BENZYLIDENEGUANIDINE DERIVATIVES AND THERAPEUTIC USE FOR THE
TREATMENT OF PROTEIN MISFOLDING DISEASES
The present invention relates to compounds that have potential therapeutic
applications in treating disorders associated with protein misfolding stress and in
particular with an accumulation of misfolded proteins. In particular, the invention
provides compounds that are capable of exhibiting a protective effect against cytotoxic
endoplasmic reticulum (E ) stress.
BACKGROUND TO THE INVENTION
The compound 2-(2,6-dichlorobenzylidene)hydrazinecarboximidamide, also referred to
as guanabenz, is an alpha agonist of the alpha-2 type that is used as an
antihypertensive drug.
Guanabenz
Various derivatives of guanabenz have also been reported. For example, US 3,982,020
(Sandoz, Inc.) discloses substituted benzylidene hydrazines and their use as
hypoglycemic-antihyperglycemic agents, anti-obesity agents and anti-inflammatory
agents. US 2004/0068017 (Bausch & Lomb Inc.) discloses substituted benzylidene
hydrazines that are capable of increasing the activity of gelatinase A in ocular cells.
The molecules have applications in the treatment of primary open angle glaucoma.
WO 2008/061647 (Acure Pharma AB) discloses the use of N-(2-chloro-3,4,-
dimethoxybenzylideneamino)guanidine as a VEGFR inhibitor and its associated
applications in the treatment or prevention of undesired blood vessel formation during
tumour growth and/or inflammatory conditions. WO 2005/031000 (Acadia
Pharmaceuticals, Inc.) discloses substituted benzylidene hydrazines and their use in
treating acute pain and chronic neuropathic pain. Finally, EP 1908464 (CNRS)
discloses guanabenz and chloroguanabenz and their use in the treatment of
polyglutamine expansion associated diseases, including Huntington's disease.
More recently it has been reported that guanabenz has therapeutic potential in a
number of other areas. Guanabenz, was recently noted to have anti-prion activity (D.
Tribouillard-Tanvier ei a/., 2008 PLoS One 3, e1981). It has been reported that its
activity in protecting against protein misfolding is surprisingly much broader and
includes attenuating accumulation of mutant Huntingtin in cell-based assays (WO
2008/041 133) and protection against the lethal effects of expression of misfolding
prone Insulin Akita mutant in the endoplasmic reticulum (ER) of Min6 and INS-1
pancreatic beta-cells (P. Tsaytler, H. P. Harding, D. Ron and A. Bertolotti, Science,
332, 1 April 201 1, 91-94).
Guanabenz has also been shown to promote survival of HeLa cells exposed to
otherwise cytotoxic ER-stress induced by the N-glycosylation inhibitor tunicamycin, in a
dose-dependent manner (P. Tsaytler, H. P. Harding, D. Ron and A. Bertolotti, Science,
332, 1 April 201 1, 91-94). Quantitative assessment of cell viability revealed that
guanabenz doubled the number of cells surviving ER stress with a median effective
concentration of ~ 0.4 mM. Neither the a2-adrenergic receptor agonist clonidine, nor the
a2-adrenergic receptor antagonist efaroxan protected cells from cytotoxic ER stress
and efaroxan did not interfere with guanabenz's protective effect (P. Tsaytler, H. P.
Harding, D. Ron and A. Bertolotti, Science, 332, 1 April 201 1, 91-94). These
observations demonstrate that guanabenz rescues cells from lethal ER stress by a
mechanism independent of the a2-adrenergic receptor. Guanabenz protects cells from
otherwise lethal accumulation of misfolded proteins by binding to a regulatory subunit
of protein phosphatase 1, PPP1 R15A (GADD34), selectively disrupting the stressinduced
dephosphorylation of the a subunit of translation initiation factor 2 (elF2a).
Guanabenz sets the translation rates in stressed cells to a level manageable by
available chaperones, thereby restoring protein homeostasis. It was reported that
Guanabenz does not bind to the constitutive PPP1 R 15B (CReP) and therefore does
not inhibit translation in non-stressed cells. (P. Tsaytler, H. P. Harding, D. Ron and A.
Bertolotti, Science, 332, 1 April 201 1, 91-94).
Failure to maintain proteostasis in the ER by mounting an adequate unfolded protein
response (UPR) is recognized as a contributing factor to many pathological conditions.
Thus, the molecules described here, which inhibit elF2a phosphatase to fine-tune
protein synthesis, may be of therapeutic benefit to a large number of diseases caused
protein misfolding stress and in particular with an accumulation of misfolded proteins.
The present invention seeks to provide alternative compounds based on a guanabenz
core structure that have potential therapeutic applications in treating disorders
associated with protein misfolding stress and in particular with an accumulation of
misfolded proteins.
STATEMENT OF INVENTION
A first aspect of the invention relates to a compound of formula (I), or a
pharmaceutically acceptable salt thereof,
wherein:
R is alkyl, CI, F or Br;
R2 is H or F;
R3 is selected from H and alkyl;
R4 is selected from H and C(0)R 6;
R5 is H;
or R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7,OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclyl and aryl, each of which is optionally substituted with one or
more Rio groups;
each R-io is independently selected from halogen, OH, CN, COO-alkyl, aralkyl, S0 2-
alkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each independently CRu, and Y is selected from CRu and N;
for use in treating a disorder associated with protein misfolding stress and in particular
with an accumulation of misfolded proteins.
Previous studies have indicated that the aryl group must be at least disubstituted in
order for the compounds to exhibit useful pharmacological activity (see for example, D.
Tribouillard-Tanvier ei a/., PLoS One 3, e1981 (2008) and EP1908464A, CNRS).
However, contrary to the results of previous studies, the present Applicant has
surprisingly found that mono-substituted aryl derivatives are also active.
Moreover, compounds of formula (I) as defined above advantageously exhibit no
activity toward the adrenergic a2A receptor relative to prior art compounds such as
Guanabenz (Figure 4). This loss in alpha-2 adrenergic activity renders the compounds
therapeutically useful in the treatment of the disorders associated with protein
misfolding stress and in particular with an accumulation of misfolded proteins, such as
Charcot Marie Tooth (CMT), retinal diseases, preferably Retinitis Pigmentosa (RP),
Alzheimer's disease, Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS),
Huntington's disease, tauopathies, prion diseases, diabetes, preferably type 2 diabetes
and cancer. The absence of alpha-2 adrenergic activity means that compounds of
formula (I) can be administered at a dosage suitable to treat the aforementioned
diseases, without any significant effect on blood pressure.
A second aspect of the invention relates to a compound of formula (II), or a
pharmaceutically acceptable salt thereof,
wherein:
R is alkyl, CI, F or Br;
R2 is H or F;
R3 is selected from H and alkyl;
R4 is selected from H and C(0)R 6;
R5 is H;
or R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7, OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclic, aryl and heteroaryl, each of which is optionally substituted
with one or more Rio groups;
each R-io is independently selected from halogen, OH, CN, COO-alkyl, aralkyl, S0 2-
alkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each independently CRu, and Y is N;
A third aspect of the invention relates to a compound of formula (III), o r a
pharmaceutically acceptable salt thereof,
wherein:
R is alkyl, CI, F o r Br;
R2 is H o r F;
R3 is selected from H and alkyl;
R4 is C(0)R 6;
R5 is H;
o r R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7, OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclic, aryl and heteroaryl, each of which is optionally substituted
with one o r more Rio groups;
each R-io is independently selected from halogen, OH, CN, COO-alkyl, aralkyl, S0 2-
alkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each independently CRu, and Y is selected from CRu and N; and
A fourth aspect of the invention relates to a compound of formula (IV), or a
pharmaceutically acceptable salt thereof,
wherein:
R is alkyl or Br;
R2 is H;
R3 is selected from H and alkyl;
R4 is selected from H and C(0)R 6;
R5 is H;
or R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7, OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclyl and aryl, each of which is optionally substituted with one or
more Rio groups;
each R-io is independently selected from halogen, OH, CN, COO-alkyl, aralkyl, S0 2-
alkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each CH and Y is CRii;
A further aspect of the invention relates to pharmaceutical compositions comprising a
compound of formula (II), (III) or (IV) as described above, admixed with a suitable
pharmaceutically acceptable diluent, excipient or carrier.
DETAILED DESCRIPTION
As used herein, the term "alkyl" includes both saturated straight chain and branched
alkyl groups which may be substituted (mono- or poly-) or unsubstituted. Preferably,
the alkyl group is a C -20 alkyl group, more preferably a C S, more preferably still a Ci_
2 alkyl group, more preferably still, a C -6 alkyl group, more preferably a C -3 alkyl
group. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Suitable substituents include, for
example, one or more R 0 groups. Preferably, the alkyl group is unsubstituted.
As used herein, the term "cycloalkyl" refers to a cyclic alkyl group which may be
substituted (mono- or poly-) or unsubstituted. Preferably, the cycloalkyl group is a C3-12
cycloalkyl group. Suitable substituents include, for example, one or more R 0 groups.
As used herein, the term "alkenyl" refers to a group containing one or more carboncarbon
double bonds, which may be branched or unbranched, substituted (mono- or
poly-) or unsubstituted. Preferably the alkenyl group is a C2-20 alkenyl group, more
preferably a C2-15 alkenyl group, more preferably still a C2-12 alkenyl group, or preferably
a C2-6 alkenyl group, more preferably a C2-3 alkenyl group. Suitable substituents include,
for example, one or more R 0 groups as defined above. The term "cyclic alkenyl" is to
be construed accordingly.
As used herein, the term "aryl" refers to a C6- 12 aromatic group which may be
substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and
naphthyl etc. Suitable substituents include, for example, one or more R 0 groups.
As used herein, the term "heterocycle" (also referred to herein as "heterocyclyl" and
"heterocyclic") refers to a substituted (mono- or poly-) or unsubstituted saturated,
unsaturated or partially unsaturated cyclic group containing one or more heteroatoms
selected from N, O and S, and which optionally further contains one or more CO
groups. Suitable substituents include, for example, one or more R 0 groups. The term
"heterocycle" encompasses both heteroaryl groups and heterocycloalkyl groups as
defined below.
As used herein, the term "heteroaryl" refers to a C2-12 aromatic, substituted (mono- or
poly-) or unsubstituted group, which comprises one or more heteroatoms. Preferably,
the heteroaryl group is a C4-12 aromatic group comprising one or more heteroatoms
selected from N, O and S. Suitable heteroaryl groups include pyrrole, pyrazole,
pyrimidine, pyrazine, pyridine, quinoline, thiophene, 1,2,3-triazole, 1,2,4-triazole,
thiazole, oxazole, iso-thiazole, iso-oxazole, imidazole, furan and the like. Again,
suitable substituents include, for example, one or more R 0 groups.
As used herein, the term "heterocycloalkyl" refers to a substituted (mono- or poly-) or
unsubstituted cyclic aliphatic group which contains one or more heteroatoms.
Preferred heterocycloalkyl groups include piperidinyl, pyrrolidinyl, piperazinyl,
thiomorpholinyl and morpholinyl. More preferably, the heterocycloalkyl group is
selected from N-piperidinyl, N-pyrrolidinyl, N-piperazinyl, N-thiomorpholinyl and Nmorpholinyl.
Again, suitable substituents include, for example, one or more R 0 groups.
As used herein, the term "aralkyl" includes, but is not limited to, a group having both
aryl and alkyl functionalities. By way of example, the term includes groups in which one
of the hydrogen atoms of the alkyl group is replaced by an aryl group, e.g. a phenyl
group optionally having one or more substituents such as halo, alkyl, alkoxy, hydroxy,
and the like. Typical aralkyl groups include benzyl, phenethyl and the like.
In one preferred embodiment, R is CI, Br, Me or F, more preferably, CI.
In one preferred embodiment, R2 is H.
In one preferred embodiment, Y is CRu.
In another preferred embodiment, Y is N.
In one preferred embodiment, R3 and R4 are both H.
In one preferred embodiment, R3 is H and R4 is C(0)R 6.
In one preferred embodiment, R6 is alkyl or alkoxy, more preferably, Me or OMe.
In one preferred embodiment, R4 and R5 are linked to form a heterocyclic group which
is optionally substituted with one or more Rio groups.
In one preferred embodiment, said compound is of formula (la), or a pharmaceutically
acceptable salt thereof,
wherein R , R2, R3 and R 0 are as defined above.
In one especially preferred embodiment, the compound of formula (I) is selected from
the following:
and pharmaceutically acceptable salts thereof.
In one highly preferred embodiment, the compound of formula (I) is selected from
Examples 1, 3, 6 and 15 as set out above.
Even more preferably, the compound of formula (I) is selected from Example 1 and
Example 15, more preferably Example 1, i.e. the compound 1-[(E)-[(2-chlorophenyl)
methylidene]amino]-guanidine.
COMPOUNDS
One aspect of the invention relates to compounds of formulae (II), (III) or (IV), or
pharmaceutically acceptable salts thereof, as defined above. Preferred aspects of the
invention apply mutatis mutandis. Particularly preferred compounds for this aspect of
the invention include Examples 7, 8, 9, 13 and 16 as described herein.
THERAPEUTIC APPLICATIONS
The Applicant has demonstrated that compounds of formula (I) have potential
therapeutic applications in treating disorders associated with accumulation of misfolded
proteins. In particular, compounds of formula (I) have been shown to have a protective
effect against cytotoxic endoplasmic reticulum (E ) stress and age related disorders.
Another aspect of the invention relates to the use of a compound of formula (I) as
defined above in the preparation of a medicament for treating a disorder associated
with protein misfolding stress and in particular with an accumulation of misfolded
proteins.
As used herein the phrase "preparation of a medicament" includes the use of one or
more of the above described compounds directly as the medicament in addition to its
use in a screening programme for further active agents or in any stage of the
manufacture of such a medicament.
Yet another aspect of the invention relates to a method of treating a disorder
associated with protein misfolding stress and in particular with an accumulation of
misfolded proteins in a subject in need thereof, said method comprising administering a
therapeutically effective amount of a compound of formula (I) as defined above to said
subject.
The term "method" refers to manners, means, techniques and procedures for
accomplishing a given task including, but not limited to, those manners, means,
techniques and procedures either known to, or readily developed from known manners,
means, techniques and procedures by practitioners of the chemical, pharmacological,
biological, biochemical and medical arts.
Herein, the term "treating" includes abrogating, substantially inhibiting, slowing or
reversing the progression of a disease or disorder, substantially ameliorating clinical
symptoms of a disease or disorder or substantially preventing the appearance of
clinical symptoms of a disease or disorder.
The term "therapeutically effective amount" refers to that amount of the compound
being administered which will relieve to some extent one or more of the symptoms of
the disease or disorder being treated.
The unfolded protein response (UPR) is a component of the cellular defence system
against misfolded proteins that adapts folding in the endoplasmic reticulum (ER) to
changing conditions. The UPR is activated in response to an accumulation of unfolded
or misfolded proteins in the lumen of the endoplasmic reticulum. In this scenario, the
UPR has two primary aims: (i) to restore normal function of the cell by halting protein
translation, and (ii) to activate the signaling pathways that lead to the increased
production of molecular chaperones involved in protein folding. If these objectives are
not achieved within a certain time frame, or the disruption is prolonged, the UPR aims
towards apoptosis.
Upstream components of the UPR are the ER-resident trans-membrane proteins IRE1 ,
ATF6, and PERK, which sense folding defects to reprogram transcription and
translation in a concerted manner and restore proteostasis. Activated IRE1 and ATF6
increase the transcription of genes involved in ER folding, such as those encoding the
chaperones BiP and GRP94. Activated PERK attenuates global protein synthesis by
phosphorylating the subunit of translation initiation factor 2 (elF2a) on Ser51 while
promoting translation of the transcription factor ATF4. The latter controls expression of
CHOP, another transcription factor, which in turn promotes expression of
PPP1 R 15A/GADD34. PPP1 R15A, an effector of a negative feedback loop that
terminates UPR signaling, recruits a catalytic subunit of protein phosphatase 1 (PP1c)
to dephosphorylate elF2a, allowing protein synthesis to resume. UPR failure
contributes to many pathological conditions that might be corrected by adequate boost
of this adaptive response. Selective inhibitors of the stressed-induced elF2a
phosphatase PPP1 R15A-PP1 delays elF2a dephosphorylation and consequently
protein synthesis selectively in stressed cells, without affecting protein synthesis in
unstressed cells. This prolongs the beneficial effects of the UPR. A transient reduction
of protein synthesis is beneficial to stressed cells because decreasing the flux of
proteins synthetized increases the availability of chaperones and thus protects from
misfolding stress (P. Tsaytler, H. P. Harding, D. Ron and A. Bertolotti, Science, 332, 1
April 201 1, 91-94). Non-selective inhibitors of the 2 elF2a phosphatases might have
undesirable effects, as persistent translation inhibition is deleterious. Indeed, genetic
ablation of both PPP1 R15A and PPP1 R15B results in early embryonic lethality in mice
indicating that inhibition of the two elF2a phosphatases PPP1 R15A-PP1 and
PPP1 R15B-PP1 is deleterious in an organismal context. In contrast, genetic ablation of
PPP1 R 15A has no harmful consequence in mice (Harding et al., 2009, Proc Natl Acad
Sci USA, 106, 1832-1837). Furthermore, specific inhibitors of PPP1 R15A are predicted
to be inert in unstressed cells, as the PPP1 R15A is not expressed in absence of stress.
Thus, selective PPP1 R15A inhibitors are predicted to be safe. Non-selective inhibitors
of the two elF2a phosphatases may also be useful to treat protein misfolding diseases,
when used at doses that result in only a partial inhibition of the phosphatases.
Cytoprotection against ER stress can be measured by a suitable assay. For example,
cytoprotection can be measured in HeLa cells in which ER stress is elicited by the
addition of media containing tunicamycin, a mixture of homologous nucleoside
antibiotics that inhibits the UDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes
and is used to induce unfolded protein response. Cell viability can be detected in the
presence and absence of inhibitor compounds after a set period of time, by measuring
the reduction of WST-8 into formazan using a standard cell viability kit (such as Cell
Viability Counting Kit-8 from Dojindo). Cytoprotection from ER stress is measured in
terms of the percentage increase in viable cells (relative to control) after ER stress.
Further details of a suitable assay are set forth in the accompanying Examples section.
In one preferred embodiment, the compound of formula (I) is capable of prolonging the
protective effect of the UPR relative to the control (i.e. in the absence of inhibitor
compound) by at least 20 %, more preferably, at least 30 %, even more preferably, at
least 40 %, at least 50 %, at least 60%, at least 70 %, at least 80 %, more preferably
still, at least 90 %.
The Applicant has demonstrated that compounds of formula (I) are inhibitors of
PPP1 R15A-PP1 interaction which induce a protective effect. Preferably, the
compound exhibits a protective effect with EC50 of less than about 5mM, even more
preferably, less than about 2mM, more preferably still, less than about 1mM. The
compound should preferably be devoid of alpha2 adrenergic activity. Thus, in one
preferred embodiment the compound does not exhibit any activity in a functional alpha-
2-adrenergic assay.
The Applicant has further demonstrated that certain compounds of formula (I)
selectively inhibit PPP1 R15A-PP1 , and thus prolong the protective effect of the UPR,
thereby rescuing cells from protein misfolding stress. Inhibitors of PPP1 R15A-PP1
described in the present invention therefore have therapeutic applications in the
treatment of a variety of diseases associated with protein misfolding stress and in
particular with an accumulation of misfolded proteins.
In one embodiment, the compound of formula (I) is capable of inhibiting PPP1 R 15A
and PPP1 R15B.
In one preferred embodiment, the compound of formula (I) is capable of selectively
inhibiting PPP1 R 15A over PPP1 R 15B.
In one preferred embodiment of the invention, the compound of formula (I) is for use in
treating neurodegenerative diseases, and more specifically where accumulation of
misfolded proteins is involved in the mode of action (Brown et al, 2012, Frontiers in
Physiology, 3, Article 263).
In one particularly preferred embodiment, the compound of formula (I) is for use in
treating a disorder selected from Charcot Marie Tooth, severe Dejerine-Sottas
syndrome (Voermans et al., 2012, J Peripher New Syst, 17(2), 223-5), a retinal disease
(such as but not restricted to retinitis pigmentosa, retinal ciliopathies, macular
degeneration, diabetic retinopathy), Alzheimer's disease, Parkinson's disease,
Amyotrophic Lateral Sclerosis (ALS), Huntington's disease, tauopathies, prion
diseases, type 2 diabetes and/or type 1 diabetes and cancer, such as but not restricted
to, multiple myeloma.
In one embodiment, the invention relates to a compound of formula (I) as defined
above for use in treating a disorder associated with the elF2a phosphorylation pathway
where accumulation of misfolded proteins is involved in the mode of action. Preferably,
the disorder is a PPP1 R15A-related disease or disorder. Examples of such disorders
include protein misfolding diseases, such as but not limited to, Charcot Marie Tooth,
severe Dejerine-Sottas syndrome and Retinitis pigmentosa.
In another embodiment, the invention relates to a compound of formula (I) as defined
above for use in treating a disorder caused by, associated with or accompanied by
elF2a phosphorylation and/or PPP1 R15A activity where accumulation of misfolded
proteins is involved in the mode of action.
In another embodiment, the invention relates to a compound of formula (I) as defined
above for use in treating UPR disorder such as, but not limited to aging (Naidoo et al.,
2008, J Neurosci, 28, 6539-48).
As used herein, "PPP1 R15A related disease or disorder" refers to a disease or disorder
characterized by abnormal PPP1 R15A activity where accumulation of misfolded
proteins is involved in the mode of action. Abnormal activity refers to: (i) PPP1 R15A
expression in cells which normally do not express PPP1 R15A; (ii) increased
PPP1 R15A expression; or, (iii) increased PPP1 R15A activity.
In another embodiment, the invention relates to a method of treating a mammal having
a disease state alleviated by the inhibition of PP1 R15A, where accumulation of
misfolded proteins is involved in the mode of action, wherein the method comprises
administering to a mammal a therapeutically effective amount of a compound of
formula (I) as defined above.
In another embodiment, the invention relates to a PPP1 R15A inhibitor of formula (I) or
a pharmaceutical acceptable salt thereof for the use in treating disorders associated
with protein misfolding stress and in particular with an accumulation of misfolded
proteins and/or UPR disorders, wherein said compound has no or reduced adrenergic
alpha 2 agonist activity in comparison with Guanabenz.
In another embodiment, the invention relates to a PPP1 R15A inhibitor of formula (I) or
a pharmaceutical acceptable salt thereof for the use in treating disorders associated
with protein misfolding stress and in particular with an accumulation of misfolded
proteins and/or UPR disorders, wherein said compound does not inhibit protein
translation in non-stressed cells expressing PPP1 R15B.
In another embodiment, the invention relates to a method of treating a disorder
characterized by ER stress response activity with an accumulation of misfolded
proteins, the method comprising administering to a patient a therapeutically effective
amount of at least one compound of formula (I) wherein said compound modulates ER
stress response.
In another embodiment, the invention relates to PPP1 R 15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating disorders associated with
protein misfolding stress and in particular with an accumulation of misfolded proteins
and/or UPR disorders, wherein said compound has a selectivity towards PPP1 R15APP1
holophosphatase, having but no or reduced activity towards PPP1 R15B-PP1
holophosphatase, and wherein the ratio (activity towards PPP1 R15A-PP1
holophosphatase / activity towards PPP1 R15B-PP1 ) for said compound is at least
equal or superior to the ratio (activity towards PPP1 R15A-PP1 holophosphatase /
activity towards PPP1 R 15B-PP1 ) for Guanabenz.
In another embodiment, the invention relates to a PPP1 R15A inhibitor of formula (I) or
a pharmaceutical acceptable salt thereof for the use in treating disorders associated
with protein misfolding stress and in particular with an accumulation of misfolded
proteins and/or UPR disorders, wherein :
said compound has an activity towards PPP1 R15A-PP1 holophosphatase but
no or reduced activity towards PPP1 R15B-PP1 holophosphatase, and ;
wherein the ratio (activity towards PPP1 R15A-PP1 holophosphatase / activity
towards PPP1 R 15B-PP1 ) for said compound is at least equal or superior to the
ratio (activity towards PPP1 R15A-PP1 holophosphatase / activity towards
PPP1 R15B-PP1 ) for Guanabenz; and
wherein said compound has no or reduced adrenergic alpha 2 agonist activity in
comparison with Guanabenz.
As used herein, the disease or disorder characterized by ER stress response activity,
and/or the disease or disorder associated with protein misfolding stress and in
particular with an accumulation of misfolded proteins and/or UPR disorders, is selected
from Charcot Marie Tooth, severe Dejerine-Sottas syndrome (Voermans et al., 2012, J
Peripher New Syst, 17(2), 223-5), a retinal disease (such as but not restricted to
retinitis pigmentosa, retinal ciliopathies, macular degeneration, diabetic retinopathy),
Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS),
Huntington's disease, diabetes, such as but not restricted to type 2 diabetes and
cancer such as but not restricted to multiple myeloma.
Charcot Marie Tooth
In one preferred embodiment, the compound of formula (I) is for use in treating Charcot
Marie Tooth.
Over a 100 mutations in the gene encoding myelin protein zero (P0), a single-pass
transmembrane protein, which is the major protein produced by myelinating Schwann
cells causes Charcot-Marie-Tooth neuropathy (D'Antonio et al., 2009, J Neurosci Res,
87, 3241-9). The mutations are dominantly inherited and cause the disease through a
gain of toxic function (D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). Deletion of
serine 63 from P0 (P0S63del) causes Charcot-Marie-Tooth 1B neuropathy in humans
and a similar demyelinating neuropathy in transgenic mice. The mutant protein
accumulates in the ER and induces the UPR (D'Antonio et al., 2009, J Neurosci Res,
87, 3241-9). Genetic ablation of CHOP, a pro-apoptotic gene in the UPR restores
motor function in Charcot-Marie-Tooth mice (Pennuto et al., 2008, Neuron, 57, 393-
405). The finding that PPP1 R15A inhibition in cells nearly abolishes CHOP expression
in ER-stressed cells indicates that genetic or pharmacological inhibition of PPP1 R15A
should reduce motor dysfunction in Charcot-Marie-Tooth mice. Recently, D'Antonio et
al. (2013 J.Exp. Med Vol. pp1-18) demonstrated that P0S63del mice treated with
salubrinal, a small molecule that increases the phosphorylation of elF2alpha (Boyce et
al. 2005 Science Vol. 307 pp935-939) regained almost normal motor capacity in
rotarod analysis and was accompanied by a rescue of morphological and electro
physiological abnormalities. Accumulation of the of CMT-related mutant in the ER
proteins is not unique to P0S63del; at least five other P0 mutants have been identified
that are retained in the ER and elicit an UPR (Pennuto et al., 2008 Neuron Vol.57
pp393-405; Saporta et al., 2012 Brain Vol.135 pp2032-2047). In addition, protein
misfolding and accumulation of misfolded protein in the ER have been implicated in
the pathogenesis of other CMT neuropathies as a result of mutations in PMP22 and
Cx32 (Colby et al., 2000 Neurobiol. Disease Vol. 7 pp561-573; Kleopa et al., 2002 J.
Neurosci. Res. Vol.68 pp522-534; Yum et al., 2002 Neurobiol. Dis. Vol. 11 pp43-52).
However, Salubrinal is toxic and can not be used to treat human patients D'Antonio et
al. (2013 J.Exp. Med Vol. pp1-18). In contrast, the PPP1 R15A inhibitors of formula (I)
are predicted to be safe and could be useful for the treatment of CMT-1A and 1B.
Retinal diseases
Recently published literature has provided evidences that the UPR is involved in the
development of retinal degeneration: inherited retinal degeneration such as retinal
ciliopathies & retinitis pigmentosa, macular degeneration, retinopathy of premarurity,
light-induced retinal degeneration, retinal detachment, diabetic retinopathy and
glaucoma (for review Gorbatyuk et Gorbatyuk 2013 - Retinal degeneration: Focus on
the unfolded protein response, Molecular Vision Vol. 19 pp1 985-1 998).
In one preferred embodiment, the compound of formula (I) is for use in treating retinal
diseases, more preferably, inherited retinal degeneration such as retinal ciliopathies &
retinitis pigmentosa, macular degeneration, retinopathy of premarurity, light-induced
retinal degeneration, retinal detachment, diabetic retinopathy and glaucoma.
Retinal ciliopathies are a group of rare genetic disorders originating from a defect in the
primary cilium of photoreceptors thus inducing retinitis pigmentosa. This defect has
been reported to induce an ER stress due to protein accumulation in the inner segment
of the photoreceptor which in turn induces the UPR (WO20 13/1 24484). Retinal
degeneration is a very common feature in ciliopathies that can be observed either in
isolated retinitis pigmentosa such as Leber's congenital amaurosis or X-linked retinitis
pigmentosa, or also in syndromic conditions like the Bardet-Biedl Syndrome (BBS) or
the Alstrom syndrome (ALMS). The retinal ciliopathy is selected from the group
consisting of Bardet-Biedl syndrome, Senior-Loken syndrome, Joubert syndrome,
Salidono-Mainzer syndrome, Sensenbrenner syndrome, Jeune syndrome, Meckel-
Gruder syndrome, Alstrom syndrome, MORM syndrome, Leber's congenital amaurosis
caused by mutation in a ciliary gene and X-linked retinitis pigmentosa caused by
mutation in the RPGR gene.
Retinitis pigmentosa is an inherited, degenerative eye disease that causes severe
vision impairment and often blindness. It is the most common cause of genetically
determined blindness. Sufferers will experience one or more of the following
symptoms: night blindness; tunnel vision (no peripheral vision); peripheral vision (no
central vision); latticework vision; aversion to glare; slow adjustment from dark to light
environments and vice versa; blurring of vision; poor color separation; and extreme
tiredness.
Emerging evidence supports a role of ER stress in retinal apoptosis and cell death
(Jing et a/., 2012, Exp Diabetes Res, 2012, 589589). Retinis pigmentosa (RP) is the
most common form of hereditary retinal degeneration caused by over 100 mutations in
the rhodopsin gene (Dryja et a/., 1991 , Proc Natl Acad Sci U S A, 88, 9370-4).
Rhodopsin is a G protein-coupled receptor that transduces light in the rod
photoreceptors and consists of a covalent complex between the transmembrane
protein opsin of 348 amino acids, covalently bound to 1 -cis retinal (Palczewski, 2006,
Annu Rev Biochem, 75, 743-67). The RP-causing rhodopsin mutations are mostly
missense mutations distributed throughout the protein (Dryja et al., 1991 , Proc Natl
Acad Sci USA, 88, 9370-4), similar to the ALS-causing SOD1 mutations (Valentine et
a/., 2005, Annu Rev Biochem, 74, 563-93). The RP-causing rhodopsin mutants have
been studied in diverse systems and results from heterologous expression of the
proteins in mammalian cells, in transgenic mice and drosophila are consistent (Griciuc
et al., 201 1, Trends Mo Med, 17, 442-51 ) . The most prevalent RP-causing rhodopsin
are misfolded, do not bind 11-c/s-retinal, do not reach the cell surface but are retained
in the ER (Griciuc e al., 201 1, Trends Mol Med, 17, 442-51 ) . Misfolding of the
rhodopsin mutants causes ER stress and rod cell death (Griciuc e al., 201 1, Trends
Mol Med, 17, 442-51 ) . This strongly suggests that the PPP1 R 15A inhibitors described
in the invention will be useful to treat RP.
Age-related macular degeneration (AMD) is the main cause of legal blindness
among those over 65 years of age in the United States. AMD was reported to account
for 54% of all current cases of blindness among the Caucasian population in the United
States. The study predicted that as a result of the rising prevalence of AMD, the
number of blind people in the US could increase by as much as 70% by 2020.
Shen et al. (201 1 Effect of Guanabenz on Rat AMD Models and Rabbit Choroidal
Blood - Vol. 5 pp27-31 ) demonstrated that Guanabenz significantly protected retinal
pigment epithelium (RPE) from Nal03-induced degeneration, inhibited the
development of choroidal neovascularization (CNV) in laser-induced rat AMD model
and increased choroidal blood flow markedly in vivo.
However, Guanabenz is an alpha2 adrenergic receptor and because of its hypotensive
activity, it can not be used to treat retinal or macular degeneration.
Compounds of the invention which are PPP1 R 15A inhibitors like Guanabenz but which
advantageously exhibit no activity toward the adrenergic alpha2A receptor will
ameliorate retinal or macular.
Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease,
tauopathies and prion diseases
In one preferred embodiment, the compound of formula (I) is for use in treating a
disease selected from Alzheimer's disease, Parkinson's disease, ALS, Huntington's
disease, tauopathies and prion diseases.
Because accumulation of misfolded proteins is a hallmark of diverse diseases and
having shown that compound of formula (I) reduces accumulation of 4 unrelated
misfolded and disease-causing proteins (FIGURE 4-6), the compound of formula (I) will
be useful to also treat other neurodegenerative diseases caused by accumulation of
misfolded proteins.
In addition, as UPR induction is a hallmark of these diseases caused by accumulation
of misfolded protein, the compound of formula (I) will be useful to treat these diseases.
(Scheper & Hoozemans 2009; Kim et al. 2008).
Guanabenz reduces the symptoms of prion infected mice (D. Tribouillard-Tanvier et al.,
2008 PLoS One 3, e1981 ) . However, Guanabenz is not useful for the treatment of
human protein misfolding diseases due to its hypotensive activity. In contrast, the
PPP1 R15A inhibitors, devoid of alpha2 adrenergic activity, and described in this
invention could be useful to treat prion diseases.
Parkinson's disease (PD)
Salubrinal inhibits the PPP1 R15A mediated dephosphorylation of elF2a (Boyce et al.
2005 Science Vol. 307 pp935-939). Recently, Colla et al. (J. of Neuroscience 2012 Vol.
32 N°10 pp3306-3320) demonstrated that Salubrinal significantly attenuates disease
manifestations in two animal models of alpha-synucleinopathy.
Without to be bound by a theory, it is anticipated that compounds of the invention which
are PPP1 R15A inhibitors will ameliorate disease manifestations of alphasyncleinopathies
such as Parkinson's disease.
Amyotrophic Lateral Sclerosis (ALS)
Saxena et al. (Nature Neuroscience 2009 Vol. 12 pp627-636) demonstrated that
Salubrinal extends the life span of a G93A-SOD1 transgenic mouse model of motor
neuron disease. Without to be bound by a theory, it is anticipated that compounds of
the invention which are PPP1 R15A inhibitors will ameliorate disease manifestations of
ALS with the SOD1 mutation G93A. More than 140, mostly missense, mutations in
the SOD1 gene cause aggregation of the affected protein in familial forms of
amyotrophic lateral sclerosis (ALS). Because diverse SOD1 mutants share common
defects (Munch et al. 2010), it is accepted that diverse SOD1 mutant cause ALS by a
common mechanism. Moreover, the clinical manifestations are shared between
sporadic and familial forms of the diseases, and it is now well recognized that protein
misfolding plays a central role in both familial and sporadic ALS. Therefore, the
compounds of formula (I) can be used to treat both familial and sporadic forms of ALS.
The Applicant has found that the cytoprotective activity of guanabenz on protein
misfolding stress is surprisingly broad as guanabenz also reduces mutant huntingtin
accumulation in cells (WO 2008/041 133). This finding is unexpected since mutant
huntingtin is either cytosolic or nuclear. However, there is evidence that mutant
huntingtin metabolism has previously been connected to the ER stress response
(Nishitoh et al., 2002, Genes Dev, 16, 1345-55; Rousseau et al., 2004, Proc Natl Acad
Sci U S A, 101 , 9648-53; Duennwald and Lindquist, 2008, Genes Dev, 22, 3308-19).
The Applicant's findings that guanabenz protects cells from cytotoxic ER stress and
reduces mutant huntingtin accumulation further supports the idea that there may be
aspects of the ER stress response that impact on mutant huntingtin accumulation.
Furthermore, dysfunction of the ER stress response has been involved in a variety of
pathologies, including type 2 diabetes and neurodegeneration (Scheper and
Hoozemans, 2009, Curr Med Chem, 16, 615-26). Thus, without wishing to be bound by
theory, it is believed that guanabenz and related compounds have a protective effect
against secondary UPR disorders, namely disorders due to an accumulation of a non-
ER resident misfolded protein, which induces the UPR.
Diabetes
In one preferred embodiment, the compound of formula (I) is for use in treating
diabetes, more preferably type 2 diabetes.
The insulin-secreting b-cells in the pancreas have a heavy and tightly regulated
biosynthetic burden consisting in insulin secretion. Thus, these cells have an important
need to maintain ER homeostasis (Back and Kaufman, 2012, Annu Rev Biochem, 8 1,
767-93). Type 2 diabetes is manifested by increased levels of blood glucose due to
insulin resistance in the adipose, muscle and liver and/or impaired insulin secretion
from pancreatic b-cells. As a response, b-cells mass increase and their function is
enhanced. Eventually, the burden on the b-cells is too high leading to their progressive
decline and death. Increasing evidence reveals that death of b-cells results from ER
stress (Back and Kaufman, 2012, Annu Rev Biochem, 8 1, 767-93). Importantly, Chop
deletion improves b-cells function in diverse models of diabetes (Song et al., 2008, J
Clin Invest, 118, 3378-89). Without wishing to be bound by theory, it is believed that
inhibitors of PPP1 R15A-PP1 will improve b-cells function in type 2 diabetes since
inhibition of PPP1 R15A-PP1 reduces the levels of the pro-apoptotic protein CHOP
during ER stress (Tsaytler et ai, 201 1, Science, 332, 91-4).
Cancer
In one preferred embodiment, the compound of formula (I) is for use in treating cancer.
Cancer cells have high metabolic requirement and their proliferation relies on efficient
protein synthesis. Translation initiation plays a crucial role in controlling protein
homeostasis, differentiation, proliferation and malignant transformation. Increasing
translation initiation contributes to cancer initiation and conversely, decreasing
translation initiation could reduce tumor growth (Donze et al., 1995, EMBO J, 14, 3828-
34; Pervin et al., 2008, Cancer Res, 68, 4862-74; Chen et al., 201 1, Nat Chem Biol, 7,
6 10-6). Without wishing to be bound by theory, it is believed that inhibiting PPP1 R 15A
could selectively reduce translation in tumor cells and thus reduce tumor growth.
Aging
Aging is known to impair stress responses and in particular, the UPR is impaired with
age (Naidoo et al., 2008, J Neurosci, 28, 6539-48). Thus, prolonging the beneficial
effect of the UPR by inhibition of elF2a phosphatase could ameliorate age-related
disorders.
PHARMACEUTICAL COMPOSITIONS
For use according to the present invention, the compounds or physiologically
acceptable salts, esters or other physiologically functional derivatives thereof,
described herein, may be presented as a pharmaceutical formulation, comprising the
compounds or physiologically acceptable salt, ester or other physiologically functional
derivative thereof, together with one or more pharmaceutically acceptable carriers
therefore and optionally other therapeutic and/or prophylactic ingredients. The
carrier(s) must be acceptable in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient thereof. The
pharmaceutical compositions may be for human or animal usage in human and
veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical
compositions described herein may be found in the "Handbook of Pharmaceutical
Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller.
Acceptable carriers or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's Pharmaceutical
Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose,
magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents
include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard
to the intended route of administration and standard pharmaceutical practice. The
pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient
or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s),
solubilising agent(s), buffer(s), flavouring agent(s), surface active agent(s),
thickener(s), preservative(s) (including anti-oxidants) and the like, and substances
included for the purpose of rendering the formulation isotonic with the blood of the
intended recipient.
Examples of suitable binders include starch, gelatin, natural sugars such as glucose,
anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and
synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl
cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium
stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the
pharmaceutical composition. Examples of preservatives include sodium benzoate,
sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents
may be also used.
Pharmaceutical formulations include those suitable for oral, topical (including dermal,
buccal, ocular and sublingual), rectal or parenteral (including subcutaneous,
intradermal, intramuscular and intravenous), nasal, intra-ocularly and pulmonary
administration e.g., by inhalation. The formulation may, where appropriate, be
conveniently presented in discrete dosage units and may be prepared by any of the
methods well known in the art of pharmacy. All methods include the step of bringing
into association an active compound with liquid carriers or finely divided solid carriers
or both and then, if necessary, shaping the product into the desired formulation.
Pharmaceutical formulations suitable for oral administration wherein the carrier is a
solid are most preferably presented as unit dose formulations such as boluses,
capsules or tablets each containing a predetermined amount of active compound. A
tablet may be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by compressing in a
suitable machine an active compound in a free-flowing form such as a powder or
granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent,
surface-active agent or dispersing agent. Moulded tablets may be made by moulding
an active compound with an inert liquid diluent. Tablets may be optionally coated and, if
uncoated, may optionally be scored. Capsules may be prepared by filling an active
compound, either alone or in admixture with one or more accessory ingredients, into
the capsule shells and then sealing them in the usual manner. Cachets are analogous
to capsules wherein an active compound together with any accessory ingredient(s) is
sealed in a rice paper envelope. An active compound may also be formulated as
dispersible granules, which may for example be suspended in water before
administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet.
Formulations suitable for oral administration wherein the carrier is a liquid may be
presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water liquid emulsion.
Formulations for oral administration include controlled release dosage forms, e.g.,
tablets wherein an active compound is formulated in an appropriate release -
controlling matrix, or is coated with a suitable release - controlling film. Such
formulations may be particularly convenient for prophylactic use.
Pharmaceutical formulations suitable for rectal administration wherein the carrier is a
solid are most preferably presented as unit dose suppositories. Suitable carriers
include cocoa butter and other materials commonly used in the art. The suppositories
may be conveniently formed by admixture of an active compound with the softened or
melted carrier(s) followed by chilling and shaping in moulds.
Pharmaceutical formulations suitable for parenteral administration include sterile
solutions or suspensions of an active compound in aqueous or oleaginous vehicles.
Pharmaceutical formulations of the invention are suitable for ophthalmic administration,
in particular for intra-ocular, topical ocular or peri-ocular administration, more preferably
for topical ocular or peri-ocular administration.
Injectible preparations may be adapted for bolus injection or continuous infusion. Such
preparations are conveniently presented in unit dose or multi-dose containers which
are sealed after introduction of the formulation until required for use. Alternatively, an
active compound may be in powder form which is constituted with a suitable vehicle,
such as sterile, pyrogen-free water, before use.
An active compound may also be formulated as long-acting depot preparations, which
may be administered by intramuscular injection or by implantation, e.g.,
subcutaneously or intramuscularly. Depot preparations may include, for example,
suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting
formulations are particularly convenient for prophylactic use.
Formulations suitable for pulmonary administration via the buccal cavity are presented
such that particles containing an active compound and desirably having a diameter in
the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient. As one
possibility such formulations are in the form of finely comminuted powders which may
conveniently be presented either in a pierceable capsule, suitably of, for example,
gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation
comprising an active compound, a suitable liquid or gaseous propellant and optionally
other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants
include propane and the chlorofluorocarbons, and suitable gaseous propellants include
carbon dioxide. Self-propelling formulations may also be employed wherein an active
compound is dispensed in the form of droplets of solution or suspension.
Such self-propelling formulations are analogous to those known in the art and may be
prepared by established procedures. Suitably they are presented in a container
provided with either a manually-operable or automatically functioning valve having the
desired spray characteristics; advantageously the valve is of a metered type delivering
a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.
As a further possibility an active compound may be in the form of a solution or
suspension for use in an atomizer or nebuliser whereby an accelerated airstream or
ultrasonic agitation is employed to produce a fine droplet mist for inhalation.
Formulations suitable for nasal administration include preparations generally similar to
those described above for pulmonary administration. When dispensed such
formulations should desirably have a particle diameter in the range 10 to 200 microns
to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of
a powder of a suitable particle size or choice of an appropriate valve. Other suitable
formulations include coarse powders having a particle diameter in the range 20 to 500
microns, for administration by rapid inhalation through the nasal passage from a
container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an
active compound in aqueous or oily solution or suspension.
Pharmaceutically acceptable carriers are well known to those skilled in the art and
include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8%
saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non
aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents
are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other
additives may also be present, such as, for example, antimicrobials, antioxidants,
chelating agents, inert gases and the like.
Formulations suitable for topical formulation may be provided for example as gels,
creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either
directly spread upon the surface of the wound or ulcer or carried on a suitable support
such as a bandage, gauze, mesh or the like which may be applied to and over the area
to be treated.
Liquid or powder formulations may also be provided which can be sprayed or sprinkled
directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as
a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and
then applied to the site to be treated.
According to a further aspect of the invention, there is provided a process for the
preparation of a pharmaceutical or veterinary composition as described above, the
process comprising bringing the active compound(s) into association with the carrier,
for example by admixture.
In general, the formulations are prepared by uniformly and intimately bringing into
association the active agent with liquid carriers or finely divided solid carriers or both,
and then if necessary shaping the product. The invention extends to methods for
preparing a pharmaceutical composition comprising bringing a compound of general
formula (I) in conjunction or association with a pharmaceutically or veterinarily
acceptable carrier or vehicle.
SALTS/ESTERS
The compounds of the invention can be present as salts or esters, in particular
pharmaceutically and veterinarily acceptable salts or esters.
Pharmaceutically acceptable salts of the compounds of the invention include suitable
acid addition or base salts thereof. A review of suitable pharmaceutical salts may be
found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with
strong inorganic acids such as mineral acids, e.g. hydrohalic acids such as
hydrochloride, hydrobromide and hydroiodide, sulfuric acid, phosphoric acid sulphate,
bisulphate, hemisulphate, thiocyanate, persulphate and sulphonic acids; with strong
organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which
are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated
or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic,
fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic,
glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or
glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (CrC 4)-alkyl- or
aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen)
such as methane- or p-toluene sulfonic acid. Salts which are not pharmaceutically or
veterinarily acceptable may still be valuable as intermediates.
Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate,
citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate,
butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate,
heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate,
picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate
and succinate, organic sulphonic acids such as methanesulphonate,
ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-
naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and ptoluenesulphonate;
and inorganic acids such as hydrochloride, hydrobromide,
hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric
and sulphonic acids.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the
functional group being esterified. Organic acids include carboxylic acids, such as
alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted
(e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid,
for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with
hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric
acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with
organic sulfonic acids, such as (CrC 4)-alkyl- or aryl-sulfonic acids which are
unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene
sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium
hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols
include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or
substituted, e.g. by a halogen).
ENANTIOMERS/TAUTOMERS
In all aspects of the present invention previously discussed, the invention includes,
where appropriate all enantiomers, diastereoisomers and tautomers of the compounds
of the invention. The person skilled in the art will recognise compounds that possess
optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The
corresponding enantiomers and/or tautomers may be isolated/prepared by methods
known in the art. Enantiomers are characterised by the absolute configuration of their
chiral centres and described by the R- and S-sequencing rules of Cahn, Ingold and
Prelog. Such conventions are well known in the art (e.g. see 'Advanced Organic
Chemistry', 3rd edition, ed. March, J., John Wiley and Sons, New York, 1985).
Compounds of formula (I) thus also include the tautomer forms of formula:
As an illustrative example, a tautomer form of example 1 is:
Compounds of the invention containing a chiral centre may be used as a racemic
mixture, an enantiomerically enriched mixture, or the racemic mixture may be
separated using well-known techniques and an individual enantiomer may be used
alone.
STEREO AND GEOMETRIC ISOMERS
Some of the compounds of the invention may exist as stereoisomers and/or geometric
isomers - e.g. they may possess one or more asymmetric and/or geometric centres
and so may exist in two or more stereoisomeric and/or geometric forms. The present
invention contemplates the use of all the individual stereoisomers and geometric
isomers of those inhibitor agents, and mixtures thereof. The terms used in the claims
encompass these forms, provided said forms retain the appropriate functional activity
(though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the agent or a
pharmaceutically acceptable salt thereof. An isotopic variation of an agent of the
present invention or a pharmaceutically acceptable salt thereof is defined as one in
which at least one atom is replaced by an atom having the same atomic number but an
atomic mass different from the atomic mass usually found in nature. Examples of
isotopes that can be incorporated into the agent and pharmaceutically acceptable salts
thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur,
fluorine and chlorine such as 2H, H, C, 4C, N, 70 , 0 , P, 2P, S, F and 6CI,
respectively. Certain isotopic variations of the agent and pharmaceutically acceptable
salts thereof, for example, those in which a radioactive isotope such as H or 4C is
incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated,
i.e., H, and carbon-14, i.e., 4C, isotopes are particularly preferred for their ease of
preparation and detectability. Further, substitution with isotopes such as deuterium, i.e.,
2H, may afford certain therapeutic advantages resulting from greater metabolic stability,
for example, increased in vivo half-life or reduced dosage requirements and hence may
be preferred in some circumstances. For example, the invention includes compounds
of general formula (I) where any hydrogen atom has been replaced by a deuterium
atom. Isotopic variations of the agent of the present invention and pharmaceutically
acceptable salts thereof of this invention can generally be prepared by conventional
procedures using appropriate isotopic variations of suitable reagents.
PRODRUGS
The invention further includes the compounds of the present invention in prodrug form,
i.e. covalently bonded compounds which release the active parent drug according to
general formula (I) in vivo. Such prodrugs are generally compounds of the invention
wherein one or more appropriate groups have been modified such that the modification
may be reversed upon administration to a human or mammalian subject. Reversion is
usually performed by an enzyme naturally present in such subject, though it is possible
for a second agent to be administered together with such a prodrug in order to perform
the reversion in vivo. Examples of such modifications include ester (for example, any of
those described above), wherein the reversion may be carried out be an esterase etc.
Other such systems will be well known to those skilled in the art.
SOLVATES
The present invention also includes solvate forms of the compounds of the present
invention. The terms used in the claims encompass these forms.
POLYMORPHS
The invention further relates to the compounds of the present invention in their various
crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within
the pharmaceutical industry that chemical compounds may be isolated in any of such
forms by slightly varying the method of purification and or isolation form the solvents
used in the synthetic preparation of such compounds.
ADMINISTRATION
The pharmaceutical compositions of the present invention may be adapted for rectal,
nasal, intrabronchial, topical (including buccal, sublingual and ophthalmic
administration, in particular for intra-ocular, topical ocular or peri-ocular administration),
vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial
and intradermal), intraperitoneal or intrathecal administration. Preferably the
formulation is an orally administered formulation. The formulations may conveniently be
presented in unit dosage form, i.e., in the form of discrete portions containing a unit
dose, or a multiple or sub-unit of a unit dose. By way of example, the formulations may
be in the form of tablets and sustained release capsules, and may be prepared by any
method well known in the art of pharmacy.
Formulations for oral administration in the present invention may be presented as:
discrete units such as capsules, gellules, drops, cachets, pills or tablets each
containing a predetermined amount of the active agent; as a powder or granules; as a
solution, emulsion or a suspension of the active agent in an aqueous liquid or a non
aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or
as a bolus etc. Preferably, these compositions contain from 1 to 250 mg and more
preferably from 10-100 mg, of active ingredient per dose.
For compositions for oral administration (e.g. tablets and capsules), the term
"acceptable carrier" includes vehicles such as common excipients e.g. binding agents,
for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone
(Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose,
hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn
starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium
phosphate, sodium chloride and alginic acid; and lubricants such as magnesium
stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid,
silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as
peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may
be desirable to add a colouring agent to make the dosage form readily identifiable.
Tablets may also be coated by methods well known in the art.
A tablet may be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by compressing in a
suitable machine the active agent in a free flowing form such as a powder or granules,
optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or
dispersing agent. Moulded tablets may be made by moulding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid diluent. The tablets
may be optionally be coated or scored and may be formulated so as to provide slow or
controlled release of the active agent.
Other formulations suitable for oral administration include lozenges comprising the
active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles
comprising the active agent in an inert base such as gelatin and glycerin, or sucrose
and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
Other forms of administration comprise solutions or emulsions which may be injected
intravenously, intraarterially, intrathecally, subcutaneously, intradermally,
intraperitoneally, intra-ocularly, topical, peri-ocularly or intramuscularly, and which are
prepared from sterile or sterilisable solutions. Injectable forms typically contain between
10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
The pharmaceutical compositions of the present invention may also be in form of
suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels,
sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For
example, the active ingredient can be incorporated into a cream consisting of an
aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can
also be incorporated, at a concentration of between 1 and 10% by weight, into an
ointment consisting of a white wax or white soft paraffin base together with such
stabilisers and preservatives as may be required.
DOSAGE
A person of ordinary skill in the art can easily determine an appropriate dose of one of
the instant compositions to administer to a subject without undue experimentation.
Typically, a physician will determine the actual dosage which will be most suitable for
an individual patient and it will depend on a variety of factors including the activity of
the specific compound employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity of the particular
condition, and the individual undergoing therapy. The dosages disclosed herein are
exemplary of the average case. There can of course be individual instances where
higher or lower dosage ranges are merited, and such are within the scope of this
invention.
In accordance with this invention, an effective amount of a compound of general
formula (I) may be administered to target a particular condition or disease. Of course,
this dosage amount will further be modified according to the type of administration of
the compound. For example, to achieve an "effective amount" for acute therapy,
parenteral administration of a compound of general formula (I) is preferred. An
intravenous infusion of the compound in 5% dextrose in water or normal saline, or a
similar formulation with suitable excipients, is most effective, although an intramuscular
bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about
100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the
concentration of drug in the plasma at an effective concentration The compounds may
be administered one to four times daily at a level to achieve a total daily dose of about
0.4 to about 400 mg/kg/day. The precise amount of an inventive compound which is
therapeutically effective, and the route by which such compound is best administered,
is readily determined by one of ordinary skill in the art by comparing the blood level of
the agent to the concentration required to have a therapeutic effect.
The compounds of this invention may also be administered orally to the patient, in a
manner such that the concentration of drug is sufficient to achieve one or more of the
therapeutic indications disclosed herein. Typically, a pharmaceutical composition
containing the compound is administered at an oral dose of between about 0.1 to about
50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral
dose would be about 0.1 to about 20 mg/kg.
No unacceptable toxicological effects are expected when compounds of the present
invention are administered in accordance with the present invention. The compounds
of this invention, which may have good bioavailability, may be tested in one of several
biological assays to determine the concentration of a compound which is required to
have a given pharmacological effect.
COMBINATIONS
In a particularly preferred embodiment, the one or more compounds of the invention
are administered in combination with one or more other active agents, for example,
existing drugs available on the market. In such cases, the compounds of the invention
may be administered consecutively, simultaneously or sequentially with the one or
more other active agents.
Drugs in general are more effective when used in combination. In particular,
combination therapy is desirable in order to avoid an overlap of major toxicities,
mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to
administer most drugs at their maximum tolerated doses with minimum time intervals
between such doses. The major advantages of combining drugs are that it may
promote additive or possible synergistic effects through biochemical interactions and
also may decrease the emergence of resistance.
Beneficial combinations may be suggested by studying the inhibitory activity of the test
compounds with agents known or suspected of being valuable in the treatment of a
particular disorder. This procedure can also be used to determine the order of
administration of the agents, i.e. before, simultaneously, or after delivery. Such
scheduling may be a feature of all the active agents identified herein.
ASSAY
A further aspect of the invention relates to the use of a compound as described above
in an assay for identifying further candidate compounds capable of inhibiting
PPP1 R15A-PP1 .
Preferably, the assay is a competitive binding assay.
More preferably, the competitive binding assay comprises contacting a compound of
the invention with PPP1 R15A-PP1 and a candidate compound and detecting any
change in the interaction between the compound according to the invention and the
PPP1 R15A-PP1 .
Preferably, the candidate compound is generated by conventional SAR modification of
a compound of the invention.
As used herein, the term "conventional SAR modification" refers to standard methods
known in the art for varying a given compound by way of chemical derivatisation.
Thus, in one aspect, the identified compound may act as a model (for example, a
template) for the development of other compounds. The compounds employed in such
a test may be free in solution, affixed to a solid support, borne on a cell surface, or
located intracellular^. The abolition of activity or the formation of binding complexes
between the compound and the agent being tested may be measured.
The assay of the present invention may be a screen, whereby a number of agents are
tested. In one aspect, the assay method of the present invention is a high through-put
screen.
This invention also contemplates the use of competitive drug screening assays in
which neutralising antibodies capable of binding a compound specifically compete with
a test compound for binding to a compound.
Another technique for screening provides for high throughput screening (HTS) of
agents having suitable binding affinity to the substances and is based upon the method
described in detail in WO 84/03564.
It is expected that the assay methods of the present invention will be suitable for both
small and large-scale screening of test compounds as well as in quantitative assays.
Preferably, the competitive binding assay comprises contacting a compound of the
invention with PPP1 R15A-PP1 in the presence of a known substrate of PPP1 R15APP1
and detecting any change in the interaction between said PPP1 R15A-PP1 and
said known substrate.
A further aspect of the invention provides a method of detecting the binding of a ligand
to PPP1 R15A-PP1 , said method comprising the steps of:
(i) contacting a ligand with PPP1 R15A-PP1 in the presence of a known substrate
(ii) detecting any change in the interaction between PPP1 R15A-PP1 and said
known substrate;
and wherein said ligand is a compound of the invention.
One aspect of the invention relates to a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more ligands capable of binding to a ligand binding domain;
and
(c) preparing a quantity of said one or more ligands.
Another aspect of the invention provides a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more ligands capable of binding to a ligand binding domain;
and
(c) preparing a pharmaceutical composition comprising said one or more ligands.
Another aspect of the invention provides a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more ligands capable of binding to a ligand binding domain;
(c) modifying said one or more ligands capable of binding to a ligand binding
domain;
(d) performing the assay method described hereinabove;
(e) optionally preparing a pharmaceutical composition comprising said one or more
ligands.
The invention also relates to a ligand identified by the method described hereinabove.
Yet another aspect of the invention relates to a pharmaceutical composition comprising
a ligand identified by the method described hereinabove.
Another aspect of the invention relates to the use of a ligand identified by the method
described hereinabove in the preparation of a pharmaceutical composition for use in
the treatment of a disorder associated with accumulation of misfolded proteins as
defined above.
The above methods may be used to screen for a ligand useful as an inhibitor of
PPP1 R15A-PP1 .
Compounds of general formula (I) are useful both as laboratory tools and as
therapeutic agents. In the laboratory certain compounds of the invention are useful in
establishing whether a known or newly discovered target contributes a critical or at
least significant biochemical function during the establishment or progression of a
disease state, a process commonly referred to as 'target validation'.
The present invention is further described with reference to the following figures,
wherein:
Figure 1 shows dose dependent protection of HeLa cells by Compound of the formula
(I), Example 1 of the invention, from ER stress induced by 6 hour exposure to
tunicamycin. See description test 1.
Figure 2 shows that Compound of the formula (I), Example 1 of the invention,
postpones translation recovery in stressed cells. More specifically, Figure 2 shows that
translation is attenuated 2h following Tunicamycin addition. Translation recovery is
noticeable in cells treated with tunicamycin only. Example 1 of the invention prolongs
translation attenuation in tunicamycin treated cells. See description test 3.
Figure 3 shows Compound of the formula (I), Example 1 of the invention, unlike
Guanabenz, has a no activity for adrenergic a2A receptor as measured by a functional
assay for the adrenergic a2A receptor. See description test 5.
Figure 4 shows that a Compound of the formula (I), Example 1 of the invention
prevents ER-retention of P0S63del, the mutant protein associated with Charcot Marie
Tooth 1B. Y axis: number of cells. UT: untreated.
Figure 5 shows that a Compound of the formula (I), Example 1 of the invention reduces
accumulation of two unrelated disease-causing, misfolded proteins: mutant huntingtin
amino-terminal fragment (Htt48Q) associated with Huntington's disease and SOD1
mutant (A4V), associated with amyotrophic lateral sclerosis. Y axis: percentage
accumulation of protein, relative to untreated cells. UT: untreated.
Figure 6 shows that a Compound of the formula (I), Example 1 of the invention reduces
accumulation of rhodopsin mutant P23H associated with retinitis pigmentosa. Y axis:
number of cells. UT: untreated.
The present invention is further described with reference to the following non-limiting
examples.
EXAMPLES
Methods & Materials
Example 1 was purchased from Chemdiv ref: 1683-6588
Example 2 was purchased from Chembridge ref: 5173161
Example 4 was purchased from Enamine ref: Z49562642
Example 6 was purchased from Chemdiv ref: 1683-6502
Preparation of the compounds according to the present invention
The reactants and commercials compounds were purchased from Acros Organics,
Sigma-Aldrich. The compounds according to the present invention can be prepared
according to the following general procedure:
General procedure A:
To a solution of benzaldehyde (1eq.) in ethanol (300ml) was sequentially added
Aminoguanidine hydrochloride (1eq.) and sodium acetate (1eq.) at 25°C. The resulting
reaction mixture was heated at 80°C for next 6 hours. Reaction completion was
monitored on TLC using dichloromethane/methanol (8/2) as mobile phase. After
completion of reaction, the reaction mixture was allowed to cool down to 25°C and
dumped in the saturated solution of NaHC0 3 (700ml). The resulting precipitate were
filtered off under vacuum and washed with water ( 100ml). The resulting solid material
was titurated with diethylether (2 x 25 ml) and dried under vacumm to provide the
desired substituted aminoguanidine derivative.
The following compounds were prepared according general procedure A:
Example 1: 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine
Prepared following general procedure A from 2-chlorobenzaldehyde. H-NMR (DMSOd6)
: d (ppm) 5.61 (s, 2H); 6.06 (s, 2H); 7.22-7.32 (m, 2H); 7.40 (dd, 1H); 8.15 (dd, 1H);
8.28 (s, 1H); MS (ESI+): m/z = 197.4 [M+H]+
Example 3 : 1-[(E)-[(2-fluorophenyl)methylidene]amino]-guanidine
Prepared following general procedure A from 2-fluorobenzaldehyde.
Example 7 : 1-[(E)-[(2-chloro-4-fluorophenyl)methylidene]amino]guanidine
Prepared following general procedure A from 2- chloro-4-fluorobenzaldehyde in 67%
yield. H-NMR (DMSO-d ) : d (ppm) 5.80 (brs, 2H); 5.84 (brs, 2H); 7.19-7.34 (m, 4H);
8.16 (s, 1H); MS (ESI+): m/z = 215.1 [M+H]+
Example 13: 1-[(E)-[(3-chloropyridin-4-yl)methylidene]amino]guanidine
Prepared following general procedure A from 3-chloroisonicotinaldehyde in 50% yield.
H-NMR (DMSO-d ) : d (ppm) 6.01 (brs, 2H); 6.33 (brs, 2H); 8.10 (d, 1H); 8.14 (s, 1H);
8.37 (dd, 1H); 8.52 (s, 1H); MS (ESI+): m/z = 198.4 [M+H]+
Example 15: 1-[(E)-[(2-chloro-6-fluorophenyl)methylidene]amino]guanidine
Prepared following general procedure A from 2-chloronicotinaldehyde in 56% yield. HNMR
(DMSO-d ) : d (ppm) 5.84 (brs, 2H); 5.88 (brs, 2H); 7.18-7.35 (m, 3H); 8.16 (s,
1H); MS (ESI+): m/z = 2 15.4 [M+H]+.
Intermediate 1: 3-chloro-5-fluoroisonicotinaldehyde
To a stirred solution of N,N-Diisopropylamine (0.864g, 0.006690mol) in THF (6ml) was
added n-buLi ( 1.6M in hexane ) (7.6ml, 0.012164mol) dropwise over a priod of 15
minutes at -78° C. The resulting reaction mixture was stirred at -78° C for 15 minutes
and then it was allowed to warm at 0° C whereby it was further stirred for 1 hour. The
resulting reaction mixture was again cooled at -78° C and a solution of 3-chloro-5-
fluoropyridine (0.8g, 0.006082mol) in THF (6 ml) was added dropwise over priod of 10
minutes. The resulting reaction mixture was strried at -78° C for 1 hour, thereafter
methyl formate (0.73g, 0.012164mol) was added dropwise at -78° C. The resulting
reaction mixture was further strried at -78° C for 1 more hour. The reaction was
monitored on TLC using Hexane : ethylaceate (5:5) as mobile phase. After completion
of reaction, the reaction mixture was dumped in saturated solution of NH4CI (50ml) and
extracted with Ethyl acetate (4 x 25ml). The combined organic extract was washed with
demineralised water (50ml), brine (25ml), dried over sodium sulphate and concentrated
under vacuo. Distillation of the organic layer provided the desired aldehyde (0.6g,
6 1.85% yield) in crude form. This crude compound was directly used for the next step
without any further treatment.
Example 16: 1-[(E)-[(3-chloro-5-fluoropyridin-4-yl)methylidene]amino]guanidine
Prepared following general procedure A from 3-chloro-5-fluoroisonicotinaldehyde in
14% yield. H-NMR (DMSO-d ) : d (ppm) 5.95-6.30 (m, 4H); 8.10 (s, 1H); 8.46-8.52 (m,
2H); MS (ESI+): m/z = 216.0 [M+H]+.
Example 8 : N-{N-[(E)-[(2-chlorophenyl)methylidene]amino]
carbamimidoyl}acetamide
To a solution of 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine (0.50g,
0.002543mol) in DMSO (10ml) was added acetic anhydride (0.26g, 0.002543mol) at
25°C. The resulting reaction mixture was stirred at 25°C for next 15 hours. Reaction
completion was monitored on TLC using dichloromethane/Methanol (9.5/0.5) as mobile
phase. After completion of reaction the reaction mixture was dumped in the water
(100ml) and extracted with ethyl acetate (2 x 150ml). The combined organic extract
was washed with brine (100ml), dried over sodium sulphate, filtered and concentrated
in vacuo. The resulting crude material was further purified by flash chromatography
using dichloromethane: methanol as mobile phase whereby the desired product eluted
at around 1.0% methanol in dichloromethane. Distillation of the pure product fractions
provided N-{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}acetamide
(0.080g, 13% yield). H-NMR (DMSO-d ) : d (ppm) 2.97 (s, 3H); 7.25-7.41 (m, 3H);
7.42-7.53 (m, 1H); 7.79 (brs, 1H); 8.22-8.29 (m, 1H); 8.48 (s, 1H); 10.58 (brs, 1H); MS
(ESI+): m/z = 239.2 [M+H]+.
Example 9 : methyl N-{N-[(E)-[(2-chlorophenyl)methylidene]amino]
carbamimidoyl}carbamate
C I
N H Methylchloroformate / / C l
N H Q
To a suspension of 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine (0.1 5g,
0.000762mol) in dichloromethane (5ml) was added triethylamine (0.32ml,
0.002288mol) at 25°C. The resulting reaction mixture was cooled to 0°C using ice/salt
bath; thereafter methylchloroformate (0.09ml, 0.001 144mol) was added in to the
reaction mixture at 0°C. The resulting reaction mixture was stirred at room temperature
for 15 hours. Reaction completion was monitored on TLC using dichloromethane/
methanol (9/1 ) as mobile phase. After completion of reaction, the reaction mixture was
dumped in saturated solution of NaHCO3 (20ml) and extracted with dichloromethane (3
x 25ml). The combined organic extract was washed with D.M. water (20ml), brine
(20ml), dried over sodium sulphate, filtered and concentrated in vacuo. The resulting
crude material was further purified by flash column chromatography using
dichloromethane: methanol as mobile phase whereby the desired product eluted at
around 1.0% methanol in dichloromethane. Distillation of the pure product fractions
provided methyl N-{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}
carbamate (0.065g, 37% yield). H-NM (DMSO- ) : d (ppm) 3.60 (s, 3H); 7.34-7.43
(m, 2H); 7.45-7.52 (m, 1H); 7.67 (brs, 1H); 7.92 (brs, 1H); 8.22-8.30 (m, 1H); 8.44 (s,
1H); 11.02 (brs, 1H); MS (ESI+): m/z = 255.4 [M+H]+
Selected compounds according to the invention are set forth in Table 1 below:
In some of the experiments below, the salt of these compounds may be used; for
example, the acetate salt of example 1 formed with acetic acid may be used.
Cytoprotection from ER stress (Test 1)
HeLa cells were cultured in Dulbecco's Modified Eagle's Media (DMEM) supplemented
with penicillin, streptomycin, containing 5% fetal bovine serum (FBS), at 37 °C in 5%
C0 2 atmosphere. Cells were plated in 24 well plates at a density of 15,000 cells/ml 24
hours prior treatment. ER stress was elicited by addition of fresh media containing 2.5
mg ml tunicamycin (Sigma-Aldrich) together with elF2a phosphatases inhibitors (0.2-
5mM) . Media were changed 6 h later with fresh media containing phosphatase
inhibitors (0.2-5 mM) . Inhibitors were dissolved in DMSO (50 mM) and DMSO was used
as a mock treatment. Cell viability was assessed by measuring the reduction of WST-8
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium]
into formazan using Cell viability Counting Kit-8 (Dojindo) according to the supplier's
recommendation, 48 h after tunicamycin treatment. Cytoprotection from ER stress is
measured in terms of the percentage increase in viable cells (relative to control) after
ER stress. The result for Example 1 of the invention is shown in Figure 1.
Assessment of translation rates in unstressed cells (Test 2)
HeLa cells (80,000 cells/ml) were plated in 12-well plates 24 h before each experiment
and either untreated or treated with compounds (50 mM) for 0.5, 1, 2.5, 5 and 7.5 h. At
the end of each time point, 30.6 m /GhI S-methionine (EasyTag, PerkinElmer) was
added to the culture medium for 10 min at 37 °C. Following labelling, cells were
washed with ice-cold PBS and lysed in 75 m I Laemmli Buffer. Lysates were sonicated,
boiled at 95 °C for 5 min and resolved on NuPAGE 4-12% gradient gels. Gels were
then stained with Coomassie Brilliant Blue R-250 and analyzed by phosphorimaging.
Assessment of translation rates in stressed cells (Test 3)
Treatments were performed as for measuring translation in unstressed cells, except
that Tunicamycin (2.5 g/ml) was added together with the compounds. The result for
Example 1 of the invention is shown in Figure 3.
Immunoprecipitations (Test 4)
HeLa cells (80,000 cells/ml) were plated the day before the indicated treatments,
transfected with GFP-PPP1 R15A or FLAG-PPP1 R15B expression plasmids using
Lipofectamine 2000 (Invitrogen) according to manufacturer's procedure. Two days
following transfection, cells were treated for 6 h with compounds (50 mM) and then
washed in PBS and lysed in IP buffer (50 mM Tris pH 7.4, 150 mM NaCI, 0.2% Triton
X-100, 10% glycerol, and EDTA-free protease inhibitor cocktail). Lysates were clarified
by centrifugation at 15,000g for 15 min at 4 °C and pre-cleared on protein G beads for
1 hour at 4 °C. Proteins were immunoprecipitated with 1.5 m I GFP antibody (JL-8,
Clontech, 632380), bound to 20 m I of protein-G-sepharose beads (GE Healthcare, 17-
0618-01). The beads were then washed 3 times with cold IP buffer and boiled in 50 m I
Laemmli Buffer (25 mM Tris-HCI pH 6.8, 1% SDS, 25 mM DTT, 7.5% Glycerol, 0.05%
Bromophenol blue). The immunoprecipitated protein complexes (17 m I) were separated
on 4-12% NuPAGE gradient gels (Invitrogen), transferred to Optitran BA-S 83
reinforced Nitrocellulose membrane and revealed with GFP and PP1 antibodies (sc-
7482, Santa Cruz).
Functional aequorin assay for Adrenergic a2A receptor (Test 5)
CHO-K1 cells coexpressing mitochondrial apoaequorin, Ga16 and recombinant human
Adrenergic a2A receptor grown to mid-log phase in culture media without antibiotics
were detached with PBS-EDTA, centrifuged and resuspended in DMEM/HAM's F12
with HEPES, without phenol red + 0.1% BSA protease free buffer at a concentration of
1x106 cells/ml. Cells were incubated at room temperature for at least 4h with
coelenterazine h. On each day of the test, reference agonist (UK14304) was tested to
evaluate the performance of the assay and determine EC50. Then, 50m I of cell
suspension was mixed with 50m I of test agonist in a 96-well plate. The resulting
emission of light was recorded using Hamamatsu Functional Drug Screening System
6000 luminometer. To standardize the emission of recorded light (determination of the
"100% signal") across plate and across different experiments, some of the wells
contained 100 mM digitonin or a saturating concentration of AT (20mM) .
Dose-response data from test compounds were analysed with XLfit ( IDBS) software
using nonlinear regression applied to a sigmoidal dose-response model.
The result for Example 1 of the invention is shown in Figure 4. Advantageously, in
contrast to Guanabenz, Example 1 is not considered to be a potent alpha-2 agonist.
This loss in alpha-2 adrenergic activity renders the compound therapeutically useful in
the treatment of the disorders claimed herein. The absence of alpha-2 adrenergic
activity means that the compound can be administered at a dosage suitable to treat the
disorders claimed herein, but without any significant effect on blood pressure, thereby
avoiding the need to co-administer with a known alpha-2 adrenergic antagonist (an
alpha blocker).
Selectivity assessment
Selectivity was inferred from the results of test 1, 2, 3, and 5:
A selective inhibitor of PPP1 R 15A should, protect cells from ER stress (Test 1) , does
not inhibit translation in non-stressed cells (Test 2), prolongs translation attenuation
after Tunicamycin (Test 3), and selectively dissociates PPP1 R15A-PP1
holophosphatase but not PPP1 R15b-PP1 holophosphatase (Test 4).
Results
The results of Tests 1 to 4 for selected compounds of the invention are shown below in
Table 1.
Table 1:
a inferred from translation inhibition in stressed/non-stressed cells
confirms selectivity towards PPP1 R15A-PP1 or lack thereof.
Cell-based assays:
Material and Methods
Cell Culture and Reagents 293T cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and transfected in 6- or
12- well plates by using the calcium phosphate method leading usually to 70%
transfection efficiency. Routinely, 45,000 cells/ml were plated before transfection as
described in (Rousseau et al. 2009). Myelin P0S63del-DSred construct was described
in (Pennuto et al. 2008), the Huntingtin construct was described in (Rousseau et al.
2009), the SOD1A4V construct is described in (Munch et al. 201 1) and the P23H
construct is described in (Mendes and Cheetham 2008).
Fluorescence Microscopy
Transfected cells were fixed with 4% paraformaldehyde and labeled with indicated
antibodies. Micrographs were taken at 100X magnification on a Leica TCS SP2AOBS
confocal microscope or Leica DMRB Fluorescence microscope.
Immunoblotting
Routinely, 70% confluent cells from a well of a 12-well plate were lysed in 140 m I of
boiling Laemmli buffer (25 mM Tris-HCI, pH 6.8, 1% SDS, 25 mM dithiothreitol, 7.5%
glycerol, 0.05% bromphenol blue) for immunoblot analysis. 18 m I of protein extracts
were loaded on 2-12% NuPAGE gels and transferred to Optitran BA-S 83 reinforced
nitrocellulose membrane (Whatman and Schleicher & Schuell). Equal loading of protein
extracts analyzed by immunoblot was controlled by Ponceau Red staining and vimentin
(data not shown). Membranes were satutated in 5% dried skimmed milk in phosphatebuffered
saline and probed with Htt 2b4 antibody or HA antibody to reveal HA-tagged
SOD1 . The appropriate secondary antibody coupled to peroxidase was revealed using
the SuperSignal West Pico Chemiluminescent kit (Pierce). Chemiluminescent images
were acquired using the Chemi-Smart 5000 (Vilber-Lourmat) allowing quantitative
detection of chemilumi-nescence. Signals of interest were quantified using ImageJ.
Assay for Charcot Marie Tooth 1B (Test 6)
Deletion of serine 63 from P0 (P0S63del) causes Charcot-Marie-Tooth 1B neuropathy
in humans and a similar demyelinating neuropathy in transgenic mice. The mutant
protein misfolds and accumulates in the ER, induces the UPR and fails to be
incorporated into myelin (D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). 293T
cells were transfected with labeled P0S63 del - P0S63del-DSred - and analyzed by
confocal microscopy, 48h post-transfection in the presence or absence of compound of
formula (I). In accordance with the methodology described in (Pennuto et al. 2008),
cells with ER-retained P0S63del-DSred were scored . Figure 4 shows that in untreated
cells, P0S63del accumulates in the ER but Example 1 prevents this accumulation.
Since accumulation of misfolded P0 causes CMT-1B and having shown that Example
1reduces accumulation of the disease-causing protein, the compound of formula (I)
should be useful to treat CMT-1B as well as other forms of CMT where the disease
causing-protein is misfolded and retained in the ER.
Assay for Huntington's disease and amyotrophic lateral sclerosis (Test 7)
We tested for accumulation of mutant huntingtin amino-terminal fragment (Htt48Q)
associated with Huntington's disease and SOD1 mutant (A4V), associated with
amyotrophic lateral sclerosis.
We used a method previously described in WO/2008/041 133. 293T cells were
transfected with plasmids encoding for Htt48 or SOD1 4V and treated with Example 1in
DMSO or DMSO alone 4 h post-transfection. SDS lysates collected 48 h posttransfection
were analyzed on a NuPAGE followed by immunoblot with Huntingtin
antibody (2B4) or HA (SOD1 ) . Figure 5 shows the quantifications of the signal on
immunoblots, normalized to untreated cells. Example 1reduces accumulation of both
proteins. Having shown that Example 1reduces accumulation of the proteins causing
Huntington's disease and Amyotrophic lateral sclerosis, the compound of formula (I)
should be useful to treat such diseases as well as other neurodegenerative diseases
caused by accumulation of misfolded proteins.
Assay for rhodopsin P23H aggregation (Test 8)
We tested for aggregation of rhodopsin associated for retinitis pigmentosa as described
(Mendes & Cheetham 2008).
293T cells were transfected with plasmid encoding the P23H mutant of rhodopsin and
treated with Example 1 in DMSO or DMSO alone 4 h post-transfection. Cells were
analyzed by microscopy. Figure 6 shows the cells with or without aggregates.
Example 1 reduces aggregates. Since accumulation of misfolded rhodopsin causes RP
and having shown that Example 1reduces accumulation of the disease-causing protein,
the compound of formula (I) should be useful to treat Retinitis Pigmentosa.
Examplesl and 6 are confirmed selective inhibitors of PPP1 R15A.
Examples 2,4,7,8 inhibit both PPP1 R 15A and B.
Various modifications and variations of the invention will be apparent to those skilled
the art without departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be unduly limited to
such specific embodiments. Indeed, various modifications of the described modes for
carrying out the invention which are obvious to those skilled in the relevant fields are
intended to be covered by the present invention.

CLAIMS
1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,
wherein:
R is alkyl, CI, F or Br;
R2 is H or F;
R3 is selected from H and alkyl;
R4 is selected from H and C(0)R 6;
R5 is H;
or R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7, OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclyl and aryl, each of which is optionally substituted with one or
more Rio groups;
each R-io is independently selected from halogen, OH, N0 2, CN, COO-alkyl, aralkyl,
S0 2-alkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each independently CRu, and Y is selected from CRu and N;
for use in treating a disorder associated with protein misfolding stress and in particular
with accumulation of misfolded proteins.
2 . A compound for use according to claim 1 wherein R is CI, Br, Me, H or F, more
preferably, CI.
3 . A compound for use according to claim 1 or claim 2 wherein R2 is H.
4 . A compound for use according to any preceding Y is CRu.
5. A compound for use according to any one of claims 1 to 3 wherein Yis N.
6. A compound for use according to any preceding claim wherein F¾ and R4 are
both H.
7. A compound for use according to any one of claims 1 to 5 wherein R3 is H and
R4 is C(0)R 6.
8. A compound for use according to claim 7 wherein Re is Me or OMe.
9. A compound for use according to any one of claims 1 to 5 wherein R and R 5
are linked to form a heterocyclic group which is optionally substituted with one or more
Rio groups.
10. A compound for use according to claim 9 wherein said compound is of formula
(la), or a pharmaceutically acceptable salt thereof,
wherein R , R2, R 3 and R10 are as defined in claim 1.
11. A compound for use according to any preceding claim which is selected from
the following:
Example 1

12. A compound for use according to claim 11 which is Example 1 or a
pharmaceutically acceptable salt thereof.
13. A compound for use according to claim 11 which is Example 15 or a
pharmaceutically acceptable salt thereof.
14. A compound for use according to claim 11 which is Example 16 or a
pharmaceutically acceptable salt thereof.
15. A compound for use according to any preceding claim wherein the disorder is
associated with PPP1 R15A-PP1 .
16. A compound for use according to any preceding claim wherein the disorder is a
neurodegenerative disorder.
17. A compound for use according to any one of claims 1 to 14 wherein the
disorder is Charcot Marie Tooth neuropathy or severe Dejerine-sottas syndrome.
18. A compound for use according to any one of claims 1 to 14 wherein the
disorder is a retinal disease, preferably retinitis pigmentosa, retinal ciliopathies,
macular degeneration or diabetic retinopathy.
19. A compound for use according to any one of claims 1 to 14 wherein the
disorder is selected from Alzheimer's disease, Parkinson's disease, ALS and
Huntingdon's disease.
20. A compound for use according to any one of claims 1 to 14 wherein the
disorder is type 2 diabetes.
2 1. A compound for use according to any one of claims 1 to 14 wherein the
disorder is cancer, preferably multiple myeloma.
22. A compound of formula (I) as defined in any one of claims 1 to 14 for use in
protecting cells from cytoxic ER stress.
A compound of formula (II), or a pharmaceutically acceptable salt thereof,
wherein:
R alkyl, CI, F or Br;
R2 is H or F;
R3 is selected from H and alkyl;
R4 is selected from H and C(0)R 6;
R5 is H;
or R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7,OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclic, aryl and heteroaryl, each of which is optionally substituted
with one or more Rio groups;
each R-io is independently selected from halogen, OH, CN, N0 2, COO-alkyl, aralkyl,
SCValkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each independently CRu, and Y is N; and
24. A compound of formula (III), or a pharmaceutically acceptable salt thereof,
wherein:
R is alkyl, CI, F or Br;
R2 is H or F;
R3 is selected from H and alkyl;
R4 is C(0)R 6;
R5 is H;
or R4 and R5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups;
R6 is selected from R7,OR7 and NR8Rg;
R7, R8 and R9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclic, aryl and heteroaryl, each of which is optionally substituted
with one or more Rio groups;
each R-io is independently selected from halogen, OH, CN, N0 2, COO-alkyl, aralkyl,
S0 2-alkyl, S0 2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl, N(alkyl) 2, CF3, alkyl and
alkoxy;
X and Z are each independently CRu, and Y is selected from CRu and N; and
25. A compound according to claim 24 of the formula (Ilia), or a pharmaceutically
acceptable salt thereof,
wherein R-i, R2, R3 and Rio are as defined in claim 2 1.
A compound of formula (IV), or a pharmaceutically acceptable salt thereof,
wherein:
R is alkyl or Br;
R 2 is H ;
R 3 is selected from H and alkyl;
R 4 is selected from H and C (0)R6;
R 5 is H ;
or R 4 and R 5 are linked to form a heterocyclic group which is optionally substituted with
one or more Rio groups; R 6 is selected from O R 7 and N R 8 Rg;
R 7 , R 8 and R 9 are each independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclyl and aryl, each of which is optionally substituted with one or
more Rio groups;
each R 0 is independently selected from halogen, O H , C N , N0 2, COO -alkyl, aralkyl,
S0 2-alkyl, S0 2-aryl, COOH , C O -alkyl, C O -aryl, N H 2 , N H -alkyl, N(alkyl)2, C F 3 , alkyl and
alkoxy;
X and Z are each C H and Y is CRu ;
27. A compound selected from the following, and pharmaceutically acceptable salts
thereof,
28. A pharmaceutical composition comprising a compound according to any one of
claims 23 to 27 admixed with a pharmaceutically acceptable diluent, excipient or
carrier.
29. Use of a compound of formula (I) as defined in any one of claims 1 to 14 in the
preparation of a medicament for treating a disorder associated with protein misfolding
stress in and particular with accumulation of misfolded proteins.
30. A method of treating a disorder associated with accumulation of misfolded
proteins in a subject in need thereof, said method comprising administering a
therapeutically effective amount of a compound of formula (I) as defined any one of
claims 1 to 14 to said subject.
3 1. A combination comprising a compound according to any one of claims 23 to 27
and a second active agent.
32. A compound of formula (I) as defined in any one of claims 1 to 14 for use in
treating a disorder associated with the PPP1 R15A pathway.