Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-INFLAMMATORY COMPOSITIONS COMPRISING IRAK AND JAK
INHIBITORS
FIELD OF THE INVENTION
[0001] The present invention relates to compositions, useful in the
prophylaxis and/or treatment of
inflammatory diseases, autoimmune diseases, proliferative diseases, allergic
diseases, transplant
rejection, diseases involving impairment of cartilage turnover, congenital
cartilage malformations,
and/or diseases associated with hypersecretion of IL6 or interferons. In a
particular aspect, the
composition comprises a first compound having IRAK inhibitory activity, and a
second compound
having JAK inhibitory activity. The present invention also provides
pharmaceutical compositions
comprising the combination of the invention and methods for the prophylaxis
and/or treatment of
diseases including inflammatory diseases, autoimmune diseases, proliferative
diseases, allergic
diseases, transplant rejection, diseases involving impairment of cartilage
turnover, congenital cartilage
malformations, and/or diseases associated with hypersecretion of IL6 or
interferons by administering
the combination of the invention.
BACKGROUND OF THE INVENTION
[0002] Kinases are involved in many essential processes of cell physiology,
for example protein
phosphorylation. In particular, protein and lipid kinases are involved in the
activation, growth,
differentiation, and survival of cells. Protein kinases can be divided between
those preferentially
phosphorylating tyrosine residues, and those preferentially phosphorylating
serine and/or threonine
residues.
[0003] Over the years, kinases have grown to become very important targets for
the development of
anti-inflammatory drugs (Cohen, 2009) . In particular, Interleukin-1 receptor-
associated kinases
(IRAK), and more particularly IRAK-4 have been identified as playing a role in
inflammation and
autoimmune diseases (Ringwood and Li, 2008; Wang et al., 2009).
[0004] IRAKs are expressed in many cell types and mediate signals from various
cell receptors
including interleukin-1 (IL-1) and toll-like receptors (TLRs). In the IRAK
family, 4 members have
been identified namely IRAK 1-4 (Wang et al., 2009), and IRAK-4, the newest
member of the family
represents an attractive therapeutic target (Li et al., 2002). Indeed, IRAK-4
is believed to be the key
protein kinase activated early downstream of the IL-1 receptor and TLRs
(except TLR3), initiating
signaling via rapid activation of IRAK-1 and IRAK-2, leading to innate immune
responses. Also,
other interleukins, such as IL-18 and IL-33, are dependent on IRAK-4 for
signaling. As such, diseases
for which these cytokines are involved in the pathogenic process (e.g.,
fibrosis (Li et al., 2014;
McHedlidze et al., 2013; Rankin et al., 2010) and atopic dermatitis (Salimi et
al., 2013)) are potential
target diseases for treatment by IRAK-4 inhibitors.
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[0005] In mice expressing an inactive IRAK-4 mutant instead of wild type,
complete resistance to
septic shock triggered by several TLR agonists as well as impaired response to
IL-1 is observed.
Furthermore, mice expressing an inactive IRAK-4 mutant instead of wild type
are partially protected
in several models of auto-immune diseases, such as rheumatoid arthritis
(Koziczak-Holbro et al.,
2009) and multiple sclerosis (Staschke et al., 2009). Interestingly, the serum
of rheumatoid arthritis
and systemic lupus erythematosus patients has been shown to activate
plasmacytoid dendritic cells in
an IRAK-4 dependent manner (Chiang et al., 2011). Finally, recurring pyogenic
bacterial infection has
been observed in children suffering from genetic defects leading to IRAK-4
inactivity. As these
pyogenic infections are not observed in adults carrying inactivating IRAK-4
mutations, the IRAK-4
signaling system appears to be redundant for certain aspects of adult innate
immunity.
[0006] The dysregulation of signaling components of the innate immune system
is also increasingly
being recognized as an important factor in cancer initiation and progression
(Rhyasen and
Starczynowski, 2015). Indeed, there is evidence that IL-1 plays a direct role
in tumor cell growth,
angiogenesis, invasion, drug resistance, and metastasis (Carmi et al., 2013;
Vidal-Vanaclocha et al.,
2000). Additionally, TLRs are involved in a multitude of protumor responses,
depending on the tumor
cell context. As essential mediators of IL-1 receptor and TLRs signaling, IRAK
family kinases
represent promising cancer drug targets. In addition, several cancer types
have been shown to be
dependent on activated forms of MYD88, an adaptor molecule downstream of the
TLR and IL-1R,
which activates IRAK-4. Activating MYD88 mutations have been identified in
e.g., diffuse large
B-cell lymphomas (DLBCL) (Ngo et al., 2011), and in Waldenstrom
macroglobulinemia (Treon et al.,
2012). Another report supports the role of IRAK-4 in the field of oncology, T-
cell acute lymphoblastic
leukemia (T-ALL) in particular (Li et al., 2015). The pharmacological
inhibition of IRAK-4 has been
shown to enhance the sensitivity of T-ALL to chemotherapeutic agents.
[0007] IL-33 has been shown to play a role in the development of fibrotic and
allergic diseases,
asthma and atopic dermatitis in particular (Nabe, 2014). As this cytokine
signals through an IRAK-4
dependent pathway (Kroeger et al., 2009) , these diseases might also represent
a target for IRAK-4
inhibitors.
[0008] Finally, several auto-inflammatory diseases have been shown to be
dependent on IL-1 activity
and, as a consequence, IL-1 blocking biologicals show some benefit to these
patients. Gout, juvenile
idiopathic arthritis, Muckle-Wells disease, familial Mediterranean fever,
Behget's disease, adult onset
Still's disease are examples of such auto-inflammatory diseases (Dinarello et
al., 2012).
[0009] The inhibition of cytokine signaling with small molecules may help in
reducing disease
outcome in immune-inflammatory diseases (Sundberg et al., 2014). In
particular, cytokines may play a
role in the defense of organisms against pathogens and infections. However,
when developing new
therapies for immune-inflammatory diseases, it is crucial on one hand to
select a target involved in a
pathway that can be inhibited without compromising the adaptive and/or innate
immune responses
since the simultaneous inhibition of multiple cytokine response pathways may
excessively weaken the
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immune system. However, drug selectivity towards kinases is difficult to
achieve (Bain et al., 2003;
Fabian et al., 2005), but is highly desirable in order to avoid off-target
associated side effects,
particularly in the context of chronic treatments (Brockman et al., 2011; Dy
and Adjei, 2013; Force
and Kolaja, 2011) .
[0010] In particular, it was recently shown that concomitant use of an IL-1
blocking agent (Anakinra)
and a TNFa blocker (Etanercept) resulted in increased risk of neutropenia and
infection. (Genovese et
al., 2003, EMEA public statement EMEA/31631/02, 05 Feb 2003). This finding
highlights that
selectivity is a crucial element when developing new medicines, and therefore,
it would be desirable to
develop compounds that are able to selectively modulate a signaling pathway
without affecting others,
in particular compounds able to selectively modulate IL-1 response, without
affecting TNFa signaling
pathways.
[0011] Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transduce
cytokine signaling from
membrane receptors to STAT transcription factors. Four JAK family members are
described, JAK1,
JAK2, JAK3 and TYK2. Upon binding of the cytokine to its receptor, JAK family
members auto-
and/or transphosphorylate each other, followed by phosphorylation of STATs
that then migrate to the
nucleus to modulate transcription. JAK-STAT intracellular signal transduction
serves the interferons,
most interleukins, as well as a variety of cytokines and endocrine factors
such as EPO, TPO, GH,
OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. et al. (2008)).
[0012] The combination of genetic models and small molecule JAK inhibitor
research revealed the
therapeutic potential of several JAKs. JAK3 is validated by mouse and human
genetics as an immune-
suppression target (O'Shea J. et al. (2004)). JAK3 inhibitors were
successfully taken into clinical
development, initially for organ transplant rejection but later also in other
immuno-inflammatory
indications such as rheumatoid arthritis (RA), psoriasis and Crohn's disease
(http://clinicaltrials.gov/).
[0013] TYK2 is a potential target for immuno-inflammatory diseases, being
validated by human
genetics and mouse knock-out studies (Levy D. and Loomis C. (2007)).
[0014] JAK1 is a novel target in the immuno-inflammatory disease area. JAK1
heterodimerizes with
the other JAKs to transduce cytokine-driven pro-inflammatory signaling.
Therefore, inhibition of
JAK1 and/or other JAKs is expected to be of therapeutic benefit for a range of
inflammatory diseases
as well as for other diseases driven by JAK-mediated signal transduction.
[0015] The current therapies are not satisfactory and therefore there remains
a need to identify further
compounds with reduced off-target related side effects that may be of use in
the prophylaxis and/or
treatment of inflammatory diseases, autoimmune diseases, proliferative
diseases, allergic diseases,
transplant rejection, diseases involving impairment of cartilage turnover,
congenital cartilage
malformations, and/or diseases associated with hypersecretion of IL6 or
interferons.
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 describes the clinical scores in a therapeutic CIA model for the
illustrative Compound 1 at
1 mg/kg bid (filled triangles), 3 mg/kg bid (crosses), 10 mg/kg bid
(asterisks), and 30 mg/kg bid (filled
circles) compared to vehicle (filed diamonds) over the period from day 31 days
to day 46.
Figure 2 describes the clinical scores in a therapeutic CIA model for the
illustrative Compound XXb
at 7.5 mg/kg bid (filled triangle), and 15 mg/kg bid (tilted crosses), and the
Compound 1 (30 mg/kg
bid) + Compound XXb (15 mg/kg bid) (filled circles) compared to vehicle
(filled diamonds) from day
31 to day 41.
SUMMARY OF THE INVENTION
[0016] The present invention is based on the identification of composition of
the invention, useful in
the prophylaxis and/or treatment of inflammatory diseases, autoimmune
diseases, proliferative
diseases, allergic diseases, transplant rejection, diseases involving
impairment of cartilage turnover,
congenital cartilage malformations, and/or diseases associated with
hypersecretion of IL6 or
interferons. In a particular aspect, the composition comprises a first
compound having IRAK
inhibitory activity, and a second compound having JAK inhibitory activity.
[0017] The present invention also provides pharmaceutical compositions
comprising the combination
of the invention and methods for the prophylaxis and/or treatment of diseases
including inflammatory
diseases, autoimmune diseases, proliferative diseases, allergic diseases,
transplant rejection, diseases
involving impairment of cartilage turnover, congenital cartilage
malformations, and/or diseases
associated with hypersecretion of IL6 or interferons by administering the
combination of the
invention.
[0018] In a particular aspect, the JAK inhibitor has JAK1 inhibitory activity.
In a more particular
embodiment, the JAK inhibitor is a JAK1 selective inhibitor.
[0019] In another particular aspect, the IRAK inhibitor has IRAK4 inhibitory
activity. In a more
particular embodiment, the IRAK inhibitor is a IRAK4 selective inhibitor.
[0020] Accordingly, in a first aspect of the invention, a composition of the
invention is provided
comprising:
a) a compound according to Formula I :
R1 ,,ON,
0
2
RNNN------N
I
Cy / N
\
,
ON
I
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wherein
Cy is
- monocyclic C3_7 cycloalkyl optionally substituted with one or more
independently selected R3, or
- 4-7 membered monocyclic heterocycloalkyl comprising one or two
heteroatoms independently
selected from N, S, and 0, optionally substituted with one or more
independently selected R3;
R1 is
-H,
- -S03H,
- -P(=0)(OH)2,
- C1_4 alkyl,
- -C(=0)-(4-7 membered monocyclic heterocycloalkyl comprising one or two
heteroatoms
independently selected from N, S, and 0), or
- -C(=0)C1_6 alkyl, which Ci_6 alkyl is optionally substituted with one or
more independently
selected R4 groups;
R2 is H or C1_4 alkyl;
each R3 is independently selected from:
-OH,
- =0,
- halo, and
- C1_4 alkyl;
each R4 is independently selected from:
- -NR5aR5b,
- -C(=0)0H,
- 4-7 membered monocyclic heterocycloalkyl comprising one or two
heteroatoms independently
selected from N, S, and 0, optionally substituted with one or more
independently selected C1_4
alkyl, and
- -NHC(=0)-C1_4 alkyl-NH2; and
R5' and R51' are independently H or C1_4 alkyl;
or a pharmaceutically acceptable salt or a solvate or the salt of a solvate
thereof; and
b) a second compound having a JAK inhibiting activity.
[0021] In a particular aspect, the compositions of the invention are provided
for use in the
prophylaxis and / or treatment of inflammatory diseases, autoimmune diseases,
proliferative diseases,
allergic diseases, transplant rejection, diseases involving impairment of
cartilage turnover, congenital
cartilage malformations, and/or diseases associated with hypersecretion of IL6
or interferons.
[0022] In one aspect, the combination of the invention may inhibit the IRAK
kinase family members,
and more particularly IRAK-4.
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[0023] In one aspect, the combination of the invention may inhibit the JAK
kinase family members,
and more particularly JAK1.
[0024] In a further aspect, the combination of the invention may show
selectivity towards IRAK-4
and JAK1, which may result in improved safety and lower off-target related
side effects. In a
particular aspect, the combination of the invention may be selective
inhibitors of IL-1.
[0025] In yet a further aspect of the invention, it has also been unexpectedly
demonstrated that the
combinations of the invention exhibit increased efficacy compared to each
member taken individually.
In a particular aspect, the combination of the invention may show a synergy,
which in turn may allow
for a reduced dosage of each component of the composition of the invention
This may be
advantageous in avoiding taking unnecessary drug amounts while maintaining
efficacy, and thus
reducing the risk of adverse drug events.
100261 In a further aspect, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and a pharmaceutical carrier, excipient or
diluent. In a particular aspect,
the pharmaceutical composition may additionally comprise further
therapeutically active ingredients
suitable for use in combination with the compounds of the invention. In a more
particular aspect, the
further therapeutically active ingredient is an agent for the treatment of
inflammatory diseases,
autoimmune diseases, proliferative diseases, allergic diseases, transplant
rejection, diseases involving
impairment of cartilage turnover, congenital cartilage malformations, and/or
diseases associated with
hypersecretion of IL6 or interferons.
[0027] Moreover, the compositions of the invention, useful in the
pharmaceutical compositions and
treatment methods disclosed herein, are pharmaceutically acceptable as
prepared and used.
[0028] In a further aspect of the invention, this invention provides a method
of treating a mammal, in
particular humans, afflicted with a condition selected from among those listed
herein, and particularly
inflammatory diseases, autoimmune diseases, proliferative diseases, allergic
diseases, transplant
rejection, diseases involving impairment of cartilage turnover, congenital
cartilage malformations,
and/or diseases associated with hypersecretion of IL6 or interferons, which
method comprises
administering an effective amount of the pharmaceutical composition or
combinations of the invention
as described herein.
[0029] The present invention also provides pharmaceutical compositions
comprising a composition of
the invention, and a suitable pharmaceutical carrier, excipient or diluent for
use in medicine. In a
particular aspect, the pharmaceutical composition is for use in the
prophylaxis and/or treatment of
inflammatory diseases, autoimmune diseases, proliferative diseases, allergic
diseases, transplant
rejection, diseases involving impairment of cartilage turnover, congenital
cartilage malformations,
and/or diseases associated with hypersecretion of IL6 or interferons.
[0030] In additional aspects, this invention provides methods for synthesizing
the compounds of
compositions of the invention, with representative synthetic protocols and
pathways disclosed later on
herein.
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[0031] Other objects and advantages will become apparent to those skilled in
the art from a
consideration of the ensuing detailed description.
[0032] It will be appreciated that compounds of the invention may be
metabolized to yield
biologically active metabolites.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0033] The following terms are intended to have the meanings presented
therewith below and are
useful in understanding the description and intended scope of the present
invention.
[0034] When describing the invention, which may include compounds,
pharmaceutical compositions
containing such compounds and methods of using such compounds and
compositions, the following
terms, if present, have the following meanings unless otherwise indicated. It
should also be understood
that when described herein any of the moieties defined forth below may be
substituted with a variety
of substituents, and that the respective definitions are intended to include
such substituted moieties
within their scope as set out below. Unless otherwise stated, the term
"substituted" is to be defined as
set out below. It should be further understood that the terms "groups" and
"radicals" can be considered
interchangeable when used herein.
[0035] The articles 'a' and 'an' may be used herein to refer to one or to more
than one (i.e. at least
one) of the grammatical objects of the article. By way of example 'an
analogue' means one analogue
or more than one analogue.
[0036] 'Alkyl' means straight or branched aliphatic hydrocarbon having the
specified number of
carbon atoms. Particular alkyl groups have 1 to 6 carbon atoms or 1 to 4
carbon atoms. Branched
means that one or more alkyl groups such as methyl, ethyl or propyl is
attached to a linear alkyl chain.
Particular alkyl groups are methyl (-CH3), ethyl (-CH2-CH3), n-propyl (-CH2-
CH2-CH3), isopropyl (-
CH(CH3)2), n-butyl (-CH2-CH2-CH2-CH3), tert-butyl (-CH2-C(CH3)3), sec-butyl (-
CH2-CH(CH3)2), n-
pentyl (-CH2-CH2-CH2-CH2-CH3), n-hexyl (-CH2-CH2-CH2-CH2-CH2-CH3), and 1,2-
dimethylbutyl
(-CHCH3)-C(CH3)H2-CH2-CH3). Particular alkyl groups have between 1 and 4
carbon atoms.
[0037] `Alkenyl' refers to monovalent olefinically (unsaturated) hydrocarbon
groups with the number
of carbon atoms specified. Particular alkenyl has 2 to 8 carbon atoms, and
more particularly, from 2 to
6 carbon atoms, which can be straight-chained or branched and having at least
1 and particularly from
1 to 2 sites of olefinic unsaturation. Particular alkenyl groups include
ethenyl (-CH=CH2), n-propenyl
(-CH2CH=CH2), isopropenyl (-C(CH3)=CH2) and the like.
[0038] `Alkylene' refers to divalent alkene radical groups having the number
of carbon atoms
specified, in particular having 1 to 6 carbon atoms and more particularly 1 to
4 carbon atoms which
can be straight-chained or branched. This term is exemplified by groups such
as methylene (-CH2-),
ethylene (-CH2-CH2-), or -CH(CH3)- and the like.
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[0039] `Alkynylene' refers to divalent alkyne radical groups having the number
of carbon atoms and
the number of triple bonds specified, in particular 2 to 6 carbon atoms and
more particularly 2 to 4
carbon atoms which can be straight-chained or branched. This term is
exemplified by groups such
as -CH2.-CC-, and -C(CH3)H-CCH-.
[0040] `Alkoxy' refers to the group 0-alkyl, where the alkyl group has the
number of carbon atoms
specified. In particular the term refers to the group -0-C1.6 alkyl.
Particular alkoxy groups are
methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-
pentoxy, n-hexoxy, and
1,2-dimethylbutoxy. Particular alkoxy groups are lower alkoxy, i.e. with
between 1 and 6 carbon
atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
[0041] 'Amino' refers to the radical -NH2.
[0042] 'Aryl' refers to a monovalent aromatic hydrocarbon group derived by the
removal of one
hydrogen atom from a single carbon atom of a parent aromatic ring system. In
particular aryl refers to
an aromatic ring structure, monocyclic or fused polycyclic, with the number of
ring atoms specified.
Specifically, the term includes groups that include from 6 to 10 ring members.
Particular aryl groups
include phenyl, and naphthyl.
[0043] `Cycloalkyl' refers to a non-aromatic hydrocarbyl ring structure,
monocyclic, fused
polycyclic, bridged polycyclic, or spirocyclic, with the number of ring atoms
specified. A cycloalkyl
may have from 3 to 12 carbon atoms, in particular from 3 to 10, and more
particularly from 3 to 7
carbon atoms. Such cycloalkyl groups include, by way of example, single ring
structures such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
[0044] `Cyano' refers to the radical -CN.
[0045] 'Halo' or 'halogen' refers to fluor (F), chloro (Cl), bromo (Br) and
iodo (I). Particular halo
groups are either fluoro or chloro.
[0046] Iletero' when used to describe a compound or a group present on a
compound means that one
or more carbon atoms in the compound or group have been replaced by a
nitrogen, oxygen, or sulfur
heteroatom. Hetero may be applied to any of the hydrocarbyl groups described
above such as alkyl,
e.g. heteroalkyl, cycloalkyl, e.g. heterocycloalkyl, aryl, e.g. heteroaryl,
and the like having from 1 to 4,
and particularly from 1 to 3 heteroatoms, more typically 1 or 2 heteroatoms,
for example a single
heteroatom.
[0047] Ileteroaryl' means an aromatic ring structure, monocyclic or fused
polycyclic, that includes
one or more heteroatoms independently selected from 0, N and S and the number
of ring atoms
specified. In particular, the aromatic ring structure may have from 5 to 9
ring members. The heteroaryl
group can be, for example, a five membered or six membered monocyclic ring or
a fused bicyclic
structure formed from fused five and six membered rings or two fused six
membered rings or, by way
of a further example, two fused five membered rings. Each ring may contain up
to four heteroatoms
typically selected from nitrogen, sulphur and oxygen. Typically the heteroaryl
ring will contain up to 4
heteroatoms, more typically up to 3 heteroatoms, more usually up to 2, for
example a single
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heteroatom. In one embodiment, the heteroaryl ring contains at least one ring
nitrogen atom. The
nitrogen atoms in the heteroaryl rings can be basic, as in the case of an
imidazole or pyridine, or
essentially non-basic as in the case of an indole or pyrrole nitrogen. In
general the number of basic
nitrogen atoms present in the heteroaryl group, including any amino group
substituents of the ring,
will be less than five.
[0048] Examples of five membered monocyclic heteroaryl groups include but are
not limited to
pyrrolyl, furanyl, thiophenyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl,
oxatriazolyl, isoxazolyl,
thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups.
[0049] Examples of six membered monocyclic heteroaryl groups include but are
not limited to
pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl.
[0050] Particular examples of bicyclic heteroaryl groups containing a five
membered ring fused to
another five-membered ring include but are not limited to imidazothiazolyl and
imidazoimidazolyl.
[0051] Particular examples of bicyclic heteroaryl groups containing a six
membered ring fused to a
five membered ring include but are not limited to benzofuranyl,
benzothiophenyl, benzoimidazolyl,
benzoxazolyl, isobenzoxazolyl, benzisoxazolyl, benzothiazolyl,
benzoisothiazolyl, isobenzofuranyl,
indolyl, isoindolyl, indolizinyl, purinyl (e.g. adenine, guanine), indazolyl,
pyrazolopyrimidinyl,
triazolopyrimidinyl, and pyrazolopyridinyl groups.
[0052] Particular examples of bicyclic heteroaryl groups containing two fused
six membered rings
include but are not limited to quinolinyl, isoquinolinyl, pyridopyridinyl,
quinoxalinyl, quinazolinyl,
cinnolinyl, phthalazinyl, naphthyridinyl, and pteridinyl groups. Particular
heteroaryl groups are those
derived from thiophenyl, pyrrolyl, benzothiophenyl, benzofuranyl, indolyl,
pyridinyl, quinolinyl,
imidazolyl, oxazolyl and pyrazinyl.
[0053] Examples of representative heteroaryls include the following:
N
$ " " ,N ,N
N
I I
Y Y Y N.N-- -.N-:-
H
.õõ...N.z....õ
N /
N N N
0 N,
I N 0 ,N \
N Y Y Y
wherein each Y is selected from >C=0, NH, 0 and S.
[0054] `Heterocycloalkyr means a non-aromatic fully saturated ring structure,
monocyclic, fused
polycyclic, spirocyclic, or bridged polycyclic, that includes one or more
heteroatoms independently
selected from 0, N and S and the number of ring atoms specified. The
heterocycloalkyl ring structure
may have from 4 to 12 ring members, in particular from 4 to 10 ring members
and more particularly
from 4 to 7 ring members. Each ring may contain up to four heteroatoms
typically selected from
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nitrogen, sulphur and oxygen. Typically the heterocycloalkyl ring will contain
up to 4 heteroatoms,
more typically up to 3 heteroatoms, more usually up to 2, for example a single
heteroatom. Examples
of heterocyclic rings include, but are not limited to azetidinyl, oxetanyl,
thietanyl, pyrrolidinyl (e.g. 1-
pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), tetrahydrofuranyl (e.g. 1-
tetrahydrofuranyl, 2-
tetrahydrofuranyl and 3-tetrahydrofuranyl), tetrahydrothiophenyl (e.g. 1-
tetrahydrothiophenyl, 2-
tetrahydrothiophenyl and 3 -tetrahydrothiophenyl), pip eridinyl (e.g. 1 -
piperidinyl, 2-pip eridinyl, 3 -
piperidinyl and 4-piperidinyl), tetrahydropyranyl (e.g. 4-tetrahydropyranyl),
tetrahydrothiopyranyl
(e.g. 4-tetrahydrothiopyranyl), morpholinyl, thiomorpholinyl, dioxanyl, or
piperazinyl.
[0055] As used herein, the term `heterocycloalkenyP means a `heterocycloalkyr,
which comprises at
least one double bond. Particular examples of heterocycloalkenyl groups are
shown in the following
illustrative examples:
C ,Z
Y Y Y Y
Z W
r z
Y Y YNõ\ /N¨Z
wherein each W is selected from CH2, NH, 0 and S; each Y is selected from NH,
0, C(=0), SO2, and
S; and each Z is selected from N or CH.
[0056] Particular examples of monocyclic rings are shown in the following
illustrative examples:
1-Dy -1, __ )
1-c, vj
,
. i--\
< . X
Y Y Y
W W Y
r
Y Y N¨w
wherein each W and Y is independently selected from -CH2-, -NH-, -0- and ¨S-.
[0057] Particular examples of fused bicyclic rings are shown in the following
illustrative examples:
7"--- ______ ---""
YL;¨-------...
Y
Y ,
L---------/ Y /
Y
wherein each W and Y is independently selected from -CH2-, -NH-, -0- and ¨S-.
[0058] Particular examples of bridged bicyclic rings are shown in the
following illustrative examples:
W
L\
c----- --,
-,---r- Y \ .-c----- -- \
Y
wherein each W and Y is independently selected from -CH2-, -NH-, -0- and ¨S-
and each Z is selected
from N or CH.
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[0059] Particular examples of spirocyclic rings are shown in the following
illustrative examples:
wherein each Y is selected from -CH2-, -NH-, -0- and ¨S-.
[0060] 'Hydroxyl' refers to the radical -OH.
[0061] `Oxo' refers to the radical =0.
[0062] 'Substituted' refers to a group in which one or more hydrogen atoms are
each independently
replaced with the same or different substituent(s).
[0063] `Sulfo' or `sulfonic acid' refers to a radical such as ¨S03H.
[0064] `Thior refers to the group -SH.
[0065] As used herein, term 'substituted with one or more' refers to one to
four substituents. In one
embodiment it refers to one to three substituents. In further embodiments it
refers to one or two
substituents. In a yet further embodiment it refers to one substituent.
[0066] `Thioalkoxy' refers to the group ¨S-alkyl where the alkyl group has the
number of carbon
atoms specified. In particular the term refers to the group -S-Ci 6 alkyl.
Particular thioalkoxy groups
are thiomethoxy, thioethoxy, n-thiopropoxy, isothiopropoxy, n-thiobutoxy, tert-
thiobutoxy, sec-
thiobutoxy, n-thiopentoxy, n-thiohexoxy, and 1,2-dimethylthiobutoxy.
Particular thioalkoxy groups
are lower thioalkoxy, i.e. with between 1 and 6 carbon atoms. Further
particular alkoxy groups have
between 1 and 4 carbon atoms.
[0067] One having ordinary skill in the art of organic synthesis will
recognize that the maximum
number of heteroatoms in a stable, chemically feasible heterocyclic ring,
whether it is aromatic or non-
aromatic, is determined by the size of the ring, the degree of unsaturation
and the valence of the
heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms
so long as the
heteroaromatic ring is chemically feasible and stable.
[0068] 'Pharmaceutically acceptable' means approved or approvable by a
regulatory agency of the
Federal or a state government or the corresponding agency in countries other
than the United States, or
that is listed in the U.S. Pharmacopoeia or other generally recognized
pharmacopoeia for use in
animals, and more particularly, in humans.
[0069] 'Pharmaceutically acceptable salt' refers to a salt of a compound of
the invention that is
pharmaceutically acceptable and that possesses the desired pharmacological
activity of the parent
compound. In particular, such salts are non-toxic may be inorganic or organic
acid addition salts and
base addition salts. Specifically, such salts include: (1) acid addition
salts, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or
formed with organic acids such as acetic acid, propionic acid, hexanoic acid,
cyclopentanepropionic
acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,
malic acid, maleic acid,
fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)
benzoic acid, cinnamic acid,
mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-
disulfonic acid, 2-
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hydroxyethanesulfonic acid, benzenesulfonic acid, 4 -
chlorob enzene sulfonic acid, 2 -
naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-
methylbicyclo[2.2.2]-oct-2-
ene- 1 -carboxylic acid, glucoheptonic acid, 3 -phenylpropionic acid,
trimethylacetic acid, tertiary
butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic
acid, stearic acid, muconic acid, and the like; or (2) salts formed when an
acidic proton present in the
parent compound either is replaced by a metal ion, e.g. an alkali metal ion,
an alkaline earth ion, or an
aluminum ion; or coordinates with an organic base such as ethanolamine,
diethanolamine,
triethanolamine, N-methylglucamine and the like. Salts further include, by way
of example only,
sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the
like; and when the
compound contains a basic functionality, salts of non-toxic organic or
inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and
the like. The term
'pharmaceutically acceptable cation' refers to an acceptable cationic counter-
ion of an acidic
functional group. Such cations are exemplified by sodium, potassium, calcium,
magnesium,
ammonium, tetraalkylammonium cations, and the like.
[0070] 'Pharmaceutically acceptable vehicle' refers to a diluent, adjuvant,
excipient or carrier with
which a compound of the invention is administered.
[0071] Trodrugs' refers to compounds, including derivatives of the compounds
of the invention,
which have cleavable groups and become by solvolysis or under physiological
conditions the
compounds of the invention which are pharmaceutically active in vivo. Such
examples include, but are
not limited to, choline ester derivatives and the like, N-alkylmorpholine
esters and the like.
[0072] 'Solvate' refers to forms of the compound that are associated with a
solvent, usually by a
solvolysis reaction. This physical association includes hydrogen bonding.
Conventional solvents
include water, Et0H, acetic acid and the like. The compounds of the invention
may be prepared e.g. in
crystalline form and may be solvated or hydrated. Suitable solvates include
pharmaceutically
acceptable solvates, such as hydrates, and further include both stoichiometric
solvates and non-
stoichiometric solvates. In certain instances the solvate will be capable of
isolation, for example when
one or more solvent molecules are incorporated in the crystal lattice of the
crystalline solid. 'Solvate'
encompasses both solution-phase and isolable solvates. Representative solvates
include hydrates,
ethanolates and methanolates.
[0073] 'Subject' includes humans. The terms 'human', 'patient' and 'subject'
are used
interchangeably herein.
[0074] 'Effective amount' means the amount of a compound of the invention
that, when administered
to a subject for treating a disease, is sufficient to effect such treatment
for the disease. The "effective
amount" can vary depending on the compound, the disease and its severity, and
the age, weight, etc.,
of the subject to be treated.
[0075] 'Preventing' or 'prevention' refers to a reduction in risk of acquiring
or developing a disease
or disorder (i.e. causing at least one of the clinical symptoms of the disease
not to develop in a subject
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that may be exposed to a disease-causing agent, or predisposed to the disease
in advance of disease
onset.
[0076] The term 'prophylaxis' is related to 'prevention', and refers to a
measure or procedure the
purpose of which is to prevent, rather than to treat or cure a disease. Non-
limiting examples of
prophylactic measures may include the administration of vaccines; the
administration of low
molecular weight heparin to hospital patients at risk for thrombosis due, for
example, to
immobilization; and the administration of an anti-malarial agent such as
chloroquine, in advance of a
visit to a geographical region where malaria is endemic or the risk of
contracting malaria is high.
[0077] 'Treating' or 'treatment' of any disease or disorder refers, in one
embodiment, to ameliorating
the disease or disorder (i.e. arresting the disease or reducing the
manifestation, extent or severity of at
least one of the clinical symptoms thereof). In another embodiment 'treating'
or 'treatment' refers to
ameliorating at least one physical parameter, which may not be discernible by
the subject. In yet
another embodiment, 'treating' or 'treatment' refers to modulating the disease
or disorder, either
physically, (e.g. stabilization of a discernible symptom), physiologically,
(e.g. stabilization of a
physical parameter), or both. In a further embodiment, "treating" or
"treatment" relates to slowing the
progression of the disease.
[0078] As used herein the term 'inflammatory disease(s)' refers to the group
of conditions including,
rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis,
psoriasis, psoriatic arthritis, ankylosing
spondylitis, allergic airway disease (e.g. asthma, rhinitis), chronic
obstructive pulmonary disease
(COPD), inflammatory bowel diseases (e.g. Crohn's disease, ulcerative
colitis), sarcoidosis,
endotoxin-driven disease states (e.g. complications after bypass surgery or
chronic endotoxin states
contributing to e.g. chronic cardiac failure), and related diseases involving
cartilage, such as that of the
joints. Particularly the term refers to rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis,
osteoarthritis, allergic airway disease (e.g. asthma), sarcoidosis, chronic
obstructive pulmonary disease
(COPD) and inflammatory bowel diseases. More particularly the term refers to
rheumatoid arthritis,
sarcoidosis, psoriatic arthritis, ankylosing spondylitis, and inflammatory
bowel diseases.
[0079] As used herein the term `autoimmune disease(s)' refers to the group of
diseases including
obstructive airways disease, including conditions such as COPD, asthma (e.g
intrinsic asthma,
extrinsic asthma, dust asthma, infantile asthma) particularly chronic or
inveterate asthma (for example
late asthma and airway hyperreponsiveness), bronchitis, including bronchial
asthma, systemic lupus
erythematosus (SLE), cutaneous lupus erythrematosus, membranous lupus
nephritis, dermatomyositis,
Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye disease, type I
diabetes mellitus and
complications associated therewith, atopic eczema (atopic dermatitis),
thyroiditis (Hashimoto's and
autoimmune thyroiditis), contact dermatitis and further eczematous dermatitis,
inflammatory bowel
disease (e.g. Crohn's disease and ulcerative colitis), atherosclerosis and
amyotrophic lateral sclerosis,
alopecia areata, vitiligo. Particularly the term refers to COPD, asthma,
cutaneous lupus
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erythrematosus, membranous lupus nephritis, alopecia areata, vitiligo, type I
diabetes mellitus arid
inflammatory bowel disease.
[0080] As used herein the term 'proliferative disease(s)' refers to conditions
such as cancer (e.g.
uterine leiomyosarcoma or prostate cancer), myeloproliferative disorders (e.g.
polycythemia vera,
essential thrombocytosis and myelofibrosis), leukemia (e.g. acute myeloid
leukaemia, acute and
chronic lymphoblastic leukemia), multiple myeloma, psoriasis, restenosis,
scleroderma or fibrosis. In
particular the term refers to cancer, leukemia, multiple myeloma and
psoriasis.
[0081] As used herein, the term 'cancer' refers to a malignant or benign
growth of cells in skin or in
body organs, for example but without limitation, breast, prostate, lung,
kidney, pancreas, stomach or
bowel. A cancer tends to infiltrate into adjacent tissue and spread
(metastasise) to distant organs, for
example to bone, liver, lung or the brain. As used herein the term cancer
includes both metastatic
tumour cell types (such as but not limited to, melanoma, lymphoma, leukaemia,
fibrosarcoma,
rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (such as but
not limited to,
colorectal cancer, prostate cancer, small cell lung cancer and non-small cell
lung cancer, breast cancer,
pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma,
primary liver cancer,
ovarian cancer, prostate cancer and uterine leiomyosarcoma). In particular,
the term 'cancer' refers to
acute lymphoblastic leukemia, acute myeloidleukemia, adrenocortical carcinoma,
anal cancer,
appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, basal cell
carcinoma, bile duct
cancer, bladder cancer, bone cancer (osteosarcoma and malignant fibrous
histiocytoma), brain stem
glioma, brain tumors, brain and spinal cord tumors, breast cancer, bronchial
tumors, Burkitt
lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous
leukemia, colon
cancer, colorectal cancer, craniopharyngioma, cutaneous T -Cell lymphoma,
embryonal tumors,
endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, ewing
sarcoma family of
tumors, eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach)
cancer, gastrointestinal
carcinoid tumor, gastrointestinal stromal tumor (GIST), gastrointestinal
stromal cell tumor, germ cell
tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular
(liver) cancer, hodgkin
lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors
(endocrine pancreas),
Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal
cancer, leukemia, Acute
lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic
myelogenous leukemia, hairy cell leukemia, liver cancer, non-small cell lung
cancer, small cell lung
cancer, Burkitt lymphoma, cutaneous T-celllymphoma, Hodgkin lymphoma, non-
Hodgkin lymphoma,
lymphoma, Waldenstrom macroglobulinemia, medulloblastoma, medulloepithelioma,
melanoma,
mesothelioma, mouth cancer, chronic myelogenous leukemia, myeloid leukemia,
multiple myeloma,
asopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung
cancer, oral
cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma of
bone, ovarian cancer,
ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant
potential tumor, pancreatic
cancer, papillomatosis, parathyroid cancer, penile cancer, pharyngeal cancer,
pineal parenchymal
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tumors of intermediate differentiation, pineoblastoma and supratentorial
primitive neuroectodermal
tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma,
pleuropulmonary blastoma, primary
central nervous system lymphoma, prostate cancer, rectal cancer, renal cell
(kidney) cancer,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing
sarcoma family of tumors,
sarcoma, kaposi, Sezary syndrome, skin cancer, small cell Lung cancer, small
intestine cancer, soft
tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,
supratentorial primitive
neuroectodermal tumors, T -cell lymphoma, testicular cancer, throat cancer,
thymoma and thymic
carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma,
vaginal cancer, vulvar
cancer, Waldenstrom macroglobulinemia, and Wilms tumor
[0082] As used herein the term 'leukemia' refers to neoplastic diseases of the
blood and blood
forming organs. Such diseases can cause bone marrow and immune system
dysfunction, which renders
the host highly susceptible to infection and bleeding. In particular the term
leukemia refers to acute
myeloid leukaemia (AML), and acute lymphoblastic leukemia (ALL) and chronic
lymphoblastic
leukaemia (CLL).
[0083] As used herein the term 'allergic disease(s)' refers to the group of
conditions characterized by
a hypersensitivity disorder of the immune system including, allergic airway
disease (e.g. asthma,
rhinitis), sinusitis, eczema and hives, as well as food allergies or allergies
to insect venom.
[0084] As used herein the term 'asthma' as used herein refers to any disorder
of the lungs
characterized by variations in pulmonary gas flow associated with airway
constriction of whatever
cause (intrinsic, extrinsic, or both; allergic or non-allergic). The term
asthma may be used with one or
more adjectives to indicate the cause.
[0085] As used herein the term 'transplant rejection' refers to the acute or
chronic rejection of cells,
tissue or solid organ allo- or xenografts of e.g. pancreatic islets, stem
cells, bone marrow, skin, muscle,
corneal tissue, neuronal tissue, heart, lung, combined heart-lung, kidney,
liver, bowel, pancreas,
trachea or oesophagus, or graft-versus-host diseases.
[0086] As used herein the term 'diseases involving impairment of cartilage
turnover' includes
conditions such as osteoarthritis, psoriatic arthritis, juvenile rheumatoid
arthritis, gouty arthritis, septic
or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy,
algodystrophy, Tietze syndrome
or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic
arthritis, arthropathy,
endemic forms of arthritis like osteoarthritis deformans endemica, Mseleni
disease and Handigodu
disease; degeneration resulting from fibromyalgia, systemic lupus
erythematosus, scleroderma and
ankylosing spondylitis.
[0087] As used herein the term 'congenital cartilage malformation(s)' includes
conditions such as
hereditary chondrolysis, chondrodysplasias and pseudochondrodysplasias, in
particular, but without
limitation, microtia, anotia, metaphyseal chondrodysplasia, and related
disorders.
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[0088] As used herein the term `disease(s) associated with hypersecretion of
IL6' includes conditions
such as Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma
and/or mesangial
proliferative glomerulonephritis.
[0089] As used herein the term `disease(s) associated with hypersecretion of
interferons includes
conditions such as systemic and cutaneous lupus erythematosis, lupus
nephritis, dermatomyositis,
Sjogren's syndrome, psoriasis, rheumatoid arthritis.
[0090] `Composition(s) of the invention', and equivalent expressions, are
meant to embrace
compounds of the Formula(e) as herein described, which expression includes the
pharmaceutically
acceptable salts, and the solvates, e.g. hydrates, and the solvates of the
pharmaceutically acceptable
salts where the context so permits. Similarly, reference to intermediates,
whether or not they
themselves are claimed, is meant to embrace their salts, and solvates, where
the context so permits.
[0091] When ranges are referred to herein, for example but without limitation,
Crs alkyl, the citation
of a range should be considered a representation of each member of said range.
[0092] Other derivatives of the compounds of this invention have activity in
both their acid and acid
derivative forms, but in the acid sensitive form often offers advantages of
solubility, tissue
compatibility, or delayed release in the mammalian organism (Bundgard, H,
1985). Prodrugs include
acid derivatives well know to practitioners of the art, such as, for example,
esters prepared by reaction
of the parent acid with a suitable alcohol, or amides prepared by reaction of
the parent acid compound
with a substituted or unsubstituted amine, or acid anhydrides, or mixed
anhydrides. Simple aliphatic or
aromatic esters, amides and anhydrides derived from acidic groups pendant on
the compounds of this
invention are particularly useful prodrugs. In some cases it is desirable to
prepare double ester type
prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters.
Particular such prodrugs
are the C1-8 alkyl, C2-8 alkenyl, C6-10 optionally substituted aryl, and (C6-
10 aryl)-(C4 alkyl) esters of
the compounds of the invention.
[0093] As used herein, the term 'isotopic variant' refers to a compound that
contains unnatural
proportions of isotopes at one or more of the atoms that constitute such
compound. For example, an
'isotopic variant' of a compound can contain one or more non-radioactive
isotopes, such as for
example, deuterium (2H or D), carbon-13 (13C), nitro (15N), or the like. It
will be understood that, in a
compound where such isotopic substitution is made, the following atoms, where
present, may vary, so
that for example, any hydrogen may be 2H/D, any carbon may be 13C, or any
nitrogen may be 15N, and
that the presence and placement of such atoms may be determined within the
skill of the art. Likewise,
the invention may include the preparation of isotopic variants with
radioisotopes, in the instance for
example, where the resulting compounds may be used for drug and/or substrate
tissue distribution
studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C,
are particularly useful for this
purpose in view of their ease of incorporation and ready means of detection.
Further, compounds may
be prepared that are substituted with positron emitting isotopes, such as 11C,
18F, 150 and 13N, and
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would be useful in Positron Emission Topography (PET) studies for examining
substrate receptor
occupancy.
[0094] It is also to be understood that compounds that have the same molecular
formula but differ in
the nature or sequence of bonding of their atoms or the arrangement of their
atoms in space are termed
'isomers'. Isomers that differ in the arrangement of their atoms in space are
termed `stereoisomers'.
[0095] Stereoisomers that are not mirror images of one another are termed
`diastereomers' and those
that are non-superimposable mirror images of each other are termed
`enantiomers'. When a compound
has an asymmetric center, for example, it is bonded to four different groups,
a pair of enantiomers is
possible. An enantiomer can be characterized by the absolute configuration of
its asymmetric center
and is described by the R- and S-sequencing rules of Cahn and Prelog, or by
the manner in which the
molecule rotates the plane of polarized light and designated as dextrorotatory
or levorotatory (i.e. as
(+) or (-)-isomers respectively). A chiral compound can exist as either
individual enantiomer or as a
mixture thereof. A mixture containing equal proportions of the enantiomers is
called a `racemic
mixture'.
[0096] l'automers' refer to compounds that are interchangeable forms of a
particular compound
structure, and that vary in the displacement of hydrogen atoms and electrons.
Thus, two structures may
be in equilibrium through the movement of z electrons and an atom (usually H).
For example, enols
and ketones are tautomers because they are rapidly interconverted by treatment
with either acid or
base. Another example of tautomerism is the aci- and nitro- forms of
phenylnitromethane, that are
likewise formed by treatment with acid or base.
[0097] Tautomeric forms may be relevant to the attainment of the optimal
chemical reactivity and
biological activity of a compound of interest.
[0098] The compounds of the invention may possess one or more asymmetric
centers; such
compounds can therefore be produced as individual (R)- or (S)- stereoisomers
or as mixtures thereof.
[0099] Unless indicated otherwise, the description or naming of a particular
compound in the
specification and claims is intended to include both individual enantiomers
and mixtures, racemic or
otherwise, thereof. The methods for the determination of stereochemistry and
the separation of
stereoisomers are well-known in the art.
[0100] It will be appreciated that compounds of the invention may be
metabolized to yield
biologically active metabolites.
THE INVENTION
[0101] The present invention is based on the identification of composition of
the invention, useful in
the prophylaxis and/or treatment of inflammatory diseases, autoimmune
diseases, proliferative
diseases, allergic diseases, transplant rejection, diseases involving
impairment of cartilage turnover,
congenital cartilage malformations, and/or diseases associated with
hypersecretion of IL6 or
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interferons. In a particular aspect, the composition comprises a first
compound having IRAK
inhibitory activity, and a second compound having JAK inhibitory activity.
[0102] The present invention also provides pharmaceutical compositions
comprising the combination
of the invention and methods for the prophylaxis and/or treatment of diseases
including inflammatory
diseases, autoimmune diseases, proliferative diseases, allergic diseases,
transplant rejection, diseases
involving impairment of cartilage turnover, congenital cartilage
malformations, and/or diseases
associated with hypersecretion of IL6 or interferons by administering the
combination of the
invention.
[0103] In a particular aspect, the JAK inhibitor has JAK1 inhibitory activity.
In a more particular
embodiment, the JAK inhibitor is a JAK1 selective inhibitor.
[0104] In another particular aspect, the IRAK inhibitor has IRAK4 inhibitory
activity. In a more
particular embodiment, the IRAK inhibitor is a IRAK4 selective inhibitor.
[0105] Accordingly, in a first aspect of the invention, a composition of the
invention is provided
comprising:
a) a compound according to Formula I :
R1.,,--,
0 /
2
RNN/N/----N
I
Cy /
,
CN
I
wherein
Cy is
- monocyclic C37 cycloalkyl optionally substituted with one or more
independently selected 123, or
- 4-7 membered monocyclic heterocycloalkyl comprising one or two
heteroatoms independently
selected from N, S, and 0, optionally substituted with one or more
independently selected ll3;
R1 is
-H,
- -S03H,
-
- Cm alkyl,
- -C(=0)-(4-7 membered monocyclic heterocycloalkyl comprising one or two
heteroatoms
independently selected from N, S, and 0), or
- -C(=0)C1,6 alkyl, which Ci_6 alkyl is optionally substituted with one or
more independently
selected R4 groups;
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R2 is H or C14 alkyl;
each R3 is independently selected from:
-OH,
- =0,
- halo, and
- Cm alkyl;
each R4 is independently selected from:
- -NR5aR5b,
- -C(=0)0H,
- 4-7 membered monocyclic heterocycloalkyl comprising one or two
heteroatoms independently
selected from N, S, and 0, optionally substituted with one or more
independently selected Ci_4
alkyl, and
- -NHC(=0)-C1_4 alkyl-NH2; and
R5a and leb are independently H or C1_4 alkyl;
or a pharmaceutically acceptable salt or a solvate or the salt of a solvate
thereof; and
b) a second compound having a JAK inhibiting activity.
[0106] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein Cy is monocyclic C3_7 cycloalkyl. In a particular
embodiment, Cy is cyclohexyl.
[0107] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein Cy is monocyclic C3_7 cycloalkyl substituted with one, two
or three independently
selected R3. In a particular embodiment, Cy is cyclohexyl substituted with
one, two or three
independently selected R3. In another particular embodiment, Cy is monocyclic
C3_7 cycloalkyl
substituted with one or two R3. In a more particular embodiment, Cy is
cyclohexyl substituted with
one or two independently selected R3.
[0108] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein Cy is 4-7 membered monocyclic heterocycloalkyl comprising
one or two
heteroatoms independently selected from N, S, and 0. In a particular
embodiment, Cy is
--
tetrahydropyranyl, or tetrahydrothiopyranyl. In a more particular embodiment,
Cy is
[0109] In one embodiment, the composition of the invention comprises the
compound according to
Formula I , wherein Cy is monocyclic 4-7 membered monocyclic heterocycloalkyl
comprising one or
two heteroatoms independently selected from N, S, and 0, substituted with one,
two or three
independently selected R3. In another embodiment, Cy is tetrahydropyranyl or
tetrahydrothiopyranyl,
each of which is substituted with one, two or three independently selected R3.
In a particular
embodiment, Cy is 4-7 membered monocyclic heterocycloalkyl comprising one or
two heteroatoms
independently selected from N, S, and 0, substituted with one or two R3. In
another particular
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embodiment, Cy is tetrahydropyranyl, or tetrahydrothiopyranyl, each of which
is substituted with one
or two independently selected R3. In a more particular embodiment, Cy is - or
s , each of
which is substituted with one or two independently selected R3.
[0110] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein each R3 is selected from OH, =0, halo, and Ci_4 alkyl. In a
particular, embodiment,
each R3 is selected from OH, =0, F, and -CH3. In a more particular embodiment,
each R3 is selected
from OH, and -CH3. In another more particular embodiment, each R3 is F. In yet
another more
particular embodiment, each R3 is =0.
[0111] In one embodiment, the composition of the invention comprises the
compound according to
Formula Ha, Hb, Hc, lid, He, or IIf:
,õ-0-,-- ..-- N .-----,...,0 ...,..,.. ,. .. =,--____N ..----..,..ØN
0 0
...,_ 1
0 1 ri, 2
N m
R2. N ." .^- () , ..-"---- ,-" N 1 Ki R2 N 4 . -- j---
rc N N N '''
0-R1 01 OR 01 0-R1 01
\
-. ..--
0 A
HO CN CN 00 CN
Ha Hb IIc
..----...õ...0 ....õ...õ-r...._. .......N ......,..õ--7,......_N ..----
..õ...0 ..õ-...., .......---:'.---..õN
0 0 0
R2 ki
, 1
'N N i'i R2.-1----õ
N N IN R2.-----õ,
N N isl
0-R1 01 0-Rib
01 0-R1 I.OH 01
0
HO
F F CN CN CN
lid He Iff
[0112] wherein R1 and R2 are as described above.
[0113] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-Hf, wherein R1 is H, -S03H, or -P(=0)(0F1)2.
[0114] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-Hf, wherein R1 is CIA alkyl. In a particular embodiment,
R1 is -CH3, -CH2CH3,
or -CH(CH3)2. In a more particular embodiment, R1 is -CH3.
[0115] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-Hf, wherein R1 is -C(=0)-(4-7 membered monocyclic
heterocycloalkyl
comprising one or two heteroatoms independently selected from N, S, and 0). In
a particular
embodiment, R1 is -C(=0)-pyffolidinyl.
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[0116] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R1 is -C(-0)C1_6 alkyl. In a particular
embodiment, R1
is -C(-0)C1_6 alkyl, which C1_6 alkyl is selected from -CH3, -CH2CH3, -
CH2CH2CH3,
or -CH2(CH(CH3)2)=
[0117] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R1 is -C(=0)C1_6 alkyl, which Ci_6 alkyl is
substituted with one or
more independently selected R4. In a particular embodiment, 121 is -C(=0)Ci_6
alkyl, which C1_6 alkyl
is selected from -CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH(CH3)2), each of which
is substituted with
one or more independently selected R4. In another particular embodiment, R1 is
-C(=0)C1_6 alkyl,
which C1_6 alkyl is substituted with one or two independently selected R4. In
a more particular
embodiment, R1 is -C(=0)C1_6 alkyl, which C1_6 alkyl is selected from -CH3, -
CH2CH3, -CH2CH2CH3,
or -CH2(CH(CI-13)2), each of which is substituted with one or two
independently selected R4.
[0118] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, and R4 is -Nlealeb, wherein each R5a and leb is
independently H or C1-4
alkyl. In a particular embodiment, each R5a and leb is independently H, -CH3,
or -CH2CH3. In a more
particular embodiment, R5a is H, and le is H, -CH3, or -CH2CH3. In a most
particular embodiment, R4
is -NH2, -NHCH3, or -N(CH3)2.
[0119] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R4 is -C(=0)0H.
[0120] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R4 is 4-7 membered monocyclic
heterocycloalkyl comprising one
or two heteroatoms independently selected from N, S, and 0, optionally
substituted with one or more
independently selected Ci_4 alkyl. In a particular embodiment, R4 is
morpholinyl, piperidinyl, or
piperazinyl, each of which is optionally substituted with one or more
independently selected C1_4 alkyl.
In another particular embodiment, R4 is 4-7 membered monocyclic
heterocycloalkyl comprising one or
two heteroatoms independently selected from N, S, and 0, optionally
substituted with one Ci_4 alkyl.
In a more particular embodiment, R4 is 4-7 membered monocyclic
heterocycloalkyl comprising one or
two heteroatoms independently selected from N, S, and 0, optionally
substituted with one -CH3. In
another more particular embodiment, R4 is morpholinyl, piperidinyl, or
piperazinyl, each of which is
optionally substituted with one or more -CH3. In a most particular embodiment,
R4 is morpholinyl,
piperidinyl, or piperazinyl, each of which is optionally substituted with one -
CH3.
[0121] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R4 is -NHC(=0)-C14 alkyl-NH2. In a
particular embodiment, R4
is -NHC(=0)-CH2-NH2.
[0122] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R1 is -C(=0)CH2NH2, -C(=0)CH2NHCH3, -
C(=0)CH2N(CH3)2,
-C(=0)CH2CH2N(CH3)2, -C(=0)CH(NH2)CH(CH3)2, -C(=0)CH2CH2C(=0)0H, -
C(=0)CH(NH2)CH2
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C(=0)0H, -C(=0)CH(NH2)CH2CH2C(=0)0H, -
C(=0)CH(CH(CH3)2)NHC(=0)CH2NH2,
N) N'Th
0 LN.
, or .
[0123] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R2 is H.
[0124] In one embodiment, the composition of the invention comprises the
compound according to
any one of Formulae I-IIf, wherein R2 is Ci_4 alkyl. In a particular
embodiment, R2 is -CH3.
[0125] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein the compound is selected from:
6- [6- [2-(2-hydroxy-ethoxy)-ethoxy]-5-(tetrahydro-pyran-4-ylamino)-
imidazo[4,5-b]pyridin-3 -y1]-
nicotinonitrile,
6- {5-( 1 , 1 -dioxo-tetrahydro-2H-thiopyran-4-ylamino)-6- [2-(2-hydroxy-
ethoxy)-ethoxy] -imidazo [4,5-
b]pyridin-3 -yll -nicotinonitrile,
6- {6- [2-(2-hydroxy-ethoxy)-ethoxy]-5-[((cis-1,4)-4-hydroxy-4-methyl-
cyclohexyl)-methyl-amino] -
imidazo[4,5-b]pyridin-3-yll -nicotinonitrile,
6- {6- [2-(2-methoxy- ethoxy)- ethoxy] -5- [methyl-(tetrahydro-pyran-4-y1)-
amino] -imidazo [4,5-
b]pyridin-3 -yll -nicotinonitrile,
6- [6- [2-(2-methoxy-ethoxy)-ethoxy]-5-(tetrahydro-pyran-4-ylamino)-imidazo
[4,5-b]pyridin-3-y1]-
nicotinonitrile,
6- {543 -hydroxy-cyclohexylamino)-642-(2-hydroxy-ethoxy)-ethoxy]-imidazo [4,5-
b]pyridin-3 -yll -
nicotinonitrile,
6- {5-(4,4-difluoro-cyclohexylamino)-6-[2-(2-hydroxy-ethoxy)-ethoxy]-
imidazo[4,5-b]pyridin-3-y1} -
nicotinonitrile,
sulfuric acid mono-(2- {243 -(5 -cyano-pyridin-2-y1)-5 -(tetrahydro-pyran-4-
ylamino)-3H-imidazo[4,5-
b]pyridin-6-yloxy]-ethoxy} -ethyl) ester,
(S)-2-amino-3-methyl-butyric acid 2- {2- [3-(5-cyano-pyridin-2-y1)-5-
(tetrahydro-pyran-4-ylamino)-
3H-imidazo [4,5-b]pyridin-6-yloxy]-ethoxy} -ethyl ester,
(S)-2-amino-3-methyl-butyric acid 2- {2- [3-(5-cyano-pyridin-2-y1)-5-
(tetrahydro-pyran-4-ylamino)-
3H-imidazo [4,5-b]pyridin-6-yloxy]-ethoxy} -ethyl ester oxalic acid salt,
6- [6- [2-(2-hydroxyethoxy)ethoxy]-5 -[[(cis-3,4)-4-hydroxytetrahydropyran-3-
yl]amino]imidazo [4,5-
b]pyridin-3 -yl]pyridine-3 -carbonitrile,
6- [6- [2-(2-hydroxyethoxy)ethoxy]-5-[((cis-1,4)-4-hydroxy-4-
methylcyclohexyeamino]imidazo [4,5-
b]pyridin-3 -yl]pyridine-3 -carbonitrile,
6- [5- [((cis-1,4)-4-hydroxy-4-methyl-cyclohexyl)-methyl-amino]-6- [242-
methoxyethoxy)ethoxy]imidazo [4,5-b]pyridin-3 -yl]pyridine-3 -carbonitrile,
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6-[5-[((cis-1,4)-4-hydroxy-4-methylcyclohexypamino]-642-(2-
methoxyethoxy)ethoxy]imidazo[4,5-
b]pyridin-3-yl]pyridine-3-carbonitrile,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-(dimethylamino)acetate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-aminoacetate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-(methylamino)acetate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl (2S)-pyrrolidine-2-carboxylate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl (2S)-2-[(2-aminoacetyl)amino]-3-methyl-butanoate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-morpholinoacetate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl] oxyethoxy] ethyl 2-(4-methylpiperazin- 1 -yl)acetate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 3-(dimethylamino)propanoate,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-(dimethylamino)acetate oxalic acid salt,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-aminoacetate oxalic acid salt,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-(methylamino)acetate oxalic acid salt,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl (2S)-pyrrolidine-2-carboxylate oxalic acid salt,
(3S)-3-amino-4-[242-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-b]pyridin-6-
yl]oxyethoxy]ethoxy]-4-oxo-butanoic acid hydrochloric acid salt,
(4S)-4-amino-5-[242-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-b]pyridin-6-
yl]oxyethoxy]ethoxy]-5-oxo-pentanoic acid hydrochloric acid salt,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl (2S)-2-[(2-aminoacetyl)amino]-3-methyl-butanoate oxalic
acid salt,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-morpholinoacetate oxalic acid salt,
2-[2-[3-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo[4,5-b]pyridin-
6-
yl]oxyethoxy]ethyl 2-(4-methylpiperazin-1-yl)acetate oxalic acid salt,
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2- [2- [3 -(5- cyano-2-pyridy1)-5- (tetrahydropyran-4-ylamino)imidazo [4,5-
b]pyridin-6-
yl] oxyethoxy] ethyl 3-(dimethylamino)propanoate oxalic acid salt, and
4- [2- [2-[3 - (5-cyano-2-pyridy1)-5-(tetrahydropyran-4-ylamino)imidazo [4,5-
b] pyridin-6-
yl] oxyethoxy] ethoxy] -4- oxo-butanoic acid.
[0126] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein the compound is 6-[6-[2-(2-hydroxy-ethoxy)-ethoxy]-5-
(tetrahydro-pyran-4-
ylamino)-imidazo [4,5-b]pyridin-3-y1]-nicotinonitrile.
[0127] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein the compound is not 6-[6-[2-(2-hydroxy-ethoxy)-ethoxy]-5-
(tetrahydro-pyran-4-
ylamino)-imidazo [4,5-b]pyridin-3-y1]-nicotinonitrile.
[0128] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein the compound is (S)-2-amino-3-methyl-butyric acid 2- {2-[3-
(5-cyano-pyridin-2-
y1)-5-(tetrahydro-pyran-4-ylamino)-3H-imidazo[4,5-b]pyridin-6-yloxy] -ethoxy} -
ethyl ester.
[0129] In one embodiment, the composition of the invention comprises the
compound according to
Formula I, wherein the compound is not (S)-2-amino-3-methyl-butyric acid 2- {2-
[3
[4,5-b]pyridin-6-yloxy] -ethoxy} -ethyl ester.
[0130] In another embodiment, the composition of the invention comprises a
compound having a
JAK inhibiting activity. In a particular embodiment, the compound having a JAK
inhibiting activity is
a compound disclosed in WO 2010/010190.
[0131] In another embodiment, the composition of the invention comprises a
compound having a
JAK1 inhibiting activity. In a particular embodiment, the compound having a
JAK1 inhibiting activity
is according to Formula XXa, or XXb:
/ -N H2
0
0
...----,.. ..----...,
N N
L=S=0 L=S=0
\\ \\
0 0
XXa XXb
[0132] In a particular embodiment, the composition of the invention comprises:
a) A compound selected from 6-[6-[2-(2-hydroxy-ethoxy)-ethoxy]-5-(tetrahydro-
pyran-4-ylamino)-
imidazo[4,5-b]pyridin-3-y1]-nicotinonitrile, and (S)-2-amino-3-methyl-butyric
acid 2- {2-[3-(5-
cyano-pyridin-2-y1)-5-(tetrahydro-pyran-4-ylamino)-3H-imidazo [4,5-b]pyridin-6-
yloxy]- ethoxy} -
ethyl ester; and
b) A compound selected from the compound according to Formula XXa and XXb
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[0133] In a particular embodiment, the composition of the invention comprises:
a) A compound selected from 6- [6- [2-(2-hydroxy-ethoxy)-ethoxy]-5 -
(tetrahydro-pyran-4-ylamino)-
imidazo [4,5-b]pyridin-3-y1]-nicotinonitrile, and (S)-2-amino-3-methyl-butyric
acid 2- {243 -(5-
cyano -pyridin-2-y1)-5 -(tetrahydro -pyran-4-ylamino)-3 H-imidazo [4,5-
b]pyridin-6-yloxy] - ethoxy} -
ethyl ester; and
b) The compound according to Formula XX])
[0134] In a more particular embodiment, the composition of the invention
comprises:
a) 6- [642-(2-hydroxy- ethoxy)- ethoxy] -5 -(tetrahydro-pyran-4-ylamino)-
imidazo [4,5-b]pyridin-3-yl] -
nicotinonitrile; and
b) the compound according to Formula ,ab
[0135] In one embodiment the compound from the composition of the invention is
not an isotopic
variant.
[0136] In one aspect the compound from the composition of the invention
according to any one of the
embodiments herein described is present as the free base.
[0137] In one aspect the compound from the composition of the invention
according to any one of the
embodiments herein described is a pharmaceutically acceptable salt.
[0138] In one aspect the compound from the composition of the invention
according to any one of the
embodiments herein described is a solvate of the compound.
[0139] In one aspect a compound of composition of the invention according to
any one of the
embodiments herein described is a solvate of a pharmaceutically acceptable
salt of a compound.
[0140] While specified groups for each embodiment have generally been listed
above separately, a
compound of the composition of the invention includes one in which several or
each embodiment in
the above Formula, as well as other formulae presented herein, is selected
from one or more of
particular members or groups designated respectively, for each variable.
Therefore, this invention is
intended to include all combinations of such embodiments within its scope.
[0141] While specified groups for each embodiment have generally been listed
above separately, a
compound of the invention may be one for which one or more variables (for
example, R groups) is
selected from one or more embodiments according to any of the Formula(e)
listed above. Therefore,
the present invention is intended to include all combinations of variables
from any of the disclosed
embodiments within its scope.
[0142] Alternatively, the exclusion of one or more of the specified variables
from a group or an
embodiment, or combinations thereof is also contemplated by the present
invention.
[0143] In certain aspects, the present invention provides prodrugs and
derivatives of the compounds
according to the formulae above. Prodrugs are derivatives of the compounds of
the composition of the
invention, which have metabolically cleavable groups and become by solvolysis
or under
physiological conditions the compounds of the invention, which are
pharmaceutically active, in vivo.
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Such examples include, but are not limited to, choline ester derivatives and
the like, N-
alkylmorpholine esters and the like.
[0144] Other derivatives of the compounds of the composition of this invention
have activity in both
their acid and acid derivative forms, but the acid sensitive form often offers
advantages of solubility,
tissue compatibility, or delayed release in the mammalian organism (Bundgard,
H, 1985). Prodrugs
include acid derivatives well known to practitioners of the art, such as, for
example, esters prepared by
reaction of the parent acid with a suitable alcohol, or amides prepared by
reaction of the parent acid
compound with a substituted or unsubstituted amine, or acid anhydrides, or
mixed anhydrides. Simple
aliphatic or aromatic esters, amides and anhydrides derived from acidic groups
pendant on the
compounds of this invention are preferred prodrugs. In some cases it is
desirable to prepare double
ester type prodrugs such as (acyloxy)alkyl esters or
((alkoxycarbonypoxy)alkylesters. Particularly
useful are the CI to C8 alkyl, C2-C8 alkenyl, aryl, C7-C12 substituted aryl,
and C7-C12 arylalkyl esters of
the compounds of the invention.
PHARMACEUTICAL COMPOSITIONS
[0145] When employed as a pharmaceutical, a composition of the invention is
typically administered
in the form of a pharmaceutical composition. Such compositions can be prepared
in a manner well
known in the pharmaceutical art and comprise at least one active compound of
the invention according
to Formula I. Generally, a composition of the invention is administered in a
pharmaceutically effective
amount. The amount of compound of the invention actually administered will
typically be determined
by a physician, in the light of the relevant circumstances, including the
condition to be treated, the
chosen route of administration, the actual compound of the invention
administered, the age, weight,
and response of the individual patient, the severity of the patient's
symptoms, and the like.
[0146] The pharmaceutical compositions of this invention can be administered
by a variety of routes
including oral, rectal, transdermal, subcutaneous, intra-articular,
intravenous, intramuscular, and
intranasal. Depending on the intended route of delivery, a compound of the
invention is preferably
formulated as either injectable or oral compositions or as salves, as lotions
or as patches all for
transdermal administration.
[0147] The compositions for oral 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, vehicle or carrier.
Typical unit dosage forms
include prefilled, premeasured ampules or syringes of the liquid compositions
or pills, tablets, capsules
or the like in the case of solid compositions. In such compositions, the
composition of the invention is
usually a minor component (from about 0.1 to about 50% by weight or preferably
from about 1 to
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about 40% by weight) with the remainder being various vehicles or carriers and
processing aids
helpful for forming the desired dosing form.
[0148] Liquid forms suitable for oral administration may include a suitable
aqueous or non-aqueous
vehicle with buffers, suspending and dispensing agents, colorants, flavors and
the like. Solid forms
may include, for example, any of the following ingredients, or compound of the
inventions of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as
starch or lactose, a disintegrating agent such as alginic acid, Primogel, or
corn starch; a lubricant such
as magnesium stearate; a glidant such as colloidal silicon dioxide; a
sweetening agent such as sucrose
or saccharin; or a flavoring agent such as peppermint or orange flavoring.
[0149] Injectable compositions are typically based upon injectable sterile
saline or phosphate-
buffered saline or other injectable carriers known in the art. As before, the
active compounds of the
composition of the invention in such compositions is typically a minor
component, often being from
about 0.05 to 10% by weight with the remainder being the injectable carrier
and the like.
[0150] Transdermal compositions are typically formulated as a topical ointment
or cream containing
the active ingredient(s), generally in an amount ranging from about 0.01 to
about 20% by weight,
preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to
about 10% by weight,
and more preferably from about 0.5 to about 15% by weight. When formulated as
an ointment, the
active ingredients will typically be combined with either a paraffinic or a
water-miscible ointment
base. Alternatively, the active ingredients may be formulated in a cream with,
for example an oil-in-
water cream base. Such transdermal formulations are well-known in the art and
generally include
additional ingredients to enhance the dermal penetration of stability of the
active ingredients or the
formulation. All such known transdermal formulations and ingredients are
included within the scope
of this invention.
[0151] A composition of the invention can also be administered by a
transdermal device.
Accordingly, transdermal administration can be accomplished using a patch
either of the reservoir or
porous membrane type, or of a solid matrix variety.
[0152] The above-described components for orally administrable, injectable or
topically
administrable compositions are merely representative. Other materials as well
as processing
techniques and the like are set forth in Part 8 of Remington's Pharmaceutical
Sciences, 17th edition,
1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated
herein by reference.
[0153] A composition of the invention can also be administered in sustained
release forms or from
sustained release drug delivery systems. A description of representative
sustained release materials can
be found in Remington's Pharmaceutical Sciences.
[0154] The following formulation examples illustrate representative
pharmaceutical compositions
that may be prepared in accordance with this invention. The present invention,
however, is not limited
to the following pharmaceutical compositions.
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Formulation 1 - Tablets
[0155] A composition of the invention comprising a compound according to
Formula I and a JAK
inhibitor may be admixed as a dry powder with a dry gelatin binder in an
approximate 1:2 weight
ratio. A minor amount of magnesium stearate may be added as a lubricant. The
mixture may be
formed into 240-270 mg tablets (80-90 mg of active compound of the invention
according to Formula
I per tablet) in a tablet press.
Formulation 2 - Capsules
[0156] A composition of the invention comprising a compound according to
Formula I and a JAK
inhibitor may be admixed as a dry powder with a starch diluent in an
approximate 1:1 weight ratio.
The mixture may be filled into 250 mg capsules (125 mg of active compositon of
the invention
according to Formula I per capsule).
Formulation 3 - Liquid
[0157] A composition of the invention comprising a compound according to
Formula I and a JAK
inhibitor (125 mg), may be admixed with sucrose (1.75 g) and xanthan gum (4
mg) and the resultant
mixture may be blended, passed through a No. 10 mesh U.S. sieve, and then
mixed with a previously
made solution of microcrystalline cellulose and sodium carboxymethyl cellulose
(11:89, 50 mg) in
water. Sodium benzoate (10 mg), flavor, and color may be diluted with water
and added with stirring.
Sufficient water may then be added with stirring. Further sufficient water may
be then added to
produce a total volume of 5 mL.
Formulation 4 - Tablets
[0158] A composition of the invention comprising a compound according to
Formula I and a JAK
inhibitor may be admixed as a dry powder with a dry gelatin binder in an
approximate 1:2 weight
ratio. A minor amount of magnesium stearate may be added as a lubricant. The
mixture may be
formed into 450-900 mg tablets (150-300 mg of active compound of the invention
according to
Formula I) in a tablet press.
Formulation 5 - Injection
[0159] A composition of the invention comprising a compound according to
Formula I and a JAK
inhibitor may be dissolved or suspended in a buffered sterile saline
injectable aqueous medium to a
concentration of approximately 5 mg/mL.
Formulation 6 - Topical
[0160] Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted at
about 75 C and then
a mixture of a composition of the invention comprising a compound according to
Formula I and a JAK
inhibitor (50 g) methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl
sulfate (10 g), and
propylene glycol (120 g) dissolved in water (about 370 g) may be added and the
resulting mixture may
be stirred until it congeals.
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METHODS OF TREATMENT
[0161] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in medicine. In a
particular embodiment, the present invention provides composition of the
invention or pharmaceutical
compositions comprising a composition of the invention, for use in the
prophylaxis and/or treatment of
inflammatory diseases, autoimmune diseases, proliferative diseases, allergic
diseases, transplant
rejection, diseases involving impairment of cartilage turnover, congenital
cartilage malformations,
and/or diseases associated with hypersecretion of IL6 or interferons.
[0162] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of inflammatory
diseases, autoimmune
diseases, proliferative diseases, allergic diseases, transplant rejection,
diseases involving impairment
of cartilage turnover, congenital cartilage malformations, and/or diseases
associated with
hypersecretion of IL6 or interferons.
[0163] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with inflammatory diseases, autoimmune
diseases,
proliferative diseases, allergic diseases, transplant rejection, diseases
involving impairment of cartilage
turnover, congenital cartilage malformations, and/or diseases associated with
hypersecretion of IL6 or
interferons, which methods comprise the administration of an effective amount
of a composition of the
invention or one or more of the pharmaceutical compositions herein described
for the treatment or
prophylaxis of said condition.
[0164] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of inflammatory diseases,
autoimmune diseases,
proliferative diseases, allergic diseases, transplant rejection, diseases
involving impairment of cartilage
turnover, congenital cartilage malformations, and/or diseases associated with
hypersecretion of IL6 or
interferons treatment agent.
[0165] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of inflammatory diseases. In a particular embodiment, the
inflammatory disease is
selected from rheumatoid arthritis, psoriatic arthritis, ankylosing
spondylitis, osteoarthritis, allergic
airway disease, sarcoidosis, chronic obstructive pulmonary disease (COPD) and
inflammatory bowel
diseases. In a more particular embodiment, the inflammatory disease is
selected from rheumatoid
arthritis, sarcoidosis, psoriatic arthritis, ankylosing spondylitis, and
inflammatory bowel diseases.
[0166] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
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a medicament for use in the prophylaxis and/or treatment of inflammatory
diseases. In a particular
embodiment, the inflammatory disease is selected from rheumatoid arthritis,
psoriatic arthritis,
ankylosing spondylitis, osteoarthritis, allergic airway disease, sarcoidosis,
chronic obstructive
pulmonary disease (COPD) and inflammatory bowel diseases. In a more particular
embodiment, the
inflammatory disease is selected from rheumatoid arthritis, sarcoidosis,
psoriatic arthritis, ankylosing
spondylitis, and inflammatory bowel diseases.
[0167] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with inflammatory diseases, which
methods comprise the
administration of an effective amount of a composition of the invention or one
or more of the
pharmaceutical compositions herein described for the treatment or prophylaxis
of said condition. In a
particular embodiment, the inflammatory disease is selected from rheumatoid
arthritis, psoriatic
arthritis, ankylosing spondylitis, osteoarthritis, allergic airway disease,
sarcoidosis, chronic obstructive
pulmonary disease (COPD) and inflammatory bowel diseases. In a more particular
embodiment, the
inflammatory disease is selected from rheumatoid arthritis, sarcoidosis,
psoriatic arthritis, ankylosing
spondylitis, and inflammatory bowel diseases.
[0168] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of inflammatory diseases. In a
particular embodiment,
the inflammatory disease is selected from rheumatoid arthritis, psoriatic
arthritis, ankylosing
spondylitis, osteoarthritis, allergic airway disease, sarcoidosis, chronic
obstructive pulmonary disease
(COPD) and inflammatory bowel diseases. In a more particular embodiment, the
inflammatory disease
is selected from rheumatoid arthritis, sarcoidosis, psoriatic arthritis,
ankylosing spondylitis, and
inflammatory bowel diseases.
[0169] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of autoimmune diseases. In a particular embodiment, the
autoimmune disease is
selected from COPD, asthma, cutaneous lupus erythrematosus, membranous lupus
nephritis, alopecia
areata, vitiligo, type I diabetes mellitus and inflammatory bowel disease.
[0170] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of autoimmune
diseases. In a particular
embodiment, the autoimmune disease is selected from COPD, asthma, cutaneous
lupus
erythrematosus, membranous lupus nephritis, alopecia areata, vitiligo, type I
diabetes mellitus and
inflammatory bowel disease.
[0171] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with autoimmune diseases, which methods
comprise the
administration of an effective amount of a composition of the invention or one
or more of the
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pharmaceutical compositions herein described for the treatment or prophylaxis
of said condition. In a
particular embodiment, the autoimmune disease is selected from COPD, asthma,
cutaneous lupus
erythrematosus, membranous lupus nephritis, alopecia areata, vitiligo, type I
diabetes mellitus and
inflammatory bowel disease.
[0172] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of autoimmune diseases. In a
particular embodiment,
the autoimmune disease is selected from COPD, asthma, cutaneous lupus
erythrematosus,
membranous lupus nephritis, alopecia areata, vitiligo, type I diabetes
mellitus and inflammatory bowel
disease.
101731 In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of proliferative diseases. In a particular embodiment, the
proliferative disease is
selected from cancer, leukemia, multiple myeloma and psoriasis.
[0174] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of proliferative
diseases. In a particular
embodiment, the proliferative disease is selected from cancer, leukemia,
multiple myeloma and
psoriasis.
[0175] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with proliferative diseases, which
methods comprise the
administration of an effective amount of a composition of the invention or one
or more of the
pharmaceutical compositions herein described for the treatment or prophylaxis
of said condition. In a
particular embodiment, the proliferative disease is selected from cancer,
leukemia, multiple myeloma
and psoriasis.
[0176] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of proliferative diseases. In
a particular embodiment, the
proliferative disease is selected from cancer, leukemia, multiple myeloma and
psoriasis.
[0177] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of allergic diseases. In a particular embodiment, the
allergic disease is eczema.
[0178] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of allergic diseases.
In a particular
embodiment, the allergic disease is eczema.
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[0179] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with allergic diseases, which methods
comprise the
administration of an effective amount of a composition of the invention or one
or more of the
pharmaceutical compositions herein described for the treatment or prophylaxis
of said condition. In a
particular embodiment, the allergic disease is eczema.
[0180] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of allergic diseases. In a
particular embodiment, the
allergic disease is eczema.
[0181] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of transplant rejection. In a particular embodiment, the
transplant rejection is graft-
versus-host diseases.
[0182] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of transplant
rejection. In a particular
embodiment, the transplant rejection is graft-versus-host diseases.
[0183] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with transplant rejection, which
methods comprise the
administration of an effective amount of a composition of the invention or one
or more of the
pharmaceutical compositions herein described for the treatment or prophylaxis
of said condition. In a
particular embodiment, the transplant rejection is graft-versus-host diseases.
[0184] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of transplant rejection. In a
particular embodiment, the
transplant rejection is graft-versus-host diseases.
[0185] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of diseases involving impairment of cartilage turnover. In a
particular embodiment,
the disease involving impairment of cartilage turnover is ankylosing
spondylitis.
[0186] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of diseases involving
impairment of cartilage
turnover. In a particular embodiment, the disease involving impairment of
cartilage turnover is
ankylosing spondylitis.
[0187] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with diseases involving impairment of
cartilage turnover,
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which methods comprise the administration of an effective amount of a
composition of the invention
or one or more of the pharmaceutical compositions herein described for the
treatment or prophylaxis
of said condition. In a particular embodiment, the disease involving
impairment of cartilage turnover is
ankylosing spondylitis.
[0188] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of diseases involving
impairment of cartilage turnover
treatment agent. In a particular embodiment, the disease involving impairment
of cartilage turnover is
ankylosing spondylitis.
[0189] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of congenital cartilage malformations.
[0190] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of congenital
cartilage malformations.
[0191] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with congenital cartilage
malformations, which methods
comprise the administration of an effective amount of a composition of the
invention or one or more
of the pharmaceutical compositions herein described for the treatment or
prophylaxis of said
condition.
[0192] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
therapeutic agent is an agent for the treatment of congenital cartilage
malformations treatment agent.
[0193] In one embodiment, the present invention provides a composition of the
invention, or
pharmaceutical compositions comprising a composition of the invention, for use
in the prophylaxis
and/or treatment of diseases associated with hypersecretion of IL6 or
interferons.
[0194] In another embodiment, the present invention provides compositions of
the invention, or
pharmaceutical compositions comprising a composition of the invention for use
in the manufacture of
a medicament for use in the prophylaxis and/or treatment of diseases
associated with hypersecretion of
IL6 or interferons.
[0195] In additional method of treatment aspects, this invention provides
methods of prophylaxis
and/or treatment of a mammal afflicted with diseases associated with
hypersecretion of IL6 or
interferons, which methods comprise the administration of an effective amount
of a composition of the
invention or one or more of the pharmaceutical compositions herein described
for the treatment or
prophylaxis of said condition.
[0196] In one embodiment, the present invention provides pharmaceutical
compositions comprising a
composition of the invention, and another therapeutic agent. In a particular
embodiment, the other
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therapeutic agent is an agent for the treatment of diseases associated with
hypersecretion of IL6 or
interferons treatment agent.
[0197] Injection dose levels range from about 0.1 mg/kg/h to at least 10
mg/kg/h, all for from about 1
to about 120 h and especially 24 to 96 h. A preloading bolus of from about 0.1
mg/kg to about 10
mg/kg or more may also be administered to achieve adequate steady state
levels. The maximum total
dose is not expected to exceed about 1 g/day for a 40 to 80 kg human patient.
[0198] For the prophylaxis and/or treatment of long-term conditions, such as
degenerative conditions,
the regimen for treatment usually stretches over many months or years so oral
dosing is preferred for
patient convenience and tolerance. With oral dosing, one to four (1-4) regular
doses daily, especially
one to three (1-3) regular doses daily, typically one to two (1-2) regular
doses daily, and most typically
one (1) regular dose daily are representative regimens. Alternatively for long
lasting effect drugs, with
oral dosing, once every other week, once weekly, and once a day are
representative regimens. In
particular, dosage regimen can be every 1-14 days, more particularly 1-10
days, even more
particularly 1-7 days, and most particularly 1-3 days.
[0199] Using these dosing patterns, each dose provides from about 1 to about
1000 mg of a
composition of the invention, with particular doses each providing from about
10 to about 500 mg and
especially about 30 to about 250 mg.
[0200] Transdermal doses are generally selected to provide similar or lower
blood levels than are
achieved using injection doses.
[0201] When used to prevent the onset of a condition, a composition of the
invention will be
administered to a patient at risk for developing the condition, typically on
the advice and under the
supervision of a physician, at the dosage levels described above. Patients at
risk for developing a
particular condition generally include those that have a family history of the
condition, or those who
have been identified by genetic testing or screening to be particularly
susceptible to developing the
condition.
[0202] A composition of the invention can be administered as the sole active
agent or it can be
administered in combination with other therapeutic agents, including other
compound of the
inventions that demonstrate the same or a similar therapeutic activity and
that are determined to be
safe and efficacious for such combined administration. In a specific
embodiment, co-administration of
two (or more) agents allows for significantly lower doses of each to be used,
thereby reducing the side
effects seen.
[0203] In one embodiment, a composition of the invention or a pharmaceutical
composition
comprising a composition of the invention is administered as a medicament. In
a specific embodiment,
said pharmaceutical composition additionally comprises a further active
ingredient.
[0204] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of a disease involving
inflammation, particular
agents include, but are not limited to, immunoregulatory agents e.g.
azathioprine, corticosteroids (e.g.
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prednisolone or dexamethasone), cyclophosphamide, cyclosporin A, tacrolimus,
mycophenolate,
mofetil, muromonab-CD3 (OKT3, e.g. Orthocolone0), ATG, aspirin, acetaminophen,
ibuprofen,
naproxen, and piroxicam.
[0205] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of arthritis (e.g.
rheumatoid arthritis), particular
agents include but are not limited to analgesics, non-steroidal anti-
inflammatory drugs (NSAIDS),
steroids, synthetic DMARDS (for example but without limitation methotrexate,
leflunomide,
sulfasalazine, auranofin, sodium aurothiomalate, penicillamine, chloroquine,
hydroxychloroquine,
azathioprine, tofacitinib, baricitinib, fostamatinib, and cyclosporin), and
biological DMARDS (for
example but without limitation infliximab, etanercept, adalimumab, rituximab,
and abatacept).
[0206] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of proliferative
disorders, particular agents
include but are not limited to: methotrexate, leukovorin, adriamycin,
prednisone, bleomycin,
cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine,
vinblastine, vinorelbine,
doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin,
anti-HER2 monoclonal
antibody (e.g. HerceptinTm), capecitabine, raloxifene hydrochloride, EGFR
inhibitors (e.g. lressa0,
Tarcevem, Erbituxim), VEGF inhibitors (e.g. AvastinTm), proteasome inhibitors
(e.g. Velcaderm),
Glivec0 and hsp90 inhibitors (e.g. 17-AAG). Additionally, the composition of
the invention
comprising a compound according to Formula I and a JAK inhibitor may be
administered in
combination with other therapies including, but not limited to, radiotherapy
or surgery. In a specific
embodiment the proliferative disorder is selected from cancer,
myeloproliferative disease or
leukaemia.
[0207] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of autoimmune diseases,
particular agents
include but are not limited to: glucocorticoids, cytostatic agents (e.g.
purine analogs), alkylating
agents, (e.g nitrogen mustards (cyclophosphamide), nitrosoureas, platinum
compound of the
inventions, and others), antimetabolites (e.g. methotrexate, azathioprine and
mercaptopurine),
cytotoxic antibiotics (e.g. dactinomycin anthracyclines, mitomycin C,
bleomycin, and mithramycin),
antibodies (e.g. anti-CD20, anti-CD25 or anti-CD3 (OTK3) monoclonal
antibodies, Atgam0 and
Thymoglobuline0), cyclosporin, tacrolimus, rapamycin (sirolimus), interferons
(e.g. IFN41), TNF
binding proteins (e.g. infliximab, etanercept, or adalimumab), mycophenolate,
fingolimod and
myriocin..
[0208] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of transplant
rejection, particular agents include
but are not limited to: calcineurin inhibitors (e.g. cyclosporin or tacrolimus
(FK506)), mTOR
inhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g.
azathioprine, mycophenolic acid),
corticosteroids (e.g. prednisolone, hydrocortisone), antibodies (e.g.
monoclonal anti-IL-2Ra receptor
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antibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.
anti-thymocyte globulin
(ATG), anti-lymphocyte globulin (ALG)).
[0209] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of asthma and/or
rhinitis and/or COPD,
particular agents include but are not limited to: beta2-adrenoceptor agonists
(e.g. salbutamol,
levalbuterol, terbutaline and bitolterol), epinephrine (inhaled or tablets),
anticholinergics (e.g.
ipratropium bromide), glucocorticoids (oral or inhaled). Long-acting I32-
agonists (e.g. salmeterol,
formoterol, bambuterol, and sustained-release oral albuterol), combinations of
inhaled steroids and
long-acting broncho dilators (e.g. flutic as one/s almeterol,
budesonide/formoterol), leukotriene
antagonists and synthesis inhibitors (e.g. montelukast, zafirlukast and
zileuton), inhibitors of mediator
release (e.g. cromoglycate and ketotifen), biological regulators of IgE
response (e.g. omalizumab),
antihistamines (e.g. ceterizine, cinnarizine, fexofenadine) and
vasoconstrictors (e.g. oxymethazoline,
xylomethazoline, nafazoline and tramazoline).
[0210] Additionally, a composition of the invention may be administered in
combination with
emergency therapies for asthma and/or COPD, such therapies include oxygen or
heliox administration,
nebulized salbutamol or terbutaline (optionally combined with an
anticholinergic (e.g. ipratropium),
systemic steroids (oral or intravenous, e.g. prednisone, prednisolone,
methylprednisolone,
dexamethasone, or hydrocortisone), intravenous salbutamol, non-specific beta-
agonists, injected or
inhaled (e.g. epinephrine, isoetharine, isoproterenol, metaproterenol),
anticholinergics (IV or
nebulized, e.g. glycopyrrolate, atropine, ipratropium), methylxanthines
(theophylline, aminophylline,
bamiphylline), inhalation anesthetics that have a bronchodilatory effect (e.g.
isoflurane, halothane,
enflurane), ketamine and intravenous magnesium sulfate.
[0211] In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of inflammatory bowel
disease (IBD), particular
agents include but are not limited to: glucocorticoids (e.g. prednisone,
budesonide) synthetic disease
modifying, immunomodulatory agents (e.g. methotrexate, leflunomide,
sulfasalazine, mesalazine,
azathioprine, 6-mercaptopurine and cyclosporin) and biological disease
modifying,
immunomodulatory agents (infliximab, adalimumab, rituximab, and abatacept).
[0212] In one embodiment, a composiotion of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of SLE, particular
agents include but are not
limited to: human monoclonal antibodies (belimumab (Benlysta)), Disease-
modifying antirheumatic
drugs (DMARDs) such as antimalarials (e.g. plaquenil, hydroxychloroquine),
immunosuppressants
(e.g. methotrexate and azathioprine), cyclophosphamide and mycophenolic acid,
immunosuppressive
drugs and analgesics, such as nonsteroidal anti-inflammatory drugs, opiates
(e.g. dextropropoxyphene
and co-codamol), opioids (e.g. hydrocodone, oxycodone, MS Contin, or
methadone) and the fentanyl
duragesic transdermal patch.
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102131 In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of psoriasis,
particular agents include but are
not limited to: topical treatments such as bath solutions, moisturizers,
medicated creams and ointments
containing coal tar, dithranol (anthralin), corticosteroids like
desoximetasone (TopicortTm),
fluocinonide, vitamin D3 analogues (for example, calcipotriol), argan oil and
retinoids (etretinate,
acitretin, tazarotene), systemic treatments such as methotrexate,
cyclosporine, retinoids, tioguanine,
hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus,
fumaric acid esters or
biologics such as AmeviveTM, EnbrelTM, HumiraTM, RemicadeTM, RaptivaTM and
ustekinumab (a IL-12
and IL-23 blocker). Additionally, a compound of the invention may be
administered in combination
with other therapies including, but not limited to phototherapy, or
photochemotherapy (e.g. psoralen
and ultraviolet A phototherapy (PUVA)).
102141 In one embodiment, a composition of the invention is co-administered
with another
therapeutic agent for the treatment and/or prophylaxis of allergic reaction,
particular agents include but
are not limited to: antihistamines (e.g. cetirizine, diphenhydramine,
fexofenadine, levocetirizine),
glucocorticoids (e.g. prednisone, betamethasone, beclomethasone,
dexamethasone), epinephrine,
theophylline or anti-leukotrienes (e.g. montelukast or zafirlukast), anti-
cholinergics and decongestants.
[0215] By co-administration is included any means of delivering two or more
therapeutic agents to
the patient as part of the same treatment regime, as will be apparent to the
skilled person. Whilst the
two or more agents may be administered simultaneously in a single formulation,
i.e. as a single
pharmaceutical composition, this is not essential. The agents may be
administered in different
formulations and at different times.
CHEMICAL SYNTHETIC PROCEDURES
General
[0216] A compound of the composition of the invention can be prepared from
readily available
starting materials using the following general methods and procedures. It will
be appreciated that
where typical or preferred process conditions (i.e., reaction temperatures,
times, mole ratios of
reactants, solvents, pressures, etc.) are given, other process conditions can
also be used unless
otherwise stated. Optimum reaction conditions may vary with the particular
reactants or solvent used,
but such conditions can be determined by one skilled in the art by routine
optimization procedures.
[0217] Additionally, as will be apparent to those skilled in the art,
conventional protecting groups
may be necessary to prevent certain functional groups from undergoing
undesired reactions. The
choice of a suitable protecting group for a particular functional group as
well as suitable conditions for
protection and deprotection are well known in the art. For example, numerous
protecting groups, and
their introduction and removal, are described in T. W. Greene and P. G. M.
Wuts, Protecting Groups
in Organic Synthesis, Wiley-Blackwell; 4th Revised edition (2006), and
references cited therein (Wuts
and Greene, 2006).
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[0218] The following methods are presented with details as to the preparation
of a compound of the
composition of the invention as defined hereinabove and the comparative
examples. A compound of
the composition of the invention may be prepared from known or commercially
available starting
materials and reagents by one skilled in the art of organic synthesis.
[0219] All reagents are of commercial grade and are used as received without
further purification,
unless otherwise stated. Commercially available anhydrous solvents are used
for reactions conducted
under inert atmosphere. Reagent grade solvents are used in all other cases,
unless otherwise specified.
Column chromatography is performed on silica standard (30-70 gm). Thin layer
chromatography is
carried out using pre-coated silica gel 60 F-254 plates (thickness 0.25 mm).
1H NMR spectra are
recorded on a 400 MHz Bruker Avance spectrometer or a 300 MHz Bruker Avance
DPX
spectrometer. Chemical shifts (6) for 1H NMR spectra are reported in parts per
million (ppm) relative
to tetramethylsilane (6 0.00) or the appropriate residual solvent peak as
internal reference.
Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet
(q), quintet (quin), multiplet (m)
and broad (br). Electrospray MS spectra are obtained on a Waters platform
LC/MS spectrometer or
with a Waters Acquity UPLC with Waters Acquity PDA detector and SQD mass
spectrometer.
Columns used: UPLC BEH C18 1.7 gm, 2.1 x 5 mm VanGuard pre-column with Acquity
UPLC BEH
C18 1.7 gm, 2.1 x 30 mm column or Acquity UPLC BEH C18 1.7 gm, 2.1 x 50 mm
column. All the
methods are using MeCN/H20 gradients. MeCN and H20 contain either 0.1% formic
acid or 0.05%
NH3. Preparative LCMS: columns used, Waters XBridge Prep C18 5 gm ODB 30 x 100
mm
(preparative column) and Waters XBridge C18 5 gm, 4.6 mm x 100 mm (analytical
column). All the
methods are using MeCN/H20 gradients. MeCN and H20 contain either 0.1% formic
acid or 0.1%
diethylamine.
Table I. List of abbreviations used in the experimental section.
Abbrevi Abbrevi
definition definition
ation ation
AcOH acetic acid DCM dichloromethane
APMA 4-aminophenylmercuric acetate dd doublet of doublets
acl= aqueous DIPEA diisopropylethylamine
atm atmosphere DMAP dimethylaminopyridine
BINAP (+/-)-2,2' -bis(diphenylphosphino)- DMF dimethylformamide
1,1'-binaphthyl
DMSO dimethylsulfoxide
Boc tert-butyloxy-carbonyl
dt doublet of triplets
hr broad signal
DTT dithiothreitol
BSA bovine serum albumin
N-(3 -di
EDCI
methylaminopropy1)-N'-eth
Cale calculated ylcarbodiimide hydrochloride
Cpd compound EDTA ethylenediaminetetraacetic acid
d doublet eq. equivalent
chemical shift ES- electrospray negative
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Abbrevi Abbrevi
definition definition
ation ation
ES+ electrospray positive N normality
Et20 diethyl ether NaBH(0
sodium triacetoxyborohydride
Ac)3
Et0Ac ethyl acetate
NaOtBu sodium tert-butylate
Et0H ethanol
NBS N-bromosuccinimide
g gram
NMR nuclear magnetic resonance
h hour
Obsvd observed
high performance liquid
HPLC
chromatography PBS phosphate-buffered saline
Hz hertz PBST phosphate-buffered saline with
Tween 20
Int intermediate
Pd(OAc)2 palladium diacetate
iPrOH isopropanol
Pd/C palladium on carbon
Li0Me lithium methoxide
PPm part-per-million
LiOtBu lithium tert-butoxide
q quadruplet
m multiplet
qd quadruplet of doublets
MeCN acetonitrile
quin quintet
Me0H methanol
r.t. room temperature
mg milligram
s singlet
Mg0Ac magnesium acetate
sat. saturated
MHz megahertz
SEM standard error of the mean
min minute
t triplet
mL millilitre
td triplet of doublets
mmol millimole
TEA triethylamine
mol mole
TFA trifluoroacetic acid
3-(N-morpholino)propanesulfonic
MOPS
acid THF tetrahydrofurane
MS mass spectrometry UPLC ultra-performance liquid
chromatography
MW molecular weight
SYNTHETIC PREPARATION OF THE COMPOUNDS OF THE INVENTION
[0220] The compound of the composition of the invention according to Formula
(X0(a) has been
extensively profiled, and data are disclosed in WO 2013/189771 (Van't Klooster
et al., 2013).
[0221] Similarly, the compound of the invention according to Formula (XXI))
has been extensively
profiled, and data are disclosed in WO 2010/149769 (Menet and Smits, 2010).
The synthesis of the
salt and suitable formulations have been described in W02015/117980, and in
W02015/117981.
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Example 1. General Synthetic Methods
1.1. Synthetic methods overview
NO2 NO,
A fft B
R,
CI N CI
RI R1 1
R
C 1
R3
o1
.., ,,,\.,r NO, D
I\I-L Br,y,-..\-.L
,r NO2
R = I
R2, R2,
N N-.L1 yH N N r
CN R1 R R1 R
I E E 1
R3
1
R2
R2NOr\ l.N ..v, 1:'
, m
N ,1
R1 R R1 R
HOõ.....õ----.0ON
R4,iØ0.0N
R2, --1, ----r, F
R, N N-i--
--N
N N '1 0 2
R1 R
R1 R
1.2. General methods
1.2.1. General method A
_3.. laNO
NO2 ....... 2
N NH
R1n N CI R1
R
[0222] To a solution of NaH (2 eq., 60% in mineral oil) in dry THF cooled at 0
C is added the
corresponding 6-amino-nicotinonitrile (1.1 to 1.2 eq.). After 30 min at 0 C,
the
2-chloro-3-nitropyridine (1 eq.) is added and the reaction is stirred at r.t.
and monitored by UPLC-MS.
If the reaction is not complete, the reaction is cooled again at 0 C and more
NaH is added followed by
more amine. The reaction mixture is poured into icy water and stirred for 2 h.
The precipitate is
filtered off, washed with H20, and air dried under vacuum to afford the
desired compound.
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Illustrative synthesis of general method A:
6-(6-chloro-3-nitro-pyridin-2-ylamino)-nicotinonitrile (Int 1)
NH2 õNO,
.NO2CINCI CI N
N NH
":7=A'N
ON
CN
[0223] To a solution of NaH (2.07 g, 51.81 mmol, 2 eq., 60% in mineral oil) in
dry THF (50 mL)
cooled at 0 C is added 6-amino-nicotinonitrile (3.4 g, 28.5 mmol, 1.1 eq.).
After 30 min at 0 C,
2,6-dichloro-3-nitro-pyridine (5 g, 25.91 mmol, 1 eq.) is added and the
reaction is stirred at r.t. for
16 h. The reaction is cooled to 0 C, NaH (0.5 g, 13 mmol, 0.5 eq.) is added
and the reaction is stirred
for 1 h at 0 C then for 2 h at r.t. The reaction mixture is poured into icy
water and stirred for 2 h. The
precipitate is filtered off, washed with H20, and air dried under vacuum. The
obtained solid is taken
up in MeCN (75 mL), stirred at r.t. for 1 h 30 min and at 0 C for 1 h. It is
then filtered and washed
with Me0H to afford the desired compound.
[0224] 1H NMR (400 MHz, DMSO-d6) 6 10.81 (1H, br s), 8.73 (1H, dd), 8.61 (1H,
d), 8.31 (1H, dd),
8.01 (1H, dd), 7.36 (1H, d).
1.2.2. General method B
NO NO2
I 2
R2. I
CI N NH N N NH
[0225] To 6-chloro-3-nitro-pyridin-2-ylamino derivative (1 eq.) in DMSO is
added the corresponding
amine (1.1 eq.) and DIPEA (2 eq.), the reaction mixture is then microwaved at
110-130 C until
completion of the reaction. The mixture is diluted with H20, the precipitate
is filtered off and air dried
under vacuum to give the desired compound.
Illustrative synthesis of general method B:
6-13-nitro-6-(tetrahydro-pyran-4-ylamino)-pyridin-2-ylaminol-nicotinonitrile
(Int 8)
No2
1
N H2 C NO2
CI N N H HNNH
0
===IL". N
0
ON
ON
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[0226] To 6-(6-chloro-3-nitro-pyridin-2-ylamino)-nicotinonitrile (Int 1, 4 g,
14.51 mmol, 1 eq.) in
DMSO (20 mL) is added tetrahydro-pyran-4-ylamine (1.65 mL, 15.96 mmol, 1.1
eq.) and DIPEA
(5.05 mL, 29.02 mmol, 2 eq.), the reaction mixture is then microwaved at 130
C for 20 min. The
mixture is diluted with H20 and Et20, the precipitate is filtered off and air
dried under vacuum to give
the desired compound.
[0227] 1H NMR (300 MHz, DMSO-d6) 6 11.38 (1H, s), 8.78 (1H, d), 8.47-8.63 (2H,
m), 8.39 (1H,
dd), 8.17 (1H, d), 6.27 (1H, d), 3.98-4.12 (1H, m), 3.91 (2H, d), 3.52 (2H,
t), 1.94 (2H, d), 1.33-1.67
(2H, m).
1.2.3. General method C
Br NO2
R2 -3- R2
N N NH N N NH
R1 R R1
[0228] NBS (1.1 to 2 eq.) is added to a solution of 3-nitro-pyridine-2,6-
diamino derivative (1 eq.) in
dry MeCN, the reaction is stirred at r.t. and monitored by UPLC-MS. If full
completion is not reached,
additional NBS is added until no starting material is left. The precipitate
formed is filtered off, washed
with Et20 and air dried under vacuum to provide the desired compound.
Illustrative synthesis of general method C:
6-15-bromo-3-nitro-6-(tetrahydro-pyran-4-ylamino)-pyridin-2-ylaminol-
nicotinonitrile (Int 11)
No2 Br nNO2
HN'¨'N NH HN N NH
cJ
0
CN CN
[0229] NBS (2.04 g, 11.46 mmol, 1.3 eq.) is added to a solution of 643-nitro-6-
(tetrahydro-pyran-4-
ylamino)-pyridin-2-ylamino]-nicotinonitrile (Int 8, 3 g, 8.81 mmol, 1 eq.) in
dry MeCN (150 mL) and
the reaction is stirred at r.t. for 4 h. NBS (0.31 g, 1.76 mmol, 0.2 eq.) is
added and the reaction is
stirred at r.t. for another 16 h. The precipitate formed is filtered off,
washed with Et20 and air dried
under vacuum to provide the desired compound.
[0230] 1H NMR (300 MHz, DMSO-d6) 6 11.10 (1H, br s), 8.80 (1H, m), 8.34-8.50
(3H, m), 7.72
(1H, d), 4.05-4.25 (1H, m), 3.93 (2H, m), 3.38-3.55 (2H, m), 1.70-1.87 (4H,
m).
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1.2.4. General method D
Br ,NO2
0
R2,N
R1 R IN- NH
R1
[0231] LiOtBu (3 eq.) is added portionwise to a solution of the corresponding
alcohol (5 eq.) in dry
1,4-dioxane, or in the corresponding alcohol used as the solvent. 2-amino-3-
bromo pyridine derivative
(1 eq.) is then added followed by CuI (0.6 eq.). The reaction is heated to 80-
120 C, or at 110-150 C
under microwaves irradiation, until completion of the reaction. The mixture is
poured into icy water or
a 1 N aqueous solution of HC1 is added. The precipitate is filtered off and
air dried under vacuum. The
residue is then purified by flash chromatography on silica gel to obtain the
desired compound.
Illustrative synthesis of general method D:
6-15-12-(2-hydroxy-ethoxy)-ethoxyl-3-nitro-6-(tetrahydro-pyran-4-ylamino)-
pyridin-2-ylaminol-
nicotinonitrile (Int 19)
NO2
HN ¨NH
HN N NH
CN
ON
[0232] LiOtBu (2.87 g, 35.8 mmol, 3 eq.) is added portionwise to a solution of
2-(2-hydroxy-ethoxy)-ethanol (5.7 mL, 59.7 mmol, 5 eq.) in dry 1,4-dioxane (50
mL). 645-bromo-3-
nitro-6-(tetrahydro-pyran-4-ylamino)-pyridin-2-ylamino]-nicotinonitrile (Int
11, 5.0 g, 11.9 mmol, 1
eq.) is added followed by CuI (1.36 g, 7.2 mmol, 0.6 eq.). The reaction is
then heated to 120 C for
4 h. The mixture is cooled to 0 C, a 1 N aqueous solution of HC1 (50 mL) is
added and the resulting
mixture is stirred at r.t. for 20 min. The precipitate is filtered and dried
under vacuum. The residue is
then purified by flash chromatography on silica gel, eluting from 0 to 5% of
Me0H in DCM to give
the desired compound.
[0233] MW (calcd): 444.45; MW (obsd): 445.18 ES+.
[0234] 1H NMR (400 MHz, DMSO-d6) 6 11.42 (1H, s), 8.75-8.79 (1H, m), 8.47-8.50
(1H, m), 8.38
(1H, dd), 7.87 (1H, d), 7.69 (1H, s), 4.58-4.72 (1H, m), 4.21-4.26 (2H, m),
4.11-4.21 (1H, m), 3.94
(2H, dd), 3.82 (2H, dd), 3.44-3.57 (6H, m), 1.81-1.90 (2H, m), 1.74 (2H, qd).
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1.2.5. General method E
R'.-,.NO2 R',....._.õ...--...õ.._, ...., N
R2N.NN 1 1
___________________________________ 3.- R2N N ., ------1,1
'1
1 1 1
R1 R R1 R
[0235] To a solution of 2,6-diamino-5-nitro-pyridin derivative (1 eq.) in dry
Me0H are added
trimethylorthoformate (roughly 0.1 mL for 0.1 mmol of 2,6-diamino-5-nitro-
pyridin derivative) and
formic acid (roughly 0.1 mL for 0.1 mmol of 2,6-diamino-5-nitro-pyridin
derivative). NH4C1 (4 eq.)
and Zn (4 to 5 eq.) are then added and the mixture is heated to 70 C until
completion of the reaction.
The reaction mixture is then cooled to r.t.
[0236] If upon cooling precipitation is observed, the solid is filtered and
submitted to aqueous
work-up with DCM/CHC13 and a 2% formic acid aqueous solution to afford the
desired compound.
[0237] If upon cooling no precipitation occurs, solvents are evaporated and
the residue is then
purified by flash chromatography on silica gel to obtain the desired compound.
Illustrative synthesis of general method E:
6-16-12-(2-hydroxy-ethoxy)-ethoxyl-5-(tetrahydro-pyran-4-ylamino)-imidazo[4,5-
blpyridin-3-yll-
nicotinonitrile (Compound 1)
1 1
HN---...N*--'N - HN---..-N-:;---01N
1.
N /I\
y..o...... ....o....
c
CN N
[0238] To a solution of 6- [5- [2-(2-hydroxy-ethoxy)-ethoxy]-3-nitro-6-
(tetrahydro-pyran-4-ylamino)-
pyridin-2-ylamino]-nicotinonitrile (Int 19, 2.3 g, 5.2 mmol, 1 eq.) in dry
Me0H (60 mL) is added
trimethylorthoformate (10 mL) and formic acid (10 mL). NH4C1 (1.1 g, 20.7
mmol, 4 eq.) and Zn (1.4
g, 20.7 mmol, 4 eq.) are then added and the mixture is heated to reflux for 2
h. Me0H (30 mL) is
added and the reaction mixture is heated to reflux for 1 h.
[0239] The mixture is cooled to r.t., the precipitate formed is filtered and
dried under vacuum. Me0H
(100 mL) and formic acid (2 mL) are added and the resulting mixture is stirred
under reflux for 1 h.
The mixture is cooled to r.t., poured into icy water and the precipitate
formed is filtered and dried
under vacuum. The solid is suspended in a mixture of DCM and CHC13, filtered
through celite and the
filtrate is washed with a 2% formic acid aqueous solution. The organic phase
is dried over Na2SO4,
filtered and concentrated to dryness to afford the desired compound.
102401 MW (calcd): 424.46; MW (obsd): 425.40 ES+
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[0241] 1H NMR (400 MHz, DMSO-d6) 6 9.00 (1H, dd), 8.89-8.95 (1H, m), 8.75 (1H,
s), 8.62 (1H,
dd), 7.59 (1H, s), 6.04 (1H, d), 4.61-4.69 (1H, m), 4.22 (2H, dd), 4.05-4.17
(1H, m), 3.90-3.98 (2H,
m), 3.83 (2H, dd), 3.50-3.60 (6H, m), 1.99 (2H, m), 1.55-1.70 (2H, m).
1.2.6. General method F
HO -.-,0 .-----N
1 O. 0
1
1\N.----N R,
N"---"se--"N
)\ _,..
)\ 0
O__
CN CN
[0242] A mixture of Compound 1, corresponding carboxylic acid (1.5 eq.), DMAP
(1.5 eq.) and
EDCI (2.25 eq.) are stirred in DCM at r.t. until completion of the reaction.
The reaction is quenched
with H20, extracted with DCM, and then the organic layer is dried over MgSO4
and evaporated to
dryness. The residue is purified by flash chromatography on silica gel to
obtain the desired compound.
Illustrative synthesis of general method F:
(S)-2-tert-butoxycarbonylamino-3-methyl-butyric acid 242-13-(5-cyano-pyridin-2-
y1)-5-(tetrahydro-
pyran-4-ylamino)-3H-imidazo[4,5-b]pyridin-6-yloxy]-ethoxy}-ethyl ester (Int
30)
`./
Ho.õ,,c).(:)...___N
HNN-____ N H
0
HN"--.1,4- N
), 0CN 0
CN
[0243] A mixture of Compound 1(42 mg, 0.1 mmol, 1 eq.), boc-(S)-valine (33 mg,
0.15 mmol, 1.5
eq.), DMAP (19 mg, 0.15 mmol, 1.5 eq.), and EDCI (45 mg, 0.225 mmol, 2.25 eq.)
is stirred in DCM
(5 mL) at r.t. for 3 h. The reaction is quenched with H20, extracted with DCM,
then the organic layer
is dried over MgSO4 and evaporated to dryness. The residue is purified by
flash chromatography on
silica gel, eluting from 0 to 100% of Et0Ac in heptanes to give the desired
compound.
1.2.7. General method G: Salification method
[0244] The starting material is dissolved in hot Et0Ac or in a hot mixture of
Et0Ac and Me0H (5/1).
Oxalic acid (0.2 M in Et0Ac, 1 eq.) is added to the hot solution. A
precipitate forms, which is filtered,
rinsed with Et20 and dried to afford the desired compound as the oxalic acid
salt of the starting
material.
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Illustrative synthesis of general method G:
(S)-2-amino-3-methyl-butyric acid 2-12-13-(5-cyano-pyridin-2-y1)-5-(tetrahydro-
pyran-4-ylamino)-
311-imidazo[4,5-blpyridin-6-yloxyl-ethoxy)-ethyl ester oxalic acid salt
(Compound 10)
H 2 N 0 -N H2N
HNNI
0
N 0
HNNN
0
HO
OH
CN 0
CN
[0245] Compound 9 (100 mg, 0.19 mmol, 1 eq.) is dissolved in hot Et0Ac (10 mL)
and oxalic acid
(0.2 M in Et0Ac, 0.96 mL, 0.19 mmol, 1 eq.) is added to the hot solution. The
formed precipitate is
filtered, rinsed with Et20 and dried to afford the desired compound.
1.2.8. Synthesis of (3,4 cis)-3-
Amino-tetrahydro-pyran-4-ol (Int 28)
HN HN
41 I HO
0 N 0 N
HO
HO HN HN NH2 NH2
iv
HO v HO HO
1.2.8.1. Step 0: 3,7-dioxa-bicyclo[4.1.0Jheptane
[0246] To a solution of m-chloroperbenzoic acid (23.51 g, 136.2 mmol, 2 eq.)
in DCM (15 mL) is
added a solution of 3,6-dihydro-2H-pyran (5.73 g, 68.1 mmol, 1 eq.) in DCM (10
mL). The reaction
mixture is allowed to stir at r.t. for 6 h, after which m-chloroperbenzoic
acid (11.76 g, 68.1 mmol,
1 eq.) is added. The reaction mixture is stirred at r.t. for 16 h and filtered
off. The filtrate is washed
with saturated solutions of Na2S03, NaHCO3, and water. The organic layer is
then dried over Na2SO4,
filtered and concentrated in vacuo to afford the desired compound, used in the
next step without
further purification.
1.2.8.2. Step ii): (3,4 trans)-3-Benzylamino-tetrahydro-pyran-4-ol
[0247] A mixture of 3,7-dioxa-bicyclo[4.1.0]heptane (2.7 mmol, 1 eq.) and
benzylamine (300 L, 2.7
mmol, 1 eq.) in Et0H (10 mL) is heated at reflux temperature for 18 h. Et0H is
then evaporated and
the crude is purified by column chromatography on silica gel, eluting with
DCM:MeOH:NH4OH
10:1:0.1, to give the desired compound.
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1.2.8.3. Step N-Benzyl-N-((3,4 trans)-4-hydroxy-tetrahydro-pyran-3-y1)-
benzamide
[0248] Benzoyl chloride (78 L, 0.68 mmol, 1 eq.) is added dropwise to an ice-
cooled solution of
(3,4 anti)-3-benzylamino-tetrahydro-pyran-4-ol from previous step (140 mg,
0.68 mmol, 1 eq.) and
TEA (280 L, 2.03 mmol, 3 eq.) in DCM (2 mL). The reaction mixture is stirred
at r.t. for 1 h. The
mixture is then washed twice with a 2 N aqueous HC1 solution. The aqueous
layers are extracted with
DCM, and the combined organic layers are then dried over Na2SO4, filtered and
concentrated in vacuo
to afford the desired compound.
1.2.8.4. Step iv): (3,4 cis)-3-Benzylamino-tetrahydro-pyran-4-ol
[0249] A solution of N-benzyl-N-((3,4 trans)-4-hydroxy-tetrahydro-pyran-3-y1)-
benzamide (220 mg,
0.71 mmol, 1 eq.) in DCM (2.5 mL) is added dropwise to thionyl chloride (195
L, 2.68 mmol, 3.8
eq.) at 0 C. The reaction mixture is stirred at r.t. for 4 h, and then
concentrated in vacuo. To the
residue is added a 6 N aqueous HC1 solution (2 mL), and the resulting mixture
is heated at reflux
temperature for 18 h. After cooling, a precipitate is filtered off, washed
with water, and the filtrate is
extracted with Et0Ac. To the aqueous layer is added Et20 and a 2 N NaOH
aqueous solution is added
to make the mixture alkaline. The phases are separated and the aqueous phase
is extracted with DCM
and Et0Ac. The combined organic layers are then dried over Na2SO4, filtered
and concentrated in
vacuo to afford the desired compound.
1.2.8.5. Step v): (3,4 cis)-3-Amino-tetrahydro-pyran-4-ol
[0250] A solution of (3,4 cis)-3-benzylamino-tetrahydro-pyran-4-ol (100 mg,
0.48 mmol, 1 eq.) in
Me0H (3 mL) is hydrogenated over 10% Pd/C (40 mg) for 1.5 h at r.t. under 1
atm of H2. The catalyst
is removed by filtration through celite, washed with Me0H and the filtrate is
evaporated to give the
desired compound.
1.2.9. Synthesis of (cis-1,4)-1-methyl-4-methylamino-cyclohexanol (Int 29)
NH NH
, H
c:)
' OH
1.2.9.1. Step i): (cis-1,4)-(4-hydroxy-4-methyl-cyclohexy0-carbamic acid tert-
butyl ester
[0251] To a suspension of cis-4-amino-1-methylcyclohexanol (1.0 g, 7.74 mmol,
1 eq.) in MeCN (15
mL) is added di-tert-butyl dicarbonate (1.85 g, 8.47 mmol, 1.1 eq.) and the
mixture is stirred at r.t. for
16 h. The precipitate is filtered, washed with hexane and dried to afford the
desired compound.
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1.2.9.2. Step ii): (cis-1,4)-1-methyl-4-methylamino-cyclohexanol
[0252] To a 2.0 M solution of LiA1H4 in THF (7 mL, 14.0 mmol, 4.9 eq.) is
added portionwise
(cis-1,4)-(4-hydroxy-4-methyl-cyclohexyl)-carbamic acid tert-butyl ester (660
mg, 2.9 mmol, 1 eq.) at
r.t. The reaction mixture is stirred at r.t. for 1 h and at reflux for 45 min.
The reaction mixture is cooled
to r.t., then water and THF are added. The precipitate is filtered off and
washed with THF. The filtrate
is concentrated to dryness, affording the desired compound.
1.2.10. Intermediate 34: 2-12-13-
(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-b]pyridin-6-ylloxyethoxylethyl (2S)-2-
[12-(tert-butoxycarbonylamino)
acetyllaminal-3-methyl-butanoate
0
H
1X,r, 0 0 N 0
YN
0 0 0
)\
CY ON
ON
[0253] Compound 9 (170 mg, 0.40 mmol, 1 eq.), 2-(tert-
butoxycarbonylamino)acetic acid
(Boc-Gly-OH, 105 mg, 0.60 mmol, 1.5 eq.), EDCI (173 mg, 0.90 mmol, 2.25 eq.)
and DMAP (73 mg,
0.6 mmol, 1.5 eq.) are mixed in DCM (4 mL) and stirred at r.t. overnight. The
reaction is quenched
with brine, extracted with DCM and the combined organic phases are evaporated
to give the desired
compound.
Table II. Intermediates used towards the compounds of the invention.
SM = Starting Material, Mtd = Method, .. MS Mes'd = Mesured
mass
Int# Structures Name SM Mtd MW MS
Mes'd
CI N N H itro-2-
6-[(6-chloro-3-
2,6-
dichloro
n
1 -3- A 275.7
276.3
pyridyeamino]pyri
nitropyri
dine-3 -carbonitrile
dine
CN
64[6-acis-3,4)-4-
hydroxytetrahydrop
HNN71\1H Intl
2 HO yran-3-yl] amino] -
3 -nitro-2- Int 28 356.3 357.4
pyridyl] amino] pyri
ON dine-3 -carbonitrile
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MS
Int# Structures Name SM Mtd MW
Mes'd
.NO2
6-[[6-[(1,1-
HN1 NNH dioxothian-4-
3 '-'
yl)amino]-3-nitro-
--L'N 2- Intl B 388.4 389.2
'S' Y pyridyl]amino]pyri
O c) CN dine-3-carbonitrile
6-[[6-[((cis-1,4)-4-
1 hydroxy-4-methyl-
Th\JNNH cyclohexyl)- Intl
4 methyl-amino]-3- + B 382.4 -
C-LN
Y nitro-2- Int 29
. pyridyl]amino]pyri
' OH CN dine-3-carbonitrile
1 NH 6- [[6- [((cis1,4)-4-
Hf\l hydroxy-4-methyl-
'N'
cyclohexyl)amino]-
Intl B 368.4 -
Cj)''N 3-nitro-2-
y pyridyl]amino]pyri
' OH ON dine-3-carbonitrile
NO2
1 6-[[6-[(3-
HN'-'f\INH hydroxycyclohexyl
6 -L )amino]-3-nitro-2- Intl B 354.4 355.4 l -)-1\1
pyridyl]amino]pyri
y
OH dine-3-carbonitrile
CN
NO2
1 , 6-[[6-[(4,4-
HN'N' 'NH difluorocyclohexyl)
7 amino]-3-nitro-2- Intl B 374.4 375.4
ctsD -'LN
.) pyridyl]amino]pyri
dine-3-carbonitrile
F F CN
-NO2
1 6-[[3-nitro-6-
HNINNH (tetrahydropyran-4-
8
)'. l'I\I ylamino)-2- Intl B 340.3 341.4
pyridyl]amino]pyri
-10 y dine-3-carbonitrile
CN
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MS
Int# Structures Name SM Mtd MW
Mes'd
...,,,...z...,....... ,NO2
I 6-[[6-
.NNN H [methyl(tetrahydro
9 ).. ,.. j.,N p3ynriatnro-42-yDamino]-
Intl B 354.4 -
pyridyl]amino]pyri
dine-3-carbonitrile
ON
Br nNO2
I 6-[[5-bromo-6-
HN N-- NH [[(cis-3,4)-4-
hydroxytetrahydrop
H Ob ,. 435.4
N yran-3-yl]amino]- Int 2 C 435.2
437.3
I 3-nitro-2-
pyridyl]amino]pyri
ON dine-3-carbonitrile
NO2
1 6-[[5-bromo-3-
HN N NH
nitro-6-
-
11 )\ (tetrahydropyran-4-
N ylamino)-2- Int 8 C 419.2 419.5
421.4
0 y pyridyl]amino]pyri
dine-3-carbonitrile
ON
Br NO2
6-[[5-bromo-6-
HN Iµ NH
[(1,1-dioxothian-4-
--r-:
yl)amino]-3-nitro- 467.2
12 Int 3 C 467.3
---- =-=71N 2- 469.3
I
,- ---y pyridyl]amino]pyri
dine-3-carbonitrile
o' '0
CN
64[5-bromo-6-[(4-
1 hydroxy-4-methyl-
N'¨'eNH cyclohexyl)-
13 ( ,) methyl-amino]-3- Int 4 C 461.3
Y
nitro-2-
pyridyl]amino]pyri
'. OH ON dine-3-carbonitrile
Br - 2 64[5-bromo-6-
1 [((cis1,4)-4-
HN''N'¨'NH hydroxy-4-methyl-
14 ,) cyclohexyl)amino]- Int 5 C 447.3 -
[] !\1
y 3-nitro-2-
pyridyl]amino]pyri
S. OH ON dine-3-carbonitrile
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MS
Int# Structures Name SM Mtd MW
Mes'd
BrNO,
I 6-[[5-bromo-6-[(3-
HN N NH hydroxycyclohexyl
433.4
15 )amino]-3-nitro-2- Int 6 C 433.3
tl N 435.4
OH
pyridyl]amino]pyri
r,
dine-3-carbonitrile
ON
Br
I 6-[[5-bromo-6-
HNNNH [(4,4-
16 r]o3eynciltorhoe2xyl) , ,..,
Int / C 453.3 453.3
-)- N admitliunO 455.3
y pyridyl]amino]pyri
dine-3-carbonitrile
F F CN
Br .1\102
I 6-[[5-bromo-6-
Th\INNH [methyl(tetrahydro
033.4
17
pyran-4-ypamino]-
Int 9 C 433.3 435.4
)"'N 3-nitro-2-
, JI pyridyl]amino]pyri
dine-3-carbonitrile
CN
rõ.OH 6-[[5-[2-(2-
hydroxyethoxy)eth
-NO2 oxy]-6- Ecis-3,4)-
0
I 4-
HN NNH
18 hydroxytetrahydrop Int 10 D 460.5 -
HO )/ N yran-3-yl]amino]-
0 y 3-nitro-2-
pyridyl]amino]pyri
CN dine-3-carbonitrile
6-[[5-[2-(2-
HO'--0(:)N 2 hydroxyethoxy)eth
,L HN N' NH oxy]-3-nitro-6-
19 N (tetrahydropyran-4- Int 11 D 444.5
445.2
a -,) ylamino)-2-
0
oN pyridyl]amino]pyri
dine-3-carbonitrile
6-[[6-[(1,1-
HO,,Ø,,ONO2 dioxothian-4-
1 HN kr NH yl)amino]-5-[2-(2-
20 --- N hydroxyethoxy)eth Int 12 D 492.5
493.6
C) I oxy]-3-nitro-2-
ON pyridyl]amino]pyri
dine-3-carbonitrile
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MS
Int# Structures Name SM Mtd MW
Mes'd
6-[[5-[2-(2-
No
1 methoxyethoxy)eth
rj NN NH oxy]-6-
[methyl(tetrahydro
21 0 k Int 17 D 472.5 473.6
N pyran-4-yl)amino]-
-- y 3-nitro-2-
0
pyridyl]amino]pyri
CN
dine-3-carbonitrile
6-[[5-[2-(2-
,0õ,-..Ø1o, ,...j
., ..No2
methoxyethoxy)eth
HN N NH oxy]-3-nitro-6-
22 N (tetrahydropyran-4- Int 11 D 458.5 --
459.5
a ylamino)-2-
0
oN pyridyl]amino]pyri
dine-3-carbonitrile
6-[[6-[(3-
hydroxycyclohexyl
HN N' NH )amino]-5-[2-(2-
23 .-- N hydroxyethoxy)eth Int 15 D 458.5
459.5
Ia
OH xo y]-3-nitro-2-
l- '.." ..I
ON pyridyl]amino]pyri
dine-3-carbonitrile
64[64(4,4-
H -.."0^.-- N 2 difluorocyclohexyl)
j,
-
HN N NH amino]-5-[2-(2-
24 N hydroxyethoxy)eth Int 16 D 478.5 479.5
cL(',1 I oxy]-3-nitro-2-
F F ON pyridyl]amino]pyri
dine-3-carbonitrile
6-[[6-[((cis1,4)-4-
o0NO2 hydroxy-4-methyl-
1 cyclohexyl)-
rj N*-NH methy1-amino]-5-
25 [242- Int 13 D 500.6 -
methoxyethoxy)eth
.. oxy]-3-nitro-2-
,
s' OH CN pyridyl]amino]pyri
dine-3-carbonitrile
64[6-[((cis1,4)-4-
....--..,.,0, NO2
hydroxy-4-methyl-
0
H HN
1
cyclohexyl)amino]-
NNH
cl 1 5-[2-(2-
Int 14 D 486.5 -
,,
N methoxyethoxy)eth
26 0
y, oxy]-3-nitro-2-
'.. OH CN pyridyl]amino]pyri
dine-3-carbonitrile
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MS
Int# Structures Name SM Mtd MW
Mes'd
6-[[5-[2-(2-
õ hydroxyethoxy)eth
HO,...-,0,=-=,,O 2
oxy]-6- [((cis1,4)-4-
HN N NH hydroxy-4-methyl-
27 ..-- N Int 14 D 472.5 -
cyclohexyl)amino]-
1
3-nitro-2-
s OH CN
pyridyl]amino]pyri
dine-3-carbonitrile
NH2 NH2
(3,4-cis)-3-Amino- 3'6-
28 HO.)H + HO,-
tetrahydro-pyran-4- dihydro- Example
-
2H- 1.2.8
117.2
ol
pyran
-.NH cis-4-
(cis-1,4)-1-methyl- amino-
1- o- Example
29 4-methylamino- 143.2 -
methylc 1.2.9
cyclohexanol
yclohex
ss OH anol
----õ-- 2-[243-(5-cyano-2-
0
pyridy1)-5-
-,i-- N 0 (tetrahydropyran-4-
vo H ylamino)imidazo[4,
5-b]pyridin-6-
30 c:r'-' '"'-''N Cpd 1 F 623.7 624.9
I yl]oxyethoxy]ethyl
HN ¨ le¨N (2S)-2-(tert-
/ butoxycarbonylami
'0' ----- no)-3-methyl-
CN butanoate
o 2-[243-(5-cyano-2-
0N,K0 pyridy1)-5-
70 El (tetrahydropyran-4-
ylamino)imidazo[4,
31 I 5-b]pyridin-6- Cpd 1 F 581.6 582.7
HNNN yl]oxyethoxy]ethyl
2-(tert-
-o' ¨ butoxycarbonylami
CN no)acetate
o 2-[2-[3-(5-cyano-2-
o
NA,0 pyridy1)-5-
.--0 1 ,--------, (tetrahydropyran-4-
ylamino)imidazo[4,
32 I ___.. 5-b]pyridin-6- Cpd 1 F 595.7 596.7
HN---'N" N yl]oxyethoxy]ethyl
2-[tert-
-,0 ¨ butoxycarbonyl(me
CN thyl)amino]acetate
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MS
Int# Structures Name SM Mtd MW
Mes'd
------ 01-tert-butyl 02-
oy---N __ [242-[3-(5-cyano-
}:) 0-----0 2-pyridy1)-5-
(tetrahydropyran-4-
c;,,,_N
33 I ylamino)imidazo[4, Cpd 1 F 621.6
622.8
HN --N" N 5-b]pyridin-6-
yl]oxyethoxy]ethyl]
o ¨ (2S)-pyrrolidine-
cN 1,2-dicarboxylate
2-[243-(5-cyano-2-
'' o pyridy1)-5-
o Fd 0
(tetrahydropyran-4-
,o " o ylamino)imidazo[4,
õo ,, N 5-b]pyridin-6- Example
34 0 '1--X Cpd 9 680.8 681.8
yl]oxyethoxy]ethyl 1.2.10
' N
[121-N z N (2S)-2-[[2-(tert-
\ butoxycarbonylami
_
CN no)acetyl]amino]-
3-methyl-butanoate
>-0 04-tert-butyl 01-
HN o
)- [2-[2-[3-(5-cyano-
2-pyridy1)-5-
oy
(tetrahydropyran-4-
o 0..-.;---..Ø< ylamino)imidazo[4,
35 Cpd 1 F 695.8 696.9
'o--- -'-------N 5-b]pyridin-6-
yl]oxyethoxy]ethyl]
HN'-'N N (2S)-2-(tert-
)'- butoxycarbonylami
o ¨ no)butanedioate
CN
>0:) 05-tert-butyl 01-
HN '---0
[2-[2-[3-(5-cyano-
0 0 2-pyridy1)-5-
(tetrahydropyran-4-
,o ylamino)imidazo[4,
36 5-b]pyridin-6-
Cpd 1 F 709.8 710.8
I , yl]oxyethoxy]ethyl]
HN -''N N (2S)-2-(tert-
butoxycarbonylami
o ¨ no)pentanedioate
CN
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Int# Structures Name SM Mtd MW MS
Mes'd
04-tert-butyl 01-
o
[242- [3 - (5-cyano-
2-pyridy1)-5-
37 ON> (tetrahydropyran-4-
Cpd 1 F 580.6 581.7
HNN N ylamino)imidazo [4,
rs\i 5-b]pyridin-6-
yl]oxyethoxy] ethyl]
CN butanedioate
Example 2. Preparation of the compounds of
the invention.
2.1. Compound 3: 6-16-12-
(2-hydroxy-ethoxy)-ethoxy]-54((cis-1,4)-4-hydroxy-4-methyl-
cyclohexyl)-methyl-aminol-imidazo[4,5-Npyridin-3-y11-nicotinonitrile
L>C.j OH CN CN
[0254] Formaldehyde (3.1 ILLL, 0.11 mmol, 1 eq.) is added to a solution of
Compound 12 (50 mg, 0.11
mmol, 1 eq.) in a mixture of TFA/DCM (2 mL, 1/1). After stirring at r.t. for
30 min, NaBH(0Ac)3 (47
mg, 0.22 mmol, 2 eq.) is added and the reaction is stirred for 1 h at r.t. The
reaction mixture is then
evaporated to dryness and the crude product is purified by preparative HPLC-MS
to obtain the desired
compound.
2.2. Compound 8: sulfuric acid mono-(2-12-13-(5-cyano-pyridin-2-y1)-5-
(tetrahydro-pyran-4-
ylamino)-3H-imidazo[4,5-blpyridin-6-yloxy]-ethoxyl-ethyl) ester
0'
HNNN
OH HNNN
CN CN
[0255] Compound 1 (84 mg, 0.2 mmol, 1 eq.) is added to a solution of pyridine-
S03 complex
(127 mg, 0.8 mmol, 4 eq.) in pyridine (5 mL) and the reaction is heated to
reflux for 16 h. The mixture
is then evaporated to dryness and purified by flash chromatography on silica
gel, eluting from 100%
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Et0Ac to 100% (DCM/Me0H/AcOH/H20: 90/10/1/1) then to 100% (DCM/Me0H/AcOH/H20:
85/15/2/2) to give the desired compound.
2.3. Compound 9: (S)-2-amino-3-methyl-butyric acid 2-1243-(5-cyano-pyridin-2-
y1)-5-
(tetrahydro-pyran-4-ylamino)-3H-imidazo[4,5-b]pyridin-6-yloxyl-ethoxy}-ethyl
ester
N 0 H2Ny0
0 0
H
C HNNN
ON CON
[0256] TFA (0.5 mL) is added to a solution of Int 30 (20 mg, 0.032 mmol, 1
eq.) in DCM (10 mL),
and the mixture is stirred for 1 h at r.t. The reaction is quenched with a
sat. aq. solution of NaHCO3
and extracted with Et0Ac. The organic layer is dried over MgSO4 and evaporated
to dryness. The
residue is recrystallized from the solvent system DCM/Et20 in pentane to
provide the desired
compound.
2.4. Compound 16: 242-13-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-
b]pyridin-6-ylloxyethoxylethyl 2-aminoacetate
0
0
H 2 N 0 0 N
0 N 0 N
0 0
N
CN CON
[0257] Int 31 (195 mg, 0.34 mmol, 1 eq.) is placed in a TFA/DCM mixture (1/5
mL) and the reaction
is stirred at r.t. for 2 h. The reaction mixture is then diluted with toluene
and evaporated to dryness.
The residue is taken up in DCM, washed with a sat. aq. NaHCO3 solution and the
organic phase is
evaporated to dryness. The residue is dissolved in the minimal amount of DCM,
a large volume of
Et20 is added and the formed solid is allowed to settle at the bottom of the
flask. Solvents are carefully
removed, leaving the solid in the flask, pentane is added and the solid is
filtered to give the desired
compound.
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2.5. Compound 17: 242-13-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-
blpyridin-6-ylloxyethoxylethyl 2-(methylamino)acetate
0
0 0 0
I 0 0 HNNN
HNN
CN CN
[0258] Int 32 (205 mg, 0.34 mmol, 1 eq.) is placed in a TFA/DCM mixture (1/5
mL) and the reaction
is stirred at r.t. for 2 h. The reaction mixture is then diluted with toluene
and evaporated to dryness.
The residue is taken up in DCM, washed with a sat. aq. NaHCO3 solution and the
organic phase is
evaporated to dryness. The residue is dissolved in the minimal amount of DCM,
a large volume of
Et20 is added and the formed solid is allowed to settle at the bottom of the
flask. Solvents are carefully
removed, leaving the solid in the flask, pentane is added and the solid is
filtered to give the desired
compound.
1.1. Compound 18: 2-12-13-(5-cyano-2-pyridy1)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-
bJpyridin-6-ylioxyethoxylethyl (2S)-pyrrolidine-2-carboxylate
0o0¨
HNN
04 0
0
0
0
CN CN
[0259] Int 33 (211 mg, 0.34 mmol, 1 eq.) is placed in a TFA/DCM mixture (1/5
mL) and the reaction
is stirred at r.t. for 2 h. The reaction mixture is then diluted with toluene
and evaporated to dryness.
The residue is taken up in DCM, washed with a sat. aq. NaHCO3 solution and the
organic phase is
evaporated to dryness. The residue is dissolved in the minimal amount of DCM,
a large volume of
Et20 is added and the formed solid is allowed to settle at the bottom of the
flask. Solvents are carefully
removed, leaving the solid in the flask, pentane is added and the solid is
filtered to give the desired
compound.
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2.6. Compound 19: 242-13-(5-cyano-2-pyridyl)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-
blpyridin-6-ylloxyethoxylethyl (2S)-2-[(2-aminoacetyl)amino]-3-methyl-
butanoate
0
H 11
H2No N o
I 11:11 H ii
H oil
0 0
HN---N--N HN-----N-----N
\
0 ¨
CN ON
[0260] Int 34 (212 mg, 0.31 mmol, 1 eq.) is placed in a TFA/DCM mixture (1/5
mL) and the reaction
is stirred at r.t. for 2 h. Toluene is then added and the solvents evaporated
to dryness. The residue is
dissolved in DCM and after addition of a sat. aq. NaHCO3 solution a solid
precipitates. It is filtered
and dried to afford the desired compound.
2.7. Compound 27: (3S)-3-amino-4-12-12-13-(5-cyano-2-pyridyl)-5-
(tetrahydropyran-4-
ylamino)imidazo[4,5-blpyridin-6-ylloxyethoxylethoxyl-4-oxo-butanoic acid
hydrochloric acid
salt
>'o OH
0.1LN 0 0 N 0
.......õ-----.. ...---..,,.0 ..,..-.,...õ__
H-ro........õ.^.Ø----..õ. ....___,--....__N
H 1 ¨.... 1
õ...---..., 0 . 0
HI\I'le---N HCI HNe"--N
tr\I )\ 0
ON CN
[0261] A solution of HC1 in 1,4-dioxane (4 M, 0.4 mL, 1.6 mmol, 5 eq.) is
added to Int 35 (222 mg,
0.32 mmol, 1 eq.) in 1,4-dioxane (4 mL). The mixture is stirred at r.t. for 3
h and then is evaporated to
dryness. The residue is taken up in an HC1 solution in 1,4-dioxane (4 M, 6
mL), the reaction is stirred
at r.t. for 2 h and more HC1 solution in 1,4-dioxane (4 M, 2 mL) is added.
After 1 h stirring at r.t. the
solvents are evaporated to dryness to give the desired compound.
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2.8. Compound 28: 48)-4-amino-5-12-12-13-(5-cyano-2-pyridyl)-5-
(tetrahydropyran-4-
ylamino)imidazo[4,5-blpyridin-6-ylloxyethoxylethoxyl-5-oxo-pentanoic acid
hydrochloric acid
salt
0 0
HO 0
0
OAN H si , 1 0 0 , 0
H2N HCI HN ¨ ¨ `-- N---N,
õ......---.õ, 0
HNN si
7---N = 0 1
)L\ 01 '¨'-----N
)\ 0
CN
CN
[0262] A solution of HC1 in 1,4-dioxane (4 M, 0.41 mL, 1.63 mmol, 5 eq.) is
added to Int 36 (232
mg, 0.33 mmol, 1 eq.) in 1,4-dioxane (4 mL). The mixture is stirred at r.t.
for 3 h and then is
evaporated to dryness. The residue is taken up in an HC1 solution in 1,4-
dioxane (4 M, 6 mL) and the
reaction is stirred at rt for 2 h. The solvents are evaporated to dryness to
give the desired compound.
2.9. Compound 33: 4-124243-(5-cyano-2-pyridyl)-5-(tetrahydropyran-4-
ylamino)imidazo[4,5-
b]pyridin-6-ylloxyethoxylethoxyl-4-oxo-butanoic acid
o o
0000
...---õ.s.õ...õ.õ,...õ---.N
HO,11.õ.___õ,--õ,y0 ¨ ¨ N
'- '0' '¨' '------"*""--..--
0 1 1
HNI\l 0"----N HNre---N
)\
0 _...
ON ON
[0263] Int 37 (90 mg, 0.16 mmol, 1 eq.) is placed in a TFA/DCM mixture (1/5
mL) and the reaction
is stirred at r.t. for 2 h. Toluene is then added and the solvents are
evaporated to dryness. The residue
is purified by flash chromatography on silica gel, eluting from 0 to 6% Me0H
in DCM, and the
obtained product is precipitated from Me0H to afford after filtration the
desired compound.
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Table III. Illustrative compounds of the invention.
SM = Starting Material, Mtd = Method, MS Mes'd = Mesured
mass
Cpd MS
Structure Name SM Method MW
# Mes'd
001-1 6-[6-[2-(2-hydroxy-
L,.0 ethoxy)-ethoxy]-5-
I \> (tetrahydro-pyran-4-
1 HN' N Int ES+-7----N ylamino)- E 424.5
imidazo [4,5- 19 425.4
¨ b]pyridin-3-y1]-
0
ON nicotinonitrile
0--,oFi 6- {5-(1,1-dioxo-
tetrahydro-2H-
ES+
thiopyran-4-
473.6
HN---N-N ylamino)-6-[2-(2- Int
2 E 472.5
/ N\I hydroxy-ethoxy)- 20
ES-
- ethoxyHmidazo [4,5-
s.s - 471.5
// \\ ON b]pyridin-3-yll-
o o nicotinonitrile
oH 6- {6-[2-(2-hydroxy-
0
N
ethoxy)-ethoxy]-5-
0
1 $ [((cis-1,4)-4-
3
N.%---- 466.5 N hydroxy-4-methyl- Cpd
Example ES+
/ N cyclohexyl)-methyl- 12 2.1 467.6
\ amino]-imidazo[4,5-
ON
b]pyridin-3-yll-
". OH nicotinonitrile
...-..õ0..,
o 6- {6-[2-(2-methoxy-
0,...N ethoxy)-ethoxy]-5-
I [methyl-(tetrahydro-
'N'N'N Int
4 pyran-4-y1)-amino]- 21 E 452.5 453.6
). 01 imidazo[4,5-
I / b]pyridin-3-yll -
(3'
CN nicotinonitrile
--------_- 0--
0 6-[6-[2-(2-methoxy-
ON ethoxy)-ethoxy]-5- ES+
(tetrahydro-pyran-4- 439.5
HN N N
,--)L Int
5 ylamino)- E 438.5
)\ -N imidazo [4,5- 22
ES-
\ /
, - b]pyridin-3-y1]- 437.5
0
ON nicotinonitrile
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Cpd MS
Structure Name SM Method MW
# Mes'd
.-----,,,OH
0 6-{5-(3-hydroxy-
0-N cyclohexylamino)-6-
I [2-(2-hydroxy-
Int ES+
HN,-------.N-:%"----N
6 ethoxy)-ethoxy]- 23 E 438.5
439.49
H / "\' imidazo [4,5-
¨ b]pyridin-3-yll -
O
CN nicotinonitrile
õ----.õ. OH
0
6-{5-(4,4-difluoro-
oN cyclohexylamino)-6- ES+
I lilt HN N [2-(2-hydroxy- 459.5
,---------;"----N
7 ethoxy)-ethoxy]-
24 E 458.5
/ N\I imidazo [4,5- ES-
- b]pyridin-3-yll - 457.4
CN nicotinonitrile
F F
0
HO_ II 0 sulfuric acid mono-
' (2- {2-[3-(5-cyano-
pyridin-2-y1)-5-
ES+
505.4
L----- -....------...--N (tetrahydro-pyran-4- Cpd Example
8 I 504.5
HN----'N''.--N ylamino)-3H- 1 2.2
)-.
ES-
imidazo [4,5-
503.4
¨ b]pyridin-6-yloxy]-
CN ethoxy{ -ethyl) ester
NH2
oy (S)-2-amino-3-
methyl-butyric acid
,--,0 2- 1243-(5-cyano- ES+
0
pyridin-2-y1)-5- 524.6
Int Example
9 I (tetrahydro-pyran-4- 30
2.3 523.6
HN-----'N N ylamino)-3H- ES-
imidazo[4,5- 522.6
\
--- b]pyridin-6-yloxy]-
... ..--
0
CN ethoxyl -ethyl ester
NH2
(S)-2-amino-3-
methyl-butyric acid
o 0
2- {243-(5-cyano-
0 N pyridin-2-y1)-5-
HN----'N-1'1 (tetrahydro-pyran-4- Cpd
' N
ylamino)-3H- G 613.6 524.4
9
\ imidazo[4,5-
1APYridiri-6-yiOnd-
CN
0
= 1 ethoxyl -ethyl ester ,
HO OH
H
oxalic acid salt
0
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Cpd MS
Structure Name SM Method MW
# Mes'd
646-[2-(2-
---õOH
0 hydroxyethoxy)ethox
y]-5-[[(cis-3,4)-4-
1 hydroxytetrahydropy
11 HN --(\f'----N ran-3- Int
E 440.5 441.3
HO / N\i yl]amino]imidazo[4, 18
-,0 ---- 5-b]pyridin-3-
ON yl]pyridine-3-
carbonitrile
6-[6-[2-(2-
H 0
o hydroxyethoxy)ethox
oN y]-5-[((cis-1,4)-4-
hydroxy-4-
Int
12 HN N IN methylcyclohexyl)a
E 452.5 453.0
27
bnli;yor]iicaid3az o [4,5-
"'. OH ON yl]pyridine-3-
carbonitrile
645-[((cis-1,4)-4-
..,,,o,
o hydroxy-4-methyl-
__N cyclohexyl)-methyl-
1 amino]-642-(2-
-..N.--.N--;----N Int
13 methoxyethoxy)etho E 480.6 481.4
/ N\I blip]iymriiddianz3o [4,5- 25
OH ON Yllpyridine-3-
carbonitrile
645-[((cis-1,4)-4-
---..,....,
o 0 hydroxy-4-
_N methylcyclohexyl)a
1 mino]-6-[2-(2-
Int
14 HN---'eN methoxyethoxy)etho 26
E 466.5 467.3
blfp] iymriiddianz3o [4,5-
/ OH ON yllpyridine-3-
carbonitrile
2-[2-[3-(5-cyano-2-
pyridy1)-5-
1 (tetrahydropyran-4-
`0---- --------N, ylamino)imidazo[4,5
15 1 , ') -Npyridin-6- Cpd
F 509.6 510.4
HI\l'-'N----N 1
yl]oxyethoxy]ethyl
\ 2-
_
,o,--
CN (dimethylamino)acet
ate
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Cpd MS
Structure Name SM Method MW
# Mes'd
o,NH2
2-[2-[3-(5-cyano-2-
o pyridy1)-5-
(tetrahydropyran-4-
16 I HN N-- N ylamino)imidazo[4,5 Int Example
481.5 482.4
-b]pyridin-6- 31 2.4
_ \ yl]oxyethoxy]ethyl
2-aminoacetate
CN
2-[2-[3-(5-cyano-2-
oN
0 H pyridy1)-5-
(tetrahydropyran-4-
ylamino)imidazo[4,5
Int Example
17 I - -b]pyridin-6- 495.5 496.3
HN---N ----N
ylloxyethoxy]ethyl 32 2.5
\ 2-
-
(methylamino)acetat
ON
e
70 2-[2-[3-(5-cyano-2-
o, pyridy1)-5-
(tetrahydropyran-4-
18
y1amino)imidazo[4,5 Int Example
cp-'-'`)---N
)D_ -b]pyridin-6- 33 1.1 521.6 522.5
HN N N
a0 Y(211s0)Xpyyetrrho7d)ineteh32T1
0 ON carboxylate
--___-- 242-13-(5-cyano-2-
, o
0,-,õ. .KõNH2
1 ri pyridy1)-5-
,o (tetrahydropyran-4-
ylamino)imidazo[4,5 603.4
19 `-o----,c1,.---N Int Example
1 -b]pyridin-6- 580.6 (M+
HN f%i 34 2.6
-'--N yl]oxyethoxy]ethyl Na)
(2S)-2-[(2-
. aminoacetyl)amino]-
ON 3-methyl-butanoate
N
242-[3-(5-cyano-2-
0 ,0 pyridy1)-5-
"co-----0------:----N (tetrahydropyran-4-
20 I
HN'N---N ylamino)imidazo[4,5 Cpd F 551.6 552.4
'-
/ N -Npyridin-6- 1
_ \ yl]oxyethoxy]ethyl
'o¨
CN 2-morpholinoacetate
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Cpd MS
Structure Name SM Method MW
# Mes'd
242-[3-(5-cyano-2-
oNTh
pyridy1)-5-
O rsL, (tetrahydropyran-4-
"(:)-----c)----N ylamino)imidazo[4,5
21 , N N -b]pyridin-6- Cpd
F 564.6 565.3
tr? yl]oxyethoxy]ethyl 1
_ \ 244-
. ,
o
ON methylpiperazin-l-
yl)acetate
I 2-[2-[3-(5-cyano-2-
N
pyridy1)-5-
,0 (tetrahydropyran-4-
ylamino)imidazo[4,5
'0---, ---------. N
22 1 -b]pyridin-6- Cpd F 523.6 524.3
HI\J- N-7----N yl]oxyethoxy]ethyl 1
/ N\I 3-
-.0 ¨ (dimethylamino)prop
CN anoate
O
2-[2-[3-(5-cyano-2-
J-oiH PYridy1)-5-
N HO (tetrahydropyran-4-
,O = 0
ylamino)imidazo[4,5
23 -b]pyridin-6- Cpd ES+ yl]oxyethoxy]ethyl 15
G 599.6
510.3
HN----N'N 2-
(dimethylamino)acet
¨ ate
CN
oxalic acid salt
= o 2-[2-[3-(5-cyano-2-
o NH2 H0¨(
OH pyridy1)-5-
-F j-
(7) o (tetrahydropyran-4-
ylamino)imidazo[4,5 Cpd ES+
24 - 0-------0------ G 571.6
-b]pyridin-6- 16 482.3
)_HN N N N yl]oxyethoxy]ethyl
/ \ 2-aminoacetate
¨
oxalic acid salt
CN
O
2-[2-[3-(5-cyano-2-
HO
OH PYridY1)-5-
N-
H . A (tetrahydropyran-4-
,o
ylamino)imidazo[4,5
'o'--- ------1 NI\ -b]pyridin-6- Cpd ES+
25 G 585.
HN N' N yl]oxyethoxy]ethyl 6 17 496.3
2-
-c) ¨ (methylamino)acetat
CN e
oxalic acid salt
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Cpd MS
Structure Name SM Method MW
# Mes'd
HN 242-[3-(5-cyano-2-
¨\ 0
0 y-L JJOH PYridY1)-5-
./
HO 1 1 (tetrahydropyran-4-
o O
ylamino)imidazo[4,5
26 '101.-- -"CIN\ -b]pyridin-6- Cpd
G 611.6 ES+
HINN' N yl]oxyethoxy]ethyl 18 522.3
) , N (2S)-pyrrolidine-2-
\
¨ carboxylate
CN
oxalic acid salt
NH, 0 (3S)-3-amino-4-[2-
0oH [2-[3-(5-cyano-2-
o pyridy1)-5-
(tetrahydropyran-4-
- ------0-------:---, N
0 ylamino)imidazo[4,5 Int Example ES+
27
HN--- e---N -b]pyridin-6- 35 2.7 576.0
540.5
= )_ / N Ha yl]oxyethoxy]ethoxy
\
¨ ]-4-oxo-butanoic
,
o
CN acid
hydrochloric acid salt
NH2 (4S)-4-amino-5-[2-
0oH [2-[3-(5-cyano-2-
11
o o pyridy1)-5-
(tetrahydropyran-4-
"0-'-' '-'- I'l ylamino)imidazo[4,5 Int Example
ES+
28
HN -"N1-7---N -b]pyridin-6- 36 2.8 590.0
554.6
= ,0\ yl]oxyethoxy]ethoxy
HCI
.- ,- ¨ ]-5-oxo-pentanoic
o
CN acid
hydrochloric acid salt
2-[2-[3-(5-cyano-2-
'---' o pyridy1)-5-
c...õõ,.õ,..11NH2
11
(tetrahydropyran-4-
,(I)
ylamino)imidazo[4,5
' --0 N -b]pyridin-6- C ES+
29 ,j1, ,j,_ pd G 670.6
o HN rsr N yl]oxyethoxy]ethyl 19 581.4
/ N (2S)-2-[(2-
Ho-1--frOH \
6 - aminoacetyl)amino]-
0
CN 3-methyl-butanoate
oxalic acid salt
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Cpd MS
Structure Name SM Method MW
# Mes'd
OINTh 242-[3-(5-cyano-2-
o o pyridy1)-5-
(tetrahydropyran-4-
ylamino)imidazo[4,5 Cpd ES+
30 0 HNN"-----N py blridin-6-
510.3
HO
OH ),õ,. / N -- - 20 G 641.6
1 \ yl]oxyethoxy]ethyl
O ,0, ¨ 2-morpholinoacetate
CN
oxalic acid salt
242-[3-(5-cyano-2-
,0 1-,.N, pyridy1)-5-
(tetrahydropyran-4-
0-'---)D'' ----N1 ylamino)imidazo[4,5
L >
31 HN 2
-- N N -b]pyridin-6- Cpd ES+ G 654.6
/ N yl]oxyethoxy]ethyl 21 565.6
. \
___ 2-(4-
o
o CN methylpiperazin-1-
HO OH yl)acetate
O
oxalic acid salt
I 2-[2-[3-(5-cyano-2-
N 0
0 -T-----,= , OH PYridY1)-5-
0 . HO (tetrahydropyran-4-
o ylamino)imidazo[4,5
-b]pyridin-6-
32 Cpd ES+
HN-----N G 613.6
" N yl]oxyethoxy]ethyl 22 524.4
)-_ / N 3-
(dimethylamino)prop
o
CN anoate
oxalic acid salt
o
4-[2-[2-[3-(5-cyano-
OH 2-pyridy1)-5-
,o
(tetrahydropyran-4-
33N ylamino)imidazo[4,5 Int Example ES+
524.5
-b]pyridin-6- 37 2.9 525.3
HN N N
yl]oxyethoxy]ethoxy
_ \ ]-4-oxo-butanoic
CN acid
Table IV. NMR data of representative compounds of the invention.
Cpd # NMR Data
IFINMR (400 MHz, DMSO-d6) 6 9.00 (1H, dd), 8.89-8.95 (1H, m), 8.75 (1H, s),
8.62
1 (1H, dd), 7.59 (1H, s), 6.04 (1H, d), 4.61-4.69 (1H, m), 4.22 (2H,
dd), 4.05-4.17 (1H,
m), 3.90-3.98 (2H, m), 3.83 (2H, dd), 3.50-3.60 (6H, m), 1.98-2.01 (2H, m),
1.55-1.70
(2H, m)
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Cpd # NMR Data
NMR (400 MHz, DMSO-d6) 6 8.87-9.06 (2H, m), 8.63 (1H), 7.65 (1H, br s), 6.38
2 (1H, d), 4.64 (1H, br s), 4.17-4.30 (3H, m), 3.80-3.88 (2H, m), 3.50 -
3.60 (4H, m),
3.40-3.48 (2H, m), 3.09-3.19 (3H, m), 2.15-2.35 (4H, m)
'H NMR (400 MHz, DMSO-d6) 6 8.98-9.03 (1H, m), 8.94 (1H), 8.87 (1H, s), 8.58
(1H,
3 dd), 7.66 (1H, s), 4.58-4.70 (1H, m), 4.13-4.22 (2H, m), 4.08 (1H,
s), 3.85-3.96 (1H,
m), 3.80 (2H, dd), 3.46-3.58 (4H, m), 2.93 (3H, s), 1.91-2.07 (2H, m), 1.62
(2H, d),
1.50 (2H, d), 1.35-1.45 (2H, m), 1.13 (3H, s)
1H NMR (300 MHz, CDC13) 6 9.02-9.11 (1H, m), 8.93 (1H, s), 8.75 (1H, d), 8.13
(1H,
4 dd), 7.51 (1H, s), 4.16-4.29 (3H, m), 4.09 (2H, dd), 3.85-3.97 (2H,
m), 3.67-3.74 (2H,
m), 3.55-3.63 (2H, m), 3.41-3.55 (2H, m), 3.40 (3H, s), 3.00 (3H, s), 2.01
(2H, qd),
1.76 (2H, dd)
'H NMR (400 MHz, DMSO-d6) 6 9.00 (1H, dd), 8.92 (1H, dd), 8.76 (1H, s), 8.62
(1H,
dd), 7.60 (1H, s), 6.00 (1H), 4.21 (2H, dd), 4.06-4.17 (1H, m), 3.90-3.97 (2H,
m),
3.80-3.84 (2H, m), 3.61-3.66 (2H, m), 3.54 (2H, td), 3.47-3.51 (2H, m), 3.27
(3H, s),
1.99 (2H, dd), 1.54-1.67 (2H, m)
11-1 NMR (400 MHz, DMSO-d6) 6 8.99 (1H, d), 8.93 (1H, d), 8.74 (1H, s), 8.58
(1H,
6 dd), 7.55 (1H, s), 6.19 (1H, d), 4.72 (1H, d), 4.61-4.68 (1H, m),
4.15-4.23 (2H, m),
3.89-4.02 (1H, m), 3.77-3.87 (2H, m), 3.67 (1H, td), 3.50-3.57 (4H, m), 2.17
(1H, d),
1.87-1.96 (1H, m), 1.71-1.86 (2H, m), 1.14-1.45 (4H, m)
11-1 NMR (400 MHz, DMSO-d6) 6 8.86-9.07 (2H, m), 8.76 (1H, br s), 8.63 (1H,
d), 7.58
7 (1H, br s), 6.13 (1H, d), 4.51-4.76 (1H, m), 4.13-4.26 (2H, m), 3.72-
3.94 (2H, m),
3.47-3.63 (5H, m), 2.09 (8H, m)
Iff NMR (300 MHz, CD30D) 6 9.03 (1H, d), 8.88-8.95 (1H, br s), 8.84 (1H, d),
8.41
8 (1H, dd), 7.46 (1H, br s), 4.14-4.36 (5H, m), 3.95-4.12 (4H, m), 3.86
(2H, dd),
3.60-3.76 (2H, m), 2.12 (2H, d), 1.64-1.85 (2H, m)
NMR (400 MHz, CDC13) 6 9.02 (1H, d), 8.83 (1H, s), 8.76 (1H, br s), 8.13 (1H,
d),
9 7.42 (1H, br s), 5.31 (1H, d), 4.36 (2H, br s), 4.24 (2H, br s), 4.18
(1H, br s), 4.08 (2H,
d), 3.91 (2H, br s), 3.81 (2H, d), 3.62 (2H, t), 3.36 (1H, d), 2.16 (2H, d),
2.05 (1H, d),
1.26 (2H, br s), 0.98 (3H, d), 0.91 (3H, d)
'H NMR (400 MHz, DMSO-d6) 6 9.02 (1H, dd), 8.93 (1H, dd), 8.78 (1H, s), 8.64
(1H,
dd), 7.60 (1H, s), 6.00 (1H, d), 4.40-4.46 (1H, m), 4.21-4.31 (3H, m), 4.09-
4.18 (1H,
m), 3.91-3.99 (2H, m), 3.85-3.90 (2H, m), 3.83 (1H, s), 3.75-3.80 (2H, m),
3.51-3.60
(2H, m), 2.04-2.15 (1H, m), 1.96-2.04 (2H, m), 1.58-1.68 (2H, m), 0.89-0.98
(6H, m)
Iff NMR (500 MHz, DMSO-d6) 6 9.00 (1H, d), 8.84 (1H, d), 8.76 (1H, s), 8.59
(1H,
11 dd), 7.60 (1H, s), 5.80 (1H, d), 5.25 (1H, br s), 4.65 (1H, br s),
4.20-4.27 (2H, m), 4.16
(1H, tt), 4.02-4.10 (1H, m), 3.79-3.84 (2H, m), 3.66-3.75 (2H, m), 3.56-3.62
(2H, m),
3.54 (4H, s), 1.82-1.94 (1H, m), 1.65-1.76 (1H, m)
NMR (600 MHz, DMSO-d6) 6 8.86-8.99 (2H, m), 8.69 (1H, s), 8.49-8.58 (1H, m),
12 7.50 (1H, s), 5.82 (1H, d), 4.58-4.71 (1H, m), 4.17 (2H, d), 4.11
(1H, s), 3.81 (2H, d),
3.68-3.77 (1H, m), 3.53 (4H, s), 1.71-1.86 (4H, m), 1.62 (2H, d), 1.44-1.53
(2H, m),
1.17 (3H, s)
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Cpd # NMR Data
NMR (500 MHz, DMSO-d6) 6 9.00 (1H, d), 8.93 (1H, d), 8.87 (1H, s), 8.58 (1H,
13 dd), 7.65 (1H, s), 4.11-4.21 (2H, m), 4.08 (1H, s), 3.89 (1H, tt),
3.72-3.82 (2H, m),
3.55-3.62 (2H, m), 3.44-3.50 (2H, m), 3.25 (3H, s), 2.93 (3H, s), 1.99 (2H,
qd), 1.62
(2H, d), 1.50 (2H, d), 1.40 (2H, td), 1.12 (3H, s)
'H NMR (300 MHz, DMSO-d6) 6 8.92-8.99 (2H, m), 8.73 (1H, s), 8.59 (1H, dd),
7.54
14 (1H, s), 5.79 (1H, d), 4.18 (2H, t), 4.08 (1H, br s), 3.80 (2H, t),
3.72-3.82 (1H, m),
3.60-3.68 (2H, m), 3.45-3.52 (2H, m), 3.26 (3H, s), 1.72-1.83 (4H, m), 1.57-
1.68 (2H,
m), 1.43-1.56 (2H, m), 1.17 (3H, s)
Iff NMR (400 MHz, CDC13) 6 9.03 (1H, dd), 8.83 (1H, s), 8.76 (1H, dd), 8.14
(1H,
15 dd), 7.40 (1H, s), 5.42 (1H, d), 4.37- 4.39 (2H, m), 4.07-4.26 (5H,
m), 3.93-3.95 (2H,
m), 3.82-3.84 (2H, m), 3.61-3.67 (2H, m), 3.24 (2H, s), 2.37 (6H, s), 2.14-
2.24 (2H,
m), 1.66-1.76 (2H, m)
Iff NMR (400 MHz, CDC13) 6 9.05 (1H, d), 8.85 (1H, s), 8.79 (1H, d), 8.16 (1H,
dd),
16 7.44 (1H, s), 5.34 (1H, d), 4.38- 4.40 (2H, m), 4.08-4.28 (5H, m),
3.94-3.96 (2H, m),
3.83-3.85 (2H, m), 3.62-3.68 (2H, m), 3.52 (2H, s), 2.37 (6H, s), 2.15-2.22
(2H, m),
1.65-1.75 (2H, m)
11-1 NMR (400 MHz, CDC13) 6 9.05 (1H, dd), 8.85 (1H, s), 8.79 (1H, dd), 8.16
(1H,
17 dd), 7.43 (1H, s), 5.36 (1H, d), 4.38- 4.41 (2H, m), 4.08-4.28 (5H,
m), 3.94-3.96 (2H,
m), 3.83-3.85 (2H, m), 3.62-3.68 (2H, m), 3.45 (2H, s), 2.48 (3H, s), 2.15-
2.22 (2H,
m), 1.68-1.76 (2H, m)
'H NMR (400 MHz, CDC13) 6 9.05 (1H, d), 8.85 (1H, s), 8.79 (1H, d), 8.16 (1H,
dd),
18 7.43 (1H, s), 5.38 (1H, d), 4.37- 4.40 (2H, m), 4.08-4.27 (5H, m),
3.94-3.96 (2H, m),
3.82-3.86 (3H, m), 3.62-3.68 (2H, m), 3.08-3.14 (1H, m), 2.91-2.97 (1H, m),
2.12-2.21
(3H, m), 1.86-1.95 (1H, m), 1.60-1.82 (4H, m)
Iff NMR (400 MHz, DMSO-d6) 6 9.02 (1H, d), 8.93 (1H, d), 8.77 (1H, d), 8.64
(1H,
19 dd), 8.30 (1H, br s), 7.61 (1H, s), 6.03 (1H, d), 5.14 (2H, br s),
4.09- 4.35 (6H, m),
3.91-3.98 (2H, m), 3.85-3.87 (2H, m), 3.74-3.76 (2H, m), 3.52-3.59 (2H, m),
3.35 (2H,
br s), 1.96-2.10 (3H, m), 1.58-1.68 (2H, m), 0.85-0.88 (6H, m)
NMR (400 MHz, CDC13) 6 9.03 (1H, d), 8.83 (1H, s), 8.77 (1H, d), 8.14 (1H,
dd),
20 7.42 (1H, s), 5.34 (1H, d), 4.36- 4.39 (2H, m), 4.07-4.26 (5H, m),
3.93-3.95 (2H, m),
3.81-3.84 (2H, m), 3.74-3.77 (4H, m), 3.61-3.67 (2H, m), 3.28 (2H, s), 2.59-
2.61 (4H,
m), 2.16-2.20 (2H, m), 1.65-1.75 (2H, m)
11-1 NMR (400 MHz, CDC13) 6 9.04 (1H, dd), 8.84 (1H, s), 8.77 (1H, dd), 8.14
(1H,
22 dd), 7.42 (1H, s), 5.35 (1H, d), 4.33- 4.36 (2H, m), 4.07-4.27 (5H,
m), 3.94-3.96 (2H,
m), 3.81-3.83 (2H, m), 3.61-3.68 (4H, m), 2.54-2.68 (4H, m), 2.27 (6H, s),
2.16-2.20
(2H, m), 1.66-1.76 (2H, m)
'H NMR (400 MHz, DMSO-d6) 6 9.00 (1H, dd), 8.92 (1H, dd), 8.77 (1H, s), 8.63
(1H,
23 dd), 7.60 (1H, s), 6.02 (1H, d), 4.21-4.27 (4H, m), 4.06-4.18 (1H,
m), 3.91-3.95 (2H,
m), 3.84-3.86 (2H, m), 3.73-3.76 (2H, m), 3.62-3.68 (2H, br s), 3.51-3.57 (2H,
m), 2.5
(6H, s), 1.96-2.01 (2H, m), 1.57-1.67 (2H, m)
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Cpd # NMR Data
NMR (400 MHz, DMSO-d6) 6 9.02 (1H, dd), 8.93 (1H, dd), 8.78 (1H, s), 8.64 (1H,
24 dd), 7.62 (1H, s), 6.04 (1H, d), 4.32-4.35 (2H, m), 4.24-4.26 (2H,
m), 4.09-4.18 (1H,
m), 3.92-3.97 (2H, m), 3.86-3.89 (2H, m), 3.84 (2H, s), 3.77-3.79 (2H, m),
3.53-3.59
(2H, m), 1.98-2.02 (2H, m), 1.58-1.68 (2H, m)
'H NMR (400 MHz, DMSO-d6) 6 9.02 (1H, dd), 8.93 (1H, dd), 8.78 (1H, s), 8.64
(1H,
25 dd), 7.62 (1H, s), 6.04 (1H, d), 4.33-4.35 (2H, m), 4.23-4.26 (2H,
m), 4.09-4.18 (1H,
m), 3.92-3.98 (4H, m), 3.86-3.89 (2H, m), 3.76-3.79 (2H, m), 3.52-3.59 (2H,
m), 2.58
(3H, s), 1.96-2.04 (2H, m), 1.58-1.68 (2H, m)
NMR (400 MHz, DMSO-d6) 6 9.02 (1H, dd), 8.93 (1H, dd), 8.78 (1H, s), 8.64 (1H,
dd), 7.62 (1H, s), 6.04 (1H, d), 4.29-4.42 (3H, m), 4.23-4.26 (2H, m), 4.10-
4.18 (1H,
26 m), 3.92-3.97 (2H, m), 3.87-3.89 (2H, m), 3.77-3.80 (2H, m), 3.53-
3.59 (2H, m),
3.12-3.25 (4H, m), 2.19-2.27 (1H, m), 1.92-2.03 (3H, m), 1.81-1.89 (2H, m),
1.58-1.68
(2H, m)
NMR (400 MHz, DMSO-d6) 6 9.04 (1H, dd), 8.92-8.94 (H, m), 8.78 (1H, s), 8.66
28 (1H, dd), 8.54 (3H, hr s), 7.59 (1H, s), 4.38-4.43 (1H, m), 4.24-4.34
(3H, m), 4.08-4.17
(2H, m), 3.87-3.97 (4H, m), 3.77-3.80 (2H, m), 3.51-3.56 (2H, m), 2.37-2.54
(2H, m),
1.97-2.06 (4H, m), 1.59-1.69 (2H, m)
NMR (400 MHz, DMSO-d6) 6 9.01 (1H, dd), 8.93 (1H, d), 8.77 (1H, s), 8.63 (1H,
30 dd), 7.61 (1H, s), 6.03 (1H, d), 4.21-4.27 (4H, m), 4.08-4.18 (1H,
m), 3.91-3.98 (2H,
m), 3.84-3.88 (2H, m), 3.72-3.76 (2H, m), 3.52-3.60 (6H, m), 3.32 (2H, s),
2.53-2.57
(4H, m), 1.96-2.04 (2H, m), 1.58-1.68 (2H, m)
'H NMR (400 MHz, DMSO-16) 6 9.02 (1H, dd), 8.93 (1H, d), 8.78 (1H, s), 8.64
(1H,
31 dd), 7.62 (1H, s), 6.03 (1H, d), 4.08-4.26 (5H, m), 3.92-3.98 (2H,
m), 3.84-3.88 (2H,
m), 3.72-3.76 (2H, m), 3.52-3.59 (2H, m), 3.36 (2H, s), 2.60-3.15 (11H, m),
1.96-2.04
(2H, m), 1.58-1.68 (2H, m)
NMR (400 MHz, DMSO-d6) 6 9.01 (1H, dd), 8.93 (1H, dd), 8.78 (1H, s), 8.63 (1H,
32 dd), 7.62 (1H, s), 6.04 (1H, d), 4.21-4.27 (4H, m), 4.09-4.18 (1H,
m), 3.91-3.98 (2H,
m), 3.84-3.89 (2H, m), 3.73-3.78 (2H, m), 3.52-3.59 (2H, m), 3.24-3.28 (2H,
t),
2.82-2.86 (2H, t), 2.73 (6H, s), 1.96-2.04 (2H, m), 1.57-1.67 (2H, m)
NMR (400 MHz, DMSO-d6) 6 12.19 (1H, hr s), 9.02 (1H, dd), 8.94 (1H, dd), 8.77
33 (1H, s), 8.64 (1H, dd), 7.61 (1H, s), 6.03 (1H, d), 4.08-4.25 (5H,
m), 3.91-3.97 (2H, m),
3.84-3.88 (2H, m), 3.70-3.75 (2H, m), 3.51-3.59 (2H, m), 2.45-2.55 (4H, m),
1.96-2.03
(2H, m), 1.58-1.68 (2H, m)
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BIOLOGICAL EXAMPLES
Example 3. In vitro assays
3.1. IC50 determination for human IRAK-4
[0264] The IC50 value for IRAK-4 is determined in a radioactive filter plate
assay. The principle of
the assay consists in the measurement of incorporated 33P into the RIP140
substrate upon
phosphorylation by the enzyme IRAK-4 using [7-3313]ATP and ATP. Unincorporated
33P is removed
by loading the samples on a filter plate (using a harvester, PerkinElmer) and
6 subsequent washing
steps. Incorporated 33P in RIP140 is measured by a scintillation counter
(Topcount, PerkinElmer) after
addition of MicroScinem-20 (PerkinElmer, 6013621) to the filter plates.
[0265] 5 L of a water dilution series of test compound (starting from 20 M
or 6.6 M highest
concentration), from a 100% DMSO stock solution, 1/5 dilution, is added to the
wells (final DMSO
concentration of 1% in reaction assay). IRAK-4 (Carna Biosciences, 09-145) and
RIP140 (SEQ ID1,
cf. Table VII) are used at a final concentration of 10 ng/mL and 4 M,
respectively. The enzyme and
substrate are diluted in 25 mM Iris pH 7.5, 0.025% Triton X-100, 5 mM MnC12,
and 2 mM DTT to a
total volume of 11 L. The reaction is started by addition of 9 L of 1 M ATP
(Sigma, A6419-5G) +
0.25 Ci [7-33P]ATP (PerkinElmer, NEG602K001MC), diluted in the same buffer as
enzyme and
substrate. The mixture is incubated at 30 C for 45 min. The reaction is
terminated by adding 25 1.(L, of
150 mM phosphoric acid (VWR, 1.00573.1000). Samples are transferred to filter
plates and
incorporated radioactivity is measured using a scintillation counter.
[0266] 10 M staurosporine (1% DMSO) is used as positive control (100%
inhibition); vehicle (water
+ I% DMSO) as negative control (0% inhibition).
Table V. In vitro human IRAK-4 IC50 of the compounds of the invention
hIRAK-4 hIRAK-4
Cpd# Cpd#
IC50 (nM) IC50 (nM)
1 6.35 8 51.4
2 25.4 9 13.5
3 21.7 11 14.3
4 523 12 0.95
5 13.8 13 39.1
6 4.85 14 3.45
7 14.3
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3.2. Kinase selectivity profiling (broad panel)
[0267] Inhibition of human kinases is determined in radiometric kinase assays
at REACTION
BIOLOGY (Reaction Biology Corp., 1 Great Valley Parkway, Suite 2 Malvern, PA
19355, USA).
[0268] To determine its IC50, a compound is tested at 10 doses starting from
10 p.M (highest
concentration), with 3-fold serial dilutions. IC50 values are derived by
fitting dose-response curves of
% Remaining Enzyme Activity (relative to DMSO controls).
3.3. Kinase selectivity profiling (focused panel)
[0269] The purpose of this assay is to determine the activity and selectivity
of a compound of the
invention on a selected range of human kinases which may result in undesirable
side-effects when
inhibited (Dy and Adjei, 2013; Force and Kolaja, 2011).
3.3.1. Assay protocol
[0270] The IC50 value for off-target kinases is determined in radioactive
filter plate assays. The principle
of the assays consists in the measurement of incorporated 33P into a peptide
substrate upon
phosphorylation by the kinase enzyme using [7-33P]ATP and ATP. Unincorporated
33P is removed by
loading the samples on a filter plate (using a harvester, PerkinElmer) and 6
subsequent washing steps.
Incorporated 33P in the peptide substrate is measured by a scintillation
counter (Topcount, PerkinElmer)
after addition of MicroScintTm-20 (PerkinElmer, 6013621) to the filter plates.
[0271] 5 ii.tL of a water dilution series of test compound (starting from 20
j.tM or 6.6 ii.tM highest
concentration), from a 100% DMSO stock solution, 1/5 dilution, is added to the
wells (final DMSO
concentration of I% in reaction assay). Enzyme and peptide substrate are used
at optimized
concentrations (cf. Table VI). The enzyme and substrate are diluted in assay
buffer to a total volume of
11 L. The reaction is started by addition of 9 L of ATP + [7-33P]ATP,
diluted in the same buffer as
enzyme and substrate. The mixture is incubated at 30 C. The reaction is
terminated by adding 25 uL of
150 mM phosphoric acid. Samples are transferred to filter plates and
incorporated radioactivity is
measured using a scintillation counter.
[0272] The incubation time, assay buffer composition, and concentrations of
ATP, enzyme and substrate
are reported in Table VI for example kinase off-target assays.
[0273] 10 0/1 staurosporine (1% DMSO) is used as positive control (100%
inhibition); vehicle (water +
1% DMSO) as negative control (0% inhibition).
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Table VI. Conditions for human kinase off-targets inhibition assays
Kinase, Substrate,
Incubation
ATP Assay buffer
[Kinase] [Substrate] time
ABL PolyGT (Sigma- 0.5 M ATP + 50 mM Tris pH 7.7 60 min
(Life Technologies, Aldrich, P0275), 0.25 Ci/25 I_, 0.03% Triton X-
100
P3049), 40 ng/mL 5 11,0111- [7-3311ATP 1 mM DTT
25 mM MgCl2
Aurora B (Carna Histone H3 peptide 1.3 M ATP + 25 mM
Tris pH 7.7 90 min
BioSciences, (SEQ ID2), 0.5 M 0.25 Ci/25 I_, 0.01% Triton X-100
05-102), 10 ng/mL [33P]ATP 5 mM MgC12
mM DTT
CDK2 (Carna Histone Hi-derived 0.1 M ATP + 8 mM MOPS
pH 7.0 60 min
Biosciences, peptide (SEQ ID3), 0.25 Ci/25 L 0.01% Brij-35
04-103), 30 ng/mL 0.36 M [133P]ATP 1 mM DTT
5 mM MnC12
CDK9 (Millipore, PDKtide (SEQ 0.25 M ATP + 20 mM MOPS pH 7.0 60 min
14-685), ID4), 0.5 M 0.125 Ci/25 tit 0.01% Triton X-100
230 ng/mL [7-3311ATP 5 mM MnC12
c-KIT (Millipore, PolyGT (Sigma- 3 M ATP
+ 16 mM Tris pH 7.0 90 min
14-559), Aldrich, P0275), 0.25 Ci/25 L 500 M EDTA
0.01 mU/ L 0.1 mg/mL [7-33F]ATP 0.01% Triton X-100
mM MnC12
1 mM DTT
10 mM Mg0Ac
GSK3b (Carna Phospho glycogen 1.5 M ATP + 50 mM
Tris pH8.0 90 min
Biosciences, synthase peptide2 0.25 Ci/25 L 0.01% Brij-35
04-141), 20 ng/mL (Millipore, 12-241), [7-33F]ATP 1 mM DTT
1.25 M
5 mM Mg0Ac
c-SRC (Carna PolyGT (Sigma- 0.25 M ATP + 25 mM MOPS pH 7.0 60 min
Biosciences, Aldrich, P0275), 0.125 Ci/25 L 10 mM MnC12
08-173), 8 ng/mL 2 ug/mL [7-33F]ATP 2.5 mM DTT
0.01% Brij35
Table VII. Peptide substrates used in human kinase off-targets inhibition
assays
Substrate SEQ ID No. Sequence Provider
RIP140 1 CYGVASSHLKTLLKKSKVK Almac Group Ltd.
peptide DQ 20 Seagoe
Industrial Estate
Craigavon BT63 5QD UK
ARTKQTARKSTGGKAPRKQ
Histone 2 AnaSpec Inc.
H3 peptide LC 34801 Campus Drive
Fremont, CA 94555 USA
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Substrate SEQ ID No. Sequence Provider
3 GGGPATPKKAKKL
Histone AnaSpec Inc.
Hl-derived 34801 Campus Drive
peptide Fremont, CA 94555 USA
PDKtide 4 KTFCGTPEYLAPEVRREPRIL Thermo Fisher Scientific Inc.
SEEEQEMFRDFDYIADWC 81 Wyman St.
Waltham, MA 02451 USA
Table VIII. In vitro human kinase off-targets IC50 of illustrative
compounds of the invention
ABL Aurora B CDK2 CDK9 GSK3b c-SRC
Cpdtt
IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
IC50 (nM)
1 >6670 >1330 >6670 >1330
2 - - >1330 712 >1330 >1330
2690 - >4000 2290 >4000 >2940
6 656 - 2670 1030 >4000 754
9 633 >4000 639 3250 286
11 - - >1330 834 >1330 >1330
12 138 >1880 944 1150 >1330 477
14 - - 1380 1390 2240 319
3.3.2. Conclusion
[0274] The data of Table VIII, in relation to those of Table V, show the lower
inhibitory potency of
compounds of the invention in kinase off-targets versus in IRAK-4. These data
confirm the selectivity of
compounds of the invention towards IRAK-4, thus limiting the risk of side
effects associated with kinase
off-targets inhibition.
3.4. Cellular assay: CL097 activated TNFa release inhibition in PBMCs
[0275] The compounds of the invention are tested in a cellular assay using
primary isolated human
peripheral blood mononuclear cells (PBMCs) to measure the secretion of the
inflammatory cytokine
TNFa upon TLR activation using the specific TLR7/8 agonist, CL097. The release
of TNFa protein in the
cell culture supernatant is quantified by a human TNFa enzyme-linked
immunosorbent assay (ELISA)
protocol.
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3.4.1. Isolation of human primary PBMCs from human bulb, coat
[0276] A human buffy coat (provided by the Croatian Institute for Transfusion
Medicine) is kept
overnight at 4 C and processed the next day for isolation of PBMCs. PBMCs are
isolated by density
gradient centrifugation using Ficoll-PaqueTM PLUS (GE HealthCare, 17-1440-02).
Equal volumes of a
buffy coat are diluted 1:4 with sterile PBS (1X) and 35 mL is carefully
layered on top of 15 mL
Ficoll-Paque PLUS in appropriate 50 mL Falcon tubes. The tubes are
centrifuged for 35 min at
1500 rpm at r.t. without acceleration or break. After centrifugation, the
upper plasma layer is removed
and the mononuclear cell ring is carefully isolated and transferred to a fresh
Falcon tube. The isolated
cell suspension is diluted in PBS up to 50 mL followed by a centrifugation
step at 1300 rpm for 10 min at
r.t. After 2 additional washing steps in PBS and cell pooling, the remaining
erythrocytes are lysed by
resuspension of the cell pellet in 50 mL of AKL lysis buffer (150 mM NH4C1, 10
mM NaHCO3, 1 mM
Na2EDTA, pH 7.4) followed by gentle mixture. The 50 mL suspension is then
centrifuged at 1300 rpm
for 10 min at r.t. followed by removal of supernatant and resuspension of the
cell pellet in culture medium
(RPMI 1640 (Gibco, 21875) + 10% fetal bovine serum (FBS, Biowest, S1810) heat
inactivated for 30
min at 56 C + Pen/Strep (Gibco, 15240)).
3.4.2. .. Compound treatment and triggering in PBMC assay
[0277] Cells are counted using a hematologic analyzer (Sysmex XS-500i) and
plated at a density of
4.0 x 105 cells per well in 160 ILIL culture medium in 96-well culture plates.
Subsequently, PBMCs are
pre-incubated with test compound by addition of 20 jiL of 10X concentrated
compound solution for 1
hour at 37 C and 5% CO2. The compounds are tested at different concentrations
and prepared by 3-fold
serial dilutions from the 10 mM stock solution in DMSO followed by a 1:50
dilution step in 2X M199
medium (Gibco, 21157-029) supplemented with 1% FBS and 1% Pen/Strep. Final
test concentrations in
the assay start from 20 j.tM, with subsequent 3-fold serial dilutions and
equal final DMSO concentrations
of 0.2%. After the compound pre-incubation step, the PBMCs are triggered by
adding 20 !Lit of a
[ig/mL CL097 solution (InvivoGen, tlrl-c97-5) to the wells with final assay
volume of 200 tit per well
and 1 ig/mL final CL097 trigger concentration. Negative controls are adjusted
with equal DMSO
concentrations without CL097 trigger. The assay plates are then incubated for
4 h in a humidified
incubator at 37 C and 5% CO2. Cell supernatants are then harvested by
transferring the cell medium into
a 384 deep well plate and immediately transferred to the ELISA plate for
quantification of human TNFa.
3.4.3. Quantification of TNFa by ELISA
[0278] The levels of secreted TNFa in the cell supernatants are quantified in
an antibody capture activity
assay (ELISA). A white Greiner LumitracTm 384-well plate is coated with 40 tiL
per well of a 1 ttg/mL
anti-human TNFa antibody solution (MAbl; BD Biosciences, 551220) diluted in
PBS for an overnight
incubation at 4 C. After washing the wells with 100 p.L PBS, the remaining
binding sites are blocked
with 100 RI, of blocking buffer (PBS + 1% bovine serum albumin + 5% sucrose)
and incubated for 4 h at
r.t. Subsequent to the blocking step, the wells are washed once with PBS with
Tween 20 (PBST),
followed by addition of samples and standards. Samples containing INFa are
diluted 1/3 in dilution
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buffer and 40 p.L is added for an overnight incubation at 4 C. The wells are
then washed 3 times, twice
with PBST and once with PBS, following addition of 35 p.L of secondary
biotinylated anti-TNFa
detection antibody (MAbll; BD Biosciences, 554511) in a 1/2000 diluted format
at final concentration of
250 ng/mL. After 2 hours of incubation at r.t. and appropriate washing steps
(2x PBST, lx PBS), the
wells are incubated with 35 gt of a 1/4000 diluted horseradish peroxidase-
conjugated streptavidin
solution (Life Technologies, SNN2004), followed by a 45 min incubation step at
r.t. in the dark. The
wells are then washed 3 times (2x PBST, lx PBS), followed by 5 min incubation
with 50 gt of
Chemiluminescence ELISA Substrate solution (Roche, 11582950001). The converted
substrate
luminescent signal is measured in a PerkinElmer EnVision 2104 Multilabel Plate
Reader.
3.4.4. Data analysis
[0279] All controls are measured within the linear range of the human TNFa
standard curve of the
ELISA. All data are checked for validity against the assay quality parameters
(signal/background > 2 and
Z' > 0.3).
[0280] Unstimulated samples (no trigger/vehicle (0.2% DMSO)) are used as
positive control (100%
inhibition). As negative control (0% inhibition), the stimulated samples
(trigger/vehicle (0.2% DMSO))
are used. The positive and negative controls are used to calculate Z' and
percent inhibition (PIN) values,
according to the following formula:
RCLUtrigger/vehicule - RCLUtest compound
[0281] PIN = x 100; with RCLU =
Relative
RCLUtrigger/vehicule - RCLUno trigger/vehicule
Chemilumine scent Light Units.
[0282] PIN values are plotted for compounds tested in concentration-response
mode, and IC50 values are
derived using the GraphPad Prism software applying a non-linear regression
(sigmoidal) curve fitting.
3.5. Cellular assay: cancer cell assays
3.5.1. Cell lines
[0283] Human lymphoma cells from the OCI-Ly3, OCI-Lyl 0, OCI-Ly7, and OCI-Ly19
cell lines (from
DSMZ, Germany or ATTC, US) are cultured in IMDM (Gibco , 21980-032)
supplemented with 10%
fetal bovine serum (Invitrogen, S7524) or 20% human serum (Invitrogen,
34005100) at 37 C in 5% CO2.
3.5.2. Cell growth assay
[0284] Lymphoma cells (2-7 x 103) are plated in 96 well plates, and treated
with different doses of test
compounds from 30 laM (1/3 dilutions, 8 points). The treated cells are
incubated for 7 days at 37 C in
5% CO2. Staurosporin (10 laM) is used as positive control.
[0285] Cell growth is determined by incubating the cells with alamarBlue
(Invitrogen, DAL 1025),
according to the manufacturer's instructions. Fluorescence is measured using a
PerkinElmer EnVision
plate reader. A percentage of growth inhibition is calculated using DMSO
vehicle values as 0% inhibition
and staurosporin values as 100% inhibition.
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3.5.3. IL-1 and TNFa response cellular assay in SW1353 cells
[0286] The aim of this assay is to evaluate the selectivity of compounds of
the invention for the activated
TLR / IRAK-4 pathway in an in vitro human cellular assay setting. SW1353 cells
are from a chondrocytic
cell line and are responsive to both the interleukin 1 (IL-1) and the TNFot
cytokine triggers. Both cytokine
triggers induce the expression of interleukin 6 (IL-6) and MMP13 by these
cells. IL-6 and MMP13
releases are used as readouts in this assay and represent a measure for the
level of inhibition of the TLR /
IRAK-4 pathway by the tested compound. The IL-1 trigger signals through an
IRAK-4 dependent
pathway, whereas TNFa does not require IRAK-4 for signaling. Therefore,
compounds selectively
inhibiting IRAK-4 only impact IL-1 driven expression of MMP13 or IL-6 by
SW1353 cells and do not
impact TNFa driven expression of these proteins.
3.5.4. Harvesting and seeding of SW1353 cells
[0287] SW1353 cells are cultured in DMEM supplemented with 10% FBS and 1%
Penicillin/Streptomycin. Cells are incubated at 37 C in a humidified
atmosphere of 5% CO2 and
subcultured twice a week. During subculturing, trypsin-EDTA is used to detach
the cells, followed by a
neutralization step with cell culture medium. After centrifugation (1,000 rpm
during 5 min), the pellet is
resuspended in cell culture medium and cells are counted using an automated
cell counter (Invitrogen
Countess).
[0288] Cells are used at passage 16 and plated at a density of 15,000 cells
per well in 120 [LI. cell culture
medium in 96-well culture plates. Cells are allowed to attach during overnight
incubation.
3.5.5. Compound treatment and triggering in SW1353 assay
[0289] SW1353 cells are pre-incubated with test compound by addition of 15
1_, of 10X concentrated
compound solution for 2 h at 37 C and 5% CO2. The compounds are tested at
different concentrations
and prepared by 3-fold serial dilutions from the 10 mM stock solution in DMSO
followed by a 1/50
dilution step in cell culture medium. Final test concentrations in the assay
start from 20 uM, with
subsequent 3-fold serial dilutions with equal final DMSO concentrations of
0.2%. After the compound
pre-incubation step, the SW1353 cells are triggered by addition of 15 tit of
10X concentrated IL-1I3
(Peprotech, 200-01B) or TNFa trigger (Peprotech, 300-01A) to the wells with
final assay volume of
150 uL per well and final trigger concentration of 1 ng/mL and 10 ng/mL,
respectively. Negative controls
are adjusted with equal DMSO concentrations without trigger. The assay plates
are then incubated in a
humidified incubator at 37 C and 5% CO2. Cell supernatants are harvested 24 h
and 48 h later by
transferring the cell medium into a V-bottom polypropylene 96-well plate and
stored at -80 C until
ELISA readout.
3.5.6. Quantification of IL-6 by ELISA
[0290] The levels of secreted IL-6 in the cell supernatants are quantified in
an enzyme-linked
immunosorbent assay (ELISA). A white LumitracTm 384-well plate is coated
overnight with 40 uL per
well of a 1 lug/mL anti-human IL-6 mouse antibody (R&D Systems, MAB206)
solution diluted in PBS at
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4 C. After washing the wells twice with 100 tL PBST and once with PBS, the
remaining binding sites
are blocked with 100 [IL of blocking buffer (1% BSA and 5% sucrose in PBS) and
incubated for 4 hat r.t.
Subsequent to the blocking step, the wells are washed once with PBST followed
by addition of either
samples or recombinant human IL-6 (R&D Systems, 206-IL-050) as standard.
Samples are diluted 1/20 in
dilution buffer and 40 iaL is added for an overnight incubation at 4 C. The
wells are then washed 3 times,
twice with PBST and once with PBS, following addition of 35 [it of secondary
biotinylated anti-IL-6
detection antibody (human IL-6 biotinylated goat polyclonal antibody (R&D
Systems, BAF206)) at a
final concentration of 50 ng/mL. After 2 h of incubation at r.t. and
appropriate washing steps (twice with
PBST and once with PBS), the wells are incubated with 35 j.iL of a 1/2,000
diluted streptavidin-HRP
solution (Invitrogen, SNN2004), followed by a 45 min incubation at r.t. in the
dark. The wells are then
washed 3 times (twice with PBST and once with PBS), followed by 5 min
incubation with a 50 !IL of
chemiluminescence ELISA substrate solution (Roche, 11 582 950 001).
Luminescence of the converted
substrate is measured with a LuminoskanTM Ascent luminometer.
3.5.7. Quantification of 111111P13 by ELISA
[0291] The levels of secreted MMP13 in the cell supernatants are quantified in
an antibody capture
activity assay. For this purpose, black Nunc MaxiSorpTM 384-well plates are
coated with 35 j.iL of a
1.5 lag/mL anti-human MMP13 antibody solution overnight at 4 C. After washing
the wells twice with
PBST, the remaining binding sites are blocked with 100 !IL 5% non-fat dried
milk in PBS for 24 h at
4 C. Subsequent to the blocking step, the wells are washed twice with PBST
followed by addition of
samples and standards. Samples are 1/5 diluted in dilution buffer and 35 p1 is
added for 4 h at r.t. The
wells are then washed twice with PBST. Subsequently, the MMP13 protein is
fully activated by addition
of 35 1 of a 1.5 mM APMA solution (Sigma-Aldrich, A9563) and incubated at 37
C for 1 h. The wells
are then washed twice with PBST and 35 tiL MMP13 substrate (OMNIMMP
fluorogenic substrate
(BIOMOL, P-126)) is added. After incubation for 1 h at 37 C, fluorescence of
the converted substrate is
measured with a PerkinElmer EnVision (excitation wavelength: 320 nm, emission
wavelength: 405 nm).
3.5.8. Data analysis and calculation
[0292] All controls are measured within the linear range of the human IL-6 and
MMP13 standard curve
of the ELISA. All data generated are validated against the assay quality
parameters (signal/background >
2 and Z' >0.3).
[0293] Unstimulated samples (no trigger/vehicle (0.2% DMSO)) are used as
positive control (100%
inhibition). As a negative control (0% inhibition), the stimulated samples
(trigger/vehicle (0.2% DMSO))
are used. The positive and negative controls are used to calculate Z' and
percent inhibition (PIN) values.
[0294] Percentage inhibition (PIN) = ((RUtrigger/veh ¨ RUtest
compound)/(RUtrigger/veh ¨ RUno
trigger/veh)*100); with RU meaning relative chemiluminescent light units or
relative fluorescence units
for IL-6 and MMP13 ELISA, respectively. PIN values are plotted for test
compounds tested in
concentration-response and IC50 values are derived using the GraphPad Prism
software applying non-
linear regression (sigmoidal) curve fitting.
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Table IX. SW1353 cellular selectivity assay results of illustrative compounds
of the invention
IL-10 trigger TNFa trigger
IL-6 IC50 (nM) MMP13 IC50 (nM) IL-6 IC50 (nM) M1VIP13 IC50 (nM)
Cpd#
[PIN at 20 uM] [PIN at 20 M] [PIN at 20 AM] [PIN at 20 iuM]
1 54 36 >20000 >20000
[89%] [81%] [54%] [26%]
12 40 29 >20000 >20000
[80%] [75%] [22%] [36%]
3.5.9. Conclusion
[0295] The data of Table IX show that compounds according to formula I of the
composition of the
invention potently inhibit IL-6 and MMP13 expression in SW1352 cells triggered
by IL-113, whereas the
effect of such compounds on TNFa triggered events is limited, both in terms of
potency and maximal
amplitude. These data confirm the selectivity of compounds according to
formula I of the composition of
the invention towards IRAK-4 driven pathways, with very limited impact on TNFa
signaling, which may
in turn limit the occurrence of treatment-associated side effects such as
neutropenia and infection.
Example 4. ADME assays
4.1. Kinetic solubility
[0296] Starting from a 3.3 mM DMSO stock solution of compound, a serial
dilution of the compound is
prepared in DMSO by performing 1/2 dilutions: 3.3, 1.6, 0.83, 0.41 and 0.21
mM. This dilution series is
transferred to a clear V-bottom 96 well plate (Greiner, 651201) and farther
diluted 1/33.5 in 0.1 M
phosphate buffer pH 7.4 or 0.1 M citrate buffer pH 3Ø Final compound
concentrations are 99.5, 49.7,
24.9, 12.4 and 6.22 M. The final DMSO concentration does not exceed 3%. As a
positive control for
precipitation, pyrene (30 mM) is added to the corner wells of each 96 well
plate. The assay plates are
sealed and incubated for 1 h at 37 C while shaking at 230 rpm. The plates are
then scanned under a white
light microscope, yielding individual pictures (50x magnification) of the
precipitate per concentration.
Each well is analyzed by image analysis software, and the highest
concentration at which the compound
appears completely dissolved is reported.
4.2. Microsomal stability
[0297] A 10 mM stock solution of compound in DMSO is diluted three-fold in
DMSO. This pre-diluted
compound solution is then diluted to 2111\4 in a 105 mM phosphate buffer (pH
7.4) in a 96 deep well plate
(Nunc, 278752 ) and pre-warmed at 37 C.
[0298] A glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001) working
stock solution
of 700 U/mL is diluted with a factor 1:700 in a 105 mM phosphate buffer, pH
7.4. A co-factor mix
containing 0.528 M MgC12.6H20 (Sigma, M2670), 0.528 M D-glucose-6-phosphate
(Sigma, G7879) and
0.208 M NADP+ (Sigma, N0505) is diluted with a factor 1:8 in a 105 mM
phosphate buffer, pH 7.4.
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[0299] A working solution is made containing 1 mg/mL liver microsomes (Tebu-
bio) of the species of
interest (e.g., human, mouse, rat, dog), 1.2 U/mL G6PDH and co-factor mix (6.6
mM MgCl2, 6.6 mM
glucose-6-phosphate, 2.6 mM NADP+). This mix is pre-incubated for 15 min, but
never more than 20
min, at r.t.
[0300] After pre-incubation, the compound dilution and the mix containing the
microsomes, are added
together in equal amount and incubated for 30 min at 300 rpm. For the 0 min
time point, two volumes of
MeCN are added to the compound dilution before the microsome mix is added. The
final concentrations
during incubation are: 1 uM test compound or control compound, 0.2% DMSO, 0.5
mg/mL microsomes,
0.6 U/mL G6PDH, 3.3 mM MgCl2, 3.3 mM glucose-6-phosphate and 1.3 mM NaDP+.
[0301] After 30 min of incubation at 37 C, the reaction is stopped with 2
volumes of MeCN.
[0302] Samples are mixed, centrifuged and the supernatant is harvested for
analysis on LC-MS/MS. The
instrument responses (i.e. peak heights) are referenced to the zero time point
samples (considered as
100%) in order to determine the percentage of compound remaining. Propranolol
and verapamil are
included as references in the assay design.
[0303] The data on microsomal stability are expressed as a percentage of the
total amount of compound
remaining after 30 min incubation.
4.3. Metabolic stability in S9 subcellular fraction
[0001] The
aim of this assay is to assess compound metabolism by aldehyde oxidase by
determination
of their in vitro metabolic stability in S9 subcellular fraction.
[0002] A 10
mM stock solution of compound in DMSO is first diluted in DMSO (40 fold) to
obtain
250 uM concentration. This compound solution is further diluted with water (5
fold) to obtain a 50 uM
compound working solution (to obtain compound final concentration of 1 uM).
Hydralazine (selective
inhibitor of aldehyde oxidase) is prepared in water at 5 mM (to obtain final
concentration of 100 M).
Incubation mixtures are prepared by adding 10 j.tL of liver S9 suspension
(human, rat, mouse, monkey,
BD GentestTM, 20 mg/mL) to 86 of 50
mM potassium phosphate buffer, pH 7.4 at 37 C (final
concentration of 2 mg protein/mL). 2 tit of 5 mM hydralazine is added for
incubations with the addition
of selective inhibitor or 2 !IL of water, for incubations without inhibitor.
After 5 min pre-warming, the
reaction is initiated by the addition of 2 !IL of 50 ,1\4 test compound to
the incubation mixture. After 0, 3,
6, 12, 18 and 30 min of incubation, the reaction (100 tit) is terminated with
300 I. of MeCN:Me0H
(2:1) with 1% acetic acid mixture containing 10 ng/mL of warfarin as
analytical internal standard.
Samples are mixed, centrifuged and the supernatant analyzed by LC-MS/MS.
Phtalazine is included as
positive control.
[0003] The
instrument responses (peak area ratios of compound and internal standard) are
referenced
to the zero time point samples (considered as 100%) in order to determine the
percentage of compound
remaining. Plots of the % of compound remaining are used to determine the half-
life and intrinsic
clearance in the S9 incubations using the GraphPad Prism software. The
following formula is used to
calculate in vitro intrinsic clearance (jIL/min/mg):
CLiiit (IaL/min/mg) = 0.693/t112 (min) * (mL of incubation/mg protein) * 1000
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[0004] Test compounds can be classified as substrates of aldehyde oxidase
if clearance by S9 is
inhibited by hydralazine. Species specific clearance of test compound may also
indicate metabolism by
aldehyde oxidase.
4.4. Metabolic stability in hepatocytes
[0304] A 10 mM stock solution of test compound in DMSO is first diluted in
DMSO to 3 mM, and then
in modified Krebs-Henseleit buffer (Sigma, K3753) to 5 M. This compound
dilution is added to a
suspension of pooled cryopreserved hepatocytes (BioreclamationIVT) at 37 C
under gentle shaking.
Final reaction conditions are: 1 M of test compound, 0.03% DMSO, 0.5 million
viable hepatocytes/mL,
and 75 gt incubation volume. Testosterone (1 laM) and 7-hydroxycoumarin (1
j.iM) are used, respectively
as phase I and phase II metabolic reaction controls.
[0305] After 0, 10, 20, 45, 90, 120 and 180 min of incubation, the reaction is
terminated with 225 1.1.L of
MeCN:Me0H (2:1) containing 10 ng/mL of warfarin sodium as analytical internal
standard. Samples are
mixed, centrifuged and the supernatant analyzed by LC-MS/MS.
[0306] The instrument responses (ratios of test compound and internal standard
peak areas) are
referenced to the zero time point samples (considered as 100%) in order to
determine the percentage of
compound remaining.
[0307] Plots of percentage compound remaining are used to determine the half-
life and intrinsic
clearance in the hepatocyte incubations using the GraphPad Prism software.
4.5. CYP inhibition
[0308] The inhibitory potential of a test compound for human cytochrome P450
isoenzymes (CYP1A2,
2C9, 2C19, 2D6 and 3A4) is assessed using cDNA-expressed human cytochrome P450
isoenzymes and
non-fluorescent substrates which are metabolized to fluorescent metabolites.
[0309] Compounds are tested at 3.3 and 10 laM, with a final DMSO concentration
of 0.3%. Compounds
are incubated for 15 min with enzyme before the cofactor-substrate mix is
added. Final reaction
concentrations in cofactor mix for the CYP3A4 (BD Biosciences, 456202), CYP2C9
(BD Biosciences,
456258), CYP2C19 (BD Biosciences, 456259) and CYP1A2 (BD Biosciences, 456203)
assays are: 0.4
U/mL glucose-6-phophate-dehydrogenase (G6PDH, Roche, 10165875001), 3.3 mM
MgCl2 (Sigma,
M2670), 3.3 mM D-glucose-6-phosphate (Sigma, G7879) and 1.3 mM NADP+ (Sigma,
N0505). For
CYP2D6 (BD Biosciences, 456217), final reaction concentrations in the assay
are 0.4 U/mL G6PDH,
0.41 mM MgCl2, 0.41 mM D-glucose-6-phosphate and 8.2 p.M NADP+. The
concentrations of enzyme
and substrate are reported in Table X. After an incubation period, the
reaction is stopped by adding a stop
solution. For experiments with DBF as substrate, a 2 N NaOH stop solution is
used, while for all other
substrates the stop solution is 80% MeCN/20% 0.5 M Tris base.
[0310] Fluorescence is read either immediately (for CEC, AMMC, BFC), or after
20 min (for CYP2C9
and CYP3A4 using DBF as substrate) on a PerkinElmer EnVision reader at the
appropriate excitation
and emission wavelength (cf. Table X).
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[0311] The percentage inhibition of CYP by the test compound is then
calculated by normalizing the
data to blank samples: 100% inhibition is the blank sample stopped before
addition of the
enzyme/substrate mix, and 0% inhibition is the blank sample stopped after the
enzymatic reaction has
occurred (50 min).
Table X. Inhibition assay
conditions used for each CYP450 isoenzyme studied
CYP3A4 CYP3A4 CYP2C19 CYP2C9 CYP1A2 CYP2D6
Substrate
(IIM)
DBF 1 - - 0.5 - -
CEC - - 35 - 4 -
AMMC - - - - - 0.5
BFC - 120 - - - -
Phosphate
buffer pH 7.4 200 90 25 25 25 25
(mM)
Enzyme
1 1.5 6 2 1.5 3
(pmol/well)
Incubation
50 50 50 50 50 50
time (min)
Positive
ketoconazole ketoconazole fluvoxamine sulfaphenazole fluvoxamine quinidine
control
Excitation
wavelength 485 400 400 485 400 380
(nm)
Emission
wavelength 530 530 460 530 460 460
(nm)
AMMC: aminoethy1-7-methoxy-4-methylcoumarin CEC: 3-cyano-7-ethoxycoumarin
BFC: 7-benzyloxy-4-trifluoromethylcoumarin DBF: dibenzylfluorescein
4.6. MDCKII-MDR1 permeability
[0312] MDCKII-MDR1 cells are Madin-Darby canine kidney epithelial cells,
overexpressing the human
multi-drug resistance (MDR1) gene, coding for P-glycoprotein (P-gp). Cells are
obtained from the
Netherlands Cancer Institute and used after a 3-4 day culture in 24-well
Millicell cell culture insert
plates (Millipore, PSRP010R5). A bi-directional MDCKII-MDR1 permeability assay
is performed as
described below.
[0313] 3 x 105 cells/mL (1.2 x 105 cells/well) are seeded in plating medium
consisting of DMEM
(Sigma, D5796) + 1% Glutamax-100 (Sigma, G8541) + 1% antibiotic/antimycotic
(Sigma, A5955) +
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10% FBS (Sigma, F7524; inactivated at 56 C for 30 min). Cells are left in CO2
incubator for 3-4 days.
The medium is changed 24 h after seeding and on the day of experiment.
[0314] Test and reference compounds (amprenavir (Moravek Biochemicals, M-
1613), diclofenac
(Sigma, D6889)) are prepared in Dulbecco's phosphate buffer saline (D-PBS, pH
7.4; Sigma, D8662) and
added to either the apical (400 L) or basolateral (800 IlL) chambers of the
Millicell cell culture plates
assembly at a fmal concentration of 10 loM (0.5 RIVI in case of amprenavir)
with a final DMSO
concentration of 1%. A receiver solution (D-PBS + 1% DMSO) is added to the
opposite chamber of the
Millicell cell culture plate.
[0315] 100 RIVI Lucifer yellow (Sigma, L0259) is added to all donor buffer
solutions, in order to assess
integrity of the cell monolayers by monitoring Lucifer yellow permeation.
Lucifer yellow is a fluorescent
marker for the paracellular transport pathway and is used as internal control
to verify tight junction
integrity of every cell monolayer during the assay.
[0316] After a 1 h incubation at 37 C while shaking on an orbital shaker at
150 rpm, 75 L aliquots are
taken from both apical and basal chambers and added to 225 p1 of MeCN:water
solution (2:1) containing
analytical internal standard (10 ng/mL warfarin) in a 96 well plate.
Aliquoting is also performed at the
beginning of the experiment from donor solutions to obtain initial
concentrations.
[0317] Concentration of compound in the samples is measured by high
performance
liquid-chromatography/mass spectroscopy (LC-MS/MS).
[0318] Lucifer yellow is measured with a Thermo Scientific Fluoroskan Ascent
FL (excitation
wavelength: 485nm, measurement wavelength: 530nm) in a 96 well plate
containing 150 L of liquid
from all receiver wells (basolateral or apical side).
Example 5. Whole blood assays
5.1. Ex vivo human TNFa release inhibition (whole blood assay)
[0319] The objective of the assay is to evaluate the activity of compounds of
the invention on the
activated TLR / IRAK-4 pathway in an ex vivo human whole blood setting. Toll-
like receptors (TLRs) are
pattern recognition receptors that recognize a wide variety of microbial
molecules, called
pathogen-associated molecular patterns (PAMPs). Human TLR7 and TLR8 recognize
imidazoquinoline
compounds (e.g., CL097) and single stranded RNAs as their natural ligands.
Activation of TLRs leads to
the production of several cytokines (e.g., INFa , IL-8, IL-6) by the TLR
agonist-treated cells. Cytokine
release is used as readout in this assay and represents a measure for the
level of inhibition of the TLR /
IRAK-4 pathway by the tested compound. It should be noted that in the context
of the complete organism,
other sources for these cytokines exist that are not dependent on the TLR /
IRAK-4 pathway, such as e.g.,
macrophages (upon activation of the Fcy receptor (Yon et al., 2012)) or T
cells (upon activation of the T
cell receptor (Brehm et al., 2005)).
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5.1.1. Experimental design
[0320] Blood is collected from healthy volunteers into lithium heparin tubes
by venipuncture, then
gently inverted several times to prevent clotting and incubated for at least
15 min at 37 C on a rocking
mixer shaker. Then, 200 !at of blood is dispensed into 2 mL-microtubes and pre-
incubated in duplicate
with DMSO 0.3% or test compound at different concentrations (from 10 to 0.01
aM, 3 fold dilutions in
RPMI 1640 without glutamine (Life Technologies, 31870)) for 15 min at 37 C.
After this pre-incubation,
blood is triggered with CL097 (2 u.g/mL from 1 mg/mL solution in water;
InvivoGen, tlrl-c97) or vehicle
(distilled water) for 3 h 30 min at 37 C. Microtubes are centrifuged at 5000
x g for 10 min at 4 C and
approximately 80 iaL of plasma is collected into a polystyrene 96-well plate.
Plasma can be analyzed
freshly or frozen at -80 C shortly after triggering. Finally, the
quantification of TNFa is performed by
diluting 40 times the plasma using the human TNF-alpha DuoSet ELISA kit (R&D
Systems, DY210),
according to manufacturer's instructions. The optical density (OD) is
determined at 450 nm on a
PerkinElmer EnVision 2102 Multilabel plate reader.
5.1.2. Data analysis
[0321] A standard curve is created by plotting the mean absorbance on the y-
axis against the
concentration on the x-axis and a best fit curve is drawn through the points
on the graph. A linear
regression analysis is performed to determine the equation (y=ax +b) and the R-
squared value. For each
blood sample replicate, the TNFa concentration is calculated, taking into
account the dilution factor using
the formula:
[0322] TNFa concentration sample 40 *( Opsample b)/a
[001] Data are then expressed as a percentage of inhibition (PIN) for each
replicate using the formula:
mean TNFa with CL097 - TNFa sample'
PIN sample1 = x 100,
mean TNFa with CL097 - mean TNFa with vehicle
where 'mean TNFa with CL097' is the mean TNFa concentration of replicate
samples triggered with
CL097; `TNFot sample 1' is the TNFa concentration of sample 1; and 'mean TNFa
with vehicle' is the
mean TNFa concentration of replicate samples treated with vehicle.
[0323] Curve fittings are generated using mean PIN SEM. Graphs and IC50
calculations are derived
using the GraphPad Prism software.
[0324]
5.2. Ex vivo rat TNFa release inhibition (whole blood assay)
[0325] The objective of the assay is to assess the activity of compounds of
the invention on the activated
TLR / IRAK-4 pathway in an ex vivo rat whole blood setting. Toll-like
receptors (TLRs) are pattern
recognition receptors that recognize a wide variety of microbial molecules,
called pathogen-associated
molecular patterns (PAMPs). While human TLR7 and TLR8 both recognize
imidazoquinoline
compounds (e.g., CL097) and single stranded RNAs as their natural ligands,
rodent TLR8 needs
additional factors such as oligodeoxynucleotides (e.g., poly(dT)) for
activation.
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5.2.1. Experimental design
[0326] Sprague Dawley rats (male, 7-8 weeks old, 200-250 g body weight) are
obtained from Janvier
Labs (France).
[0327] Blood, obtained by exsanguination, is collected from at least 2 rats
into lithium heparinate tubes
and then pre-incubated for at least 15 min at 37 C on a rocking mixer shaker.
Blood from all rats is
mixed into a 50 mL polypropylene tube to get a unique blood batch. Then, 200
L of blood is dispensed
into 2 mL-microtubes and incubated in duplicate with DMSO 0.3% or test
compound at different
concentrations (from 10 to 0.01 M, 3 fold dilutions in RPMI 1640 without
glutamine (Life
Technologies, 31870)) for 15 min at 37 C. After this pre-incubation, blood is
triggered with CL097 (10
ug/mL from 1 mg/mL solution in water; InvivoGen, tlrl-c97) and poly(dT) (1 04
from 100 uM solution
in water; InvivoGen, tlrl-pt17) or vehicle (distilled water) for 3 h 30 min at
37 C. Microtubes are
centrifuged at 5000 x g for 10 min at 4 C and approximately 80 L of plasma
is collected into a
polystyrene 96-well plate. Plasma can be analyzed freshly or frozen at -80 C
shortly after triggering.
Finally, the quantification of TNFa is performed on plasma (1:3 diluted) using
the rat TNF-alpha
Quantikine ELISA kit (R&D Systems, SRTA00), according to manufacturer's
instructions. The optical
density (OD) is determined at 450 nm on a PerkinElmer EnVision 2102 Multilabel
plate reader.
5.2.2. Data analysis
[0328] A standard curve is created by plotting the mean absorbance on the y-
axis against the
concentration on the x-axis and a best fit curve is drawn through the points
on the graph. A linear
regression analysis is performed to determine the equation (y=ax +b) and the R-
squared value. For each
blood sample replicate, the TNFa concentration is calculated, taking into
account the dilution factor using
the formula:
[0329] TNFa concentration sample 1¨ 40 *( Opsample 1 - b)/a
[0330] Data are then expressed as a percentage of inhibition (PIN) for each
replicate using the formula:
mean TNFa with CL097 - TNFa samplel
PIN sample' = x 100,
mean TNFa with CL097 - mean TNFa with vehicle
where 'mean TNFa with CL097' is the mean TNFa concentration of replicate
samples triggered with
CL097 + poly(dT); `TNFa samplel' is the TNFa concentration of sample 1; and
'mean TNFa with
vehicle' is the mean TNFa concentration of replicate samples treated with
vehicle.
[0331] Curve fittings are generated using mean PIN SEM. Graphs and IC50
calculations are derived
using the GraphPad Prism software.
Example 6. In vivo assays
6.1. Murine model of psoriatic-like epidermal hyperplasia induced by topical
applications of
imiquimod, a TLR7/8 agonist.
6.1.1. Materials
103321 Aldara 5% imiquimod cream is obtained from MEDA.
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[0333] Anti-mouse IL-12/IL-23 p40 FG purified antibody (C17.8) is obtained
from Affymetrix
eBioscience (cat no. 16-7123-85).
6.1.2. Animals
[0334] Balb/cJ mice (female, 18-20 g body weight) are obtained from Janvier
Labs (France). Mice are
kept on a 12 h light/dark cycle (07:00 - 19:00). Temperature is maintained at
22 2 C, food and water
are provided ad libitum.
6.1.3. Study design
[0335] The design of the study is adapted from Van der Fits L. et al. (van der
Fits et al., 2009).
[0336] On the first day, the mice are shaved around the two ears under light
anaesthesia with isoflurane.
[0337] 30 mg of commercially available imiquimod cream (Aldara 5% cream) are
applied on both
internal and external surfaces of each ear for 4 consecutive days, translating
in a daily dose of 1.5 mg of
the active compound. Control animals received the same quantity of vaseline.
[0338] From day 1 to day 5, mice are dosed with test compound, 10 or 30 mg/kg,
p.o., b.i.d. in methyl
cellulose 0.5%, before application of imiquimod (on day 5, the mice are dosed
only once, 2 h before
euthanasia).
[0339] In a positive reference group, the animals receive two intraperitoneal
injections of anti-mouse
IL-12/IL-23 p40 antibody, 10 mg/kg, on day 1 and 3 days before day 1.
6.1.4. Assessment of disease
[0340] The thickness of both ears is measured daily with a thickness gage
(Mitutoyo, Absolute
Digimatic, 547-321). Body weight is assessed at initiation of the experiment
and at sacrifice. At day 5, 2 h
after the last dosing, the mice are sacrificed. The pinnae of the ear are cut,
excluding cartilage. The pinnae
are weighed and then immersed in a vial containing 1 mL of RNAlater solution
to assess gene
expression or in formalin for histology.
[0341] There are 14 mice per group. The results are expressed as mean SEM
and statistical analysis is
performed using one-way ANOVA followed by Dunnett's post-hoc test versus
imiquimod-vehicle group.
6.1.5. Histology
[0342] After sacrifice, ears are collected and fixed in 3.7% formaldehyde
before embedding in paraffin.
2 gm thick sections are cut and stained with haematoxylin and eosin. Ear
epidermis thickness is measured
by image analysis (SisNcom software) with 6 images per ear captured at 20x
magnification. Data are
expressed as mean SEM and statistical analysis is performed using one-way
ANOVA followed by
Dunnett's post-hoc test versus imiquimod-vehicle group.
6.1.6. Gene expression analysis
[0343] Ears are removed from the RNAlater solution and put in Trizol after
disruption with 1.4 mm
ceramic beads in a Precellys device. Total RNA is then purified using
NucleoSpin RNA kit. cDNA is
prepared and quantitative PCR is performed with gene-specific primers from
Qiagen using SYBR Green
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technology in a ViiA7 real-time PCR system (Applied Biosystems). Expression
levels of each gene
(IL17A, IL1B, IL22, LCN2, S100A8 and S100A9) are calculated relative to the
cyclophilin A
housekeeping gene expression level. Data are expressed as mean SEM of the
relative quantity (RQ=
ACT, where ACT= CT sample - CT cyclophilin A). The statistical test used is
ANOVA analysis of variance
with Dunnett's post-hoc test versus imiquimod-vehicle group.
6.2. Marine model of psoriatic-like epidermal hyperplasia induced by
intradermal injections of
IL-23
6.2.1. Materials
[0344] Mouse recombinant IL-23, carrier free (14-8231, CF) is provided by e-
Bioscience.
6.2.2. Animals
[0345] Balb/c mice (female, 18-20g body weight) are obtained from CERJ
(France). Mice are kept on a
12 h light/dark cycle (07:00 ¨ 19:00). Temperature is maintained at 22 C,
food and water are provided ad
libitum.
6.2.3. Study design
[0346] The design of the study is adapted from Rizzo HL. et al. (Rizzo et al.,
2011).
[0347] On the first day (D1), the mice are shaved around the two ears.
[0348] For 4 consecutive days (D1 to D4), the mice receive a daily intradermal
dose of mouse
recombinant IL-23 (1 jig/20 1_, in PBS/0.1% BSA) in the right pima ear and 20
gL of PBS/0.1% BSA in
the left pinna ear under anesthesia induced by inhalation of isoflurane.
[0349] From D1 to D5, mice are dosed with test-compound (10, 30, or 100 mg/kg,
p.o., q.d. in
methylcellulose 0.5%) or with vehicle, 1 h prior IL-23 injection.
6.2.4. Assessment of disease
[0350] The thickness of both ears is measured daily with an automatic caliper.
Body weight is assessed
at initiation and at sacrifice. On fifth day, 2 h after the last dosing, the
mice are sacrificed. The pinnae of
the ear are cut, excluding cartilage. The pinnae are weighed and then, placed
in a vial containing 1 mL of
RNAlater solution or in formaldehyde.
[0351] At D4, blood samples are also collected from the retro-orbital sinus
for PK profiling just before
dosing (TO) and 1 h, 3 h, 6 h post-dosing.
[0352] There are 8 mice per group. The results are expressed as mean SEM and
statistical analysis is
performed using one-way ANOVA followed by Dunnett's post-hoc test versus IL-23
vehicle groups.
6.2.5. Histology
[0353] After sacrifice, ears are collected and fixed in 3.7% formaldehyde
before embedding in paraffin.
2 gm thick sections are done and stained with hematoxylin and eosin. Ear
epidermis thickness is
measured by image analysis (Sis'Ncom software) with 6 images per ear captured
at magnification x20.
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Data are expressed as mean SEM and statistical analysis is performed using
one-way ANOVA followed
by Dunnett's post-hoc test versus IL-23 vehicle groups.
6.2.6. Gene expression analysis
[0354] Half ears are removed from RNAlater solution and put in Trizol after
disruption with 1.4 mm
ceramic beads in a Precellys device. Total RNA is then purified using
NucleoSpin RNA kit. cDNA is
prepared and quantitative PCR is performed with gene-specific primers from
Qiagen using SYBR Green
technology in a ViiA7 real-time PCR system (Applied Biosystems). Expression
levels of each gene
(IL17A, IL1B, IL22, LCN2, S100A8 and S100A9) are calculated relative to the
cyclophilin A
housekeeping gene expression level. Data are expressed as mean SEM of the
relative quantity (RQ =
ACT, where ACT = CT sample - Cr cyclophilin A). The statistical test used is
ANOVA analysis of variance
with Dunnett's post-hoc test versus the IL-23 vehicle group.
6.3. PK/PD model: TNFa release induced by CL097, a specific TLR7/8 agonist
[0005] The aim of this assay is to determine the relationship between the
inhibition of an IRAK-4
dependent event in vivo upon administration of a compound of the invention and
the circulating
concentration levels of this compound.
6.3.1. Materials
[0355] CL097 (cat no. tlrl-c97) and poly(dT) (cat no. tlrl-ptl 7) are obtained
from InvivoGen.
[0356] AlphaLISA mouse TNFa kits are obtained from Perkin-Elmer (cat no.
AL505C).
6.3.2. Animals
[0357] DBAI 1J mice (male, 18-20 g body weight) are obtained from Janvier Labs
(France). Mice are
kept on a 12 h light/dark cycle (07:00 ¨ 19:00). Temperature is maintained at
22 + 2 C, food and water
are provided ad libitum.
6.3.3. Study design
[0358] The mice receive an oral dose of test-compound. A group of intact
animals which does not
receive any dosing is used as the t = 0 time point.
[0359] Two blood samples obtained by intra-cardiac sampling (under isoflurane
anesthesia) are collected
into lithium heparinate tubes at 30 min, 1 h, 3 h, 8 h or 24 h post-dosing.
One is used for
pharmacokinetics (PK) analysis and the second for pharmacodynamic (PD) marker
quantification.
6.3.4. Quantification of compound levels in plasma
[0360] Whole blood samples are centrifuged at 5000 rpm for 10 min and the
resulting plasma samples
are stored at -20 C pending analysis. Plasma concentrations of each test
compound are determined by an
LC-MS/MS method.
6.3.5. Determination of pharmacokinetic parameters
103611 Pharmacokinetic parameters are calculated using WinNonlin (Pharsight ,
United States).
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6.3.6. Quantification of PD marker
[0362] Each blood sample is stimulated with CL097 and poly(dT) for 2 h at 37
C. Then, plasma is
collected and analyzed for TNFa by AlphaLISA according to the manufacturer's
instructions.
103631 There are 6 mice per group. The results are expressed as TNFa
concentration (pg/mL), or as
percentage of inhibition (PIN) relative to the t = 0 time point. The data are
presented as mean SEM and
statistical analysis is performed using one-way ANOVA followed by Dunnett's
post-hoc test versus
vehicle group of the corresponding time point.
6.4. Murine prophylactic model of atopic dermatitis induced by topical
application of MC903
6.4.1. Materials
[0364] Methylcellulose 0.5% is obtained from VWR (cat no. AX021233). MC903
(calcipotriol) is
obtained from Tocris Bioscience (cat no. 2700/50). ProSense 680 is obtained
from PerkinElmer (cat no.
NEV10003). RNAlater is obtained from Ambion (cat no. AM7021). Imalgene 1000
(Merial) and
Rompun 2% (Bayer) are obtained from Centravet (cat no. IMA004-6827812 and
ROM001-6835444).
6.4.2. Animals
[0365] BALB/cN mice (female, 18-20 g body weight) or CD1/Swiss mice (female,
24-26 g body weight)
are obtained from Janvier Labs (France). Mice are kept on a 12 h light/dark
cycle (07:00 ¨ 19:00).
Temperature is maintained at 22 2 C, food and water are provided ad
libitum.
6.4.3. Study design
[0366] The design of the study is adapted from Li M. et al. (Li et al., 2006).
[0367] On the first day (D1), the mice are anesthetized with an
intraperitoneal injection of Imalgene and
Rompun (7.5%/ 2.5%; 0.1 mL/10 g) and shaved around the two ears.
[0368] As of D1, either 20 'IL Et0H or 2 nmol of MC903 (in 20 pt Et0H) are
topically applied on both
ears of mice for five consecutive days.
[0369] From D1 to D8, the mice are dosed with test compound (15 or 30 mg/kg,
p.o., b.i.d. in
methylcellulose 0.5%) or dexamethasone (5 mg/kg, p.o., q.d. in methylcellulose
0.5%), or with vehicle.
6.4.4. Quantifkation of compound levels in plasma
[0370] Plasma concentrations of each test compound are determined by an LC-
MS/MS method in which
the mass spectrometer is operated in positive or negative electrospray mode.
6.4.5. Determination of pharmacokinetic parameters
[0371] Pharmacokinetic parameters are calculated using Phoenix WinNonlin
(Pharsight , United
States).
6.4.6. Assessment of disease
[0372] The thickness of both ears is measured (after anaesthesia induced by
isoflurane inhalation) at
initiation of the study, every other day and at sacrifice using a thickness
gage (Mitutoyo, Absolute
Digimatic, 547-321).
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[0373] Body weight is assessed at initiation of the study, every other day and
at sacrifice.
[0374] On D4, mice from all groups receive ProSense 680 probe (0.8 nmo1/10 g,
IP). On D5, the mice
are anesthetized with an intraperitoneal injection of Imalgene and Rompun
(7.5% / 2.5%; 0.1 mL/10 g).
Granulocyte infiltration is measured using in vivo molecular imaging (Bruker
In-Vivo Xtreme imaging
system, excitation wavelength: 630 nm, emission wavelength: 700 nm,
acquisition time: 5 seconds).
[0375] On D8, 2 h after the last dosing, mice are sacrificed and total blood
is collected on EDTA-coated
tubes and plasma is frozen for further measurements (including circulating
compound). A sample of
blood is also collected in heparin-coated tubes.
[0376] The pinnae of the ears are collected and weighed. One ear is cut
longitudinally into 2 halves. One
half is fixed in formaldehyde buffer 4% for histology; the other one is
immersed in RNAlater to assess
gene expression.
[0377] There are 8 mice per group. The results are expressed as mean SEM and
statistical analysis is
performed using one-way ANOVA followed by Dunnett's post-hoc test versus MC903
vehicle groups for
ear thickness and weight, versus Et0H vehicle group for body weight.
6.4.7. Histology
[0378] After sacrifice, half ears are collected and fixed in 3.7% formaldehyde
before embedding in
paraffin. 4 j.tm thick sections are immunostained by immunohistochemistry with
specific cell marker
antibody: CD3 for T cells and EPX for eosinophils. The immunostained cell
areas from a whole section
per mouse are measured by image analysis (CaloPix software, TRIBVN
Healthcare). Data are expressed
as mean SEM and statistical analysis is performed using one-way ANOVA followed
by Dunnett's post-
hoc test versus MC903 vehicle groups.
6.4.8. .. Gene expression analysis
[0379] Ears are removed from RNAlater solution and placed in Trizol after
disruption with 1.4 mm
ceramic beads in a Bertin Instruments Precellys homogenizer. Total RNA is
then extracted using a
phenol/chloroform protocol and purified with a QIAcube using an RNeasy 96
QIAcube HT Kit
(Qiagen, cat no. 74171). cDNA is prepared and quantitative PCR performed with
gene-specific primers
from Qiagen using SYBR Green technology in a ViiA 7 real-time PCR system
(Applied Biosystems).
Expression levels of each gene (IL4, IL5, IL13, TSLP, IL33, S12, IL25, IL31,
IFN7, IL6, IL10, LCN2,
S100A8 and S100A9) are calculated relative to the HPRT, GAPDH and I3-actin
housekeeping gene
expression levels. Data are expressed as mean SEM
of the relative quantity (RQ = TAcT, where
ACT = CT sample ¨ average (CT HPRT, CT GAPDH, CT 13-actin). The statistical
test used is ANOVA
analysis of variance with Dunnett's post-hoc test versus the Et0H/MC903
vehicle group.
6.5. Marine therapeutic model of atopic dermatitis induced by topical
application of MC903
6.5.1. Materials
[0380] Methylcellulose 0.5% is obtained from VWR (cat no. AX021233). MC903
(calcipotriol) is
obtained from Tocris Bioscience (cat no. 2700/50). ProSense 680 is obtained
from PerkinElmer (cat no.
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NEV10003). RNAlater is obtained from Ambion (cat no. AM7021). Imalgene 1000
(Merial) and
Rompun 2% (Bayer) are obtained from Centravet (cat no. IMA004-6827812 and
ROM001-6835444).
6.5.2. Animals
[0381] BALB/cN mice (female, 18-20 g body weight) or CD1/Swiss mice (female,
24-26 g body weight)
are obtained from Janvier Labs (France). Mice are kept on a 12 h light/dark
cycle (07:00 ¨ 19:00).
Temperature is maintained at 22 2 C, food and water are provided ad
libitum.
6.5.3. Study design
[0382] The design of the study is adapted from Li M. et al. (Li et al., 2006).
[0383] On the first day (D1), the mice are anesthetized with an
intraperitoneal injection of Imalgene and
Rompun (7.5%/ 2.5%; 0.1 mL/10 g) and shaved around the two ears.
[0384] As of D1, either 20 'IL Et0H or 2 nmol of MC903 (in 20 j.iL Et0H) are
topically applied on both
ears of mice up to D9, Dll or D15 (except during the weekend).
[0385] From D5, the mice are dosed with test compound (15 or 30 mg/kg, p.o.,
b.i.d. in methylcellulose
0.5%) or dexamethasone (5 mg/kg, p.o., q.d. in methylcellulose 0.5%), or with
vehicle, until D10, D12, or
D16.
6.5.4. Quantification of compound levels in plasma
[0386] Plasma concentrations of each test compound are determined by an LC-
MS/MS method in which
the mass spectrometer is operated in positive or negative electrospray mode.
6.5.5. Determination of pharmacokinetic parameters
[0387] Pharmacokinetic parameters are calculated using Phoenix WinNonlin
(Pharsight , United
States).
6.5.6. Assessment of disease
[0388] The thickness of both ears is measured (after anaesthesia induced by
isoflurane inhalation), prior
to application of MC903, at initiation of the study, three times a week and at
sacrifice using a thickness
gage (Mitutoyo, Absolute Digimatic, 547-321).
[0389] Body weight is assessed at initiation of the study, three times a week
and at sacrifice.
[0390] On D8, D10 or D11, mice from all groups receive ProSense 680 probe
(0.8 nmo1/10 g, IP). On
the next day (D9, D1 1 or D12), the mice are anesthetized with an
intraperitoneal injection of Imalgene
and Rompun (7.5% / 2.5%; 0.1 mL/10 g). Granulocyte infiltration is then
measured using in vivo
molecular imaging (Bruker In-Vivo Xtreme imaging system, excitation
wavelength: 630 nm, emission
wavelength: 700 nm, acquisition time: 5 seconds).
[0391] On D10, D12, or D16, 2 h after the last dosing, the mice are
sacrificed; total blood is collected on
EDTA-coated tubes and plasma is frozen for further measurements (including
circulating compound).
[0392] The pinnae of the ears are collected. One ear is cut longitudinally
into 2 halves. One half is fixed
in formaldehyde buffer 4% for histology; the other one is immersed in RNAlater
to assess gene
expression.
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[0393] There are 8 mice per group. The results are expressed as mean SEM and
statistical analysis is
performed using one-way ANOVA followed by Dunnett's post-hoc test versus MC903
vehicle groups for
ear thickness and weight, versus Et0H vehicle group for body weight.
6.5.7. Histology
[0394] After sacrifice, half ears are collected and fixed in 3.7% formaldehyde
before embedding in
paraffin. 4 gm thick sections are immunostained by immunohistochemistry with
anti-CD3 antibody. The
immunostained cell areas from a whole section per mouse are measured by image
analysis (CaloPix
software, TRIBVN Healthcare). Data are expressed as mean SEM and statistical
analysis is performed
using one-way ANOVA followed by Dunnett's post-hoc test versus MC903 vehicle
groups.
6.5.8. .. Gene expression analysis
[0006] Ears are removed from RNAlater solution and placed in Trizol after
disruption with 1.4 mm
ceramic beads in a Bertin Instruments Precellys homogenizer. Total RNA is
then extracted using a
phenol/chloroform protocol and purified with a QIAcube using an RNeasy 96
QIAcube HT Kit
(Qiagen, cat no. 74171). cDNA is prepared and quantitative PCR performed with
gene-specific primers
from Qiagen using SYBR Green technology in a ViiA 7 real-time PCR system
(Applied Biosystems).
Expression levels of each gene of interest (GOT = IL4, IL5, IL13, TSLP, IL33,
ST2, IL25, IL31, IFNy,
IL6, IL10, LCN2, S100A8 and S100A9) are calculated relative to the HPRT, GAPDH
and ft-actin
housekeeping gene expression levels.
[0007] All qPCR data are expressed as mean SEM of the normalized relative
quantity (NRQ =
2^(ACq GOI)/Geomean (2^(ACq HPRT), 2^(ACq GAPDH), 2^(ACq ft-actin)) where ACq
= Cq average ¨
Cq sample. The statistical test used is ANOVA analysis of variance with
Dunnett's post-hoc test versus
the Et0H/MC903 vehicle group.
6.6. Marine model of systemic lupus erythematosus induced by epicutaneous
applications of
imiquimod
6.6.1. Materials
[0395] Aldara 5% imiquimod cream is obtained from MEDA.
[0396] Mouse anti-double-stranded DNA antibodies ELISA kits are obtained from
Alpha Diagnostic
International (cat no. 5120). Mouse urinary albumin ELISA kits are obtained
from Abeam (cat no.
ab108792). Urine creatinine assay kits are obtained from Abnova (cat no.
KA4344).
6.6.2. Animals
[0397] BALB/cJ mice (female, 18-20 g body weight) are obtained from Janvier
Labs (France). Mice are
kept on a 12 h light/dark cycle (07:00 ¨ 19:00). Temperature is maintained at
22 + 2 C, food and water
are provided ad libitum.
6.6.3. Study design
103981 The design of the study is adapted from Yokogawa M. et al. (Yokogawa et
al., 2014).
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[0399] On the first day (D1), the mice are shaved around the right ears.
[0400] The mice receive an epicutaneous application of 1.25 mg of imiquimod 3
times per week on the
right pinna ear for 12 consecutive weeks (D1 to D86). The control group
receives the same quantity of
vaseline.
[0401] From D1 to D86, mice are dosed with test compound (30 mg/kg, p.o., q.d.
in methylcellulose
0.5%) or with vehicle (10 mL/kg).
6.6.4. Assessment of disease
[0402] The thickness of the ears is measured once a week with an automatic
gage (Mitutoyo, Absolute
Digimatic, 547-321).
[0403] Body weight is assessed at initiation and once a week until sacrifice.
At necropsy, the spleen
weight is also measured. The mice are sacrificed 2 h after the last dosing.
[0404] At different time points (e.g., on days D28, D56 and D84), the mice are
individually placed in a
metabolic cage to perform urinalysis and assess proteinuria (albumin to
creatinine ratio).
[0405] Serums are collected at different time points (e.g., on D28, D56 and
D86) to assess anti-double
stranded-DNA IgG levels.
[0406] At D13, blood samples are also collected from the retro-orbital sinus
for PK profiling just before
dosing (TO) and 1 h, 3 h, 6 h post-dosing.
[0407] There are 8-19 mice per group. The results are expressed as mean SEM
and statistical analysis
is performed using one-way ANOVA followed by Dunnett's post-hoc test versus
imiquimod vehicle
groups.
6.6.5. .. Quantification of compound levels in plasma
[0408] Plasma concentrations of each test compound are determined by an LC-
MS/MS method in which
the mass spectrometer is operated in positive or negative electrospray mode.
6.6.6. Determination of pharmacokinetic parameters
[0409] Pharmacokinetic parameters are calculated using Phoenix WinNonlin
(Pharsight , United
States).
6.6.7. .. Histology
[0410] After sacrifice, left kidneys are collected and cut longitudinally into
2 parts. One part is fixed in
3.7% formaldehyde before embedding in paraffin. 4 p.m thick sections are made
and stained with Period
acid-Schiff(PAS) or immunostained with CD3 (T cells), CD20 (B cells) and F4/80
(macrophages).
6.6.7.1. Histopathology
[0411] In each glomerulus, 4 different readouts including
mesangioproliferation, endocapillary
proliferation, mesangial matrix expansion and segmental sclerosis are graded
on a scale of 0 to 2 and then
summed. For each kidney, about 50 glomeruli are scored and then averaged
giving one glomerular lesion
score (Yokogawa et al., 2014). Data are expressed as mean SEM and
statistical analysis is performed
using the Kruskal-Wallis test followed by Dunn's post-hoc test versus
imiquimod vehicle group.
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6.6.7.2. Cellular quantifications
[0412] For each cell type, immunohistochemical analysis is performed using
image analysis (CaloPix
software, TRIBVN Healthcare) on the whole tissue section at a magnification of
x20. Data are expressed
as mean + SEM and statistical analysis is performed using one-way ANOVA
followed by Dunnett's post-
hoc test versus imiquimod vehicle group.
6.6.7.3. Gene expression analysis
[0413] At sacrifice, the second part of the left kidneys is placed in tubes
containing 1.4 mm ceramic
beads and disrupted in 1% DTT RLT lysis buffer (Qiagen, cat no. 79216) with a
Bertin Instruments
Precellys homogenizer. Total RNA is then purified with a QIAcube using an
RNeasy 96 QIAcube HT
Kit (Qiagen, cat no. 74171). cDNA is prepared and quantitative PCR performed
with gene-specific
primers from Qiagen using SYBR Green technology in a ViiA 7 real-time PCR
system (Applied
Biosystems). Expression levels of each gene of interest (GOI = CD3, CD68,
CD20, OAS1, Mxl, IFIT1,
CXCL11 and Usp18) are calculated relative to the cyclophilin, GAPDH and [3-
actin housekeeping gene
expression levels.
[0414] At sacrifice, one-third of the spleen is placed into tubes containing
1.4 mm ceramic beads and
disrupted in Trizol with a Bertin Instruments Precellys homogenizer. Total
RNA is extracted using a
phenol/chloroform process and then purified with a QIAcube using an RNeasy 96
QIAcube HT Kit
(Qiagen, cat no. 74171). cDNA is prepared and quantitative PCR performed with
gene-specific primers
from Qiagen using SYBR Green technology in a ViiA 7 real-time PCR system
(Applied Biosystems).
Expression levels of each gene of interest (GOT = CD20, IRF7, OAS1, Mx 1,
IFIT1, CXCL11, Usp18,
BCL6, CXCL13, CXCR5, MAF, ICOSL, PDCD1, SH2D1a) are calculated relative to the
cyclophilin,
GAPDH and 13-actin housekeeping gene expression levels.
[0008] All qPCR data are expressed as mean SEM of the normalized relative
quantity (NRQ =
2^(ACq G01)/Geomean (2^(ACq cyclophilin), 2^(ACq GAPDH), 2^(ACq I3-actin))
where ACq= Cq
average ¨ Cq sample. The statistical test used is ANOVA analysis of variance
with Dunnett's post-hoc
test versus imiquimod vehicle group.
6.7. Marine model of psoriatic arthritis induced by overexpression of IL-23
6.7.1. Materials
[0415] Mouse IL-23 enhanced episomal expression vector (EEV) is obtained from
System Biosciences
(cat no. EEV651A-1). Ringers solution tablets are obtained from Sigma-Aldrich
(cat no. 96724-100TAB).
Mouse IL-23 Quantikine ELISA Kits are obtained from R&D Systems (cat no.
M2300). ProSense 680
and OsteoSense 750EX are obtained from PerkinElmer (cat no. NEV10003 and
NEV10053EX).
RNAlater is obtained from Ambion (cat no. AM7021). Imalgene 1000 (Merial)
and Rompun 2%
(Bayer) are obtained from Centravet (cat no. IMA004-6827812 and ROM001-
6835444).
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6.7.2. Animals
[0416] B 10.RIII mice (male, 8-week old) are obtained from Charles River
(France). Mice are kept on a
12 h light/dark cycle (07:00 ¨ 19:00). Temperature is maintained at 22 2 C,
food and water are
provided ad libitum.
6.7.3. Study design
[0417] The design of the study is adapted from Sherlock JP. et al. (Sherlock
et al., 2012).
[0418] On the first day (D1), the mice undergo a hydrodynamic injection of
Ringer or IL-23 EEV in
Ringer into the tail vein (3 gg/2.1 mL, IV injected over a period of 4-6 sec).
[0419] As of D5, twice a week, the mice are scored for clinical symptoms until
the end of the
experiment.
[0420] On D5, blood is collected by puncture in the submandibular vein to
assess the serum IL-23
concentration.
[0421] On D9, mice from all groups receive ProSense 680 probe (0.8 nmo1/10 g,
IP). On D10, the mice
are anesthetized with an intraperitoneal injection of Imalgene and Rompun
(7.5% / 2.5%; 0.1 mL/10 g).
Granulocyte infiltration is then measured using in vivo molecular imaging
(Bruker In-Vivo Xtreme
imaging system, excitation wavelength: 630 nm, emission wavelength: 700 nm,
acquisition time: 5
seconds).
[0422] On D11, randomization is performed according to ProSense 680 molecular
imaging and scoring.
[0423] As of D12, mice are dosed with test compound (30 mg/kg, p.o., b.i.d. in
methylcellulose 0.5%) or
with vehicle.
[0424] On D19, blood is sampled at time TO, Tlh, T3h and T6h after last
dosing. Plasma is separated
and kept at 20 C until bioanalysis.
[0425] On D36, mice from all groups are sacrificed 2 h after last
administration of compound. The
following is collected:
[0426] Heels around enthesis (without skin) of the left hindlimb are
immediately snap frozen in Precellys
tubes. Fingers are collected in tubes containing RNAlater . The right hindlimb
is immediately fixed in
formaldehyde buffer 4% for histology evaluation. X-ray measurement is
performed 48 h after fixation.
[0427] One ear is collected in tube containing RNAlater for transcript
analysis.
[0428] Total blood is collected in a serum blood tube and mixed by gentle
inversion 8-10 times. After
clotting, blood samples are centrifuged 10 min at 1800 x g. After
centrifugation, serum is stored at -
80 C.
[0429] Part of the colon (1 cm distal colon) is immediately snap frozen in
Precellys tube for transcript
analysis. Another part (1 cm distal colon) is immediately fixed in
formaldehyde buffer 4% for further
histology analysis.
6.7.4. Assessment of disease
104301 Body weight is assessed at initiation of the study, then twice a week
and at sacrifice.
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[0431] Twice weekly, clinical signs of inflammation are scored: 0 for normal
paw; 1 if swelling of one
digit; 2 if swelling of two or more digits ; 3 if swelling of the entire paw.
The scores of all limbs are
summed up to produce a global score.
[0432] On D23, mice from all groups receive ProSense 680 probe (0.8 nmo1/10
g, IP). On D24, the
mice are anesthetized with an intraperitoneal injection of Imalgene and Rompun
(7.5% / 2.5%; 0.1
mL/10 g). Granulocyte infiltration is then measured using in vivo molecular
imaging (Bruker In-Vivo
Xtreme imaging system, excitation wavelength: 630 nm, emission wavelength: 700
nm, acquisition time:
5 seconds).
[0433] On D32, mice from all groups receive ProSense 680 probe (0.8 nmo1/10
g, IP) and OsteoSense
750EX probe (0.8 nmo1/10 g, IP). On D33, the mice are anesthetized with an
intraperitoneal injection of
Imalgene and Rompun (7.5% / 2.5%; 0.1 mL/10 g). Granulocyte infiltration and
bone remodelling are
measured using in vivo molecular imaging (Bruker In-Vivo Xtreme imaging
system; excitation
wavelength: 630 nm, emission wavelength: 700 nm, acquisition time: 5 seconds
for ProSense 680 probe;
excitation wavelength: 720 nm, emission wavelength: 790 nm, acquisition time:
5 seconds for
0 steo S ens e 75 OEX probe).
[0434] There are 10 mice per group. The results are expressed as mean SEM
and statistical analysis is
performed using one-way ANOVA followed by Dunnett's post-hoc test versus
diseased vehicle group for
scoring and imaging analysis, versus sham vehicle group for body weight.
6.8. CIA model
6.8.1. Materials
[0435] Completed Freund's adjuvant (CFA) and incomplete Freund's adjuvant
(IFA) were purchased
from Difco. Bovine collagen type II (CII), lipopolysaccharide (LPS), and
Enbrel was obtained from
Chondrex (Isle d'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett
(25mg injectable
syringe, France) Acros Organics (Palo Alto, CA), respectively. All other
reagents used were of reagent
grade and all solvents were of analytical grade.
6.8.2. Animals
[0436] Dark Agouti rats (male, 7-8 weeks old) were obtained from Harlan
Laboratories (Maison-Alfort,
France). Rats were kept on a 12 hr light/dark cycle (0700 - 1900). Temperature
was maintained at 22 C,
and food and water were provided ad libitum.
6.8.3. Collagen induced arthritis (CIA)
[0437] One day before the experiment, CH solution (2 mg/mL) was prepared with
0.05 M acetic acid and
stored at 4 C. Just before the immunization, equal volumes of adjuvant (IFA)
and CII were mixed by a
homogenizer in a pre-cooled glass bottle in an ice water bath. Extra adjuvant
and prolonged
homogenization may be required if an emulsion is not formed. 0.2 mL of the
emulsion was injected
intradermally at the base of the tail of each rat on day 1, a second booster
intradermal injection (CII
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solution at 2 mg/mL in CFA 0.1 mL saline) was performed on day 9. This
immunization method was
modified from published methods (Sims et al, 2004; Jou et al., 2005).
6.8.4. Study design
[0438] The therapeutic effects of the compounds were tested in the rat CIA
model. Rats were randomly
divided into equal groups and each group contained 10 rats. All rats were
immunized on day 1 and
boosted on day 9. Therapeutic dosing lasted from day 16 to day 30. The
negative control group was
treated with vehicle (MC 0.5%) and the positive control group with Enbrel (10
mg/kg, 3x week., s.c.). A
compound of interest was typically tested at 4 doses, e.g. 1, 3, 10, 30 mg/kg,
bid
6.8.5. Clinical assessment of arthritis
[0439] Arthritis is scored according to the method of Khachigian 2006, Lin et
al 2007 and Nishida et al.
2004). The swelling of each of the four paws is ranked with the arthritic
score as follows: 0-no
symptoms; 1-mild, but definite redness and swelling of one type of joint such
as the ankle or wrist, or
apparent redness and swelling limited to individual digits, regardless of the
number of affected digits; 2-
moderate redness and swelling of two or more types of joints; 3-severe redness
and swelling of the entire
paw including digits; 4-maximally inflamed limb with involvement of multiple
joints (maximum
cumulative clinical arthritis score 16 per animal) (Nishida et al., 2004).
[0440] To permit the meta-analysis of multiple studies the clinical score
values were normalised as
follows:
[0441] AUC of clinical score (AUC score): The area under the curve (AUC) from
day 1 to day 14 was
calculated for each individual rat. The AUC of each animal was divided by the
average AUC obtained
for the vehicle in the study from which the data on that animal was obtained
and multiplied by 100 (i.e.
the AUC was expressed as a percentage of the average vehicle AUC per study).
[0442] Clinical score increase from day 1 to day 14 (End point score): The
clinical score difference for
each animal was divided by the average clinical score difference obtained for
the vehicle in the study
from which the data on that animal was obtained and multiplied by 100 (i.e.
the difference was expressed
as a percentage of the average clinical score difference for the vehicle per
study).
6.8.5.1. Change in body weight (%) after onset of arthritis
[0443] Clinically, body weight loss is associated with arthritis (Shelton et
al., 2005; Argiles et al., 1998;
Rall, 2004; Walsmith et al., 2004). Hence, changes in body weight after onset
of arthritis can be used as a
non-specific endpoint to evaluate the effect of therapeutics in the rat model.
The change in body weight
(%) after onset of arthritis was calculated as follows:
Body Weight (week6) ¨ Body Weight(week5)
X 100%
[0444] Mice: Body Weight .(week5)
Body Weight .(week4) Body Weight .(week3)
X 100%
104451 Rats: Body We ight(week3)
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6.8. 5.2. Radiology
[0446] X-ray photos were taken of the hind paws of each individual animal. A
random blind identity
number was assigned to each of the photos, and the severity of bone erosion
was ranked by two
independent scorers with the radiological Larsen's score system as follows: 0-
normal with intact bony
outlines and normal joint space; 1- slight abnormality with any one or two of
the exterior metatarsal bones
showing slight bone erosion; 2-definite early abnormality with any three to
five of the exterior metatarsal
bones showing bone erosion; 3-medium destructive abnormality with all the
exterior metatarsal bones as
well as any one or two of the interior metatarsal bones showing definite bone
erosions; 4-severe
destructive abnormality with all the metatarsal bones showing definite bone
erosion and at least one of the
inner metatarsal joints completely eroded leaving some bony joint outlines
partly preserved; 5-mutilating
abnormality without bony outlines. This scoring system is a modification from
Salvemini et al., 2001;
Bush et al., 2002; Sims et al., 2004; Jou et al., 2005.
6.8. 5.3. Histology
[0447] After radiological analysis, the hind paws of mice were fixed in 10%
phosphate-buffered
formalin (pH 7.4), decalcified with rapid bone decalcifiant for fine histology
(Laboratories Eurobio) and
embedded in paraffin. To ensure extensive evaluation of the arthritic joints,
at least four serial sections (5
gm thick) were cut and each series of sections were 100 gm in between. The
sections were stained with
hematoxylin and eosin (H&E). Histologic examinations for synovial inflammation
and bone and cartilage
damage were performed double blind. In each paw, four parameters were assessed
using a four-point
scale. The parameters were cell infiltration, pannus severity, cartilage
erosion and bone erosion. Scoring
was performed according as follows: 1-normal, 2-mild, 3-moderate, 4-marked.
These four scores are
summed together and represented as an additional score, namely the 'RA total
score'.
6.8.5.4. Micro-computed tomography (uCT) analysis of calcaneus (heel bone):
[0448] Bone degradation observed in RA occurs especially at the cortical bone
and can be revealed by
gCT analysis (Sims NA et al., Arthritis Rheum. 50 (2004) 2338-2346: Targeting
osteoclasts with
zoledronic acid prevents bone destruction in collagen-induced arthritis; Oste
L et al., ECTC Montreal
2007: A high throughput method of measuring bone architectural disturbance in
a murine CIA model by
micro-CT morphometry). After scanning and 3D volume reconstruction of the
calcaneus bone, bone
degradation is measured as the number of discrete objects present per slide,
isolated in silico
perpendicular to the longitudinal axis of the bone. The more the bone is
degraded, the more discrete
objects are measured. 1000 slices, evenly distributed along the calcaneus
(spaced by about 10.8 gm), are
analyzed.
6.8.5.5. Steady State PK
[0449] At day 7 or 11, blood samples were collected at the retro-orbital sinus
with lithium heparin as
anti-coagulant at the following time points: predose, 1, 3 and 6 hrs. Whole
blood samples were
centrifuged and the resulting plasma samples were stored at -20 C pending
analysis. Plasma
concentrations of each test compound were determined by an LC-MS/MS method in
which the mass
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98
spectrometer was operated in positive electrospray mode. Pharmacokinetic
parameters were calculated
using Winnonlin0 (Pharsight0, United States) and it was assumed that the
predose plasma levels were
equal to the 24 hrs plasma levels.
6.8.6. Results
Table XI. Clinical score
Cpd 1 Cpd 1 Cpd 1 Cpd 1
Day CIA Vehicle
(1 mg/kg) (3 mg/kg) (10 mg/kg) (30
mg/kg)
31 2.3 0.448 2.3 2.2 2.2 2.3
32 3 0.537 2.6 2.6 2.5 2
33 3.7 0.578 3.1 2.7 2.4 2
34 4.6 0.562 3.7 2.8 2 1.7
35 5.5 0.637 4.5 3 2 1.8
38 7.5 0.86 6.5 4.1 4.1 3.9
39 7.9 0.849 7.5 4.9 4.8 4.1
40 7.8 0.827 7.6 5.3 6.4 4.4
41 8.2 0.964 7.4 5.3 6.4 5.4
42 7.9 0.948 7.6 5.6 6.6 5.9
45 7.9 0.752 8.4 7.1 6.7 7
46 7.8 0.786 8.2 7.3 6.3 7.1
Table XII. Clinical score (p values)
Cpd XXb Cpd XXb Cpd 1 + Cpd XXb
Day Vehicle
(7,5 mg/kg bid) (15 mg/kg bid) (30 + 15 mg/kg)
31 2.9 (1) 3 (0.889) 3 (0.889) 3 (0.889)
32 3.5 (1) 3.6 (0.8664) 3.8 (0.6661) 3.2
(0.1964)
34 4.1 (1) 3.8 (0.6338) 3.9 (0.7869) 2.5
(0.1387)
35 4.4 (1) 3.7 (0.3327) 3.9 (0.5456) 2 (0.2409)
38 5.8 (1) 4.6 (0.2354) 4.2 (0.1094) 1.9
(0.0003)
39 7.1 (1) 4.7 (0.0712) 4.5 (0.0467) 2.2
(0.0001)
40 8 (1) 5.1 (0.038) 4.6 (0.0152) 2.6
(0.0267)
41 8.1 (1) 4.7 (0.0185) 4.3 (0.0085) 2.3
(0.0002)
42 8.3 (1) 4.7 (0.012) 4.6 (0.0104) 2.1
(0.0001)
45 10.1 (1) 5.3 (0.0024) 6.1 (0.0080) 2.9
(0.0001)
6.8.7. Conclusions
[0450] As may be seen above, when taken together the combination of the IRAK
and JAK inhibitor
results in a better effect than for each compound taken individually.
[0451] This in turn may allow achieving the same or better therapeutic effect
with a lower amount of
drugs. This may particularly be beneficial in avoiding taking unnecessary drug
amounts while
maintaining efficacy, and thus reducing the risk of drug induced side effects.
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FINAL REMARKS
[0452] It will be appreciated by those skilled in the art that the foregoing
descriptions are exemplary and
explanatory in nature, and intended to illustrate the invention and its
preferred embodiments. Through
routine experimentation, an artisan will recognize apparent modifications and
variations that may be
made without departing from the spirit of the invention. All such
modifications coming within the scope
of the appended claims are intended to be included therein. Thus, the
invention is intended to be defined
not by the above description, but by the following claims and their
equivalents.
[0453] All publications, including but not limited to patents and patent
applications, cited in this
specification are herein incorporated by reference as if each individual
publication are specifically and
individually indicated to be incorporated by reference herein as though fully
set forth.
[0454] It should be understood that factors such as the differential cell
penetration capacity of the various
compounds can contribute to discrepancies between the activity of the
compounds in the in vitro
biochemical and cellular assays.
[0455] At least some of the chemical names of compound of the invention as
given and set forth in this
application, may have been generated on an automated basis by use of a
commercially available chemical
naming software program, and have not been independently verified.
Representative programs
performing this function include the Lexichem naming tool sold by OpenEye
Scientific Software, Inc.
and the Autonom Software tool sold by MDL Information Systems, Inc. In the
instance where the
indicated chemical name and the depicted structure differ, the depicted
structure will control.
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