DIAZASPIROALKANEONE-SUBSTITUTED OXAZOLE DERIVATIVES AS
SPLEEN TYROSINE KINASE INHIBITORS
The present disclosure discloses substituted oxazole derivatives that selectively
modulate, regulate, and/or inhibit signal transduction mediated by certain native
and/or mutant protein kinases implicated in a variety of human and animal diseases
such as cell proliferative, metabolic, autoimmune, allergic, and degenerative disorders.
In particular, several of these compounds are potent and selective spleen tyrosine
kinase (Syk) inhibitors.
Background
Protein Kinases are receptor type or non-receptor type proteins, which transfer the
terminal phosphate of ATP to aminoacid residues, such as tyrosine, threonine, serine
residues, of proteins, thereby activating or inactivating signal transduction pathways.
These proteins are known to be involved in many cellular mechanisms, which in case
of disruption, lead to disorders such as abnormal cell proliferation and migration as
well as inflammation.
As of today, there are over 500 known Protein kinases. Included are the well-known
Abl, Aktl, Akt2, Akt3, ALK, Alk5, A-Raf, Axl, B-Raf, Brk, Btk, Cdk2, Cdk4, Cdk5, Cdk6,
CHK1, c-Raf-1, Csk, EGFR, EphAl, EphA2, EphB2, EphB4, Erk2, Fak, Fes, Fer, FGFR1,
FGFR2, FGFR3, FGFR4, Flt-3, Fms, Frk, Fyn, Gsk3a, Gsk3 HCK, Her2/Erbb2,
Her4/Erbb4, IGF1R, IKK beta, Irak4, Itk, Jakl, Jak2, Jak3, Jnkl, Jnk2, Jnk3, KDR, Kit,
Lck, Lyn, MAP2K1, MAP2K2, MAP4K4, MAPKAPK2, Met, Mer, MNK1, MLK1, mTOR, p38,
PDGFRa, PDGFRp, PDPK1, PI3Ka, RBKb, RI3Kd, RBKg, Piml, Pim2, Pim3, PKC alpha,
PKC beta, PKC theta, Plkl, Pyk2, Ret, ROCK1, ROCK2, RON, Src, Stk6, Syk, TEC, Tie2,
TrkA, TrkB, Tyk2, VEGFRl/Flt-1, VEGFR2/Kdr, VEGFR3/Flt-4, Yes, and Zap70.
Spleen tyrosine kinase (Syk), an intracellular protein tyrosine kinase, is a key mediator
of immunoreceptor signalling in a host of inflammatory cells including B cells, mast
cells, macrophages, and neutrophils (Wong Br et al (2004), Expert Opin. Investig.
Drugs, 13, 743-762). Syk is also widely expressed in nonhematopoietic cells like
fibroblasts, breast cancer cells, colonic carcinoma cells, hepatocytes, neuronal cells,
and vascular endothelial cells (Okamura S et al (1999), Oncol. Res. 11, 281-285).
Originally, Syk was thought to function primarily in signaling of immunoreceptors such
as Fc receptor (FcR) and B cell receptor (BCR). However, recent studies demonstrated
the crucial role of Syk in the cell signaling of diverse cellular stimuli including IL-1,
tumor necrosis factor-a (TNFa), lipopolysaccharide, and bΐ -integrin (Yamada T et al
(2001), J. Immunol., 167, 283-288). For instance, Syk can be activated by TNFa,
resulting in MAPK phosphorylation and NF-kB translocation in hematopoietic cell lines
(Takada Y and Aggarwal BB (2004), J. Immunol., 173, 1066-1077). IL-l-induced
chemokine production in fibroblasts of nasal polyps is also mediated by Syk activation
(Yamada T et al (2001), J. Immunol., 167, 283-288). Syk has emerged as a potential
therapeutic target for treatment of allergic and autoimmune disorders.
Detailed description
Existing compounds active on protein kinases are not always endowed with satisfactory
properties such as potency and selectivity. Additionally, existing compounds active on
protein kinases are not always endowed with satisfactory in vivo bioavailability. The
present disclosure discloses compounds that display potent and selective inhibitory
activity on wild type and/or mutated protein kinase, in particular wild type and/or
mutated tyrosine kinase, and more particularly Syk. I n particular, the present
disclosure discloses a method and compounds for selectively modulating, regulating,
and/or inhibiting signal transduction mediated by certain native and/or mutant protein
kinase, and in particular tyrosine kinases implicated in a variety of human and animal
diseases such as cell proliferative, metabolic, autoimmune, allergic, and degenerative
disorders. More particularly, these compounds are potent and selective Syk inhibitors.
More in particular, the inventors have discovered that compounds displaying specific
substitutions in oxazole derivatives are potent and selective inhibitors of Syk tyrosine
kinase.
I n a first aspect, the present disclosure relates to compounds of formula (I), which
may represent either free base forms of the substances or pharmaceutically acceptable
salts thereof:
I )
Wherein:
Rl, R2, R3 and R4 are each independently selected from:
hydrogen,
cyano,
CF3,
halogen (selected from F, CI, Br or I),
an alkyl group optionally substituted with an heterocycle,
an alkoxy group optionally substituted with an heterocycle,
a solubilising group,
a heterocycle,
-CO-NRR',
-S0 -NRR',
-NRR',
-NR-CO-R' and
-NR-SO2R' group
wherein R and R are each independently hydrogen or alkyl group;
W is aryl or heteroaryl group, unsubstituted or substituted by one or more (for example
from one to four, such as one or two or three, for example one) substituents selected
from:
cyano,
CF3,
halogen (selected from F, CI, Br or I),
an alkyl group optionally substituted with an heterocycle,
a cycloalkyl group,
an alkoxy group optionally substituted with an heterocycle,
an aryl group,
a heteroaryl group,
a heterocycloalkyl group,
a solubilising group,
-CO-NRR',
-S0 2-NRR',
-NRR',
-NR-CO-R' and
-NR-S0 R' group
wherein R and R' are each independently hydrogen or alkyl group;
X is selected from 0 , S, N(R5), N[C(=0)R6] and (CH )n wherein n is 0, 1 or 2, R5 and
R6 are each independently H or Cl-4alkyl group;
Y is (CH2)m wherein m is 1, 2, 3 or 4;
Z is (CH2)p wherein p is 1 or 2.
The present disclosure discloses compounds wherein X may be (CH2)n, n may be 0, 1
or 2 and m and p may be 1. For example, n is 0 and m and p are 1 (thereby obtaining
a cyclopropyl). Alternatively, n is 1 and m and p are 1 (thereby obtaining a cyclobutyl).
Alternatively, n is 2 and m and p are 1 (thereby obtaining a cyclopentyl).
The present disclosure discloses compounds wherein W may be a substituted, such as
monosubstituted, heteroaryl or a substituted, such as monosubstituted, aryl. For
example, W is as monosubstituted heteroaryl.
When W is a heteroaryl, the heteroaryl may be a 5-8 membered monocyclic ring. That
ring may contain at least one, such as from one to three, for example one or two
nitrogen atoms. For example, the heteroaryl is pyrimidine such as pyrimidin-2-yl. An
example of W is 4-substituted pyrimidin-2-yl.
The present disclosure discloses compounds wherein each of the substituents of W
may be independently selected from the group consisting of cyano, CF3, halogen, an
alkyl group optionally substituted with a heterocycle (such as an unsubstituted C1-C3
alkyl, for example methyl, ethyl, propyl), a cycloalkyl group, an alkoxy group optionally
substituted with an heterocycle, an aryl group (for example phenyl), an heteroaryl
group (for example thiophene or pyridine), and an heterocycloalkyl group (for example
morpholine). For example, each substituent of W may be independently selected from
the group consisting of cyano, CF3, an alkyl group optionally substituted with a
heterocycle (such as an unsubstituted C1-C3 alkyl, for example methyl, ethyl, propyl),
an aryl group (for example phenyl), an heteroaryl group (for example thiophene or
pyridine), and an heterocycloalkyl group (for example morpholine). For example, each
substituent of W may independently be an alkyl group optionally substituted with a
heterocycle (such as an unsubstituted C1-C3 alkyl, for example methyl, ethyl and
propyl). An example of W is 4-(Cl-3)alkyl pyrimidin-2-yl.
The present disclosure discloses compounds wherein Rl, R2, R3 and R4 may each be
independently selected from the group consisting of hydrogen, halogen, an alkyl group
optionally substituted with a heterocycle, an alkoxy group optionally substituted with a
heterocycle and a solubilising group. For example, at least three of Rl, R2, R3 and R4
are hydrogen. For example R3 and R4 are hydrogen, one of Rl and R2 is hydrogen
and the other is selected from the group consisting of hydrogen, halogen, an alkyl
group optionally substituted with a heterocycle and an alkoxy group optionally
substituted with a heterocycle. For example, Rl, R2, R3 and R4 are all hydrogen.
The present disclosure discloses compounds of the following formula (II) or a
pharmaceutically salt thereof:
( )
Wherein W, Rl, R2, R3, R4 and X are as defined above. For example, W, Rl, R2, R3,
R4 are as defined above, X is (CH2)n and n is 0, 1 or 2, such as 0. For example, in
compounds of formula (II), at least three of Rl to R4, for example each one of Rl to
R4 is hydrogen, W is a monosubstituted aryl or a monosubstituted heteroaryl, such as
monosubstituted heteroaryl, X is (CH )n and n is 0, 1 or 2, such as 0.
The present disclosure discloses compounds of the following formula (III) or a
pharmaceutically salt thereof:
(III)
Wherein Rl, R2, R3, R4 and X are as defined above and R7 is selected from the group
consisting of:
hydrogen,
cyano,
CF3,
halogen (selected from F, CI, Br or I),
an alkyl group,
a cycloalkyl group,
an alkoxy group,
an aryl group,
a heteroaryl group,
a heterocycloaikyl group,
a solubilising group and
-N R' group wherein R and R' are each independently selected from hydrogen or alkyl
group.
For example, in compounds of formula (III), Rl to R4 and R7 are as defined above, X
is (CH )n and n is 0, 1 or 2, such as 0. For example, Rl to R4 are as defined above, R7
is an alkyl group (such as C1-C3 alkyl, for example methyl, ethyl or propyl), X is (CH2)n
and n is 0, 1 or 2, such as 0. For example R3 and R4 are hydrogen, one of Rl and R2
is hydrogen and the other is selected from the group consisting of hydrogen, halogen,
an alkyl group optionally substituted with a heterocycle and an alkoxy group optionally
substituted with a heterocycle, X is (CH2)n, n is 0, 1 or 2, such as 0, and R7 is C1-C3
alkyl, for example methyl, ethyl or propyl.
Unless otherwise specified, the below terms used herein are defined as follows.
As used herein, the term "alkyl" or "alkyl group" means a saturated straight chain or
branched non-cyclic hydrocarbon. Unless otherwise indicated, alkyl groups may have
from 1 to 10, such as from 1 to 6, or from 1 to 4 carbon atoms, for example from 1 to
3 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, npropyl,
n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl; while
saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-
methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-
methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-
dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-
dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-
dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-
ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-
ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-
diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl and 3,3-diethylhexyl. Alkyl groups
included in compounds of this invention may be unsubstituted or substituted with one
or more (for example from one to five, such as one) substituents. An optional
substituent may be a solubilising group.
As used herein, the term "aryl" or "aryl group" means a monocyclic or polycyclicaromatic
hydrocarbon radical. Unless otherwise indicated, aryl groups may have from 6
to 14 carbon atoms. Examples of suitable aryl groups include phenyl, tolyl,
anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused
carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. An aryl group can be
unsubstituted or substituted with one or more (for example from one to five, such as
from one to four, for example one or two or three) substituents. The optional
substituent may be a solubilising group.
The term "cycloalkyl" or "cycloalkyl group" means a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly. This includes substituted
or unsubstituted cycloalkyl groups. For example, cycloalkyl group may be a C3-C10
cycloalkyl group, such as C3 or C4 cycloalkyl group, such as a cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl group.
As used herein, the term "alkoxy" or "alkoxy group" refers to an alkyl group as defined
above which is attached to another moiety by an oxygen atom. Examples of alkoxy
groups include methoxy, isopropoxy, ethoxy, tert-butoxy.
As used herein, the term "heterocycle" refers collectively to heterocycloalkyl groups
and heteroaryl groups.
As used herein, the term "heterocycloalkyl" or "heterocycloalkyl group" means a
monocyclic or polycyclic group having at least one (for example from one to five, such
as one or two or three or four) heteroatom selected from 0 , N or S, and which may be
saturated or unsaturated, but is not aromatic. A heterocycloalkyl may have from 2 to
11 carbon atoms. Examples of heterocycloalkyl groups including: piperidinyl,
piperazinyl, N-methylpiperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
4-piperidonyl, pyrrolidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,
tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyrindinyl, tetrahydropyrimidinyl,
tetrahydrothiopyranyl sulfone, tetrahydrothiopyranyl sulfoxide, morpholinyl,
thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane,
tetrahydrofuranyl, dihydrofuranyl-2-one, tetrahydrothienyl, and tetrahydro-1,1-
dioxothienyl. Typically, monocyclic heterocycloalkyl groups have 3 to 7 ring atoms.
Preferred 3 to 7 membered monocyclic heterocycloalkyl groups have 5 or 6 ring atoms.
A heteroatom may be substituted with a protecting group known to those of ordinary
skill in the art, for example, the hydrogen on a nitrogen may be substituted with a tertbutoxycarbonyl
group. Furthermore, heterocycloalkyl groups may be unsubstituted or
substituted with one or more (such as from one to four, such as one or two)
substituents. In addition, the point of attachment of a heterocyclic ring to another
group may be at either a carbon atom or a heteroatom of a heterocyclic ring.
As used herein, the term "heteroaryl" or "heteroaryl group" means a monocyclic or
polycyclic heteroaromatic ring comprising carbon atom ring members and one or more
heteroatom ring members (such as, for example, oxygen, sulfur or nitrogen). Typically,
heteroaryl groups may have from 5 to 14, such as from 5 to 8 ring members. Typically,
a heteroaryl group has from 1 to 5, such as one or two or three or four, heteroatom
ring members. Typically may have from 1 to about 14 carbon atom ring members.
Representative heteroaryl groups include pyridyl, 1-oxo-pyridyl, furanyl,
benzo[l,3]dioxolyl, benzo[l,4]dioxinyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl,
isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl,
imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl,
benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl,
purinyl, pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl, imidazo[l,2-a]pyridyl, and
benzo(b)thienyl. A heteroatom may be substituted with a protecting group known to
those of ordinary skill in the art, for example, the hydrogen on nitrogen may be
substituted with a tert-butoxycarbonyl group. Heteroaryl groups may be unsubstituted
or substituted with one or more substituents. In addition, nitrogen or sulfur
heteroatom ring members may be oxidized. The heteroaromatic ring may be a 5-8
membered monocyclic heteroaryl ring. The point of attachment of a heteroaromatic or
heteroaryl ring to another group may be at either a carbon atom or a heteroatom of
the heteroaromatic or heteroaryl rings.
As used herein the term "substituent" or "substituted" means that a hydrogen radical
on a compound or group is replaced with any desired group that is substantially stable
to reaction conditions in an unprotected form or when protected using a protecting
group. Examples of substituents are those found in the exemplary compounds and
embodiments disclosed herein, as well as halogen, alkyl or aryl groups as defined
above, hydroxyl, alkoxy as defined above, nitro, thiol, heterocycloalkyl groups,
heteroaryl groups, cyano, cycloalkyl groups as defined above, as well as a solubilising
group, -NRR', -NR-CO-R', -CONRR', -S0 2NRR' group wherein R and R' are each
independently hydrogen or alkyl as defined above. Examples of substituents are
halogen, C1-C10 unsubstituted alkyl, C6-C14 unsubstituted aryl, hydroxyl, C1-C10
unsubstituted alkoxy, nitro, thiol, unsubstituted 3-7 membered heterocycloalkyl,
unsubstituted 3-7 membered heteroaryl, cyano, C1-C10 unsubstituted cycloalkyl, a
solubilising group, -NRR', -NR-CO-R', -CONRR', -S0 2NRR' group wherein R and R' are
each independently hydrogen or C1-C10 unsubstituted alkyl.
As used herein, the term "solubilising" group means a group which has a hydrophilic
character sufficient to improve or increase the water-solubility of the compound in
which it is included, as compared to an analog compound that does not include the
group. The hydrophilic character can be achieved by any means, such as by the
inclusion of functional groups that ionize under the conditions of use to form charged
moieties (e.g., carboxylic acids, sulfonic acids, phosphoric acids, amines, etc.); groups
that include permanent charges (e.g., quaternary ammonium groups); and/or
heteroatoms or heteroatomic groups.
Examples of "heteroatomic groups" are N-(CH )zR", N-(CH2)z-C(0)R", N-(CH2)z-
C(0)OR", N-(CH )z-S(0) 2R", N-(CH2)z-S(0) OR", N-(CH )z-C(0)NR"R"', where z is an
integer ranging from 0 to 6, such as 0 or 1 or 2 or 3 or 4 or 5 or 6, R" and R'" are each
independently selected from the group consisting of:
hydrogen,
a C1-C10 alkyl group which is optionally substituted with one or more hetereoatoms
such as halogen (selected from F, CI, Br or I), oxygen, and nitrogen,
a C1-C10 alkoxy group,
an unsubstituted aryl, and
an unsubstituted heteroaryl group.
The solubilising group may be a moiety having one of the following structures:
wherin
L is selected from the group consisting of CH and N,
M is selected from the group consisting of -CH(R")-, -CH -, -0-, -S-, -NH-, -IN(-(CH2)z-
R")-, -N(-(CH )z-C(0)R")-, -N(-(CH2)z-C(0)OR")-, -N(-(CH )z-S(0) R")-, -N(-(CH )z-
S(0) 2OR")- and -N(-(CH2)z-C(0)NR"R'")-, where z is an integer ranging from 0 to 6, R"
and R'" are each independently selected from:
hydrogen,
a C1-C10 alkyl group which is optionally substituted with one or more hetereoatoms
such as halogen (selected from F, CI, Br or I), oxygen, and nitrogen,
a C1-C10 alkoxy group,
an unsubstituted aryl, and
an unsubstituted heteroaryl,
or the group -NR"R"' is a group -NRR group wherein Ra and Rb are each independently
selected from hydrogen or unsubstituted alkyl;
with the proviso that L and are not both simultaneously CH and CH2, respectively.
Examples of solubilising groups are morpholinyl, piperidinyl, pyrrolidinyl, N-(C1-C6)alkyl
piperidinyl, in particular N-methyl piperidinyl and N-ethyl piperidinyl, N-(4-
piperidinyl)piperidinyl, 4-(l-piperidinyl)piperidinyl, 1-pyrrolidinylpiperidinyl, 4-
morpholinopiperidinyl, 4-(N-methyl-l-piperazinyl)piperidinyl, piperazinyl, N-(C1-
C6)alkylpiperazinyl, in particular N-methyl piperazinyl and N-ethyl piperazinyl, N-(C3-
C6)cycloalkyl piperazinyl, in particular N-cyclohexyl piperazinyl, pyrrolidinyl, N-(C1-
C6)alkyl pyrrolidinyl, in particular N-methyl pyrrolidinyl and N-ethyl pyrrolidinyl,
diazepinyl, N-(C1-C6)alkyl azepinyl, in particular N-methyl azepinyl and N-ethyl
azepinyl, homopiperazinyl, N-methyl homopiperazinyl, N-ethyl homopiperazinyl,
imidazolyl.
The compounds of formula (I) may be used in the form of salts derived from
pharmaceutically acceptable inorganic or organic acids. Unless otherwise indicated,
"pharmaceutically acceptable salt" refers to a salt prepared by combining a compound
of formula (I) with an acid whose anion, or a base whose cation, is generally
considered suitable for human consumption. Pharmaceutically acceptable salts are
particularly useful as products of the methods of the present invention because of their
greater aqueous solubility relative to the parent compound. For use in medicine, the
salts of the compounds of this invention are non-toxic "pharmaceutically acceptable
salts." Salts encompassed within the term "pharmaceutically acceptable salts" refer to
non-toxic salts of the compounds of this invention which are generally prepared by
reacting the free base with a suitable organic or inorganic acid. Suitable
pharmaceutically acceptable acid addition salts of the compounds of the present
invention when possible include those derived from inorganic acids, such as
hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric,
metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as
acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic,
isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic,
succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable organic acids
generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic,
heterocyclic, carboxylic, and sulfonic classes of organic acids. Specific examples of
suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate,
glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate,
glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic
acid, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate
(pamoate), methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate,
toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, b-
hydroxybutyrate, galactarate, galacturonate, adipate, alginate, butyrate, camphorate,
camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate,
glycerophosphate, heptanoate, hexanoate, nicotinate, 2-naphthalesulfonate, oxalate,
palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, and
undecanoate. Furthermore, where the compounds of the invention carry an acidic
moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal
salts, i.e., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or
magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary
ammonium salts. In another embodiment, base salts are formed from bases which
form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine,
diolamine, glycine, lysine, meglumine, olamine, tromethamine and zinc salts. Organic
salts may be made from secondary, tertiary or quaternary amine salts, such as
tromethamine, diethylamine, L/ ,L/'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (/V-methylglucamine), and procaine.
Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl
(CrCe) halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides),
dialkyl sulfates (i.e., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides
(e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl
halides (e.g., benzyl and phenethyl bromides), and others. Hemisalts of acids and
bases may also be formed, for example, hemisulfate and hemicalcium salts.
Unless otherwise indicated, the language "compounds of formula (I)" include all forms
of the compound of formula I, including hydrates, solvates isomers, crystalline and
non-crystalline forms, isomorphs, polymorphs, and metabolites thereof. For example,
the compounds of formula (I), or pharmaceutically acceptable salts thereof, may exist
in unsolvated and solvated forms. When the solvent or water is tightly bound, the
complex will have a well-defined stoichiometry independent of humidity. When,
however, the solvent or water is weakly bound, as in channel solvates and hygroscopic
compounds, the water/solvent content will be dependent on humidity and drying
conditions. In such cases, non-stoichiometry will be the norm. Stereoisomers of the
compounds of formula (I) include cis and trans isomers, optical isomers such as R and
S enantiomers, diastereomers, geometric isomers, rotational isomers, conformational
isomers, and tautomers of the compounds of the invention, including compounds
exhibiting more than one type of isomerism; and mixtures thereof (such as racemates
and diastereomeric pairs). Unless otherwise indicated, the language "compounds of
formula (I)" include the tautomeric forms of compounds. Where structural isomers are
interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can
occur. This can take the form of proton tautomerism in compounds of the invention
containing, for example, an imino, keto, or oxime group, or so-called valence
tautomerism in compounds which contain an aromatic moiety. It follows that a single
compound may exhibit more than one type of isomerism. The various ratios of the
tautomers in solid and liquid form is dependent on the various substituents on the
molecule as well as the particular crystallization technique used to isolate a compound.
The compounds of the present invention may be prepared using the general protocols
as follows:
General Synthetic Procedures
Compounds of the invention can be prepared by several methods including methods
outlined in Schemes 1-4, wherein the substituents are as defined in formula (I) above,
except where further noted. The synthetic methods described below are merely
exemplary, and the compounds of the invention may be synthesized by alternate
routes as appreciated by persons of ordinary skill in the art.
Piperazinones (2) (Scheme 1) were prepared by reacting ethylene diamine with 2-
bromo ester (1) using the method described by A. Benjahad et al {Tetrahedron Letters,
(1994), 9545-9548).
1 2
Scheme 1
The piperazinones (2) may alternatively be prepared via /V-Nosyaziridine (4) according
to the protocol outlined in Scheme 2. /V-Nosyaziridine intermediate (4) is prepared from
2-chloroethylamine hydrochloride by first reacting with /?-nitrosulfonyl chloride to give
the /V-Nosylamine (3) which is subsequently cyclised with potassium hydroxide to
afford (4) using a method adapted from Iwaki et al {Bioorganic & Medicinal Chemistry
Letters, (2012), 2798-2802). Ring-opening with an amino acid ethyl ester
hydrochloride (5) gives the acyclic aminoester (6) which is cyclised in 2 steps: Ndeprotection
with thiophenol then heating to afford piperazinones (2) as described by
Maligres et al {Tetrahedron Letters, (1997), 5253-5256).
Scheme 2
Aromatic aldehydes (7) (Scheme 3) were reacted with ^toluenesulfonylmethyl
isocyanide (TosMIC) to prepare the corresponding 5-arylsubstitued oxazoles (8) using
the method of Van Leusen et al {Tetrahedron Letters, (1972), 2369-2372). The noncommercial
aldehydes (7) were prepared using literature methods. Deprotonation of
the oxazole moiety (8) by a suitable organic base such as lithium hexamethyldisilazide
(LiHMDS) and subsequent electrophilic chlorination was used to prepare the 2-
chlorooxazole coumpounds (9). This allowed access to compounds (10) by substitution
of the chloride by sustituted piperazinones (2). This substitution was performed either
by heating in the presence of solvent such as isopropanol or heating under solvent-free
conditions. In certain cases and in the presence of solvent, compounds (10) can be
obtained by using an acid such as hydrochloric acid. Nitro compound (10) is reduced to
form the corresponding aniline (11). Preferably, the reduction reaction is performed in
the presence of hydrogen with a catalyst, such as a palladium on carbon 10% by wt..
Compounds (11) were used to prepare further analogues (12) of formula (I) by a
direct nucleophilic displacement reaction in the presence of a suitable solvent such as
alcohol and with heating in elevated temperature, where X of W-X can be F, I , Br or CI.
Presence of an acid such as hydrochloric acid may or may not be necessary to drive
the reaction to completion or to obtain improved yields. In certain cases compounds
(12) can be obtained by using known metal-catalysed /V-arylation protocols with a
suitable combination of ligand and inorganic base.
Scheme 3
Following the reaction scheme depicted in Scheme 4, compounds (12) of formula (I)
were obtained by using the same protocols described above.
Scheme 4
In a second aspect, the present disclosure discloses a pharmaceutical composition
comprising a compound of formula (I) as defined above, such as a compound of
formula (II) or (III), or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient and/or carrier. In the pharmaceutical
composition, a compound of formula (I) may be the sole pharmaceutically active
ingredient or it may be combined with one or more distinct pharmaceutically active
ingredients.
Suitable carriers and excipients are widely known in the art and are commonly used for
example to facilitate the processing of the active compounds into preparations which
can be used pharmaceutically. Further details on techniques for formulation and
administration may be found in the latest edition of Remington's Pharmaceutical
Sciences (Maack Publishing Co., Easton, Pa.).
Various forms of excipients can be used depending on the desired mode of
administration and some of them can improve or tailor the effectiveness of the active
compound, e.g. by promoting a release profile rendering this active compound overall
more effective for the treatment desired. The pharmaceutical compositions of the
invention are suitable to be administered in various forms, for example in an injectable,
pulverizable or ingestible form, for example via the intramuscular, intravenous,
subcutaneous, intradermal, oral, topical, rectal, vaginal, ophthalmic, nasal, transdermal
or parenteral route.
The pharmaceutical composition presently disclosed may be intended for oral
administration. In this case, the composition may be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
The compositions presently disclosed may be a pharmaceutical or cosmetic
composition. They may be intended for topical administration. Such compositions may
be presented in the form of a gel, paste, ointment, cream, lotion, liquid suspension,
aqueous-alcoholic or oily solutions, or dispersions of the lotion or serum type, or
anhydrous or lipophilic gels, or emulsions of liquid or semi-solid consistency of the milk
type, obtained by dispersing a fatty phase in an aqueous phase or vice versa, or of
suspensions or emulsions of soft, semi-solid consistency of the cream or gel type, or
alternatively of microemulsions, of microcapsules, of microparticles or of vesicular
dispersions to the ionic and/or nonionic type. These compositions may be prepared
according to standard methods.
The compositions presently defined may comprise any ingredient commonly used in
dermatology and cosmetics. It may comprise at least one ingredient selected from
hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents,
preservatives, emollients, viscosity enhancing polymers, humectants, surfactants,
preservatives, antioxidants, solvents, perfumes, fillers, screening agents, bactericides,
odor absorbers and coloring matter. As oils which can be used in the invention, mineral
oils (liquid paraffin), vegetable oils (liquid fraction of shea butter, sunflower oil), animal
oils, synthetic oils, silicone oils (cyclomethicone) and fluorinated oils may be
mentioned. Fatty alcohols, fatty acids (stearic acid) and waxes (paraffin, carnauba,
beeswax) may also be used as fatty substances. Emulsifiers which can be used in the
invention include, for example, glycerol stearate, polysorbate 60 and the PEG-6/PEG-
32/glycol stearate mixture. Hydrophilic gelling agents which can be used in the
invention include, for example, carboxyvinyl polymers (carbomer), acrylic copolymers
such as acrylate/alkylacrylate copolymers, polyacrylamides, polysaccharides such as
hydroxypropylcellulose, clays and natural gums., Lipophilic gelling agents which can be
used in the invention include, for example modified clays such as bentones, metal salts
of fatty acids such as aluminum stearates and hydrophobic silica, or alternatively
ethylcellulose and polyethylene. As hydrophilic active agents, proteins or protein
hydrolysates, amino acids, polyols, urea, allantoin, sugars and sugar derivatives,
vitamins, starch and plant extracts, in particular those of Aloe Vera may be used. As
lipophilic active, agents, retinol (vitamin A) and its derivatives, tocopherol (vitamin E)
and its derivatives, essential fatty acids, ceramides and essential oils may be used.
These agents add extra moisturizing or skin softening features when utilized. In
addition, a surfactant can be included in the composition so as to provide deeper
penetration of the compound capable of depleting mast cells, such as a tyrosine kinase
inhibitor. Among the contemplated ingredients, one may chose penetration enhancing
agents selected for example from the group consisting of mineral oil, water, ethanol,
triacetin, glycerin and propylene glycol; cohesion agents selected for example from the
group consisting of polyisobutylene, polyvinyl acetate and polyvinyl alcohol, and
thickening agents. Chemical methods of enhancing topical absorption of drugs are well
known in the art. For example, compounds with penetration enhancing properties
include sodium lauryl sulfate (Dugard, P. H. and Sheuplein, R. 1 , "Effects of Ionic
Surfactants on the Permeability of Human Epidermis: An Electrometric Study," J. Ivest
Dermatol., V.60, (1973), pp. 263-69), lauryl amine oxide (Johnson et a/, US
4,411,893), azone (Rajadhyaksha, US 4,405,616 and 3,989,816) and decylmethyl
sulfoxide (Sekura, D. L. and Scala, J., "The Percutaneous Absorption of Alkylmethyl
Sulfides," Pharmacology of the Skin, Advances In Biology of Skin, (Appleton-Century
Craft) V. 12, (1972), pp. 257-69). It has been observed that increasing the polarity of
the head group in amphoteric molecules increases their penetration-enhancing
properties but at the expense of increasing their skin irritating properties (Cooper, E. R.
and Berner, B., "Interaction of Surfactants with Epidermal Tissues: Physiochemical
Aspects," Surfactant Science Series, V. 16, Reiger, . . ed. (Marcel Dekker, Inc.),
(1987), pp. 195-210). Chemical enhancers may also be co-solvents. These materials
are absorbed topically relatively easily, and, by a variety of mechanisms, achieve
permeation enhancement for some drugs. Ethanol (Gale et a/f US Pat. No. 4,615,699
and Campbell et al., US Pat. Nos. 4,460,372 and 4,379,454), dimethyl sulfoxide (US
3,740,420 and US 3,743,727, and US 4,575,515), and glycerin derivatives (US
4,322,433) are a few examples of compounds which have shown an ability to enhance
the absorption of various compounds.
The pharmaceutical compositions presently disclosed can also be intended for
administration with aerosolized formulation to target areas of a patient's respiratory
tract. Devices and methodologies for delivering aerosolized bursts of a formulation of a
drug is disclosed in US 5,906,202. Formulations are preferably solutions, e.g. aqueous
solutions, ethanolic solutions, aqueous/ethanolic solutions, saline solutions, colloidal
suspensions and microcrystalline suspensions. For example aerosolized particles
comprise the active ingredient mentioned above and a carrier, (e.g., a
pharmaceutically active respiratory drug and carrier) which are formed upon forcing
the formulation through a nozzle which nozzle is preferably in the form of a flexible
porous membrane. The particles have a size which is sufficiently small such that when
the particles are formed they remain suspended in the air for a sufficient amount of
time such that the patient can inhale the particles into the patient's lungs. Suitable
devices for the administration of the present compounds to a patient's respiratory tract
are discussed for example in US 5,556,611:
- liquid gas systems (a liquefied gas is used as propellant gas e.g. low-boiling FCHC or
propane, butane in a pressure container),
- suspension aerosol (the active substance particles are suspended in solid form in the
liquid propellant phase),
- pressurized gas system (a compressed gas such as nitrogen, carbon dioxide,
dinitrogen monoxide, or air is used.
Thus, the pharmaceutical composition presently disclosed is made in that the active
substance is dissolved or dispersed in a suitable nontoxic medium and said solution or
dispersion atomized to an aerosol, i.e. distributed extremely finely in a carrier gas. This
is technically possible for example in the form of aerosol propellent gas packs, pump
aerosols or other devices known per se for liquid misting and solid atomizing which in
particular permit an exact individual dosage. Therefore, to the present disclosure also
discloses aerosol devices comprising a compound as defined above and such a
formulation, preferably with metered dose valves.
The pharmaceutical compositions presently disclosed can also be intended for
intranasal administration. In this regard, pharmaceutically acceptable carriers for
administering the compound to the nasal mucosal surfaces will be readily appreciated
by the ordinary artisan. These carriers are described in the Remington's Pharmaceutical
Sciences" 16th edition, (1980), Ed. By Arthur Osol, the disclosure of which is
incorporated herein by reference.
For administration via the upper respiratory tract, the composition can be formulated
into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a
suspension, for intranasal administration as drops or as a spray. Preferably, such
solutions or suspensions are isotonic relative to nasal secretions and of about the same
pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers
should be physiologically compatible and include, simply by way of example, phosphate
buffers. For example, a representative nasal decongestant is described as being
buffered to a pH of about 6.2 (Remington's, Id. at page 1445). Of course, the ordinary
artisan can readily determine a suitable saline content and pH for an innocuous
aqueous carrier for nasal and/or upper respiratory administration. Common intranasal
carriers include nasal gels, creams, pastes or ointments with a viscosity of, e.g., from
about 10 to about 3000 cps, or from about 2500 to 6500 cps, or greater, may also be
used to provide a more sustained contact with the nasal mucosal surfaces. Such carrier
viscous formulations may be based upon, simply by way of example, alkyicelluloses
and/or other biocompatible carriers of high viscosity well known to the art (see e.g.,
Remington's, cited supra. A preferred alkylcellulose is, e.g., methylcellulose in a
concentration ranging from about 5 to about 1000 or more mg per 100 ml of carrier. A
more preferred concentration of methyl cellulose is, simply by way of example, from
about 25 to about 150 mg per 100 ml of carrier. Other ingredients, such as known
preservatives, colorants, lubricating or viscous mineral or vegetable oils, perfumes,
natural or synthetic plant extracts such as aromatic oils, and humectants and viscosity
enhancers such as, e.g., glycerol, can also be included to provide additional viscosity,
moisture retention and a pleasant texture and odor for the formulation. For nasal
administration of solutions or suspensions, various devices are available in the art for
the generation of drops, droplets and sprays.
A premeasured unit dosage dispenser including a dropper or spray device containing a
solution or suspension for delivery as drops or as a spray is prepared containing one or
more doses of the drug to be administered. Also disclosed is a kit containing one or
more unit dehydrated doses of a compound of formula (I) as presently disclosed,
together with any required salts and/or buffer agents, preservatives, colorants and the
like, ready for preparation of a solution or suspension by the addition of a suitable
amount of water.
Another aspect of the present disclosure is directed to a compound of formula (I) as
defined above, such as a compound of formula (II) or (III), or a pharmaceutically
acceptable salt thereof, for use as a medicament.
The compounds of formula (I) or pharmaceutically salts thereof as presently disclosed
(also jointly referred to as "compounds of formula (I)") are endowed with Syk tyrosine
kinase inhibiting activity. I n particular, they may inhibit (thereby regulating) the signal
transduction mediated by Syk.
Accordingly, in one aspect the present disclosure discloses a method for treating a
disease or disorder associated with unregulated or deregulated Syk activity, said
method comprising administering an effective amount of a compound of formula (I) to
a subject (such as a human or animal subject) in need of such treatment. For example,
the present disclosure discloses a method for treating a disease or disorder associated
with signal transduction mediated by SYK, the present disclosure discloses a method
for treating a disease or disorder associated.
Effective amounts of the compounds of formula (I) are generally comprised between
0.1 mg and 2 g of the compound per day and per kilogram of body weight.
In another aspect, the present disclosure discloses a method for modulating,
regulating, and/or inhibiting, in cells, the signal transduction mediated by Syk protein
kinase. Said method comprises administering to cells at least one compound of formula
(I) as defined above, such as a compound of formula (II) or (III), or a
pharmaceutically acceptable salt thereof.
The present disclosure discloses the use of at least one compound of formula (I) or a
pharmaceutically acceptable salt thereof, for the in vitro or in vivo selective inhibition
of Syk.
The methods presently disclosed may be for treating a hematological, an inflammatory,
an autoimmune, a proliferative, a metabolic, an allergic and/or degenerative disease or
disorder in a patient.
In one embodiment, said subject or patient has been diagnosed as having
hematological disorders, allergic disorders, metabolic disorders, inflammatory
disorders, autoimmune disorders and/or proliferative disorders.
Diseases and disorders known to be associated with unregulated or deregulated signal
transduction mediated by Syk are for example:
hematological disorders such as Non-Hodgkin Lymphoma and leukemia
including Diffuse large B-cell lymphoma (DLBCL) Follicular lymphoma (FL), Mantle cell
lymphoma (MCL), B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic
lymphoma (SLL), Waldenstrom's macroglbulinemia (WM), Marginal zone lymphoma
(MZL), Burkitt lymphoma and peripheral T-cell lymphomas (PTCL), as well as multiple
myeloma (MM), myelodysplatic syndrome (MDS), myelodysplasia with myelofibrosis,
neoplastic diseases such as mastocytosis, solid tumours including head and
neck cancer, hepatocellular carcinoma, and human gastrointestinal disorders.
metabolic diseases such diabetes mellitus and its chronic complications, obesity,
diabetes type II, hyperlipidemias and dyslipidemias, atherosclerosis; hypertension and
cardiovascular disease.
allergic diseases such as asthma, allergic rhinitis, allergic sinusitis, anaphylactic
syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis,
erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis and insect
bite skin inflammation and blood sucking parasitic infestation.
- bone resorption (osteoporosis).
angiogenesis
inflammatory diseases such as rheumatoid arthritis, conjunctivitis, rheumatoid
spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions.
autoimmune diseases such as multiple sclerosis, psoriasis, intestine
inflammatory disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis and
polyarthritis, local and systemic scleroderma, systemic lupus erythematosus, discoid
lupus erythematosus, cutaneous lupus, dermatomyositis, polymyositis, Sjogren's
syndrome, nodular panarteritis, autoimmune enteropathy, as well as proliferative
glomerulonephritis and T-cell mediated autoimmune diabetes.
- graft-versus-host disease or graft rejection for allogeneic hematopoietic cell
transplantation for the treatment of leukemia and lymphoma, cardiac allograft and in
any organ transplantation such as kidney, pancreas, liver, and lung.
Other autoimmune diseases embraced by the invention include active chronic
hepatitis and chronic fatigue syndrome.
- vasculitis.
viral infection.
fungal infection.
bacterial infection.
CNS disorders such as Nasu-Hakola disease, psychiatric disorders, migraine,
pain, memory loss and nerve cells degeneracy. More particularly, the method according
to the invention is useful for the treatment of the following disorders: depression
including dysthymic disorder, cyclothymic disorder, bipolar depression, severe or
"melancholic" depression, atypical depression, refractory depression, seasonal
depression, anorexia, bulimia, premenstrual syndrome, post-menopause syndrome,
other syndromes such as mental slowing and loss of concentration, pessimistic worry,
agitation, self-deprecation, decreased libido, pain including, acute pain, postoperative
pain, chronic pain, nociceptive pain, cancer pain, neuropathic pain, psychogenic pain
syndromes, anxiety disorders including anxiety associated with hyperventilation and
cardiac arrhythmias, phobic disorders, obsessive-compulsive disorder, post-traumatic
stress disorder, acute stress disorder, generalized anxiety disorder, psychiatric
emergencies such as panic attacks, including psychosis, delusional disorders,
conversion disorders, phobias, mania, delirium, dissociative episodes including
dissociative amnesia, dissociative fugue and dissociative identity disorder,
depersonalization, catatonia, seizures, severe psychiatric emergencies including suicidal
behaviour, self-neglect, violent or aggressive behaviour, trauma, borderline personality,
and acute psychosis, schizophrenia including paranoid schizophrenia, disorganized
schizophrenia, catatonic schizophrenia, and undifferentiated schizophrenia,
- neurodegenerative diseases including Alzheimer's disease, Parkinson's disease,
Huntington's disease, the prion diseases, Motor Neurone Disease (MND), and
Amyotrophic Lateral Sclerosis (ALS).
Cerebral ischemia
Retinal ischemia
- Ischemic stroke
Fibrosis.
Hematological malignancies may be non-Hodgkin lymphoma (NHL) including BCLL/
SLL, DLBCL, FL, MCL and WM, peripheral T-cell lymphoma and myelodysplastic
syndromes (MDS). Proliferative disorder may be cancer. Autoimmune disorders may be
multiple sclerosis, psoriasis, intestine inflammatory disease, ulcerative colitis, Crohn's
disease, rheumatoid arthritis and polyarthritis, local and systemic scleroderma,
systemic lupus erythematosus, discoid lupus erythematosus, cutaneous lupus,
dermatomyositis, polymyositis, Sjogren's syndrome, nodular panarteritis, autoimmune
enteropathy, atopic dermatitis and/or proliferative glomerulonephritis. Allergic diseases
may be asthma, allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria,
angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum,
erythema multiforme, cutaneous necrotizing venulitis and insect bite skin inflammation
and/or blood sucking parasitic infestation. Neurologic diseases may be Huntington's
disease, schizophrenia, Parkinson's disease and/or Alzheimer's disease.
I n one particular embodiment, the methods presently disclosed may be for preventing
or treating a disease or disorder selected form rheumatoid arthritis, asthma, multiple
sclerosis, atopic dermatitis, Crohn's disease, interstitial cystitis, ankylosing spondylitis,
chronic obstructive pulmonary disease, psoriasis, mastocytosis, B-cell malignancies,
colorectal carcinoma, lung cancer, gastric carcinoma, glioblastoma, gastrointestinal
stromal tumor (GIST), melanoma, breast cancer, triple negative breast cancer, gastric
carcinoma, oesogastric carcinoma, pancreatic cancer, prostate cancer, multiple
myeloma, T-cell lymphoma, head and neck cancer, hepatocellular carcinoma, Hodgkin
lymphoma, ischemic hepatitis, hepatitis B, amyotrophic lateral sclerosis (ALS),
Parkinson's disease, muscular dystrophy (de Duchene), progressive supranuclear palsy
(PSP), cerebral ischemia, addiction, cocaine addiction, depression, mood disorders
associated to major depression or dysthymic disorder, and Alzheimer's disease.
A compound of formula (I), such as a compound of formula (II) or (III), or a
pharmaceutically acceptable salt thereof may be used for treating a disease or disorder
disclosed above such as hematological disorders, proliferative disorders, autoimmune
disorders, metabolic disorders, inflammatory disorders and/or allergic disorders.
In the methods presently disclosed, the compound of formula (I) or a pharmaceutically
acceptable salt thereof, may be used as sole active pharmaceutical ingredient or in
combination with another active pharmaceutical ingredient.
The present disclosure discloses a method for preventing or treating a disease or
disorder selected form hematological disorders, proliferative disorders, metabolic
disorders, inflammatory disorders, autoimmune disorders and allergic disorders, that
method comprising simultaneously or sequentially administering to a human or animal
subject in need thereof at least one compound of formula (I) or a pharmaceutically
acceptable salt thereof in combination with another active pharmaceutical ingredient,
in sufficient amounts to provide a therapeutic effect.
The present disclosure discloses a pharmaceutical composition comprising a compound
of formula (I) such as a compound of formula (II) or (III), or a pharmaceutically
acceptable salt thereof, and another active pharmaceutical agent as a combined
preparation for sequential, simultaneous or separate use in the treatment of a disease
or disorder selected from the group consisting of hematological disorders, proliferative
disorders, autoimmune disorders, inflammatory disorders, and allergic disorders.
The present disclosure discloses the use of a compound of formula (I) such as a
compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof
optionally in combination with another pharmaceutically active agent, for the
manufacture of a medicament for the treatment of a a disease or disorder selected
from the group consisting of a hematological disorder, a proliferative disorder, a
metabolic disorder, an autoimmune disorder, an inflammatory disorder, and an allergic
disorder.
Although methods and uses disclosed above refer to a compound of formula (I), such
as a compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof,
whenever technically compatible they are to be understood to equally refer to
pharmaceutical compositions including the same compounds.
The invention is now illustrated by Examples which represent currently preferred
embodiments which make up a part of the invention but which in no way are to be
used to limit the scope of it.
Examples of Compound Synthesis
The invention will be more fully understood by reference to the following preparative
examples, but they should not be construed as limiting the scope of the invention.
General: All chemicals used were commercial reagent grade products. Solvents were of
anhydrous commercial grade and were used without further purification. The progress
of the reactions was monitored by thin layer chromatography using precoated silica gel
60F 254, Merck TLC plates, which were visualized under UV light. Multiplicities in
NM spectra are indicated as singlet (s), broad singlet (br s), doublet (d), triplet (t),
quadruplet (q), and multiplet (m) and the NMR spectrum were performed either on a
Bruker 300 or 400 MHz spectrometer. Liquid chromatography-Mass Spectrometry
(LCMS) was run on an Ultra-high performance liquid chromatography (UPLC) ACQUITY
Waters instrument coupled to a TQD mass spectrometer. The gradient used was:
starting at t=0.0min with 5% of CH3CN+0,1% Formic acid in Water+0,1% Formic acid
until t=0.5min ; then a linear gradient from t=0.5min to t=7.0min reaching 100%
CH3CN+0,1% Formic acid ; then staying at this state from t=7.0min until t=10.0min.
The column used was a Waters HSS C18 I .dm i, 2.1 x 50mm. The detection
instrument used was the triple quadrupole mass spectrometer (TQD) using
electrospray ionisation (ESI) in positive mode. Chemical names were generated using
ChemDraw Ultra Version 7.0.1
Abbreviations
CDCI3 Deuterochloroform
Cone. HCI Concentrated hydrochloric acid (37%)
Cs2C0 3 Cesium carbonate
DCM Dichloromethane
D SO- Hexadeuterodimethyl sulfoxide
EtOAc Ethyl acetate
EtOH Ethanol
Et3N Triethylamine
Fe(acac) 3 Tris(acetylacetonato) iron(III)
h Hour(s)
iPrOH 2-Propanol
K2C0 3 Potassium carbonate
KOH Potassium hydroxide
LiHMDS Lithium bis(trimethylsilyl)amide
MeCN Acetonitrile
MeOH Methanol
MgS04 Magnesium sulfate
Mins Minutes
NaCI Sodium Chloride
Na2C0 3 Sodium carbonate
NaHC0 3 Sodium hydrogencarbonate
Ns Nosyl or p-nitrophenylsulfonyl
Pd2(dba) 3 Tris(dibenzylideneacetone)dipalladium(0)
Pd(PPh 3)4 Tetrakis(triphenylphosphine)palladium(0)
RT Room temperature
Si0 2 Silica gel
TosMIC -Toluenesulfonylmethyl isocyanide
THF Tetrahydrofuran
tR Retention time
Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene
Example 001:
Synthetic approach of compound 001
Preparation of 4,7-Diaza-spiro[2.5]octan-8-one (le)
Synthetic approach of intermediate (le)
H,
8h
le lh
Preparation of N-(2-Chloro-ethyl)-4-nitro-benzenesulfonamide (If)
A stirred solution of 2-chloroethylamine hydrochloride (l.OOg, 8.62mmol) and Et 3N
(3.60ml, 25.9mmol) in dry DCM (25ml) at 0°C was treated with a solution of nosyl
chloride (1.91g, 8.62mmol) in dry DCM (25ml) dropwise. On complete addition, the
solution was warmed to ambient temperature and stirred overnight. The solution was
evaporated and the residue purified by column chromatography (Si0 2, 20% EtOAc to
30% EtOAc in cyclohexane) to afford the title compound as a white solid (2.03g, 89%).
H NM (400 MHz, DMSO-d ) d 8.45 - 8.38 (m, 3H), 8.09 - 8.04 (m, 2H), 3.59 (t, J =
6.0 Hz, 2H), 3.17 (s, 2H).
Preparation of l-(4-Nitro-benzenesulfonyl)-aziridine (Ig)
Ns
N
Intermediate Ig was prepared according to the method of Iwaki et al, Bioorganic &
Medicinal Chemistry Letters, (2012), 2798-2802. A stirred slurry of If (2.00g,
7.56mmol) in toluene (100ml) was treated with a solution of KOH (2.54g, 45.3mmol) in
water (12ml) in one portion then stirred at ambient temperature for 3h. The solution
was diluted with EtOAc and the organics separated, washed with saturated aqueous
NaCI, dried (MgS0 ), filtered and evaporated to afford the title compound as a pale
yellow solid (1.41g, 82%). H N R (400 MHz, CDCI3) d 8.47 - 8.26 (m, 2H), 8.24 -
8.10 (m, 2H), 2.48 (s, 4H).
Preparation of l-[2-(4-nitro-benzenesulfonylamino)-ethylamino]-cyclopropane
carboxylic acid ethyl ester (Ih)
A mixture of the Ns-aziridine Ig (6.89g, 30.2mmol), 1-amino-cyclopropanecarboxylic
acid ethyl ester hydrochloride (5.00g, 30.2mmol) and Na2C0 3 (3.20g, 30.2mmol) in dry
acetonitrile (120ml) was heated to reflux for 3h. The mixture was cooled, filtered and
evaporated. The residue was purified by column chromatography (Si0 2, 5% acetone to
10% acetone in DCM) to afford the title compound as a pale yellow solid (6.58g, 61%).
H NMR (400 MHz, DMSO-d6) d 8.41 (d, J = 8.8 Hz, 2H), 8.04 (d, J = 8.8 Hz, 2H), 7.84
(br s, 1H), 4.02 (q, J = 7.1 Hz, 2H), 2.84 (s, 2H), 2.72-2.58 (m, 3H), 1.14 (t, J = 7.1
Hz, 3H), 1.07 (dd, J = 7.0, 3.7 Hz, 2H), 0.81 (dd, J = 7.0, 3.8 Hz, 2H).
Preparation of 4,7-Diaza-spiro[2.5]octan-8-one (Ie)
Prepared largely according to the method of Maligres et al, Tetrahedron Letters,
(1997), 5253-5256. A solution of the protected aminoester Ih (5.45g, 15.3mmol) in dry
acetonitrile (250ml) was treated with K C0 3 (8.95g, 64.8mmol) and thiophenol
(4.96ml, 48.6mmol) and stirred at 50°C overnight. The mixture was evaporated under
vacuum and the residue purified by column chromatography (Si0 2, 10:90:1
EtOH:DCM:NH4OH by volume) to afford the title compound as an off-white solid
(1.14g, 59%). H NMR (400 MHz, DMSO-d6) d 7.53 (s, 1H), 3.26 (s, 2H), 2.84 (s, 3H),
1.01 (dd, J = 6.1, 3.1 Hz, 2H), 0.60 (dd, J = 6.1, 3.1 Hz, 2H).
Preparation of 5-(3-Nitro-phenyl)-oxazole (la)
A solution of 3-nitrobenzaldehyde (15. Og, 99.3mmol) in methanol (400ml) was treated
with TosMIC (21. 3g, 109mmol) and K2C0 3 (16.5g, 119mmol) and heated to reflux for
30mins. The cooled solution was concentrated and treated with water (400ml) to form
copious precipitate and was filtered. The filter cake was washed with water, then the
solid was taken up in EtOAc and dried over MgS0 . The solution was filtered and
evaporated and the resultant solid was dried under vacuum to give the title compound
as a beige solid (18.0g, 95%). H NMR (400 MHz, DMSO-d6) d 8.56 (s, 1H), 8.50 (t, J
= 1.9 Hz, 1H), 8.21 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 8.17 (ddd, = 7.8, 1.6, 1.0 Hz,
1H), 7.99 (s, 1H), 7.78 (t, J = 8.0 Hz, 1H).
Preparation of 3-Oxazol-5-yl-phenylamine (lb)
A solution of intermediate l a (3.52g, 18.5mmol) in absolute ethanol (210ml) was
treated with water (21ml) then SnCI2.2H 0 (20.9g, 92.6mmol) and cone. HCI (15ml,
180mmol). After stirring at room temperature overnight, the solution was taken to pH
7 with 10% aqueous NaOH solution and extracted repeatedly with EtOAc. The organics
were dried (MgS0 4), filtered and evaporated to afford the title compound as a pale
orange powder (2.74g, 93%). H N R (400 MHz, DMSO-d6) d 8.37 (s, 1H), 7.49 (s,
1H), 7.10 (t, J = 7.8 Hz, 1H), 6.90 (t, J = 1.8 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 6.59 -
6.54 (m, 1H), 5.25 (s, 2H).
Preparation of (4-Methyl-pyrimidin-2-yl)-(3-oxazol-5-yl-phenyl)-amine (Ic)
A solution of intermediate l b (l.OOg, 6.24mmol) in 2-propanol (50ml) was treated with
2-chloro-4-methylpyrimidine (800mg, 6.22mmol) and 1.25M HCI solution in ethanol
(7.5ml, 9.38mmol) and heated to reflux for 40h. The solvent was evaporated and the
residue made basic with saturated aqueous NaHC03 and extracted with EtOAc. The
organics were dried (MgS04), filtered and evaporated to afford the title compound as a
beige solid (1.04g, 67%). H NMR (400 MHz, DMSO-d6) d 9.71 (s, 1H), 8.45 (s, 1H),
8.37 (d, J = 4.9 Hz, 1H), 8.23 (s, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.60 (s, 1H), 7.37 (t, J
= 7.8 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H), 6.77 (d, J = 4.9 Hz, 1H), 2.38 (s, 3H).
Preparation of [3-(2-Chloro-oxazol-5-yl)-phenyl]-(4-methyl-pyrimidin-2-yl)-amine (Id)
A solution of intermediate Ic (1.23g, 4.88mmol) in dry THF (60ml) under argon at -
78°C was treated with 1 LiHMDS solution in THF (7.20mmol, 7.20mmol) dropwise.
After 45 mins at -78°C, hexachloroethane (1.39g, 5.87mmol) was added in one portion
and stirring continued for a further 40 mins before warming to RT. The solution was
then cooled to -78°C once more, treated with 1M LiHMDS (7.20ml, 7.20mmol)
dropwise and immediately allowed to warm to room temperature. The solution was
treated with water and extracted with EtOAc. The combined organics were washed
with brine, dried (MgS0 4), filtered and evaporated. The residue was purified by column
chromatography (Si0 2, 20% t o 30% EtOAc in cyclohexane) to afford the title
compound as a pale yellow solid (1.17g, 84%). H NMR (400 MHz, DMSO-d6) d 9.74 (s,
1H), 8.37 (d, J = 5.0 Hz, 1H), 8.19 (t, J = 1.8 Hz, 1H), 7.79 (dd, J = 8.0, 1.8 Hz, 1H),
7.70 (s, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 5.0 Hz,
1H), 2.38 (s, 3H).
Preparation of 4-{5-[3-(4-Methyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one (Compound 001)
A mixture of intermediate Id (800mg, 2.79mmol) and Ie (704mg, 5.58mmol) was
ground together and heated to 140°C for lh. The cooled solid residue was taken up in
a little hot ethanol, treated with NaHC03 solution (sat aqu) and extracted with 10%
EtOH in DCM. The combined organics were washed with brine, dried (MgS0 ), filtered
and evaporated. The residue was purified by column chromatography (Si0 2, 10% EtOH
in DCM) to afford the title compound as a beige solid (691mg, 66%). H NMR (400
MHz, DMSO-de) d 9.63 (s, 1H), 8.35 (d, J = 5.0 Hz, 1H), 8.09 (t, J = 1.8 Hz, 1H), 7.77
(s, 1H), 7.66 (dd, J = 8.1, 1.3 Hz, 1H), 7.31 - 7.26 (m, 2H), 7.14 (d, J = 7.8 Hz, 1H),
6.76 (d, J = 5.0 Hz, 1H), 3.85 (t, J = 5.7 Hz, 2H), 3.44 (td, J = 5.7, 1.5 Hz, 2H), 2.38
(s, 3H), 1.46 (dd, J = 7.8, 4.5 Hz, 2H), 1.32 (dd, J = 7.7, 4.4 Hz, 2H).
Example 002:
Synthetic approach of compound 002
Preparation of 2-Chloro-5-(3-nitro-phenyl)-oxazole (Ila)
Prepared as for Intermediate Id above from Intermediate l a followed by purification by
column chromatography (Si0 2, 20% EtOAc in cyclohexane) to afford the title
compound as a pale yellow solid (77%).*H NMR (400 MHz, CDCI3) d 8.45 - 8.43 (m,
1H), 8.21 (ddd, J = 8.2, 2.2, 0.9 Hz, 1H), 7.91 (ddd, J = 7.8, 1.5, 1.0 Hz, 1H), 7.64 (t,
J = 8.0 Hz, 1H), 7.46 (s, 1H).
Preparation of 4-[5-(3-Nitro-phenyl)-oxazol-2-yl]-4,7-diaza-spiro[2.5]octan-8-one (lib)
A solution of intermediate Ila (500mg, 2.23mmol) and 4,7-diaza-spiro[2.5]octan-8-one
Ie (842mg, 6.68mmol) in 2-propanol (100ml) was heated to reflux for 10 days. The
mixture was cooled to ambient temperature, concentrated under vacuum and the
yellow precipitate formed removed by filtration and dried in a dessicator to give the
title compound as a yellow solid (410mg, 59%). H NMR (400 MHz, DMSO-d ) d 8.33
(s, 1H), 8.08 (dd, J = 8.1, 1.9 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.80 (s, 1H), 7.75 -
7.66 (m, 2H), 3.90 (t, J = 5.6 Hz, 2H), 3.42 (t, J = 4.6 Hz, 2H), 1.45 (dd, J = 7.7, 4.5
Hz, 2H), 1.32 (dd, J = 7.7, 4.4 Hz, 2H).
Preparation of 4-[5-(3-Amino-phenyl)-oxazol-2-yl]-4,7-diaza-spiro[2.5]octan-8-one
(lie)
A slurry of the nitrooxazole lib (360mg, 1.15mmol) and 10% Pd/C (50mg) in THF
(60ml) and methanol (40ml) was stirred under an atmosphere of hydrogen at ambient
temperature and atmospheric pressure for 16h. The solution was filtered and
evaporated under vacuum before purification by column chromatography (Si0 2, 5%
EtOH in DC ) to afford the title compound as a white solid (230mg, 79%). H N R
(400 MHz, DMSO-d ) d 7.79 (s, 1H), 7.19 (s, 1H), 7.04 (t, J = 7.8 Hz, 1H), 6.74 - 6.69
(m, 2H), 6.46 (dd, J = 7.9, 1.9 Hz, 1H), 5.19 (s, 2H), 3.82 (t, J = 5.6 Hz, 2H), 3.43 -
3.38 (m, 2H), 1.43 (dd, J = 7.8, 4.5 Hz, 2H), 1.27 (dd, J = 7.7, 4.4 Hz, 2H).
Preparation of 2-Chloro-4-cyanopyrimidine (lid)
Prepared largely according to the method described in WO2005/075468. A solution of
4-methyl-l H-pyrimidin-2-one hydrochloride (14.7g, lOOmmol) in 50% aqueous acetic
acid (100ml) at 15°C was treated with sodium nitrite in one portion (10.4g, 150mmol)
with vigorous stirring causing an exothermic reaction (40°C). A yellow precipitate was
filtered off, washed with cold water and dried in a vacuum dessicator to afford the 2-
hydroxy-pyrimidine-4-carbaldehyde oxime intermediate as a pale yellow solid (13. lg,
94%) H NMR (400 MHz, DMSO-d6) d 12.44 (s, 1H), 11.87 (br s, 1H), 7.92 (d, J = 6.3
Hz, 1H), 7.77 (d, J = 0.4 Hz, 1H), 6.66 (dd, J = 6.4, 0.9 Hz, 1H). The oxime was
treated with phosphorus oxychloride (20ml) and warmed slowly to 45°C. Warming was
stopped as the temperature rose suddenly to 70°C and the mixture stirred for 3h.
Diisopropylethylamine (2ml) was added and the mixture refluxed for 30mins before
pouring into ice and extraction with DCM. The organics were washed with water then
NaHC03 (sat aqu) then again with water, dried (MgS0 4), filtered and evaporated to
afford Intermediate lid as a yellow oil which crystallized on standing (1.51g, 30%).
H NMR (400 MHz, DMSO-d6) d 9.15 (d, J = 4.9 Hz, 1H), 8.25 (d, J = 4.9 Hz, 1H).
Preparation of 2-{3-[2-(8-Oxo-4,7-diaza-spiro[2.5]oct-4-yl)-oxazol-5-yl]-phenylamino}-
pyrimidine-4-carbonitrile (Compound 002)
A solution of Intermediate lie (45mg, 0.158mmol) and 2-chloro-4-cyanopyrimidine lid
(66mg, 0.474mmol) in 2-propanol (3ml) was heated to reflux for 40h. The formed
precipitate was filtered and treated with NaHC03 solution (sat aqu) and extracted with
10% EtOH in DCM (50 mL). The combined organics were dried (MgS0 4), filtered and
then partially concentrated under vacuum. The precipitate was collected by filtration,
washed with ether and dried to afford (compound 002 as a yellow solid (39mg,
64%).^ NMR (400 MHz, DMSO-d6) d 10.31 (s, 1H), 8.79 (d, J = 4.7 Hz, 1H), 7.95 (s,
1H), 7.81 (s, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 4.8 Hz, 1H), 7.39 - 7.33 (m,
2H), 7.25 (d, J = 7.7 Hz, 1H), 3.85 (t, J = 5.6 Hz, 2H), 3.44 (t, J = 6.0 Hz, 2H), 1.46
(dd, J = 7.8, 4.5 Hz, 2H), 1.32 (dd, J = 7.6, 4.4 Hz, 2H).
Non-commercially available 2-chloro-4-alkylpyrimidines intermediates which were used
to prepare compounds listed in the Compound Table, were prepared according to the
method of Jorgensen et al{J. Am. Chem. Soc, (2011), 15686-15696).
Preparation of 2-Chloro-4-ethylpyrimidine (Ilia)
A mixture of 2,4-dichloropyrimidine (2.00g, 13.4mmol) and Fe(acac)3 (954mg,
2.70mmol) in dry THF (24ml) at -78°C under argon was treated with a solution of
ethylmagnesium bromide (lm in THF, 16.2ml, 16.2mmol) dropwise. After stirring at -
78°C for 30mins, the mixture was warmed to ambient temperature and stirred for a
further hour. The mixture was again cooled to -78°C and treated with ethylmagnesium
bromide solution (10ml, lOmmol) and warmed to RT. The mixture was diluted with
water, extracted with EtOAc and the organics dried (MgS04), filtered and evaporated.
The residue was purified by column chromatography (Si0 2, 20% EtOAc in cyclohexane)
to afford the title compound as a clear liquid (642mg, 34%). H NMR (400 MHz, DMSOd
6) d 8.64 (d, J = 5.1 Hz, 1H), 7.47 (d, J = 5.1 Hz, 1H), 2.76 (q, J = 7.6 Hz, 2H), 1.21
(t, J = 7.6 Hz, 3H).
The 2-chloro-4-aryl-2-ylpyrimidine and 2-chloro-4-heteroaryl-2-ylpyrimidine
intermediates which were used to prepare compounds listed in the Compound Table,
were prepared by Suzuki coupling methods (See for example, N. Miyaura and A.
Suzuki, Chemical Reviews, (1995), 2457-2483).
Preparation of 2-Chloro-4-thiophen-2-ylpyrimidine (Illb)
A mixture of 2,4-dichloropyrimidine (l.OOg, 6.71mmol), 2-thiopheneboronic acid
(430mg, 3.36mmol), Na2C0 3 (0.4M solution in water, 20ml, 8.06mmol) and Pd(PPh3)4
(78mg, 0.067mmol) in THF (20ml) was heated to 90°C overnight. The cooled mixture
was diluted with water, extracted with DCM and the organics dried (MgS0 ) , filtered
and evaporated. The residue was purified by column chromatography (Si0 2, 20%
EtOAc in cyclohexane) to afford the title compound as a white solid (591mg, 89%). H
NMR (400 MHz, DMSO-d6) d 8.73 (d, J = 5.3 Hz, 1H), 8.16 (dd, J = 3.8, 1.1 Hz, 1H),
8.04 (d, J = 5.3 Hz, 1H), 7.95 (dd, J = 5.0, 1.1 Hz, 1H), 7.29 (dd, J = 5.0, 3.8 Hz, 1H).
4-Amino-2-chloro-pyrimidines intermediates, which were used to prepare compounds
listed in the Compound Table, were prepared according to the method below largely
based on that described in US2006/199804.
Preparation of 4-(2-Chloro-pyrimidin-4-yl)-morpholine (IIIc)
A stirred solution of 2,4-dichloropyrimidine (5.00g, 36.5mmol) and
diisopropylethylamine (14.0ml, 80.4mmol) in EtOH (60ml) at 0°C was treated with
morpholine (3.18ml, 36.5mmol) and allowed to warm to ambient temperature
overnight. The solution was poured into brine and extracted with DCM. The organics
were dried (MgS0 ), filtered and evaporated. The residue was purified by column
chromatography (Si0 , 5% EtOH in DCM) to afford the title compound IIIc as a white
solid (1.3g, 36%). H NMR (300 MHz, DMSO-d6) d 8.10 (d, J = 6.2 Hz, 1H), 6.83 (d, J
= 6.2 Hz, 1H), 3.72 - 3.49 (m, 8H).
Preparation of 4-{5-[3-(4-Methyl-pyridin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one (Compound 021) :
021
4-[5-(3-Bromo-phenyl)-oxazol-2-yl]-4,7-diaza-spiro[2.5]octan-8-one (IVa) was
prepared from 3-bromobenzaldehyde in 3 steps as described for intermediates la, Ila
and lib. H N R (400 MHz, , DMSO-d6) d 7.81 (s, 1H), 7.77 (t, J = 1.7 Hz, 1H), 7.58 -
7.54 (m, 1H), 7.54 (s, 1H), 7.44 (ddd, J = 8.0, 1.7, 1.1 Hz, 1H), 7.37 (t, J = 7.9 Hz,
1H), 3.87 (t, J = 5.7 Hz, 2H), 3.40 (td, J = 5.5, 1.6 Hz, 2H), 1.42 (dd, J = 7.8, 4.5 Hz,
2H), 1.29 (dd, J = 7.7, 4.4 Hz, 2H).
A solution of Intermediate IVa (lOOmg, 0.287mmol) in degassed dioxane (5ml) and in
a sealed tube was treated with 2-amino-4-methylpyridine (47mg, 0.431mmol),
Pd2(dba) 3 (5mg, 0.00574mmol), xantphos (7mg, 0.0115mmol) and Cs2C0 3 (140mg,
0.431mmol). The tube was sealed and heated to 100°C overnight. After a further
addition of Pd2(dba) 3 (20mg, 0.0218mmol) and xantphos (28mg, 0.0484mmol) the
mixture was heated for a further 24h. The cooled mixture was treated with water and
extracted with DCM and the organics dried (MgS0 4), filtered and evaporated. The
residue was purified first by column chromatography (Si0 2, 5%-10% EtOH in DCM)
then by trituration with EtOAc to afford compound 021 as a cream colored solid (32mg,
30%). H NMR (400 MHz, , DMSO-d6) d 9.01 (s, 1H), 8.04 (d, J = 5.1 Hz, 1H), 7.95 (s,
1H), 7.79 (s, 1H), 7.54 (dd, J = 8.2, 1.2 Hz, 1H), 7.29 (s, J = 2.7 Hz, 1H), 7.26 (t, J =
8.0 Hz, 1H), 7.08 (d, J = 7.7 Hz, 1H), 6.66 (s, 1H), 6.62 (d, J = 5.2 Hz, 1H), 3.85 (t, J
= 5.5 Hz, 2H), 3.44 (t, J = 4.4 Hz, 2H), 2.24 (s, 3H), 1.46 (dd, J = 7.7, 4.4 Hz, 2H),
1.32 (dd, J = 7.6, 4.4 Hz, 2H).
Preparation of 4-{5-[3-(Thiazol-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one (Compound 022) :
IVb Compound 022
A solution of intermediate l b (1.50g, 9.37mmol) in EtOH (45ml) and water (5ml) was
treated with 2-bromothiazole (1.69ml, 18.7mmol) and cone. H (1.61ml, 187mmol)
and stirred at 100°C for 6h. Aftre addition of further 2-bromothiazole (1.69ml,
18.7mmol) the solution was heated for a further 24h then cooled and taken to pH14
with aqueous NaOH solution. After extraction with 10% EtOH in DCM then DCM, the
organics were dried (MgS0 4), filtered and evaporated. The residue was purified by
column chromatography (Si0 2, 5% acetone in DCM) to afford intermediate IVb as a
white solid (493mg, 22%). H NMR (300 MHz, DMSO-d6) d 10.36 (s, 1H), 8.46 (s, 1H),
8.10 (t, J = 1.8 Hz, 1H), 7.63 (s, 1H), 7.56 (ddd, J = 8.1, 2.2, 1.1 Hz, 1H), 7.39 (t, J =
7.9 Hz, 1H), 7.33 - 7.29 (m, 2H), 6.95 (d, J = 3.7 Hz, 1H).
Compound 022 was then prepared from intermediate IVb as described for intermediate
Id and Compound 001 above : H NMR (300 MHz, DMSO-d ) d 10.29 (s, 1H), 7.84 (s,
1H), 7.81 (s, 1H), 7.54 (dd, J = 8.1, 1.3 Hz, 1H), 7.37 - 7.29 (m, 2H), 7.27 (d, J = 3.6
Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 3.7 Hz, 1H), 3.85 (t, J = 5.5 Hz, 2H),
3.43 (s, 2H), 1.46 (dd, J = 7.7, 4.5 Hz, 2H), 1.32 (dd, J = 7.6, 4.5 Hz, 2H).
By repeating the methods described above using the appropriate starting materials and
conditions, the following additional analogues in the compound Table 1 were prepared
and characterized.
Compound Table 1:

IC50: Concentration inhibiting 50% of protein kinase.
The Syk activities given in the Compound Table above are expressed as:
+++: IC50 < 500 nM
++: 500>IC50 < 2000 nM
+ : IC50 > 2000 nM
PHARMACOLOGICAL EXAMPLES:
1) I n vitro SYK inhibition assays
Protocol inhibition assays
SYK kinase was purified as a full length protein in a baculovirus system near
homogeneity. All kinase assays were performed with the Kinase TK (tyrosine kinase)
HTRF (Homogeneous Time Resolved Fluorescence) assay developed by Cisbio
international. These assays were carried out at room temperature in 96-wells half-area
white plates in a final volume of 25 m I of kinase buffer (10 m MgCI2; 2 m nCI2; 50
m Sodium-HEPES pH 7.8; BRD-35 0.01%, 1 m substrate) containing ATP at a
concentration of at least twice the Km for each enzyme and an appropriate amount of
recombinant enzyme to ensure a linear reaction rate. Reactions were initiated upon
introduction of the enzyme and terminated with the addition of one reaction volume
(25 m ) of HTRF detection buffer. Plates were incubated for one hour at room
temperature and the time resolved Fluorescence resonance energy transfer signal was
measured in a Pherastar FS microplate reader (BMG Labtech). All data were the
average of triplicate results with a standard deviation < 10%.
Experimental results
The experimental results for various compounds according to the invention using
above-described protocols are set forth at the compound Table 1.
Comments on the experiments and results
The inventors observed a very effective inhibition of SYK by the class of compounds of
formula (I) as presently disclosed. The listed compounds in the Compound Table are
well representing the class of compounds of formula (I).
The below references to compound 001 referred to the compound of the same number
in the Compound Table 1 above. Compound 001 is compared to R406, the active
metabolite of fostamatinib/R788, a Rigel Pharmaceutical SYK inhibitor
(WO2006/078846A1; Drugs Future, (2011), 36(4): 273-280).
2) I n vitro anti-SYK kinase activity and selectivity
Activity and selectivity towards SYK target were determined by screening test
compounds against various tyrosine kinases using both in vitro recombinant kinase
assays and cell-based proliferation assays.
Protocol inhibition assays
Cell-based proliferation and viability assay
CellTiter-Bleue cell-based survival/proliferation assay (Promega G8080) was performed
on BaF3 models.
A total of 1.104 cells/well/50pl were seeded in a 96-wells plate. Treatment was initiated
by addition of a 2x drug solution of V serial dilutions ranging from 0 to IOmM. Cells
were grown for 48-72 h at 37°C and then incubated with 10 m I/well of Promega
CellTiter-Bleue reagent for 4 h at 37°C. The amount of formazan dye formed was
quantified by its absorbance at 450 nm using a scanning multiwell spectrophotometer
(POLARstar Omega, BMG labtech, France). A blank well without cells was used as a
background control for the spectrophotometer and all assays were performed in
duplicates and the experiments were repeated at least twice.
I n vitro kinase assays with recombinant SYK and various protein kinases
Cloning and expression of kinases
Most of the kinases tested in this study were cloned, expressed and purified in the
facilities of the inventors. They were expressed either as N-terminus hexahistidine-,
hexahistidine-asparagine- or GST-tagged enzymes in a baculovirus or in a Colibacille
expression system. Remaining enzymes (JAKs) were purchased from Millipore or
Proqinase. For each enzyme, steady state kinetic parameters were determined and
validated with known inhibitors. All experiments were performed in a large excess of
substrate and with an ATP concentration corresponding at least to 2*Km with respect
to ATP.
HTRF Kinase assay
The analysis of the effect of compounds on kinase activity was assessed with the
HTRF® KinEASE assay (Cisbio International), an immuno-assay based on the
quantification of the level of phosphorylation of a biotinylated peptide substrate using
anti-phospho-specific antibody labeled with Europium (Eu3 +) cryptate. This assay
comprises two steps: -an enzymatic step, during which the peptide substrate, the
kinase, ATP, Mg + and/or Mn + are incubated with varying concentration of drug (from
0 to 10 mM); -a detection step, at the end of the reaction (stopped by addition of EDTA
which chelates Mg +), the antibody anti-phospho peptide-Eu 3 + (emission 620 nm) and
streptavidin XL-665 (emission 665 nm) are added to the reaction mix. After incubation,
the obtained signal is proportional to the concentration of phosphorylated peptide in
the sample. All measurements were performed on a BMG Labtech Pherastar FS
apparatus. Results are expressed in delta fluorescence (DF) unit defined as follow DF
%=[(ratio-ratio blank)/(ratio blank)]*100, where ratio=(665 nm/620 nm)*10 4. Each
experiment was performed in duplicate and repeated two or three times.
Experimental results
Table 2: Activity and selectivity of anti-SYK compounds (IC 0 mM)
Protein kinases Compound 001 R406
TEL-SYK 0.27 1
Jakl* 3.4 0.01
Jak2* 0.9 0.006
Jak3* 20 2.5
Class III receptor tyrosine kinases
KIT wild-type 10 0.5
KIT D816V 10 0.1
PDGFR - 0.15
PDGFRa - 0.1
Flt3 6 0.1
c-fms* - 5
Other receptor tyrosine kinases
VEGFR1* 7.5 0.35
VEGFR2* 20 0.06
VEGFR3* - < 1
EGFR WT / del - 3 / 1
ERBB2 - 3
FGFR 1 - 0.25
FGFR 3 - 0.15
c- et * 10 20
TrkB - 2
IGFR1 - 0.9
c-Ret WT / mut* 10 / - 0.5 / 0.5
Non receptor tyrosine kinases
BCR-ABL 10 5
FAK* - 0.4
Src* 2.6 0.1
Lyn B* - 1
Fyn* - 0.05
Lck*
Hck* - 15
Btk* 20 0.1
Bmx* - 0.12
Fes* - 5
*enzymatic determination of kinase inhibitory activity of test compounds (otherwise cell-based
assay), R406 is the active metabolite of R788/fostamatinib
Comments on the experiments and results
The above in vitro data demonstrated good anti-SYK activity of compound 001. This
compound was found more potent than R406 on Ba/F3 TEL-SYK model and exhibited
very good selectivity in both cell-based and kinase assays in contrast to the multikinase
inhibitor R406.
3) I n vitro anti-SYK activity in murine bone marrow mast degranulation
assay
Protocol inhibition assays
I n vitro mast cell degranulation assay monitored by released of b-hexosaminidase.
Murine bone marrow mast cells (BMMCs) are obtained by flushing bone marrow cells
from the femurs of C57BL/6J mice, then cultured for 3-4 weeks in RPMI containing
mouse recombinant IL-3 (30ng/ml). RBL-2H3 is a rat basophile cell line maintained in
monolayer culture in EMEM supplemented with 20% FCS, ImM Glutamine and I X
antibiotics. Cells were sensitized overnight with 0.2pg/ml anti-DNP-IgE (Sigma-Aldrich
D8406). Cells were washed extensively in Tyrode buffer (lOmM Hepes, 130mM NaCI,
6.2mM D-Glucose, 3mM KCI, 1.4mM CaCI2, ImM MgCI2 and 0.1% BSA. Cells (5.10 4
cells/well/90Ml) were plated in triplicates in 96 wells plates. Treatment with SYK kinase
inhibitors was initiated by adding IOmI of 10X concentrated dilutions to obtain 0.01,
0.1, 1 and IOmM final concentrations. After 2 hours drug treatment, cells were
stimulated for 90min with 125 ng/ml DNP-HAS (Sigma-Aldrich A6661) in a final volume
of IIOmI . For measurement of b-hexosaminidase activity, 50m I of supernatant were
collected and incubated with 50m I of 3.7mM p-nitrophenol-N-acetyl-p-Dglucosaminidine
(PNAG, Sigma-Aldrich N9376) prepared in citrate buffer (pH 4.5). After
incubation 90min at 37°C, the reaction was quenched by addition of IOOmI of sodium
carbonate buffer (0.1M Na C0 3/NaHC03 pH 10.0). b-hexosaminidase activity was
quantified by reading plate absorbance at 405nm with reference at 620nm using a
PolarSTAR reader plate (BMG labech).
Experimental results
The activity of anti-SYK compounds was tested in an IgE-mediated mast cell
degranulation assay. SYK has been identified to be critical for the initiation of mast cell
mediator release following the aggregation of FcsRI. Anti-SYK compound 001 and R406
were compared in their ability to block IgE-mediated release of the granule component
b-hexosaminidase. The results are expressed as % of b-hexosaminidase residual
activity compared to untreated cells (100%) as shown in Table 3 below.
Table 3 : Effect of anti-SYK on b-hexosaminidase release/activity
Drug b-hexosaminidase activity Kinase activity profile
(% control untreated cells) ICso (nM)
0.01 mM 0.1 mM 1 mM Btk Syk Tel-Syk* Src JAK 2
R406 78±11 16±6 3±3 95 34 1000 100 6
(n=ll) (n=14) (n=14)
Drug b-hexosaminidase activity Kinase activity profile
(% control untreated cells) IC50 (nM)
Compound 88±14 32±9 3±5 20000 41 272 2600 920
001 (n=9) (n=ll) (n=ll)
*Cell-based assay of test compounds (otherwise enzymatic determination of kinase inhibitory
activity).
Comments on the experiments and results
Compound 001 was efficient at inhibiting mast cell degranulation (IC 0=50 nM) in
agreement with in vitro kinase assay on purified SYK and cell-based proliferation assay
on Ba/F3 tel-SYK cell line. In these experiments, the SYK inhibitor compound 001 was
equally potent as the multi-kinase inhibitor R406, at inhibiting mast cell degranulation.
4 ) I n vitro anti-SYK activity in murine bone marrow mast cytokine
production
Protocol inhibition assays
Murine bone marrow mast cells (BMMCs) are obtained by flushing bone marrow cells
from the femurs of C57BL/6J mice, and then cultured for 3-4 weeks in RPMI containing
mouse recombinant IL-3 (30ng/ml). Cells were sensitized overnight in IL3-depleted
culture medium with 0.1-0.2pg/ml anti-DNP-IgE (Sigma-Aldrich D8406). Cells were
preincubated with inhibitors for 30 min, and then stimulated with lOng/ml antigen (Ag)
and/or Stem Cell Factor (SCF) (30ng/ml). The cells were incubated for 6h. The
supernatants were harvested, and the cytokine content measured using CISBIO IL6
and TNFa HTRF assay (cat* 63ADKEBU043 and 6FMTNPEB respectively).
Experimental results
To explore the effect of compound 001 on cytokine production, the production of IL-6
and TNFa in murine bone marrow mast cells (BMMCs) were examined (Table 4 and
Table 5).
Table 4: FcsRI and Kit-induced TNFa cytokine release in BMMCs.
TNFa Cytokine Release
(Relative Fluorescence Units) (% relative to
control Ag + SCF)
Ag* -22
Controls SCF** 29
Ag + SCF 491 (100%)
Ag + SCF + O.OI m of compound 468 (95%)
Compound
Ag + SCF + O.ImM of compound 158 (32%)
001
Ag + SCF + ImM of compound 9.5 (2%)
Ag + SCF + O.OI m of compound 568.5 (120%)
R406 Ag + SCF + O.ImM of compound 171 (35%)
Ag + SCF + I m of compound -29 (0%)
* Ag = antigen; ** SCF = Stem Cell Factor
Table 5 : FcsRI and Kit-induced IL6 cytokine release in BMMCs
IL6 Cytokine Release
(Relative Fluorescence Units) ) (% relative to
control Ag + SCF)
Ag* -10
Controls SCF** 123
Ag + SCF 849 (100%)
Ag + SCF + O.OImM of compound 756.5 (89%)
Compound
Ag + SCF + O.ImM of compound 249 (29%)
001
Ag + SCF + ImM of compound 54 (6%)
Ag + SCF + O.OImM of compound 830 (98%)
R406 Ag + SCF + O.ImM of compound 137 (16%)
Ag + SCF + ImM of compound 5 (1%)
* Ag = antigen; ** SCF = Stem Cell Factor
Comments on the experiments and results
As previously reported, IgE-stimulated FcsRI aggregation in the absence of SCF
minimally induced cytokine production in BMMCs. However, in the presence of SCF,
there was a marked increase in the production and release of IL-6 and TNFa. The
release of these cytokines was efficiently blocked by compound 001 (IC5 ~50 nM). In
these experiments compound 001 was equally potent as R406 at blocking cytokine
release with inhibitory effect observed at concentrations as low as 0.1 m .
5) In vivo anti-SYK activity in mouse model of asthma
Protocol assays
Balb/c male mice, 8 weeks old, were purchased from Janvier (Le Genest-Saint-Isle;
FRANCE) and bred in the animal facility for 4 weeks. Drugs were dissolved in drinking
solvent (80% water; 10% Tween 80 and 10% isopropanediol). It was aliquoted and
stored at -20°C. Each mouse received drugs twice a day. The weight of animals was
calculated before and after treatment.
Sensitization and treatment
Mice, 9 weeks of age, were sensitized with 50 g of ovalbumin (Sigma-Aldrich,
Germany) adsorbed to 2mg AI(OH)3 (Prolabo, France) and diluted in physiological
saline (0.9% NaCI). Ovalbumin was injected intraperitoneally on days 1 and 7.
Sensitization was followed by intranasal challenges with 10 g of ovalbumin diluted in
0.9% NaCI, each day from day 18 to day 21. On days 17 to 21, animals treated with
drugs received IOOmI of a solution at 15mg/ml, at 7.5mg/ml or at 3.75mg/ml
administered per os twice a day during 5 days. These treatments corresponded to
concentration of 60mg/ mouse kg, 30mg/ mouse kg and 15mg/ mouse kg respectively.
Assessment of airway responsiveness and lung function
The airway responsiveness was measured using the invasiveness FlexiVent® (SCIREQ)
technique. Briefly, mice were weighed and anesthetized by i.p. with 6mg/kg of the
xylasine solution (Bayer, France) and after 10 minutes with 6mg/kg of the
pentobarbital solution (CEVA). Each anesthetic was diluted in physiological saline
solution. Once the mouse was deeply anesthetized, the trachea was canulated and
connected to the computer-controlled small animal ventilator (FlexiVent®). When
connected, the computer controls the mechanical ventilation with a variety of volume
and pressure controlled maneouvres to obtain accurate, reproducible measurement of
respiratory mechanics as airway resistance, elastance and compliance. The baseline
values were measured after saline solution nebulization and airway
hyperresponsiveness evaluated after nebulization of a 0.26 metacholine solution
(50mg/ml).
Assessment of airway inflammation
Bronchio-alveolar lavage (BAL) was performed after the animals were disconnected
from the FlexiVent®. The airways were lavaged with 5 ml (10 times 0.5ml) ice cold
0.9%NaCI 2.6m EDTA. BAL samples were centrifuged at 1200rpm for 5 minutes at
4°C. Pellet cells were collected and erythrocytes lysed by adding 1.5ml deionized water
for 30 seconds. After neutralization with 0.5ml of 0.6M KCI, cells were centrifuged at
1200rpm for 5 minutes at 4°C. BAL samples were used to count the total cell number
and to perform cytospins (shandon, Pontoise). Cell types were identified by
morphological criteria after Hemacolor (Merck) staining.
Experimental results
Compound 001 was evaluated for its impact on airway inflammation and
responsiveness in a murine model of asthma, in comparison to Syk inhibitor R406 and
the anti-Kit inhibitor Masitinib, an AB Science drug candidate for treating asthma
(Allergy, (2009), 64(8): 1194-201). Mice were sensitized and challenged with ovalbumin
as described in protocol assays above. The mice were distributed in groups of 5-6
animals that were treated twice daily with 7.5 to 60mg/Kg of anti-SYK/ KGG drugs (PO).
A summary of the data obtained in several experiments is presented in Table 6.
Table 6: Comparison of compound 001 activity with Masitinib and R406 in murine
model of asthma
Compounds Dose Percentage of Eosinophils Statistical
(mg/kg/day) recruitment in Bronchio-alveolar Analysis
PO lavage (BAL)
Experiment # 1
Masitinib 2 x 7.5 -15% p>0.05
Compounds Dose Percentage of Eosinophils Statistical
(mg/kg/day) recruitment in Bronchio-alveolar Analysis
PO lavage (BAL)
2 x 25 -35% p<0.05
2 x 30 -56% p<0.05
R406 2 x 30 -31% p>0.05
Experiment # 2
Masitinib 2 30 -47% p<0.05
(n=12)
Compound 001 2 x 15 -14% p>0.05
(n=12) 2 x 30 -47% p<0.05
Comments on the experiments and results
Sensitization and challenges with ovalbumin induced a marked increase in the total
number of cells, mainly macrophages and eosinophils. Results showed that compound
001 induced a significant decrease of eosinophils recruitment in a dose-dependent
manner. C-kit inhibitor Masitinib and compound 001 induced a significant and similar
decrease in the number of eosinophils in BAL from sensitized mice at the dose of 30
mg/kg (Table 6, experiment #2), while Masitinib was more potent than R406 in this
model of asthma (Table 6, experiment #1). Indeed with a lower dose of Masitinib or
Compound 001, the Same effect as R406 was Obtained (Erreur ! Source du renvoi
introuvabie.6, experiment # 1 and #2). In accordance with airway inflammation analysis,
compound 001 treatment reduced bronchial hyper-responsiveness in a dose-dependent
manner.
6) In vivo anti-SYK activity in mouse model of Rheumatoid Arthritis
Protocol assays
K/BxN serum pools were prepared from arthritic mice at 8 weeks of age. The mice
were pre-treated with SYK inhibitors by two per os administration for two days before
induction of arthritis. Arthritis was induced in the C57BI/6 mice (8 weeks of age) by
intraperitoneal injection (7.5 m I serum per g weight) at days 0 and 2.
The treatment with SYK inhibitors was continued for 13 days. The control mice were
injected with solvent before the induction of arthritis and during the course of the
disease. The compounds were dissolved in a solution containing 10% of Tween 80 and
10% 1-2 propandiol. It was prepared before administration.
Ankle thickening measure, being defined as the difference in ankle thickness from the
day 0, was measured using a precision calliper. Arhritis/clinical score was defined by
sum of scores of each lim (0 no disease; 1 mild swelling of paw or of just of few digits;
2 clear joint inflammation; 3 severe joint inflammation) maximum score=12.
Myeloperoxidase Activity
Myeloperoxidase (MPO) is the most abundant enzyme in primary neutrophils and has
been shown to be a useful reliable marker for neutrophil infiltration in inflammatory
diseases. MPO activity was determined following published protocol (American Journal
of Pathology, (2000), 156 (6):2169-2177). Briefly, tissue samples were weighed and
suspended in 50 mmol/L potassium phosphate buffer (pH 6.0) containing 5 mg/ml
hexadecyltrimethylammonium bromide (Sigma Chemical Co.) at a ratio of 50 g tissue
to 1 ml of buffer. Tissues were homogenized by a polytron tissue homogenizer for 1
min, and 1 ml was decanted into sterile Eppendorf tubes and centrifuged at 12,000
rpm for 15 minutes. Using a microtiter plate scanner, 200 m I of the reaction mixture
(containing 16.7 mg of o-dianisidine (Sigma Chemical Co.), 90 m I of distilled H20 , 10 m I
of potassium-phosphate buffer, and 50 m I of 1% H20 2) was added to each well
containing 7 m I of sample in a standard 96-well plate and three absorbance readings at
5 min intervals at 450 nm were recorded.
Experimental results
K/BxN was a murine model of spontaneous rheumatoid arthritis that mimics many of
the clinical and histological features of human disease with synovitis predominantly in
the distal small joints. Arthritis was induced by intraperitoneal injection of arthritis
serum of K/BxN mice at days 0 and 2 as described in protocol assays above.
Compound 001 was tested at 15 and 50 mg/kg/day b.i.d. and compared with Masitinib
at 50 mg/kg/day b.i.d., an AB Science drug candidate for treating rheumatoid arthritis
(Arthritis Res Ther., (2009), 11(3):R95). Ankle thickening and arthritis score were used
to monitor the in vivo activity of the compounds. Data showed that compound 001
significantly improves the arthritis symptoms in a dose-dependent manner (Table 7).
Table 7: Comparison of compound 001 and Masitinib in murine model of arthritis
Compounds Ankle Thickening Score Arthritis Score
Masitinib 50 mg/kg b.i.d. -27% P<0.05 -15%
Compound 001, 15 mg/kg b.i.d. -17% -23%
Compound 001, 50 mg/kg b.i.d. -52% P<0.005 -68% P<0.005
Myeloperoxidase (MPO) is the most abundant enzyme in primary neutrophils and has
been shown to be a useful reliable marker for neutrophil infiltration in inflammatory
diseases. MPO activity has been measured with different doses of compound 001
(Table 8).
Table 8: MPO dosage following treatment with compound 001
Comments on the experiments and results
The results of Table 7 has shown that compound 001 administered at 2x50 mg/kg/day
was very efficient at decreasing arthritis score with 52 and 68% reduction of ankle
thickening and arthritis score respectively with a dose-dependent effect. Compound
001 was clearly and significantly more potent in this model of rheumatoid arthritis
when compared with Masitinib at the same dosage.
An increase in MPO activity (U/ml) was measured in KBxN mice compared to control
mice that was significantly reduced by compound 001 treatment at dosage 50 mg/kg
b.i.d. (Table 8).
7) Cardiotoxicity: cell proliferation and viability assay on cardiomyocytes
Protocol assays
WST-1 cell survival/proliferation assay (Roche Diagnostic ref N°1644807) was
performed on both primary human adult cardiomyocytes and rat neonatal cardiac
myocytes.
Human cardiomyocytes were isolated from normal human ventricle tissue of the adult
heart and were able to proliferate for a number of passage. Neonatal ventricular
Clonetics® Rat Cardiac Myocytes (Pl-3) retained their contractile property and were
electrophysiological^ active in culture. 1.104 primary rat cells or 2-2.5 104 adult human
cells were plated per well of a 96 wells plates. The cells were allowed to adhere to the
plates for 5 days before drug treatment. Treatment was initiated by addition of a 2X
drug solution of serial dilutions ranging from 0 to IOmM. Cells were grown for 48 h
at 37°C and then incubated with 10 m I/well of WST-1 reagent for 4 h at 37°C. The
amount of formazan dye formed was quantified by its absorbance at 450 nm using a
scanning multiwell spectrophotometer (MultiSkan MS, Thermo-LabSystems, France). A
blank well without cells was used as a background control for the spectrophotometer
and all assays were performed in triplicate.
Experimental results
Cardiac toxicity of drugs can be determined in vitro using primary cardiomyocytes. The
cytotoxicity of tests substances was analyzed on rat and human cardiomyocytes using
an in vitro proliferation and survival assay. Results are reported in Table .
Table 9: Drug-induced cytotoxicity of human and rat cardiomyocytes
Drug-induced cytotoxicity (IC 0 mM)
(% cell viable at 1 mM)
Drugs Hu-CM Rat-CM
Compound 001 10 (+14%) 10 (-2%)
R406 2.6 (-18%) < 1 (-60%)
Hu-CM=Human Cardiac Myocyte, Rat-CM=Rat Cardiac Myocyte
Comments on the experiments and results
The above data clearly showed that compound 001 exhibited no cytotoxic effect on
both human and rat cardiomyocytes at concentrations up to 10 mM. I n contrast, R406
was toxic to both human and rat cardiomyocytes with an IC50 <2.6 m .
8) Cardiotoxicity: functional hERG/Kvll.l Potassium Chanel
Protocol assays
The activity of the test compounds on the hERG potassium channel was evaluated
using a binding assay provided by Invitrogen (Predictor hERG Fluorescence Polarization
assay Kit PV5365). The test was performed at a single concentration of 3m of drug.
E-4031, a well-known selective inhibitor of hERG potassium channel, is used as positive
control. The data are expressed as a percentage of hERG inhibition, 100%
corresponding to the inhibition obtained with E-4031 and 0% with the solvent DMSO
(negative control).
Experimental results
Cardiotoxicity effects may arise through undesired blockage of the human Ether-a-gogo-
related (hERG) potassium channel. Therefore, evaluating the effect on hERG
channel function is essential in the development of small-molecule therapy in order to
predict its potential cardiotoxic side-effects. The binding assay, the "predictor hERG
fluorescence polarization assay" (Invitrogen PV5365), was used to determine the
activity of compound 001 on hERG function (Table 10).
Table 10: Drug-induced inhibition of hERG function
Drugs hERG
% inhibition at 3 mM
Compound 001 0
R406 5
Comments on the experiments and results
Compound 001 had no effect on hERG function.
9) Intracellular ROS in cardiomyocytes
Protocol assays
2xl0 3 human cardio myocytes (Promocell C-12810) or 4xl0 4 rat neonatal cardiac
myocytes (R-CM-561 Lonza) were seeded per well of a 96-well plate (black wall for
fluorimetry usage (655090 Greiner Bio one)) 24hours before drug treatment.
Treatment was initiated by addition of a drug solution for a IOmM final concentration.
Doxorubicin was used at 5mM as positive control for the induction of ROS. After 8 hours
treatment with drugs, cells were loaded with IOmM of CM-H2DCFDA (C6827 Invitrogen)
for 1 hour, followed by 2 washes in PBS. CM-H2DCFDA is a membrane permeable
reagent that can be enzymatically converted to dichlorodihydrofluorescein (DCF) in the
presence of ROS. Fluorescent DCF was detected using a fluorescence
spectrophotometer (BMG Labtech) with excitation of 485nm and emission of 560nm.
The results were expressed as a relative percent of DCF-fluorescence in control cells.
Experimental results
Monitoring drug-induced increase in the production of reactive oxygen species (ROS) in
cardiomyocyte may be important to predict the cardiotoxic side effects of a drug (Table
11). The production of ROS in human and rat cardiomyocytes was studied following 8
hours drug treatment at concentration of 10 mM. The results were expressed as a
percent relative to the production of ROS induced by the cardiotoxic chemotherapeutic
agent doxorubicin (100%).
Table 1: Drug-induced ROS production in human and rat cardiomyocytes
Activity relative to doxorubicine (100%)
Drugs Hu-CM Rat-CM
Compound 001 0 0
Hu-CM=Human Cardiac Myocyte, Rat-CM=Rat Cardiac Myocyte
Comments on the experiments and results
These results demonstrated that compound 001 did not induce significant increase in
cardiac production of ROS.
10) Mitochondrial function
Protocol assays
Drug-induced mitochondrial toxicity was evaluated by monitoring oxygen consumption
using a Clark oxygen electrode on mitochondria isolated from heart of healthy mouse
(Mitologics S.A.S, France). Briefly, isolated mitochondria are placed at 37°C in a sealed
chamber that is exposed to the surface of a Clark oxygen electrode. The presence of
oxygen caused the electrode to deliver a current to the oxygen monitor, which
amplified the current and converted it to a voltage output that was directly
proportional to the concentration of oxygen in the chamber. Drug-induced
mitochondrial toxicity was also evaluated by monitoring mitochondrial ATP production
using a cell-based assay (Mitochondrial ToxGloTM assay, Promega Corporation
(cat#G8000)). This multiplex assay measured concurrently cell membrane integrity as
a function of cytotoxicity, and mitochondrial function via ATP production, thus
distinguishing between compounds that exhibited mitochondrial toxicity versus overt
cytotoxicity. General toxicity was characterized by a decrease in ATP production and a
loss of membrane integrity whereas mitochondrial toxicity resulted in decreased ATP
production with little to no change in membrane integrity. The test was performed on
HepG2 tumor cell line grown in galactose medium in order to force the cancer cells to
produce ATP via oxidative phosphorylation (crabtree effect). Cells were treated for 2
hours with 20mM drug before analysis of ATP detection (Luminescent signal) and
cytotoxicity (Fluorescent signal) as described by manufacturers using PHERAstar and
POLARstar OMEGA microplate readers respectively (BMG Labteck Sari).
Experimental results
Mitochondrial dysfunction is a major mechanism of drug-induced toxicity. Oxygen
consumption is one of the most informative and direct measures of mitochondrial
function. In order to evaluate the potential mitochondrial toxicity of test compounds,
0 2 consumption measurement was performed in the presence of drugs using an
oxygen-sensitive probe (MitoXpress-Xtra, Luxcel Biosciences).
The oxygen consumption was analyzed during 20 min of drug treatment at
concentrations of 40 and 80 mM (Table ) .
Table 2: Drug-induced inhibition of 0 2 consumption
Drugs ATP production relative to Cytotoxicity relative to DMSO
DMSO (100%) (0%)
Mito ToxGlo™ Mito ToxGlo™
Compound 001 104% 11%
R406 105% 15%
Comments on the experiments and results
Data showed that compound 001 did not exhibit inhibition of cardiac mitochondria ATP
production (104% compare to 100% DMSO control) with low cytotoxicity (11%).
These results showed that compound 001 did not affect mitochondrial function in
HepG2 cells in the experimental conditions used.
11) AMES Mutagenic assay
Protocol assays
Mutagenic activity of test compounds and their metabolites (produced by rat liver S9
fraction) was evaluated in the Salmonella typhimurium strains TA100 and TA98
according to the Ames assay (Toxicology in vitro, (2001), 15: 105-114). Both strains
were treated with various concentrations of test compounds ranging from 0.5 to
1000pg and incubated overnight at 37°C. Analysis of mutagenicity was then performed
against TA100 and TA98 strains in comparison to positive controls 2-Nitrofluorene
(TA98) and Sodium Azide (TA100) without S9 fraction, and 2-Anthramine with rat S9
mix (TA98 and TA100). The results are expressed as a mutagenicity ratio in
comparison to solvent controls. A test substance was considered as active if
mutagenicity ratio is >2.
Experimental results
The numbers of revertant colonies per plate in TA98 and TA100 strains treated with
compound 001 are reported in Table and Table respectively. The results are
expressed as a ratio of the number of colonies over the number of colonies in the
negative control (DMSO). The test substance was active if mutagenicity ratio is >2.
Table 3: Revertant colony numbers per plate using TA98 strain treated with compound
001
Compound Dose level per S-9 Mean revertant SD Ratio
well ^g) fraction colony counts treated/solvent
DMSO - 4.3 3.2
0.5 - 7.3 3.2 1.7
1.4 - 4.3 1.2 1.0
4.1 - 6.3 1.5 1.5
Compound 12.3 - 5.3 0.6 1.2
001 37 - 8.3 3.5 1.9
111.1 - 6.3 1.5 1.5
333.3 - 4.3 3.2 1.0
1000 - 3.7 4.0 0.8
2-NF 0.25 - 59.7 8.5 13.8
DMSO + 2.7 0.6
0.5 + 10.3 1.2 3.9
1.4 + 7.3 2.1 2.8
4.1 + 9.3 1.5 3.5
Compound 12.3 + 6.7 4.0 2.5
001 37 + 5.7 0.6 2.1
111.1 + 7.3 3.2 2.8
333.3 + 6.3 5.1 2.4
1000 + 5.0 1.7 1.9
2-AM 1 + 368.3 61.4 138.1
Table 4 : Revertant colony numbers per plate using TA100 strain treated with
compound 001
Compound Dose level per S-9 Mean revertant SD Ratio
well^g) fraction colony counts treated/solvent
DMSO - 26.3 6.4
0.5 - 32.0 4.4 1.2
1.4 - 24.7 11.7 0.9
4.1 - 23.7 1.5 0.9
Compound 12.3 - 21.0 7.8 0.8
001 37 - 21.0 3.6 0.8
111.1 - 26.7 2.5 1.0
333.3 - 23.7 2.3 0.9
1000 - 32.0 4.6 1.2
NAN3 1 - 206.0 21.3 7.8
DMSO + 25.7 5.0
0.5 + 26.7 10.7 1.0
1.4 + 26.0 8.7 1.0
4.1 + 26.7 5.0 1.0
Compound 12.3 + 25.3 4.2 1.0
001 37 + 20.7 4.2 0.8
111.1 + 28.0 7.0 1.1
333.3 + 28.0 3.5 1.1
1000 + 19.0 13.9 0.7
2-AM 1 + 463.3 129.8 18.1
- Absence of S-9, + Presence of S-9, 2-NF= 2-Nitrofluorene, NAN3= Sodium Azide, 2-AM= 2-
Anthramine
Comments on the experiments and results
Noteworthy increases in the number of revertants were observed in the TA98 strain
with S9 mix at all tested doses. The increases exceeded the threshold of 2 fold the
vehicle control value but they were not dose-related. More over the number of
revertants and the corresponding individual revertants colony counts observed at these
dose-levels remained within the historical range for the corresponding vehicle control.
Consequently, these increases were not considered biologically relevant.
Under the experimental conditions of the studies, the test compound 001 did not show
any mutagenic activity with or without liver metabolic activation system (S9 mix).
12) Early bioavailability determination
Protocol assays
Early screen was conducted to estimate plasma concentrations of test compounds
obtained after oral or intravenous administration to Sprague Dawley rats. DMSO was
used as vehicle for oral and intravenous administration. In short, 4 male Sprague
Dawley rats around 5 weeks old were used. Drugs were administered PO (lOmg/kg) or
IV (2mg/kg). Blood samples were collected at defined time schedule and analysed
using LC-MS/MS determination for PK parameters calculations.
Experimental results
PK parameters were calculated based on mean data and are presented in Table .
Table 5: PK parameters calculated based on mean data
AUCt(ng/mL*h)= area under the plasma concentration-time curve from administration up to the
last quantifiable concentration at time t , Absolute bioavailability^ F(%) = (AUC PO/dose PO)/
(AUC IV/dose IV)*100.
Comments on the experiments and results
Compound 001 had excellent pharmacokinetic properties with a bioavailability of
97.5%.
I n these in vitro and in vivo studies, the inventor observed a very effective inhibition of
SYK kinase activity by the class of compounds of formula (I) as presently disclosed.
Using established models in toxicological and physiological test systems to evaluate in
vitro cardiotoxicity, mutagenicity and biodisponibility, the inventors demonstrated that
anti-SYK compounds of formula (I) had good safety prolife.
While the present invention has been described with reference to the specific examples
thereof, it should be understood by those skilled in the art that various changes may
be made and equivalents may be substituted without departing from the true spirit and
scope of the invention. In addition, many modifications may be made to adapt a
particular situation, material, composition of matter, process, process step or steps, to
the objective, spirit and score of the present invention. All such modifications are
intended to be within the scope of the claims appended hereto.

Claims:
1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:
I )
wherein
Rl, R2, R3 and R4 are each independently selected from:
hydrogen,
cyano,
CF3,
halogen,
an alkyl group optionally substituted with a heterocycle,
an alkoxy group optionally substituted with a heterocycle,
a solubilising group,
a heterocycle,
-CO-NRR',
-S02-NRR',
-NRR',
-NR-CO-R' and
-NR-S02R' group
wherein R and R' are each independently hydrogen or alkyl group;
W is aryl or heteroaryl group, unsubstituted or substituted by one or more substituents
selected from:
cyano,
CF3,
halogen,
an alkyl group optionally substituted with a heterocycle,
a cycloalkyl group,
an alkoxy group optionally substituted with a heterocycle,
an aryl group,
a heteroaryl group,
a heterocycloalkyl group,
a solubilising group,
-CO-NRR',
-S02-NRR',
-NRR',
-NR-CO-R' and
-NR-S02R' group,
wherein R and R' are each independently hydrogen or alkyl group.
X is selected from the group consisting of 0 , S, N(R5), N[C(=0)R6] and (CH2)n
wherein n is 0, 1 or 2, R5 and R6 are each independently hydrogen or Cl-4alkyl group;
Y is (CH2)m wherein m is 1, 2, 3 or 4;
Z is (CH2)p wherein p is 1 or 2.
2. The compound according to claim 1 or a pharmaceutically acceptable salt
thereof, wherein X is (CH )n, n is 0, 1 or 2 and m and p are 1.
3. The compound according to claims 1 or 2 or a pharmaceutically acceptable salt
thereof, wherein W is a substituted heteroaryl.
4. The compound according to claim 3 or a pharmaceutically acceptable salt
thereof, wherein the heteroaryl is a 5-8 membered monosubstituted, monocyclic ring
containing at least one nitrogen atom.
5. The compound according to claim 4 or a pharmaceutically acceptable salt
thereof, wherein the heteroaryl is pyrimidin-2-yl.
6. The compound according to any one or more of the preceding claims or a
pharmaceutically acceptable salt thereof, wherein each of the one or more substituents
of W is independently selected in the group consisting of:
cyano,
CF3,
halogen,
an alkyl group optionally substituted with a heterocycle,
a cycloalkyl group,
an alkoxy group optionally substituted with a heterocycle,
an aryl group,
a heteroaryl group and
a heterocycloalkyl group.
7. The compound according to any one or more of the preceding claims or a
pharmaceutically acceptable salt thereof, wherein at least three of Rl, R2, R3 and R4
are hydrogen.
8. The compound according to any one or more of the preceding claims having
formula (II) or a pharmaceutically acceptable salt thereof,
wherein Rl, R2, R3, R4 and W are as defined in any one or more of claims 1 to 7, X is
(CH2)n and n is 0, 1 or 2.
9. The compound according to claim 8 having formula (III) or a pharmaceutically
acceptable salt thereof,
(III)
wherein Rl, R2, R3, R4 and X are as defined in claim 8, and R7 is selected from the
group consisting of:
hydrogen,
cyano,
CF3
halogen,
an alkyl group,
a cycloalkyl group,
an alkoxy group,
an aryl group,
a heteroaryl group,
a heterocycloalkyl group,
a solubilising group, and
-NRR' group wherein R and R' are each independently hydrogen or an alkyl group.
10. The compound according to claim 1 selected from the group consisting of:
4-{5-[3-(4-Methyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-
8-one;
2-{3-[2-(8-Oxo-4,7-diaza-spiro[2.5]oct-4-yl)-oxazol-5-yl]-phenylamino}-pyrimidine-4-
carbonitrile;
4-{5-[3-Morpholin-4-ylmethyl-5-(4-trifluoromethyl-pyrimidin-2-ylamino)-phenyl]-oxazol-
2-yl}-4,7-diaza-spiro[2.5]octan-8-one;
4-{5-[2-(2-Morpholin-4-yl-ethoxy)-5-(4-thiophen-2-yl-pyrimidin-2-ylamino)-phenyl]-
oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-8-one;
4-{5-[3-Methyl-5-(4-trifluoromethyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7
diaza-spiro[2.5]octan-8-one;
4-{5-[3-(4-Methyl-pyrimidin-2-ylamino)-5-morpholin-4-ylmethyl-phenyl]-oxazol-2-yl}-
4,7-diaza-spiro[2.5]octan-8-one;
4-{5-[3-(4-Ethyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-8-
one;
4-{5-[3-(4-Trifluoromethyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
6-{5-[3-(4-Methyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-6,9-diaza-spiro[4.5]decan-
10-one;
4-{5-[3-(4-Phenyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-
8-one;
5-{5-[3-(4-Isopropyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-5,8-diazaspiro[
3.5]nonan-9-one;
5-{5-[3-(4-Methyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-5,8-diazaspiro[
3.5]nonan-9-one;
4-{5-[3-(4-Isopropyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
4-{5-[3-(4-Morpholin-4-yl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
4-{5-[3-(4-Thiophen-2-yl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
5-{5-[3-(4-Trifluoromethyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-5,8-diazaspiro[
3.5]nonan-9-one;
4-{ 5-[2-(2-Morpholin-4-yl-ethoxy)-5-(4-t rifl uoromethyl-pyrimidin-2-ylamino)-phenyi]
oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-8-one;
4-{5-[2-Fluoro-5-(4-methyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
4-{5-[3-(4-Pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
4-{5-[2-Methyl-3-(4-methyl-pyrimidin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diazaspiro[
2.5]octan-8-one;
4-{5-[3-(4-Methyl-pyridin-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diaza-spiro[2.5]octan
one;
4-{5-[3-(Thiazol-2-ylamino)-phenyl]-oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-8-one;
and a pharmaceutically acceptable salt thereof.
11. The compound according to claim 10 which is 4-{5-[3-(4-Methyl-pyrimidin-2-
ylamino)-phenyl]-oxazol-2-yl}-4,7-diaza-spiro[2.5]octan-8-one or a pharmaceutically
acceptable salt thereof.
12. A pharmaceutical composition comprising a compound according to any one or
more of the preceding claims, or a pharmaceutically acceptable salt thereof and one or
more pharmaceutically acceptable excipients and/or carriers.
13. The pharmaceutical composition according to claim 12, comprising a compound
as defined in any one or more of claims 1 to 11, or a pharmaceutically acceptable salt
thereof as sole active pharmaceutical ingredient or in combination with another active
pharmaceutical ingredient.
14. A process for the manufacture of a compound as defined in any one or more of
claims 1 to 11, or a pharmaceutically acceptable salt thereof, said process comprising a
step of reacting a compound of formula (i)
with a compound of formula W-G,
wherein Rl to R4, W, X, Y and Z are as defined in an one or more of claims 1 to 9, and
G is halogen.
15. A process for the manufacture of a compound as defined in any one or more of
claims 1 to 11, or a pharmaceutically acceptable salt thereof, said process comprising a
step of reacting a compound of formula (ii)
with a compound of formula (iii)
wherein Rl to R4, W, X, Y and Z are as defined in an one or more of claims 1 to 9.
16. A compound according to any one or more of claims 1 to 11, or a
pharmaceutically acceptable salt thereof, for use as a medicament.
17. A compound according to any one or more of claims 1 to 11, or a
pharmaceutically acceptable salt thereof, for use in treating a disease or disorder
associated with unregulated or deregulated tyrosine kinase activity.
18. A compound according to any one or more of claims 1 to 11, or a
pharmaceutically acceptable salt thereof, for use in treating a disease or disorder
associated with signal transduction mediated by SYK.
19. The compound according to claim 17 or 18, wherein the disease or disorder is
selected from the group consisting of hematological disorders, proliferative disorders,
autoimmune disorders, metabolic disorders, inflammatory diseases, allergic diseases
and neurological diseases.
20. The pharmaceutical composition according to claim 13, comprising a compound
defined in any one or more of claims 1 to 11, or a pharmaceutically acceptable salt
thereof, and another active pharmaceutical ingredient as a combined preparation for
sequential, simultaneous or separate use in the treatment of a disease or disorder
selected from the group consisting of hematological disorders, proliferative disorders,
autoimmune disorders, metabolic disorders, inflammatory diseases, allergic diseases
and neurological diseases.