment
paragraph. After 48 h, cells were fixed with 4% paraformaldehyde in
0.1 M phosphate buffer (PBS) for 15 min at room temperature. Cells
were washed 3 times with ice cold PBS and blocked with PBS
supplemented with 1% BSA. Antibodies were incubated overnight at
4.degree. C. in PBS-BSA, rinsed 3-times with ice cold-PBS and
incubated with a secondary FTIC or Rhodamine-conjugated secondary
antibody (Lifesciences Technologies). The coverslips were mounted
onto slides with Vectashield with DAPI mounting medium (Vector
laboratories). Images were acquired with a Zeiss Apotome microscope
or Leica confocal microscope. All data were analyzed using
Photoshop Element 6 Software (Adobe) without any modification of
raw images.
[0135] Immunohistochemistry
[0136] Serial coronal sections (n=3) of the same brain coordinates
(George Paxinos et al.) were incubated with the phospho-dependent
AT100 or AT8 antibodies. They respectively recognized
phospho-epitope of tau at residues Thr 212-Ser 214 and Ser 202-Thr
205. Quantification of labeled neurons was achieved using the
NIH-Image J software and expressed in arbitrary units (A.U).
[0137] Statistical Analyses
[0138] Results were reported as means SEM. Differences between mean
values were evaluated using the Student's t-test, one Way-ANOVA
followed by a post-hoc Fisher's LSD test using GraphPad Prism 6
Software. P values<0.05 were considered significant.
[0139] Results
[0140] Modulatory Effect on Tau Phosphorylation and Tau
Degradation
[0141] The effect of the compound of Formula I on Tau
phosphorylation was analyzed in vitro and in vivo. Neuroblastoma
SKNSH-SY5Y cells expressing the human Tau isoform 412 (with the
exon 2 and 10 encoding sequences) were treated with 10 .mu.M of the
compound of Formula I.
[0142] In Vitro Experiments
[0143] Expression and total phosphorylation was assessed by ID and
2D gel electrophoresis using phospho-dependent and pan-Tau
antibodies (FIGS. 1 and 2). The phospho-dependant Tau antibody
S199P is directed against the phosphorylated serine 199 residue
(numbering according to the longest human Tau isoform) and is an
early marker of Tau hyperphosphorylation during neurofibrillary
degeneration (Maurage et al., 2003). The S396 antibody is directed
to an epitope comprising the phosphorylated serine 396 of Tau
(numbering according to the longest brain tau isoform of 441 amino
acids). Tau expression is induced by tetracycline treatment and
cells were treated afterwards with the compound of Formula I.
Change of Tau expression or phosphorylation is compared to the
induced but non-treated condition.
[0144] No significant change of Tau expression was observed with
the compound of Formula I although Tau phosphorylation at serines
199 and 396 was reduced in cells treated with the compound of
Formula I (FIG. 1).
[0145] These results were corroborated using 2D gel
electrophoresis. In control conditions, isovariants of Tau were
resolved between pi 6.5 and 10.5. Following treatment with the
compound of Formula I, a reduction of the isovariants at the
neutral pi was observed with Tau396 phospho-dependent antibody
(S396) as well as with both N- and C-terminal directed pan-Tau
antibodies (FIG. 2).
[0146] In Vivo Experiments
[0147] To further determine the effect of the compound of Formula I
on Tau metabolism, wild-type C57/1BL6 animals were treated with 0.5
and 1 mg/Kg of the compound of Formula I for 1 month and endogenous
MAPT expression was assessed by 2D gel electrophoresis. Tau protein
isovariants consist of three series ranging from pi 10 to 6. The
major and most basic isovariants correspond to the mouse Tau
isoform comprising the sole exon 10 encoding sequence. The two
other series of isoforms of molecular weight of 64 and 69 kDa are
isovariants of Tau isoforms which, in addition to the exon 10,
contain exon 2 or exon 2+3 encoding sequences, respectively.
[0148] As shown at FIG. 3 with the Tau-Cter antibody, the
distribution of Tau isovariants did not significantly change in
treated animals although low molecular weight Tau catabolic
products (indicated by arrows on FIG. 3) increased in number and
intensity at 0.5 mg/Kg and even more significantly at 1 mg/Kg of
the compound of Formula I.
[0149] Interestingly when the antibody SI99 is used, 5 isovariants
of Tau are detected in the non-treated condition and the number of
isovariants stained diminished to 3 and 2 isovariants at both
dosages (0.5 mg/Kg and 1 mg/Kg) of the compound of Formula I,
respectively. The surface of the spots was also reduced suggesting
a lower abundance of the phospho-site after treatment, although the
overall 2D profile of the full-length Tau isoforms is preserved
after treatment.
[0150] Altogether, these results suggest that the compound of
Formula I modulates the endogenous Tau metabolism, in particular by
reducing Tau phosphorylation and by increasing Tau proteolysis.
[0151] Preserved Spatial Memory and Reduced Tau Pathology in
Thy-Tau22 Transgenic Animals
[0152] The hippocampal development of NFT in Thy-Tau22 is
correlated to an impairment of the spatial memory (Laurent et al.,
2015). The spatial memory can be preserved by voluntary exercise
(Belarbi et al, 2011) or immunotherapy (Troquier et al., 2012) thus
making Thy-Tau22 a good model to test beneficial or detrimental
effect of drugs. Thy-Tau22 mice were treated for 4 months with 0.5
mg/kg of Compound I.HCl and the spatial memory was assessed using
the morris water maze.
[0153] As shown at FIG. 4A, the learning phase is not significantly
different between the animal groups in the upper panel. After 5
days of learning, the distance to reach the platform is reduced
showing that mice have successfully learnt. In the probe test,
performed after 72 hours following the last learning day, the
Compound of Formula I had no significant effect whereas in
Thy-Tau22 mice the spatial memory was preserved.
[0154] In wild-type animals, the animal had a significant
preference for the target quadrants than the three other quadrants
independently of the treatment (FIG. 4B). Thus, the Compound of
Formula I did not impact the cognitive performance of wild-type
animals.
[0155] Thy-Tau22 animals spent equal % of time in the target
quadrant (where the platform is positioned during the learning
phase) versus the others. The lack of distinction is interpreted as
a loss of spatial memory. In Thy-Tau22 treated animal, mice spent
more time in the target quadrant and therefore showed a preserved
spatial memory. The ANOVA statistical analysis showed that a
treatment with the compound of Formula I protected or preserved the
Thy-Tau22 mice from developing spatial memory impairments.
[0156] The burden of NFT in the hippocampal CA1 was investigated by
immunohistochemistry and quantification. As mentioned above, AT100
recognizes pathological sites of Tau (residues Thr 212-Ser 214),
and AT8 is used to detect the presence of phosphorylated tau
(residues Ser 202-Thr 205). As shown at FIG. 5, the average surface
of staining measured after AT100 staining was significantly reduced
in Compound I.HCl treated animal whereas no significant differences
were observed after AT8 immuno staining. AT100 recognized a
pathological Tau epitope suggesting that Compound I.HQ impacts on
the development of tau pathology but not necessarily on
phosphorylated Tau proteins labeled by AT8.
[0157] To further assess this hypothesis, Tau expression and
phosphorylation was further investigated by western-blotting (FIG.
6).
[0158] Brain proteins (of hippocampus) from untreated and Compound
I.HCl treated thy-tau22 animals were resolved by ID SDS-PAGE and
Tau proteins were labeled with Pan-Tau antibodies (N-ter, C-ter).
Catabolites detected with these antibodies are indicated as f-Nter
and f-Cter. Phosphorylated variants of Tau were stained with the
pSerl99 (against phosphorylated serine 199), pSer262 (against the
phosphorylated serine 262), pSer396 (against the phosphorylated
serine 396), pSer404 (against the phosphorylated serine 404),
pSer422 (against the phosphorylated serine 422) and Tau-1 (against
unphosphorylated epitope comprised between amino acid residues
198-205). The quantification of each phospho-epitote is expressed
as the ratio of phospho-epitope upon the Total hTau signal
determined by the N-ter staining. This ratio is given as the % of
variation to the control condition (untreated Thy-Tau22 animals)
whose value is arbitrary given the value of 100%.
[0159] A significant reduction of Tau phosphorylation at
phospho-sites 396 and 404 was observed whereas other phospho-sites
were not significantly modified (FIG. 6). More interestingly, Tau
C-terminal catabolites products at 43 kDa were significantly
increased whereas the N-terminal catabolites were significantly
decreased (FIG. 6). These results suggest that a treatment with the
Compound I-HCl reduces the burden of Tau pathology by reducing the
phosphorylation of Tau and increasing the catabolism of Tau which
is sufficient to preserve the Thy-Tau22 animal model of
neurofibrillary degeneration from development of spatial memory
impairment.
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