Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Treating Demyelinating Disease
[001] Introduction
[002] Wallerian degeneration is a process that occurs at the distal segment of
an injured nerve;
it results in the axon degeneration and demyelination of nerves distal to the
injury site. Myelin,
which surrounds the axon to provide insulation for nerve electronic
conduction, is composed of
the tightly packed, multi-layered specialized plasma membranes (myelin
sheaths) of Schwann
cells in the peripheral nervous system (PNS). Axon growth inhibitory proteins
such as myelin
associated glycoprotein (MAG) are known to be present in peripheral myelin
sheaths, and it has
been demonstrated that efficient demyelination is necessary before an injured
nerve can start to
regrow. After nerve injury, demyelination occurs in peripheral nerves in two
stages: an early
Schwann cell-mediated myelin clearance followed by immune cell infiltration
and macrophage-
mediated clearance of myelin debris. The known roles of Schwann cells in
demyelination
include processing myelin debris by phagocytosis and helping recruitment of
macrophages.
However, how the Schwann cells break down of their own intact myelin structure
in the first
place in response to nerve injury remains a mystery.
[003] Our laboratory has been conducting research on a seemingly-unrelated
cellular process,
necroptosis, a form of regulated necrosis triggered by the tumor necrosis
factor (TNF) family of
cytokines and the ligands of Toll-like receptors (TLRs); the death signal is
transduced by a pair
of receptor-interacting kinases (RIP1 and RIP3) and executed by the RIP3
kinase substrate (the
pseudokinase MLKL). In response to the activation of TNF receptors, RIP1
kinase is recruited
from the cytosol to become part of the receptor complex at the plasma
membrane. This complex
subsequently binds to RIP3 via interactions between the homophilic RIP
homotypic interaction
motif (RHIM) domains of RIP1 and RIP3, leading to RIP3 activation. Similarly,
in response to
the activation of TLRs (including TLR3 and 4), another RH1M-domain containing
protein,
TRIP, is able to bind and activate R1P3. Activated RIP3 then phosphorylates
mouse MLKL at its
serine 345/serine 347/threonine 349 sites (or human MLKL at its serine
357/threonine 358
sites). These phosphorylation events release the auto-inhibition imposed by
the pseudokinase
domain on the N-terminal membrane-disrupting helix bundle of MLKL, which then
causes
monomeric MLKL to form oligomers that translocate from the cytosol to
membranes by binding
to negatively charged phospho-lipids. The membrane-bound oligomeric MLKL
disrupts
membrane integrity and causes cells to undergo necrotic cell death.
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[004] Here, we report that following sciatic nerve injury the mixed lineage
kinase domain-like
protein (MLKL), a pseudokinase known to rupture cell membranes during
necroptotic cell
death, is induced and targets the myelin sheath membrane of Schwann cells to
promote myelin
breakdown. The function of MLKL in disrupting myelin sheaths requires injury-
induced
phosphorylation of serine 441, an activation signal distinct from the
necroptosis-inducing
phosphorylation by RIP3 kinase. Mice with their Mild specifically knocked out
in Schwann cells
showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve
regeneration
following injury, while overexpression of MLKL accelerated myelin breakdown
and promoted
the regeneration of axons, and pharmacological inhibition of MLKL inhibits
demyelination.
[005] Summary of the Invention
[006] The invention provides methods and compositions for inhibiting or
treating
demyelination (or treating demyelinating disease). In an aspect the invention
provides a method
of inhibiting or treating demyelination comprising administering to a person
in need thereof an
MLKL inhibitor.
[007] In embodiments:
[008] - the MLKL inhibitor is selected from:
(2E)-N-[4-[[(3-Methoxy-2-pyrazinypamino]sulfonyllphenyl]-3-(5-nitro-2-thieny1)-
2-
propenamide (Necrosulfonamide);
1,3,7-tri methy1-8-(methylsulfony1)-1H-puri ne-2,6(3H,7H)-di one (TC13-4);
8-(2,5-dimethoxybenzylsulfony1)-1,3,7-tritnethy1-1H-purine-2,6(3H,7H)-dione
(TC13-58);
7-ethyl-1,3-dimethy1-8-(methylsulfony1)-1H-purine-2,6(3H,7H)-dione (TC13-74);
1,7-dimethy1-8-(methylsulfony1)-3-(prop-2-yny1)-1H-purine-2,6(3H,7H)-dione
(TC13-106);
2-(1,7-dimethy1-8-(methylsulfony1)-2,6-dioxo-IH-purin-3(2H,6H,7H)-
y1)acetonitrile (TC13-
107);
3-(3-(3-chlorophenyl)prop-2-yn-1-y1)-8-((cyclopropylmethyl)sulfony1)-1,7-
dimethyl-3,7-
dihydro-1H-purine-2,6-di one (TC13-119);
84(2,5-dimethoxybenzypsulfony1)-1,7-dimethy1-3-(3-(2-(methylamino)pyridin-4-
ypprop-2-yn-
1-y1)-3,7-dihydro-IH-purine-2,6-dione (TC13-127);
3-(3-(3-hydroxyphenypprop-2-yn-l-y1)-1,7-dimethyl-8-(methylsulfonyl)-3,7-
dihydro-lH-
purine-2,6-dione (TC13-172); and
3-04-(methyl(4-(3-(4-(trifluoromethoxy)phenyOureido)phenypamino)pyrimidin-2-
ypamino)benzenesulfonamide (Compound 1).
[009] - the MLKL-inhibitor is a compound of formula I:
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0
R2
R3
> _________________ S(0)R1
wherein:
each of RI -R4 is independently H, or optionally substituted, optionally
hetero-, optionally cyclic
Cl-C18 hydrocarbyl, or optionally substituted heteroatom, and RI and R2 are
optionally joined
to form a ring;
n is 0, 1 or 2; and
or a pharmaceutically acceptable salt, hydride or stereoisomer the compound.
[010] In embodiments:
[011] - (a) one of R1-R4 is alkylcarbocylic, such as methylcyclopropyl;
(b) one of RI -R4 comprises a fluoroalkyl., such as CF3;
(c) R4 is alkylcyano or alkylCR, such as CH2CR wherein R is H, or optionally
substituted, optionally hetero-, optionally cyclic CI-C18 hydrocarbyl; and/or
(d) R1 and R2 are joined to form a ring, such as Rl/R2 are Me/Me or Me/Et
joined to
form a ring;
[012] - one, two or three of R1-R4 is Me;
n is 2;
one of R1-R4 is alkylcyano or alkylCR, such as CH2CR wherein R is H, or
optionally
substituted, optionally hetero-, optionally cyclic Cl-C18 hydrocarbyl; and/or
[013] - each of R1, R2 and R3 is Me;
n is 2; and
R4 is alkylcyano or alkylCR, such as CH2CR, wherein R is H, or optionally
substituted,
optionally hetero-, optionally cyclic CI-C18 hydrocarbyl,
[014] - wherein RI and R2 are joined to form a ring, such as Rl/R2 are Me/Me
or Me/Et
joined to form a ring.
[015] - the optionally substituted, optionally hetero-, optionally cyclic Cl-
C18 hydrocarbyl is
optionally-substituted, optionally hetero-, optionally cyclic alkyl, alkenyl
or alkynyl, or
optionally-substituted, optionally hetero- aryl; and/or
the optionally substituted heteroatom is halogen, optionally substituted
hydroxyl
(alkoxy), optionally substituted acyl (formyl, alkanoyl, carbamoyl, carboxyl,
amido), optionally
substituted amino (amino, alkylamino, dialkylamino, ami.do, sulfamidyl.),
optionally substituted
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thiol, (mercapto, alkylthiol, aryl thiol), optionally substituted sulfinyl or
sulfonyl (alkylsulfinyl,
arylsulfinyl, alkyl sulfonyl, arylsulfonyl), nitro, or cyano.
[016] - the compound comprises a formula of Table 1 or 2.
[017] - the method further comprising the antecedent step of diagnosing the
demyelination, or
the subsequent step of detecting a resultant amelioration of the
demyelination.
[018] - the method further comprises administering to the person a second,
different drug for
treating or inhibiting the demyelination.
[019] The invention encompasses all combinations of the particular embodiments
recited
herein.
[020] Description of Particular Embodiments of the Invention
[021] The following descriptions of particular embodiments and examples are
provided by
way of illustration and not by way of limitation. Those skilled in the art
will readily recognize a
variety of noncritical parameters that could be changed or modified to yield
essentially similar
results. The invention provides myriad embodiments.
[022] Unless contraindicated or noted otherwise, in these descriptions and
throughout this
specification, the terms "a" and "an" mean one or more, the term "or" means
and/or and
polynucleotide sequences are understood to encompass opposite strands as well
as alternative
backbones described herein.
[023] The methods and compositions may employ the compounds in any suitable
form and
dosage unit, including salts, prodrugs, stereoisomers, amorphous forms, etc.
[024] Demyelinating diseases include demyelinating myelinoclastic diseases and
demyelinating leukodystrophic diseases; demyelination may be localized to the
central nervous
system or the peripheral nervous system, and may be inflammatory or non-
inflammatory.
Demyelinating disorders of the CNS include: myelinoclastic disorders, standard
multiple
sclerosis, Devic's disease, CNS neuropathies, central pontine myelinolysis,
myelopathies like
tabes dorsalis (syphilitic myelopathy), leukoencephalopathies,
leukodystrophies, etc., and can be
associated with optic neuritis and transverse myelitis. Demyelinating diseases
of the peripheral
nervous system include Guillain¨Barre syndrome and its chronic counterpart,
chronic
inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy,
Charcot¨Marie¨
Tooth disease and its counterpart hereditary neuropathy with liability to
pressure palsy, copper
deficiency associated conditions (peripheral neuropathy, myelopathy, and
rarely optic
neuropathy) and progressive inflammatory neuropathy.
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[025] The term "pharmaceutically acceptable salts" is meant to include salts
of the active
compounds which are prepared with relatively nontoxic acids or bases,
depending on the
particular substituents found on the compounds described herein. When
compounds of the
invention contain relatively acidic functionalities, base addition salts can
be obtained by
contacting the neutral form of such compounds with a sufficient amount of the
desired base,
either neat or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition
salts include sodium, potassium, calcium, ammonium, organic amino, or
magnesium salt, or a
similar salt. When compounds of the invention contain relatively basic
functionalities, acid
addition salts can be obtained by contacting the neutral form of such
compounds with a
sufficient amount of the desired acid, either neat or in a suitable inert
solvent. Examples of
pharmaceutically acceptable acid addition salts include those derived from
inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric,
hydriodic,
or phosphorous acids and the like, as well as the salts derived from
relatively nontoxic organic
acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,
succinic, suberic,
fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,
tartaric, methanesulfonic,
and the like. Also included are salts of amino acids such as arginate and the
like, and salts of
organic acids like glucuronic or galactunoric acids and the like. Certain
specific compounds of
the invention contain both basic and acidic functionalities that allow the
compounds to be
converted into either base or acid addition salts.
[026] The neutral forms of the compounds may be regenerated by contacting the
salt with a
base or acid and isolating the parent compound in the conventional manner. The
parent form of
the compound differs from the various salt forms in certain physical
properties, such as
solubility in polar solvents, but otherwise the salts are equivalent to the
parent form of the
compound for the purposes of the invention.
[027] In addition to salt forms, the invention provides compounds which are in
a prodrug
form. Prodrugs of the compounds described herein are those compounds that
undergo chemical
changes under physiological conditions to provide the compounds of the
invention.
Additionally, prodrugs can be converted to the compounds of the invention by
chemical or
biochemical methods in an ex vivo environment. For example, prodnigs can be
slowly converted
to the compounds of the invention when placed in a transdermal patch reservoir
with a suitable
enzyme or chemical reagent. Prodrugs are often useful because, in some
situations, they may be
easier to administer than the parent drug. They may, for instance, be more
bioavailable by oral
administration than the parent drug. The prodrug may also have improved
solubility in
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pharmacological compositions over the parent drug. A wide variety of prodrug
derivatives are
known in the art, such as those that rely on hydrolytic cleavage or oxidative
activation of the
prodrug. An example, without limitation, of a prodrug is a compound of the
invention which is
administered as an ester (the "prodrug"), but then is metabolically hydrolyzed
to the carboxylic
acid, the active entity. Additional examples include peptidyl derivatives of a
compound of the
invention.
[028] Certain compounds of the invention can exist in unsolvated forms as well
as solvated
forms, including hydrated forms. In general, the solvated forms are equivalent
to unsolvated
forms and are intended to be encompassed within the scope of the invention.
Certain compounds
of the invention may exist in multiple crystalline or amorphous forms. In
general, all physical
forms are equivalent for the uses contemplated by the invention and are
intended to be within
the scope of the invention.
[029] Certain compounds of the invention possess asymmetric carbon atoms
(optical centers)
or double bonds; the racemates, diastereomers, geometric isomers and
individual isomers are all
intended to be encompassed within the scope of the invention.
[030] The term "therapeutically effective amount" refers to the amount of the
subject
compound that will elicit, to some significant extent, the biological or
medical response of a
tissue, system, animal or human that is being sought by the researcher,
veterinarian, medical
doctor or other clinician, such as when administered, is sufficient to prevent
development of, or
alleviate to some extent, one or more of the symptoms of the condition or
disorder being treated.
The therapeutically effective amount will vary depending on the compound, the
disease and its
severity and the age, weight, etc., of the mammal to be treated.
[031] The invention also provides pharmaceutical compositions comprising the
subject
compounds and a pharmaceutically acceptable excipient, particularly such
compositions
comprising a unit dosage of the subject compounds, particularly such
compositions copackaged
with instructions describing use of the composition to treat an applicable
disease or condition
(herein).
[032] The compositions for administration can take the form of bulk liquid
solutions or
suspensions, or bulk powders. More commonly, however, the compositions are
presented in unit
dosage forms to facilitate accurate dosing. The term "unit dosage forms"
refers to physically
discrete units suitable as unitary dosages for human subjects and other
mammals, each unit
containing a predetermined quantity of active material calculated to produce
the desired
therapeutic effect, in association with a suitable pharmaceutical excipient.
Typical unit dosage
forms include prefilled, premeasured ampules or syringes of the liquid
compositions or pills,
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tablets, capsules, losenges or the like in the case of solid compositions. In
such compositions, the
compound is usually a minor component (from about 0.1 to about 50% by weight
or preferably
from about 1 to about 40% by weight) with the remainder being various vehicles
or carriers and
processing aids helpful for forming the desired dosing form.
[033] Suitable excipients or carriers and methods for preparing administrable
compositions are
known or apparent to those skilled in the art and are described in more detail
in such
publications as Remington:s Pharmaceutical Science, Mack Publishing Co, NJ
(1991). In
addition, the compounds may be advantageously used in conjunction with other
therapeutic
agents as described herein or otherwise known in the art. Hence the
compositions may be
administered separately, jointly, or combined in a single dosage unit.
[034] The amount administered depends on the compound formulation, route of
administration, etc. and is generally empirically determined in routine
trials, and variations will
necessarily occur depending on the target, the host, and the route of
administration, etc.
Generally, the quantity of active compound in a unit dose of preparation may
be varied or
adjusted from about 1, 3, 10 or 30 to about 30, 100, 300 or 1000 mg, according
to the particular
application. In a particular embodiment, unit dosage forms are packaged in a
multipack adapted
for sequential use, such as blisterpack, comprising sheets of at least 6, 9 or
12 unit dosage forms.
The actual dosage employed may be varied depending upon the requirements of
the patient and
the severity of the condition being treated. Determination of the proper
dosage for a particular
situation is within the skill of the art. Generally, treatment is initiated
with smaller dosages
which are less than the optimum dose of the compound. Thereafter, the dosage
is increased by
small amounts until the optimum effect under the circumstances is reached. For
convenience, the
total daily dosage may be divided and administered in portions during the day
if desired.
[035] The compounds can be administered by a variety of methods including, but
not limited
to, parenteral, topical, oral, or local administration, such as by aerosol or
transdermally, for
prophylactic and/or therapeutic treatment. Also, in accordance with the
knowledge of the skilled
clinician, the therapeutic protocols (e.g., dosage amounts and times of
administration) can be
varied in view of the observed effects of the administered therapeutic agents
on the patient, and
in view of the observed responses of the disease to the administered
therapeutic agents.
[036] The therapeutics of the invention can be administered in a
therapeutically effective
dosage and amount, in the process of a therapeutically effective protocol for
treatment of the
patient. For more potent compounds, microgram (ug) amounts per kilogram of
patient may be
sufficient, for example, in the range of about 1, 10 or 100 ug/kg to about
0.01, 0.1, 1, 10, or 100
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mg/kg of patient weight though optimal dosages are compound specific, and
generally
empirically determined for each compound.
[037] In general, routine experimentation in clinical trials will determine
specific ranges for
optimal therapeutic effect, for each therapeutic, each administrative
protocol, and administration
to specific patients will also be adjusted to within effective and safe ranges
depending on the
patient condition and responsiveness to initial administrations. However, the
ultimate
administration protocol will be regulated according to the judgment of the
attending clinician
considering such factors as age, condition and size of the patient as well as
compounds potency,
severity of the disease being treated. For example, a dosage regimen of the
compounds can be
oral administration of from 10 mg to 2000 mg/day, preferably 10 to 1000
mg/day, more
preferably 50 to 600 mg/day, in two to four (preferably two) divided doses.
Intermittent therapy
(e.g., one week out of three weeks or three out of four weeks) may also be
used.
[038] The subject compounds may be employed alone or in combination with other
therapeutic agents. Combination therapies thus comprise the administration of
at least one
pharmaceutically acceptable crystalline or amorphous form of the compounds and
at least one
other therapeutically active agent. The subject compounds and the other
therapeutically active
agent(s) may be administered together in a single pharmaceutical composition
or separately and,
when administered separately this may occur simultaneously or sequentially in
any order. The
amounts of the subject compounds and the other therapeutically active agent(s)
and the relative
timings of administration will be selected in order to achieve the desired
combined therapeutic
effect. Thus in a further aspect, there is provided a combination comprising a
pharmaceutically
acceptable crystalline or amorphous form of the compounds together with one or
more other
therapeutically active agents.
[039] The compounds of the invention may be administered by any suitable route
of
administration, including both systemic administration and topical
administration. Systemic
administration includes oral administration, parenteral administration,
transdermal
administration, rectal administration, and administration by inhalation.
Parenteral administration
refers to routes of administration other than enteral, transdermal, or by
inhalation, and is
typically by injection or infusion. Parenteral administration includes
intravenous, intramuscular,
and subcutaneous injection or infusion. Inhalation refers to administration
into the patient's lungs
whether inhaled through the mouth or through the nasal passages. Topical
administration
includes application to the skin.
[040] The compounds of the invention may be administered once or according to
a dosing
regimen wherein a number of doses are administered at varying intervals of
time for a given
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period of time. For example, doses may be administered one, two, three, or
four times per day.
Doses may be administered until the desired therapeutic effect is achieved or
indefinitely to
maintain the desired therapeutic effect. Suitable dosing regimens for a
compound of the
invention depend on the pharmacokinetic properties of that compound, such as
absorption,
distribution, and half-life, which can be determined by the skilled artisan.
In addition, suitable
dosing regimens, including the duration such regimens are administered, for a
compound of the
invention depend on the disease or disorder being treated, the severity of the
disease or disorder
being treated, the age and physical condition of the patient being treated,
the medical history of
the patient to be treated, the nature of concurrent therapy, the desired
therapeutic effect, and like
factors within the knowledge and expertise of the skilled artisan. It will be
further understood by
such sidled artisans that suitable dosing regimens may require adjustment
given an individual
patient's response to the dosing regimen or over time as individual patient
needs change. Total
daily dosages range from 1 mg to 2000 mg.
[041] It is understood that the examples and embodiments described herein are
for illustrative
purposes only and that various modifications or changes in light thereof will
be suggested to
persons skilled in the art and are to be included within the spirit and
purview of this application
and scope of the appended claims. All publications, patents, and patent
applications cited herein,
including citations therein, are hereby incorporated by reference in their
entirety for all purposes.
[042] Examples
[043] We used surgical nerve injury (axotomy) in wild type, Rip3 knockout, and
Mlkl
knockout mice to induce axon degeneration and myelin breakdown. We found that
MLKL
protein expression was drastically elevated in the Schwann cells in the
injured nerves and
underwent a novel RIP3-independent phosphorylation event that activated MLKL
to target
myelin membranes to promote the initial breakdown of the myelin sheath
following nerve
injury. Our results demonstrate that MLKL functions in Wallerian degeneration,
that that
targeted inhibition of MLKL, including both genetic and pharmacological
inhibition, Wallerian
degeneration and associated demyelination.
[044] We disclose:
a) Knockout of Mlkl, but not Rip3, inhibits axotomy-induced m.yelin breakdown
b) Myelin breakdown following sciatic nerve injury requires expression of MLKL
in Schwann
cells;
c) Binding of MLKL to myelin sheath membrane requires an injury-induced
activation signal
d) Nerve injury-induced activation of MLKL requires phosphorylation of its
serine 441 residue
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e) Mimicking phosphorylation on S441 of MLKL breaks down myelin without nerve
injury
d) MLKL-dependent myelin breakdown promotes sciatic nerve regeneration
g) Overexpression of MLKL accelerated myelin breakdown and promoted the
regeneration of
axons, wherein ectopic expression of MLKL (e.g. by AAV) accelerates the
functional recovery
of injured nerves; and
h) Pharmacological inhibition of MLKL inhibits demyelination, as detailed
below.
[045] MLKL inhibitors
[1:146] Cell, 2012, 148, 213-227; Med. Chem. Comm., 2014. 5, 333-337:
(2E)-N44-[[(3-Methoxy-2-pyrazinyflamino]sulfonyl]phenyl]-3-(5-nitro-2-thieny1)-
2-
propenamide (Necrosulfonamide).
[047] Chem. Comm., 2017, 53, 3637-3640; PCT/CN2017/075248. 1,3,7-trimethy1-8-
(methylsulfony1)-1H-purine-2.6(3H,7H)-dione(TC13-4);
58-(2,5-dimethoxybenzylsulfony1)-1,3,7-trimethy1-1H-purine-2,6(3H,7H)-dione
(TC13-58)
7-ethyl-1,3-dimethy1-8-(methylsulfony1)-1H-purine-2,6(3H,7H)-dione (TC13-74)
1,7-dimethy1-8-(methylsulfony1)-3-(prop-2-yny1)-1H-purine-2,6(3H,7H)-dione
(TC13-106)
2-(1,7-dimethy1-8-(methylsulfony1)-2,6-dioxo-1H-purin-3(2H,6H,7H)-
ypacetonitrile (TC13-
107)
3-(3-(3-chlorophenyl)prop-2-yn-l-y1)-8-((cyclopropylmethypsulfony1)-1,7-
dimethyl-3,7-
dihydro-1H-purine-2,6-dione (TC13-119)
8-((2,5-dimethoxybenzypsulfony1)-1,7-dimethy1-3-(3-(2-(methylamino)pyridin-4-
ypprop-2-yn-
1-y1)-3,7-dihydro-1H-purine-2,6-dione (TC13-127)
3-(3-(3-hydroxyphenyl)prop-2-yn-1-y1)-1,7-dimethy1-8-(methylsulfony1)-3,7-
dihydro-1H-
purine-2,6-dione (TC13-172).
[048] Proc Natl Acad Sci USA, 2014, 111, 15072-15077; W02015172203:
3-04-(methyl(4-(3-(4-(trifluoromethoxy)phenyOureido)phenypamino)pyrimidin-2-
yl)amino)benzenesulfonamide (Compound 1)
[049] Additional active MLKL inhibitors are disclosed in PCT/CN2018/077464,
including
compounds of Tables 1 and 2, below.
[OM Table 1. Series 1 MLKL inhibitors
o
/
'N'ItXr`j
),-NJ NI-SH ONJ I rl-S I
ON N 0 0 N 0
3 4
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O 1 --------------- 0 --------------- 0 , 0 ,
/ ,
N)LINi N'ILCI\lµ N )(Xi N \ /¨ N
/2--S/¨
O N N 0 N N \\0 I /)¨
0 N N 0 0 N N
I I I I
6 7 8
o o o 0
/
/
/ N N .I)LN¨sr-.<1
CD''N
O N N 0 0 N N N N \o
I I I I
9 10 11 12
O 1 o o
Nµ
I //¨ ..;c) -',N._s/-0F3 "-N--11=-=1\1___si=0F3
O N N o 0 N N 6 0
1 o N N \\0
1 1
13 14 15
r _pH
0 , A 0 , ___________________________________________________________
/
/
O , /
s.
I
= I 1
17
16
0.... ________________________________ o
o o
N 11
N * *
N'ILK õ.0
N'it'l 0.' H.sc)
/
0 N 'ILK ____
''''N N 0 N
= 1 e's N N 0 I y0
19 I 0.'N N 0
21 I
20 2'7
0
11 0 . o ______________
i_7 11 N . 1 r\L II
/ N
N. = Nf 0
I e
0 - y-it ,.()
0 "--4- 0-...)
'N N 0 /
N
23 I
H
:24 0."". N N 0
II...NH
I
H
N--,----\
o=K,L;S
N
I-I
F F 0
O / * 0 * /
/ N * F
'FµI)LX1\1\ Th\1N\ Ni1)1/>_,0
ce-N N 0
O N N 0 0 N N 0 1
I I 28
26 27
11
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ci CI 0 Br
''1\l'i, Nµ . CI /
lik
N lik N'jN> . j I /)-,õ
0-'''-' N N C''' r\l'1, Nµ
,.L I /)-c)
y-i'LT _,, j
0-'N N/ e I 0'"" -N N 0
0 I I
I 31
30 32
29
Br 0 / 0-
O / 0 0
/ . -,-N N * Br 0 /
N
1AK o A ,,,0 ,
0'.- -N II
0-'-'N N 0 I 0.'" -N N 0
I o N N I o
I 36
34
33
0 02N 0 /
/
N
. 0' 0 r /I O
0-"-N N-
a_ 1 0 )Ni N 4.
1 _s,r,
` 0 y)1 ___.*
8 L'
1 I
37 38 39
0 NNNN '
CF3
o / 'y'icN 1,
cF3
8'-
1 41
NC CN 0 NH2
O 0 /
/
=
C
N I* N 0 -y)No
Ce'N N 0 NI
1
44
42 43 45
0 0 ________________________________ 0
NH2 / 0 /
0 /4)
/ 'roc" II NH2 'y--IKN \
N
m II 0
Nil")LI 0
(:).'N N r
Ce."NI N 0 I I
I
47
48
46
e o -------------------------- 0 a /
oe i
o / pi=\/-01 o N
'.=NrNH,/.7-0N1
8'-
A.,
()- -N N 0 I
0 ..'"N N 0-o I
I I
49 51 52
12
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o ci a a a
0 0 1 0
1N-0 /
,k.....isi
. /
. ,
O N N 0 '.sy)IXN_s
I I
ON N r 0 N N 0 CI 0 N N 0 CI
I I 1
53 54- 55 56
CI Me0 OMe Me0
O 1 0 0 0
/ /
. i
'''N)L1, N \ ris, let
=.'"-ItiN * ¨..,D//-1:., OMe
0.. . \I I 1/-1 Br
4.0
0-' - N N O'cb O''N N 0 OMe 0 N N 0 0 . N 0
I I I
57 58 59
i i i c 1
0 0¨ 0 0 C I
0
N' Br
. f,, ,
O N N 0 *,,i, 1 , .'¨ .'1.0 ,.., .,i=- /7--10
0 N N 0
I 0
1 \
61
62 63
64
Et P r0 MO Me0
0 9 , o o /
N "AX N'/- 11,iµ
1 I 14=0 J 1 ,.)---S, *
0 N N 0 0 M1A e 0 .c.).=,,N, N CO .... 1 /7¨h) j., I
//¨=;(:)
I OMe0,1
E1 0 N N 0 OPr
I 1 I
67 68
66
/ Me0 Me0
0 Br 0 0 0
O H
/
CI F \.õ ,
/7---h_,
7--11x
jõ r , 0 ¨N N 0' h, - OMe 0' - OMe
0 N N 000 tZ 11'0
I \ I I 0-....'N '' -- 0
69 70 71 I
72
,
o r¨ 0
r--- 0 0
-N-111---N / - N N / ...... ====..
A N)-1--
C31'''N"---1\1 N, 0 N N 0
I
I
I I
.73 74 75 76
o o
\.) \si
/
/ 0
i /
/>¨sS
O '1\1`---N 8.'`-',-, oN'"---N y-11" ,,>_s J
I ,>---S-;==
I I c"."'N N Nb 0
0 N N =''' "
0
I I
77 78
79 80
o 0 0
"s) 0 N
r-I
-.... N /¨ /---.<
N
,s -..N
N /
0 N /
O.-N1 N N N õ 8 ¨
1 1 N 0 0
81 82 I I
13
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PCT/CN2019/091478
83 84
o o 0 / 0
o rk
NH, 0 ri(
NH .)-1...,1\i / AN
HN 1 HN /
/
N)L---H N \ N N , 1 ---S
,i , ---,S,---
0¨NI'----N N 0-,¨N---N o'o 0 N N 0"--N N 0
I I I
85 86 87 88
o / o
/ o
/Ni
/ \ /
01\1"---N r (D" "N N (:;.¨ 0-'NJ"---N S.-
r,
0.--N-----N O'õ''
1 1 1
I
90 91.
89 92
o ._-- ------------- o ------ 0
/
rft. N 1 _ gt
,$)N ,J, g--0 ,A,
I I 0
.1111- ONi ,N N 0 0 NI, - 0 0 T
N 0
94
93 96
(D,' no o / n y
0-N N
/
NoN N
I o N ¨
IC 0 '1\l'iCN /
8 N I
A, 1 n ON I IN.I 0
¨S* II 0
b' = - '
I H
98
97 99
o o i o .. o
1 /
/
NN /
KI / ,,_..ni A -. 1 /
N 1 N 1 \ 1 \J____ 40
01\1----N O'C) 0 N N (:) 0 0 N N 0 0 N '---- NH. 0 0
H L'7 40
100
101 102 103
\,0 o
11 /
S;
\N--( N)
\ K
0,L1rN o N "õ, 0
Sq
L' \
1-1 SQ
H
NNN,N,N Nfi = NH
"NVNO/N/Nr`s
0 0 liN--0 o HN
o
104 105
14
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0 / 0 0
-. N K, / =-=, N / N /
ON -.-N 0'
L.,,,,....õ
1--,..,. ...,
...-- N
106 0
107
108
o / 0 / o 0 /
.*INFAXKI / N)Li=N \ /
0 N N
O N No ON NO 0 N N 0 -
=-,õ 0.'"" WI
\ , ta6
0. Ali
* lir 0,1 I.
F
OH
109 110 111 112
O / o o , 0
N \ _ / N )\, 1\ il /
---
K... 0 N N /¨<1 --. N ,
6' 0 (:)
0 N N 0 0 N N 0
Mr ,....õ
0 N,,
N...õ N.,.
N.õ.
N H2 1110 110
H N \
113 115 OH
114
116
O o 0 0
.- NNl'Iµ /¨<1 0
O N
_...4....N '1(plir-H.,c)
)1X N_C---<H
0 0 N
,0
.XILIX ,Hc..,
11
N 0o 0 N 0
',..'. # ,,,
-,,,
110 10
-=,.,
0
....-
F CI F
117
118 119 120
O o o 0
N N r¨K1
..'N'IL----- NI ir---(1
A ...0
1
oNN0 ---õ,õ,
oNN0 0 N N 0
C;N----N O0'
..õ
,..
,,,,._ -.. nV'ivi
-...,
= CI
110
1. Br OyNH
121 122 ,...0 NH2
124
123
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O / 0 ------------------------------------------
0/
),;', 9 j>.
0 1 1
oNo 0
ON N N
140 0 /
'N-M=xN 'i 11
-.
I ON I 1-0 /0
N ..,
126 H.T.D.....".--)-,
N ---
NO2 -- =-,
I
N .'
125
127
oi m e 0
0
/
y
0
--11.X õ>___,(1--
N C?
NII)- ___is 0 0-0- N N 3 0 OM e
/
Of%1 N ilk OM s
H.10,1
...- ====., Me() 11111"
I
N 128 129
0 0 o __ /
/ /
ni /
,.. N 9 ,.
1AL, NH/c) N ,
N)tx ____s_ a, 1 ¨1.,.0
0." -N N 0
Ce'N N 0 0 N N 0
'--= ..............1.i ti
",..----
iµ1"=:-"N 0
130 131 132
o / -------------- o ----------- 0
=-, N /,¨K1 ------------ o j
N'A'---- /
/
N)IsK i.,.?z...
I H(:) _
0 0 0 N NN / __ <
0 0 N N 0 0.1.
-:"'N N 0
0 110 IP
F F CI
F
133 135 136
134
1\ /¨ 0 0 0 /
,N_4
,t 1 ../¨w.:0
-11), .L1\1 /¨<1
N 1 NA`---"N / ¨cl N N / --------
¨<1
)1X /
0';'"'NH.c, I Hc) 11'0
0 N N 0 0 N N 0 ON N 0
I
-...,(-
a 40 40 40
CI Br
137 138 139 Br
140
o o 0 o
_nrk y )c N\_ /¨<1 N , N /¨< '-wN /¨<
0=2--N N O - 10.N1 N 8,-,- o N N o o N N 8 s-)
40 t
Br N / LT) tIN
N
141 142 1
143 44
16
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0 0 o / OH 0
0
/ '.=N)-L,....- N / Mv)1X, NI\ /---/
N)=.,-- Rf'-1
N-S.
O'c' ,L .,--,0 0 N N 0 ONN 0
0 N N\ 0
I
-.., -..,
145 146 0 ....,
1101
OH
OH
147
148
o o o / 0
\
\ / N¨ N¨
N 11'11, _\ (----:0 IV H N
N \ /--/
J ---N.C.---) H2 ,k)
Or'\ N /
0
ss
N N 0 0
N 0 0 N N = -... N N (3
o 0
-.., ..,
...,, -....,
...,
I.Si 101
1101
OH OH
OH
HO
150 151 152
149
0_ 1-0H / 0 _/ 0
0 / 0 0
/
/ 0 N N NH2
1)11 H=0
0 N N 0 - ,), I -i--:.=.0 0 N NO
0 0 N N 0
0 N N
\--- RP ia., --õ,
-..., -.....
OH 0 01
OH OH
OH
153 154 156
155
O o o 0
/ \ / /OH
N
N NH N ,N¨ /--' 1\11 /
¨<1
trill
1, / \
-Kx
xN
, ,
,
0 0 N 0 N N
N 0 0
0., -...,
====,, -.......
-...,...
0,
=0 ill Oi
OH HNO H N ......0
OH
157
158
-,=k,.. L.....,
159 160
O o 0 3c 0 /
0
'N-1N\ i¨< -.... N / 0 ,,IL KJ /
I /7¨S .., N '.1'1 ._ N 1 0 /
.'' N N i'zc- cy0."- N N d 0
O'N N 0
".....'"- 0 ,,, i ii¨r0
,,,
II I 10 -...,
-.,
HNO
0 ---,
S
H N ,i I
0
L',.. HN / N
H N
163
17
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161 162 164
o / 0 / o / 0
-. -1(,...Ni / `rs1) / \ ,IL,NI / \ /
N /
N , \ * ;
I S,
i 1 ii---S,,, ON No I o )-11
O1µr-N u O o N N o o N N '0 o
L
W.-
iaõ
L,..,,c) ..,,
-,, il, oili Br
I HN
N 0 N
4
H 10 H
NH2
165 167 168
166
0 0 0 o
`NA):Nh N
\ / -.. -11,-Ni /---.<
, \
O N N cj 0 0-"- -N N 0 - 0--- -N N 0 -
I /1¨:=0
0 N N 0
¨
'===õ''' nail NH \ IP At \
LIP
\ ris
IIW Y\--
N=NINI--%
\
HN HN NH2
169
170 171 172.
o o o 0
N)---, N\ /¨<1 .N)1¨, NI\ / 0-'N N
N 1 \
0
I 0
01\J --'- N CYr
8 _..1, I //-1;=
"- -N N 0 -r,
N N 8 u
" - 0
\ S ----
\ r
=N 1 / W/
N I NH
HN¨
HNõ L......,,N,,
HN,, C)
1'75
174
173 176
[051] Table 2, Series 2 MLKL inhibitors
----------------------------------------------------------------- ,
1 2 3 4
O / OH HO
ON N 00
I cp'.N N 6 so
I I I
6 7 8
OH \ \o 0
0
O 0 0 N r\
).--li /
I ¨5,0
/
N N / 0 N N
i-s
y).H. ; . -.N)---, N, /
ON N 8 ON .m 11'0
, 0 I 0 =,.
I I 0 1\1---'N
1 110
OH
9 10 11 12
18
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0 0 0 0
/ 1 / 1
)c.....-N/ I .,0
, µ
N (:) N N \ 1,.
y--il-- / .......%
Ce's' N N 0 NN (31.."N N
ONIN
\. ti,.,
I. 1. ) ir IW''
NH 40 NH
HN if 0.),"
0 .õ.Ø..,.........",,o,",õ.-
NH
rh-3¨ 14 15 16
0 0 0 0
IN
N 11,0 .`,1,1)1,õN/ I ,C) '1,,,, N/ c,0 ', N N -
;,0 *)t / .,. .....%
N N 0 N N 0 N N 0 N N
-,,, ==,,
0
IVNH r.,..N.......,.......AH -. N.,,-.,,,,, NH
õ............s,,,,,.....õ NH
0.) I
17 18 ' 19 20
0 0 0
/ 0
'.' N A"---1\j/ I %C) '.1\l'it---"N I ===() -.
..k.,_. ./ e 1-0
1N
I 4\ --- %
Ci"¨ N N
ONN 0 N N ONN
'`=.
-..,
F
--,,.
r-
r.....N.,...-..y. NH 0 ,,õNH .Ni...---....,...õNH
N---ir NH
0,,.) 0
0 ,...,,..I 0 ),,.)
21 ' 2) 23 ' 24
o o 0
i ,),
N 1 ,,0
N e
ce'' N N .._...so
0..-1\1 N
ON N
0 N N
'.... At, OMe \
SP
\ r.,, ........, CI
'..,
e.,-...N....-....,_,AH
0 HN--iNH I
N ,...,- N
(3)
N H 0 1
r----N-Thr NH2
0,,J o
25 26 27 28
19
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o ---------------------------------- 0 --------------------------- 0 ,
A /
r 1 / 0
N I ,0 `ThriLx N i 1 ,0
N K.,-- N/ I,o
ilK -itxN, e
0 N N 0 N N 0 N N
H
0..' N 0
H H --... H
, N 0 . .'C ,,,,
4,6 N 0 , N 0
0 1 '-'1
Mr I 0
OyNH 1\ õ.0
ONH OINH Oy N H L,0
i--- N )
29
Oj 0
30 31 32
0
i
N./ N/ e e Ni
N zc.:1
0 H 0 N N
".==== 0 N
) N Cl'-. N N
L
(NH )13
. N H 0 c
I K I
ON H
0 I
NI
0,NH
OyNH
)
A re-N
0)
33 ' 34 35 ' 36
0
o o 0
N
N/ r..,) ,.. NI/ . e NiL,...-. N / .1::
C i>..,0 N
Ce' N N 0 N N ..... H
0 T
'1,,,., .,..,,, ci ''' I L =\,,,,,,c N '''')
OyANH 1,......,0
N 1
HN .1A H N ,i H N ,,,,,,=
1
37 38 39 40
0 o 0 0
/ 1 / 1 / 1
/ c0 N 1,0 N (3
N N
)13: ,>____ N'jtX
[ 1*
11 ._...,0
(DN N
H H .,`,,, H
,..,
AI N NH
0 -,õ
wi N ''''. rõ
il -V
0 1
(NH 1 (DINH
OINH OxNH
A
41 42 43 44
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o o 0 o ,
/ 1
N 1-0
j
/ i
0...''N N O''''N N ONN e''N N 0
'..
'e o '=
'. * 0 o ,,,, '''' 106 cF3
o 1
WI
N e N
ONH 1
OINH
OX
NH IN H
A
45 46 47 48
0 0
O 0
/ (A/ rA N/ 1 N/ 1
y-'1x N
y'jjrsiõ),g,00
,1)11
e''N N Ce"N N 0 N N
\
\ H
C F3 1W1
46 N 0
''''.-ci.,CF3
I I
I N ,, N
()INN
O NH
ONH OxNH y
A A
49 50 51 52
o 0 0
N/ 1 'I' N/ (0
- IV)N 0 4
y
IrjLK ___s..0 ".11..
I %
(2N N
o N N
,=,, ,=,, H H
ris6 CF3
1W.' W AtI
N 0
itr 1 =,', H
- N o
1401
OINH axNH Ox NH 0INH
53 54 ' 55 56
0
O o ,
/ ) /
NN r
g
00- N N c,
Ni'Lri HN N HN,
0.'N N rrilx
... H
WI 1 1
-, ra6 N 0
lir 1 oxNH
INN
57 58 .
59 60
o , o 0
NN H N -...../ --N-JLIN I-I / \ 0 /,N¨Z--
0
1 ,>---,0
0-N N 0 0 N NJ 0
I 1 ON N 8
1 1
61 62 63 64
21
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'
F3C 0 0 0
0
/ HN)
-... N 1õ0 MiNi -'/N) 1 ,---S..-:=0 'YT'
N'AT ,..._s,0
ON N ON"/----N 6 o'1\1 N dN
o-N N
65
1 1 1
66 67 68
o o 0 0
'-.N-k..-Nr--1 eN Nr--1,0 li-jtXN/---ko N
11--- 'C) 0 N N
0NN ICIN N H
.,.
-, i=.,, N,0
0 ir A
,=,., \,
0 0 r/N.Ns.NH OINH
0,,)
OH OH
69 70 71 72
o o o
S / / ,
N 1 0 Th\J)10 N 'k--"" N/ 1 %.0
/
N
/ O
--., 0 N N 0 N N Ce'N N
0 N Ni F
,,\ H =,\. H
L IP
, 14,6, A N,,0 -, ra.6.
FIP I F A
oxNH OX NH
Ox NH
73 ' 74 75 ' 76
0 0 0 / ,
/ A 1
N 1...0
N' N N HN N N i,
N N
1
H
s=-=.,. N 0 0
0.INH 0,xNH
0,xNH OINH
77 78 79 80
/ 0 0 / oF3 o
cF,
----CIN__I:c00 10 ''''N'A'N s* --"c
N N '-'0
0 N N
FNI,e0
IP A
OINH OINH OINH OINH
81 ' 82 83 ' 84
2_,-,1
,
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O HN -- n \
.... N -- o
o
Ni I ,o NI I ,o
y N r
I .0 1 õ0
--1" ;
)----co itx N
0..... N N 0.....N N O''' N N
H ''' Fti 0
,....., '...' N NI ,,.0 -. AI N 0 N
I / A
WI I
0.1 NH ONH Ox NH Ox. NH
85 86 87 88
o 0 D
0 r\ 0 r,,
µ1\1H
H--,,c,
N 0 0 N N 0
(=,,r.,,,r,
ilk N .....e0
1115 A -.., H
'", N 0
OX
N H 0,x NH ONH
Allikk OyA NH
----------------------------------------------------------------- ,
89 90 91 92
o D o o
/ 1
N/ , , 0 N'ItX, NLL P
N AI
-0
N HN, N N
-..., H ONN ...-., F
=-..,, iith N
VP' A ,.....õ H
N 0 --- 0 ..õ,.Ø-.. -
..' 0
0
()INN 0 I Ox NH Ox NH
Oy N H
A
93 94 95 96
o o o 0 CF
1 0 ,
/ , / N , .0 N 1, '''' N A N ---c k N
I \ 1(. )I '-"SO *'
N N 0 N N 0 N N c)
0 N
0,, 0..
-...,
0,,,,... ..-- -....,,
õ--...0/ 0 0 -,,, 5
0INN Ox NH
0.1 NH Ox NH
97 98 99 100
23
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----------------------------------------------------------------- -.
0
N/ (D0
NN >H
//- H
_I_ 1 eo
0-"" 'N N 0 N
0 0
0 ..,,T, , -,.õ
.,,,. .., 0
N N 0 '.
: 0.õõ..---,, ...-= 0 0, 0 0,,,
ax NH Oy NH Ox NH Ox NH
A
----------------------------------------------------------------- ,
101 102 103 104
0 c D3 0 0
i ,. ,
D3C,N N ic D3 D3C.N N,C D3 pa3
0
A ,,.. I ,,,, ,CD
D3C,N...ix[\( ,cD3
0 N N 0 0 0 N N 0
0....N 1 r \,]>¨%0
',....õ `....... ',......
dis6
0 11,011 ',...... 11
*
HN,...õ,0,,,o,,
OH 0 NH revNH
/
0,)
105 106 107 108
N N/ i m,
0 o
µ: 0 o
osc,
CD / C D3 ..õ,
D3C,N.,11xNif N /
---, H N, H ..õ...
Ali N 0 '`, 0
Mr
, N
ax N H OINH NH OX
NH
tc A
109 110 111 112
O c D3 0 ,
/ 0
/ 0
)*L_ /
NA 1 D3C. N /
Irjt1X
">--1:-0 m 0
0 N N 0
0
0-'- 'N N 0
--, N 0 ....,
',.., NN 0 /
,...,
0 0
o OINH
(DINH
HN1>
/
HN
113 114 115 116
o o o o
A ">"*"11=''0
1)IX />"1,4''ID jijIX .>-1=zb 21:04
N H Li.S/ H1 ii0 H 1.,,,,c_N H
Nz---/ 0 N/0
0 NO N 1 N/J * Nr N/i . Nr
0INH
ax NH OINH (DINH
24
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117 118 119 120
0 0 0 0
,c0 1
N N g D3c, ,ruu3
N N 0 N NO 0 s
'I1X, N" 1] , ). 1N NO ).X (DI N NI>-- 3
OH(,/,!
/ so NO N NJ /pi (j'.1
OINH OINH OINH INN
[052] Synthesis
[053] Synthetic schemes for the Series 1 and Series 2 MLKL inhibitors are
disclosed in
PCT/CN2018/077464.
[054] Pharmacological Activity Assays
[055] To evaluate hMLKL inhibitors we used AAV to deliver hMLKL into Mk' KO
mice, and
we confirmed that the hMLKL functions normally in myelin breakdown as iriMLKL.
We then
evaluated the relative activities of hMLKL inhibitors to block myelin
breakdown of hMLKL,
and confirmed efficacy of our inhibitors in this model. Our fluorescent
micrography results
show myelin breakdown of untreated nerves and inhibition by hMLKL inhibitor
NSA
(Necrosulfonamide), and non-inhibition by a negative control, and our
quantitative data
measuring intact myelin sheathing demonstrated that our MLKL inhibitor
substantially inhibited
d.emyelination,
[056] To evaluate the relative activities of our hMLKL inhibitors we generated
humanized
mice by inserting a hMLKL cDNA to the loci of mouse MTh' gene, and bred them
into
homozygous. Using the homozygous mice, whose niMLKL is not expressed, but
hMLKL
expressing well, we confirmed that hM1_,KL. functions normally in myelin
breakdown as
[057] The sciatic nerve functional recovery assay was performed as previously
described
(Baptista et al., 2007). Ex vivo cultured sciatic nerves were treated with
DIVISO or with the
MLKL inhibitors. Cross sections of nerves were immunostained with antibodies
against MBR
Quantification shows that MLKL inhibitors block myelin breakdown.
[058] We then evaluated the relative activities of hM1_,K1... inhibitors to
block myelin
breakdown. of hMl...K-1.õ and confirmed efficacy of our inhibitors M this
model. Fluorescent
micrography results demonstrated myelin breakdown of untreated nerves at day 0
(DO) and day
3 (D3), inhibition by inhibitor compounds 172 (1)3+172) and. 249 (D3+236),
and. non-inhibition
by control compound 235 (1)3+236), and our quantitative data measuring intact
myelin
sheathing demonstrated that our MUM, inhibitors substantially inhibited
demyelination.
CA 03104504 2020-12-18
WO 2019/242576 PCT/CN2019/091478
0
Ni 0 =J 0 `-=
11õ, 11 11 .-'"Is40
1
'skNk,
.***3
OH 0y, ,i4111 NH
es's
TC013172 TC013249 T00132313
CO2 rM C50t11 .3 rttx4 105021:73 uM
[059] We also established and validated two animal models treating
demyelination or treating
demyelinating disease and confirming efficacy of our hMLKL inhibitors.
Experimental
Autoimmune Encephalomyelitis (EAE) is a widely-accepted model of demyelinating
diseases,
such as multiple sclerosis (MS). We initially used an EAE model (see, McCarthy
et al., Methods
Mol Biol. 2012 ; 900: 381-401) to demonstrate that genetically inhibiting MLKL
decreases
disease symptoms in this multiple sclerosis disease models by monitoring
disease progression in
WT and mike- mice. We also used a chemically induced demyelination model,
confirming
these results, wherein we evaluated immunofluorescence analysis of brain
sections from
cuprizone induced demyelination model in WT and mike- mice. In pilot studies
our hMLK
inhibitors similarly inhibit demyelination and disease progression in both the
EAE and
cuprizone induced demyelination models.
[060] Series 1 compound activity, sciatic nerve recovery assay.
1 0.1-10pM 2 0.1-10pM 3 0.1-10p M. 4 0.1-
10pM
0.1-10pM 6 10-100pM 7 10-100pM 8 1-100nM
9 1-100nM 10 1-100nM 11 1-100nM 12 1-100nM
13 1-10011M 14 1-100nM 15 1-100nM 16 0.1-10pM
17 1-100nM 18 0.1-10pM 19 1-100nM 20 1-100nM
21 1 -100nM 22 1-100nM 23 0.1-10p M 24 1- 100nM
25 1-100nM 26 1-100nM 27 1-100nM 28 1- 1000/1
29 1-100nM 30 1-100nM 31 1-100nM 32 1-100nM
33 1-100nM 34 1-100nM 35 1-100nM 36 1-100nM
37 1-100nM 38 1-100nM 39 1-1.00nM 40 1-100nM
41 1-100nM 42 1-100n M 43 1-1.00nM 44 1-100nM
45 1-100nM 46 0.1-10p M 47 0.1-10p M 48 0.1-10pM
26
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49 1 -1000p M 50 1-100n M. 51 0.1-10pM 52 0.1-10pM
53 0.1-10pM 54 1-100nM 55 10-100p M 56 10-100p M
57 10- 100p M 5= 8 10-100p M 59 10-100p M 60 1= 0-100p M
61 10-100p M 62 0.1-10p M 63 0.1-10p M 64 0.1-1.0p M
65 1-100nM 66 1-1 00n M 67 1-100nM 68 10-100p M
69 0.1-10p M 70 1 -1000nM 71 1-1. 000nM. 72 1-1000n M
73 1-1000nM 74 1 -1000nM 75 1-1. 000nM. 76 1-1000nM
77 1-1000n M 78 1 - 1000n M 79 1-1000n M 80 1-1000nM
81 1-1000n M 82 1-1000n M 83 1- 1()00n M 84 1-1000nM
1
85 1-1000n M 86 1-1000n M 87 1- 1()00n M 88 1-1000nM
89 1 -1000n M 90 1-1000n M 91 1-1000n M 92 1-
1.000nM
93 1 -1000n M 94 1-1000n M 95 1 -1000n M 96 1- 1000nM.
97 1 -1000nM 98 1-1000nM 99 1-1000nM 100 1-1000nM.
101 1 -1000n M 102 1-1000nM 103 1-1000n M 104 1- 1000n
M
105 1-1000n M 1= 06 1-1000nM 107 1-1.000n M 108 1= -
10(X)nM
109 1-1000n M 110 1-1000nM 111 1-1000nM 112 1-1000nM
113 1-1000nM 114 1-1000nM 115 1-1000nM 116 1-1000nM
117 1-1000nM 118 1-1000nM 119 1-1.000nM. 120 1-1000nM
[061] Series 2 compound activity; sciatic nerve recovery assay.
1 0.1-10p M 2 0.1-10p M 3 0.1-10p M 4 0.1-10p M
0.1-10pM 6 10-100p M 7 10-100p M 8 1-1.00nM
9 1-100nM 10 1-100nM 11 1-100nM 12 1-1.00nM
13 1-100nM 14 1-100nM. 15 1-1001(1M 16 0.1-10pM
17 1 -100nM 1= 8 0.1-10p M 19 1-100nM 20 1-= 100nM
21 1-100nM 2= 2 1-100nM 23 0.1-10p M 24 1= -100nM
25 1-100n M 26 1-100n M 27 1-1.00n M 28 1-100nM
29 1-100n M 30 1-1 00n M 31 1-100nM 32 1-100nM
33 1-100n M. 34 1 -100nM 35 1-1.00nM 36 1-100nM
37 1-100nM. 38 1-100nM 39 1-1. 00n M 40 1-100nM
41 1-100n M 42 1 - 100n M 43 1-100nM 44 1-100nM
45 1-100n M 46 0.1-10p M 47 0.1-10p M 48 0.1-10p M
1
49 1-1000p M 50 1-100n M 51 0.1-10p M 52 0.1-10p M
27
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53 0.1-10p. M 54 1-100n M. 55 10-100p M. 56 10-
10011M
57 10-100p M 58 10-100p M 59 10-100p M 60 10-100p M
61 10-100p M 62 0.1-10p M 63 0.1-10p M 64 0.1-10p M
65 1-100n M 66 1-100n M 67 1-1 00n M 68 10- 1()Op M
69 0.1-1.0p M 70 1-1000nM 71 1-1000nM 72 1-1000n M
73 1-1000nM 74 1 -1000n M 75 1-1. 000n M. 76 1-1000nM
77 1-1000nM 78 1 -1000n M 79 1-1. 000n M. 80 1-1000nM
81 1-1000n M 82 1 - 1000n M 83 1-1000n M 84 1-1000nM
85 1-1000n M 86 1-1000nM 87 1-1()00nM 88 1-1000nM
1
89 1-1000n M 90 1-1000nM 91 1-1()00nM 92 1-1000nM
93 1 -1000n M 94 1-1000n M 95 1-1000n M 96 1-1. 000nM
97 1 -1000n M 98 1-1000n M 99 1 -1000n M 100 1-1000nM.
101 1 -1000n M 102 1-1000nM 103 1 -1000n M 104 1- 1000n
M.
105 1 -1000n M 106 1-1000nM 107 1-1000n M 108 1- 1000n
M
109 1-1000n M 110 1-1000nM 111 1-1000n M 112 1-1((X)nM
113 1-1000n M 114 1-1000nM 115 1-1000nM 116 1-1000n M
117 1-1000nM 118 1-1000nM 119 1-1000nM 120 1-1000n M
28
